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el autor hace mención los ejes fundamentales de la investigación en donde habla del tema, problema y la metodología lo cual es muy preciso y claro para tener la misma idea que el autor.

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es muy importante resaltar que el autor menciona que una de las principales dificultades al iniciar una investigación o una tesis es saber escoger el nombre del título y concuerdo con el ya que como hemos mencionado en clase guía a nosotros como investigadores a relacionarlo con todo el proyecto.

Comentario #1: Para mí es muy interesante lo que se menciona acerca del proceso de investigación científica, ya que menciona que la investigación es una estrategia para conocer y actuar. Tiene mucho sentido para mí la forma en que se define, ya que la investigación a nosotros como seres humanos no ha ayudado a enriquecernos y ampliar nuestra capacidad de pensamiento.

Comentario #2: "Toda indagación debe surgir de una motivación del alumno que la realiza". Esta parte para mí es la parte más importante para que se lleve a cabo una investigación, ya que si no hay interés en el tema probablemente la investigación no será fructífera.

Comentrio #3: Se menciona que el punto de partida de una investigación es el problema, y para mí además de que sea el punto de partida también es en donde la investigación se debería de enfocar al 100% con el fin de resolver la hipótesis de la problemática.

Comentario 1: El texto inicia con una muy buena explicación sobre lo que es una investigación científica dando a entender que una forma de conocer las perspectivas y manejando 3 dimensiones complejas que informan, posponen y relacionan.

Comentario 2: Muestra el proceso de una investigación que resalta el hacer preguntas iniciales hasta realizar las hipótesis, trabajo de campo, la recolección y análisis de los datos obtenidos, la figura 1 muestra a detalle la relación de todo el proceso con los objetivos.

Comentario 3: Nos muestra que los objetivos de dicha investigación es lo que se desea obtener y que estos deben de estar formulados con la claridad para que sean coherentes, creo es de suma importancia resaltar que es lo que se desea y quiere mostrar una investigación

COMENTARIO 2: Hace mucho énfasis en diversos tipos de metodologías y eso es muy valioso la verdad, pero la falta de una estructura más organizada provoca que el mensaje principal la importancia de elegir bien el tema y plantear correctamente el problema se pierda entre tantas explicaciones.

COMENTARIO 1: El texto explica muy bien el proceso de investigación, pero resulta demasiado extenso y cargado de información esto puede hacer que uno como lector se abrume o nos perdamos un poco y no nos quede muy claro

Responder a las preguntas sobre la importancia, relevancia y beneficios del problema permite no solo darle sentido al trabajo, sino también mostrar el impacto que puede tener en la sociedad, en un área del conocimiento o en la práctica profesional. En mi opinión, una buena justificación le da credibilidad al estudio y motiva tanto al investigador como a quienes consulten sus resultados.

Coincido con Ander-Egg respecto a que la investigación realmente nace de la necesidad de resolver dudas o problemáticas que enfrentamos en nuestro día a día. El hecho de que todo proceso investigativo parta de una situación-problema nos recuerda que la investigación no es algo lejano ni exclusivo de los académicos, sino que está presente en cualquier persona que se cuestiona el porqué de las cosas y busca respuestas.

Coincido con este punto ya que una vez definido el tema se tiene que profundizar buscando información en diferentes sitios o diferentes fuentes esto ayuda a delimitar y sustentar el problema, es de gran ayuda ya que podemos evitar la duplicación de trabajos.

Aquí se presentan dos aspectos sumamente importantes el objetivo y la justificación, se sabe que los objetivos son nuestros guías para saber hacia dónde llevar nuestra investigación, por otra parte, la justificación responde el porqué del trabajo o investigación, en general estos dos apartandos garantizan que nuestra investigacion tenga un proposito.

Primero se tiene que tener un interés del investigador ya que esto sirve de motivación para saber hacia donde va su trabajo o hacia donde lo quiere llevar, este interés despertara curiosidad por aprender más le invertirá tiempo y esfuerzo para profundizar y alcanzar su objetivo.

El texto describe como la investigación no es un proceso lineal, sino una dinámica más compleja y avanzada y se va corrigiendo según lo encontrado se decide si se corrige o si se sigue avanzando, en este aspecto se resalta la importancia de la reflexión contante y la adaptación a la investigación.

La investigación como herramienta fundamental para comprender y transformar la realidad, la investigación no solo cumple un papel científico académico, sino también social ya que nos permite desenvolvernos de mejor manera en las dimensiones que se marcan.

Tenemos que hacer un análisis sobre el problema que estamos planteando para conocer si realmente es un problema. Ya que en esta primera investigación se va a describir el contexto ya sea el ámbito, la duración y la relevancia, para la formulación de este debe ser precisa y viable.

por lo que entendi del tema de La situación y el campo problemático son la base para poner en contexto o en situacion una investigación, ya que permiten entender de dónde surge el problema y cuáles son los factores que lo rodean

El proceso de investigación científica es fundamental porque permite transformar dudas o problemas en conocimiento válido y confiable

Me gusta que tambien nos da una guía clara sobre cómo plantear un problema de investigación, pero lo aborda de manera muy técnica y rígida. En la práctica, este proceso no solo es seguir pasos, sino también un ejercicio de reflexión crítica y creatividad que conecta teoría

Este texto plantea un problema real y recurrente en la formación universitaria: la dificultad que enfrentan los estudiantes para seleccionar un tema de investigación y formular adecuadamente el planteamiento del problema y tiende a enfocarse más en señalar las carencias del estudiante que en analizar críticamente el papel de la institución y del docente en este proceso

1.- El texto destaca que, a pesar de la diversidad de enfoques, todos los procesos de investigación comparten tres pilares esenciales: tema, problema y metodología.

2.- Se identifican obstáculos comunes que enfrentan los alumnos, como la confusión entre conocimiento científico y sentido común.

3.- El escrito subraya que un mal planteamiento del problema puede bloquear todo el proceso investigativo.

Al cierre del texto se resalta que la investigación no concluye de manera definitiva, sino que deja abiertas nuevas preguntas y caminos para futuros estudios. Este punto suele pasar desapercibido, pero es fundamental porque muestra que el conocimiento científico en las ciencias sociales nunca se agota. Más que ofrecer respuestas cerradas, la investigación abre horizontes y fomenta una actitud crítica y reflexiva en el investigador.

El artículo explica que no se avanza de forma lineal, sino que se retrocede constantemente para replantear preguntas, ajustar objetivos o redefinir enfoques. Esto refleja que la investigación no es estática, sino dinámica, y permite ver los retrocesos como parte del aprendizaje y no como errores definitivos.

El texto subraya que la elección del tema de investigación es el punto de partida esencial en cualquier proyecto académico. No se trata únicamente de seleccionar algo que interese al estudiante, sino de considerar factores como la disponibilidad de bibliografía, la comprensión de los conceptos implicados y la factibilidad metodológica. De esta manera, la investigación no solo será significativa en lo personal, sino también realizable en la práctica y con valor científico.

Otra parte que me parece sumamente interesante es la manera en la que el texto nos indica como se debe plantear una pregunta, el cual es uno de los pasos principales al comenzar una investigación. Se menciona que las principales preguntas son demasiado abstractas y generales, ya que al comenzar solo se tiene una pequeña noción sobre el tema que se quiere investigar, sin embargo es de suma importancia que estas preguntas iniciales se enfoquen a la búsqueda de una respuesta más puntual, es decir, debemos aprender a realizar preguntas más especificas y especializadas, debido a que está pregunta funcionará como uno de los puntos principales sobre los cuáles vamos a comenzar a investigar.

La representación gráfica de como debe ser un correcto proceso de investigación me parece sumamente útil, pues muchas veces pensamos y concebimos a la investigación como un proceso lineal, en el cual se busca solucionar una problemática, para posteriormente realizar una investigación y obtener una respuesta, sin embargo la figura nos muestra que muchas veces el proceso de investigación puede ser repetitivo, pues a medida que vamos investigando y obteniendo más información, podemos ver el tema de investigación con un enfoque más crítico, generando nuevas interrogantes, hipótesis, planteamientos y conclusiones.

En base a mi propia experiencia coincido completamente con lo que expresa el texto, pues muchas veces el problema al realizar el desarrollo de una investigación no es la investigación en si (ya que al plantear una metodología clara se puede tener una mejor noción sobre como se va a trabajar y los objetivos que se busca alcanzar) pues el principal problema es plantear la problemática inicial de la investigación, así como encontrar soluciones creativas, originales e innovadoras que puedan dar una base solida y sustento a nuestra investigación.

Dice que plantear el problema de investigación no es solo tener una idea, sino estructurarla de manera clara, concreta y organizada. Me parece muy útil la recomendación de formular preguntas relacionadas con el tema, jerarquizarlas y construir una red conceptual, porque así se clarifica el objeto de estudio y se delimita la investigación.

Ayuda a entender que investigar es un proceso ordenado y reflexivo, donde identificar la situación problemática y formular el problema correctamente es fundamental. Me parece interesante que resalte que todo conocimiento científico surge de preguntas claras y que, sin una buena formulación del problema, es difícil avanzar en la investigación de manera sólida.

Por eso es importante conocer la historia para poder entender los problemas, buscar soluciones y no repetir los errores cometidos en el pasado.

Es importante tener claro el tema, el problema y la metodología en una investigación. Nos podemos llegar a confundir porque hay muchas formas de hacer investigación, pero al final todo depende de plantear bien el problema y seguir un proceso ordenado. También me parece interesante el proceso en espiral de la investigación donde se aprende y se mejora constantemente.

En la parte del texto donde se dice: "Antes, tiene que for mular el problema específico en términos concretos y explícitos", es decir que debes de conocer primeramente que es lo que estas buscando con exactitud y en qué grado. Cómo resultado encontraras una respuesta más satisfaria y aterrizada a lo que encuentras.

Es muy importante tomas en cuentas estas tres dimensiones cuando se quiere investigar ya que los seres humanos somos seres sociales y estas dimensiones nos pueden ayudar mucho a entender como nos relacionamos y a su vez nos permite entender el porque de muchos problemas que se originan.

Una de las principales ventajas que adquieres al investigar es conocer la realidad y los antecedentes que preceden los sucesos. Conocer todo esto es una ventaja, principalmente para no cometer los mismos errores que las personas que nos prescindieron, si no que tambien es una forma de impulsarnos a tomar una perspectiva diferente a las problemáticas que se pudieran encontrar en función a lo previamente establecido.

No sabia el significado de epistemología, así que la busque, significa ciencia que indaga lo científico, reflexionando la profundidad del conocimiento, su origen, su forma y como debería ser.

Creo que una de las fundamentales cosas que se tiene que hacer a la hora de investigar un tema es conocer del mismo, como resultado de eso se tiene que hacer una investigación previa en la cual puedas conocer todo acerca del tema, no es necesario volverse un experto, pero tienes que entender de lo que estás hablando para saber qué es lo que estás haciendo y el porque de tu investigación.

la hipótesis central del presente trabajo es que los actuales planes de estudio de las licenciaturas en Administración en México están articulados en función de los procesos administrativos tradicionales y el estudio de cuatro o cinco de las áreas denominadas "funcionales" de la administración. Esta estructura curricular se complementa con una serie de materias básicas de carácter cuasipropedéutico y otro conjunto de materias especializadas, ya sean optativas o pertenecientes a un núcleo introductorio, que dependen más del capricho de las autoridades universitarias en turno o de las modas administrativas, que de un análisis sustentado en las necesidades de los mercados de trabajo o las realidades locales, regionales o nacionales. Pág. 4 Pdf y Pág. 125

$$Opinión Personal: $$El planteamiento de este articulo es acertado, la administración, al estar tan vinculada con la práctica profesional, debe actualizarse constantemente según las demandas del entorno, social, económico y profesional. Mas sin embargo, los programas universitarios tienden a ser sistemas cerrados ya que repiten esquemas que ya no corresponden a la realidad. En mi opinión, el artículo señala un problema que trasciende incluso hoy en día muchas universidades aún forman administradores con un perfil demasiado teórico y poco eficaz, dejando de lado habilidades requeridas en este campo, pensamiento crítico, visión global y responsabilidad social. Para responder a la globalización, se necesita un modelo más flexible, práctico e interdisciplinario.

La Administración es una disciplina relativamente reciente en México, con poca tradición en investigación y orientada principalmente hacia las áreas docentes. Se enseñan generalmente modelos idealizados desde la optimización hasta la excelencia ; los cuales representan la parte "dura", "científica" relativamente fácil de transferir, pero difícil de operar. La Administración está asentada en organizaciones concretas, alejadas de las condiciones de idealidad de dichos modelos. No se puede estudiar la Administración en abstracto, ésta tiene que ser referida siempre a las condiciones particulares de las organizaciones que aplican esos dispositivos. Pág. 7 Pdf y Pág. 128

$$$$

¿Qué opina el autor acerca del liderazgo?

El autor opina que el liderazgo es una habilidad esencial para los administradores en el nuevo entorno laboral, ya que con este es de las partes clave que debería formar parte central de los programas de Administración, junto con la toma de decisiones, creatividad y solución de problemas.

Opinión de la hipótesis: Considero que la hipótesis es acertada porque demuestra que la formación en administración sigue centrada en lo tradicional y no prepara lo suficiente en competencias como liderazgo e innovación, esenciales en el contexto actual. Pienso que sí es necesario que se reestructure para que además de lo básico, nos den herramientas que nos permitan ser líderes, propositivos y capaces de adaptarnos a entornos dinámicos.

Luis Montaño: La afirmación de Luis Montaño al señalar que la Administración en México es una disciplina relativamente reciente, con poca tradición en investigación y más enfocada en la docencia. Esta situación limita la posibilidad de que la Administración se consolide como un campo académico fuerte y con identidad propia en el país, ya que dependemos mucho de modelos y marcos conceptuales extranjeros. Sería necesario fortalecer la investigación en esta área para que, además de formar profesionales competentes, también se produzca conocimiento que aporte soluciones innovadoras y contextualizadas a los problemas organizacionales y sociales de México.

¿Qué opina el autor acerca del liderazgo? Él autor considera que el liderazgo es una competencia fundamental para la formación de administradores en el contexto actual. Para él, formar en liderazgo implica preparar a los estudiantes no solo para dirigir equipos, sino también para ser agentes de cambio, capaces de adaptarse, innovar y tomar decisiones en entornos complejos y globalizados. En su opinión, el liderazgo debe enseñarse como una práctica que combina valores, iniciativa y responsabilidad, no como un simple complemento académico.

hipótesis es que los actuales planes de estudio de las licenciaturas de administración en México están articulados en funciones de los procesos administrativos tradicionales y el estudio de cuatro o cinco de las áreas denominadas "funcionales" de la administración y esas están en estructura curricular es una serie de materias básicas y un conjunto de materias especializadas ya sea en operativas o pertenecientes a un núcleo introductorio. Mi opinión es de que ese plan de estudio no esta alineado con las necesidades del mercado laboral actual lo que le causaría problema a los recién egresados.

Luis Montaño que tiene poca tradición en investigación y se basan en procesos administrativos y tradicionales si base solida en investigación y análisis critico, la orientación va enfocada solo a la docencia.

¿Qué opina el autor acerca del liderazgo? La capacidad de liderar y gestionar equipos de manera efectiva en entornos complejos y globales, se trata de habilidades que permiten a los profesionales de la administración .

la hipótesis del autor es que las carreras de Administración en México todavía se basan mucho en lo tradicional, o sea en el proceso administrativo clásico (planeación, organización, dirección y control), y casi no incluyen materias más actuales como liderazgo, creatividad, toma de decisiones o trabajo en equipo. Lo que él dice es que por eso los estudiantes no salen tan preparados para el mundo globalizado en el que vivimos. Yo pienso que tiene razón, porque si solo nos enseñan lo viejo y no se actualiza el plan de estudios, al salir puede que no tengamos las herramientas necesarias para enfrentar lo que de verdad pasa en las empresas.

Sobre Luis Montaño, él afirma que la Administración en México es una disciplina relativamente nueva, con poca tradición en investigación, y que está más enfocada en la parte de dar clases que en generar conocimiento propio. También critica que se usan muchos modelos “ideales” que vienen de otros países, y que no siempre sirven para la realidad mexicana. Eso me parece cierto, porque muchas veces lo que vemos en libros extranjeros no encaja con lo que pasa en nuestras empresas o nuestra cultura.

Finalmente, acerca del liderazgo, el autor dice que en la mayoría de los programas de estudio casi no se enseña, aparece solo como optativa o muy de lado. Sin embargo, él lo considera súper importante, porque el administrador no solo debe saber de números o procesos, también necesita dirigir personas, motivar y trabajar con valores. Yo estoy de acuerdo, porque sin liderazgo un administrador difícilmente va a poder guiar a un equipo o generar un buen ambiente de trabajo.

¿Qué opina el autor acerca del liderazgo? El autor considera que el liderazgo, tal como se enseña tradicionalmente en la administración, suele ser un concepto idealizado y alejado de la realidad. Para él, el liderazgo debe entenderse como una práctica contextualizada, que requiere habilidades concretas y adaptabilidad para enfrentar situaciones complejas y específicas del entorno, en lugar de seguir un modelo rígido o universal.

El autor nos da a entender que la administración es una disciplina relativamente reciente en México, en gran parte porque se ha enfocado más en la enseñanza que en la investigación académica. Los planes de estudio están basados en modelos tradicionales e importados que no se adaptan a las particularidades del contexto nacional, lo cual limita el desarrollo de estudios relevantes y una formación ajustada a las necesidades del mercado local.

El autor critica que los planes de estudio de la carrera de Administración en México están estancados. Básicamente, se enfocan en lo mismo de siempre: procesos administrativos clásicos y unas ciertas áreas como finanzas, mercadotecnia o recursos humanos. Además, muchas materias que se agregan parecen elegirse por moda o por decisión de los que están a cargo, más que por un análisis real de lo que el país o el mercado laboral necesitan. Considero que su critica es válida, ya que la formación profesional debe responder a las transformaciones sociales, económicas y organizacionales actuales. De lo contrario, se corre el riesgo de formar profesionales poco preparados para enfrentar los retos del entorno real.

los actuales planes de estudio de las licenciaturas en Administración en México están articulados en función de los procesos administrativos tradicionales y el estudio de cuatro o cinco de las áreas denominadas "funcionales" de la administración. Esta estructura curricular se complementa con una serie de materias básicas de carácter cuasipropedéutico y otro conjunto de materias especializadas, ya sean optativas o pertenecientes a un núcleo introductorio... - página 4 del .pdf y 125 del artículo

En paráfrasis la hipótesis que expone el autor es que el sistema educativo ha tomado un enfoque más centrado en formas obsoletas de enseñar la administración, esto en vez de adaptarse a los contextos temporales, económicos y políticos que le concierne a nuestra sociedad.

Se enseñan generalmente modelos idealizados desde la optimización hasta la excelencia ; los cuales representan la parte "dura", "científica" relativamente fácil de transferir, pero difícil de operar. La Administración está asentada en organizaciones concretas, alejadas de las condiciones de idealidad de dichos modelos. No se puede estudiar la Administración en abstracto, ésta tiene que ser referida siempre a las condiciones particulares de las organizaciones que aplican esos dispositivos. - página 7 del .pdf y 128 del artículo

Este fragmento indica que a la hora de realizarse el proceso de enseñanza los subtemas de administración es en base a presunciones de utopías organizacionales, mientras que la materia debería de estar aterrizada en las aplicaciones reales en rubros laborales.

La dirección por valores (DpV) es una herramienta de liderazgo que va más allá de los conceptos de dirección por instrucciones y dirección por objetivos que la precedieron, y pretende darle un sentido más ético y emocional a la construcción de empresas. - página 10 del .pdf y 131 del artículo

Aquí el autor plantea un enfoque innovador acerca del liderazgo, consiste de un acercamiento al empleado en base a los axiomas de moral social, con reciprocidad, como se sobreentiende, en lugar de dar el enfoque anticuado de presentar indicaciones y objetivos. Sin embargo quiero recalcar que no se debe de confundir un enfoque humanístico de una empresa con ciertas practicas de manipulación emocional que toman determinadas compañías para atar a sus empleados, esta propuesta del autor busca un compromiso real para generar una relación con limites establecidos entre empleador y empleado donde se tenga siempre presente el contrato entre ambas partes (obligaciones y derechos laborales) y asegurarse de que se cumplan mediante el uso de esta estrategia novedosa.

los actuales planes de estudio de las licenciaturas en Administración en México están articulados en función de los procesos administrativos tradicionales y el estudio de cuatro o cinco de las áreas denominadas "funcionales" de la administración. Esta estructura curricular se complementa con una serie de materias básicas de carácter cuasipropedéutico y otro conjunto de materias especializadas, ya sean optativas o pertenecientes a un núcleo introductorio, que dependen más del capricho de las autoridades universitarias en turno o de las modas administrativas, que de un análisis sustentado en las necesidades de los mercados de trabajo o las realidades locales, regionales o nacionales.

La Administración está asentada en organizaciones concretas, alejadas de las condiciones de idealidad de dichos modelos. No se puede estudiar la Administración en abstracto, ésta tiene que ser referida siempre a las condiciones particulares de las organizaciones que aplican esos dispositivos. Pag. 128 del artículo

La hipótesis del autor, José Luis Pariente Fragoso, es que los planes de estudio actuales de las licenciaturas en Administración en México no están alineados con las tendencias y exigencias del nuevo entorno globalizado. El contenido bibliográfico que nos llega de otros lados como seria Estados Unidos, no se adapta a nuestra realidad aquí en México; además de que las organizaciones piden habilidades y conocimientos muy alejados a lo que podremos ver en la escuela. Por último, el autor nos dice que el liderazgo es una cualidad que solo se menciona, pero no se ejerce de manera tanta práctica, es una cualidad secundaria y opcional. Al final, la escuela está estructurada para crear empleados, no para crear visionarios y gente líder.

1. Hipótesis del autor y justificación Hipótesis: La formación de los administradores en México no se ajusta a las demandas del entorno internacional actual, ya que los planes de estudio se estructuran en torno al proceso administrativo tradicional y las áreas funcionales, dejando de lado habilidades clave como liderazgo, toma de decisiones, creatividad y conocimiento intercultural.

Justificación:

El autor analizó 22 programas de licenciatura en Administración acreditados por CACECA.

Encontró que la mayoría de las materias se centran en:

Proceso administrativo (planeación, organización, dirección, control).

Áreas funcionales (mercadotecnia, finanzas, producción, recursos humanos).

Las materias relacionadas con habilidades blandas (liderazgo, toma de decisiones, ética, etc.) aparecen de forma marginal o optativa.

Critica la dependencia de bibliografía extranjera (principalmente estadounidense), que no siempre se adapta a la realidad mexicana.

Señala que los egresados no están preparados para enfrentar problemas complejos, multiculturales y no cuantificables.

Opinión: La hipótesis es sólida y está bien sustentada. El autor evidencia una desconexión entre la formación académica y las necesidades reales del mercado global. Es crucial que los programas de administración integren más contenidos sobre habilidades transversales, contexto local e internacional, y ética profesional.

1. Luis Montaño: ¿Por qué afirma que la Administración en México es una disciplina reciente con poca investigación? Luis Montaño (citado por Pariente) argumenta que:

La administración en México tiene poca tradición investigadora y está orientada principalmente a la docencia.

Se enseñan modelos idealizados (optimización, excelencia) que son difíciles de aplicar en la realidad organizacional mexicana.

Existe una falta de contextualización: los modelos extranjeros no siempre se adaptan a las condiciones sociales, económicas y culturales de México.

Hay una escasa producción académica nacional en administración, lo que obliga a depender de literatura traducida, often poco adecuada.

Reflejo en el texto: Pariente respalda esta visión al señalar la ausencia de investigaciones y publicaciones de autores mexicanos, así como la falta de materias que aborden la realidad nacional y regional.

1. ¿Qué opina el autor acerca del liderazgo? El autor considera que el liderazgo es una de las habilidades esenciales que los administradores deben desarrollar para enfrentar los nuevos entornos internacionales. Sin embargo, destaca que:

El liderazgo es una materia poco frecuente en los planes de estudio analizados (solo aparece en el 20% de los programas).

Cuando se incluye, often forma parte de bloques optativos o complementarios, no del núcleo central.

Propone que el liderazgo debe integrarse junto con otras habilidades como la toma de decisiones, la creatividad y el trabajo en redes.

Además, el autor valora el enfoque de dirección por valores (Blanchard y O’Connor) como una herramienta clave para un liderazgo ético y efectivo.

Hipótesis del autor y opinión personal

-los actuales planes de estudio de las licenciaturas en Administración en México están articulados en función de los procesos administrativos tradicionales y el estudio de cuatro o cinco de las áreas denominadas "funcionales" de la administración…- Página 4.

Los autores Pariente Fragoso y José Luis proponen como su hipótesis que, las licenciaturas de administración de México no se encuentran actualizadas antes las nuevas situaciones y factores que pasa el país y el mundo, manteniendo una enseñanza obsoleta. Hipótesis la cual yo me encuentro de acuerdo, debido a que, es muy usual que varias licenciaturas se vean en la misma posición de solo centrarse en la enseñanza teórica e introductoria (como ejemplos de licenciaturas podemos usar, pedagogía, filosofía, historia, etc) afectando no solo al aprendizaje de los estudiantes y egresados al momento de tener que enfrentarse al campo laboral real, no solo eso, si no que también afectando al mismo sistema (ya sea económico, politico y/o el social) que nos rige debido a la ineficiencia que ellos presentan, creando un ciclo de ineptitud de progresión en nuestro país.

¿Por qué Luis Montaño afirma que la Administración es una disciplina relativamente reciente en México, con poca tradición en investigación y orientada principalmente hacia las áreas docentes?

-con poca tradición en investigación y orientada principalmente hacia las áreas docentes. Se enseñan generalmente modelos idealizados…- Página 7.

Porque Luis Montaño explica que la forma la forma en la que se lleva a cabo la enseñanza de la administración es debido a solamente se enseña de forma aislada de situaciones reales (las cuales cuentan con cambios graduales o repentinos), solo ofreciendo la vista de un modelo idealizado y aislado. Es decir, que solo se enseña lo que es la administración como un concepto, forma que solo funciona cuando se trata de enseñar para la formación básica o futura con un enfoque de docente, y que se encuentra errónea cuando se supone que el objetivo es la enseñanza de este para su aplicación general y no exclusivamente enfocada en una area de desempeño laboral; teniendo que incluir de forma eficiente otras areas para ejercer, sus condiciones y cambios que se puedan presentar en el mundo laboral.

¿Qué opina el autor acerca del liderazgo?

-las materias de vanguardia que deberían integrar la currícula de los estudios administrativos como liderazgo, solución de problemas y toma de decisiones, creatividad, trabajo en redes, conocimiento del entorno e incluso calidad total sólo se encuentran de manera aislada y marginal en la mayor parte de todos los programas estudiados…- Página 15.

Los autores opinan que el liderazgo se trata de una competencia que deben de contar los egresados de cualquier licenciatura de administración, la cual Mexico cuenta pero no formando de forma clave en las aptitudes de los egresados, al tratarse de una materia opcional que se puede o no cursar. Contradiciendo no solo la demanda si no que también, las mismas habilidades que debe de poseer un administrador.

Opinión hipótesis: Se comenta que siguen un plan de estudios tradicional con materias que se incluyen por obligación que por necesidad . Considero que es acertada, ya que los programas deberían estar más enfocados a la realidad laboral actual. Adaptarlos a las demandas del entorno profesional que permitiría formar y preparar estudiantes, con mayores herramientas para diferentes retos.

LUIS MONTOÑA: Lo señala así porque la Administración en México aún no ha consolidado una base fuerte de investigación y su desarrollo ha estado más centrado en la enseñanza teórica que en la práctica dentro de las organizaciones reales.

¿Qué opina el autor acerca del liderazgo? El autor opina que el liderazgo es un tema importante que debería estar bien integrado en los planes de estudio, pero en realidad casi no se toma en cuenta y solo aparece de manera muy aislada.

El autor plantea que las universidades se enfocan demasiado en lo técnico y lo tradicional, descuidando formar líderes. Esto es crucial, porque aunque se domine la teoría, sin capacidad de dirigir equipos o decidir bajo presión, se corre el riesgo de quedar rezagado frente a otros.

¿Qué opina el autor acerca del liderazgo? Ve el liderazgo como una habilidad clave para el administrador moderno, aunque casi no se enseña en los programas. Además, menciona nuevas corrientes como la dirección por valores, que plantean un liderazgo más humano y ético, que no solo mande o fije metas, sino que inspire y dé sentido a las organizaciones.

• Hay una escasez de investigaciones originales y publicaciones académicas nacionales.

• Predomina el uso de traducciones de textos extranjeros, sobre todo de Estados Unidos, esto dificulta la generación de conocimiento adaptado a la realidad mexicana.

• No hay suficientes revistas, artículos o libros hechos por investigadores nacionales; eso limita el debate teórico.

• Se produce muy poco conocimiento propio, lo cual limita que la administración se consolide como disciplina académica.

La hipótesis se justifica porque muchos planes de estudio de administración en México siguen un esquema tradicional, centrado en procesos clásicos y unas cuantas áreas funcionales, complementadas con materias básicas y optativas que a menudo responden más a decisiones del momento o modas que a un análisis serio de las necesidades del mercado laboral. Provoca que los egresados tengan una formación general pero poco adaptada a los retos del momento. En mi opinión, esta crítica es válida, mientras la administración evoluciona con temas como digitalización o sostenibilidad, limitan la preparación real de los estudiantes.

La hipótesis se enfoca principalmente en como el estudio de la Administración en México es tradicional y como se complementa con materias básicas y personalmente creo que el seguir toda esta base o estructura es bastante importante ya que seguimos una estructura ya previamente analizada y trabajada

Luis Montaño nos dice que relativamente es nueva la administración y que tiene poca tradición en investigación y la orientación dirigida principalmente a docentes y que es fácil de transferir pero difícil de operar

¿Qué opina el autor acerca del liderazgo? es la toma de decisiones y el conocimiento hacia los objetivos

El autor nos da a entender que los programas de estudio están mal idealizados ya que te enseñan los temas de manera fácil pero al momento de lo practico es difícil llevarlo a cabo, ya que las organizaciones exigen habilidades y conocimientos que no tienen nada que ver a como te lo enseñaron en la escuela. Nos dice que el liderazgo es algo muy importante que se debe de tomar en cuenta en los planes de estudio, ya que al estar en una empresa tu debes de saber como dirigir personas, porque aspiras a tener un mejor puesto lo que trae mas responsabilidades como liderar un equipo de trabajo, por lo que debes de saber como guiarlos y motivarlos.

Estoy de acuerdo en la parte de innovar la educación con el tiempo ya que muchas veces se mantienen planes de estudio que cuando el alumno egresa son obsoletos en el mercado, solo que creo que se deberían de basar mejor en lo actual y preservar en el plan nuevo solo lo que se considere necesario del plan anterior, no que se enseñe a las nuevas generaciones lo que aprendieron generaciones de 20 años atrás, porque el mercado de trabajo no es el mismo.

El autor dice que el liderazgo es una materia fundamental y de vamguardia que es algo que debe formar parte de la preparcion de los administradores. Tambien menciona que que la mayoria de los programas de estudio de Mexico el liderazgo no se le da esa importancia que requiere lo cual deja ver lo desactualizado de los planes de estudio frente a exigencias reales

Esto podemos verlo por partes: Es reciente en Mexico la misma trayectora que otras diciplinas, como carrera universitaria es nueva esto hace que aun tenga poca tradicion de investigacion y este mas enfocada en la enseñanza , las universidades transmiten modelos teoricos idealizados los cuales son faciles de transmitir pero dificiles de aplicar en condiciones reales

El autor hace una critica a los planes de estudios y el sistema educatico obsoleto ya que llevan mas de 50 años con la misma estrategia, tambièn menciona sobre los docentes que tienen poca experiencia practica lo cual hace que los egresados esten poco preparados para problemas reales y complejos. A mi parecer tiene razon en lo que dice, creo que cosas tan importantes como lo es la educacion deben evolucionar junto con las nuevas tecnologias, creando nuevas estrategias para asi poder adaptarnos al entorno real

El autor dice básicamente que las escuelas siguen demasiado enfocadas en lo técnico y lo tradicional, pero se olvidan de enseñar a los futuros administradores a ser líderes. Lo cual es importante ya que puedes saber mucha teoría, pero si no sabes guiar un equipo o tomar decisiones bajo presión, te vas a quedar atrás en comparación con algunas personas

En mi opinión, pienso que el autor tiene razón ya que suena lógico que si se sigue enseñando como hace muchos años los egresados puedan salir no tan preparados. Ya que el mundo cambio bastante desde ese entonces ahora las empresas piden mas requisitos y muchos programas siguen con lo mismo de antes pienso que deberían de implementar nuevas materias que ayuden al egresado a prepararse mejor como lo pueden ser materia de liderazgo o de innovación

Aquí esta un poco de la opinión del liderazgo. Dice que a comparación de todas las materias que tienen el plan de estudios de la administración, el liderazgo no se encuentra del todo presente y en opinión del autor el liderazgo debería estar mas presente para el uso en el mercado mas actual.

Aquí esta de que la disciplina es reciente en México. Y en pocas palabras es por que falta una tradición investigadora propia en México, se privilegia la enseñanza sobre la investigación y por que se usan modelos teóricos idealizados que no son adaptados a las condiciones reales de las organizaciones mexicanas.

Aquí esta la hipótesis. Y estoy en un punto medio de estar de acuerdo con el porque siento que esta bien que en los planes de estudio se vean materias que se relacionen con los procesos administrativos tradicionales ya que esto seria como la base que deberían de ver. Pero al igual que el estoy de acuerdo con que también deberían de dar enseñanza a las necesidades mas actuales al mercado y a lo que es el mundo real y no solo lo que seria en teoria.

Esto explica la debilidad investigativa de la administración en México. Se repiten modelos extranjeros en lugar de generar conocimiento contextualizado. Considero que esto limita mucho la autonomía académica del país.

Esto refuerza la crítica del autor. Las universidades mexicanas no están formando a los administradores con las competencias que demanda la realidad laboral. En mi opinión, actualizar el currículo es urgente para no rezagarse frente a los estándares internacionales.

El autor critica que los planes de estudio estén centrados en un modelo obsoleto. Coincido en que esto limita a los futuros administradores frente a un mundo globalizado, pues hoy se requieren competencias como liderazgo, creatividad y manejo intercultural que no aparecen de forma sólida en los programas.

Explicación o justificación: para poder consolidar un verdadero y sustantivo cambio en la formación de nuestros futuros administradores es imprescindible partir de un nuevo paradigma que deseche radicalmente los obsoletos enfoques de funciones y proceso administrativo además deberá articularse con una visión realista de los entornos multiculturales y globaliza dotes de la sociedad en la que se desenvolverán los futuros profesionales de la administración en México y en el mundo.

Luis Montaño: La Administración es una disciplina relativamente reciente en México, con poca tradición en investigación y orientada principalmente hacia las áreas docentes. Se enseñan generalmente modelos idealizados desde la optimización hasta la excelencia; los cuales representan la parte "dura", "científica" relativamente fácil de transferir, pero difícil de operar. La Administración está asentada en organizaciones concretas, alejadas de las condiciones de idealidad de dichos modelos. No se puede estudiar la Administración en abstracto, ésta tiene que ser referida siempre a las condiciones particulares de las organizaciones que aplican esos dispositivos.

¿Qué opina el autor acerca del liderazgo?: el autor opina que se debería añadir al currículo de los estudios administrativos, objeto de ser propositivos y de plantear sólo algunos apuntes preliminares para una solución a esta preocupante problemática para el desarrollo de la administración en el país. Prácticamente dice que se debería de tomar con mayor importancia el liderazgo.

HIPÓTESIS: la hipótesis central del presente trabajo es que los actuales planes de estudio de las licenciaturas en Administración en México están articulados en función de los procesos administrativos tradicionales y el estudio de cuatro o cinco de las áreas denominadas "funcionales" de la administración. Esta estructura curricular se complementa con una serie de materias básicas de carácter casi propedéutico y otro conjunto de materias especializadas, ya sean optativas o pertenecientes a un núcleo introductorio, que dependen más del capricho de las autoridades universitarias en turno o de las modas administrativas, que de un análisis sustentado en las necesidades de los mercados de trabajo o las realidades locales, regionales o nacionales.

Mi opinión: de acuerdo a lo ya leído es que se necesitan nuevos enfoques de enseñanza que cada cierto tiempo vayan actualizándose y cambiando de acuerdo a como cambian los tiempos y las personas ya que así se tendría una mejor enseñanza y una más clara definición de lo que es o será ser un administrador ya sea en las diferentes áreas en las que se puede administrar o que tiene relación con la administración.

Utilidad didáctica Resulta muy útil como material formativo en cursos de metodología de la investigación. Facilita que los nosotros como estudiantes comprendamos como se construye un título correcto y de manera más precisa a partir del problema y el objetivo que se plantean, también ante esto refleja un mayor valor científico.

El articulo resalta de forma muy convincente que la precisión es más importante que la extensión en un artículo de investigación. Enfatiza la relación entre problema, objetivo y titulo.

3

Menciona la importancia del impacto que puede tener el título y que debe de ser caracterizado de un brote de fasciolasis hepatica y que es para para conocer el objetivo de la investigación

2

la relaccion que mencionan los dos autores respecto al titulo hace alusion ah que un buen titulo puede ser una buena investigacion y que el metodo de escoger un titulo es de las caracteristicas mas importantes que tiene

Un título bien formulado actúa como una síntesis estratégica del planteamiento, orientando la lectura y evitando interpretaciones erróneas sobre el alcance o propósito del trabajo Esta coherencia asegura que el lector comprenda desde el inicio qué se investiga, por qué y en qué contexto.

La formulación de un buen título requiere partir del planteamiento del problema y del objetivo de investigación. En algunos casos, el título puede definirse con mayor precisión al concluir el estudio, cuando se ha logrado una comprensión más profunda del fenómeno analizado.

No sabía que un título de algún documento científico, podría llegar a ser tan extenso teniendo entre 15 a 20 palabras aunque hay que tomar en cuenta que son necesarias a veces un poco más, por ejemplo: “Evolución de indicadores de salud seleccionados en la provincia de Cienfuegos durante el decenio 1991-2000”<br /> indica tiempo y espacio, mientras que: “Evolución de indicadores de salud seleccionados” tiene información incompleta que se podría prestar a otros tiempos y otro espacio o podría ser inútil ya que no te serviría como referencia.

Es importante tomar en cuenta la calidad y no la cantidad, siempre cuando se mantengan los estándares para poder ser fuente de consulta confiable

Los términos son claves para la creación de títulos ya que son breves, claros y representativos, estos facilitaran a los lectores comprender de que trata el tema, despertara interés al momento de la lectura y será más fácil su búsqueda

El titulo es de suma importancia definirlo de una manera clara ya que este nos ayuda a entender el porque o el objetivo de la investigación, ya que todos están conectados para que los lectores comprenda la idea de la investigación

Finalmente luego de un análisis y desarrollo de como es que se debe crear un buen titulo para nuestra investigación, se llega a la conclusión de que el tamaño del mismo no es precisamente lo mas importante, ya que lo que verdaderamente importa es la precisión del titulo, que este contenga información útil y precisa sobre el contenido que se puede encontrar dentro de la investigación.

Es importante tener bien definido el titulo del caso ya que para este sirve como primer atractivo de la investigación, tiene que ser claro y sobre todo tener coherencia con la investigación

Como vimos en el texto anterior donde se muestra una representación gráfica de como es el proceso de investigación, este no es lineal, lo cual incluye también el desarrollo del titulo, pues es necesario comenzar a delimitar ciertas bases de nuestra investigación, como lo son los objetivos y la problemática inicial, para poder definir un correcto titulo para nuestra investigación, pues a partir de conocer cuales son los limites y alcances de nuestra investigación, podemos encontrar un titulo adecuado que represente que es lo que estamos tratando de encontrar y transmitir mediante nuestro trabajo,

En los primeros párrafos de la lectura podemos encontrar una frase que me parece de suma importancia en el momento de elegir un titulo para nuestra investigación, "lo verdaderamente relevante, es su precisión y no su extensión", haciendo alusión a la famosa frase "de la vista nace el amor" considero que dentro de la lectura podemos encontrar algo similar, pues un buen titulo, corto, preciso y bien definido puede transmitir un buen mensaje sobre el contenido que se encuentra dentro de nuestra investigación.

Aportación 1: Considero que es un tema del que se habla poco y por lo tanto se me hace relevante ya que habla de la calidad del titulo. Generalmente se da mayor énfasis a la extensión, cuando en realidad lo central es la precisión, pues esta permite reflejar de forma fiel el objeto de estudio y orientar al lector sobre la pertinencia del trabajo.

Aportación 2: El texto se apoya en ejemplos concretos como el de la investigación sobre leucemias agudas o el estudio del impacto de la COVID-19 para demostrar cómo la reducción de palabras en un título puede sacrificar información clave. Estos fortalecen el argumento y evidencian que la correspondencia entre problema, objetivo y título debe prevalecer sobre una regla rígida de extensión.

Aportación 3:La conclusión del artículo es práctica y aplicable: un título no debe juzgarse por ser corto o largo, sino por su capacidad de expresar con exactitud el objeto de estudio. Este enfoque resulta útil para investigadores, tesistas y editores, ya que orienta hacia una valoración más justa y científica de los títulos.

1-Se enfatiza que un buen título no puede formularse correctamente si no se ha definido antes el problema y el objetivo de la investigación. El título debe surgir como una consecuencia lógica de estos elementos, asegurando que exista una correspondencia clara entre ellos. 2- El texto sostiene que, al evaluar la calidad de un título en una investigación, la prioridad debe ser la precisión, es decir, qué tan bien refleja el contenido del estudio, y no su brevedad o número de palabras. Un título largo puede ser perfectamente válido si expresa claramente el objeto del estudio. 3-Aunque es deseable que los títulos no sean innecesariamente extensos, no se debe recortar información.

se logra apreciar en el texto que un título bien formulado funciona como una resumen claro y directo del objeto de estudio, y esto facilita al lector comprender de inmediato el enfoque y la intención de la investigación. No solo le da identidad a la investigación, sino también coherencia y sustento ante los lectores APORTACIÓN 3

Esto es muy rescatable y de mucha ayuda, ya que podemos ver que existe una flexibilidad en la construcción de los títulos y que esta abre la posibilidad de enfocarnos en lo importante, y que no forzar la inclusión del tiempo o espacio nos da la oportunidad que el título sea más universal y que el lector se centre en el problema investigado, sin ponerle un límite con un contexto de título no necesario. APORTACIÓN 2

Esto es interesante ya que menciona la idea de que primero hay que entender bien el problema y los objetivos de una investigación, y ya después crear el título. Esto nos ayuda a que el trabajo que realizamos sea más coherente, organizado y sólido. APORTACIÓN 1

APORTACIONES:

1. Resalta que la forma de ser del título, que se más extenso y exprese lo que realmente quiere dar a entender la investigación

2. De igual forma expresa la forma en que da a entender un título tentativo ósea no uno original como el anterior, podemos observar que nos relata el ejemplo del articulo con el título menos impreciso a lo que describe el contenido del mismo

3. Como podemos observan nos dan un contexto más exacto y coherente sobre la estructura del título, puede que no sea largo, pero debe ser claro en lo que expresa con las palabras suficientes, no sirve de nada un título extenso si no da a entender nada

APORTACIÓN 3.

Como podemos observan nos dan un contexto más exacto y coherente sobre la estructura del título, puede que no sea largo, pero debe ser claro en lo que expresa con las palabras suficientes, no sirve de nada un título extenso si no da a entender nada

Aportación 1:

Resalta que la forma de ser del título, que se más extenso y exprese lo que realmente quiere dar a entender la investigación

1. El documento es claro y puntual a lo que nos quiere dar a entender, realmente por mi parte nunca le había dado la importancia que merece a un "Titulo", es cierto que este es el primer gancho a la vista que los lectores tendrán y por medio de este se decide si continuar la lectura o no.
2. El hecho de que la lectura me de ejemplos de manera extensa, recalcándome que no siempre más es más me gusto mucho ya que me ayuda a una mejor comprensión para poder aplicarlo en mis futuras investigación y saber como redactar de manera correcta un titulo.
3. En la ultima parte del texto se menciona que el responder en el titulo a las preguntas ¿Cuándo? y ¿Donde? es algo importante ya que se detalla la investigación y pareciera que tiene mas detalle y no solo es un tema al azar.

Me parece un texto muy claro, va directo al punto y te hace pensar en algo que normalmente se da por hecho: que lo importante de un título no es solo cuántas palabras tiene, sino lo que transmite

De igual modo, me gustaría que se indagará más en el objetivo a modo de ejemplo para ver que NO considerar poner en el titulo y mejor ponerlo en esa parte del texto (el objetivo).

Consejo valido, sintetizar el titulo y explicar más en el resumen. Me gustaría que indagara más que clase de cosas puede contribuir el reasumen para también evitarlo en el titulo.

Es la tercera vez que en e texto resalta la importancia de ser precisos, más allá de extensos, pero me gustaría un desarrollo más extenso y sencillo de como lograr eso. Entiendo los ejemplos, pero son demasiado especifico, tal vez algún consejo para lograrlo de manera practica y personal sería más útil.

En la conclusión se enfatiza que la precisión es más relevante que la extensión. Sin embargo, también sería útil señalar que un título demasiado técnico podría dificultar su comprensión, por lo que se debe buscar un equilibrio entre exactitud y claridad.

El ejemplo de la investigación sobre leucemias ilustra bien cómo un título puede derivarse con precisión del problema y del objetivo. Esto resalta que la coherencia entre estos tres elementos es clave para evitar títulos vagos o poco informativos.

1.- El título no debe ser un punto de partida improvisado, sino el resultado de un proceso reflexivo.

2.- Un título extenso no es un defecto en sí, si aporta claridad y especificidad. Sin embargo, la extensión debe ser funcional, evitando redundancias o detalles irrelevantes.

3.- Diferenciación entre objeto de estudio y variables. Esto ayuda a evitar títulos que mencionan solo el fenómeno general sin especificar qué se está midiendo o analizando.

Comentario #1: Es muy importante tener en cuenta el título de la investigación ya que, si este mismo es claro y conciso, el lector se podría interesar mucho más en la investigación.

Comentario #2: Aunque el título es muy importante, en mi opinión formular correctamente la problematica es un gran paso para considerar cuál va a ser el título de la investigación y obtener los objetivos.

Comentario #3: Un buen titulo de investigación debe tener en cuenta la presición de la investigación, de lo contario, puede estar sujeto a ausencia de informacion y esto podría desorientar al lector.

Me parece valioso resaltar que no es imprescindible que un título contenga referencia directa con el contexto de la investigación. En base a esto se puede mencionar que no debe ser una exigencia rutinaria y brinda una mayor libertad en relación a lo que se quiere contar,

Cuando se trata de una investigación se espera que el titutlo tenga presicion y fuerte impacto, es decir que se diga de que es la investigació y que impacto tiene, la afirmación de que muchos autores pasan por alto tener un buen titulo es cierta, después de todo, es normal que no todos los titulos sean adecuados. ¿Podrían ser mejores?, si, pero depende de enfoque siempre.

Adecuar correctamente el objetivo de una investigación y todo lo que implica esta, antes de ponerle nombre al titulo es más importante en una escala. El titulo muchas veces busca llamar la atención pero no representa en su totalidad la idea de lo que tienes y como lo tienes en general, pero te puedes dar una idea de como quieres llevar a cabo tu proyecto y en que dirección va si comienzas por los objetivos y esta bien fomrmulado.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Summary:

The authors use high-throughput gene editing technology in larval zebrafish to address whether microexons play important roles in the development and functional output of larval circuits. They find that individual microexon deletions rarely impact behavior, brain morphology, or activity, and raise the possibility that behavioral dysregulation occurs only with more global loss of microexon splicing regulation. Other possibilities exist: perhaps microexon splicing is more critical for later stages of brain development, perhaps microexon splicing is more critical in mammals, or perhaps the behavioral phenotypes observed when microexon splicing is lost are associated with loss of splicing in only a few genes.

A few questions remain:

(1) What is the behavioral consequence for loss of srrm4 and/or loss-of-function mutations in other genes encoding microexon splicing machinery in zebrafish?

It has been established that srrm4 mutants exhibit no overt morphological phenotypes and are not visually impaired (Ciampi et al., 2022). We are coordinating our publication with Lopez-Blanch et al. (https://doi.org/10.1101/2024.10.23.619860), which shows that srrm4 mutants also have minimal behavioral phenotypes. In contrast, srrm3 mutants have severe vision loss, early mortality, and numerous neural and behavioral phenotypes (Ciampi et al., 2022; Lopez-Blanch et al., 2024). We now point out the phenotypes of srrm3/srrm4 mutants in the manuscript.

We chose not to generate and characterize the behavior and brain activity of srrm3/srrm4 mutants for two reasons: 1) we were aware of two other labs in the zebrafish community that had generated srrm3 and/or srrm4 mutants (Ciampi et al., 2022 and Gupta et al., 2024, https://doi.org/10.1101/2024.11.29.626094; Lopez-Blanch et al., 2024, https://doi.org/10.1101/2024.10.23.619860), and 2) we were far more interested in determining the importance of individual microexons to protein function, rather than loss of the entire splicing program. Microexon inclusion can be controlled by different splicing regulators, such as srrm3 (Ciampi et al., 2022) and possibly other unknown factors. Genetic compensation in srrm4 mutants could also result in microexons still being included through actions of other splicing regulators, complicating the analysis of these regulators. We mention srrm4 in the manuscript to point out that some selected microexons are adjacent to regulatory elements expected of this pathway. We did not, however, choose microexons to mutate based on whether they were regulated by Srrm4, making the characterization of srrm3/srrm4 mutants disconnected from our overarching project goal.

We have edited the Introduction as follows to clarify our goal: “Studies of splicing regulators such as srrm4 impact the entire splicing program, making it impossible to determine the importance of individual microexons to protein function. Further, microexons could still be differentially included in a regulatory mutant via compensation by other splicing factors ...”

(2) What is the consequence of loss-of-function in microexon splicing genes on splicing of the genes studied (especially those for which phenotypes were observed).

We are unclear whether “microexon splicing genes” refers to the splicing regulators srrm3/srrm4, which we choose not to study in this work (see response to point #1 above), or the genes that contain microexons. The severe visual phenotypes of srrm3 mutants confounds the study of microexon splicing in this line because altered splicing levels could be due to downstream changes in this significantly different developmental context. A detailed discussion of splicing consequences on removal of microexons from microexoncontaining genes is in the response to point #4 below.

(3) For the microexons whose loss is associated with substantial behavioral, morphological, or activity changes, are the same changes observed in loss-of-function mutants for these genes?

In the first version of the manuscript, we had included two explicit comparisons of microexon loss with a standard loss-of-function allele, one with a phenotype and one without, in Figure S1 (now Figures S3 and S4) of this manuscript. Beyond the two pairs we had included, Lopez-Blanch et al. (https://doi.org/10.1101/2024.10.23.619860) described mild behavioral phenotypes for a microexon removal for kif1b, and we showed developmental abnormalities for the kif1b loss-of-function allele (now Figure S3). We have now added a predicted protein-truncating allele for ppp6r3. This new line has phenotypes that are similar but slightly stronger in brain activity and structure than the mutant that lacks only the microexon. The prior Figure S1 (now Figures S3 and S4) was only briefly mentioned in the first version of the manuscript, and we now clarify this point in the Results: “Protein-truncating mutations in eleven additional genes that contain microexons revealed developmental and neural phenotypes in zebrafish (Figure S3, Figure S4), indicating that the genes themselves are involved in biologically relevant pathways. Three of these genes– tenm4, sptan1, and ppp6r3 – are also in our microexon line collection.”

Additionally, we can draw expected conclusions from the literature, as some genes with our microexon mutations have been studied as typical mutants in zebrafish or mice. We have modified our manuscript to include a discussion of both loss-of-function zebrafish and mouse mutants. See the response to below point #4.

(4) Do "microexon mutations" presented here result in the precise loss of those microexons from the mRNA sequence? E.g. are there other impacts on mRNA sequence or abundance?

We acknowledge that unexpected changes to the mRNA of the tested mutants could occur following microexon removal. In particular, all regulatory elements should be removed from the region surrounding the microexon, as any remaining elements could drive the inclusion of unexpected exons that result in premature stop codons.

First, we have clarified our generated mutant alleles by adding a figure (Figure S1) that details the location of the gRNA cut sites in relation to the microexon, its predicted regulatory elements, and its neighboring exons.

Second, we have experimentally determined whether the mRNA was modified as expected for a subset of mutants with phenotypes. In all eight tested lines (Figure S2), the microexon was precisely eliminated without causing any other effects on the sequence of the transcript in the neighboring region. We did, however, observe an effect on transcript abundance for one homozygous mutant (vav2). It is possible that complex forms of genetic regulation are occurring that are not induced by unexpected isoforms or premature stop codons. Interestingly, Lopez-Blanch et al. (https://doi.org/10.1101/2024.10.23.619860) eliminated a different microexon in vav2 and also observed a subtle well center preference. If their allele from an entirely different intronic region also results in transcript downregulation, it would support the hypothesis of genetic compensation through atypical pathways. If not, it is likely this phenotype is due specifically to removal of the microexon protein sequence. Not all mutants with phenotypes could be assessed with qRT-PCR because some were no longer present in the lab. All lines were generated in a similar way, however, removing both the microexon and neighboring regulatory elements while avoiding the neighboring exons. Accordingly, we now also explicitly point out those where the clean loss of the microexon was confirmed (eif4g3b, ppp6r3, sptan1, vti1a, meaf6, nrxn1a, tenm3) and those with possibly interesting phenotypes that were not confirmed (ptprd-1, ptprd-2, rapgef2, dctn4, dop1a, mapk8ip3).

Third, we have further emphasized in the manuscript that these observed phenotypes are extremely mild compared to those observed in over one hundred protein-truncating mutations we have assessed in previous (Thyme et al., 2019; Capps et al., 2024) and unpublished ongoing work. We showed data for one mutant, tcf7l2, which we consider to have moderately strong neural phenotypes, and we have extended this comparison in the revision (new Figure 3G). Additionally, loss-of-function alleles for some microexoncontaining genes have strong developmental phenotypes, as we showed in Figure S1 (now Figures S3 and S4) of this manuscript in addition to our published work (Thyme et al., 2019; Capps et al., 2024). It is known from the literature that the loss-of-function mutants for mapk8ip3 are stronger than we observed here (Tuttle., et al., 2019), suggesting that only the microexon is removed in our line. The microexons in Ptprd are also well-studied in mice, and we expect that only the microexon was removed in our lines. Both Dctn4 and Rapgef2 are completely lethal prior to weaning in mice (the International Mouse Phenotyping Consortium).

(5) Microexons with a "canonical layout" (containing TGC / UC repeats) were selected based on the likelihood that they are regulated by srrm4. Are there other parallel pathways important for regulating the inclusion of microexons? Is it possible to speculate on whether they might be more important in zebrafish or in the case of early brain development?

The microexons were not selected based on the likelihood that they were regulated by Srrm4. We have clarified the manuscript regarding this point. There are parallel pathways that can control the inclusion of microexons, such as Srrm3 (Ciampi et al., 2022). It is wellknown that loss of srrm3 has a stronger impact on zebrafish development than srrm4 (Ciampi et al., 2022). The goal of our work was not to investigate these splicing regulators but instead to determine the individual importance of these highly conserved protein changes.

Strengths:

(1) The authors provide a qualitative analysis of splicing plasticity for microexons during early zebrafish development.

(2) The authors provide comprehensive phenotyping of microexon mutants, addressing the role of individual microexons in the regulation of brain morphology, activity, and behavior.

We thank the reviewer for their support. The pErk brain activity mapping method is highly sensitive, significantly minimizing the likelihood that the field has simply not looked hard enough for a neural phenotype in these microexon mutants. In our published work (Thyme et al., 2019), we show that brain activity can be drastically impacted without manifesting in differences in those behaviors assessed in a typical larval screen (e.g., tcf4, cnnm2, and more).

Weaknesses:

(1) It is difficult to interpret the largely negative findings reported in this paper without knowing how the loss of srrm4 affects brain activity, morphology, and behavior in zebrafish.

See response to point 1.

(2) The authors do not present experiments directly testing the effects of their mutations on RNA splicing/abundance.

See response to point 4.

(3) A comparison between loss-of-function phenotypes and loss-of-microexon splicing phenotypes could help interpret the findings from positive hits.

See response to points 3 and 4.

Reviewer #2 (Public review):

Summary:

The manuscript from Calhoun et al. uses a well-established screening protocol to investigate the functions of microexons in zebrafish neurodevelopment. Microexons have gained prominence recently due to their enriched expression in neural tissues and misregulation in autism spectrum disease. However, screening of microexon functionality has thus far been limited in scope. The authors address this lack of knowledge by establishing zebrafish microexon CRISPR deletion lines for 45 microexons chosen in genes likely to play a role in CNS development. Using their high throughput protocol to test larval behaviour, brain activity, and brain structure, a modest group of 9 deletion lines was revealed to have neurodevelopmental functions, including 2 previously known to be functionally important.

Strengths:

(1) This work advances the state of knowledge in the microexon field and represents a starting point for future detailed investigations of the function of 7 microexons.

(2) The phenotypic analysis using high-throughput approaches is sound and provides invaluable data.

We thank the reviewer for their support.

Weaknesses:

(1) There is not enough information on the exact nature of the deletion for each microexon.

To clarify the nature of our mutant alleles, we have added a figure (Figure S1) that details the location of the microexon in relation to its predicted neighboring exons, deletion boundaries, guide RNAs, and putative regulatory elements.

(2) Only one deletion is phenotypically analysed, leaving space for the phenotype observed to be due to sequence modifications independent of the microexon itself.

We have determined whether the mRNA is impacted in unanticipated ways for a subset of mutants with mild phenotypes (see point #4 responses to Reviewer 1 for details). Our findings for three microexon mutants (ap1g1, vav2, and vti1a) are corroborated by LopezBlanch et al. (https://doi.org/10.1101/2024.10.23.619860). We have also already compared the microexon removal to a loss-of-function mutant for two lines (Figures S3 and S4), and we have made this comparison more obvious as well as increasing the discussion of the expected phenotypes from typical loss-of-function mutants (see point #3 response to reviewer 1).

Unlike protein-coding truncations, clean removal of the microexon and its regulatory elements is unlikely to yield different phenotypic outcomes if independent lines are generated (with the exception of genetic background effects). When generating a proteintruncating allele, the premature stop codon can have different locations and a varied impact on genetic compensation. In previous work (Capps et al., 2024), we have observed different amounts of nonsense-mediated decay-induced genetic compensation (El-Brolosy, et al., 2019) depending on the location of the mutation. As they lack variable premature stop codons (the expectation of a clean removal), two mutants for the same microexons should have equivalent impacts on the mRNA.

We now address the concern of subtle genetic background effects in the Methods: “Even with using sibling controls and collecting multiple biological replicates from individual parents, the possibility remains that linked genetic variation may have contributed to the mild phenotypes we observed, as only a single line was generated.”

Reviewer #3 (Public review):

Summary:

This paper sought to understand how microexons influence early brain function. By selectively deleting a large number of conserved microexons and then phenotyping the mutants with behavior and brain activity assays, the authors find that most microexons have minimal effects on the global brain activity and broad behaviors of the larval fish-- although a few do have phenotypes.

Strengths:

The work takes full advantage of the scale that is afforded in zebrafish, generating a large mutant collection that is missing microexons and systematically phenotyping them with high throughput behaviour and brain activity assays. The work lays an important foundation for future studies that seek to uncover the likely subtle roles that single microexons will play in shaping development and behavior.

We thank the reviewer for their support.

Weaknesses:

The work does not make it clear enough what deleting the microexon means, i.e. is it a clean removal of the microexon only, or are large pieces of the intron being removed as well-- and if so how much? Similarly, for the microexon deletions that do yield phenotypes, it will be important to demonstrate that the full-length transcript levels are unaffected by the deletion. For example, deleting the microexon might have unexpected effects on splicing or expression levels of the rest of the transcript that are the actual cause of some of these phenotypes.

To clarify the nature of our mutant alleles, we have added a figure (Figure S1) that details the location of the microexon in relation to its predicted neighboring exons, deletion boundaries, guide RNAs, and putative regulatory elements. We have determined whether the mRNA is impacted in unanticipated ways for a subset of mutants with mild phenotypes (see point #4 responses to Reviewer 1 for details).

Reviewer #1 (Recommendations for the authors):

(1) For most ME mutations, 4 guide sequences are provided. More description / a diagram could be helpful to interpret how ME mutations were generated.

We have added diagrams to the Supplementary Materials (new Figure S1) to show where the guide RNAs, cut sites, and putative regulatory elements are in relationship to the microexon and its neighboring exons. We have also added the following point to the text: “Four guide RNAs were used, two on each side of the microexon (Table S2, Figure S1).”

(2) Figure 1 indicates that there are 45 microexons (MEs) but the text initially indicates that there are 44 that exist in a canonical layout (the text later indicates there are 45). This could be made more clear.

The 45 refers to the mutants that were generated, not the microexons with putative Srrm4 regulatory elements. We did not choose microexons to mutate based on whether they were regulated by Srrm4. We have clarified these points in the manuscript as follows: “Of these 95 microexons, 42 exist in a canonical layout in the zebrafish genome, with both a UGC and UC repeat – or similar polypyrimidine tract – directly upstream of the alternatively spliced exon (Gonatopoulos-Pournatzis et al., 2018) (Table S1), indicating that Srrm4 likely controls their inclusion. Of the remaining microexons, 44 are organized similarly to the canonical layout, typically with either a UGC or UC repeat. Thus, they may also be regulated by Srrm4.” and “Using CRISPR/Cas9, we generated lines that removed 45 conserved microexons  (Table S2) and assayed larval brain activity, brain structure, and behavior (Figure 1A). Four guide RNAs were used, two on each side of the microexon (Table S2, Figure S1). For microexons with upstream regulatory elements that are likely important for splicing, these elements were also removed (Figure S1).”

(3) The description of the "canonical layout" as containing TGC / UC repeats could be rewritten as either "containing a UGC motif and UC repeats" or "containing a TGC motif and TC repeats."

This error has been corrected.

(4) Why was tcf7l2 selected as a control for MAP mapping?

The mutant for tcf7l2 is an example of a moderately strong phenotype from a recent study we completed (Capps et al., 2025). This mutant was selected because it has both increased and decreased activity and structure and is ideal for setting the range of the graph. We now include a comparison to additional mutants from this study of autism genes (Capps et al., 2025) to further demonstrate how mild the phenotypes are in the microexon removal mutants (new Figure 3G). We also include the activity and structure maps of tcf7l2 mutants in Supplementary Figures 9 and 11.

(5) What does it mean that of the remaining microexons, most are similar to canonical layout?

Typically, they would have one of the two regulatory elements instead of both or the location of the possible elements would be slightly farther away than expected. We have clarified this point in the manuscript as follows: “Of these 95 microexons, 42 exist in a canonical layout in the zebrafish genome, with both a UGC and UC repeat  or similar polypyrimidine tract – directly upstream of the alternatively spliced exon (Gonatopoulos-Pournatzis et al., 2018) (Table S1), indicating that Srrm4 likely controls their inclusion. Of the remaining microexons, 44 are organized similarly to the canonical layout, typically with either a UGC or UC repeat. Thus, they may also be regulated by Srrm4.”

(6) Figure 2A is very difficult to see - most are either up or down - suggest splitting into 2 figures - one = heat map, second can summarize values that were both up and down.

We prefer to retain this information for accuracy. The bubble location is offset to effectively share the box between the orange (decreased) and purple (increased) measures. For example, and as noted in the methods and now expanded upon, a measure can change between 4 and 6 dpf or a measure such as bout velocity could be increased while the distance traveled is decreased (both are magnitude measures). The offset of the bubbles is consistently 0.2 data units in x and y from the center of the box.

(7) The authors apply rigorous approaches to testing the importance of microexons. I especially appreciate the inclusion of separate biological replicates in the main figures!

We thank the reviewer for their positive feedback.

(8) Page 5 line 5 - suggest "compared to homozygous mutants".

The change has been made.

(9) For Eif5g3b dark flash phenotype, it's not clear what "p-values are not calculated for response plots" means. A p-value is provided in the plot for ppp6r3 response freq.

The eif4g3b plot is the actual response trace measuring through pixel changes whereas the ppp6r3 is the frequency of response. While informative, the response plot is time-based data with a wide dynamic range, making the average signal across the entire time window meaningless. We include the p-values for a related measure, the latency for the first 10 dark flashes in block 1 (day6dpfdf1a_responselatency) in the legend.

(10) The ptprd phenotype in 2D is not described in the text.

The change has been made.

(11) Page 7 line 7: "mild" is repeated.

This error has been corrected.

Reviewer #2 (Recommendations for the authors):

Specific points for needed improvement:

(1) The title should be adjusted to more accurately describe the results. The term 'minimal' is under-representing the findings. 9/45 (20%) of targets in their screen have some phenotype, indicating that a significant number have indeed an important function. Moreover, the phenotypic analysis is limited, leaving space for missed abnormalities (as discussed by the authors). I would therefore suggest a more neutral title such as 'Systematic genetic deletion of microexons uncovers their roles in zebrafish brain development and larval behaviour'.

While some microexon mutants do have repeatable phenotypes, these phenotypes are far milder than phenotypes observed in other mutant sets. We now include a comparison to additional mutants from this study of autism genes (Capps et al.,2025) to further demonstrate how mild the phenotypes are in the microexon removal mutants (new Figure 3G). The title states that these microexons have a minimal impact on larval zebrafish brain morphology and function, leaving room for the possibility of adult phenotypes. Thus, we prefer to retain this title.

(2) Do the 45 chosen microexons correspond to the 44 with a canonical layout with TGC and UC repeats? If so, it needs to be explicitly stated in the text that exons were chosen for mutation based on the potential for SRRM4 regulation. If not, then the rationale for the choice of the 45 mutants from the 95 highly conserved events needs to be explained further.

The 45 refers to the mutants that were generated, not the microexons with putative Srrm4 regulatory elements. We did not choose microexons to mutate based on whether they were regulated by Srrm4. We have clarified these points in the manuscript as follows: “Of these 95 microexons, 42 exist in a canonical layout in the zebrafish genome, with both a UGC and UC repeat – or similar polypyrimidine tract – directly upstream of the alternatively spliced exon (Gonatopoulos-Pournatzis et al., 2018) (Table S1), indicating that Srrm4 likely controls their inclusion. Of the remaining microexons, 44 are organized similarly to the canonical layout, typically with either a UGC or UC repeat. Thus, they may also be regulated by Srrm4.” and “Using CRISPR/Cas9, we generated lines that removed 45 conserved microexons (Table S2) and assayed larval brain activity, brain structure, and behavior (Figure   1A). Four guide RNAs were used, two on each side of the microexon (Table S2, Figure S1). For microexons with upstream regulatory elements that are likely important for splicing, these elements were also removed (Figure S1).”

There was no clear rationale for those that were selected. We attempted to generate all 95 and some mutants were not successfully generated in our initial attempt. As we found minimal phenotypes, we elected to not continue to make the remaining ones on the list.

(3) More detail regarding the design of guides for CRISPR is required in the text in the methods section. From Table S2, 4 guides were used per microexon. Were these designed to flank the microexon? How far into the intronic sequence were the guides designed? Were the splicing regulatory sequences (polypyrimidine tract, branchpoint) also removed? The flanking sequences of each of the 45 deletion lines need to be provided.

We have added diagrams to the Supplementary Materials (new Figure S1) to show where the guide RNAs, cut sites, and putative regulatory elements are in relationship to the microexon and its neighboring exons. We removed the microexon and the surrounding area that contains the putative regulatory elements.

(4) Following on from the previous point, to ascertain that the phenotype observed is truly due to lack of microexon (rather than other event linked to removed intronic sequences) - for the 7 exons newly identified as functionally important, at least one added deletion line has to be shown, presenting the same phenotype. If making 7 more lines can't be achieved in a reasonable time (we are aware this is a big ask), a MO experiment blocking microexon splicing needs to be provided (may not be ideal for analysis at 6 dpf). For the existing mutants and the new ones (or morphants), sequencing of the mRNAs for the 7 genes in mutants and siblings also needs to be added to check any possible change in other variants.

Unlike protein-coding truncations, clean removal of the microexon and its regulatory elements is unlikely to yield different phenotypic outcomes if independent lines are generated (with the exception of genetic background effects). When generating a protein-truncating allele, the premature stop codon can have different locations and a varied impact on genetic compensation. In previous work (Capps et al., 2024), we have observed different amounts of nonsense-mediated decay-induced genetic compensation (El-Brolosy, et al., 2019) depending on the location of the mutation. As they lack variable premature stop codons (the expectation of a clean removal), two mutants for the same microexons should have equivalent impacts on the mRNA. We acknowledge that we inadequately described the generation of these alleles, and we now provide Figure S1 to show the microexon’s relationship to possible regulatory elements that impact splicing in unexpected ways if they remain.

We now acknowledge the concern of subtle genetic background effects in the Methods: “Even with using sibling controls and collecting multiple biological replicates from individual parents, the possibility remains that linked genetic variation may have contributed to the mild phenotypes we observed, as only a single line was generated.”

Given the caveats of MOs and transient microinjection for the study of 6 dpf phenotypes, we disagree that this suggested experiment would provide value. The phenotypic assays we use are highly sensitive, and we would not even trust CRISPANTs to yield reliable data. We have added an additional loss-of-function allele for ppp6r3 from the Sanger knockout project, which has a similar but stronger size change to the ppp6r3 microexon-removal line. In addition, our findings for three microexon mutants (ap1g1, vav2, and vti1a) are corroborated by Lopez-Blanch et al. (https://doi.org/10.1101/2024.10.23.619860).

To support that these we generated clean removal of these microexons, we experimentally determined whether the mRNA is impacted in unanticipated ways for a subset of mutants with mild phenotypes (see the point #4 public response to Reviewer 1). We also have already compared the microexon removal to a loss-offunction mutant for two lines (Figure S1), and we have made that outcome more obvious as well as increasing the discussion of the expected phenotypes from typical loss-of-function mutants (see point #3 public response to Reviewer 1).

(5) Figure 3: An image of control tcf7l2 mutant brain activity as a reference should be included.

We now include the activity and structure maps of tcf7l2 mutants in Supplementary Figures 9 and 11.

(6) Figure 3a/b. The gene names on the y-axis of the pERK and structure comparisons should be reordered to be alphabetical so that phenotypes can be compared by the reader for the same microexon across the two assays.

These data are clustered so that any similarities between maps can be recognized. We prefer to retain the clustering to compare lines to each other.

(7) Figure S6 legend. Including graph titles like "day3msdf_dpix_numberofbouts_60" is not comprehensible to the reader so should be replaced with more descriptive text. As should jargon such as "combo plot" and"habituation_day5dpfhab1post_responsefrequency_1_a1f1000d5p" etc.

The legend has been edited to describe the experiments. Subsections of the prior names are maintained in parentheses to enable the reader to connect the plots in this figure to the specific image and underlying data in Zenodo.

(8) Page 2 line 21 "to enable proper".

The change has been made.

(9) Page 7 line 7. Repeatable phenotypes were mild mild.

This error has been corrected.

Reviewer #3 (Recommendations for the authors):

(1) Figure 1B is confusingly laid out.

We are unclear how to modify Figure 1B, as it is a bar plot. We have modified several figures to improve clarity.

(2) Figure 1E-there are some pictures of zebrafish but to what end? They aren't labelled. The dark "no expression" looks really similar to the dark green, "high expression".

The zebrafish images represent the ages assessed for microexon inclusion. We have added labels to clarify this point.

(3) The main text says "microexons were removed by Crispr" but there is no detail in the main text about this at all-- and barely any in the methods. What does it mean to be removed? Cleanly? Or including part of the introns on either side? Etc. How selected, raised, etc? I can glean some of this from the Table S2 if I do a lot of extra work, but at least some notes about this would be important.

We have added diagrams to the Supplementary Materials (new Figure S1) to show where the guide RNAs, cut sites, and putative regulatory elements are in relationship to the microexon and its neighboring exons. We removed the microexon and the surrounding area that contains the putative regulatory elements.

(4) Figure 2 - There are no Ns, at least for the plots on the right. The reader shouldn't have to dig deep in Table S2 to find that. It is also unclear why heterozygous fish are not included in these analyses, since there are sibling data for all. Removed for readability of the plots might be warranted, but this should be made explicitly clear.

The Ns for these plots have been added to the legend. The legend was also modified as follows: “Comparisons to the heterozygous larvae are removed for clarity and available in the Supplementary Materials, as they often have even milder phenotypes than homozygous.”

(5) Needed data: for those with phenotypes, some evidence should be presented that the full-length transcripts that encode proteins without the microexons are still expressed at the same level and without splicing errors/NMD. Otherwise, some of these phenotypes that were found could be due to knockdown or LOF (or I suppose even overexpression) of the targeted gene.

We have added a new Supplementary Figure S2 confirming clean removal of the microexons with RT-PCR for a subset of mutants with phenotypes. This figure also includes qRT-PCR for the same subset. We now discuss these findings: Results: “For eight mutant lines, we confirmed that the microexon was eliminated from the transcripts as expected (Figure S2). Although our genomic deletion did not yield unexpected isoforms, qRT-PCR on these eight lines revealed significant downregulation for the homozygous vav2 mutant (Figure S2), indicating possibly complex genetic regulation.”

Author response:

The following is the authors’ response to the previous reviews

Reviewer # 1 (Public review)

This study aims to elucidate the mechanisms by which stress-induced α2A-adrenergic receptor (α2A-AR) internalization leads to cytosolic noradrenaline (NA) accumulation and subsequent neuronal dysfunction in the locus coeruleus (LC). While the manuscript presents an interesting but ambitious model involving calcium dynamics, GIRK channel rundown, and autocrine NA signaling, several key limitations undermine the strength of the conclusions. 

(1) First, the revision does not include new experiments requested by reviewers to validate core aspects of the mechanism. Specifically, there is no direct measurement of cytosolic NA levels or MAO-A enzymatic activity to support the link between receptor internalization and neurochemical changes. The authors argue that such measurements are either not feasible or beyond the scope of the study, leaving a significant gap in the mechanistic chain of evidence. 

Although the reviewer #1 commented that “The authors argue that such measurements are either not feasible or beyond the scope of the study, leaving a significant gap in the mechanistic chain of evidence”, we believe that this comment may be unfair. 

It may be unfair for the reviewer #1 to neglect our responses to the original reviewer comments regarding the direct measurement of cytosolic NA levels. It is true that none of the recommended methods to directly measure cytosolic NA levels are not feasible as described in the original authors’ response (see the original authors’ response to the comment raised by the Reviewer #1 as Recommendations for the authors (2)). To measure extracellular NA with GRAB-NE photometry, α2A-ARs must be expressed in the cell membrane. GRAB-NE photometry is not applicable unless α2A-ARs are expressed, whereas increases in cytosolic NA levels are caused by internalization of α2A-ARs in our study.

In our study, we elaborated to detect the change in MAO-A protein with Western blot method, instead of examining MAO-A enzymatic activity. Because the relative quantification of active AEP and Tau N368 proteins by Western blot analysis should accurately reflect the change in the MAO-A enzymatic activity, enzymatic assay may not be necessarily required while we admit the necessity of enzymatic assay to better demonstrate the MAO-A activities as discussed in the previously revised manuscript (R1, page 10, lines 314-315). 

We used the phrase “beyond the scope of the current study” for “the mechanism how Ca²⁺ activates MAO-A” as described in the original authors’ responses (see the original authors’ response to the comment raised by the Reviewer #1 as Weakness (3)). We do not think that this mechanism must be investigated in the present study because the Ca²⁺ dependent nature of MAO-A activity is already known (Cao et al., 2007). 

On the other hand, because it is not possible to measure cytosolic NA levels with currently available methods, the quantification of the connection between α2A-AR internalization and increased cytosolic NA levels must be considered outside the scope of the study. However, our study demonstrated the qualitative relationship between α2A-AR internalization and active-AEP/TauN-368 reflecting increased cytosolic NA levels, leaving “a small gap in the mechanistic chain of evidence.” Therefore, it may be unreasonable to criticize our study as “leaving a significant gap in the mechanistic chain of evidence” with the phrase “beyond the scope of the current study.” 

(2) Second, the behavioral analysis remains insufficient to support claims of cognitive impairment. The use of a single working memory test following an anxiety test is inadequate to verify memory dysfunction behaviors. Additional cognitive assays, such as the Morris Water Maze or Novel Object Recognition, are recommended but not performed.

As described in the original authors’ response (see the original authors’ response to the comment raised by the Reviewer #1 as Weakness (4)), we had already done another behavioral test using elevated plus maze (EPM) test. By combining the two tests, it may be possible to more accurately evaluate the results of Y-maze test by differentiating the memory impairment from anxiety. However, the results obtained by these behavioral tests showed that chronic RS mice displayed both anxiety-like and memory impairment-like behaviors. Accordingly, we have softened the implication of anxiety and memory impairment (page 13, lines 396-399) and revised the abstract (page 2, line 59) in the revised manuscript (R2).  

(3) Third, concerns regarding the lack of rigor in differential MAO-A expression in fluorescence imaging were not addressed experimentally. Instead of clarifying the issue, the authors moved the figure to supplementary data without providing further evidence (e.g., an enzymatic assay or quantitative reanalysis of Western blot, or re-staining of IF for MAO-A) to support their interpretation.

Because the quantification of MAO-A expression can be performed with greater accuracy by means of Western blot than by immunohistochemistry, we have moved the immunohistochemical results (shown in Figure 5) to the supplemental data (Figure S8) following the suggestion made by the Reviewer #3. As the relative quantification of active AEP and Tau N368 proteins by Western blot analysis may accurately reflect changes in the MAO-A enzymatic activity which is consistent with the result of Western blot analysis of MAO-A, enzymatic assay or re-staining of immunofluorescence for MAO-A may not be necessarily required. We do not think that a new experiment of Western blot analysis is necessary to re-evaluate MAO-A just because of the lack of the less-reliable quantification of immunohistochemical staining.

(4) Fourth, concerns regarding TH staining remain unresolved. In Figure S7, the α2A-AR signal appears to resemble TH staining, and vice versa, raising the possibility of labeling errors. It is recommended that the authors re-examine this issue by either double-checking the raw data or repeating the immunostaining to validate the staining.

The reviewer #3 is misunderstanding Figure S7. In Figure S7, there are two types of α2A-AR expressing neurons; one is TH-positive LC neuron and the other is TH-negative neuron in mesencephalic trigeminal nucleus (MTN). This clearly indicates that TH staining is specific. Furthermore, α2A-AR staining was much more extensive in MTN neurons than in LC neurons. Thus, α2A-AR signal is not similar to TH signal and there are no labeling errors, which is also evident in the merged image (Figure S7C).

(5) Overall, the manuscript offers a potentially interesting framework but falls short in providing the experimental rigor necessary to establish causality. The reliance on indirect reasoning and reorganizing of existing data, rather than generating new evidence, limits the overall impact and interpretability of the study.

Overall, the reviewer #1 was not satisfied with our revision regardless of the authors’ responses. As detailed above in our responses to the replies (1)~(4), we believe that in the original authors’ responses and in the above-described responses we effectively responded to the criticisms by the reviewer #1.

Reviewer #2 (Public review): 

Comments on revisions: 

The authors have addressed all of the reviewers' comments.

We appreciate constructive and helpful comments made by the reviewer #2.

Reviewer #3 (Public review): 

Weaknesses:  

Nevertheless, the manuscript currently reads as a sequence of discrete experiments rather than a single causal chain. Below, I outline the key points that should be addressed to make the model convincing.

Please see the responses to the recommendation for the authors made by reviewer #3.

Reviewer #3 (Recommendations for the authors):

(1) Causality across the pathway  

Each step (α2A internalisation, GIRK rundown, Ca²⁺ rise, MAO-A/AEP upregulation) is demonstrated separately, but no experiment links them in a single preparation. Consider in vivo Ca²⁺ or GRAB NE photometry during restraint stress while probing α2A levels with i.p. clonidine injection or optogenetic over excitation coupled to biochemical readouts. Such integrated evidence would help to overcome the correlational nature of the manuscript to a more mechanistic study. 

Authors response: It is not possible to measure free cytosolic NA levels with GRAB NE photometry when α2A AR is internalized as described above (see the response to the comment made by reviewer #1 as the recommendation for the authors).

The core idea behind my comment, as well as that of Reviewer 1, was to encourage integrating your individual findings into a more cohesive in vivo experiment. Using GRAB-NE to measure extracellular NA could serve as an indirect readout of NA uptake via NAT, and ultimately, cytosolic NA levels. Connecting these experiments would significantly strengthen the manuscript and enhance its overall impact. 

It may be true that the measurement of extracellular NA could serve as an indirect readout of NA uptake via NAT, and ultimately cytosolic NA levels. However, the reviewer #3 is still misunderstanding the applicability of GRAB-NE method to detect NE in our study. As described in the original authors’ response, there appeared to be no fluorescence probe to label cytosolic NA at present. Especially, the GRAB-NE method recommended by the reviewers #1 and #3 is limited to detect NA only when α2A-AR is expressed in the cell membrane.Therefore, when increases in cytosolic NA levels are caused by internalization of α2A-ARs, NA measurement with GRAB-NE photometry is not applicable.

(2) Pharmacology and NE concentration  

The use of 100 µM noradrenaline saturates α and β adrenergic receptors alike. Please provide ramp measurements of GIRK current in dose-response at 1-10 µM NE (blocked by atipamezole) to confirm that the rundown really reflects α2A activity rather than mixed receptor effects. 

Authors response: It is true that 100 µM noradrenaline activates both α and β adrenergic receptors alike. However, it was clearly showed that enhancement of GIRK-I by 100 µM noradrenaline was completely antagonized by 10 µM atipamezole and the Ca²⁺ dependent rundown of NA-induced GIRK-I was prevented by 10 µM atipamezole. Considering the Ki values of atipamezole for α2A AR (=1~3 nM) (Vacher et al., 2010, J Med Chem) and β AR (>10 µM) (Virtanen et al., 1989, Arch Int Pharmacodyn Ther), these results really reflect α2A AR activity but not β AR activity (Figure S5). Furthermore, because it is already well established that NA-induced GIRK-I was mediated by α2A AR activity in LC neurons (Arima et al., 1998, J Physiol; Williams et al., 1985, Neuroscience), it is not necessarily need to re-examine 1-10 µM NA on GIRK-I.

While the milestone papers by Williams remain highly influential, they should be re-evaluated in light of more recent findings, given that they date back over 40 years. Advances in our understanding now allow for a more nuanced interpretation of some of their results. For example, see McKinney et al. (eLife, 2023). This study demonstrates that presynaptic β-adrenergic receptors-particularly β2-can enhance neuronal excitability via autocrine mechanisms. This suggests that your post-activation experiments using atipamezole may not fully exclude a contribution of β-adrenergic signaling. Such a role might become apparent when conducting more detailed titration experiments.

The reviewer #3 may be misunderstanding the report by McKinney et al. (eLife, 2013). This paper did not demonstrate that presynaptic β-adrenergic receptors-particularly β2- can enhance neuronal excitability via autocrine mechanisms. It is impossible for LC neurons to increase their excitability by activating β-adrenergic receptors, as we have clearly shown that enhancement of GIRK-I by 100 µM noradrenaline was completely antagonized by 10 µM atipamezole. Considering the difference in Ki values of atipamezole for α2-AR (= 2~4 nM) (Vacher et al., 2010, J Med Chem) and β-AR (>10 µM) (Virtanen et al., 1989, Arch Int Pharmacodyn Ther), such a complete antagonization (of 100 µM NA-induced GIRK-I) by 10 µM atipamezole really reflect α2A-AR activity but not β-AR activity (Figure S5). Furthermore, it is already well established that NA-induced GIRK-I was mediated by α2-AR activity in LC neurons (Arima et al., 1998, J Physiol). McKinney et al. (eLife, 2023) have just found the absence of lateral inhibition on adjacent LC neurons by NA autocrine caused respective spike activity. This has nothing to do with autoinhibition.

(4) Age mismatch and disease claims 

All electrophysiology and biochemical data come from juvenile (< P30) mice, yet the conclusions stress Alzheimer-related degeneration. Key endpoints need to be replicated in adult or aged mice, or the manuscript should soften its neurodegenerative scope. 

Authors response: As described in the section of Conclusion, we never stress Alzheimer-related degeneration, but might give such an impression. To avoid such a misunderstanding, we have added a description “However, the present mechanism must be proven to be valid in adult or old mice, to validate its involvement in the pathogenesis of AD.” (R1, page 14, lines 448-450).

It would be great to see this experiment performed in aged mice-you are the one who has everything in place to do it right now! 

In our future separate studies, we would like to prove that the present mechanism is valid in aged mice, to validate its involvement in the pathogenesis of AD. This is partly because the patch-clamp study in aged mice is extremely difficult and takes much time.

Authors response: In the abstract, you suggest that internalization of α2A-adrenergic receptors could represent a therapeutic target for Alzheimer's disease. "...Thus, it is likely that internalization of α2A-AR increased cytosolic NA, as reflected in AEP increases, by facilitating reuptake of autocrine-released NA. The suppression of α2A-AR internalization may have a translational potential for AD treatment."

α2A-AR internalization was involved in the degeneration of LC neurons. Because we confirmed that spike-frequency adaptation reflecting α2A-AR-mediated autoinhibition can be induced in adult mice as prominently as in juvenile mice (Figure S10), it is not inadequate to suggest that the suppression of α2A-AR internalization may have a translational potential for anxiety/AD treatment (see Discussion; R2, page 14, lines 445-449).

(6) Quantitative histology  

Figure 5 presents attractive images, but no numerical analysis is provided. Please provide ROI-based fluorescence quantification (with n values) or move the images to the supplement and rely on the Western blots. 

Author response: We have moved the immunohistochemical results in Fig. 5 to the supplement, as we believe the quantification of immunohistochemical staining is not necessarily correct.   

What do you mean by that " ...immunohistochemical staining is not necessarily correct."  

It is evident that in terms of quantification, Western blot analysis is a more accurate method than immunohistochemical staining. In this sense, it is the contention of our study that the ROI-based fluorescence quantification of immunohistochemical staining is not necessarily an accurate or correct procedure, compared to the quantification by Western blot analysis.

Author response:

Notes to Editors

We previously received comments from three reviewers at Biological Psychiatry, which we have addressed in detail below. The following is a summary of the reviewers’ comments along with our responses.

Reviewers 1 and 2 sought clearer justification for studying the cognition-mental health overlap (covariation) and its neuroimaging correlates. In the revised manuscripts, we expanded the Introduction and Discussion to explicitly outline the theoretical implications of investigating this overlap with machine learning. We also added nuance to the interpretation of the observed associations.

Reviewer 1 raised concerns about the accessibility of the machine learning methodology for readers without expertise in this field. We revised the Methods section to provide a clearer, step-by-step explanation of our machine learning approach, particularly the two-level machine learning through stacking. We also enhanced the description of the overall machine learning design, including model training, validation, and testing.

In response to Reviewer 2’s request for deeper interpretation of our findings and stronger theoretical grounding, we have expanded our discussion by incorporating a thorough interpretation of how mental health indices relate to cognition, material that was previously included only in supplementary materials due to word limit constraints. We have further strengthened the theoretical justification for our study design, with particular emphasis on the importance of examining shared variance between cognition and mental health through the derivation of neural markers of cognition. Additionally, to enhance the biological interpretation of our results, we included new analyses of feature importance across neuroimaging modalities, providing clearer insights into which neural features contribute most to the observed relationships.

Notably, Reviewer 3 acknowledged the strength of our study, including multimodal design, robust analytical approach, and clear visualization and interpretation of results. Their comments were exclusively methodological, underscoring the manuscript’s quality.

Reviewer 1:

The authors try to bridge mental health characteristics, global cognition and various MRI-derived (structural, diffusion and resting state fMRI) measures using the large dataset of UK Biobank. Each MRI modality alone explained max 25% of the cognitionmental health covariance, and when combined together 48% of the variance could be explained. As a peer-reviewer not familiar with the used methods (machine learning, although familiar with imaging), the manuscript is hard to read and I wonder what the message for the field might be. In the end of the discussion the authors state '... we provide potential targets for behavioural and physiological interventions that may affect cognition', the real relevance (and impact) of the findings is unclear to me.

Thank you for your thorough review and practical recommendations. We appreciate your constructive comments and suggestions and hope our revisions adequately address your concerns.

Major questions

(1) The methods are hard to follow for people not in this specific subfield, and therefore, I expect that for readers it is hard to understand how valid and how useful the approach is.

Thank you for your comment. To enhance accessibility for readers without a machine learning background, we revised the Methods section to clarify our analyses while retaining important technical details needed to understand our approach. Recognizing that some concepts may require prior knowledge, we provide detailed explanations of each analysis step, including the machine learning pipeline in the Supplementary Methods.

Line 188: “We employed nested cross-validation to predict cognition from mental health indices and 72 neuroimaging phenotypes (Fig. 1). Nested cross-validation is a robust method for evaluating machine-learning models while tuning their hyperparameters, ensuring that performance estimates are both accurate and unbiased. Here, we used a nested cross-validation scheme with five outer folds and ten inner folds.

We started by dividing the entire dataset into five outer folds. Each fold took a turn being held out as the outerfold test set (20% of the data), while the remaining four folds (80% of the data) were used as an outer-fold training set. Within each outer-fold training set, we performed a second layer of cross-validation – this time splitting the data into ten inner folds. These inner folds were used exclusively for hyperparameter tuning: models were trained on nine of the inner folds and validated on the remaining one, cycling through all ten combinations.

We then selected the hyperparameter configuration that performed best across the inner-fold validation sets, as determined by the minimal mean squared error (MSE). The model was then retrained on the full outer-fold training set using this hyperparameter configuration and evaluated on the outer-fold test set, using four performance metrics: Pearson r, the coefficient of determination ( R²), the mean absolute error (MAE), and the MSE. This entire process was repeated for each of the five outer folds, ensuring that every data point is used for both training and testing, but never at the same time. We opted for five outer folds instead of ten to reduce computational demands, particularly memory and processing time, given the substantial volume of neuroimaging data involved in model training. Five outer folds led to an outer-fold test set at least n = 4 000, which should be sufficient for model evaluation. In contrast, we retained ten inner folds to ensure robust and stable hyperparameter tuning, maximising the reliability of model selection.

To model the relationship between mental health and cognition, we employed Partial Least Squares Regression (PLSR) to predict the g-factor from 133 mental health variables. To model the relationship between neuroimaging data and cognition, we used a two-step stacking approach [15–17,61] to integrate information from 72 neuroimaging phenotypes across three MRI modalities. In the first step, we trained 72 base (first-level) PLSR models, each predicting the g-factor from a single neuroimaging phenotype. In the second step, we used the predicted values from these base models as input features for stacked models, which again predicted the g-factor. We constructed four stacked models based on the source of the base predictions: one each for dwMRI, rsMRI, sMRI, and a combined model incorporating all modalities (“dwMRI Stacked”, “rsMRI Stacked”, “sMRI Stacked”, and “All MRI Stacked”, respectively). Each stacked model was trained using one of four machine learning algorithms – ElasticNet, Random Forest, XGBoost, or Support Vector Regression – selected individually for each model (see Supplementary Materials, S6).

For rsMRI phenotypes, we treated the choice of functional connectivity quantification method – full correlation, partial correlation, or tangent space parametrization – as a hyperparameter. The method yielding the highest performance on the outer-fold training set was selected for predicting the g-factor (see Supplementary Materials, S5).

To prevent data leakage, we standardized the data using the mean and standard deviation derived from the training set and applied these parameters to the corresponding test set within each outer fold. This standardization was performed at three key stages: before g-factor derivation, before regressing out modality-specific confounds from the MRI data, and before stacking. Similarly, to maintain strict separation between training and testing data, both base and stacked models were trained exclusively on participants from the outer-fold training set and subsequently applied to the corresponding outer-fold test set.

To evaluate model performance and assess statistical significance, we aggregated the predicted and observed g_factor values from each outer-fold test set. We then computed a bootstrap distribution of Pearson’s correlation coefficient (_r) by resampling with replacement 5 000 times, generating 95% confidence intervals (CIs) (Fig. 1). Model performance was considered statistically significant if the 95% CI did not include zero, indicating that the observed associations were unlikely to have occurred by chance.”

(2) If only 40% of the cognition-mental health covariation can be explained by the MRI variables, how to explain the other 60% of the variance? And related to this %: why do the author think that 'this provides us confidence in using MRI to derive quantitative neuromarkers of cognition'?

Thank you for this insightful observation. Using the MRI modalities available in the UK Biobank, we were able to account for 48% of the covariation between cognition and mental health. The remaining 52% of unexplained variance may arise from several sources. One possibility is the absence of certain neuroimaging modalities in the UK Biobank dataset, such as task-based fMRI contrasts, positron emission tomography, arterial spin labeling, and magnetoencephalography/electroencephalography. Prior research from our group and others has consistently demonstrated strong predictive performance from specific task-based fMRI contrasts, particularly those derived from tasks like the n-Back working memory task and the face-name episodic memory task, none of which is available in the UK Biobank.

Moreover, there are inherent limitations in using MRI as a proxy for brain structure and function. Measurement error and intra-individual variability, such as differences in a cognitive state between cognitive assessments and MRI acquisition, may also contribute to the unexplained variance. According to the Research Domain Criteria (RDoC) framework, brain circuits represent only one level of neurobiological analysis relevant to cognition. Other levels, including genes, molecules, cells, and physiological processes, may also play a role in the cognition-mental health relationship.

Nonetheless, neuroimaging provides a valuable window into the biological mechanisms underlying this overlap – insights that cannot be gleaned from behavioural data alone. We have now incorporated these considerations into the Discussion section.

Line 658: “Although recent debates [18] have challenged the predictive utility of MRI for cognition, our multimodal marker integrating 72 neuroimaging phenotypes captures nearly half of the mental health-explained variance in cognition. We demonstrate that neural markers with greater predictive accuracy for cognition also better explain cognition-mental health covariation, showing that multimodal MRI can capture both a substantial cognitive variance and nearly half of its shared variance with mental health. Finally, we show that our neuromarkers explain a substantial portion of the age- and sex-related variance in the cognition-mental health relationship, highlighting their relevance in modeling cognition across demographic strata.

The remaining unexplained variance in the relationship between cognition and mental health likely stems from multiple sources. One possibility is the absence of certain neuroimaging modalities in the UK Biobank dataset, such as task-based fMRI contrasts, positron emission tomography, arterial spin labeling, and magnetoencephalography/electroencephalography. Prior research has consistently demonstrated strong predictive performance from specific task-based fMRI contrasts, particularly those derived from tasks like the n-Back working memory task and the face-name episodic memory task, none of which is available in the UK Biobank [15,17,61,69,114,142,151].

Moreover, there are inherent limitations in using MRI as a proxy for brain structure and function. Measurement error and intra-individual variability, such as differences in a cognitive state between cognitive assessments and MRI acquisition, may also contribute to the unexplained variance. According to the RDoC framework, brain circuits represent only one level of neurobiological analysis relevant to cognition [14]. Other levels, including genes, molecules, cells, and physiological processes, may also play a role in the cognition-mental health relationship.

Nonetheless, neuroimaging provides a valuable window into the biological mechanisms underlying this overlap – insights that cannot be gleaned from behavioural data alone. Ultimately, our findings validate brain-based neural markers as a fundamental neurobiological unit of analysis, advancing our understanding of mental health through the lens of cognition.”

Regarding our confidence in using MRI to derive neural markers for cognition, we base this on the predictive performance of MRI-based models. As we note in the Discussion (Line 554: “Consistent with previous studies, we show that MRI data predict individual differences in cognition with a medium-size performance (r ≈ 0.4) [15–17, 28, 61, 67, 68].”), the medium effect size we observed (r ≈ 0.4) agrees with existing literature on brain-cognition relationships, confirming that machine learning leads to replicable results. This effect size represents a moderate yet meaningful association in neuroimaging studies of aging, consistent with reports linking brain to behaviour in adults (Krämer et al., 2024; Tetereva et al., 2022). For example, a recent meta-analysis by Vieira and colleagues (2022) reported a similar effect size (r = 0.42, 95% CI [0.35;0.50]). Our study includes over 15000 participants, comparable to or more than typical meta-analyses, allowing us to characterise our work as a “mega-analysis”. And on top of this predictive performance, we found our neural markers for cognition to capture half of the cognition-mental health covariation, boosting our confidence in our approach.

Krämer C, Stumme J, da Costa Campos L, Dellani P, Rubbert C, Caspers J, et al. Prediction of cognitive performance differences in older age from multimodal neuroimaging data. GeroScience. 2024;46:283–308.

Tetereva A, Li J, Deng JD, Stringaris A, Pat N. Capturing brain cognition relationship: Integrating task‐based fMRI across tasks markedly boosts prediction and test‐retest reliability. NeuroImage. 2022;263:119588.

(3) Imagine that we can increase the explained variance using multimodal MRI measures, why is it useful? What does it learn us? What might be the implications?

We assume that by variance, Reviewer 1 referred to the cognition-mental health covariation mentioned in point 2) above.

If we can increase the explained cognition-mental health covariation using multimodal MRI measures, it would mean that we have developed a reasonable neuromarker that is close to RDoC’s neurobiological unit of analysis for cognition. RDoC treats cognition as one of the main basic functional domains that transdiagnostically underly mental health. According to RDoC, mental health should be studied in relation to cognition, alongside other domains such as negative and positive valence systems, arousal and regulatory systems, social processes, and sensorimotor functions. RDoC further emphasizes that each domain, including cognition, should be investigated not only at the behavioural level but also through its neurobiological correlates. This means RDoC aims to discover neural markers of cognition that explain the covariation between cognition and mental health. For us, we approach the development of such neural markers using multimodal neuroimaging. We have now explained the motivation of our study in the first paragraph of the Introduction.

Line 43: “Cognition and mental health are closely intertwined [1]. Cognitive dysfunction is present in various mental illnesses, including anxiety [2, 3], depression [4–6], and psychotic disorders [7–12]. National Institute of Mental Health’s Research Domain Criteria (RDoC) [13,14] treats cognition as one of the main basic functional domains that transdiagnostically underly mental health. According to RDoC, mental health should be studied in relation to cognition, alongside other domains such as negative and positive valence systems, arousal and regulatory systems, social processes, and sensorimotor functions. RDoC further emphasizes that each domain, including cognition, should be investigated not only at the behavioural level but also through its neurobiological correlates. In this study, we aim to examine how the covariation between cognition and mental health is reflected in neural markers of cognition, as measured through multimodal neuroimaging.”

More specific issues:

Introduction

(4) In the intro the sentence 'in some cases, altered cognitive functioning is directly related to psychiatric symptom severity' is in contrast to the next sentence '... are often stable and persist upon alleviation of psychiatric symptoms'.

Thank you for pointing this out. The first sentence refers to cases where cognitive deficits fluctuate with symptom severity, while the second emphasizes that core cognitive impairments often remain stable even during symptom remission. To avoid this confusion, we have removed these sentences.

(5) In the intro the text on the methods (various MRI modalities) is not needed for the Biol Psych readers audience.

We appreciate your comment. While some members of our target audience may have backgrounds in neuroimaging, machine learning, or psychiatry, we recognize that not all readers will be familiar with all three areas. To ensure accessibility for those who are not familiar with neuroimaging, we included a brief overview of the MRI modalities and quantification methods used in our study to provide context for the specific neuroimaging phenotypes. Additionally, we provided background information on the machine learning techniques employed, so that readers without a strong background in machine learning can still follow our methodology.

(6) Regarding age of the study sample: I understand that at recruitment the subjects' age ranges from 40 to 69 years. At MRI scanning the age ranges between about 46 to 82. How is that possible? And related to the age of the population: how did the authors deal with age in the analyses, since age is affecting both cognition as the brain measures?

Thank you for noticing this. In the Methods section, we first outline the characteristics of the UK Biobank cohort, including the age at first recruitment (40-69 years). Table 1 then shows the characteristics of participant subsamples included in each analysis. Since our study used data from Instance 2 (the second in-person visit), participants were approximately 5-13 years older at scanning, resulting in the age range of 46 to 82 years. We clarified the Table 1 caption as follows:

Line 113: “Table 1. Demographics for each subsample analysed: number, age, and sex of participants who completed all cognitive tests, mental health questionnaires, and MRI scanning”

We acknowledge that age may influence cognitive and neuroimaging measures. In our analyses, we intentionally preserved age-related variance in brain-cognition relationships across mid and late adulthood, as regressing out age completely would artificially remove biologically meaningful associations. At the same time, we rigorously addressed the effects of age and sex through additional commonality analyses quantifying age and sex contributions to the relationship between cognition and mental health.

As noted by Reviewer 1 and illustrated in Figure 8, age and sex shared substantial overlapping variance with both mental health and neuroimaging phenotypes in explaining cognitive outcomes. For example, in Figure 8i, age and sex together accounted for 43% of the variance in the cognition-mental health relationship:

(2.76 + 1.03) / (2.76 + 1.03 + 3.52 + 1.45) ≈ 0.43

Furthermore, neuromarkers from the all-MRI stacked model explained 72% of this age/sexrelated variance:

2.76 / (2.76 + 1.03) ≈ 0.72

This indicates that our neuromarkers captured a substantial portion of the cognition-mental health covariation that varied with age and sex, highlighting their relevance in age/sex-sensitive cognitive modeling.

In the Methods, Results, and Discussion, we say:

Methods

Line 263: “To understand how demographic factors, including age and sex, contribute to this relationship, we also conducted a separate set of commonality analyses treating age, sex, age2, age×sex, and age2×sex as an additional set of explanatory variables (Fig. 1).”

Results

Line 445: “Age and sex shared substantial overlapping variance with both mental health and neuroimaging in explaining cognition, accounting for 43% of the variance in the cognition-mental health relationship. Multimodal neural marker of cognition based on three MRI modalities (“All MRI Stacked”) explained 72% of this age and sex-related variance (Fig. 8i–l and Table S21).”

Discussion

Line 660: “We demonstrate that neural markers with greater predictive accuracy for cognition also better explain cognition-mental health covariation, showing that multimodal MRI can capture both a substantial cognitive variance and nearly half of its shared variance with mental health. Finally, we show that our neuromarkers explain a substantial portion of the age- and sex-related variance in the cognition-mental health relationship, highlighting their relevance in modeling cognition across demographic strata.”

(7) Regarding the mental health variables: where characteristics with positive value (e.g. happiness and subjective wellbeing) reversely scored (compared to the negative items, such as anxiety, addition, etc)?

We appreciate you noting this. These composite scores primarily represent standard clinical measures such as the GAD-7 anxiety scale and N-12 neuroticism scale. We did not reverse the scores to keep their directionality, therefore making interpretability consistent with the original studies the scores were derived from (e.g., Davis et al., 2020; Dutt et al., 2022). Complete descriptive statistics for all mental health indices and detailed derivation procedures are provided in the Supplementary Materials (S2). On Page 6, Supplementary Methods, we say:

Line 92: “Composite mental health scores included the Generalized Anxiety Disorder (GAD-7), the Posttraumatic Stress Disorder (PTSD) Checklist (PCL-6), the Alcohol Use Disorders Identification Test (AUDIT), the Patient Health Questionnaire (PHQ-9) [12], the Eysenck Neuroticism (N-12), Probable Depression Status (PDS), and the Recent Depressive Symptoms (RDS-4) scores [13, 14]. To calculate the GAD-7, PCL-6, AUDIT, and PHQ-9, we used questions introduced at the online follow-up [12]. To obtain the N-12, PDS, and RDS-4 scores [14], we used data collected during the baseline assessment [13, 14].

We subcategorized depression and GAD based on frequency, current status (ever had depression or anxiety and current status of depression or anxiety), severity, and clinical diagnosis (depression or anxiety confirmed by a healthcare practitioner). Additionally, we differentiated between different depression statuses, such as recurrent depression, depression triggered by loss, etc. Variables related to self-harm were subdivided based on whether a person has ever self-harmed with the intent to die.

To make response scales more intuitive, we recorded responses within the well-being domain such that the lower score corresponded to a lesser extent of satisfaction (“Extremely unhappy”) and the higher score indicated a higher level of happiness (“Extremely happy”). For all questions, we assigned the median values to “Prefer not to answer” (-818 for in-person assessment and -3 for online questionnaire) and “Do not know” (-121 for in-person assessment and -1 for online questionnaire) responses. We excluded the “Work/job satisfaction” question from the mental health derivatives list because it included a “Not employed” response option, which could not be reasonably coded.

To calculate the risk of PTSD, we used questions from the PCL-6 questionnaire. Following Davis and colleagues [12], PCL-6 scores ranged from 6 to 29. A PCL-6 score of 12 or below corresponds to a low risk of meeting the Clinician-Administered PTSD Scale diagnostic criteria. PCL-6 scores between 13 and 16 and between 17 and 25 are indicative of an increased risk and high risk of PTSD, respectively. A score of above 26 is interpreted as a very high risk of PTSD [12, 15]. PTSD status was set to positive if the PCL-6 score exceeded or was equal to 14 and encompassed stressful events instead of catastrophic trauma alone [12].

To assess alcohol consumption, alcohol dependence, and harm associated with drinking, we calculated the sum of the ten questions from the AUDIT questionnaire [16]. We additionally subdivided the AUDIT score into the alcohol consumption score (questions 1-3, AUDIT-C) and the score reflecting problems caused by alcohol (questions 4-10, AUDIT-P) [17]. In questions 2-10 that followed the first trigger question (“Frequency of drinking alcohol”), we replaced missing values with 0 as they would correspond to a “Never” response to the first question.

An AUDIT score cut-off of 8 suggests moderate or low-risk alcohol consumption, and scores of 8 to 15 and above 15 indicate severe/harmful and hazardous (alcohol dependence or moderate-severe alcohol use disorder) drinking, respectively [16, 18]. Subsequently, hazardous alcohol use and alcohol dependence status correspond to AUDIT scores of ≥ 8 and ≥ 15, respectively. The “Alcohol dependence ever” status was set to positive if a participant had ever been physically dependent on alcohol. To reduce skewness, we logx+1-transformed the AUDIT, AUDIT-C, and AUDIT-P scores [17].”

Davis KAS, Coleman JRI, Adams M, Allen N, Breen G, Cullen B, et al. Mental health in UK Biobank – development, implementation and results from an online questionnaire completed by 157 366 participants: a reanalysis. BJPsych Open. 2020;6:e18.

Dutt RK, Hannon K, Easley TO, Griffis JC, Zhang W, Bijsterbosch JD. Mental health in the UK Biobank: A roadmap to selfreport measures and neuroimaging correlates. Hum Brain Mapp. 2022;43:816–832.  

(8) In the discussion section (page 23, line 416-421), the authors refer to specific findings that are not described in the results section > I would add these findings to the main manuscript (including the discussion / interpretation).

We appreciate your careful reading. We agree that our original Results section did not explicitly describe the factor loadings for mental health in the PLSR model, despite discussing their implications later in the paper. We needed to include this part of the discussion in the Supplementary Materials to meet the word limit of the original submission. However, in response to your suggestion, we have now added the results regarding factor loadings to the Results section. We also moved the discussion of the association between mental health features and general cognition from the Supplementary Material to the manuscript’s Discussion.

Results

Line 298: “On average, information about mental health predicted the g-factor at  R²_mean = 0.10 and r_mean \= 0.31 (95% CI [0.291, 0.315]; Fig. 2b and 2c and Supplementary Materials, S9, Table S12). The magnitude and direction of factor loadings for mental health in the PLSR model allowed us to quantify the contribution of individual mental health indices to cognition. Overall, the scores for mental distress, alcohol and cannabis use, and self-harm behaviours relate positively, and the scores for anxiety, neurological and mental health diagnoses, unusual or psychotic experiences, happiness and subjective well-being, and negative traumatic events relate negatively to cognition.”

Discussion

Line 492: “Factor loadings derived from the PLSR model showed that the scores for mental distress, alcohol and cannabis use, and self-harm behaviours related positively, and the scores for anxiety, neurological and mental health diagnoses, unusual or psychotic experiences, happiness and subjective well-being, and negative traumatic events related negatively to the g-factor. Positive PLSR loadings of features related to mental distress may indicate greater susceptibility to or exaggerated perception of stressful events, psychological overexcitability, and predisposition to rumination in people with higher cognition [72]. On the other hand, these findings may be specific to the UK Biobank cohort and the way the questions for this mental health category were constructed. In particular, to evaluate mental distress, the UK Biobank questionnaire asked whether an individual sought or received medical help for or suffered from mental distress. In this regard, the estimate for mental distress may be more indicative of whether an individual experiencing mental distress had an opportunity or aspiration to visit a doctor and seek professional help [73]. Thus, people with better cognitive abilities and also with a higher socioeconomic status may indeed be more likely to seek professional help.

Limited evidence supports a positive association between self-harm behaviours and cognitive abilities, with some studies indicating higher cognitive performance as a risk factor for non-suicidal self-harm. Research shows an inverse relationship between cognitive control of emotion and suicidal behaviours that weakens over the life course [73,74]. Some studies have found a positive correlation between cognitive abilities and the risk of nonsuicidal self-harm, suicidal thoughts, and suicidal plans that may be independent of or, conversely, affected by socioeconomic status [75,76]. In our study, the magnitude of the association between self-harm behaviours and cognition was low (Fig. 2), indicating a weak relationship.

Positive PLSR loadings of features related to alcohol and cannabis may also indicate the influence of other factors. Overall, this relationship is believed to be largely affected by age, income, education, social status, social equality, social norms, and quality of life [79–80]. For example, education level and income correlate with cognitive ability and alcohol consumption [79,81–83]. Research also links a higher probability of having tried alcohol or recreational drugs, including cannabis, to a tendency of more intelligent individuals to approach evolutionary novel stimuli [84,85]. This hypothesis is supported by studies showing that cannabis users perform better on some cognitive tasks [86]. Alternatively, frequent drinking can indicate higher social engagement, which is positively associated with cognition [87]. Young adults often drink alcohol as a social ritual in university settings to build connections with peers [88]. In older adults, drinking may accompany friends or family visits [89,90]. Mixed evidence on the link between alcohol and drug use and cognition makes it difficult to draw definite conclusions, leaving an open question about the nature of this relationship.

Consistent with previous studies, we showed that anxiety and negative traumatic experiences were inversely associated with cognitive abilities [90–93]. Anxiety may be linked to poorer cognitive performance via reduced working memory capacity, increased focus on negative thoughts, and attentional bias to threatening stimuli that hinder the allocation of cognitive resources to a current task [94–96]. Individuals with PTSD consistently showed impaired verbal and working memory, visual attention, inhibitory function, task switching, cognitive flexibility, and cognitive control [97–100]. Exposure to traumatic events that did not reach the PTSD threshold was also linked to impaired cognition. For example, childhood trauma is associated with worse performance in processing speed, attention, and executive function tasks in adulthood, and age at a first traumatic event is predictive of the rate of executive function decline in midlife [101,102]. In the UK Biobank cohort, adverse life events have been linked to lower cognitive flexibility, partially via depression level [103].

In agreement with our findings, cognitive deficits are often found in psychotic disorders [104,105]. We treated neurological and mental health symptoms as predictor variables and did not stratify or exclude people based on psychiatric status or symptom severity. Since no prior studies have examined isolated psychotic symptoms (e.g., recent unusual experiences, hearing unreal voices, or seeing unreal visions), we avoid speculating on how these symptoms relate to cognition in our sample.

Finally, negative PLSR loadings of the features related to happiness and subjective well-being may be specific to the study cohort, as these findings do not agree with some previous research [107–109]. On the other hand, our results agree with the study linking excessive optimism or optimistic thinking to lower cognitive performance in memory, verbal fluency, fluid intelligence, and numerical reasoning tasks, and suggesting that pessimism or realism indicates better cognition [110]. The concept of realism/optimism as indicators of cognition is a plausible explanation for a negative association between the g-factor and friendship satisfaction, as well as a negative PLSR loading of feelings that life is meaningful, especially in older adults who tend to reflect more on the meaning of life [111]. The latter is supported by the study showing a negative association between cognitive function and the search for the meaning of life and a change in the pattern of this relationship after the age of 60 [112]. Finally, a UK Biobank study found a positive association of happiness with speed and visuospatial memory but a negative relationship with reasoning ability [113].”

(9) In the discussion section (page 24, line 440-449), the authors give an explanation on why the diffusion measure have limited utility, but the arguments put forward also concern structural and rsfMRI measures.

Thank you for this important observation. Indeed, the argument about voxel-averaged diffusion components (“… these metrics are less specific to the properties of individual white matter axons or bundles, and instead represent a composite of multiple diffusion components averaged within a voxel and across major fibre pathways”) could theoretically apply across other MRI modalities. We have therefore removed this point from the discussion to avoid overgeneralization. However, we maintain our central argument about the biological specificity of conventional tractography-derived diffusion metrics as their particular sensitivity to white matter microstructure (e.g., axonal integrity, myelin content) may make them better suited for detecting neuropathological changes than dynamic cognitive processes. This interpretation aligns with the mixed evidence linking these metrics to cognitive performance, despite their established utility in detecting white matter abnormalities in clinical populations (e.g., Bergamino et al., 2021; Silk et al., 2009). We clarify this distinction in the manuscript.

Line 572: “The somewhat limited utility of diffusion metrics derived specifically from probabilistic tractography in serving as robust quantitative neuromarkers of cognition and its shared variance with mental health may stem from their greater sensitivity and specificity to neuronal integrity and white matter microstructure rather than to dynamic cognitive processes. Critically, probabilistic tractography may be less effective at capturing relationships between white matter microstructure and behavioural scores cross-sectionally, as this method is more sensitive to pathological changes or dynamic microstructural alterations like those occurring during maturation. While these indices can capture abnormal white matter microstructure in clinical populations such as Alzheimer’s disease, schizophrenia, or attention deficit hyperactivity disorder (ADHD) [117–119], the empirical evidence on their associations with cognitive performance is controversial [114, 120–126].”

Bergamino M, Walsh RR, Stokes AM. Free-water diffusion tensor imaging improves the accuracy and sensitivity of white matter analysis in Alzheimer’s disease. Sci Rep. 2021;11:6990.

Silk TJ, Vance A, Rinehart N, Bradshaw JL, Cunnington R. White-matter abnormalities in attention deficit hyperactivity disorder: a diffusion tensor imaging study. Hum Brain Mapp. 2009;30:2757–2765.

Reviewer 2:

This is an interesting study combining a lot of data to investigate the link between cognition and mental health. The description of the study is very clear, it's easy to read for someone like me who does not have a lot of expertise in machine learning.

We thank you for your thorough review and constructive feedback. Your insightful comments have helped us identify conceptual and methodological aspects that required improvement in the manuscript. We have incorporated relevant changes throughout the paper, and below, we address each of your points in detail.

Comment 1: My main concern with this manuscript is that it is not yet clear to me what it exactly means to look at the overlap between cognition and mental health. This relation is r=0.3 which is not that high, so why is it then necessary to explain this overlap with neuroimaging measures? And, could it be that the relation between cognition and mental health is explained by third variables (environment? opportunities?). In the introduction I miss an explanation of why it is important to study this and what it will tell us, and in the discussion I would like to read some kind of 'answer' to these questions.

Thank you. It’s important to clarify why we investigated the relationship between cognition and mental health, and what we found using data from the UK Biobank.

Conceptually, our work is grounded in the Research Domain Criteria (RDoC; Insel et al., 2010) framework. RDoC conceptualizes mental health not through traditional diagnostic categories, but through core functional domains that span the full spectrum from normal to abnormal functioning. These domains include cognition, negative and positive valence systems, arousal and regulatory systems, social processes, and sensorimotor functions. Within this framework, cognition is considered a fundamental domain that contributes to mental health across diagnostic boundaries. Meta-analytic evidence supports a link between cognitive functioning and mental health (Abramovitch, et al., 2021; East-Richard, et al., 2020). In the context of a large, population-based dataset like the UK Biobank, this implies that cognitive performance – as measured by various cognitive tasks – should be meaningfully associated with available mental health indicators.

However, because cognition is only one of several functional domains implicated in mental health, we do not expect the covariation between cognition and mental health to be very high. Other domains, such as negative and positive valence systems, arousal and regulatory systems, or social processing, may also play significant roles. Theoretically, this places an upper bound on the strength of the cognition-mental health relationship, especially in normative, nonclinical samples.

Our current findings from the UK Biobank reflect this. Most of the 133 mental health variables showed relatively weak individual correlations with cognition (mean r \= 0.01, SD = 0.05, min r \= –0.08, max r \= 0.17; see Figure 2). However, using a PLS-based machine learning approach, we were able to integrate information across all mental-health variables to predict cognition, yielding an out-of-sample correlation of r = 0.31 [95% CI: 0.29, 0.32].  

We believe this estimate approximates the true strength of the cognition-mental health relationship in normative samples, consistent with both theoretical expectations and prior empirical findings. Theoretically, this aligns with the RDoC view that cognition is one of several contributing domains. Empirically, our results are consistent with findings from our previous mega-analysis in children (Wang et al., 2025). Moreover, in the field of gerontology, an effect size of r = 0.31 is not considered small. According to Brydges (2019), it falls around the 70th percentile of effect sizes reported in gerontological studies and approaches the threshold for a large effect (r \= 0.32). Given that most studies report within-sample associations, our out-of-sample results are likely more robust and generalizable (Yarkoni & Westfall, 2017).

To answer, “why is it then necessary to explain this overlap with neuroimaging measures”, we again draw on the conceptual foundation of the RDoC framework. RDoC emphasizes that each functional domain, such as cognition, should be studied not only at the behavioural level but also across multiple neurobiological units of analysis, including genes, molecules, cells, circuits, physiology, and behaviour.

MRI-based neural markers represent one such level of analysis. While other biological systems (e.g., genetic, molecular, or physiological) also contribute to the cognition-mental health relationship, neuroimaging provides unique insights into the brain mechanisms underlying this association – insights that cannot be obtained from behavioural data alone.

In response to the related question, “Could the relationship between cognition and mental health be explained by third variables (e.g., environment, opportunities)?”, we note that developing a neural marker of cognition capable of capturing its relationship with mental health is the central aim of this study. Using the MRI modalities available in the UK Biobank, we were able to account for 48% of the covariation between cognition and mental health.

The remaining 52% of unexplained variance may stem from several sources. According to the RDoC framework, neuromarkers could be further refined by incorporating additional neuroimaging modalities (e.g., task-based fMRI, PET, ASL, MEG/EEG, fNIRS) and integrating other units of analysis such as genetic, molecular, cellular, and physiological data.

Once more comprehensive neuromarkers are developed, capturing a greater proportion of the cognition-mental health covariation, they may also lead to new research direction – to investigate how environmental factors and life opportunities influence these markers. However, exploring those environmental contributions lies beyond the scope of the current study.

We discuss these considerations and explain the motivation of our study in the revised Introduction and Discussion.

Line 481: “Our analysis confirmed the validity of the g-factor [31] as a quantitative measure of cognition [31], demonstrating that it captures nearly half (39%) of the variance across twelve cognitive performance scores, consistent with prior studies [63–68]. Furthermore, we were able to predict cognition from 133 mental health indices, showing a medium-sized relationship that aligns with existing literature [69,70]. Although the observed mental health-cognition association is lower than within-sample estimates in conventional regression models, it aligns with our prior mega-analysis in children [69]. Notably, this effect size is not considered small in gerontology. In fact, it falls around the 70th percentile of reported effects and approaches the threshold for a large effect at r = 0.32 [71]. While we focused specifically on cognition as an RDoC core domain, the strength of its relationship with mental health may be bounded by the influence of other functional domains, particularly in normative, non-clinical samples – a promising direction for future research.”

Line 658: “Although recent debates [18] have challenged the predictive utility of MRI for cognition, our multimodal marker integrating 72 neuroimaging phenotypes captures nearly half of the mental health-explained variance in cognition. We demonstrate that neural markers with greater predictive accuracy for cognition also better explain cognition-mental health covariation, showing that multimodal MRI can capture both a substantial cognitive variance and nearly half of its shared variance with mental health. Finally, we show that our neuromarkers explain a substantial portion of the age- and sex-related variance in the cognition-mental health relationship, highlighting their relevance in modeling cognition across demographic strata.

The remaining unexplained variance in the relationship between cognition and mental health likely stems from multiple sources. One possibility is the absence of certain neuroimaging modalities in the UK Biobank dataset, such as task-based fMRI contrasts, positron emission tomography, arterial spin labeling, and magnetoencephalography/electroencephalography. Prior research has consistently demonstrated strong predictive performance from specific task-based fMRI contrasts, particularly those derived from tasks like the n-Back working memory task and the face-name episodic memory task, none of which is available in the UK Biobank [15,17,61,69,114,142,151].

Moreover, there are inherent limitations in using MRI as a proxy for brain structure and function. Measurement error and intra-individual variability, such as differences in a cognitive state between cognitive assessments and MRI acquisition, may also contribute to the unexplained variance. According to the RDoC framework, brain circuits represent only one level of neurobiological analysis relevant to cognition [14]. Other levels, including genes, molecules, cells, and physiological processes, may also play a role in the cognition-mental health relationship.

Nonetheless, neuroimaging provides a valuable window into the biological mechanisms underlying this overlap – insights that cannot be gleaned from behavioural data alone. Ultimately, our findings validate brain-based neural markers as a fundamental neurobiological unit of analysis, advancing our understanding of mental health through the lens of cognition.”

Introduction

Line 43: “Cognition and mental health are closely intertwined [1]. Cognitive dysfunction is present in various mental illnesses, including anxiety [2, 3], depression [4–6], and psychotic disorders [7–12]. National Institute of Mental Health’s Research Domain Criteria (RDoC) [13,14] treats cognition as one of the main basic functional domains that transdiagnostically underly mental health. According to RDoC, mental health should be studied in relation to cognition, alongside other domains such as negative and positive valence systems, arousal and regulatory systems, social processes, and sensorimotor functions. RDoC further emphasizes that each domain, including cognition, should be investigated not only at the behavioural level but also through its neurobiological correlates. In this study, we aim to examine how the covariation between cognition and mental health is reflected in neural markers of cognition, as measured through multimodal neuroimaging.”

Discussion

Line 481: “Our analysis confirmed the validity of the g-factor [31] as a quantitative measure of cognition [31], demonstrating that it captures nearly half (39%) of the variance across twelve cognitive performance scores, consistent with prior studies [63–68]. Furthermore, we were able to predict cognition from 133 mental health indices, showing a medium-sized relationship that aligns with existing literature [69,70]. Although the observed mental health-cognition association is lower than within-sample estimates in conventional regression models, it aligns with our prior mega-analysis in children [69]. Notably, this effect size is not considered small in gerontology. In fact, it falls around the 70th percentile of reported effects and approaches the threshold for a large effect at r = 0.32 [71]. While we focused specifically on cognition as an RDoC core domain, the strength of its relationship with mental health may be bounded by the influence of other functional domains, particularly in normative, non-clinical samples – a promising direction for future research.”

Line 658: “Although recent debates [18] have challenged the predictive utility of MRI for cognition, our multimodal marker integrating 72 neuroimaging phenotypes captures nearly half of the mental health-explained variance in cognition. We demonstrate that neural markers with greater predictive accuracy for cognition also better explain cognition-mental health covariation, showing that multimodal MRI can capture both a substantial cognitive variance and nearly half of its shared variance with mental health. Finally, we show that our neuromarkers explain a substantial portion of the age- and sex-related variance in the cognition-mental health relationship, highlighting their relevance in modeling cognition across demographic strata.

The remaining unexplained variance in the relationship between cognition and mental health likely stems from multiple sources. One possibility is the absence of certain neuroimaging modalities in the UK Biobank dataset, such as task-based fMRI contrasts, positron emission tomography, arterial spin labeling, and magnetoencephalography/electroencephalography. Prior research has consistently demonstrated strong predictive performance from specific task-based fMRI contrasts, particularly those derived from tasks like the n-Back working memory task and the face-name episodic memory task, none of which is available in the UK Biobank [15,17,61,69,114,142,151].

Moreover, there are inherent limitations in using MRI as a proxy for brain structure and function. Measurement error and intra-individual variability, such as differences in a cognitive state between cognitive assessments and MRI acquisition, may also contribute to the unexplained variance. According to the RDoC framework, brain circuits represent only one level of neurobiological analysis relevant to cognition [14]. Other levels, including genes, molecules, cells, and physiological processes, may also play a role in the cognition-mental health relationship.

Nonetheless, neuroimaging provides a valuable window into the biological mechanisms underlying this overlap – insights that cannot be gleaned from behavioural data alone. Ultimately, our findings validate brain-based neural markers as a fundamental neurobiological unit of analysis, advancing our understanding of mental health through the lens of cognition.”

Insel T, Cuthbert B, Garvey M, Heinssen R, Pine DS, Quinn K, et al. Research Domain Criteria (RDoC): Toward a New Classification Framework for Research on Mental Disorders. AJP. 2010;167:748–751.

Abramovitch, A., Short, T., & Schweiger, A. (2021). The C Factor: Cognitive dysfunction as a transdiagnostic dimension in psychopathology. Clinical Psychology Review, 86, 102007.

East-Richard, C., R. -Mercier, A., Nadeau, D., & Cellard, C. (2020). Transdiagnostic neurocognitive deficits in psychiatry: A review of meta-analyses. Canadian Psychology / Psychologie Canadienne, 61(3), 190–214.

Wang Y, Anney R, Pat N. The relationship between cognitive abilities and mental health as represented by cognitive abilities at the neural and genetic levels of analysis. eLife. 2025.14:RP105537.

Brydges CR. Effect Size Guidelines, Sample Size Calculations, and Statistical Power in Gerontology. Innovation in Aging. 2019;3(4):igz036.

Yarkoni T, Westfall J. Choosing Prediction Over Explanation in Psychology: Lessons From Machine Learning. Perspect Psychol Sci. 2017;12(6):1100-1122.

Comment 2 Title: - Shouldn't it be "MRI markers" (plural)?

We used the singular form (“marker”) intentionally, as it refers to the composite neuroimaging marker derived from all three MRI modalities in our stacked model. This multimodal marker represents the combined predictive power of all modalities and captures the highest proportion of the mental health-cognition relationship in our analyses.

Comment 3: Introduction - I miss an explanation of why it is useful to look at cognition-mental health covariation

We believe we have sufficiently addressed this comment in our response to Reviewer 2, comment 1 above.

Comment 4: - "Demonstrating that MRI-based neural indicators of cognition capture the covariation between cognition and mental health will thereby support the utility of such indicators for understanding the etiology of mental health" (page 4, line 56-58) - how/why?

Previous research has largely focused on developing MRI-based neural indicators that accurately predict cognitive performance (Marek et al., 2022; Vieira et al., 2020). Building on this foundation, our findings further demonstrate that the predictive performance of a neural indicator for cognition is closely tied to its ability to explain the covariation between cognition and mental health. In other words, the robustness of a neural indicator – its capacity to capture individual differences in cognition – is strongly associated with how well it reflects the shared variance between cognition and mental health.

This insight is particularly important within the context of the RDoC framework, which seeks to understand the etiology of mental health through functional domains (such as cognition) and their underlying neurobiological units of analysis (Insel et al., 2010). According to RDoC, for a neural indicator of cognition to be informative for mental health research, it must not only predict cognitive performance but also capture its relationship with mental health.

Furthermore, RDoC emphasizes the integration of neurobiological measures to investigate the influence of environmental and developmental factors on mental health. In line with this, our neural indicators of cognition may serve as valuable tools in future research aimed at understanding how environmental exposures and developmental trajectories shape mental health outcomes. We discuss this in more detail in the revised Discussion.

Line 481: “Our analysis confirmed the validity of the g-factor [31] as a quantitative measure of cognition [31], demonstrating that it captures nearly half (39%) of the variance across twelve cognitive performance scores, consistent with prior studies [63–68]. Furthermore, we were able to predict cognition from 133 mental health indices, showing a medium-sized relationship that aligns with existing literature [69,70]. Although the observed mental health-cognition association is lower than within-sample estimates in conventional regression models, it aligns with our prior mega-analysis in children [69]. Notably, this effect size is not considered small in gerontology. In fact, it falls around the 70th percentile of reported effects and approaches the threshold for a large effect at r = 0.32 [71]. While we focused specifically on cognition as an RDoC core domain, the strength of its relationship with mental health may be bounded by the influence of other functional domains, particularly in normative, non-clinical samples – a promising direction for future research.”

Line 658: “Although recent debates [18] have challenged the predictive utility of MRI for cognition, our multimodal marker integrating 72 neuroimaging phenotypes captures nearly half of the mental health-explained variance in cognition. We demonstrate that neural markers with greater predictive accuracy for cognition also better explain cognition-mental health covariation, showing that multimodal MRI can capture both a substantial cognitive variance and nearly half of its shared variance with mental health. Finally, we show that our neuromarkers explain a substantial portion of the age- and sex-related variance in the cognition-mental health relationship, highlighting their relevance in modeling cognition across demographic strata.

The remaining unexplained variance in the relationship between cognition and mental health likely stems from multiple sources. One possibility is the absence of certain neuroimaging modalities in the UK Biobank dataset, such as task-based fMRI contrasts, positron emission tomography, arterial spin labeling, and magnetoencephalography/electroencephalography. Prior research has consistently demonstrated strong predictive performance from specific task-based fMRI contrasts, particularly those derived from tasks like the n-Back working memory task and the face-name episodic memory task, none of which is available in the UK Biobank [15,17,61,69,114,142,151].

Moreover, there are inherent limitations in using MRI as a proxy for brain structure and function. Measurement error and intra-individual variability, such as differences in a cognitive state between cognitive assessments and MRI acquisition, may also contribute to the unexplained variance. According to the RDoC framework, brain circuits represent only one level of neurobiological analysis relevant to cognition [14]. Other levels, including genes, molecules, cells, and physiological processes, may also play a role in the cognition-mental health relationship.

Nonetheless, neuroimaging provides a valuable window into the biological mechanisms underlying this overlap – insights that cannot be gleaned from behavioural data alone. Ultimately, our findings validate brain-based neural markers as a fundamental neurobiological unit of analysis, advancing our understanding of mental health through the lens of cognition.”

Marek S, Tervo-Clemmens B, Calabro FJ, Montez DF, Kay BP, Hatoum AS, et al. Reproducible brain-wide association studies require thousands of individuals. Nature. 2022;603:654–660.

Vieira S, Gong QY, Pinaya WHL, et al. Using Machine Learning and Structural Neuroimaging to Detect First Episode Psychosis: Reconsidering the Evidence. Schizophr Bull. 2020;46(1):17-26.

Insel T, Cuthbert B, Garvey M, Heinssen R, Pine DS, Quinn K, et al. Research Domain Criteria (RDoC): Toward a New Classification Framework for Research on Mental Disorders. AJP. 2010;167:748–751.

Comment 5: - The explanation about the stacking approach is not yet completely clear to me. I don't understand how the target variable can be the dependent variable in both step one and two. Or are those different variables? It would be helpful to also give an example of the target variable in line 88 on page 5

Thank you for this excellent question. In our stacking approach, the same target variable, the g-factor, is indeed used across both modeling stages, but with a key distinction in how predictions are generated and integrated.

In the first-level models, we trained separate Partial Least Squares Regression (PLSR) models for each of the 72 neuroimaging phenotypes, each predicting the g-factor independently. The predicted values from these 72 models were then used as input features for the second-level stacked model, which combined them to generate a final prediction of the g-factor. This twostage framework enables us to integrate information across multiple imaging modalities while maintaining a consistent prediction target.

To avoid data leakage, both modeling stages were conducted entirely within the training set for each cross-validation fold. Only after the second-level model was trained was it applied to the outer-fold test participants who were not involved in any part of the model training process.

To improve accessibility, we have revised the Methods section (see Page 10) to clarify this approach, ensuring that the description remains technically accurate while being easier to follow.

Line 188: “We employed nested cross-validation to predict cognition from mental health indices and 72 neuroimaging phenotypes (Fig. 1). Nested cross-validation is a robust method for evaluating machine-learning models while tuning their hyperparameters, ensuring that performance estimates are both accurate and unbiased. Here, we used a nested cross-validation scheme with five outer folds and ten inner folds.

We started by dividing the entire dataset into five outer folds. Each fold took a turn being held out as the outerfold test set (20% of the data), while the remaining four folds (80% of the data) were used as an outer-fold training set. Within each outer-fold training set, we performed a second layer of cross-validation – this time splitting the data into ten inner folds. These inner folds were used exclusively for hyperparameter tuning: models were trained on nine of the inner folds and validated on the remaining one, cycling through all ten combinations.

We then selected the hyperparameter configuration that performed best across the inner-fold validation sets, as determined by the minimal mean squared error (MSE). The model was then retrained on the full outer-fold training set using this hyperparameter configuration and evaluated on the outer-fold test set, using four performance metrics: Pearson r, the coefficient of determination ( R²), the mean absolute error (MAE), and the MSE. This entire process was repeated for each of the five outer folds, ensuring that every data point is used for both training and testing, but never at the same time. We opted for five outer folds instead of ten to reduce computational demands, particularly memory and processing time, given the substantial volume of neuroimaging data involved in model training. Five outer folds led to an outer-fold test set at least n = 4 000, which should be sufficient for model evaluation. In contrast, we retained ten inner folds to ensure robust and stable hyperparameter tuning, maximising the reliability of model selection.

To model the relationship between mental health and cognition, we employed Partial Least Squares Regression (PLSR) to predict the g-factor from 133 mental health variables. To model the relationship between neuroimaging data and cognition, we used a two-step stacking approach [15–17,61] to integrate information from 72 neuroimaging phenotypes across three MRI modalities. In the first step, we trained 72 base (first-level) PLSR models, each predicting the g-factor from a single neuroimaging phenotype. In the second step, we used the predicted values from these base models as input features for stacked models, which again predicted the g-factor. We constructed four stacked models based on the source of the base predictions: one each for dwMRI, rsMRI, sMRI, and a combined model incorporating all modalities (“dwMRI Stacked”, “rsMRI Stacked”, “sMRI Stacked”, and “All MRI Stacked”, respectively). Each stacked model was trained using one of four machine learning algorithms – ElasticNet, Random Forest, XGBoost, or Support Vector Regression – selected individually for each model (see Supplementary Materials, S6).

For rsMRI phenotypes, we treated the choice of functional connectivity quantification method – full correlation, partial correlation, or tangent space parametrization – as a hyperparameter. The method yielding the highest performance on the outer-fold training set was selected for predicting the g-factor (see Supplementary Materials, S5).

To prevent data leakage, we standardized the data using the mean and standard deviation derived from the training set and applied these parameters to the corresponding test set within each outer fold. This standardization was performed at three key stages: before g-factor derivation, before regressing out modality-specific confounds from the MRI data, and before stacking. Similarly, to maintain strict separation between training and testing data, both base and stacked models were trained exclusively on participants from the outer-fold training set and subsequently applied to the corresponding outer-fold test set.

To evaluate model performance and assess statistical significance, we aggregated the predicted and observed gfactor values from each outer-fold test set. We then computed a bootstrap distribution of Pearson’s correlation coefficient (r) by resampling with replacement 5 000 times, generating 95% confidence intervals (CIs) (Fig. 1). Model performance was considered statistically significant if the 95% CI did not include zero, indicating that the observed associations were unlikely to have occurred by chance.”

Comment 6: Methods - It's not clear from the text and Figure 1 which 12 scores from 11 tests are being used to derive the g-factor. Figure 1 shows only 8 bullet points with 10 scores in A and 13 tests under 'Cognitive tests' in B. Moreover, Supplement S1 describes 12 tests and 14 measures (Prospective Memory test is in the text but not in Supplementary Table 1).

Thank you for identifying this discrepancy. In the original Figure 1b and in the Supplementary Methods (S1), the “Prospective Memory” test was accidentally duplicated, while it was present in the Supplementary Table 1 (Line 53, Supplementary Table 1). We have now corrected both figures for consistency. To clarify: Figure 1a presents the global mental health and cognitive domains studied, while Figure 1b now accurately lists 1) the 12 cognitive scores from 11 tests used to derive the g-factor (with the Trail Making Test contributing two measures – numeric and alphabetic trails) and 2) the three main categories of mental health indices used as machine learning features.

We also corrected the Supplementary Materials to remove the duplicate test from the first paragraph. In Supplementary Table 1, there were 11 tests listed, and for the Trail Making test, we specified in the “Core measures” column that this test had 2 derivative scores: duration to complete the numeric path (Trail 1) and duration to complete the alphabetic path (Trail 2).

Supplementary Materials, Line 46: “We used twelve scores from the eleven cognitive tests that represented the following cognitive domains: reaction time and processing speed (Reaction Time test), working memory (Numeric Memory test), verbal and numerical reasoning (Fluid Intelligence test), executive function (Trail Making Test), non-verbal fluid reasoning (Matrix Pattern Completion test), processing speed (Symbol Digit Substitution test), vocabulary (Picture Vocabulary test), planning abilities (Tower Rearranging test), verbal declarative memory (Paired Associate Learning test), prospective memory (Prospective Memory test), and visual memory (Pairs Matching test) [1].”

Comment 7: - For the mental health measures: If I understand correctly, the questionnaire items were used individually, but also to create composite scores. This seems counterintuitive, because I would assume that if the raw data is used, the composite scores would not add additional information to that. When reading the Supplement, it seems like I'm not correct… It would be helpful to clarify the text on page 7 in the main text.

You raise an excellent observation regarding the use of both individual questionnaire items and composite scores. This dual approach was methodologically justified by the properties of Partial Least Squares Regression (PLSR), our chosen first-level machine learning algorithm, which benefits from rich feature sets and can handle multicollinearity through dimensionality reduction. PLSR transforms correlated features into latent variables, meaning both individual items and composite scores can contribute unique information to the model. We elaborate on PLSR's mathematical principles in Supplementary Materials (S5).

To directly address this concern, we conducted comparative analyses showing that the PLSR model (a single 80/20% training/test split), incorporating all 133 mental health features (both items and composites), outperformed models using either type alone. The full model achieved superior performance (MSE = 0.458, MAE = 0.537, R² \= 0.112, Pearson r = 0.336, p-value = 6.936e-112) compared to using only composite scores (93 features; MSE = 0.461, MAE = 0.538, R² = 0.107, Pearson r = 0.328, p-value = 5.8e-106) or only questionnaire items (40 features; MSE = 0.499, MAE = 0.561, R² = 0.033, Pearson r = 0.184, p-value = 2.53e-33). These results confirm that including both data types provide complementary predictive value. We expand on these considerations in the revised Methods section.

Line 123: “Mental health measures encompassed 133 variables from twelve groups: mental distress, depression, clinical diagnoses related to the nervous system and mental health, mania (including bipolar disorder), neuroticism, anxiety, addictions, alcohol and cannabis use, unusual/psychotic experiences, traumatic events, selfharm behaviours, and happiness and subjective well-being (Fig. 1 and Tables S4 and S5). We included both selfreport questionnaire items from all participants and composite diagnostic scores computed following Davis et al. and Dutt et al. [35,36] as features in our first-level (for explanation, see Data analysis section) Partial Least Squares Regression (PLSR) model. This approach leverages PLSR’s ability to handle multicollinearity through dimensionality reduction, enabling simultaneous use of granular symptom-level information and robust composite measures (for mental health scoring details, see Supplementary Materials, S2). We assess the contribution of each mental health index to general cognition by examining the direction and magnitude of its PLSR-derived loadings on the identified latent variables”

Comment 8: - Results - The colors in Figure 4 B are a bit hard to differentiate.

We have updated Figure 4 to enhance colour differentiation by adjusting saturation and brightness levels, improving visual distinction. For further clarity, we split the original figure into two separate figures.

Comment 9: - Discussion - "Overall, the scores for mental distress, alcohol and cannabis use, and self-harm behaviours relate positively, and the scores for anxiety, neurological and mental health diagnoses, unusual or psychotic experiences, happiness and subjective well-being, and negative traumatic events relate negatively to cognition," - this seems counterintuitive, that some symptoms relate to better cognition and others relate to worse cognition. Could you elaborate on this finding and what it could mean?

We appreciate you highlighting this important observation. While some associations between mental health indices and cognition may appear counterintuitive at first glance, these patterns are robust (emerging consistently across both univariate correlations and PLSR loadings) and align with previous literature (e.g., Karpinski et al., 2018; Ogueji et al., 2022). For instance, the positive relationship between cognitive ability and certain mental health indicators like help-seeking behaviour has been documented in other population studies (Karpinski et al., 2018; Ogueji et al., 2022), potentially reflecting greater health literacy and access to care among cognitively advantaged individuals. Conversely, the negative associations with conditions like psychotic experiences mirror established neurocognitive deficits in these domains.

As was initially detailed in Supplementary Materials (S12) and now expanded in our Discussion, these findings likely reflect complex multidimensional interactions. The positive loadings for mental distress indicators may capture: (1) greater help-seeking behaviour among those with higher cognition and socioeconomic resources, and/or (2) psychological overexcitability and rumination tendencies in high-functioning individuals. These interpretations are particularly relevant to the UK Biobank's assessment methods, where mental distress items focused on medical help-seeking rather than symptom severity per se (e.g., as a measure of mental distress, the UK Biobank questionnaire asked whether an individual sought or received medical help for or suffered from mental distress).

Line 492: “Factor loadings derived from the PLSR model showed that the scores for mental distress, alcohol and cannabis use, and self-harm behaviours related positively, and the scores for anxiety, neurological and mental health diagnoses, unusual or psychotic experiences, happiness and subjective well-being, and negative traumatic events related negatively to the g-factor. Positive PLSR loadings of features related to mental distress may indicate greater susceptibility to or exaggerated perception of stressful events, psychological overexcitability, and predisposition to rumination in people with higher cognition [72]. On the other hand, these findings may be specific to the UK Biobank cohort and the way the questions for this mental health category were constructed. In particular, to evaluate mental distress, the UK Biobank questionnaire asked whether an individual sought or received medical help for or suffered from mental distress. In this regard, the estimate for mental distress may be more indicative of whether an individual experiencing mental distress had an opportunity or aspiration to visit a doctor and seek professional help [73]. Thus, people with better cognitive abilities and also with a higher socioeconomic status may indeed be more likely to seek professional help.

Limited evidence supports a positive association between self-harm behaviours and cognitive abilities, with some studies indicating higher cognitive performance as a risk factor for non-suicidal self-harm. Research shows an inverse relationship between cognitive control of emotion and suicidal behaviours that weakens over the life course [73,74]. Some studies have found a positive correlation between cognitive abilities and the risk of nonsuicidal self-harm, suicidal thoughts, and suicidal plans that may be independent of or, conversely, affected by socioeconomic status [75,76]. In our study, the magnitude of the association between self-harm behaviours and cognition was low (Fig. 2), indicating a weak relationship.

Positive PLSR loadings of features related to alcohol and cannabis may also indicate the influence of other factors. Overall, this relationship is believed to be largely affected by age, income, education, social status, social equality, social norms, and quality of life [79–80]. For example, education level and income correlate with cognitive ability and alcohol consumption [79,81–83]. Research also links a higher probability of having tried alcohol or recreational drugs, including cannabis, to a tendency of more intelligent individuals to approach evolutionary novel stimuli [84,85]. This hypothesis is supported by studies showing that cannabis users perform better on some cognitive tasks [86]. Alternatively, frequent drinking can indicate higher social engagement, which is positively associated with cognition [87]. Young adults often drink alcohol as a social ritual in university settings to build connections with peers [88]. In older adults, drinking may accompany friends or family visits [89,90]. Mixed evidence on the link between alcohol and drug use and cognition makes it difficult to draw definite conclusions, leaving an open question about the nature of this relationship.

Consistent with previous studies, we showed that anxiety and negative traumatic experiences were inversely associated with cognitive abilities [90–93]. Anxiety may be linked to poorer cognitive performance via reduced working memory capacity, increased focus on negative thoughts, and attentional bias to threatening stimuli that hinder the allocation of cognitive resources to a current task [94–96]. Individuals with PTSD consistently showed impaired verbal and working memory, visual attention, inhibitory function, task switching, cognitive flexibility, and cognitive control [97–100]. Exposure to traumatic events that did not reach the PTSD threshold was also linked to impaired cognition. For example, childhood trauma is associated with worse performance in processing speed, attention, and executive function tasks in adulthood, and age at a first traumatic event is predictive of the rate of executive function decline in midlife [101,102]. In the UK Biobank cohort, adverse life events have been linked to lower cognitive flexibility, partially via depression level [103].

In agreement with our findings, cognitive deficits are often found in psychotic disorders [104,105]. We treated neurological and mental health symptoms as predictor variables and did not stratify or exclude people based on psychiatric status or symptom severity. Since no prior studies have examined isolated psychotic symptoms (e.g., recent unusual experiences, hearing unreal voices, or seeing unreal visions), we avoid speculating on how these symptoms relate to cognition in our sample.

Finally, negative PLSR loadings of the features related to happiness and subjective well-being may be specific to the study cohort, as these findings do not agree with some previous research [107–109]. On the other hand, our results agree with the study linking excessive optimism or optimistic thinking to lower cognitive performance in memory, verbal fluency, fluid intelligence, and numerical reasoning tasks, and suggesting that pessimism or realism indicates better cognition [110]. The concept of realism/optimism as indicators of cognition is a plausible explanation for a negative association between the g-factor and friendship satisfaction, as well as a negative PLSR loading of feelings that life is meaningful, especially in older adults who tend to reflect more on the meaning of life [111]. The latter is supported by the study showing a negative association between cognitive function and the search for the meaning of life and a change in the pattern of this relationship after the age of 60 [112]. Finally, a UK Biobank study found a positive association of happiness with speed and visuospatial memory but a negative relationship with reasoning ability [113].”

Karpinski RI, Kinase Kolb AM, Tetreault NA, Borowski TB. High intelligence: A risk factor for psychological and physiological overexcitabilities. Intelligence. 2018;66:8–23.

Ogueji IA, Okoloba MM. Seeking Professional Help for Mental Illness: A Mixed-Methods Study of Black Family Members in the UK and Nigeria. Psychol Stud. 2022;67:164–177.

Comment 10: - All neuroimaging factors together explain 48% of the variance in the cognition-mental health relationship. However, this relationship is only r=0.3 - so then the effect of neuroimaging factors seems a lot smaller… What does it mean?

Thank you for raising this critical point. We have addressed this point in our response to Reviewer 1, comment 2, Reviewer 1, comment 3 and Reviewer 2, comment 1.

Briefly, cognition is related to mental health at around r = 0.3 and to neuroimaging phenotypes at around r = 0.4. These levels of relationship strength are consistent to what has been shown in the literature (e.g., Wang et al., 2025 and Vieira et al., 2020). We discussed the relationship between cognition and mental health in our response to Reviewer 2, comment 1 above. In short, this relationship reflects just one functional domain – mental health may also be associated with other domains such as negative and positive valence systems, arousal and regulatory systems, social processes, and sensorimotor functions. Moreover, in the context of gerontology research, this effect size is considered relatively large (Brydges et al., 2019).

We conducted a commonality analysis to investigate the unique and shared variance of mental health and neuroimaging phenotypes in explaining cognition.  As we discussed in our response to Reviewer 1, comment 2, we were able to account for 48% of the covariation between cognition and mental health using the MRI modalities available in the UK Biobank. The remaining 52% of unexplained variance may arise from several sources.

One possibility is the absence of certain neuroimaging modalities in the UK Biobank dataset, such as task-based fMRI contrasts, positron emission tomography, arterial spin labeling, and magnetoencephalography/electroencephalography. Prior research from our group and others has consistently demonstrated strong predictive performance from specific task-based fMRI contrasts, particularly those derived from tasks like the n-Back working memory task and the face-name episodic memory task, none of which is available in the UK Biobank (Tetereva et al., 2025).

Moreover, there are inherent limitations in using MRI as a proxy for brain structure and function. Measurement error and intra-individual variability, such as differences in a cognitive state between cognitive assessments and MRI acquisition, may also contribute to the unexplained variance. According to RDoC framework, brain circuits represent only one level of neurobiological analysis relevant to cognition. Other levels, including genes, molecules, cells, and physiological processes, may also play a role in the cognition-mental health relationship.

We have now incorporated these considerations into the Discussion section.

Line 481: “Our analysis confirmed the validity of the g-factor [31] as a quantitative measure of cognition [31], demonstrating that it captures nearly half (39%) of the variance across twelve cognitive performance scores, consistent with prior studies [63–68]. Furthermore, we were able to predict cognition from 133 mental health indices, showing a medium-sized relationship that aligns with existing literature [69,70]. Although the observed mental health-cognition association is lower than within-sample estimates in conventional regression models, it aligns with our prior mega-analysis in children [69]. Notably, this effect size is not considered small in gerontology. In fact, it falls around the 70th percentile of reported effects and approaches the threshold for a large effect at r = 0.32 [71]. While we focused specifically on cognition as an RDoC core domain, the strength of its relationship with mental health may be bounded by the influence of other functional domains, particularly in normative, non-clinical samples – a promising direction for future research.”

Line 658: “Although recent debates [18] have challenged the predictive utility of MRI for cognition, our multimodal marker integrating 72 neuroimaging phenotypes captures nearly half of the mental health-explained variance in cognition. We demonstrate that neural markers with greater predictive accuracy for cognition also better explain cognition-mental health covariation, showing that multimodal MRI can capture both a substantial cognitive variance and nearly half of its shared variance with mental health. Finally, we show that our neuromarkers explain a substantial portion of the age- and sex-related variance in the cognition-mental health relationship, highlighting their relevance in modeling cognition across demographic strata.

The remaining unexplained variance in the relationship between cognition and mental health likely stems from multiple sources. One possibility is the absence of certain neuroimaging modalities in the UK Biobank dataset, such as task-based fMRI contrasts, positron emission tomography, arterial spin labeling, and magnetoencephalography/electroencephalography. Prior research has consistently demonstrated strong predictive performance from specific task-based fMRI contrasts, particularly those derived from tasks like the n-Back working memory task and the face-name episodic memory task, none of which is available in the UK Biobank [15,17,61,69,114,142,151].

Moreover, there are inherent limitations in using MRI as a proxy for brain structure and function. Measurement error and intra-individual variability, such as differences in a cognitive state between cognitive assessments and MRI acquisition, may also contribute to the unexplained variance. According to the RDoC framework, brain circuits represent only one level of neurobiological analysis relevant to cognition [14]. Other levels, including genes, molecules, cells, and physiological processes, may also play a role in the cognition-mental health relationship.

Nonetheless, neuroimaging provides a valuable window into the biological mechanisms underlying this overlap – insights that cannot be gleaned from behavioural data alone. Ultimately, our findings validate brain-based neural markers as a fundamental neurobiological unit of analysis, advancing our understanding of mental health through the lens of cognition.”

Wang Y, Anney R, Pat N. The relationship between cognitive abilities and mental health as represented by cognitive abilities at the neural and genetic levels of analysis. eLife. 2025.14:RP105537.

Vieira S, Gong QY, Pinaya WHL, et al. Using Machine Learning and Structural Neuroimaging to Detect First Episode Psychosis: Reconsidering the Evidence. Schizophr Bull. 2020;46(1):17-26.

Brydges CR. Effect Size Guidelines, Sample Size Calculations, and Statistical Power in Gerontology. Innovation in Aging. 2019;3(4):igz036.

Tetereva A, Knodt AR, Melzer TR, et al. Improving Predictability, Reliability and Generalisability of Brain-Wide Associations for Cognitive Abilities via Multimodal Stacking. Preprint. bioRxiv. 2025;2024.05.03.589404.

Reviewer 3:

Buianova et al. present a comprehensive analysis examining the predictive value of multimodal neuroimaging data for general cognitive ability, operationalized as a derived g-factor. The study demonstrates that functional MRI holds the strongest predictive power among the modalities, while integrating multiple MRI modalities through stacking further enhances prediction performance. The inclusion of a commonality analysis provides valuable insight into the extent to which shared and unique variance across mental health features and neuroimaging modalities contributes to the observed associations with cognition. The results are clearly presented and supported by highquality visualizations. Limitations of the sample are stated clearly.

Thank you once more for your constructive and encouraging feedback. We appreciate your careful reading and valuable methodological insights. Your expertise has helped us clarify key methodological concepts and improve the overall rigour of our study.

Suggestions for improvement:

(1) The manuscript would benefit from the inclusion of permutation testing to evaluate the statistical significance of the predictive models. This is particularly important given that some of the reported performance metrics are relatively modest, and permutation testing could help ensure that results are not driven by chance.

Thank you, this is an excellent point. We agree that evaluating the statistical significance of our predictive models is essential.

In our original analysis, we assessed model performance by generating a bootstrap distribution of Pearson’s r, resampling the data with replacement 5,000 times (see Figure 3b). In response to your feedback, we have made the following updates:

(1) Improved Figure 3b to explicitly display the 95% confidence intervals.

(2) Supplemented the results by reporting the exact confidence interval values.

(3) Clarified our significance testing procedure in the Methods section.

We considered model performance statistically significant when the 95% confidence interval did not include zero, indicating that the observed associations are unlikely to have occurred by chance.

We chose bootstrapping over permutation testing because, while both can assess statistical significance, bootstrapping additionally provides uncertainty estimates in the form of confidence intervals. Given the large sample size in our study, significance testing can be less informative, as even small effects may reach statistical significance. Bootstrapping offers a more nuanced understanding of model uncertainty.

Line 233: “To evaluate model performance and assess statistical significance, we aggregated the predicted and observed g-factor values from each outer-fold test set. We then computed a bootstrap distribution of Pearson’s correlation coefficient (r) by resampling with replacement 5 000 times, generating 95% confidence intervals (CIs) (Fig. 1). Model performance was considered statistically significant if the 95% CI did not include zero, indicating that the observed associations were unlikely to have occurred by chance.”

(2) Applying and testing the trained models on an external validation set would increase confidence in generalisability of the model.

We appreciate this excellent suggestion. While we considered this approach, implementing it would require identifying an appropriate external dataset with comparable neuroimaging and behavioural measures, along with careful matching of acquisition protocols and variable definitions across sites. These challenges extend beyond the scope of the current study, though we fully agree that this represents an important direction for future research.

Our findings, obtained from one of the largest neuroimaging datasets to date with training and test samples exceeding most previous studies, align closely with existing literature: the predictive accuracy of each neuroimaging phenotype and modality for cognition matches the effect size reported in meta-analyses (r ≈ 0.4; e.g., Vieira et al., 2020). The ability of dwMRI, rsMRI and sMRI to capture the cognition-mental health relationship is, in turn, consistent with our previous work in pediatric populations (Wang et al., 2025; Pat et al., 2022).

Vieira S, Gong QY, Pinaya WHL, et al. Using Machine Learning and Structural Neuroimaging to Detect First Episode Psychosis: Reconsidering the Evidence. Schizophr Bull. 2020;46(1):17-26.

Wang Y, Anney R, Pat N. The relationship between cognitive abilities and mental health as represented by cognitive abilities at the neural and genetic levels of analysis. eLife. 2025.14:RP105537.

Pat N, Wang Y, Anney R, Riglin L, Thapar A, Stringaris A. Longitudinally stable, brain-based predictive models mediate the relationships between childhood cognition and socio-demographic, psychological and genetic factors. Hum Brain Mapp. 2022;43:5520–5542.

(3) The rationale for selecting a 5-by-10-fold cross-validation scheme is not clearly explained. Clarifying why this structure was preferred over more commonly used alternatives, such as 10-by-10 or 5-by-5 cross-validation, would strengthen the methodological transparency.

Thank you for this important methodological question. Our choice of a 5-by-10-fold crossvalidation scheme was motivated by the need to balance robust hyperparameter tuning with computational efficiency, particularly memory and processing time. Retaining five outer folds allowed us to rigorously assess model performance across multiple data partitions, leading to an outer-fold test set at least n = 4 000 and providing a substantial amount of neuroimaging data involved in model training. In contrast, employing ten inner folds ensured robust and stable hyperparameter tuning that maximizes the reliability of model selection. Thus, the 5-outer-fold with our large sample provided sufficient out-of-sample test set size for reliable model evaluation and efficient computation, while 10 inner folds enabled robust hyperparameter tuning. We now provide additional rationale for this design decision on Page 10.

Line 188: “We employed nested cross-validation to predict cognition from mental health indices and 72 neuroimaging phenotypes (Fig. 1). Nested cross-validation is a robust method for evaluating machine-learning models while tuning their hyperparameters, ensuring that performance estimates are both accurate and unbiased. Here, we used a nested cross-validation scheme with five outer folds and ten inner folds.

We started by dividing the entire dataset into five outer folds. Each fold took a turn being held out as the outerfold test set (20% of the data), while the remaining four folds (80% of the data) were used as an outer-fold training set. Within each outer-fold training set, we performed a second layer of cross-validation – this time splitting the data into ten inner folds. These inner folds were used exclusively for hyperparameter tuning: models were trained on nine of the inner folds and validated on the remaining one, cycling through all ten combinations.

We then selected the hyperparameter configuration that performed best across the inner-fold validation sets, as determined by the minimal mean squared error (MSE). The model was then retrained on the full outer-fold training set using this hyperparameter configuration and evaluated on the outer-fold test set, using four performance metrics: Pearson r, the coefficient of determination ( R²), the mean absolute error (MAE), and the MSE. This entire process was repeated for each of the five outer folds, ensuring that every data point is used for both training and testing, but never at the same time. We opted for five outer folds instead of ten to reduce computational demands, particularly memory and processing time, given the substantial volume of neuroimaging data involved in model training. Five outer folds led to an outer-fold test set at least n = 4 000, which should be sufficient for model evaluation. In contrast, we retained ten inner folds to ensure robust and stable hyperparameter tuning, maximising the reliability of model selection.”

(4) A more detailed discussion of which specific brain regions or features within each neuroimaging modality contributed most strongly to the prediction of cognition would enhance neurobiological relevance of the findings.

Thank you for this thoughtful suggestion. To address this point, we have included feature importance plots for the top-performing neuroimaging phenotypes within each modality (Figure 5 and Figures S2–S4), demonstrating the relative contributions of individual features to the predictive models. While we maintain our primary focus on cross-modality performance comparisons in the main text, as this aligns with our central aim of evaluating multimodal MRI markers at the integrated level, we outline the contribution of neuroimaging features with the highest predictive performance for cognition in the revised Results and Discussion.

Methods

Line 255: “To determine which neuroimaging features contribute most to the predictive performance of topperforming phenotypes within each modality, while accounting for the potential latent components derived from neuroimaging, we assessed feature importance using the Haufe transformation [62]. Specifically, we calculated Pearson correlations between the predicted g-factor and scaled and centred neuroimaging features across five outer-fold test sets. We also examined whether the performance of neuroimaging phenotypes in predicting cognition per se is related to their ability to explain the link between cognition and mental health. Here, we computed the correlation between the predictive performance of each neuroimaging phenotype and the proportion of the cognition-mental health relationship it captures. To understand how demographic factors, including age and sex, contribute to this relationship, we also conducted a separate set of commonality analyses treating age, sex, age², age×sex, and age²×sex as an additional set of explanatory variables (Fig. 1).”

Results

dwMRI

Line 331: “Overall, models based on structural connectivity metrics performed better than TBSS and probabilistic tractography (Fig. 3). TBSS, in turn, performed better than probabilistic tractography (Fig. 3 and Table S13). The number of streamlines connecting brain areas parcellated with aparc MSA-I had the best predictive performance among all dwMRI neuroimaging phenotypes (R²_mean = 0.052, r_mean = 0.227, 95% CI [0.212, 0.235]). To identify features driving predictions, we correlated streamline counts in aparc MSA-I parcellation with the predicted g_factor values from the PLSR model. Positive associations with the predicted _g-factor were strongest for left superior parietal-left caudal anterior cingulate, left caudate-right amygdala, and left putamen-left hippocampus connections. The most marked negative correlations involved left putamen-right posterior thalamus and right pars opercularis-right caudal anterior cingulate pathways (Fig. 5 and Supplementary Fig. S2).”

rsMRI

Line 353: “Among RSFC metrics for 55 and 21 ICs, tangent parameterization matrices yielded the highest performance in the training set compared to full and partial correlation, as indicated by the cross-validation score. Functional connections between the limbic (IC10) and dorsal attention (IC18) networks, as well as between the ventral attention (IC15) and default mode (IC11) networks, displayed the highest positive association with cognition. In contrast, functional connectivity between the limbic (IC43, the highest activation within network) and default mode (IC11) and limbic (IC45) and frontoparietal (IC40) networks, between the dorsal attention (IC18) and frontoparietal (IC25) networks, and between the ventral attention (IC15) and frontoparietal (IC40) networks, showed the highest negative association with cognition (Fig. 5 and Supplementary Fig. S3 and S4)”

sMRI

Line 373: “FreeSurfer subcortical volumetric subsegmentation and ASEG had the highest performance among all sMRI neuroimaging phenotypes (R²_mean = 0.068, r_mean = 0.244, 95% CI [0.237, 0.259] and R²_mean = 0.059, r_mean = 0.235, 95% CI [0.221, 0.243], respectively). In FreeSurfer subcortical volumetric subsegmentation, volumes of all subcortical structures, except for left and right hippocampal fissures, showed positive associations with cognition. The strongest relations were observed for the volumes of bilateral whole hippocampal head and whole hippocampus (Fig. 5 and Supplementary Fig. S5 for feature importance maps). Grey matter morphological characteristics from ex vivo Brodmann Area Maps showed the lowest predictive performance (R²_mean = 0.008, r_mean = 0.089, 95% CI [0.075, 0.098]; Fig. 3 and Table S15).”

Discussion

dwMRI

Line 562: “Among dwMRI-derived neuroimaging phenotypes, models based on structural connectivity between brain areas parcellated with aparc MSA-I (streamline count), particularly connections with bilateral caudal anterior cingulate (left superior parietal-left caudal anterior cingulate, right pars opercularis-right caudal anterior cingulate), left putamen (left putamen-left hippocampus, left putamen-right posterior thalamus), and amygdala (left caudate-right amygdala), result in a neural indicator that best reflects microstructural resources associated with cognition, as indicated by predictive modeling, and more importantly, shares the highest proportion of the variance with mental health-g, as indicated by commonality analysis.”

rsMRI

Line 583: “We extend findings on the superior performance of rsMRI in predicting cognition, which aligns with the literature [15, 28], by showing that it also explains almost a third of the variance in cognition that mental health captures. At the rsMRI neuroimaging phenotype level, this performance is mostly driven by RSFC patterns among 55 ICA-derived networks quantified using tangent space parameterization. At a feature level, these associations are best captured by the strength of functional connections among limbic, dorsal attention and ventral attention, frontoparietal and default mode networks. These functional networks have been consistently linked to cognitive processes in prior research [127–130].”

sMRI

Line 608: “Integrating information about brain anatomy by stacking sMRI neuroimaging phenotypes allowed us to explain a third of the link between cognition and mental health. Among all sMRI neuroimaging phenotypes, those that quantified the morphology of subcortical structures, particularly volumes of bilateral hippocampus and hippocampal head, explain the highest portion of the variance in cognition captured by mental health. Our findings show that, at least in older adults, volumetric properties of subcortical structures are not only more predictive of individual variations in cognition but also explain a greater portion of cognitive variance shared with mental health than structural characteristics of more distributed cortical grey and white matter. This aligns with the Scaffolding Theory that proposes stronger compensatory engagement of subcortical structures in cognitive processing in older adults [138–140].”

(5) The formatting of some figure legends could be improved for clarity - for example, some subheadings were not formatted in bold (e.g., Figure 2 c)

Thank you for noticing this. We have updated the figures to enhance clarity, keeping subheadings plain while bolding figure numbers and MRI modality names.

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Briefing : Réunion des Parents d'Élèves de Première Générale au Lycée Louis Vincent

Résumé

• Cette note de synthèse résume les points clés de la réunion destinée aux parents des 308 élèves de première générale du lycée Louis Vincent.

L'objectif était de présenter le déroulement de l'année, les enjeux du baccalauréat et l'importance de l'anticipation pour l'orientation post-bac.

Les principaux points à retenir sont :

• • Une année charnière pour l'orientation : Bien que l'unique choix d'orientation de l'année consiste à abandonner l'une des trois spécialités pour la terminale, la classe de première est identifiée comme un moment crucial pour entamer la réflexion sur le projet post-bac et Parcoursup.
• • Rigueur sur l'assiduité : Une politique très stricte sera appliquée concernant les absences et les retards.

En raison du poids du contrôle continu (40 % de la note finale du baccalauréat), l'assiduité est primordiale et l'établissement n'hésitera pas à contacter les familles de manière insistante pour garantir la présence des élèves.

• • Nouveauté au baccalauréat : Une épreuve anticipée de mathématiques, d'un coefficient 2 et se déroulant sans calculatrice, est introduite dès la fin de l'année de première.

Elle s'ajoute à la traditionnelle épreuve anticipée de français.

• • Excellence académique et accompagnement : Le lycée affiche des résultats supérieurs à la moyenne académique, avec 98,44 % de réussite au baccalauréat général et 74 % de mentions.

L'accompagnement pour Parcoursup est également un point fort, avec 100 % des élèves de terminale ayant reçu une proposition d'affectation l'année précédente.

• • Cadre scolaire et pédagogique : L'accent est mis sur le bien-être des élèves face à la pression scolaire, la nécessité d'un usage raisonné des outils numériques (téléphones, IA) et le maintien de méthodes de travail fondamentales comme la prise de notes manuscrite et la lecture.

1. L'Année de Première : Une Année Stratégique

L'année de première générale est présentée comme "relativement cool" en termes de décisions d'orientation immédiates, contrastant avec les choix de filières en seconde et les choix de formations supérieures en terminale. Cependant, son importance stratégique est fortement soulignée.

Objectif Principal et Calendrier

• Choix de Spécialités : Le seul choix d'orientation de l'année interviendra au troisième trimestre, lorsque les élèves devront indiquer laquelle de leurs trois spécialités ils souhaitent abandonner pour la classe de terminale.

• Calendrier de l'Orientation :

◦ Dès octobre : Des bilans de mi-trimestre seront organisés pour faire des points d'étape avec les équipes pédagogiques et proposer des rendez-vous pour affiner le projet de l'élève.

◦ Novembre : Participation à la Semaine Nationale de l'Orientation.

◦ Troisième trimestre : Accélération du processus avec le choix final de la spécialité à abandonner.

L'Importance de l'Anticipation pour Parcoursup

L'année de première est le moment idéal pour commencer à préparer les choix de l'enseignement supérieur.

Il est rappelé qu'en terminale, le temps pour choisir parmi les 60 000 formations supérieures (dont 24 000 sur Parcoursup) est très court.

Il est donc conseillé aux familles d'initier la discussion sur l'avenir, même si les élèves n'ont pas de projet précis.

Une approche suggérée est d'identifier ce que les élèves ne veulent pas faire pour affiner progressivement leurs centres d'intérêt.

2. Organisation et Vie Scolaire

• Équipe Pédagogique et Administrative

• L'encadrement des neuf classes de première (601 à 609) est assuré par une équipe de référents dédiés.

Rôle

Personnes en charge

• Proviseur Adjoint
• Proviseur
• Professeurs Principaux
• Un professeur principal par classe
• CPE Référentes
• Psychologues de l'Éducation Nationale
• Disponibles sur rendez-vous pour affiner les choix d'orientation
• Secrétariat Pédagogique
• Mme x, pour les dossiers scolaires et les demandes d'aménagements
• Règlement Intérieur et Discipline
• Deux points du règlement intérieur sont particulièrement mis en avant.
• • Assiduité (Absences et Retards) :
• ◦ Enjeu majeur : Le contrôle continu compte pour 40 % de la note du bac. Chaque note obtenue en cours est donc importante.
• ◦ Politique stricte : L'établissement sera très vigilant, contactant les parents par téléphone, mail ou SMS ("on va vous harceler"). Le caractère justifié ou non des motifs d'absence sera évalué par l'administration et pourra figurer sur le bulletin, document essentiel pour Parcoursup.
• ◦ Lutte contre les stratégies d'évitement : Les absences stratégiques lors des devoirs seront combattues.

Un système de rattrapage des devoirs sera mis en place sur 24 samedis dans l'année.

Si les notes ne sont pas représentatives du niveau de l'élève, une épreuve ponctuelle au baccalauréat pourra être imposée. * • Usage du Numérique : * ◦ Téléphones portables : Interdits dans tous les bâtiments, sauf autorisation explicite d'un adulte. La dépendance et la distraction causées par les notifications sont considérées comme des freins majeurs à l'apprentissage. * ◦ Intelligence Artificielle et Triche : L'équipe pédagogique est consciente des difficultés posées par des outils comme ChatGPT pour les travaux à la maison (rédactions).

La copie entre élèves est également surveillée.

L'accent est mis sur la nécessité d'un travail personnel.

Santé et Bien-être des Élèves

• L'année de première peut être une source d'angoisse pour les élèves en raison de la pression des notes, du baccalauréat et de l'orientation future.

Les parents sont encouragés à contacter l'établissement (CPE, professeurs) s'ils observent un changement de comportement ou un mal-être chez leur enfant.

Il est rappelé qu'un travail régulier est plus productif et moins anxiogène que des révisions de dernière minute.

3. Le Baccalauréat : Modalités et Épreuves

Structure et Coefficients

• La note finale du baccalauréat est composée à 40 % du contrôle continu et à 60 % des épreuves terminales. Des ajustements de coefficients ont été annoncés.

Épreuve

Voie Générale - Coefficient * Moment * Contrôle Continu (ensemble des matières du tronc commun) * 40 % * Première et Terminale * Enseignement de Spécialité 1 * 16 * Terminale * Enseignement de Spécialité 2 * 16 * Terminale * Philosophie * 8 * Terminale * Grand Oral * 8 (anciennement 10) * Terminale * Épreuve Anticipée de Français (Écrit + Oral) * 5 * Première * Épreuve Anticipée de Mathématiques (Nouveau) * 2 * Première

Les Épreuves Anticipées en Fin de Première * • Français : * ◦ Écrit : 4 heures (commentaire ou dissertation). * ◦ Oral : Basé sur les textes étudiés pendant l'année. * ◦ Préparation : La lecture des quatre œuvres au programme (et des lectures cursives) est indispensable. L'établissement organise un bac blanc écrit et un bac blanc oral. * • Mathématiques (Nouveauté) : * ◦ Format : Épreuve sur 20 points (6 points d'automatismes, 14 points sur le programme de l'année). * ◦ Contrainte majeure : La calculatrice est interdite. L'objectif est de redonner du sens au calcul et au raisonnement. * ◦ Sujets : Trois sujets distincts seront proposés (voie technologique, voie générale sans spécialité maths, voie générale avec spécialité maths). * ◦ Préparation : Une épreuve blanche sera organisée en avril ou mai.

Résultats et Enjeux exemple au Lycée Louis Vincent

• • Taux de réussite : 98,44 % au bac général (session 2025).
• • Taux de mentions : 74 % des élèves ont obtenu une mention. Ces résultats, supérieurs aux attendus académiques, montrent que l'obtention du baccalauréat est à la portée des élèves qui travaillent régulièrement.

Le véritable enjeu est donc de bien réussir son baccalauréat afin d'obtenir une mention.

4. Parcoursup : Préparer l'Avenir dès la Première

• Une Réflexion à Long Terme
• Les bulletins de la classe de première ont une importance capitale dans le dossier Parcoursup.

Une réflexion précoce permet aux élèves de se motiver et de cibler les matières dans lesquelles ils doivent obtenir de bons résultats pour accéder aux formations souhaitées.

Des outils comme le site Parcoursup lui-même ou le site Suptracker (pour les statistiques d'admission) sont recommandés. * Un Accompagnement Efficace * L'année dernière, 100 % des élèves de terminale du lycée ont reçu une affectation via Parcoursup, témoignant de la qualité de l'accompagnement des équipes.

Le système est défendu comme une opportunité pour les élèves de postuler à des formations diverses sans hiérarchiser leurs vœux initialement, ce qui ouvre le champ des possibles.

5. Outils de Communication et Questions Pratiques

• Plateformes Numériques : * ◦ Mon Bureau Numérique (MBN) : Principal outil pour la communication par mail avec les enseignants (via EduConnect) et pour consulter le cahier de textes. * ◦ Pronote : Outil de référence pour l'emploi du temps et la consultation des notes. Il est synchronisé et accessible via MBN. * • La "Pause Numérique" : Une directive ministérielle prévoit de bloquer l'accès aux environnements numériques après 20h et le week-end.

La Région Grand Est a suspendu cette mesure jusqu'en décembre 2023, notamment en raison de l'accès aux manuels scolaires numériques.

L'avenir de cette mesure est incertain.

• • Accompagnement Personnalisé (AP) : En français et en mathématiques, les enseignants décident quels élèves doivent y assister en fonction des besoins.

Si un élève est convoqué, le cours d'AP apparaît directement dans son emploi du temps sur Pronote.

• • Absences des Professeurs : Le lycée a réalisé sa rentrée avec un effectif complet, une situation favorable qui limite le risque de non-remplacement en cas d'absence, contrairement à d'autres académies.

• • Activités Sportives (UNSS) : Les compétitions ont lieu le mercredi après-midi. Les élèves participants sont excusés mais doivent rattraper les cours manqués.

L'établissement obtient d'excellents résultats, participant régulièrement aux championnats de France.

• • Complexité des Emplois du Temps : La réforme et le système de spécialités génèrent une grande complexité, avec 12 à 14 emplois du temps différents au sein d'une même classe de 35 élèves.

Les élèves sont invités à consulter Pronote chaque matin pour vérifier les éventuelles modifications (salles, absences).

Incluir el bienestar me parece clave: no solo se gestiona información, también se gestiona el “estado del usuario”. En la disciplina archivística hablamos de contexto vital y social de los documentos; aquí, el propio estado emocional se vuelve parte del registro.

Esto me recuerda a la noción de sistemas híbridos en la Ciencia de la Información: un documento puede cumplir simultáneamente varias funciones. Aquí, un simple archivo personal se convierte en agenda, memoria y registro de productividad.

El journaling aquí cumple una función de control de calidad personal, similar a las auditorías en los sistemas de información: identificar “tiempos muertos” y medir su impacto. Esto ayuda a mejorar la eficiencia y la gestión de la atención.

Esta combinación me resulta muy útil porque articula distintos niveles de metadatos: metas, emociones, reflexiones y tareas. En archivística y bibliotecología hablamos de “contexto” y “contenido”; aquí el journaling los integra en un mismo registro.

Me parece curiosa esta parte y es que muestra cómo el diario intersticial convierte hasta lo más simple como es la hora, una tarea o una idea en algo organizado. Sin imaginar, terminas con un registro completo de tu día sin tener que hacer demasiado y eso es lo que buscamos la mayoría algo practico.

Como estudiante de Ciencia de la Información, me llama la atención cómo esta técnica une tres funciones centrales: registro, organización y control de tiempos. Esto recuerda a los sistemas integrados de gestión documental, donde cada acción debe quedar trazada y relacionada.

Esto me recuerda a cuando estoy saturada de información y escribo notas rápidas en papel. El journaling digital ordena esas ideas sin interrumpir la concentración.

Ejemplos: (https://escribelavida.com/apps-para-escribir-un-diario-digital/)

Me gusta porque no se queda solo en un simple diario, sino que mezcla varias cosas útiles en un solo lugar. Y lo mas práctico es que no se necesita nada complicado, con algo tan simple como un archivo de texto ya funciona.

Lo que me gusta de la idea del diario es que mezcla varias cosas a la vez como por ejemplo lo que planeas hacer, como te sientes, si has avanzado o aun no, y lo mas importante lo que queda pendiente. Me parece una forma sencilla de volver más conscientes los descansos.

Me parece maravilloso, es como ir dejando pequeñas huellas de tu día ya que apuntas lo que haces, cuándo lo haces y cómo te sientes. Eso ayuda a ver en qué inviertes tu tiempo, tambien te ayuda a retomar el foco rápido y a tener un registro honesto de tu jornada sin complicarte.

He leído sobre el diario intersticial y me llama mucho la atención. Me gusta que sea una forma sencilla de unir notas, tareas y tiempo en un solo flujo. Todavía no lo he probado, pero creo que podría ser una herramienta muy útil para organizarme mejor y darme más claridad en el día a día.

Es interesante la intensión del autor que es permitirnos auto pensarnos y revisar que cosas mejorar en como obligarnos a detenernos y registrar qué estamos haciendo y hacia dónde vamos. Podría funcionar como un a terapia contra la falta de concentración que se dan por las redes sociales. También las pausas para reflexionar, aparte de esforzar la memoria, permite aprender mas allá de lo común, también se ve como autorregula el aprendizaje autónomo esto requiere disciplina pero los resultados pueden ser buenos, para conocernos y optimizar todo mejor, pensarlo como una bitácora de detalles es interesante por que nos permite auto conocernos y autocriticarnos para mejorar, eso ayuda a conocernos como aprendemos mejor y puede optimizar nuestro foco de atenciones para poder concentrarse mejor.

Yo intente lo mismo con mi calendario donde incorpore alarmas y notas pero no funcionó mucho, me funciono mas llevar una agenda y con pos-it ir llevando notas de tareas, en este mundo trajinado es bueno tener herramientas que nos ayuden a conocernos y a como aprendemos mejor y quedarnos con esa opción para sentirnos realizados y que podemos servir mejor a nuestra familia y sociedad.

Feuille de Route Stratégique pour l'Année de Terminale : Un Modèle pour la Réussite

1.0 Introduction : Les Enjeux Stratégiques de l'Année de Terminale

• L'année de Terminale constitue une année charnière, un véritable pivot dans le parcours d'un élève, marquée par un double enjeu stratégique.

D'une part, elle représente l'aboutissement des années lycée avec l'objectif tangible d'obtenir le baccalauréat.

D'autre part, elle est le théâtre d'une préparation active et décisive de l'avenir, matérialisée par les choix d'orientation vers l'enseignement supérieur via la plateforme Parcoursup.

Cette feuille de route est conçue comme un guide généraliste, synthétisant les meilleures pratiques et les informations clés pour structurer l'accompagnement des élèves et de leurs familles tout au long de cette année dense et déterminante.

Le premier pilier de cette réussite est la maîtrise du cadre d'évaluation du baccalauréat.

2.0 Le Baccalauréat : Structure, Préparation et Évaluation

• La réussite au baccalauréat repose sur une compréhension claire de sa structure d'évaluation et sur une préparation méthodique tout au long de l'année.

Il ne s'agit plus seulement de viser un succès lors des examens finaux, mais de construire sa réussite de manière continue.

La performance globale de l'élève est le fruit d'un équilibre entre le travail régulier, validé par le contrôle continu, et la capacité à se mobiliser pour les épreuves terminales.

2.1 Analyse de la Structure d'Évaluation

• La note finale du baccalauréat général est une somme pondérée qui reflète à la fois le parcours de l'élève et sa performance lors des examens finaux.

La répartition des coefficients est la suivante : 40 % pour le contrôle continu et 60 % pour les épreuves terminales.

• Les épreuves terminales constituent le poids le plus important de la note finale. Leur structure en voie générale se décompose comme suit :

Épreuve Terminale Coefficient en Voie Générale * Philosophie= 8 * Enseignement de Spécialité 1= 16 * Enseignement de Spécialité 2= 16 * Grand Oral= 10

À ces épreuves s'ajoutent les notes des épreuves anticipées de français (écrit, coefficient 5, et oral, coefficient 5), passées en fin de Première, qui complètent la note des épreuves terminales pour atteindre le total de 60%.

• Le contrôle continu, quant à lui, est basé sur les moyennes annuelles de l'ensemble des disciplines du cycle terminal (Première et Terminale), qui sont officiellement validées lors des conseils de classe de fin d'année.

2.2 Le Grand Oral : Un Levier de Réussite à Fort Potentiel

• L'une des épreuves les plus stratégiques du nouveau baccalauréat est sans conteste le Grand Oral.

Avec son coefficient élevé de 10 en voie générale, il représente un levier majeur pour la note finale.

Il s'agit d'une épreuve à "rendement de notes" particulièrement intéressant ;

il est en effet plus courant pour un élève bien préparé d'y obtenir une note maximale que dans certaines disciplines écrites traditionnelles.

Les compétences évaluées lors du Grand Oral sont fondamentales :

• • Apprendre à s'exprimer en public de manière claire et convaincante.
• • Démontrer des capacités d'argumentation et un esprit critique.
• • Faire preuve de clarté dans son expression et de maîtrise de ses connaissances.
• L'épreuve se déroule en 20 minutes face à un jury. L'élève prépare en amont deux questions en lien avec ses enseignements de spécialité.

Le jour de l'épreuve, il dispose de 10 minutes pour présenter sa réponse à l'une des questions, suivies de 10 minutes d'échange et d'approfondissement avec le jury.

Au-delà de son poids dans l'examen, cette épreuve est essentielle car elle développe des compétences oratoires cruciales pour la poursuite d'études (entretiens d'admission) et pour l'ensemble de la vie professionnelle.

2.3 Garantir l'Intégrité du Contrôle Continu : Stratégies et Cadre

• Pour assurer un contrôle continu juste et représentatif du niveau réel des élèves, les établissements mettent en place des mécanismes de régulation précis.

Chaque lycée dispose d'un "projet d'évaluation" qui vise à garantir une égalité de traitement entre tous les candidats.

• Face à la tentation de l'absentéisme stratégique (éviter un devoir après avoir obtenu une bonne note pour préserver sa moyenne), une politique claire est appliquée.

L'établissement offre systématiquement la possibilité de rattraper une évaluation manquée, souvent lors de sessions organisées le samedi matin.

Cette mesure a pour but de contrecarrer ces tactiques et d'assurer que la moyenne reflète un travail régulier.

• Si, malgré tout, les notes d'un élève ne sont pas jugées "robustes" – c'est-à-dire non représentatives de son niveau réel en raison d'un nombre insuffisant d'évaluations –, l'établissement peut organiser une épreuve ponctuelle individuelle pour valider ses compétences.

2.4 Un Calendrier de Préparation Structuré

• La préparation aux épreuves finales est rythmée par des dispositifs d'entraînement organisés par l'établissement tout au long de l'année.

Ces moments sont cruciaux pour familiariser les élèves avec les conditions d'examen.

• • Devoirs communs : Des sessions d'évaluation sont organisées, fréquemment le samedi matin, pour simuler les conditions réelles des épreuves écrites (durée, format, environnement).

• • Épreuves blanches : Des examens blancs complets sont mis en place, incluant des journées banalisées (par exemple en avril) pour les épreuves de spécialité, permettant une immersion totale.

• • Oral blanc : Un entraînement spécifique au Grand Oral est organisé. Il permet aux élèves de se tester et de bénéficier de retours constructifs de la part des évaluateurs pour affiner leur prestation.

• S'exercer en conditions réelles est indispensable. Cela permet aux élèves d'apprendre à gérer leur temps sur une épreuve de 4 heures, à maîtriser leur stress dans l'environnement d'une grande salle d'examen, et à se familiariser avec le format officiel.

Cette préparation met en confiance et réduit l'imprévu le jour J.

• L'obtention du baccalauréat est la première étape vers la réussite.

La seconde, tout aussi cruciale, consiste à préparer activement son avenir et à concrétiser son projet d'orientation via Parcoursup.

3.0 Parcoursup : Naviguer Stratégiquement vers l'Enseignement Supérieur

• Parcoursup est l'outil central et incontournable de l'orientation post-bac en France.

Loin d'être une simple plateforme d'inscription, son utilisation efficace requiert de l'anticipation, une recherche approfondie et une stratégie réfléchie.

Une démarche bien menée permet aux élèves de choisir leur avenir plutôt que de le subir, en alignant leurs aspirations avec les réalités et les attendus de l'enseignement supérieur.

3.1 Le Calendrier en Trois Étapes Clés

• Le processus Parcoursup se déroule selon un calendrier national précis, articulé en trois grandes phases.

• 1. Phase 1 (Décembre - Janvier) : Découverte des Formations. Le site Parcoursup ouvre pour consultation.

À partir de la mi-décembre, les informations sur les milliers de formations disponibles sont mises à jour pour la rentrée suivante. C'est la période de recherche, d'exploration et de première sélection.

• 2. Phase 2 (Mi-Janvier - Début Avril) : Inscription et Formulation des Vœux. Cette phase est consacrée à la création du dossier de candidature et à la formulation des vœux (et sous-vœux).

Une date butoir est fixée mi-mars pour ajouter des vœux, et une seconde début avril pour finaliser chaque dossier avec les éléments requis (projets de formation motivés, etc.).

• 3. Phase 3 (Début Juin - Début Juillet) : Réception et Gestion des Propositions. C'est la phase des résultats.

Les élèves reçoivent les réponses des formations et doivent gérer les propositions d'admission en y répondant dans les délais impartis.

3.2 Le Dossier : Un Portrait Complet du Candidat

• Le dossier Parcoursup est bien plus qu'un simple relevé de notes.

Les commissions d'examen, composées d'équipes pédagogiques et non d'intelligences artificielles, analysent les dossiers pour identifier des profils d'élèves investis, sérieux et motivés.

Les appréciations des professeurs sur les bulletins scolaires sont d'une importance capitale.

Elles fournissent un contexte qualitatif aux notes et donnent des indications précieuses sur le sérieux de l'élève, son implication en classe, sa progression et son potentiel de réussite dans le supérieur.

Il est crucial de noter qu'obtenir des appréciations positives est à la portée de tout élève : l'implication, l'attention en classe et la démonstration d'efforts sont des qualités que les professeurs valorisent et signalent systématiquement.

À l'inverse, les absences, les retards et les remarques sur le comportement sont des facteurs rédhibitoires pour de nombreuses formations.

Comme l'expliquent les évaluateurs, face à des milliers de dossiers, un bulletin affichant "régulièrement absent" est souvent immédiatement mis de côté.

Entre deux candidats aux résultats similaires, le choix se portera toujours sur celui qui a démontré son assiduité et son sérieux.

3.3 Outils d'Orientation et d'Aide à la Décision

• Plusieurs ressources sont à la disposition des élèves et de leurs familles pour éclairer leurs choix et construire un projet solide.
• • Les PsyEN (Psychologues de l'Éducation Nationale) : Il est vivement recommandé de prendre rendez-vous avec un PsyEN dès le premier trimestre.

Leurs plannings se saturent rapidement, et une consultation précoce permet d'engager une réflexion accompagnée avant les échéances de Parcoursup.

• • Les Salons et Forums : Des événements comme le salon "Réaction", qui présente près de 5000 formations, ou le forum des formations organisé au sein même du lycée, sont des moments clés.

Ils permettent de rencontrer des représentants d'écoles, des étudiants et des professionnels pour poser des questions concrètes.

• • SupTracker : Cet outil en ligne est indispensable pour consulter des statistiques détaillées sur les profils des candidats admis dans chaque formation les années précédentes.

Par exemple, il permet de voir quelles combinaisons de spécialités sont les plus représentées parmi les admis en PASS (parcours d'accès spécifique santé), aidant ainsi l'élève à évaluer la cohérence de son profil avec les formations visées.

• • Parcoursup : La plateforme elle-même est une mine d'informations.

Chaque fiche de formation détaille les "attendus" (compétences et connaissances requises), les critères d'analyse des candidatures, et souvent les statistiques de l'année précédente, comme la moyenne du dernier admis.

3.4 Élaborer une Stratégie de Vœux Intelligente

• Une stratégie de vœux réussie est une stratégie anticipée et bien construite.

• 1. Commencer la réflexion tôt : Il est impératif de ne pas attendre les dernières semaines pour réfléchir à son orientation. La recherche doit commencer dès le début de l'année pour éviter des décisions prises dans l'urgence.

• 2. Diversifier et sécuriser ses vœux : Il est stratégique de formuler un nombre suffisant de vœux pour couvrir plusieurs scénarios, des plus ambitieux aux plus sécurisés.

Après la date butoir de mi-mars, aucun ajout n'est possible. Il est donc préférable d'inclure des formations "de sécurité" ou des alternatives en cas d'incertitude, plutôt que de risquer de limiter ses options.

• 3. Faire des choix cohérents : Les vœux doivent être en adéquation avec le profil académique de l'élève (spécialités suivies, résultats scolaires, compétences). Consulter les statistiques sur Parcoursup et SupTracker permet d'ajuster sa stratégie.

• 4. Comprendre les listes d'attente : Il ne faut pas se décourager face à une position lointaine sur une liste d'attente.

Celles-ci évoluent très rapidement. Consulter le rang du dernier admis de l'année précédente donne une indication précieuse, bien que non garantie, sur ses chances d'être finalement accepté.

• Le succès de ce parcours complexe ne repose pas uniquement sur l'élève, mais sur la mobilisation de tout l'écosystème qui l'entoure.

4.0 L'Écosystème de la Réussite : Le Rôle des Équipes, des Élèves et des Familles

• La réussite en Terminale est une entreprise collective.

Elle dépend d'une collaboration étroite et d'un engagement partagé entre l'établissement scolaire, qui fournit le cadre et l'accompagnement, l'élève, qui est l'acteur principal de son parcours, et sa famille, qui offre un soutien indispensable.

4.1 L'Accompagnement par l'Équipe Éducative

• Au sein du lycée, un réseau de soutien est spécifiquement structuré pour accompagner les élèves de Terminale.

Le système de double professeur principal par classe est une des clés de cet accompagnement. Généralement, l'un est désigné "professeur référent", plus spécifiquement axé sur les questions d'orientation et Parcoursup, tandis que l'autre se concentre davantage sur la gestion de la vie de la classe et le suivi scolaire global.

• Ces professeurs sont les premiers interlocuteurs des élèves et des familles.

Ils sont les mieux placés pour répondre aux questions, conseiller et orienter tout au long de l'année. À leurs côtés, les CPE (Conseillers Principaux d'Éducation) et l'équipe de direction assurent un suivi global et interviennent en appui.

4.2 L'Engagement Indispensable des Familles

• Le rôle des parents est crucial pour accompagner l'élève avec bienveillance et efficacité. Voici quelques conseils pratiques pour les familles :

• • Engager le dialogue sur l'orientation : Il est souvent plus productif d'aborder le sujet non seulement par la question "Que veux-tu faire plus tard ?" mais aussi par son inverse : "Qu'est-ce que tu ne veux absolument pas faire ?".

Cette approche par élimination permet de cerner plus facilement les centres d'intérêt et les rejets.

• • Accompagner sans décider à la place : Les parents peuvent jouer un rôle de facilitateur en se familiarisant avec les outils comme Parcoursup et SupTracker.

Ils peuvent ainsi aider leur enfant dans ses recherches, discuter des options et l'aider à structurer sa réflexion, sans imposer leurs propres choix.

• • Assurer un suivi de l'assiduité : Une vigilance particulière est recommandée concernant les absences, notamment les jours de devoir.

Il est important de dialoguer avec son enfant pour comprendre les raisons d'une éventuelle démotivation et de soutenir les politiques de l'établissement visant à garantir un travail régulier.

• • Anticiper la tension des résultats : La première semaine de juin, lors de la publication des réponses de Parcoursup, est une période de stress intense et inévitable.

Les listes d'attente peuvent être source d'angoisse. Les parents doivent se préparer à cette phase, comprendre que c'est une étape normale du processus, et être prêts à soutenir leur enfant dans un contexte qui, comme le souligne l'équipe du lycée, peut rendre "l'ambiance en famille... chaude".

5.0 Conclusion : Une Année de Préparation et de Transition

En définitive, l'année de Terminale doit être envisagée moins comme une fin en soi que comme une transition activement préparée vers l'avenir.

Le double objectif de l'obtention du baccalauréat et de la réussite de son orientation est parfaitement atteignable lorsque la démarche est structurée.

Une compréhension claire des attentes, une communication ouverte entre l'élève, sa famille et l'équipe éducative, ainsi qu'un travail régulier et une préparation sérieuse sont les garants d'une double réussite : un diplôme obtenu avec succès et une orientation post-bac choisie, ambitieuse et épanouissante.

À partir du document fourni, voici une liste des types d'informations transmises, généralisées à tout type de lycée, ainsi que des informations potentiellement manquantes qui pourraient être ajoutées pour une rentrée plus complète et sereine.

Informations Transmises (Généralisées à tout type de Lycée) : Présentation de l'établissement et de son identité : Histoire du lycée, son évolution (ex: de technique à général et technologique), sa taille (nombre d'élèves, de niveaux et de filières comme les BTS et prépas), et ses valeurs (ex: rigueur). Informations logistiques et calendrier de rentrée : Horaires spécifiques de rentrée pour chaque niveau (seconde, première, terminale, post-bac). Modalités d'accueil (pas de pression le premier jour pour les secondes, accueil des internes). Procédure pour connaître les classes (affichage, accompagnement par les professeurs principaux). Règles d'accès et de sortie de l'établissement (portail spécifique, contrôle des sacs, vérification d'identité avec livret d'accueil et photo). Déroulement des journées de cours (après-midi de cours standard). Communication avec les familles et réunions importantes : Dates et lieux des réunions de rentrée par niveau (avec des spécificités pour les secondes). Envoi régulier d'informations par courriel ("Info Parents"). Invitation aux réunions d'information générales sur le fonctionnement du lycée et sur l'engagement parental. Environnement numérique et ressources pédagogiques : Distribution d'ordinateurs portables gratuits par la région (pour les nouveaux élèves) avec explications sur leur usage, entretien et conditions de prêt. Accès WiFi dans l'établissement. Fourniture de manuels scolaires numériques (dispense d'achat). Utilisation de plateformes numériques (Mon Bureau Numérique, EduConnect, Pronote, Scolen Go) pour les emplois du temps, cahiers de texte, notes, bourses, etc. Mise à disposition gratuite de logiciels (Microsoft Office). Support pour l'utilisation des outils numériques et tests de positionnement. Absence de liste de fournitures scolaires extraordinaires (cahiers ou classeurs selon les enseignants). Résultats académiques et exigences scolaires : Présentation des taux de réussite aux examens (BTS, Baccalauréat général et technologique) et des mentions obtenues. Objectif de réussite et d'exigence de l'établissement. Règlement intérieur et vie scolaire : Attentes en matière de comportement (respect, calme dans les couloirs). Règles concernant l'utilisation du téléphone portable (interdit dans les bâtiments sauf permission). Politique stricte sur les retards et les absences, nécessité de les justifier de manière rigoureuse. Horaires d'ouverture de l'établissement et heures de cours. Ouverture du lycée certains samedis matins pour devoirs, rattrapages ou sanctions (colles). Orientation et préparation aux examens : Information sur les calculatrices spécifiques exigées au Baccalauréat et procédure de commande groupée. Nouveautés comme l'Épreuve Anticipée de Mathématiques en première. Accompagnement personnalisé et aide aux examens (ex: Grand Oral). Présentation des dispositifs comme Pix (certification numérique) et SNU (Service National Universel). Présentation détaillée de Parcoursup, avec l'encouragement à rencontrer les psychologues de l'éducation nationale (PsyEN) tôt dans l'année. Soutien à l'orientation par les professeurs principaux. Stages de seconde (dates, modalités de recherche, aide de l'établissement). Services de restauration et aides financières : Accès à la cantine pour les demi-pensionnaires et modalités pour les externes. Tarifs de la restauration scolaire et de l'internat (avec les aides régionales). Dispositifs d'aides financières (fonds sociaux, aide à la restauration scolaire) et contacts pour les solliciter. Implication des parents dans la vie du lycée : Importance de la participation des parents délégués aux conseils de classe et au conseil d'administration. Présentation des associations de parents d'élèves (FCPE, PEEP) et invitation à leurs réunions. Explication du rôle des parents élus et des formations proposées. Infrastructure et aménagements spécifiques : Visite virtuelle des locaux (bureaux, salles de classe, CDI, salle de permanence, salle d'honneur). Accessibilité de l'établissement (ascenseurs). Description des abords du lycée (arrêts de bus, carrefour, parvis de l'église, futurs travaux du métis). Nouveaux aménagements (abri vélo autonome et connecté) et équipements spécifiques (casiers pour besoins particuliers). Bureaux des personnels (vie scolaire, secrétariat, direction, PsyEN, informatique). Informations Potentiellement Manquantes : Pour une information encore plus complète et généralisable à d'autres lycées, les éléments suivants pourraient être ajoutés :

Calendrier scolaire détaillé et événements clés : Une vision plus exhaustive des dates importantes, y compris les vacances scolaires, les jours fériés, les journées pédagogiques, les périodes d'évaluation communes, les conseils de classe, et les éventuelles journées portes ouvertes ou événements culturels du lycée. Offre d'activités périscolaires : Une présentation des clubs, associations sportives (UNSS), ateliers culturels, projets (théâtre, musique, etc.) et voyages scolaires qui enrichissent la vie de l'élève en dehors des cours. Cela aiderait les élèves à s'intégrer et à trouver des centres d'intérêt. Services de santé et d'aide psychologique : Bien que les PsyEN soient mentionnés pour Parcoursup et l'infirmerie pour le mal de ventre, des détails sur les services de santé disponibles, le rôle de l'infirmière scolaire, les programmes de prévention (harcèlement, addictions) et le soutien psychologique plus général (au-delà de l'orientation) seraient précieux. Consignes de sécurité détaillées : Au-delà du contrôle des sacs et de la vérification d'identité, une explication des plans d'évacuation en cas d'incendie ou d'autres urgences, des points de rassemblement, et des procédures en cas de confinement ou d'alerte intrusion. Détails sur l'offre pédagogique et les options : Une vue d'ensemble plus précise des enseignements de spécialité et des options facultatives disponibles pour les premières et terminales, avec des informations sur les programmes et les débouchés. Cela est crucial pour les choix d'orientation des élèves. Transports scolaires spécifiques : Des informations plus complètes sur les lignes de bus desservant le lycée, les cartes de transport, les horaires des bus scolaires (si différents des bus de ville), et les possibilités de stationnement pour les parents accompagnants ou les élèves motorisés. Conseils méthodologiques pour le lycée : Des recommandations pour les élèves de seconde en particulier sur la gestion du travail personnel, l'organisation, les méthodes d'apprentissage, et l'autonomie requise au lycée, qui diffère souvent du collège. Guide de connexion aux outils numériques : Un tutoriel simple et détaillé pour la première connexion aux plateformes clés (EduConnect, Mon Bureau Numérique, Pronote) pour les élèves et les familles qui n'auraient jamais utilisé ces outils ou rencontreraient des difficultés. Informations pratiques pour les internes : Des détails sur la vie quotidienne à l'internat (règles de vie, horaires d'étude, activités, menus, équipements disponibles, encadrement) au-delà de la simple invitation à une réunion. Détails sur la restauration scolaire : Des informations sur les menus proposés, la prise en compte des régimes alimentaires spécifiques (allergies, végétarisme), les modes de paiement pour les repas occasionnels et les règles de fonctionnement du réfectoire. Informations sur l'équipe éducative (au-delà des responsables) : Une brève présentation des coordinateurs de niveau, des professeurs principaux (même si les détails sont donnés par classe), ou d'autres personnels clés (comme le documentaliste du CDI).

Author Response :

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Summary:

This work shows that a specific adenosine deaminase protein in Dictyostelium generates the ammonia that is required for tip formation during Dictyostelium development. Cells with an insertion in the ADGF gene aggregate but do not form tips. A remarkable result, shown in several different ways, is that the ADGF mutant can be rescued by exposing the mutant to ammonia gas. The authors also describe other phenotypes of the ADGF mutant such as increased mound size, altered cAMP signalling, and abnormal cell type differentiation. It appears that the ADGF mutant has defects in the expression of a large number of genes, resulting in not only the tip defect but also the mound size, cAMP signalling, and differentiation phenotypes.

Strengths:

The data and statistics are excellent.

Weaknesses

(1) The key weakness is understanding why the cells bother to use a diffusible gas like ammonia as a signal to form a tip and continue development.

Ammonia can come from a variety of sources both within and outside the cells and this can be from dead cells also. Ammonia by increasing cAMP levels, trigger collective cell movement thereby establishing a tip in Dictyostelium. A gaseous signal can act over long distances in a short time and for instance ammonia promotes synchronous development in a colony of yeast cells (Palkova et al., 1997; Palkova and Forstova, 2000). The slug tip is known to release ammonia probably favouring synchronized development of the entire colony of Dictyostelium. However, after the tips are established ammonia exerts negative chemotaxis probably helping the slugs to move away from each other ensuring equal spacing of the fruiting bodies (Feit and Sollitto, 1987).

It is well known that ammonia serves as a signalling molecule influencing both multicellular organization and differentiation in Dictyostelium (Francis, 1964; Bonner et al., 1989; Bradbury and Gross, 1989). Ammonia by raising the pH of the intracellular acidic vesicles of prestalk cells (Poole and Ohkuma, 1981; Gross et al, 1983), and the cytoplasm, is known to increase the speed of chemotaxing amoebae (Siegert and Weijer, 1989; Van Duijn and Inouye, 1991), inducing collective cell movement (Bonner et al., 1988, 1989), favoring tipped mound development.

Ammonia produced in millimolar concentrations during tip formation (Schindler and Sussman, 1977) could ward off other predators in soil. For instance, ammonia released by Streptomyces symbionts of leaf-cutting ants is known to inhibit fungal pathogens (Dhodary and Spiteller, 2021). Additionally, ammonia may be recycled back into amino acids, as observed during breast cancer proliferation (Spinelli et al., 2017). Such a process may also occur in starving Dictyostelium cells, supporting survival and differentiation. These findings suggest that ammonia acts as both a local and long-range regulatory signal, integrating environmental and cellular cues to coordinate multicellular development.

(2) The rescue of the mutant by adding ammonia gas to the entire culture indicates that ammonia conveys no positional information within the mound.

Ammonia reinforces or maintains the positional information by elevating cAMP levels, favoring prespore differentiation (Bradbury and Gross, 1989; Riley and Barclay, 1990; Hopper et al., 1993). Ammonia is known to influence rapid patterning of Dictyostelium cells confined in a restricted environment (Sawai et al., 2002). In adgf mutants that have low ammonia levels, both neutral red staining (a marker for prestalk and ALCs) (Figure. S3) and the prestalk marker ecmA/ ecmB expression (Figure. 7D) are higher than the WT and the mound arrest phenotype can be reversed by exposing the adgf mutant mounds to ammonia.

Prestalk cells are enriched in acidic vesicles, and ammonia, by raising the pH of these vesicles and the cytoplasm (Davies et al 1993; Van Duijn and Inouye 1991), plays an active role in collective cell movement during tip formation (Bonner et al., 1989).

(3) By the time the cells have formed a mound, the cells have been starving for several hours, and desperately need to form a fruiting body to disperse some of themselves as spores, and thus need to form a tip no matter what.

Exposure of adgf mounds to ammonia, led to tip development within 4 h (Figure. 5). In contrast, adgf controls remained at the mound stage for at least 30 h. This demonstrates that starvation alone is not the trigger for tip development and ammonia promotes the transition from mound to tipped mound formation.

Many mound arrest mutants are blocked in development and do not proceed to form fruiting bodies (Carrin et al., 1994). Further, not all the mound arrest mutants tested in this study were rescued by ADA enzyme (Figure. S4A), and they continue to stay as mounds.

(4) One can envision that the local ammonia concentration is possibly informing the mound that some minimal number of cells are present (assuming that the ammonia concentration is proportional to the number of cells), but probably even a minuscule fruiting body would be preferable to the cells compared to a mound. This latter idea could be easily explored by examining the fate of the ADGF cells in the mound - do they all form spores? Do some form spores?

Or perhaps the ADGF is secreted by only one cell type, and the resulting ammonia tells the mound that for some reason that cell type is not present in the mound, allowing some of the cells to transdifferentiate into the needed cell type. Thus, elucidating if all or some cells produce ADGF would greatly strengthen this puzzling story.

A fraction of adgf mounds form bulkier spore heads by the end of 36 h as shown in Figure. 2H. This late recovery may be due to the expression of other ADA isoforms. Mixing WT and adgf mutant cell lines results in a chimeric slug with mutants occupying the prestalk region (Figure. 8) and suggests that WT ADGF favours prespore differentiation. However, it is not clear if ADGF is secreted by a particular cell type, as adenosine can be produced by both cell types, and the activity of three other intracellular ADAs may vary between the cell types. To address whether adgf expression is cell type-specific, prestalk and prespore cells will be separated by fluorescence activated cell sorter (FACS), and thereafter, adgf expression will be examined in each population.

Reviewer #1 (Recommendations for the authors):

(1) Lines: 47,48 - "The gradient of these morphogens along the slug axis determines the cell fate, either as prestalk (pst) or as prespore (psp) cells." - many workers have shown that this is not true - intrinsic factors such as cell cycle phase drive cell fate.

Thank you for pointing this out. We have removed the line and rephrased as “Based on cell cycle phases, there exists a dichotomy of cell types, that biases cell fate as prestalk or prespore (Weeks and Weijer, 1994; Jang and Gomer, 2011).

(2) Line 48 - PKA - please explain acronyms at first use.

Corrected

(3) Line 56 - The relationship between adenosine deaminase and ADGF is a bit unclear, please clarify this more.

Adenosine deaminase (ADA) is intracellular, whereas adenosine deaminase related growth factor (ADGF) is an extracellular ADA and has a growth factor activity (Li and Aksoy, 2000; Iijima et al., 2008).

(4) Figure 1 - where are these primers, and the bsr cassette, located with respect to the coding region start and stop sites?

The primer sequences are mentioned in the supplementary table S2. The figure legend is updated to provide a detailed description.

(5) Line 104 - 37.47% may be too many significant figures.

Corrected

(6) Line 123 - 1.003 Å may be too many significant figures.

Corrected

(7) Line 128 - Since the data are in the figure, you don't need to give the numbers, also too many significant figures.

Corrected

(8) Figure 3G - did the DCF also increase mound size? It sort of looks like it did.

Yes, the addition of DCF increases the mound size (now Figure. 2G).

(9) Figure 3I - the spore mass shown here for ADGF - looks like there are 3 stalks protruding from it; this can happen if a plate is handled roughly and the spore masses bang into each other and then merge

Thank you for pointing this out. The figure 3I (now Figure. 2I) is replaced.

(10) Lines 160-162 - since the data are in the figure, you don't need to give the numbers, also too many significant figures.

Corrected.

(11) Line 165 - ' ... that are involved in adenosine formation' needs a reference.

Reference is included.

(12) Line 205 - 'Addition of ADA to the CM of the mutant in one compartment.' - might clarify that the mutant is the ADGF mutant

Yes, revised to 'Addition of ADA to the CM of the adgf mutant in one compartment.

(13 Lines 222-223 need a reference for caffeine acting as an adenosine antagonist.

Reference is included.

(14) Figure 8B - left - use a 0-4 or so scale so the bars are more visible.

Thank you for the suggestion. The scale of the y-axis is adjusted to 0-4 in Figure. 7B to enhance the visibility of the bars.

Reviewer #2 (Public review):

Summary:

The paper describes new insights into the role of adenosine deaminase-related growth factor (ADGF), an enzyme that catalyses the breakdown of adenosine into ammonia and inosine, in tip formation during Dictyostelium development. The ADGF null mutant has a pre-tip mound arrest phenotype, which can be rescued by the external addition of ammonia. Analysis suggests that the phenotype involves changes in cAMP signalling possibly involving a histidine kinase dhkD, but details remain to be resolved.

Strengths:

The generation of an ADGF mutant showed a strong mound arrest phenotype and successful rescue by external ammonia. Characterization of significant changes in cAMP signalling components, suggesting low cAMP signalling in the mutant and identification of the histidine kinase dhkD as a possible component of the transduction pathway. Identification of a change in cell type differentiation towards prestalk fate

(1) Weaknesses: Lack of details on the developmental time course of ADGF activity and cell type type-specific differences in ADGF expression.

adgf expression was examined at 0, 8, 12, and 16 h (Figure. 1), and the total ADA activity was assayed at 12 and 16 h (Figure. 3). Previously, the 12 h data was not included, and it’s been added now (Figure. 3A). The adgf expression was found to be highest at 16 h and hence, the ADA assay was carried out at that time point. Since the ADA assay will also report the activity of other three isoforms, it will not exclusively reflect ADGF activity.

Mixing WT and adgf mutant cell lines results in a chimeric slug with mutants occupying the prestalk region (Figure. 8) suggesting that WT adgf favours prespore differentiation. To address whether adgf expression is cell type-specific, prestalk and prespore cells will be separated by fluorescence activated cell sorter (FACS), and thereafter, adgf expression will be examined in each population.

(2) The absence of measurements to show that ammonia addition to the null mutant can rescue the proposed defects in cAMP signalling.

The adgf mutant in comparison to WT has diminished acaA expression (Fig. 6B) and reduced cAMP levels (Fig. 6A) both at 12 and 16 h of development. The cAMP levels were measured at 8 h and 12 h in the mutant.

We would like to add that ammonia is known to increase cAMP levels (Riley and Barclay, 1990; Feit et al., 2001) in Dictyostelium. Exposure to ammonia increases acaA expression in WT (Figure. 7B) and is likely to increase acaA expression/ cAMP levels in the mutant also (Riley and Barclay, 1990; Feit et al., 2001) thereby rescuing the defects in cAMP signalling. Based on the comments, cAMP levels will also be measured in the mutant after the rescue with ammonia.

(3) No direct measurements in the dhkD mutant to show that it acts upstream of adgf in the control of changes in cAMP signalling and tip formation.

cAMP levels will be quantified in the dhkD mutant after treatment with ammonia. The histidine kinases dhkD and dhkC are reported to modulate phosphodiesterase RegA activity, thereby maintaining cAMP levels (Singleton et al., 1998; Singleton and Xiong, 2013). By activating RegA, dhkD ensures proper cAMP distribution within the mound, which is essential for the patterning of prestalk and prespore cells, as well as for tip formation (Singleton and Xiong, 2013). Therefore, ammonia exposure to dhkD mutants is likely to regulate cAMP signalling and thereby tip formation.

Reviewer #2 (Recommendations for the authors):

The paper describes new insights into the role of ADGF, an enzyme that catalyses the breakdown of adenosine in ammonia and inosine, in tip formation in Dictyostelium development.

A knockout of the gene results in a tipless mound stage arrest and the mounds formed are somewhat larger in size. Synergy experiments show that the effect of the mutation is non-cell autonomous and further experiments show that the mound arrest phenotype can be rescued by the provision of ammonia vapour. These observations are well documented. Furthermore, the paper contains a wide variety of experiments attempting to place the observed effects in known signalling pathways. It is suggested that ADGF may function downstream of DhkD, a histidine kinase previously implicated in ammonia signalling. Ammonia has long been described to affect different aspects, including differentiation of slug and culmination stages of Dictyostelium development, possibly through modulating cAMP signalling, but the exact mechanisms of action have not yet been resolved. The experiments reported here to resolve the mechanistic basis of the mutant phenotype need focusing and further work.

(1) The paper needs streamlining and editing to concentrate on the main findings and implications.

The manuscript will be revised extensively.

Below is a list of some more specific comments and suggestions.

(2) Introduction: Focus on what is relevant to understanding tip formation and the role of nucleotide metabolism and ammonia (see https://doi.org/10.1016/j.gde.2016.05.014).leading). This could lead to the rationale for investigating ADGF.

The manuscript will be revised extensively

(3) Lines 36-38 are not relevant. Lines 55-63 need shortening and to focus on ADGF, cellular localization, and substrate specificity.

The manuscript will be revised accordingly. Lines 36-38 will be removed, and the lines 55-63 will be shortened.

In humans, two isoforms of ADA are known including ADA1 and ADA2, and the Dictyostelium homolog of ADA2 is adenosine deaminase-related growth factor (ADGF). Unlike ADA that is intracellular, ADGF is extracellular and also has a growth factor activity (Li and Aksoy, 2000; Iijima et al., 2008). Loss-of-function mutations in ada2 are linked to lymphopenia, severe combined immunodeficiency (SCID) (Gaspar, 2010), and vascular inflammation due to accumulation of toxic metabolites like dATP (Notarangelo, 2016; Zhou et al., 2014).

(4) Results: This section would benefit from better streamlining by a separation of results that provide more mechanistic insight from more peripheral observations.

The manuscript will be revised and the peripheral observations (Figure. 2) will be shifted to the supplementary information.

(5) Line 84 needs to start with a description of the goal, to produce a knockout.

Details on the knockout will be elaborated in the revised manuscript. Line number 84 (now 75). Dictyostelium cell lines carrying mutations in the gene adgf were obtained from the genome wide Dictyostelium insertion (GWDI) bank and were subjected to further analysis to know the role of adgf during Dictyostelium development.

(6) Knockout data (Figure 1) can be simplified and combined with a description of the expression profile and phenotype Figure 3 F, G, and Figure 5. Higher magnification and better resolution photographs of the mutants would be desirable.

Thank you, as suggested the data will be simplified (section E will be removed) and combined with a description of the expression profile and, the phenotype images of Figure 3 F, G, and Figure 5 ( now Figure. 2 F, G, and Figure. 4) will be replaced with better images/ resolution.

(7) It would also be relevant to know which cells actually express ADGF during development, using in-situ hybridisation or promoter-reporter constructs.

To address whether adgf expression is cell type-specific, prestalk and prespore cells will be separated by fluorescence activated cell sorter (FACS), and thereafter, adgf expression will be examined in each population.

(8) Figure 2 - Information is less directly relevant to the topic of the paper and can be omitted (or possibly in Supplementary Materials).

Figure. 2 will be moved to supplementary materials.

(9) Figures 4A, B - It is shown that as could be expected ada activity is somewhat reduced and adenosine levels are slightly elevated. However, the fact that ada levels are low at 16hrs could just imply that differentiation of the ADGF- cells is blocked/delayed at an earlier time point. To interpret these data, it would be necessary to see an ada activity and adenosine time course comparison of wt and mutant, or to see that expression is regulated in a celltype specific manner that could explain this (see above). It would be good to combine this with the observation that ammonia levels are lower in the ADGF- mutant than wildtype and that the mutant phenotype, mound arrest can be rescued by an external supply of ammonia (Figure 6).

In Dictyostelium four isoforms of ADA including ADGF are present, and thus the time course of total ADA activity will also report the function of other isoforms. Further, a number of pathways, generate adenosine (Dunwiddie et al., 1997; Boison and Yegutkin, 2019). ADGF expression was examined at 0, 8, 12 and 16 h (Fig 1) and the ADA activity was assayed at 12 h, the time point where the expression gradually increases and reaches a peak at 16 h. Earlier, we have not shown the 12 h activity data which will be included in the revised version. ADGF expression was found to be highly elevated at 16 h and adenosine/ammonia levels were measured at the two points indicated in the mutant.

(10) Panel 4C could be combined with other measurements trying to arrive at more insight in the mechanisms by which ammonia controls tip formation.

Panel 4C (now 3C) illustrates the genes involved in the conversion of cAMP to adenosine. Since Figure. 3 focuses on adenosine levels and ADA activity in both WT and adgf mutants, we have retained Panel 3C in Figure. 3, for its relevance to the experiment.

(11) There is a large variety of experiments attempting to link the mutant phenotype and its rescue by ammonia to cAMP signalling, however, the data do not yet provide a clear answer.

It is well known that ammonia increases cAMP levels (Riley and Barclay, 1990; Feit et al., 2001) and adenylate cyclase activity (Cotter et al., 1999) in D. discoideum, and exposure to ammonia increases acaA expression (Fig 7B) suggesting that ammonia regulates cAMP signaling. To address the concerns, cAMP levels will be quantified in the mutant after ammonia treatment.

(12) The mutant is shown to have lower cAMP levels at the mound stage which ties in with low levels of acaA expression (Figures 7A and B), also various phosphodiesterases, the extracellular phosphodiesterase pdsa and the intracellular phosphodiesterase regA show increased expression. Suggesting a functional role for cAMP signalling is that the addition of di cGMP, a known activator of acaA, can also rescue the mound phenotype (Figure 7E). There appears to be a partial rescue of the mound arrest phenotype level by the addition of 8Br-cAMP (fig 7C), suggesting that intracellular cAMP levels rather than extracellular cAMP signalling can rescue some of the defects in the ADGF- mutant. Better images and a time course would be helpful.

The relevant images will be replaced and a developmental time course after 8-Br-cAMP treatment will be included in the revised manuscript (Figure. 6D).

(13) There is also the somewhat surprising observation that low levels of caffeine, an inhibitor of acaA activation also rescues the phenotype (Figure 7F).

With respect to caffeine action on cAMP levels, the reports are contradictory. Caffeine has been reported to increase adenylate cyclase expression thereby increasing cAMP levels (Hagmann, 1986) whereas Alvarez-Curto et al., (2007) found that caffeine reduced intracellular cAMP levels in Dictyostelium. Caffeine, although is a known inhibitor of ACA, is also known to inhibit PDEs (Nehlig et al., 1992; Rosenfeld et al., 2014). Therefore, if caffeine differentially affects ADA and PDE activity, it may potentially counterbalance the effects and rescue the phenotype.

(14) The data attempting to asses cAMP wave propagation in mounds (Fig 7H) are of low quality and inconclusive in the absence of further analysis. It remains unresolved how this links to the rescue of the ADGF- phenotype by ammonia. There are no experiments that measure any of the effects in the mutant stimulated with ammonia or di-cGMP.

The relevant images will be replaced (now Figure. 6H). Ammonia by increasing acaA expression (Figure. 7B), and cAMP levels (Figure. 7C) may restore spiral wave propagation, thereby rescuing the mutant.

(15) A possible way forward could also come from the observation that ammonia can rescue the wobbling mound arrest phenotype from the histidine kinase mutant dhkD null mutant, which has regA as its direct target, linking ammonia and cAMP signalling. This is in line with other work that had suggested that another histidine kinase, dhkC transduces an ammonia signal sensor to regA activation. A dhkC null mutant was reported to have a rapid development phenotype and skip slug migration (Dev. Biol. (1998) 203, 345). There is no direct evidence to show that dhkD acts upstream of ADGF and changes in cAMP signalling, for instance, measurements of changes in ADA activity in the mutant.

cAMP levels will be quantified in the dhkD mutant after ammonia treatment and accordingly, the results will be revised.

(16) The paper makes several further observations on the mutant. After 16 hrs of development the adgf- mutant shows increased expression of the prestalk cell markers ecmA and ecmB and reduced expression of the prespore marker pspA. In synergy experiments with a majority of wildtype, these cells will sort to the tip of the forming slug, showing that the differentiation defect is cell autonomous (Fig 9). This is interesting but needs further work to obtain more mechanistic insight into why a mutant with a strong tip/stalk differentiation tendency fails to make a tip. Here again, knowing which cells express ADGF would be helpful.

The adgf mutant shows increased prestalk marker expression in the mound but do not form a tip. It is well known that several mound arrest mutants form differentiated cells but are blocked in development with no tips (Carrin et al., 1994). This is addressed in the discussions (539). To address whether adgf expression is cell type-specific, prestalk and prespore cells will be separated by fluorescence activated cell sorter (FACS), and thereafter, adgf expression will be examined in each population.

(17) The observed large mound phenotype could as suggested possibly be explained by the low ctn, smlA, and high cadA and csA expression observed in the mutant (Figure 3). The expression of some of these genes (csA) is known to require extracellular cAMP signalling. The reported low level of acaA expression and high level of pdsA expression could suggest low levels of cAMP signalling, but there are no actual measurements of the dynamics of cAMP signalling in this mutant to confirm this.

The acaA expression was examined at 8 and 12 h (Figure. 6B) and cAMP levels were measured at 12 and 16 h in the adgf mutants (Figure. 6A). Both acaA expression and cAMP levels were reduced, suggesting that cells expressing adgf regulate acaA expression and cAMP levels. This regulation, in turn, is likely to influence cAMP signaling, collective cell movement within mounds, ultimately driving tip development. Exposure to ammonia led to increased acaA expression (Figure. 7B) in in WT. Based on the comments above, cAMP levels will be measured in the mutant before and after rescue with ammonia.

(18) Furthermore, it would be useful to quantify whether ammonia addition to the mutant reverses mound size and restores any of the gene expression defects observed.

Ammonia treatment soon after plating or six hours after plating, had no effect on the mound size (Figure. 5G).

(19) There are many experimental data in the supplementary data that appear less relevant and could be omitted Figure S1, S3, S4, S7, S8, S9, S10.

Figure S8, S9, S10 are omitted. We would like to retain the other figures

Figure S1 (now Figure. S2): It is widely believed that ammonia comes from protein (White and Sussman, 1961; Hames and Ashworth, 1974; Schindler and Sussman, 1977) and RNA (Walsh and Wright, 1978) catabolism. Figure. S2 shows no significant difference in protein and RNA levels between WT and adgf mutant strains, suggesting that adenosine deaminaserelated growth factor (ADGF) activity serves as a major source of ammonia and plays a crucial role in tip organizer development in Dictyostelium. Thus, it is important to retain this figure.

Figure S3 (now Figure. S4): The figure shows the treatment of various mound arrest mutants and multiple tip mutants with ADA enzyme and DCF, respectively, to investigate the pathway through which adgf functions. Additionally, it includes the rescue of the histidine kinase mutant dhkD with ammonia, indicating that dhkD acts upstream of adgf via ammonia signalling. Therefore, it is important to retain this figure.

Figure S4 (now Figure. S5): This figure represents the developmental phenotype of other deaminase mutants. Unlike adgf mutants, mutations in other deaminases do not result in complete mound arrest, despite some of these genes exhibiting strong expression during development. This underscores the critical role of adenosine deamination in tip formation. Therefore, let this figure be retained.

Figure S7 (now Figure. S8): Figure S8 presents the transcriptomic profile of ADGF during gastrulation and pre-gastrulation stages across different organisms, indicating that ADA/ADGF is consistently expressed during gastrulation in several vertebrates (Pijuan-Sala et al., 2019; Tyser et al., 2021). Notably, the process of gastrulation in higher organisms shares remarkable similarities with collective cell movement within the Dictyostelium mound (Weijer, 2009), suggesting a previously overlooked role of ammonia in organizer development. This implies that ADA may play a fundamental role in regulating morphogenesis across species, including Dictyostelium and vertebrates. Therefore, we would like to retain this figure.

(20) Given the current state of knowledge, speculation about the possible role of ADGF in organiser function in amniotes seems far-fetched. It is worth noting that the streak is not equivalent to the organiser. The discussion would benefit from limiting itself to the key results and implications.

The discussion is revised accordingly by removing the speculative role of ADGF in organizer function in amniotes. The lines “It is likely that ADA plays a conserved, fundamental role in regulating morphogenesis in Dictyostelium and other organisms including vertebrates” have been removed.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

The paper sets out to examine the social recognition abilities of a 'solitary' jumping spider species. It demonstrates that based on vision alone spiders can habituate and dishabituate to the presence of conspecifics. The data support the interpretation that these spiders can distinguish between conspecifics on the basis of their appearance.

We appreciate the reviewer’s summary. We indeed aimed at investigating the social recognition abilities of the solitary jumping spider (Phidippus regius), using visual cues alone. By employing a habituation-dishabituation paradigm, well-established in developmental psychology, we found support for the interpretation that these spiders can distinguish between conspecifics based on their appearance, as the reviewer noted.

Strengths:

The study presents two experiments. The second set of data recapitulates the findings of the first experiment with an independent set of spiders, highlighting the strength of the results. The study also uses a highly quantitative approach to measuring relative interest between pairs of spiders based on their distance.

We appreciate the reviewer's acknowledgement of the strengths of our study. The second set of data underscores the robustness and reliability of the results. Additionally, however, the second experiment served the purpose of disentangling whether the habituation effect observed over sessions was caused by ‘physical’ or ‘cognitive’ fatigue by employing ‘long-term’ dishabituation trials at the end of Session 3. These trials are critical in our study as they help to differentiate between recognition of individual identities versus recognition of familiar individuals (as opposed to unfamiliar ones) and to determine if the observed effects are due to ‘general habituation’ or ‘specific recognition’. We will elaborate on this further below in this revision.

As stated by the reviewer, we employed a highly quantitative approach to measure relative interest between pairs of spiders based on their distance, providing precise and objective data to support our conclusions.

Weaknesses:

The study design is overly complicated, missing key controls, and the data presented in the figures are not clearly connected to the study. The discussion is challenging to understand and appears to make unsupported conclusions.

While we acknowledge that the study design is indeed complex, this complexity is essential for conducting a well-controlled and balanced experiment regarding the experimental conditions.  

The habituation-dishabituation paradigm is a well-established paradigm in developmental psychology with non-verbal infants. It is understood that during the habituation phase, an individual's attention to a repeated stimulus decreases as they engage in information processing and form a mental representation of it. As the stimulus becomes familiar, it loses its novelty and interest. When a new stimulus is introduced, a recovery of attention suggests that the individual has compared this new stimulus to the stored memory of the habituation stimulus and detected a difference. This process suggests that the individual not only remembered the original stimulus but also recognized the new one as distinct (for a review Kavšek & Bornstein, 2010).

This paradigm has also been extensively applied in animal research, where, like infants, nonverbal subjects rely on recognition and discrimination processes to demonstrate their cognitive abilities. The use of this paradigm dates back to seminal studies such as Humphrey (1974), which explored the perceptual world of monkeys, illustrating how species and individuals are perceived and recognized. In another previous study (Dahl, Logothetis, and Hoffman, 2007), we utilized an even more complex experimental design that incorporated dedicated baseline trials for both habituation and dishabituation phases, which was well-received despite its complexity. In the current study, we contrast dishabituation and habituation trials directly, creating a sequential cascade where each trial is evaluated against the preceding one as its baseline.

On the basis of these arguments, we respectfully decline the claim that this paradigm is inappropriate or lacks key controls. Our study design, though complex, is rigorously grounded in established methodologies and offers a robust framework for exploring individual recognition in Phidippus regius.

However, we take the reviewer’s comments seriously and are committed to identifying and addressing the aspects in our manuscript that may have led to misunderstandings. We clarify these areas in our revision of the manuscript. Modifications were made in the Introduction, Methods, and Discussion sections.

Dahl, C. D., Logothetis, N. K., & Hoffman, K. L. (2007). Individuation and holistic processing of faces in rhesus monkeys. Proceedings of the Royal Society B: Biological Sciences, 274(1622), 2069-2076.

Humphrey, N. K. (1974). Species and individuals in the perceptual world of monkeys. Perception, 3(1), 105-114.

Kavšek, M., & Bornstein, M. H. (2010). Visual habituation and dishabituation in preterm infants: A review and meta-analysis. Research in developmental disabilities, 31(5), 951-975.

(1) Study design: The study design is rather complicated and as a result, it is difficult to interpret the results. The spiders are presented with the same individual twice in a row, called a habituation trial. Then a new individual is presented twice in a row. The first of these is a dishabituation trial and the second is another habituation trial (but now habituating to a second individual). This is done with three pairings and then this entire structure is repeated over three sessions. 

While we acknowledge that the design is complex, this complexity is essential for conducting a well-controlled experiment, as described earlier. As the reviewer noted, our design involves presenting the same individual to the focal spider twice in a row (habituation trial), followed by a new individual (dishabituation trial), and then repeating this structure. This approach is fundamental to the habituation-dishabituation paradigm, which allows us to systematically compare the responses to a familiar individual with those elicited by a novel one. If the spiders exhibit different behaviours in terms of the distance they maintain when encountering the same individual versus a new one, it indicates that they are processing the stimuli differently, consistent with recognition memory. This differential response is a key indicator that the spiders can distinguish between familiar and unfamiliar individuals, demonstrating not only a decrease in interest or engagement due to repeated exposure but also a cognitive process where the lack of a matching memory template triggers a distinct behavioural response when confronted with novel stimuli.

By repeating this sequence two more times (Session 2 and 3), we aim to assess the consistency of this recognition process over time. If the focal spider does not remember the individuals from the previous session (one hour ago), we expect consistent behavioural responses across sessions. Conversely, if there is a decrease in response magnitude but the overall response patterns are maintained, we can infer that the focal spider recognizes the previously presented individuals and exhibits habituation, reflected in reduced response intensity. In other words, over sessions and repeated exposure to the same individuals, the memory traces become more firmly established, leading to a situation where a dishabituation trial introduces less novelty, as the spider's recognition of previously encountered individuals becomes more robust and consistent to the point where “habituation” and “dishabituation” trials become indistinguishable, as observed in Session 3. This method allows us to assess the duration of identity recognition in these spiders, indicating how long the memory of specific individuals persists. 

All of these outcomes were anticipated before we began Experiment 1. Given that the results aligned with our predictions, we then sought to determine whether the observed reduction in the magnitude of the effect (i.e., the difference between habituation and dishabituation trials) was due to a physical fatigue effect, where the spiders might simply be getting tired, or a cognitive fatigue effect, where the spiders recognized the individuals and as a result did not exhibit any novelty response. To address this, we replicated the experiment with a new group of spiders and introduced special (long-term dishabituation) trials at the end, where the focal spider was presented with a novel spider. 

These extra trials allowed us to disentangle the nature of the diminishing response across repeated sessions: a lack of dishabituation (remaining distant) would suggest general physical fatigue, whereas a strong dishabituation response (approaching closely) to the novel spider would indicate cognitive fatigue, thereby confirming that the spiders were indeed recognizing the familiar individuals throughout the experiment. 

In light of these considerations, we believe that the complexity of our design is not only justified but absolutely necessary to rigorously test the cognitive capabilities of the spiders. Nonetheless, we understand the need for clarity in presenting our findings and are committed to refining our manuscript to better communicate the rationale and results of our study.

The data appear to show the strong effects of differences between habituation and dishabituation trials in the first session. The decrease in differential behavior between the socalled habituation and dishabituation trials in sessions 2 and 3 is explained as a consequence of the spiders beginning to habituate in general to all of the individuals. 

The key question, as mentioned above, is to determine the underlying cause of this general habituation across sessions. Specifically, we aim to differentiate between two potential causes: physical fatigue, where the spiders may simply become less responsive due to the demands of the three-hour testing period, or cognitive fatigue, where the repeated exposure to the same individuals leads to a decreased response because the spiders have started to recognize these individuals over multiple repetitions.

To address this, we replicated the experiment and introduced each focal spider to a new individual in what we termed "long-term dishabituation" trials. By comparing the spiders' responses to these novel individuals with their responses in earlier trials, we sought to better understand the underlying mechanisms of habituation and the duration of individual recognition. The strong dishabituation response observed in these trials is indicative of cognitive fatigue, supporting the presence of recognition memory rather than a general physical fatigue effect.

The claim that the spiders remember specific individuals is somewhat undercut because all of the 'dishabituation' trials in session 2 are toward spiders they already met for 14 minutes previously but seemingly do not remember in session 2. 

We appreciate the reviewer’s comment regarding the claim that spiders do not remember specific individuals. This assessment does not align with the rationale of our experiment. The reviewer noted that the dishabituation trials in session 2 involved spiders previously encountered and suggested that the lack of a clear memory response might undercut the claim of specific individual recognition. 

However, as we explained earlier, we expect habituation in Session 2 relative to Session 1 precisely because spiders recognize each other in Session 2. If there were no such habituation in Sessions 2 or 3, it would suggest that the spiders’ recognition memory does not persist beyond one hour. 

Additionally, it is important to correct the timing noted by the reviewer: each individual spider reencounters the same spider exactly one hour later, not 14 minutes. This is detailed in Table 2 of the manuscript, which outlines that each trial lasts 7 minutes, with a 3-minute visual separation between trials. With six trials per session, this totals to 1 hour per session. Thus, every pair of spiders re-encounters exactly 1 hour after their last interaction.

Again, it is important to clarify that the observed decrease in differential behaviour is not indicative of a failure to remember specific individuals. Rather, it reflects a systematic pattern of habituation, which is a common and expected outcome in such paradigms. This systematic decrease in response strength suggests that the spiders recognize the previously encountered individuals and becoming less responsive over repeated exposures, consistent with the process of habituation. In different terms, the repeated exposure to the same individuals leads to more firmly established memory traces, leading to a situation where a dishabituation trial introduces less novelty, as the spider's recognition of previously encountered individuals becomes more robust and consistent.

Based on the explanations provided above, we respectfully reject the claim that “the spiders remember specific individuals is somewhat undercut […]”. In contrast, this claim is incorrect, as the exact opposite is true. The very strength of our study lies in demonstrating that spiders possess robust recognition memory, as evidenced by a clear dissociation of habituation and dishabituation trials in Session 1, followed by a gradually diminishing effect over Session 2 and 3 as the spiders are increased exposed to the same individuals: Furthermore, the strong rebound from habituation observed in long-term dishabituation trials, where the spiders were exposed to novel individuals. 

This misunderstanding suggests that we should take additional care in the revised manuscript to clarify our explanations and provide more detail, ensuring that the rationale behind our experimental design and findings are communicated effectively.

In session 3 it is ambiguous what is happening because the spiders no longer differentiate between the trial types. This could be due to fatigue or familiarity. 

The reviewer proposes that the absence of differentiation between 'habituation' and 'dishabituation' trials in Session 3 might be attributed to either fatigue or familiarity. We interpret "fatigue" as what we have termed the “physical fatigue effect” and "familiarity" as “cognitive fatigue effect.” In this context, we concur with the reviewer’s observation, and this very line of reasoning prompted us to conduct a further experiment following the outcome of Experiment 1.

A second experiment is done to show that introducing a totally novel individual, recovers a large dishabituation response, suggesting that the lack of differences between 'habituation' and 'dishabituation' trials in session 3 is the result of general habituation to all of the spiders in the session rather than fatigue. As mentioned before, these data do support the claim that spiders differentiate among individuals.

As the reviewer rightly noted, we addressed these possibilities in our second experiment by introducing a completely novel individual to the spiders, which resulted in a strong dishabituation response. This outcome suggests that the lack of differentiation in Session 3 is more likely due to cognitive habituation rather than physical fatigue. The robust response to novel individuals demonstrates that the spiders are capable of distinguishing between familiar and unfamiliar individuals, suggesting that the reduced differentiation is a consequence of habituation from repeated encounters with the same individuals. 

We appreciate the reviewer's recognition that these findings support the conclusion that spiders are capable of differentiating between individual conspecifics.

Additionally, it is important to clarify the structure of our sessions. Each of the 6 trials lasts 7 minutes with a 3-minute visual separation, resulting in a total of 1 hour per session. This ensures that each pair of spiders is encountered exactly one hour later, which controls for the timing and allows us to evaluate the spiders' recognition memory over repeated sessions.

In summary, while the data show a decrease in differential behaviour between habituation and dishabituation trials in Session 2 and 3, the results from our second experiment support the interpretation that this is due to ‘cognitive habituation’ (familiarization) rather than ‘physical fatigue’ (general habituation). This habituation effect underscores the spiders' ability to recognize and become familiar with specific individuals over time, reinforcing our conclusion that they can differentiate among individuals.

The data from session 1 are easy to interpret. The data from sessions 2 and 3 are harder to understand, but these are the trials in which they meet an individual again after a substantial period of separation. 

The data from Session 1 are straightforward to interpret, showing clear differences between habituation and dishabituation trials. However, the data from Sessions 2 and 3 are more complex, as these sessions involve the spiders re-encounter individuals after a 1-hour period of separation. Importantly, the outcome is not an artefact in our experiment, but the consequence of a deliberate choice in the experimental design to assess whether spiders can recognise each other after this duration. We believe that this complexity aligns with our expectations, based on the assumption that spiders can recognise each other after one hour. The observed pattern of habituation in Sessions 2 and 3 suggests that the spiders retain memory of the individuals, leading to decreased responsiveness upon repeated encounters. This interpretation is further supported by the Experiment 2, which introduced a novel individual and elicited a strong dishabituation response. This finding confirms that the reduced differentiation in later sessions is due to cognitive habituation rather than physical fatigue, supporting the conclusion that recognition memory last at least one hour.

We hope this explanation clarifies our findings and the rationale behind our relatively complex experimental design choice. 

Other studies looking at recognition in ants and wasps (cited by the authors) have done a 4 trial design in which focal animal A meets B in the first trial, then meets C in the second trial, meets B again in the third trial, and then meets D in the last trial. In that scenario trials 1, 2, and 4 are between unfamiliar individuals and trial 3 is between potentially familiar individuals. In both the ants and wasps, high aggression is seen in species with and without recognition on trial 1, with low aggression specifically for trials with familiar individuals in species with recognition. Across different tests, species or populations that lack recognition have shown a general reduction in aggression towards all individuals that become progressively less aggressive over time (reminiscent of the session 2 and 3 data) while others have maintained modest levels of aggression across all individuals. The 4 session design used in those other studies provides an unambiguous interpretation of the data while controlling for 'fatigue'. 

We acknowledge that there are multiple ways to design experiments to test recognition memory. In fact, we considered using the paradigm similar to the one proposed by the reviewer and used in studies like Dreier et al., which involves a series of trials with unfamiliar and familiar individuals over extended intervals. We then, however, opted for a more complex design to rigorously assess how habituation and recognition memory develop over repeated sessions with shorter intervals.

In the following, we would like to describe the advantages and disadvantages of both paradigms and outline how we ended up using the more complex version:

Advantages of our paradigm: 

As pointed out, by repeating the sequence in exactly similar manner (every same pair of spiders reoccurs after exactly 1 and 2 hours), we can comprehensively evaluate the effect of habituation over multiple exposures. This allows us to assess the extent of the spiders’ memory, when a spider shows stronger habituation to individuals that were novel in Session 1 but “familiar” by the time they encounter them again in Session 2. To achieve this, we need to ensure that each trial and visual separation is precisely timed, ensuring consistent intervals between encounters. As a consequence, each individual spider undergoes the exact same experimental protocol. Most critically, however, are the novel individuals presented after Session 3 (long-term dishabituation trials) that help differentiate between cognitive habituation and physical fatigue.  Disadvantages of our paradigm:

The sequences of habituation and dishabituation trials may make the design more complex, as pointed out by the reviewer. As a consequence, the interpretation will become more difficult. However, the data perfectly align with our predictions, and the outcomes were as anticipated in two independently run experiments with two groups of spiders. This highlights the reliability of our experimental design and robustness of our findings.

Advantages of the 4-trial paradigm proposed by the reviewer:

Clearly, the structure of the proposed design is simpler, making interpretation easier. The paradigm also accommodates longer intervals between trials (e.g., 24 hours). Longer intervals could theoretically have been applied in our study. (However, we chose not to leave the spiders in the experimental box longer than necessary, opting instead to return them to their home containers for the night to ensure their well-being. And, a 24-hour interval targets a different phase in the process of long-term memory, but more to this topic further below.)

Disadvantages of the 4-trial paradigm proposed by the reviewer:

Strictly replicating the 4-trial design would result in one familiar encounter versus three unfamiliar ones. This imbalance might introduce bias and limit the robustness of the measurements. Additionally, the design provides less data overall, as the focal individual will be confronted with three other individuals, who will then be excluded from further testing as focal subjects themselves. In contrast, our design ensures a balanced number of familiar0020(habituation) and novel encounters (dishabituation) for each focal individual, allowing for more efficient and comprehensive data collection without excluding individuals from further testing.

Given the aforementioned considerations, we determined that the advantages of our experimental design, in particular the assessment of a cognitive fatigue effect when encountering the same individuals again, outweigh those of the proposed 4-trial design. The mentioned limitations of the 4-trial design, such as the potential for bias and less comprehensive data collection, do not justify re-running the study, especially when the best case scenario is fewer insights than our already existing findings. Our current paradigm yielded results that align perfectly with our predictions, offering a thorough and reliable understanding of recognition memory and habituation in spiders. Therefore, we believe our approach provides a more complete and robust answer to our research questions.

However, we acknowledge that there might be insufficient information in the manuscript addressing the rationale behind our design choices, and we will revise the manuscript to provide a clearer explanation of why our approach is well suited to answering the research questions at hand.

That all trials in sessions 2 and 3 are always with familiar individuals makes it challenging to understand how much the spiders are habituating to each other versus having some kind of associative learning of individual identity and behavior.

We understand the reviewer's concern that having all trials in Sessions 2 and 3 involve familiar individuals could make it challenging to distinguish between general habituation and associative learning of individual identities. In our study, we contrast habituation and dishabituation trials: If general habituation were occurring, we would expect uniformly reduced responses (around the zero line) to all individuals over time, indicating that the spiders are getting used to any individual regardless of their specific identity. However, this is not the case. Our data show that while the responses in Session 2 are reduced in effect size compared to Session 1, they are not flat (around the zero line). This indicates that the spiders still differentiate between a repetition of a spider identity (habituation trials) and two different spider identities (dishabituation trials), albeit with a reduced response strength. The systematicity in the data suggests that the spiders are not merely habituating to any individual, but are instead retaining some level of recognition between specific individuals.

Only by Session 3 do the spiders fully habituate to the point where the responses to habituation and dishabituation trials converge, indicating a complete habituation effect. The introduction of novel individuals in our long-term dishabituation trials further supports the idea that the spiders are recognizing specific individuals rather than exhibiting general habituation. If the spiders were experiencing general habituation, we would not expect the strong dishabituation response observed in our study.

The data presentation is also very complicated. How is it the case that a negative proportion of time is spent? The methods reveal that this metric is derived by comparing the time individuals spent in each region relative to the previous time they saw that individual. 

We understand the reviewer's concern regarding the complexity of the data presentation and the calculation of the negative proportion of time. Regarding the complexity of the design, we have already justified our choice of a more intricate experimental setup. This complexity is necessary for accurately assessing recognition memory and habituation over repeated sessions. 

The metric is derived by comparing the time individuals spent in each region (relative to the transparent front panel) in the current trial (n) relative to the previous trial (n-1). With multiple trials, this results in a cascade of trials and conditions. This method was established in

Humphrey’s and our previous study (Humphrey, 1974; Dahl, Logothetis, Hoffman, 2007), where we demonstrated its effectiveness in assessing individuation of faces in macaque monkeys.  

Also in our current experimental design, each current trial is contrasted with the preceding one, allowing us to compare distributions of distances taken in two trials. In this context, every preceding trial serves as baseline for every current trial. 

Figure 1 of the manuscript, illustrates the structure and analysis of the trials,

Panel a depicts the baseline, habituation, and dishabituation trials, where spiders are exposed to different conspecifics.

Baseline (left panel, red): When two spiders are visually exposed to each other for the first time, it is expected that they will explore each other closely, exhibiting high levels of proximity (initial exploratory behaviour).

Habituation (centre panel, green): When the same spiders are reintroduced in a subsequent round of exposure, it is anticipated that they will exhibit reduced exploratory behaviour and maintain a greater distance compared to the baseline trial, if they recognize each other from the previous encounter (indicative of habituation).

Panel b (upper and middle panels; red and green): Demonstrates the theoretical assumptions and expected changes in behaviour:

By subtracting the distribution of distances in the baseline trial from the habituation trial, we generate a delta distribution. This delta distribution reveals negative values near the transparent panel (indicating reduced proximity in the habituation trial) and positive values at mid- to fardistances (indicating increased distancing behaviour). This delta distribution is also what is reported in Figure 2. 

Dishabituation: In this trial, a new spider (different from the one in the habituation trial) is introduced. The dishabituation trial will be considered in contrast to the habituation trial described above. If the spider recognizes the new individual as different, it is expected to show increased exploratory behaviour and reduced distance, similar to the initial baseline trial.

By subtracting the distribution of distances in the habituation trial from the dishabituation trial, we obtain another delta distribution. This delta distribution should reveal positive values near the transparent panel (indicating increased proximity in the dishabituation trial) and negative values at mid- to far-distances (indicating decreased proximity compared to the habituation trial).

We hope this clarifies the rationale behind our data presentation and the methodological approach we employed. We have revised the figure to enhance its clarity and make it more intuitive for the reader.

Dahl, C. D., Logothetis, N. K., & Hoffman, K. L. (2007). Individuation and holistic processing of faces in rhesus monkeys. Proceedings of the Royal Society B: Biological Sciences, 274(1622), 2069-2076.

Humphrey, N. K. (1974). Species and individuals in the perceptual world of monkeys. Perception, 3(1), 105-114.

At the very least, data showing the distribution of distances from the wall would be much easier to interpret for the reader.

We understand the reviewer's concern that data showing the distribution of distances from the wall would be much easier to interpret for the reader. We initially consider that but came to the conclusion that this approach is not straightforward. For instance, if both spiders are positioned at the very front but in different corners, the distance to the panel would be very small, but the distance between the spiders would be large. Thus, using distances from the wall could misrepresent the actual spatial distribution between the spiders.

(2) "Long-term social memory": It is not entirely clear what is meant by the authors when they say 'long-term social memory', though typically long-term memory refers to a form of a memory that requires protein synthesis.  

To address this conceptually, we used the term "long-term social memory" to describe the spiders' ability to recognize and remember individual conspecifics over multiple experimental sessions. While social memory refers to the ability of an individual to recognize other individuals within a social context, long-term memory typically involves the retention of information over extended periods. Recognizing that the term “long-term social memory” is not commonly used, we have revised the manuscript to use the more standard term “long-term memory.”

While the precise timing of memory formation varies across species and contexts, a general rule is that long-term memory should last for > 24 hours (e.g., Dreier et al 2007 Biol Letters). The longest time that spiders are apart in this trial setup is something like an hour. There is no basis to claim that spiders have long-term social memory as they are never asked to remember anyone after a long time apart.

We appreciate the reviewer’s feedback regarding the term "long-term social memory." The statement "long-term memory should last for > 24 hours" is a generalisation in discussions about memory. It oversimplifies a more complex topic. That is, long-term memory is typically distinguished from short-term memory by its persistence over time, often lasting from hours to a lifetime. However, the exact duration that qualifies memory as "long-term" varies depending on the context, model species, and type of memory. In studies involved in synaptic plasticity (LTP), the object might indeed be to look at memory that persists for at least 24 hours as a criterion for long-term memory. In studies of cellular and/or molecular mechanisms where the stabilization and consolidation of memory traces over time are key areas of interest this 24-hour interval is very common. But, defining long-term memory strictly by a 24-hour duration is by no means universally accepted nor does it apply across all fields of study.

To clarify, long-term memory is a process involving consolidation starting within minutes to hours after learning. Clearly, full consolidation can take longer, while memory persisting 24 hours is considered fully consolidated. But this does not mean that memory lasting less than 24 hours are not part of long-term memory. 

In fact, Atkinson and Shiffrin (1969) proposed that information entering short-term memory remains there for about 20 to 30 seconds before being displaced due to space limitations. During this brief interval, initial encoding processes begin transferring information to long-term memory, establishing an initial memory trace. This transfer is not indicative of full consolidation but represents the initial "laying down" of the memory trace (encoding). In our study, the focal spider’s brain forms initial memory traces of the individuals it encounters. This process continues during the period of visual separation. Upon re-encountering the same individual a few minutes later, the spider accesses the initial memory trace stored in long-term memory. This trace is fragile and not fully consolidated. The re-encounter acts as a rehearsal, reactivating specific memory traces and potentially strengthening them through additional encoding processes, allowing the spider to recognize the individual even an hour later.

According to Markowitsch (2013), initial encoding in long-term memory begins within seconds to minutes. It is also important to note that we argue for identity recognition rather than identity recall. Recognition involves correctly identifying a stimulus when it is presented again, while recall requires the volitional generation of information without an external stimulus. Thus, recall may rely on deeper forms of memory consolidation than recognition.

Is protein synthesis required for long-term memory? 

The role of protein synthesis in long-term memory has been extensively studied. According to Castellucci et al. (1978), explicit memory comprises a short-term phase that does not require protein synthesis and a long-term phase that does. Hebbian learning in its initial phase (early LTP) does not necessarily require protein synthesis. This phase involves the rapid strengthening of synapses through existing proteins and signaling pathways, such as the activation of NMDA receptors and the influx of Ca2+ ions. For the changes to persist (late LTP), protein synthesis is important. This phase involves the production of new proteins that contribute to long-term structural changes at the synapse, such as the growth of new synaptic connections or the stabilization of existing ones.

This differentiation between the early and late phases of LTP highlights that long-term memory can begin forming without immediate protein synthesis. Our study focuses on this early phase of memory encoding, which involves the initial formation of memory traces that do not yet depend on protein synthesis. 

It is however worth noting that recent research suggests that there is an early phase of protein synthesis (within minutes to hours) through the activation of immediate early genes (IEGs) and transcription factors. In this context, protein synthesis supports initial synaptic modifications. What the reviewer refers to is the consolidation phase (late phase), where continued synthesis of proteins induces structural changes at synapses, leading to the formation of new synaptic connections. In our study, it is plausible to assume that an early form of protein synthesis may contribute to stabilizing the initial memory traces during the encoding phase. However, whether or not protein synthesis occurred in our spiders is beyond the scope of this investigation and was not specifically addressed.

The critical aspect of our study is that the information transitioned from short-term memory to long-term memory during an early encoding phase, allowing recall after an hour. Due to the inherent limitations and transient nature of the short-term memory, it is implausible for spiders to retain these memory representations solely within the short-term memory for such durations. Our findings suggest that the initial encoding processes were robust enough to transfer these experiences into long-term memory, where they were stabilized and could be accessed later. 

In sum, it is important to note that long-term memory is a dynamic process, and while testing after 24 hours is a convention in some studies, this timing is arbitrary and not universally applicable to all contexts or species. The more critical consideration here is that we are dealing with a species where no prior evidence of long-term memory exists. Debating a 24-hour delay or the specifics of protein synthesis, while potentially interesting for future studies, detracts from the true significance of our findings. Our study is the first to show something akin to long-term memory representations in this species and this should remain in our focus.

Shiffrin, R. M., & Atkinson, R. C. (1969). Storage and retrieval processes in long-term memory. Psychological review, 76(2), 179. 

Markowitsch, H. J. (2013). Memory and self–Neuroscientific landscapes. International Scholarly Research Notices, 2013(1), 176027.

Castellucci, V. F., Carew, T. J., & Kandel, E. R., 1978. Cellular analysis of long-term habituation of the gill-withdrawal reflex of Aplysia californica. Science, 202(4374), 1306-1308.

The odd phrasing of the 'long-term dishabutation' trial makes it seem that it is testing a longterm memory, but it is not. The spiders have never met. The fact that they are very habituated to one set of stimuli and then respond to a new stimulus is not evidence of long-term memory. To clearly test memory (which is the part really lacking from the design), the authors would need to show that spiders - upon the first instance of re-encountering a previously encountered individual are already 'habituated' to them but not to some other individuals. The current data suggest this may be the case, but it is just very hard to interpret given the design does not directly test the memory of individuals in a clear and unambiguous manner.

While we appreciate the reviewer's feedback, we believe there may have been some misunderstanding regarding the term “long-term dishabituation.” The introduction of novel individuals at the end of Session 3 was not intended to test long-term memory by having spiders recognize these novel individuals. Instead, it aimed to investigate the nature of the habituation observed over the three sessions.

The novel individuals introduced at the end of Session 3 serve the purpose to differentiate between general habituation (a decline in response due to repeated exposure to any stimuli) and specific habituation (recognition and reduced response to previously encountered individuals). The novel spiders have never been encountered before, so the focal spiders cannot have prior representations of them. Thus, the strong dishabituation response to these novel individuals indicates that the habituation observed earlier is not due to a general fatigue effect or loss of interest but rather a specific habituation effect to the familiar individuals. By showing such strong and increased response to novel individuals, the study demonstrates that the spiders' increasingly reduced responses in Sessions 2 and 3 are not merely due to a general decrease in responsiveness but suggest cognitive habituation. This cognitive habituation implies that the spiders remember the familiar individuals (as each of them occurred three times across the three sessions), a process that relies on long-term memory. Therefore, while the novel spiders themselves are not a direct test of long-term memory, the use of these novel spiders helps us infer that the habituation observed over the three sessions is indeed due to the formation of long-term memory traces.

In other words, the organism detects and processes the novel stimulus as different from the habituated one. In our study, if a spider showed a strong dishabituation response to a novel individual introduced at the end of Session 3, it would indicate that the spider had formed specific representations of the individuals they encountered during the three sessions. These representations allow the spiders to recognise the novel individuals as different, leading to renewed interest and a stronger behavioural response. It is the absence of a prior representation for the novel spiders that triggers this dishabituation response. Since the novel spider does not match any stored representations of the previously encountered spiders, the focal spider responds more strongly.

The introduction of novel individuals at the end of Session 3 helps clarify that the increasing habituation observed in Session 2 and 3 is specific to familiar individuals, indicating cognitive habituation. This supports the presence of long-term memory processes in the spiders, as they can distinguish between previously encountered individuals and new ones. The habituationdishabituation paradigm thus effectively demonstrates the spiders' ability to form and reactivate encoded memory traces, providing clear evidence of recognition memory. 

For these reasons, we are convinced that our interpretation is accurate and hope this clarification renders the additional request for an entirely new experiment unnecessary.

(3) Lack of a functional explanation and the emphasis on 'asociality': It is entirely plausible that recognition is a pleitropic byproduct of the overall visual cognition abilities in the spiders. 

We agree with the reviewer that it is essential to consider the broader context of individual recognition and its potential adaptive significance. The possibility that recognition in jumping spiders could be a pleiotropic byproduct of their advanced visual cognition abilities is indeed a plausible explanation and has been discussed in our manuscript.

However, the discussion that discounts territoriality as a potential explanation is not well laid out. First, many species that are 'asocial' nevertheless defend territories. It is perhaps best to say such species are not group living, but they have social lives because they encounter conspecifics and need to interact with them.

The reviewer also correctly points out that many 'asocial' species still defend territories and have social interactions. Our use of the term 'asocial' was meant to indicate that jumping spiders do not live in cohesive social groups, but we acknowledge that they do have social lives in terms of interactions with conspecifics. It is more accurate to describe these spiders as non-groupliving, yet socially interactive species. A better term is “non-social” to refer to the jumping spider as a species that do not live in stable social groups and do not exhibit associated behaviours, such as cooperative behaviours. This also would imply that individuals still interact with conspecifics, especially in contexts like mating, territorial disputes or aggression. We, thus, change the term from “asocial” to “non-social” in the manuscript.  

Indeed, there are many examples of solitary living species that show the dear enemy effect, a form of individual recognition, towards familiar territorial neighbors. The authors in this case note that territorial competition is mediated by the size or color of the chelicerae (seemingly a trait that could be used to distinguish among individuals). Apparently, because previous work has suggested that territorial disputes can be mediated by a trait in the absence of familiarity has led them to discount the possibility that keeping track of the local neighbors in a potentially cannibalistic species could be a sufficient functional reason. In any event, the current evidence presented certainly does not warrant discounting that hypothesis.

The “dear enemy effect”, where solitary living species recognize and show reduced aggression towards familiar territorial neighbors, is a relevant consideration. This effect demonstrates that individual recognition can have significant functional implications even in species that are not group-living. We will elaborate on this effect in the revised manuscript to provide a more comprehensive discussion.

The reviewer mentioned that territorial disputes can be mediated by the size or color of the chelicerae, potentially serving as a feature for individual recognition. Our intention was not to discount the role of such traits but to highlight that the level of identity recognition we observed represents subordinate classification. This is different from the basic-level classification, such as distinguishing between male and female based on chelicerae colour. While we acknowledge that colour can be an important feature for identity discrimination, our findings suggest that individual recognition in jumping spiders goes beyond simple colour differentiation. 

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors investigated whether a salticid spider, Phidippus regius, recognizes other individuals of the same species. The authors placed each spider inside a container from which it could see another spider for 7 minutes, before having its view of the other spider occluded by an opaque barrier for 3 minutes. The spider was then either presented with the same individual again (habituation trial) or a different individual (dishabituation trial). The authors recorded the distance between the two spiders during each trial. In habituation trials, the spiders were predicted to spend more time further away from each other and, in dishabituation trials, the spiders were predicted to spend more time closer to each other. The results followed these predictions, and the authors then considered whether the spiders in habituation trials were generally fatigued instead of being habituated to the appearance of the other spider, which may have explained why they spent less time near the other individual. The authors presented the spiders with a different (novel) individual after a longer period of time (which they considered to be a long-term dishabituation trial), and found that the spiders switched to spending more time closer to the other individual again during this trial. This suggested that the spiders had recognized and had habituated to the individual that they had seen before and that they became dishabituated when they encountered a different individual.

We appreciate the reviewer's detailed summary of our study. The reviewer's summary accurately captures the essence of our experimental design, predictions, and findings.

Strengths:

It is interesting to consider individual recognition by Phidippus regius. Other work on individual recognition by an invertebrate has been, for instance, known for a species of social wasp, but Phidippus regius is a different animal. Importantly and more specifically, P. regius is a salticid spider, and these spiders are known to have exceptional eyesight for animals of their size, potentially making them especially suitable for studies on individual recognition. In the current study, the results from experiments were consistent with the authors' predictions, suggesting that the spiders were recognizing each other by being habituated to individuals they had encountered before and by being dishabituated to individuals they had not encountered before. This is a good start in considering individual recognition by this species.

We appreciate the reviewer's positive summary and acknowledgment of the strengths of our study. We would like to point out some more details: 

While the exceptional eyesight of salticid spiders is indeed a significant factor, our study reaches deeper in terms of processing. We do not argue at the level of sensation rather than at the level of perception. Even more, identity recognition is a higher-level perceptual process. This distinction is crucial: we are not merely examining the spiders' sensory capabilities (such as good eye sight), but rather how their brains interpret and represent what they “see”. This involves a cognitive process where the sensory input (sensation) is processed and integrated into meaningful constructs (perception) and memorised in form of representations. 

Our study also suggests that P. regius engages in “higher-level” perceptual processes. This most-likely involves complex representations of individual conspecifics, which in mammalian brains are associated with regions such as the central inferior temporal (cIT) and anterior inferior temporal (aIT) areas. We provide evidence that these spiders do not just sense visual stimuli but interpret and recognize individual identities, indicating sophisticated perceptual and cognitive abilities. In other words, the spiders do not merely respond to visual stimuli in a reflexive manner, but rather engage in sophisticated perceptual and cognitive processes that allow them to recognize and distinguish between individual identities. This indicates that the spiders are not simple Braitenberg vehicles reacting to stimuli, but are thinking organisms capable of complex mental representations. This resonates with current trends in animal cognition research, which increasingly recognize some level of consciousness and advanced cognitive abilities across a wide range of animal species. Moreover, this aligns with the growing interest and recognition of spider cognition, where research begins to provide evidence for the cognitive complexity and perceptual capabilities of these often underestimated creatures (Jackson and Cross, 2011). 

Jackson, R. R., & Cross, F. R. (2011). Spider cognition. Advances in insect physiology, 41, 115174.

Weaknesses:

The experiments in this manuscript (habituation/dishabituation trials) are a good start for considering whether individuals of a salticid species recognize each other. I am left wondering, however, what features the spiders were specifically paying attention to when recognizing each other. The authors cited Sheehan and Tibbetts (2010) who stated that "Individual recognition requires individuals to uniquely identify their social partners based on phenotypic variation." Also, recognition was considered in a paper on another salticid by Tedore and Johnsen (2013).

Tedore, C., & Johnsen, S. (2013). Pheromones exert top-down effects on visual recognition in the jumping spider Lyssomanes viridis. The Journal of Experimental Biology, 216, 1744-1756. doi: 10.1242/jeb.071118 

In this elegant study, the authors presented spiders with manipulated images to find out what features matter to these spiders when recognizing individuals.

The reviewer raises an important point regarding the specific features that Phidippus regius might be paying attention to when recognizing individual conspecifics. Our study indeed cited Sheehan and Tibbetts (2010) to highlight the importance of phenotypic variation in individual recognition. Additionally, we referenced the work by Tedore and Johnsen (2013) on visual recognition in another salticid species, which suggests that multiple sensory modalities, including visual and pheromonal cues, may be involved in the recognition process. While our current study focused on demonstrating that Phidippus regius can recognize individual conspecifics, we acknowledge that it does not specifically identify the phenotypic features involved in this recognition. 

Part of the problem with using two living individuals in experiments is that the behavior of one individual can influence the behavior of the other, and this can bias the results.  

We appreciate the reviewer's observation regarding the potential bias introduced by using two living individuals in experiments, as the behaviour of one individual can indeed influence the behaviour of the other. We shared this concern initially; however, the consistency of the data with our hypotheses suggests that this potential bias did not adversely affect the validity of our findings, rendering the concern largely illusory at least in the context of our study.

We opted for the living-individual paradigm for the following reasons:

There is a growing trend in ethological as well as animal cognition research towards more ecologically valid and biologically relevant settings, while simultaneously advancing the precision and quantification of the data collected. This is referred to as computational ethology.

This approach advocates for assessing behaviour in environments that more closely resemble natural conditions, rather than relying solely on sterile and artificial experimental setups. The rationale is that such naturalistic arenas allow animals to exhibit a broader range of behaviours and interactions, providing a more accurate reflection of their cognitive and social abilities. The challenge, however, lies in navigating the inherent tradeoff between the strict control offered by standardized procedures and the ecological validity of more naturalistic interactions.

By allowing two spiders to confront each other, we aimed to capture authentic behavioural responses while maintaining a degree of experimental standardization through the use of a controlled setup. Our approach ensures that the behaviours observed are not merely artifacts of an artificial environment but are representative of genuine social interactions. Also, to minimize potential biases arising from mutual behavioural influences, we employed a controlled and repeatable experimental environment. 

We believe that the chosen approach provides a meaningful balance (in the above-mentioned trade-off) between ecological validity and experimental rigour. By combining a standardized environment with the naturalistic interaction of real spiders, we ensured that our findings are both scientifically robust and biologically relevant.

However, this issue can be readily avoided because salticids are well known, for example, to be highly responsive to lures (e.g. dead prey glued in lifelike posture onto cork disks) and to computer animation. 

While it is true that salticid spiders are responsive to lures and computer animations, we carefully considered the most appropriate and ecologically valid approach for our study. Our aim was to capture genuine behavioural patterns in a context that closely mimics the natural encounters these spiders experience.

Additionally, creating comparable video stimuli of spiders presents its own set of challenges: Video recordings or computer animations may not fully capture the nuanced behaviours and subtle variations that occur during real-life interactions. There is also a risk that such stimuli could be perceived differently by the spiders, potentially introducing new biases or confounding factors.

Scientific progress is not made by merely relying on previously established paradigms, especially when they may not be suitable for the specific context of a study. While alternative methods like lures or computer animations can be valuable in certain situations, our approach was deliberately chosen to best capture the naturalistic and interactive aspects of spider behaviour.

These methods have already been successful and helpful for standardizing the different stimuli presented during many different experiments for many different salticid spiders, and they would be helpful for better understanding how Phidippus regius might recognize another individual on the basis of phenotypic variation. There are all sorts of ways in which a salticid might recognize another individual. Differences in face or body structure, or body size, or all of these, might have an important role in recognition, but we won't know what these are using the current methods alone. Also, I didn't see any details about whether body size was standardized in the current manuscript.

As mentioned previously, the goal of our study was to demonstrate that identity recognition occurs in spiders. This alone is of significant importance, as it challenges existing assumptions about the cognitive capabilities of small-brained animals. We did not aim at providing a proximate explanation (mechanism) for identity recognition in spiders.

The problem with what the reviewer suggested is this: As long as we do not have conclusive evidence that spiders recognize individual conspecifics, any attempt to design and manipulate stimuli would lack a solid foundation. Without understanding whether spiders have this capability, we cannot make informed decisions about which features or characteristics to manipulate in stimuli. In other words, this uncertainty means we lack a starting point for our assumptions, making it nearly impossible to create stimuli that would be useful or relevant in testing identity recognition.

Additionally, it is nearly impossible to artificially generate a stimulus set that encompasses the natural variance in features that spiders use for visual individuation. There is no guarantee that artificial stimuli, such as lures or computer animations, would capture the relevant features that spiders use in natural interactions.

In other words, the question how Phidippus regius recognizes another individual will be subject of further investigation. In this study, we focus on whether or not they individuate others.  

For another perspective, my thoughts turn to a paper by Cross et al.

Cross, F. R., Jackson, R. R., & Taylor, L. A. (2020). Influence of seeing a red face during the male-male encounters of mosquito-specialist spiders. Learning & Behavior, 48, 104-112. doi: 10.3758/s13420-020-00411-y

These authors found that males of Evarcha culicivora, another salticid species that is known to have a red face, become less responsive to their own mirror images after having their faces painted with black eyeliner than if their faces remained red. In all instances, the spiders only saw their own mirror images and never another spider, and these results cannot be interpreted on the basis of habituation/dishabituation because the spiders were not responding differently when they simply saw their mirror image again. Instead, it was specifically the change to the spider's face which resulted in a change of behavior. The findings from this paper and from Tedore and Johnsen can help give us additional perspectives that the authors might like to consider. On the whole, I would like the authors to further consider the features that P. regius might use to discern and recognize another individual.

We acknowledge that identifying the specific features used by P. regius for identity recognition is a valuable direction for future research. However, we must emphasise that without first establishing whether spiders are capable of individuating each other, it would be premature and challenging to determine the specific features they rely on for this process. A lack of response to certain features could either suggest that those features are not relevant or, more critically, that the spider does not recognize individual identities at all. Thus, our initial focus on demonstrating identity recognition is essential before delving into the specific cues or characteristics involved.

While the call for addressing the proximate causation of identity recognition in jumping spiders is valid, we need to also reiterate the significance of our findings and why they stand on their own merit:

Our study demonstrates for the first time that Phidippus regius can systematically individuate conspecifics, showing habituation within short intervals (10 minutes) and over longer intervals (1 hour). This behaviour is not due to general habituation or physical fatigue but is a result of cognitive habituation, as illustrated by the spiders' response to novel individuals introduced after repeated encounters with familiarized ones. 

What are the implications of this? Our findings indicate that these spiders possess long-term memory and form representations that can be reactivated after an hour. While this is most-likely not fully consolidated memory formation (see our reply to Reviewer 1), it represents an encoded long-term memory. This implies that small-brained animals can remember, represent, and potentially build internal mental images, which are crucial for sophisticated cognitive processing. 

Reviewer #3 (Public Review):

Summary:

Jumping spiders (family Salticidae) have extraordinarily good eyesight, but little is known about how sensitive these small animals might be to the identity of other individuals that they see. Here, experiments were carried out using Phidippus regius, a salticid spider from North America. There were three steps in the experiments; first, a spider could see another spider; then its view of the other spider was blocked; and then either the same or a different individual spider came into view. Whether it was the same or a different individual that came into view in the third step had a significant effect on how close together or far apart the spiders positioned themselves. It has been demonstrated before that salticids can discriminate between familiar and unfamiliar individuals while relying on chemical cues, but this new research on P. regius provides the first experimental evidence that a spider can discriminate by sight between familiar and unfamiliar individuals.

Clark RJ, Jackson RR (1995) Araneophagic jumping spiders discriminate between the draglines of familiar and unfamiliar conspecifics. Ethology, Ecology and Evolution 7:185-190

We appreciate the reviewer's comprehensive summary and acknowledgment of the significance of our findings.

Strengths:

This work is a useful step toward a fuller understanding of the perceptual and cognitive capacities of spiders and other animals with small nervous systems. By providing experimental evidence for a conclusion that a spider can, by sight, discriminate between familiar and unfamiliar individuals, this research will be an important milestone. We can anticipate a substantial influence on future research.

We appreciate the reviewer’s recognition of the strengths and significance of our study. We are pleased that the reviewer considers our research an important milestone. Our findings indeed suggest that even animals with relatively simple nervous systems can perform complex cognitive tasks, which has substantial implications for the broader study of animal cognition.

As pointed out by the reviewer, we also hope that our study will have a substantial influence on future research. By establishing a methodology and providing clear evidence of visual discrimination, we aim to encourage further investigations into the cognitive abilities of jumping spiders and other arthropods. Future research can build on our findings to explore the specific visual cues and mechanisms involved in individual recognition (as Reviewer 2 pointed out), as well as the ecological and evolutionary implications of these abilities.

Weaknesses:

(1) The conclusions should be stated more carefully.

We agree that clarity in our conclusions is paramount. We will revise the manuscript to ensure that our conclusions are presented with precision and appropriately reflect the data. Specifically, we will emphasize the evidence supporting our findings of visual individual recognition and clarify the limitations and scope of our conclusions to avoid any potential overstatements.

(2) It is not clearly the case that the experimental methods are based on 'habituation (learning to ignore; learning not to respond). Saying 'habituation' seems to imply that certain distances are instances of responding and other distances are instances of not responding but, as a reasonable alternative, we might call distance in all instances a response. However, whether all distances are responses or not is a distracting issue because being based on habituation is not a necessity.

We appreciate the reviewer's feedback and understand the concern regarding the use of the term 'habituation.' We agree that all distances maintained by the spiders are active responses and reflect their behavioral decisions based on perception and recognition of the other individual. We recognize that all distances are responses and interpret these as the spiders’ “active decisions”, modulated by their recognition of the same or different individuals. 

The terms 'habituation' and 'dishabituation' are used to label trial types for ease of discussion and to describe the expected behavioural modulation.

(3) Besides data related to distances, other data might have been useful. For example, salticids are especially well known for the way they communicate using distinctive visual displays and, unlike distance, displaying is a discrete, unambiguous response.

We appreciate the reviewer’s suggestion to incorporate data on visual displays, which are indeed well-known communication methods among salticids. We agree that visual displays are discrete and unambiguous responses that could provide additional insights into the spiders' recognition abilities.

Our primary focus on distance measurements was driven by the need to quantify behaviour in a continuous and scalable manner, that is, how spiders modulate their proximity based on familiarity with other individuals.

We acknowledge the potential value of including visual display measurments; however, in our study, we aimed to establish a foundational understanding of recognition behaviour through proximity measures first. Also, capturing diplays requires a different experimental paradigm, where the displays are clearly visible and analyzable. 

(4) Methods more aligned with salticids having extraordinarily good eyesight would be useful. For example, with salticids, standardising and manipulating stimuli in experiments can be achieved by using mounts, video playback, and computer-generated animation.

There is no doubt that salticids have excellent eyesight. However, our study focuses on higherlevel perceptual processes that require complex brain analysis, not just visual acuity. The goal was to investigate whether spiders can individuate and recognize conspecifics, which involves interpreting visual information and forming long-term representations.

Clearly, methods like video playback and computer animations are useful in controlled settings, where the spider is mounted, but they pose challenges for our specific research question. At this stage of research, we lack precise knowledge of which visual features are critical for individual recognition in spiders, making it difficult to design effective artificial stimuli. 

Our primary objective was to determine if spiders can individuate others. Before exploring the proximate mechanisms of how they individuate others, it was essential to establish that they have this capability. This foundational question needed to be addressed before delving into more detailed mechanistic studies.

(5) An asocial-versus-social distinction is too imprecise, and it may have been emphasised too much. With P. regius, irrespective of whether we use the label asocial or social, the important question pertains to the frequency of encounters between the same individuals and the consequences of these encounters.

Our intent was to convey that P. regius does not live in cohesive social groups but does engage in individual interactions that can have significant behavioral consequences. We will revise the manuscript to reduce the emphasis on the asocial-versus-social distinction. As discussed above, we also will change the term “asocial” to “non-social” in the manuscript.

(6) Hypotheses related to not-so-strictly adaptive factors are discussed and these hypotheses are interesting, but these considerations are not necessarily incompatible with more strictly adaptive influences being relevant as well.

We appreciate the reviewer's observation regarding the discussion of hypotheses related to notso-strictly adaptive factors. We agree that our considerations of these factors do not preclude the relevance of more strictly adaptive influences.

We will revise the manuscript to explicitly discuss how our findings can be interpreted in the context of adaptive hypotheses. This will provide a more comprehensive understanding of the evolutionary significance of individual recognition in P. regius. Modifications were made in the Discussion section.

In the following, we comment on issues not mentioned in the “public reviews” section.

Reviewer #1 (Recommendations For The Authors):

(1) I would suggest conducting experiments that actually test for recognition memory, as this seems to be a claim that the authors make. Following the ant studies by Dreier cited in this manuscript would be sufficient to test for memory. Given the relative simplicity of the measures being taken (location of spiders), this would seem like a very simple addition that would provide a much stronger and more readily interpreted dataset.

As previously explained in our detailed responses (public reviews), we believe that the current design effectively addresses the questions at hand. Our approach, using a habituationdishabituation paradigm, provides robust evidence for recognition memory within the framework of early long-term memory.

Additionally, we have explained why using the distance to the panel as a measure is not appropriate in this context. Specifically, using such a measure can misrepresent the actual interests of the spiders in each other.

While we acknowledge the merits of the ant studies by Dreier, our current design allows for a detailed understanding of the spiders' recognition capabilities over short (10 min) and slightly longer intervals (up to one hour). This is sufficient to demonstrate the presence of recognition memory without the necessity of further experiments. The observed patterns of habituation and dishabituation responses in our study clearly indicate that the spiders can distinguish between familiar and novel individuals, which supports our claims.

Given these points, we respectfully maintain that the current data and experimental design are adequate to support our findings and provide a comprehensive understanding of recognition memory in Phidippus regius.

(2) The writing is rather impenetrable. The results explain the basic finding in terms of statistical variables rather than simply stating the results. A clear and straightforward statement such as 'the spiders showed reduced interest upon habituation trials, indicating xyz' (and then citing the stats) is preferable to the introduction of results as a statistical model. The statistical model is a means of assessing the results. It is not the result. Describe the data.

We tried to improve that in the current version.

(3) Showing more straightforward data such as distance from the joint barrier would make the paper much easier to understand.

This paper has been on bioRxiv for some time and my guess is that it has ended up here because it is having trouble in review. Collecting new data that more directly test the question at hand, presenting the data in a more direct manner, and more critically evaluating your own claims will improve the paper.

While it is true that the paper has been on bioRxiv for a while, this submission marks the first instance where it has undergone peer review. Prior to this, the manuscript was submitted to other journals but was not reviewed.

We hope the explanations provided in the “public reviews” section, along with the revised manuscript, sufficiently clarify our study and its conclusions. We believe the current data robustly address the research questions, and as outlined in our detailed responses, we have critically evaluated our claims and presented the data clearly. Given these clarifications, we do not see the necessity for new experiments as the existing data adequately support our findings. We trust that these revisions and explanations will clarify any misunderstandings.

I am totally sold that the spiders are paying attention to identity at some level. The key now is to understand what that actually means in terms of recognition (i.e. memory of individuals) not just habituation.

We appreciate the reviewer’s emphasis on the distinction between habituation and memorybased individual recognition. As detailed in the preceding discussion, we have taken great care to clarify how our paradigm distinguishes simple habituation effects from true memory for individual identity. We trust that the preceding sections make clear how our findings go beyond simple habituation to establish genuine individual recognition.

Reviewer #2 (Recommendations For The Authors):

Aside from the comments in the public review, I have some additional comments that the authors may wish to consider.

Numerous times in the manuscript, the authors mentioned that recognizing individuals requires recognition memory. This seems rather obvious, and I wonder if the authors could instead be more precise about what they mean by 'recognition memory'?

Recognition memory refers to the cognitive ability to identify a previously encountered stimulus, an individual, or events as familiar. It involves both encoding and retrieval processes, allowing an organism to distinguish between novel and familiar stimuli. This form of memory is a fundamental component of cognitive functioning and is supported by neural mechanisms that, in the mammal brain, involve the hippocampus and other brain regions associated with memory processing. 

In our study, we aimed to test whether Phidippus regius recognizes conspecifics, or, in other words, utilizes recognition memory to distinguish between familiar and unfamiliar conspecifics. With the habituation - dishabituation paradigm, we assessed the spiders' ability to recognize previously encountered individuals and demonstrate memory retention over short (10 min) and extended periods (1 hour).

Encoding: In the initial trial, when a spider encounters an individual for the first time (Figure 1A, “Baseline” or “Dishabituation” for every following trial), it encodes the visual information related to that specific individual. This encoding process involves creating a memory trace of the individual's phenotypic characteristics.

Storage: During the visual separation period, this encoded information is stored in the spider's memory system. The memory trace, though initially fragile, starts to stabilize over the separation period. Whether or not this leads to some form of consolidated memory remains unaddressed. This aspect was highlighted by the first reviewer, but our focus is on the early process rather than on late processes, such as consolidation. 

Retrieval: In the subsequent trial, when the same individual is presented again, the spider retrieves the stored memory trace. If the spider recognizes the individual, its behaviour reflects habituation, indicating memory retrieval. Conversely, when a novel individual is introduced, the lack of stored memory trace triggers a different behavioural response, indicating dishabituation. This differential response demonstrates the spider's ability to distinguish between familiar and unfamiliar individuals. This differential response is also key to understanding the nature of habituation over the three sessions, as introducing novel spiders leads to a significant dishabituation response after the three sessions in Experiment 2.

In Line 39, the authors state that they used "a naturalistic experimental procedure". I would like to know how this experiment is 'naturalistic'. The authors' use of an arena does not appear naturalistic, or something the spiders would encounter in the wild.

We appreciate the reviewer's comment regarding our use of the term 'naturalistic'. We acknowledge that the experimental arena itself does not replicate the conditions found in the wild. Our approach aimed to incorporate elements of natural behaviour by allowing two spiders to freely move and interact within the controlled environment. This approach aligns with principles from computational ethology, which seeks to balance the trade-off between repeatability/standardization and observing free, naturalistic behaviour. By using this paradigm, we aimed to capture behaviours that closely resemble those exhibited in their natural habitat. This setup was chosen to balance the need for ecological validity with the requirements for standardized data collection. 

Also, and this point has been raised above, by observing the spiders' natural interactions without restraining them or using artificial stimuli like computer animations, we aimed to capture behaviours that closely resemble their natural responses to conspecifics. In contrast, we would not have any clear expectations regarding responses to arbitrarily designed artificial stimuli. This method provides a more ecologically valid assessment of the spiders' recognition abilities.

There are a few details wrong in Line 41. 'Salticidae' is a family name and shouldn't be italicized. Also, the sentence suggests that there is a spider called a 'jumping spider' in the family Salticidae, which is technically called Phidippus regius. To clarify, all spiders in the family Salticidae are known as jumping spiders, and one species of jumping spiders is called Phidippus regius.

We will correct this in the manuscript to accurately reflect the classification and terminology. Thank you for pointing out these inaccuracies.

A manuscript on individual recognition by a salticid should include citations to earlier papers that have already considered individual recognition by salticids. As well as the paper by Tedore and Johnsen (2013), the authors should be aware of the following papers.

Clark, R. J., & Jackson, R. R. (1994). Portia labiata, a cannibalistic jumping spider, discriminates between its own and foreign egg sacs. International Journal of Comparative Psychology, 7, 3843.

Clark, R. J., & Jackson, R. R. (1994). Self-recognition in a jumping spider: Portia labiata females discriminate between their own draglines and those of conspecifics. Ethology, Ecology & Evolution, 6, 371-375.

Clark, R. J., & Jackson, R. R. (1995). Araneophagic jumping spiders discriminate between the draglines of familiar and unfamiliar conspecifics. Ethology, Ecology & Evolution, 7, 185-190.

We appreciate the reviewer's suggestion to include citations to these earlier papers. We will add the recommended references to provide a comprehensive background.

In Line 203, I would not consider "interaction with human caretakers and experimenters" to be a form of behavioral enrichment. This kind of interaction has the potential to be stressful for the spiders, rather than enriching. I suggest deleting that part of the sentence.

We appreciate the reviewer's feedback and agree that interactions with human caretakers and experimenters might not always be enriching and could potentially be stressful for the spiders. We will remove that part of the sentence to better reflect the intended meaning.

Reviewer #3 (Recommendations For The Authors):

This manuscript is useful and interesting, and I predict that it will be influential, but more attention should be given to stating the objective and conclusion accurately and clearly. As I understand it, the objective was to investigate a specific hypothesis: that Phidippus regius has a capacity to identify conspecific individuals as particular individuals (i.e., individual identification). Strong evidence supporting this hypothesis being true would be especially remarkable because I am unaware of any published work having shown evidence of a spider expressing this specific perceptual capacity.

Thank you for recognizing the significance and potential influence of our manuscript. We agree that clearly stating the objective and conclusions is essential for conveying the importance of our findings. Our results provide robust evidence supporting the hypothesis that Phidippus regius can recognize and remember individual conspecifics. We will revise the manuscript to more clearly highlight the objective and our conclusions, emphasizing the novel evidence for individual identification in these spiders.

Based on reading this manuscript and based on my understanding of the meaning of 'individual identification', it seems to me that the hypothesis that P. regius has a capacity for individual identification might or might not be true, and the experiments in this manuscript cannot tell us which is the case. 

We respectfully disagree with the reviewer's assessment. Our experiments were carefully designed to test whether P. regius has the capacity for individual identification, and our results provide clear evidence supporting this hypothesis. The systematic differences in the spiders' behaviour when encountering familiar versus novel individuals indicate that they can recognize and remember specific conspecifics. We will revise the manuscript to ensure that the evidence and conclusions are stated more clearly to address any potential misunderstandings.

Determining which is the case would have required research that made better use of the literature, and displayed more critical thinking. addressed credible alternative hypotheses and adopted experimental methods that focused more strictly on individual identification. 

The distinction between whether P. regius has a capacity for individual identification is not ambiguous in our study. Our findings clearly demonstrate this capacity through systematic behavioural responses to familiar versus novel individuals. As pointed out above, the experimental procedure might be complex, but results are systematic despite this complexity. The experiments were designed to directly address the hypothesis of individual identification, and the data robustly support our conclusions. While considering alternative hypotheses is important, the results we present provide a coherent and compelling case for individual identification in P. regius. We will ensure our manuscript clearly articulates this narrative and the supporting evidence.

At the same time, I also appreciate that asking for all of that at once would be asking for too much. As I see it, this manuscript tells us about research that moves us closer to a clear focus on the details and questions that will matter in the context of considering a hypothesis that is strictly about individual identification. More importantly, I think this research reveals a perceptual capacity that is remarkable even if it is not strictly a capacity for individual identification.

We understand the desire for a more focused exploration of individual identification with paradigms more familiar to the reviewers and we acknowledge that further detailed studies could enhance our understanding of this capacity. However, our findings do indeed suggest that Phidippus regius exhibits a remarkable perceptual capacity for recognizing and remembering individual conspecifics. The systematic behavioural responses observed in our experiments strongly indicate that these spiders possess the ability for individual recognition. While our study may not have explored every potential detail (e.g. which features are most crucial for the memory matching processes), the evidence we present robustly supports the conclusion of individual identification.

We acknowledge that it is indeed valuable to follow established paradigms and build upon the frameworks that have been used successfully in similar species and studies. These paradigms provide a solid foundation for scientific inquiry and allow for comparability across different research efforts. However, it is equally important to acknowledge and explore alternative approaches. Scientific progress is driven not only by replication but also by innovation. By employing new paradigms, researchers can uncover novel insights and push the boundaries of current understanding. The paradigm we used in our study, while different from those traditionally applied to similar research, is not an invention but a well-established method in various domains. It represents an innovative application in the context of our specific research questions, offering a fresh perspective and contributing to the advancement of the field.

As I understand it, 'individual identification' means identifying another individual as being a particular individual instead of a member of a larger set (or 'class') of individuals. An 'individual' is a set containing a single individual. Interesting examples of identifying members of larger sets include discriminating between familiar and unfamiliar individuals. In the context of the specific experiments in this manuscript, familiar-unfamiliar discrimination means discriminating between recently-seen and not-so-recently-seen individuals. My impression is that the experiments in this manuscript have given us a basis for concluding that P. regius has a capacity for familiarunfamiliar (recently seen versus not so recently seen) discrimination. If this is the case, then I think this is the conclusion that should be emphasised. This would be an important conclusion.

I appreciate that, depending on how we use the words, familiar-unfamiliar discrimination might be construed as being 'individual identification'. An individual is identified as 'the individual recently seen'. As a casual way of speaking, it can be reasonable to call this 'individual identification'. The difficulty comes from the way calling this 'individual identification' can suggest something more than has been demonstrated. To navigate through this difficulty, we need an expression to use for a capacity that goes beyond familiar-unfamiliar discrimination. In the context of this manuscript about P. regius, we need expressions that will make it easy to consider two things. One of these things is a capacity for familiar-unfamiliar discrimination. The other is the capacity to identify another individual as being a particular individual.

We appreciate the reviewer's insightful comments on the distinction between familiar-unfamiliar discrimination and individual identity recognition. Our study indeed focuses on demonstrating that Phidippus regius can recognize and remember individual conspecifics, providing evidence for individual identity recognition.

Two specific behavioural hallmarks that speak against familiarity recognition:

First, the significant dishabituation response to novel individuals introduced after multiple sessions underscores the specificity of the recognition. This shows that the spiders' habituation is not general but specific to familiar individuals. 

Second, the pattern of habituation over the sessions provides further evidence: We observed the strongest systematic modulation in Session 1, a reduced modulation in Session 2, and a further diminished effect in Session 3. If the spiders were only responding based on familiarity, we would expect a more drastic decrease, resulting in a washed-out non-effect by Session 2. However, the continued, though diminishing, differentiation between habituation and dishabituation trials across sessions indicates that the spiders are not merely responding to a general sense of familiarity but are engaging in individual recognition. In other words, the spiders' ability to distinguish between familiar and novel individuals even after repeated exposures suggests that they are not just recognizing a familiar status but are identifying specific individuals.

Things people do might help clarify what this means. People have an extraordinary capacity for identifying other individuals as particular individuals. Often this is based on giving each other names. Imagine we are letting somebody see photographs and asking them to identify who they see. The answer might be, 'somebody familiar' or 'somebody I saw recently' (familiar-unfamiliar discrimination); or the question might be answered by naming a particular individual (individual identification).

We appreciate the reviewer's efforts to clarify the distinction between familiar-unfamiliar discrimination and individual recognition using human examples. However, we believe this comparison might not fully capture the complexity of individual recognition in non-human animals. 

Familiarity recognition refers to recognizing someone as having been seen or encountered before without necessarily distinguishing them from others in the same category. On the other hand, identity recognition involves recognizing a specific individual based on unique characteristics (or features). In humans, this often involves naming, but more critically, like in most animals, it involves recognizing visual, auditory, chemical or other sensory cues. In animals, including spiders, individual recognition does not involve and let alone rely on naming but on the ability to distinguish between individuals based on sensory cues and learnt associations. This is a valid and well-documented form of individual recognition across many species.

Individual recognition does not require naming or the assignment of a referential label. Animals can distinguish between specific individuals based on previously perceived and stored features and characteristics. Naming is the exception rather than the rule in the animal kingdom. Only a few species, such as humans and maybe certain cetaceans, use naming for identity recognition. This is an evolutionary rarity and not the standard mechanism for individual recognition, which primarily relies on sensory cues and learnt associations. Furthermore, the mechanism of recognition in both humans and animals involves a complex process of matching incoming sensory and perceptual information with stored memory representations. Naming is merely a tool for communication, allowing us to convey which individual we are referring to. It is not the mechanism by which recognition occurs. The core of individual recognition is this matching process, where sensory cues (visual, auditory, chemical, etc.) are compared to memory traces of previously encountered individuals. Therefore, the suggestion that individual identification necessitates naming misrepresents the actual cognitive processes involved. 

We can think of individual identification being based on more fine-grained discrimination (with this, set size = one), with familiar-unfamiliar discrimination being more coarse-grained discrimination (with this, set size can be more than one). Restricting the expression 'individual identification' to instances of having the capacity to identify another individual as being a particular individual (set size = one) is better aligned with normal usage of this expression.

Absolutely, the distinction between fine-grained and coarse-grained discrimination aligns with the concept of different category levels, such as basic and subordinate levels, put forward by Eleanor Rosch (e.g. Rosch, 1973). In the context of individual recognition, fine-grained discrimination (where set size = one) refers to the ability to identify a specific individual based on unique characteristics. This is referred to as subordinate level categorization. Coarse-grained discrimination (where set size can be more than one) refers to recognizing someone as familiar without distinguishing them from others in the same category, more similar to basic level categorization. 

Rosch, E.H. (1973). "Natural categories". Cognitive Psychology. 4 (3): 328–50.doi:10.1016/0010-0285(73)90017-0

There is a strong emphasis on an asocial-social distinction in this manuscript. It seems to me that this needs to be focused more clearly on the specific factors that would make a capacity for individual identification beneficial. In the context of this manuscript, the term 'social' may suggest too much. It seems to me that the issue that matters the most is whether individuals live in situations where important encounters occur frequently between the same individuals. Irrespective of whether other notions of the meaning of 'social' also apply, there are salticids that live in aggregated situations where they frequently have important encounters with each other. This is the case with Phidippus regius in the field in Florida, but I realize that there may not be much published information about the natural history of this salticid. Even so, there are salticids to which the word 'social' has been applied in published literature.

We appreciate the reviewer's comments on the asocial-social distinction and we agree that this terminology might need refinement. Our intent was not to categorize Phidippus regius rigidly but to explore the contextual factors influencing the benefits of individual identification. The critical factor in our study is indeed the frequency and importance of encounters between individuals, rather than a broader social structure. We will revise the manuscript to reflect this more nuanced perspective, focusing on the ecological validity of our experimental design and the adaptive significance of individual recognition in environments where repeated encounters can occur.

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Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review): 

Summary: 

This paper sets out to achieve a deeper understanding of the effects of hydrogen sulfide on C. elegans behavior and physiology, with a focus on behavior, detection mechanism(s), physiological responses, and detoxification mechanisms.

Strengths: 

The paper takes full advantage of the experimental tractability of C. elegans, with thorough, welldesigned genetic analyses. 

Some evidence suggests that H<SUB>2</SUB>S may be directly detected by the ASJ sensory neurons.  The paper provides interesting and convincing evidence for complex interactions between responses to different gaseous stimuli, particularly an antagonistic role between H<SUB>2</SUB>S and O2 detection/response.  Intriguing roles for mitochondria and iron homeostasis are identified, opening the door to future studies to better understand the roles of these components and processes. 

We thank the reviewer for the supportive comments.

Weaknesses: 

The claim that worms' behavioral responses to H<SUB>2</SUB>S are mediated by direct detection is incompletely supported. While a role for the chemosensory neuron ASJ is implicated, it remains unclear whether this reflects direct detection. Other possibilities, including indirect effects of ASJ and the guanylyl cyclase daf-11 on O2 responses, are also consistent with the authors' data. 

We thank the reviewer for the insightful comment and agree that the role of ASJ neurons in H<SUB>2</SUB>S detection was not clear. We included new experiments and revised our text to make it clearer.

Since our initial analyses suggest a role of ASJ neurons in H<SUB>2</SUB>S-evoked locomotory responses (Figure 2F and G), We thought that this would offer us a starting point to dissect the neuronal circuit involved in H<SUB>2</SUB>S responses. Expression of the tetanus toxin catalytic domain in ASJ, which blocks neurosecretion, inhibited H<SUB>2</SUB>S-evoked locomotory speed responses (Figure 2H), suggesting that neurosecretion from ASJ promotes H<SUB>2</SUB>S-evocked response (Lines 162–165). We then performed calcium imaging of ASJ neurons in response to H<SUB>2</SUB>S exposure. However, while we observed CO<SUB>2</SUB>-evoked calcium transients in ASJ using GCaMP6s, we did not detect any calcium response to H<SUB>2</SUB>S, under several conditions, including animals on food, off food, and with different H<SUB>2</SUB>S concentrations and exposure times (Figure2—Figure supplement 2E and F) (Lines 166–170). Since signaling from ASJ neurons regulates developmental programs that modify sensory functions in C. elegans (Murakami et al., 2001), the involvement of ASJ neurons is not specific to H<SUB>2</SUB>S and ASJ neurons are unlikely to serve as the primary H<SUB>2</SUB>S sensor (Discussed in Line 449–458). Therefore, the exact sensory neuron, circuit and molecular triggers mediating acute H<SUB>2</SUB>S avoidance remain to be elucidated.

Our subsequent investigation on mitochondrial components suggests that a burst of mitochondrial ROS production may be the trigger for H<SUB>2</SUB>S avoidance, as transient exposure to rotenone substantially increases baseline locomotory speed (Figure 7E) (Line 391–396). However, to initiate avoidance behavior to H<SUB>2</SUB>S, mitochondrial ROS could potentially target multiple neurons and cellular machineries, making it challenging to pinpoint specific sites of action. Nevertheless, we agree that further dissection of the neural circuits and mitochondrial signaling in H<SUB>2</SUB>S avoidance will be important and should be explored in future studies.

The role of H<SUB>2</SUB>S-mediated damage in behavioral responses, particularly when detoxification pathways are disrupted, remains unclear. 

We thank the reviewer for the insightful comment and fully agree with the concern raised. The same issue was also noted by the other reviewers. We agree that decreased locomotory responses in H<SUB>2</SUB>S-sensitized animals can arise from distinct causes, either systemic toxicity or behavioral adaptation, and distinguishing between these is critical. We have included new experiments and revised the text to clarify this issue.

Our data suggest that increased initial omega turns and a rapid loss of locomotion in hif-1 and detoxification-defective mutants including sqrd-1 and ethe-1 likely reflect an enhanced sensitivity to H<SUB>2</SUB>S toxicity due to their failure to induce appropriate adaptative responses (Figure 5D–F, Figure 5J–L, Figure 5—Figure supplement 1F–P).  Supporting this, hif-1 mutants become less responsive to unrelated stimuli (near-UV light) after 30 minutes of H<SUB>2</SUB>S exposure (Figure 5I).

In contrast, egl-9 and SOD-deficient animals show reduced initial omega-turn and reduced speed responses (Figure 5B, Figure 7G, Figure 5—Figure supplement 1A and B, and Figure 7—Figure supplement 1F and G), although both egl-9 and sod mutants respond normally to the other stimuli prior or after H<SUB>2</SUB>S exposure (Figure 5I, Figure 5—Figure supplement 1C, and Figure 7—Figure supplement 1H). Since disrupting egl-9 stabilizes HIF-1 and upregulates the expression of numerous genes involved in cellular defense against H<SUB>2</SUB>S toxicity, the enhanced detoxification capacity in egl-9 mutants likely increases animals’ tolerance to H<SUB>2</SUB>S, thereby reducing avoidance to otherwise toxic H<SUB>2</SUB>S levels. Similarly, persistently high ROS in SOD deficient animals activates a variety of stress-responsive signaling pathways, including HIF-1, NRF2/SKN-1 and DAF-16/ FOXO signaling (Lennicke & Cocheme, 2021; Patten et al., 2010), facilitating cellular adaptation to redox stress and reducing animals’ responsiveness to toxic H<SUB>2</SUB>S levels. Taken together, these findings support the view that reduced locomotory speed during H<SUB>2</SUB>S exposure can arise from distinct mechanisms: early systemic toxicity in hif-1 and detoxificationdefective mutants, versus enhanced cellular adaptation in egl-9 and SOD mutants. We have integrated the relevant information across the result section and discussed this in Lines 485-536. 

The findings of the paper are somewhat disjointed, such that a clear picture of the relationships between H<SUB>2</SUB>S detection, detoxification mechanisms, mitochondria, and iron does not emerge from these studies. Most importantly, the relative roles of H<SUB>2</SUB>S detection and integration, vs. general and acute mitochondrial crisis, in generating behavioral responses are not convincingly resolved.  

We thank the reviewer for this comment and agree that our presentation did not fully connect different findings into a cohesive picture. To address this, we have acquired new data, and revised the abstract, results and discussion sections to clarify two phases of H<SUB>2</SUB>S-evoked responses: an initial avoidance behavior upon H<SUB>2</SUB>S exposure, followed by a later phase of adaption and detoxification when the escape is not successful.

In brief, we began with the basic characterization of H<SUB>2</SUB>S-induced locomotory speed response, followed by a candidate gene screen to identify key molecules and pathways involved in initial speed response to H<SUB>2</SUB>S. Subsequently, we focused on three major intersecting pathways that contributed to the acute behavioral response to H<SUB>2</SUB>S. These include cGMP signaling, which led to the identification of ASJ neurons; nutrient-sensitive pathways that modulate behavioral responses to both H<SUB>2</SUB>S and CO2; and O2sensing signaling, whose activation inhibits responses to H<SUB>2</SUB>S. However, the molecules and neurons in these pathways, including ASJ, likely play modulatory roles and are unlikely to serve as the primary H<SUB>2</SUB>S sensors. Our subsequent analysis, however, suggests that mitochondria play a critical role in triggering avoidance behavior upon H<SUB>2</SUB>S exposure. Brief treatment with rotenone, a potent inducer of ROS, led to marked increase in locomotory speed (Figure 7E). This suggests the possibility that a burst of ROS production triggered toxic levels of H<SUB>2</SUB>S (Jia et al., 2020) may initiate the avoidance behavior.

When the initial avoidance fails, H<SUB>2</SUB>S detoxification programs are induced as a long-term survival strategy. The induction of detoxification programs appears to enhance tolerance to H<SUB>2</SUB>S exposure and contributes to the gradual decrease of locomotory speed in H<SUB>2</SUB>S. We now provide a clearer image of how different pathways modulate H<SUB>2</SUB>S detoxification and adaptation (see our responses to other comments). Briefly, mutants defective in detoxification, such as hif-1 and other detoxification-defective mutants, showed stronger initial omega-turn response and a rapid loss of locomotion. This loss of locomotion is likely caused by early cellular toxicity as the mutants failed to respond to other unrelated stimuli (nearUV light) after 30 minutes of H<SUB>2</SUB>S exposure (Figure 5I). Likewise, smf-3 mutants and BP-treated animals were hypersensitive to H<SUB>2</SUB>S (Figure 6D and E, and Figure 6—Figure supplement 1G and I), likely due to impaired H<SUB>2</SUB>S detoxification under low iron conditions, as iron is a co-factor required for the activity of the H<SUB>2</SUB>S detoxification enzyme ETHE-1 (Figure 5K and Figure 5—Figure supplement 1E).

In contrast, reduced locomotion and response in other contexts such as egl-9 mutants and SODdeficient animals reflect H<SUB>2</SUB>S-induced adaptive mechanism rather than toxicity as they remain responsive to the other stimuli after H<SUB>2</SUB>S exposure. Since disrupting egl-9 stabilizes HIF-1 and upregulates the expression of numerous genes involved in cellular defense against H<SUB>2</SUB>S toxicity, the enhanced detoxification capacity in egl-9 mutants likely increases animals’ tolerance to H<SUB>2</SUB>S, thereby reducing avoidance to otherwise toxic H<SUB>2</SUB>S levels. Similarly, persistently high ROS in SOD deficient animals activates a variety of stress-responsive signaling pathways, including HIF-1, NRF2/SKN-1 and DAF-16/ FOXO signaling (Lennicke & Cocheme, 2021; Patten et al., 2010), facilitating cellular adaptation to redox stress and reducing animals’ responsiveness to toxic H<SUB>2</SUB>S levels. Therefore, different animals decline their locomotory speed to the effects of H<SUB>2</SUB>S through distinct mechanisms. We have integrated the relevant information across the result section and discussed this in Lines 485-536.

Reviewer #2 (Public Review): 

Summary: 

H<SUB>2</SUB>S is a gas that is toxic to many animals and causes avoidance in animals such as C. elegans. The authors show that H<SUB>2</SUB>S increases the frequency of turning and the speed of locomotion. The response was shown to be modulated by a number of neurons and signaling pathways as well as by ambient oxygen concentrations. The long-term adaptation involved gene expression changes that may be related to iron homeostasis as well as the homeostasis of mitochondria. 

Strengths: 

Overall, the authors provide many pieces that will be important for solving how H<SUB>2</SUB>S signals through neuronal circuits to change gene expression and physiological programs. The experiments rely mostly on a behavioral assay that measures the increase of locomotion speed upon exposure to H<SUB>2</SUB>S. This assay is then combined with manipulations of environmental factors, different wild-type strains, and mutants. The mutants analyzed were obtained as candidates from the literature and from transcriptional profiling that the authors carried out in worms that were exposed to H<SUB>2</SUB>S. These studies imply several genetic signaling pathways, some neurons, and metabolism-related factors in the response to H<SUB>2</SUB>S. Hence the data provided should be useful for the field.  

We thank the reviewer for the supportive comments.

Weaknesses: 

On the other hand, many important aspects of the underlying mechanisms remain unsolved and the reader is left with many loose ends. For example, it is not clear how H<SUB>2</SUB>S is actually sensed, how sensory neurons are activated and signal to downstream circuits, and what the role of ciliated and RMG neurons is in this circuit. It remains unclear how signals lead to gene expression and physiological changes such as metabolic rewiring. Solving all this would clearly be beyond the scope of a single manuscript. Yet, the manuscript also does not focus on understanding one of these central aspects and rather is all over the place, which makes it harder to understand for readouts that are not in this core field. Multiple additional methods and approaches exist to dig deeper into these mechanisms in the future, such as neuronal calcium imaging, optogenetics, and metabolic analysis. To generate a story that will be interesting to a broad readership substantial additional experimentation would be required. Further, in the current manuscript, it is often difficult to understand the rationales of the experiments, why they were carried out, and how to place them into a context. This could be improved in terms of documentation, narration/explanation, and visualization.  

We thank the reviewer for the comment, which has also been raised by the other reviewers. We agree that our initial submission was poorly presented. We also acknowledge the fact that some aspects, such as detailed neural circuit and sensory transduction, still remain unresolved. We have now included additional experiments and revised the manuscript to clarify the logic of our experiments, provided better context for our findings, and improved both the narrative flow and data visualization to make the manuscript more accessible to readers. We now provide a clearer image of how different pathways interact to modulate the initial avoidance response, and the H<SUB>2</SUB>S detoxification and behavioral habituation during prolonged H<SUB>2</SUB>S exposure. The following response is similar to the one for reviewer #1.

In brief, we began with the basic characterization of H<SUB>2</SUB>S-induced locomotory speed response, followed by a candidate gene screen to identify key molecules and pathways involved in initial speed response to H<SUB>2</SUB>S. Subsequently, we focused on three major intersecting pathways that contributed to the acute behavioral response to H<SUB>2</SUB>S. These include cGMP signaling, which led to the identification of ASJ neurons; nutrient-sensitive pathways that modulate behavioral responses to both H<SUB>2</SUB>S and CO2; and O2sensing signaling, whose activation inhibits responses to H<SUB>2</SUB>S. However, the molecules and neurons in these pathways, including ASJ, likely play modulatory roles and are unlikely to serve as the primary H<SUB>2</SUB>S sensors. Our subsequent analysis, however, suggests that mitochondria play a critical role in triggering avoidance behavior upon H<SUB>2</SUB>S exposure. Brief treatment with rotenone, a potent inducer of ROS, led to marked increase in locomotory speed (Figure 7E). This suggests the possibility that a burst of ROS production triggered toxic levels of H<SUB>2</SUB>S (Jia et al., 2020) may initiate the avoidance behavior.

When the initial avoidance fails, H<SUB>2</SUB>S detoxification programs are induced as a long-term survival strategy. The induction of detoxification programs appears to enhance tolerance to H<SUB>2</SUB>S exposure and contributes to the gradual decrease of locomotory speed in H<SUB>2</SUB>S. We now provide a clearer image of how different pathways modulate H<SUB>2</SUB>S detoxification and adaptation (see our responses to other comments). Briefly, mutants defective in detoxification, such as hif-1 and other detoxification-defective mutants, showed stronger initial omega-turn response and a rapid loss of locomotion. This loss of locomotion is likely caused by early cellular toxicity as the mutants failed to respond to other unrelated stimuli (nearUV light) after 30 minutes of H<SUB>2</SUB>S exposure (Figure 5I). Likewise, smf-3 mutants and BP-treated animals were hypersensitive to H<SUB>2</SUB>S (Figure 6D and E, and Figure 6—Figure supplement 1G and I), likely due to impaired H<SUB>2</SUB>S detoxification under low iron conditions, as iron is a co-factor required for the activity of the H<SUB>2</SUB>S detoxification enzyme ETHE-1 (Figure 5K and Figure 5—Figure supplement 1E).

In contrast, reduced locomotion and response in other contexts such as egl-9 mutants and SODdeficient animals reflect H<SUB>2</SUB>S-induced adaptive mechanism rather than toxicity as they remain responsive to the other stimuli after H<SUB>2</SUB>S exposure. Since disrupting egl-9 stabilizes HIF-1 and upregulates the expression of numerous genes involved in cellular defense against H<SUB>2</SUB>S toxicity, the enhanced detoxification capacity in egl-9 mutants likely increases animals’ tolerance to H<SUB>2</SUB>S, thereby reducing avoidance to otherwise toxic H<SUB>2</SUB>S levels. Similarly, persistently high ROS in SOD deficient animals activates a variety of stress-responsive signaling pathways, including HIF-1, NRF2/SKN-1 and DAF-16/ FOXO signaling (Lennicke & Cocheme, 2021; Patten et al., 2010), facilitating cellular adaptation to redox stress and reducing animals’ responsiveness to toxic H<SUB>2</SUB>S levels. Therefore, different animals decline their locomotory speed to the effects of H<SUB>2</SUB>S through distinct mechanisms. We have integrated the relevant information across the result section and discussed this in Lines 485-536.

Reviewer #3 (Public Review): 

Summary: 

The manuscript explores the behavioral responses of C. elegans to hydrogen sulfide, which is known to exert remarkable effects on animal physiology in a range of contexts. The possibility of genetic and precise neuronal dissection of responses to H<SUB>2</SUB>S motivates the study of responses in C. elegans. The manuscript is well-written in communicating the complex physiology around C. elegans behavioral responses to H<SUB>2</SUB>S and in appropriately citing prior and related relevant work. 

There are three parts to the manuscript.

In the first, an immediate behavioral response-increased locomotory rate-upon exposure to H<SUB>2</SUB>S is characterized. The experimental conditions are critical, and data are obtained from exposure of animals to 150ppm H<SUB>2</SUB>S at 7% O2. The authors provide evidence that this is a chemosensory response to H<SUB>2</SUB>S, showing a requirement for genes encoding components of the cilia apparatus and implicating a role for tax-4 and daf-11. Neuron-specific rescue in the ASJ neurons suggests the ASJ neurons contribute to the response to H<SUB>2</SUB>S. One caveat is that previous work has shown that the dauer-constitutive phenotype of daf-11 mutants can be suppressed by ASJ ablation, suggesting that there may be pervasive changes in animal nervous system signaling that are ASJ-dependent in daf-11 mutants, which may indirectly alter chemosensory responses to H<SUB>2</SUB>S. More direct methods to assess whether ASJ senses H<SUB>2</SUB>S, e.g. using calcium imaging, would better assess a direct role for the ASJ neurons in a behavioral response to H<SUB>2</SUB>S. The authors also point out interesting parallels between the response to H<SUB>2</SUB>S and CO2 though provide some genetic data separating the two responses. Importantly, the authors note that when aerotaxis (O2sensing and movement) in the presence of bacterial food is intact, as in npr-1 215F animals, the response to H<SUB>2</SUB>S is abrogated. Mutation in gcy-35 in the npr-1 215F background restores the H<SUB>2</SUB>S chemosensory response. 

There is a second part of the paper that conducts transcriptional profiling of the response to H<SUB>2</SUB>S that corroborates and extends prior work in this area. 

The final part of the paper is the most intriguing, but for me, also the most problematic. The authors examine how H<SUB>2</SUB>S-evoked locomotory behavioral responses are affected in mutants defective in the stress and detoxification response to H<SUB>2</SUB>S, most notably hif-1. Prior genetic studies have established the pathways leading to HIF-1 activation/stabilization, as well as potential downstream mechanisms. The authors conduct logical genetic analysis to complement studies of the hif-1 mutant and in part motivated by their transcriptional profiling studies, examine the role of iron sequestration/free iron in the locomotory response to H<SUB>2</SUB>S, and further speculate on how the behavior of mutants defective in mitochondrial function might be affected by exposure to H<SUB>2</SUB>S. 

In some regard, this part of the manuscript is interesting because the analysis begins to connect how the behavior of an animal to a toxic compound is affected by mutations that affect sensitivity to the toxic compound. However, what is unclear is what is being studied at this point. In the context, of noting that H<SUB>2</SUB>S at 150ppm is known to be lethal, its addition to mutants clearly sensitized to its effects would be anticipated to have pervasive effects on animal physiology and nervous system function. The authors note that the continued increased locomotion of wild-type animals upon H<SUB>2</SUB>S exposure might be due to the byproducts of detoxification or the detrimental effects of H<SUB>2</SUB>S. The latter explanation seems much more likely, in which case what one may be observing is the effects of general animal sickness, or even a bit more specifically, neuronal dysfunction in the presence of a toxic compound, on locomotion. As such, what is unclear is what conclusions can be taken away from this part of the work.  

Strengths: 

(1) Characterization of a motor behavior response to H<SUB>2</SUB>S 

(2) Transcriptional profiling of the response to H<SUB>2</SUB>S corroborating prior work.  

We thank the reviewer for the supportive comments.

Weaknesses: 

Unclear significance and experimental challenges regarding the study of locomotory responses to animals sensitized to the toxic effects of H<SUB>2</SUB>S under exposure to H<SUB>2</SUB>S. 

We thank the reviewer for the comment, which has also been raised by the other reviewers. We agree that our initial submission left several important questions open, and we acknowledge the fact that some aspects, such as detailed neural circuit and sensory transduction, still remain unresolved. Nevertheless, we acquired new data and revised our text, aiming to clarify the distinct mechanisms underlying the reduced locomotion in different mutants during prolonged H<SUB>2</SUB>S exposure.

Our data suggest that increased initial omega turns and a rapid loss of locomotion in hif-1 and detoxification-defective mutants including sqrd-1 and ethe-1 likely reflect an enhanced sensitivity to H<SUB>2</SUB>S toxicity due to their failure to induce appropriate adaptative responses (Figure 5D–F, Figure 5J–L, Figure 5—Figure supplement 1F–P).  Supporting this, hif-1 mutants become less responsive to unrelated stimuli (near-UV light) after 30 minutes of H<SUB>2</SUB>S exposure (Figure 5I).

In contrast, egl-9 and SOD-deficient animals show reduced initial reorientation and reduced speed responses (Figure 5B, Figure 7G, Figure 5—Figure supplement 1A and B, and Figure 7—Figure supplement 1F and G), although both egl-9 and sod mutants respond normally to the other stimuli prior or after H<SUB>2</SUB>S exposure (Figure 5I, Figure 5—Figure supplement 1C, and Figure 7—Figure supplement 1H). Since disrupting egl-9 stabilizes HIF-1 and upregulates the expression of numerous genes involved in cellular defense against H<SUB>2</SUB>S toxicity, the enhanced detoxification capacity in egl-9 mutants likely increases animals’ tolerance to H<SUB>2</SUB>S, thereby reducing avoidance to otherwise toxic H<SUB>2</SUB>S levels. Similarly, constant high ROS in SOD deficient animals activates a variety of stress-responsive signaling pathways, including HIF-1, NRF2/SKN-1 and DAF-16/ FOXO signaling (Lennicke & Cocheme, 2021; Patten et al., 2010), facilitating cellular adaptation to redox stress and reducing animals’ responsiveness to toxic H<SUB>2</SUB>S levels. Taken together, these findings support the view that reduced locomotory speed during H<SUB>2</SUB>S exposure can arise from distinct mechanisms: early systemic toxicity in hif-1 and detoxification-defective mutants, versus enhanced cellular adaptation in egl-9 and SOD mutants. We have integrated the relevant information across the result section and discussed this in Lines 485-536.

Reviewer #1 (Recommendations For The Authors): 

To better substantiate a role for H<SUB>2</SUB>S detection, it would be useful for the authors to image Ca responses to H<SUB>2</SUB>S in ASJ in WT and unc-13, and to rule out the possibility that the requirement for daf-11 in ASJ reflects a role in O2 rather than H<SUB>2</SUB>S detection. 

We thank the reviewer for this comment. As suggested, we performed calcium imaging of ASJ neurons using GCaMP6s. As previously described, 3% CO<SUB>2</SUB> evoked a calcium transient in ASJ (Figure 2—figure supplement 2F). To investigate whether H<SUB>2</SUB>S evoked a calcium transient in ASJ neurons, we tested several conditions, including animals on food or off food, with different H<SUB>2</SUB>S concentrations (~75 or ~150ppm), and different exposure time (4 or 8 mins). However, we did not detect a calcium response to H<SUB>2</SUB>S in ASJ under any of the conditions tested (Figure2—figure supplement 2E) (Lines 166–168). Given that neuronspecific rescue of daf-11 or tax-4 mutants pointed to a role of ASJ neurons in promoting H<SUB>2</SUB>S responses, we sought to determine how ASJ neurons were involved. Expression of the tetanus toxin catalytic domain in ASJ neurons, which blocks neurosecretion, inhibited H<SUB>2</SUB>S-evoked locomotory speed responses (Figure 2H), similar to the phenotypes observed in daf-11 and daf-7 mutants (Figure 2C and D) (Lines 162–165). These results confirm that ASJ activity and neurosecretion contribute to the H<SUB>2</SUB>S responses, although ASJ is unlikely to serve as the primary H<SUB>2</SUB>S sensor. One potential explanation is that DAF-7 released by ASJ controls the starvation program, which in turn modulates the animal’s response to H<SUB>2</SUB>S. We also discussed this in Lines 449–458.

The paper would be significantly strengthened by testing the possibility (as the authors acknowledge in lines 348-52) that disruption of detoxification mechanisms reduces sustained behavioral responses to H<SUB>2</SUB>S because of physiological damage. Authors use acute exposure to high O2 for this purpose earlier in the paper, but not to probe the consequences of loss of hif-1 and detoxification factors.  

We thank the reviewer for the valuable suggestion. As the reviewer highlighted, we attributed the brief locomotory speed responses to H<SUB>2</SUB>S observed in hif-1 mutants to the lack of detoxification response, leading to the rapid intoxication of the animals. Several lines of evidence support this conclusion. First, we observed that hif-1 and the detoxification mutants displayed a stronger initial reorientation response (omega turns) and a more rapid decline in speed and reversals compared to wild type (Figure 5 D–F). Second, to test if hif-1 mutants were indeed more susceptible to H<SUB>2</SUB>S toxicity, we exposed WT and hif-1 animals to H<SUB>2</SUB>S for 30 mins and subsequently tested their ability to respond to near-UV light. Unlike WT animals, the speed response to near-UV light was inhibited in hif-1 mutants (Figure 5I), suggesting that exposure to H<SUB>2</SUB>S for 30 min causes a stronger toxicity in animals deficient of HIF-1 signaling. Third, hif-1 and detoxification mutants displayed a sustained high speed in response to 1% O<SUB>2</SUB> , suggesting the specific impairment of H<SUB>2</SUB>S response. The data were presented in Lines 318–347, and were further discussed this in Lines 485–508.

To better understand whether mitochondrial damage has a role in H<SUB>2</SUB>S-evoked behavior, it might be useful for the authors to determine whether general ROS response pathways are important for H<SUB>2</SUB>S behavioral responses.

We thank the reviewer for this insightful comment. As suggested, we investigated whether ROS detoxification pathways contribute to H<SUB>2</SUB>S-evoked locomotory speed responses by analyzing mutants in the superoxide dismutase (SOD) family. These experiments, together with other observations, suggest that mitochondrial ROS play a dual role in H<SUB>2</SUB>S-evoked locomotion. The relevant results were presented in Lines 401–425, and were further discussed in Lines 509–536.

First, we found that increased mitochondrial ROS formation, either induced pharmacologically by rotenone or genetically in mitochondrial electron transport chain (ETC) mutants (Ishii et al., 2013; Ochi et al., 2016; Ramsay & Singer, 1992; Yang & Hekimi, 2010; Zorov, Juhaszova, & Sollott, 2014), suppressed the behavioral response to toxic H<SUB>2</SUB>S (Figure 7A–E). This indicates that mitochondrial ROS plays a significant role in H<SUB>2</SUB>S-evoked responses. One likely explanation is that high ROS formation may dampen the H<SUB>2</SUB>S-triggered ROS spike, or may impair other H<SUB>2</SUB>S signaling processes required to initiate avoidance. Second, consistent with previous reports (Onukwufor et al., 2022), we observed that shortterm rotenone exposure (<1 hour) significantly increased baseline locomotory speed. Given that toxic H<SUB>2</SUB>S levels promote ROS formation (Jia et al., 2020), our findings suggest that acute mitochondrial ROS production by toxic levels of H<SUB>2</SUB>S exposure may serve as a trigger for the avoidance response.

In contrast, animals with sustained mitochondrial ROS production do not have an increased baseline locomotory speed. This effect was observed after 2 hours of rotenone exposure, in mitochondrial ETC mutants, and in animals lacking all SOD enzymes (Figures 7A–K). A likely explanation for the reduced basal locomotory speed during sustained mitochondrial ROS production is the activation of ROSresponsive signaling pathways including HIF-1, NRF2/SKN-1, and DAF-16/FOXO (Lennicke & Cocheme, 2021; Patten, Germain, Kelly, & Slack, 2010), which may promote adaptation to prolonged oxidative stress (Figure 7H). Notably, unlike hif-1 mutants, SOD-deficient animals remained as responsive as WT to other stimuli after 30 minutes of H<SUB>2</SUB>S exposure (Figure 7—figure supplement 1H), indicating that elevated ROS levels do not compromise overall viability or the ability to detoxify H<SUB>2</SUB>S.

Taken together, these results support a model in which mitochondrial ROS exerts a biphasic effect on H<SUB>2</SUB>S-induced avoidance. It enhances detection and avoidance under acute stress but contributes to locomotory suppression when ROS levels remain elevated chronically.

Reviewer #2 (Recommendations For The Authors):

The way the manuscript is presented could be improved without much effort by rewriting/editing. For the reader, it is hard at present to understand the rationales of the experiments, why they were carried out, and how to place them into a context. This could be improved on three levels:

(1) Documentation 

(2) Narration/Explanation 

(3) Visualization 

(1) Documentation

Not all of the results in the text are well documented. The results should be described with more details in the written text and improved documentation and quantification of the results. Example: 

Turning behavior is mentioned as an important aspect of the response to H<SUB>2</SUB>S. There is no citation given but this effect is not well documented. The authors image the animals and could provide video footage of the effect, could quantify eg turning/pirouettes, and provide the data. At the moment the manuscript largely relies on measuring the increase in speed, but the reader is left wondering what other behavioral effects occur and how this is altered in all of the mutant and other conditions tested. Just quantifying speed reduces the readout and seems like an oversimplification to characterize the behavioral response.  

We are grateful for this comment. We now provide a video footage of the H<SUB>2</SUB>S effects (Figure 1—Video 1). As suggested, we analyzed the recordings to extract reorientation (omega-turns) and reversals. These analyses are now included in the Supplemental file 1 with representative panels displayed in Figure 5 and supplements to Figures 2, 3, 5, 6 and 7. Even though the mutant effects on omega-turns were often subtle, and reversal responses showed considerable variability (likely due to differences in population density, food availability, or animals’ physiological state prior to the assay), this analysis has proven valuable for distinguishing mutants that exhibit adaptation from those that display hypersensitivity to H<SUB>2</SUB>S toxicity. For instance, although both SOD-deficient and BP-treated animals failed to increase their locomotory speed in H<SUB>2</SUB>S (Figure 6E and Figure 7G), they exhibited distinct omega-turn responses (Figure 6—figure supplement 1I and Figure 7—figure supplement 1F), suggesting that different mechanisms likely underlie the locomotory defects of these two animals. We have integrated the omega-turn and reversal data into the text and discussed under relevant contexts.

(2) Narration/Description.

Generally, the description of the results part is very brief and it is often not clear why a certain experiment was carried out and how. Surely it is possible to check the methods but this interrupts the flow of reading and it would be easier for the reader to be guided through the results with more information what the initial motivation for an experiment is, what the general experimental outline is, and what specific experiments are carried out. 

We apologize for the lack of clarity and logical structure in the initial submission. In the revised manuscript, we have thoroughly revised the text to improve its organization and readability.

Examples: 

Line 97ff: The authors performed a candidate screen yet it is not described why which genes were chosen. Are there also pathways that were tested that turned out to not be involved? 

We thank the reviewer for the suggestion. To address this, we have added a new section, explaining the rationale for selecting genes and pathways in our candidate screen. Briefly, we focused on genes known or predicted to be involved in sensory responses to gaseous stimuli in C. elegans and mammals, including globins and guanylate cyclases (21% O<SUB>2</SUB> sensing), potassium channels (acute hypoxia), and nutrientsensitive pathways (CO<SUB>2</SUB> responses). We also included mutants defective in sensory signal transduction and neurotransmission. In addition, mitochondrial mutants were analyzed because mitochondria play a central role in H<SUB>2</SUB>S detoxification. The pathways that contributed to the acute H<SUB>2</SUB>S response included cGMP, insulin, and TGF-β signaling, as well as mitochondrial components. In contrast, globins, potassium channels, and biogenic amine signaling did not appear to play significant roles under our assay conditions. The results of the candidate screen are described in Lines 106–138 and summarized in Supplementary File 1.

line 262ff: the paragraph starts with explaining ferritin genes that are important for iron control but the reader does not yet know why. Then it is explained that a ferritin gene is DE in the H<SUB>2</SUB>S transcriptomes. then a motivation to look into the labile iron pool is described. Why not first explain what genes are strongly regulated and why they are selected based on their DE? Then explain what is known about these genes and pathways, and then motivate a set of experiments. 

We agree with the reviewer that our initial description could have been more logically organized. We reframed this section to first present the RNA-seq data, followed by an explanation of their known biological functions and the motivation for the subsequent experiments (Lines 350–357).

nhr-49 appears suddenly in the results part and it is not clear why it was tested and how the result links. Is nhr-49 a key transcription factor that is activated by H<SUB>2</SUB>S sensory or physiological response, and does it control the signaling or protective changes induced by H<SUB>2</SUB>S?  

We thank the reviewer for the comment. As suggested, we revised the text to present the information more clearly. In our candidate gene screen, a set of mutants exhibiting reduced speed responses to H<SUB>2</SUB>S has previously been shown to be defective in response to CO<SUB>2</SUB> stimulation (Hallem & Sternberg, 2008). These included animals deficient in nutrient-sensitive pathways, including insulin, TGF-beta, and NHR49, which were reported by Sternberg’s lab to exhibit dampened responses to CO<SUB>2</SUB> (Hallem & Sternberg, 2008) (Lines 173–179). We also included a simply cartoon to further illustrate this (Figure 3C).

The nuclear hormone receptor NHR-49 has been implicated in a variety of stress responses, including starvation (Van Gilst, Hadjivassiliou, & Yamamoto, 2005), bacterial pathogen (Naim et al., 2021; Wani et al., 2021), and hypoxia (Doering et al., 2022). The nhr-49 mutants exhibited a rapid decline in locomotory speed during H<SUB>2</SUB>S exposure, implicating a role in sustaining high speed in the presence of H<SUB>2</SUB>S. Furthermore, we observed that fmo-2, a well-characterized target gene of NHR-49, was significantly upregulated after 1 hour of exposure to 50 and 150 ppm H<SUB>2</SUB>S (Supplementary file 2), suggesting that NHR-49 signaling is rapidly activated by H<SUB>2</SUB>S exposure. Exactly how NHR-49 contributes to H<SUB>2</SUB>S response requires further investigation.

(3) Visualization 

Adding a model/cartoon summary that describes the pathways tested and their interaction would be helpful in some of the figures for the reader to keep an overview of the pathways that were tested. Also, a final summary cartoon that integrates all the puzzle pieces into one larger picture would be helpful. Such a final cartoon overview could also point to the key open questions of the underlying mechanisms. 

We thank the reviewer for this suggestion. We have added a series of models/cartoons to illustrate the different pathways and their interactions. These include starvation regulatory mechanisms (Figure 3C), 21% O<SUB>2</SUB> sensing mechanisms (Figure 3G), HIF-1 signaling and detoxification (Figure 5—figure supplement 1E), HIF-1 signaling and the regulation of labile iron (Figure 6H), as well as ROS signaling and regulation (Figure 7L). To help interpretation and to elaborate on these models, we have also included explanatory sentences in the corresponding figure legends.

Other comments: 

Introduction and line 93: The authors mention that 50 ppm H<SUB>2</SUB>S has beneficial effects on lifespan yet does not have a detectable phenotype." Are there any concentrations of H<SUB>2</SUB>S that cause attraction of C. elegans and what is the preferred range if it exists? Could this be measured in an H<SUB>2</SUB>S gradient? 

We thank the reviewer for the insightful comment. We performed an H<SUB>2</SUB>S gradient assay, which suggests that wild type animals are attracted toward low concentrations of H<SUB>2</SUB>S around 40 ppm (Figure 1G and H) (Lines 95–104). These results suggest that H<SUB>2</SUB>S acts as a strong repellent for C. elegans at high concentrations but as an attractant at low levels. This dual role may be ecologically relevant, as wild C. elegans lives in complex and dynamic environments where H<SUB>2</SUB>S levels likely fluctuate over short distances (Adams, Farwell, Pack, & Bamesberger, 1979; Budde & Roth, 2011; Morra & Dick, 1991; Patange, Breen, Arsuffi, & Ruvkun, 2025; Rodriguez-Kabana, Jordan, & Hollis, 1965; Romanelli-Cedrez, Vairoletti, & Salinas, 2024).

Line 146: "Local H<SUB>2</SUB>S concentrations could also be significantly higher in decomposing substances where wild C. elegans thrives" please provide a citation.

As suggested, we included a set of references that have described the H<SUB>2</SUB>S enrichment in the natural environment in early field studies (Adams et al., 1979; Morra & Dick, 1991; Rodriguez-Kabana et al., 1965), as well as references that have discussed and implied this in C. elegans studies (Budde & Roth, 2011; Patange et al., 2025; Romanelli-Cedrez et al., 2024). They can be found in the introduction (Lines 59–62) and in the result (Lines 197–199).

Line 311 "Wild C. elegans isolates thrive in the decomposing matters, where the local concentrations of O2 are low while the levels of CO2 and H<SUB>2</SUB>S could be high. These animals have adapted their behavior in such an environment, displaying increased sensitivity to high O2 exposure but dampened responses to CO2." Please provide citations for these statements.  

As suggested, we cited the relevant articles or books describing the variation of O<SUB>2</SUB> and CO<SUB>2</SUB> levels in the decomposing matters including several C. elegans papers that mentioned this in Lines 197–199 (Bretscher, Busch, & de Bono, 2008; Gea, Barrena, Artola, & Sanchez, 2004; Hallem & Sternberg, 2008; Oshins, Michel, Louis, Richard, & Rynk, 2022), and the above-mentioned articles for H<SUB>2</SUB>S (Adams et al., 1979; Budde & Roth, 2011; Morra & Dick, 1991; Patange et al., 2025; Rodriguez-Kabana et al., 1965; Romanelli-Cedrez et al., 2024).

For C. elegans’ sensitivity to O2 and CO2, these articles were cited in Lines 201–203 (Beets et al., 2020; Bretscher et al., 2008; Carrillo, Guillermin, Rengarajan, Okubo, & Hallem, 2013; Hallem & Sternberg, 2008; Kodama-Namba et al., 2013; McGrath et al., 2009).

Reviewer #3 (Recommendations For The Authors): 

More work could be conducted establishing the neuronal circuitry involved in the initial, tractable response to H<SUB>2</SUB>S. 

We thank the reviewer for the insightful comment. Since our initial analyses suggest a role of ASJ neurons in H<SUB>2</SUB>S-evoked locomotory responses (Figure 2F and G), We thought that this would offer us an entry point to dissect the neuronal circuit involved in H<SUB>2</SUB>S responses. Expression of the tetanus toxin catalytic domain in ASJ, which blocks neurosecretion, inhibited H<SUB>2</SUB>S evoked locomotory responses (Figure 2H), suggesting that neurosecretion from ASJ promotes the speed response to H<SUB>2</SUB>S (Lines 162– 165). We then performed calcium imaging of ASJ neurons in response to H<SUB>2</SUB>S exposure. However, while we observed CO<SUB>2</SUB> -evoked calcium transients in ASJ using GCaMP6s, we did not detect any calcium response to H<SUB>2</SUB>S, under several conditions, including animals on food, off food, and with different H<SUB>2</SUB>S concentrations and exposure times (Figure2—Figure supplement 2E and 2F) (Lines 166–168). Since signaling from ASJ neurons regulates developmental programs that modify sensory functions in C. elegans, including CO<SUB>2</SUB> and O<SUB>2</SUB> responses (Murakami, Koga, & Ohshima, 2001), the involvement of ASJ neurons is not specific to H<SUB>2</SUB>S responses and ASJ neurons are unlikely to serve as a primary H<SUB>2</SUB>S sensor (Discussed in Line 449–458). Therefore, the exact sensory neuron, circuit and molecular triggers mediating acute H<SUB>2</SUB>S avoidance behavior remain to be elucidated.

Our subsequent investigation on mitochondrial components suggests that a burst of mitochondrial ROS production may be the trigger for H<SUB>2</SUB>S avoidance, as transient exposure to rotenone substantially increases baseline locomotory activity (Figure 7E) (Line 391–396). However, mitochondrial ROS could potentially target multiple neurons and cellular machineries to initiate avoidance behavior to H<SUB>2</SUB>S, making it challenging to pinpoint specific sites of action. Nevertheless, we agree that further dissection of the neural circuits and mitochondrial signaling in H<SUB>2</SUB>S avoidance will be important and should be explored in future studies. We discussed this in Lines 509–536. 

I am not sure how to overcome the challenges involved in reaching conclusions from the decreased locomotory responses of animals that are sensitized to the effects of H<SUB>2</SUB>S. Perhaps this conundrum could be discussed in more detail in the text. 

We thank the reviewer for this important comment. We agree that decreased locomotory speed during H<SUB>2</SUB>S exposure can arise from distinct causes, either systemic toxicity or adaptation, and distinguishing between these is critical. We have included new experiments and revised the text to clarify this issue.

Our data suggest that increased initial omega turns and a rapid loss of locomotion in hif-1 and detoxification-defective mutants including sqrd-1 and ethe-1 likely reflect an enhanced sensitivity to H<SUB>2</SUB>S toxicity due to their failure to induce appropriate adaptative responses (Figure 5D–F, Figure 5J–L, Figure 5—Figure supplement 1F–P).  Supporting this, hif-1 mutants become less responsive to unrelated stimuli (near-UV light) after 30 minutes of H<SUB>2</SUB>S exposure (Figure 5I).

In contrast, egl-9 and SOD-deficient animals show reduced initial reorientation and reduced speed responses (Figure 5B, Figure 7G, Figure 5—Figure supplement 1A and B, and Figure 7—Figure supplement 1F and G), although both egl-9 and sod mutants respond normally to the other stimuli prior or after H<SUB>2</SUB>S exposure (Figure 5I, Figure 5—Figure supplement 1C, and Figure 7—Figure supplement 1H). Since disrupting egl-9 stabilizes HIF-1 and upregulates the expression of numerous genes involved in cellular defense against H<SUB>2</SUB>S toxicity, the enhanced detoxification capacity in egl-9 mutants likely increases animals’ tolerance to H<SUB>2</SUB>S, thereby reducing avoidance to otherwise toxic H<SUB>2</SUB>S levels. Similarly, persistently high ROS in SOD deficient animals activates a variety of stress-responsive signaling pathways, including HIF-1, NRF2/SKN-1 and DAF-16/ FOXO signaling (Lennicke & Cocheme, 2021; Patten et al., 2010), facilitating cellular adaptation to redox stress and reducing animals’ responsiveness to toxic H<SUB>2</SUB>S levels. Taken together, these findings support the view that reduced locomotory speed during H<SUB>2</SUB>S exposure can arise from distinct mechanisms: early systemic toxicity in hif-1 and detoxificationdefective mutants, versus enhanced cellular adaptation in egl-9 and SOD mutants. We have integrated the relevant information across the result section and discussed this in Lines 485–536. 

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DOI: 10.1073/pnas.1821863116

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Curator: @scibot

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DOI: 10.1016/j.molcel.2025.08.017

Resource: None

Curator: @scibot

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DOI: 10.1016/j.devcel.2025.08.009

Resource: (Abcam Cat# ab157524, RRID:AB_2721036)

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Reviewer #2 (Public review):

This manuscript from Hariharan, Shi, Viner, and Guan presents x-ray crystallographic structures of membrane protein MelB and HDX-MS analysis of ligand-induced dynamics. This work improves on the resolution of previously published structures, introduces further sugar-bound structures, and utilises HDX to explore in further depth the previously observed positive cooperatively to cotransported cation Na+. The work presented here builds on years of previous study and adds substantial new details into how Na+ binding facilitates melibiose binding and deepens the fundamental understanding of the molecular basis underlying the symport mechanism of cation-coupled transporters. However, the presentation of the data lacks clarity, and in particular, the HDX-MS data interpretation requires further explanation in both methodology and discussion.

Comments on Crystallography and biochemical work:

(1) It is not clear what Figure 2 is comparing. The text suggests this figure is a comparison of the lower resolution structure to the structure presented in this work; however, the figure legend does not mention which is which, and both images include a modelled water molecule that was not assigned due to poor resolution previously, as stated by the authors, in the previously generated structure. This figure should be more clearly explained.

(2) It is slightly unclear what the ITC measurements add to this current manuscript. The authors comment that raffinose exhibiting poor binding affinity despite having more sugar units is surprising, but it is not surprising to me. No additional interactions can be mapped to these units on their structure, and while it fits into the substrate binding cavity, the extra bulk of additional sugar units is likely to reduce affinity. In fact, from their listed ITC measurements, this appears to be the trend. Additionally, the D59C mutant utilised here in structural determination is deficient in sodium/cation binding. The reported allostery of sodium-sugar binding will likely influence the sugar binding motif as represented by these structures. This is clearly represented by the authors' own ITC work. The ITC included in this work was carried out on the WT protein in the presence of Na+. The authors could benefit from clarifying how this work fits with the structural work or carrying out ITC with the D59C mutant, or additionally, in the absence of sodium.

Comments on HDX-MS work:

While the use of HDX-MS to deepen the understanding of ligand allostery is an elegant use of the technique, this reviewer advises the authors to refer to the Masson et al. (2019) recommendations for the HDX-MS article (https://doi.org/10.1038/s41592-019-0459-y) on how to best present this data. For example:

(1) The Methodology includes a lipid removal step. Based on other included methods, I assumed that the HDX-MS was being carried out in detergent-solubilised protein samples. I therefore do not see the need for a lipid removal step that is usually included for bilayer reconstituted samples. I note that this methodology is the same as previously used for MelB. It should be clarified why this step was included, if it was in fact used, aka, further details on the sample preparation should be included.

(2) A summary of HDX conditions and results should be given as recommended, including the mean peptide length and average redundancy per state alongside other included information such as reaction temperature, sequence coverage, etc., as prepared for previous publications from the authors, i.e., Hariharan et al., 2024.

(3) Uptake plots per peptide for the HDX-MS data should be included as supporting information outside of the few examples given in Figure 6.

(4) A reference should be given to the hybrid significance testing method utilised. Additionally, as stated by Hageman and Weis (2019) (doi:10.1021/acs.analchem.9b01325), the use of P < 0.05 greatly increases the likelihood of false positive ΔD identifications. While the authors include multiple levels of significance, what they refer to as high and lower significant results, this reviewer understands that working with dynamic transporters can lead to increased data variation; a statement of why certain statistical criteria were chosen should be included, and possibly accompanied by volcano plots. The legend of Figure 6 should include what P value is meant by * and ** rather than statistically significant and highly statistically significant.

(5) Line 316 states a significant difference in seen in dynamics, how is significance measured here? There is no S.D. given in Table S4. Can the authors further comment on the potential involvement in solvent accessibility and buried helices that might influence the overall dynamics outside of their role in sugar vs sodium binding? An expected low rate of exchange suggests that dynamics are likely influenced by solvent accessibility or peptide hydrophobicity? The increased dynamics at peptides covering the Na binding site on overall more dynamic helices suggests that there is no difference between the dynamics of each site.

(6) Previously stated HDX-MS results of MelB (Hariharan et al., 2024) state that the transmembrane helices are less dynamic than polypeptide termini and loops with similar distributions across all transmembrane bundles. The previous data was obtained in the presence of sodium. Does this remove the difference in dynamics in the sugar-binding helices and the cation-binding helices? Including this comparison would support the statement that the sodium-bound MelB is more stable than the Apo state, along with the lack of deprotection observed in the differential analysis.

(7) Have the authors considered carrying out an HDX-MS comparison between the WT and the D59C mutant? This may provide some further information on the WT structure (particularly a comparison with sugar-bound). This could be tied into a nice discussion of their structural data.

(8) Have the authors considered utilising Li+ to infer how cation selectivity impacts the allostery? Do they expect similar stabilisation of a higher-affinity sugar binding state with all cations?

(9) MD of MelB suggests all transmembrane helices are reorientated during substrate translocation, yet substrate and cotransporter ligand binding only significantly impacts a small number of helices. Can the authors comment on the ensemble of states expected from each HDX experiment? The data presented here instead shows overall stabilisation of the transporter. This data can be compared to that of HDX on MFS sugar cation symporter XylE, where substrate binding induces a transition to OF state. There is no discussion of how this HDX data compares to previous MFS sugar transporter HDX. The manuscript could benefit from this comparison rather than a comparison to LacY. It is unlikely that there are universal mechanisms that can be inferred even from these model proteins. Highlighting differences instead between these transport systems provides broader insights into this protein class. Doi: 10.1021/jacs.2c06148 and 10.1038/s41467-018-06704-1.

(10) Additionally, the recent publication of SMFS data (by the authors: doi:10.1016/j.str.2022.11.011) states the following: "In the presence of either melibiose or a coupling Na+-cation, however, MelB increasingly populates the mechanically less stable state which shows a destabilized middle-loop C3." And "In the presence of both substrate and co-substrate, this mechanically less stable state of MelB is predominant.". It would benefit the authors to comment on these data in contrast to the HDX obtained here. Additionally, is the C3 loop covered, and does it show the destabilization suggested by these studies? HDX can provide a plethora of results that are missing from the current analysis on ligand allostery. The authors instead chose to reference CD and thermal denaturation methods as comparisons.

Author response:

Reviewer #1:

While the structure of the melibiose permease in both outward and inward-facing forms has been solved previously, there remain unanswered questions regarding its mechanism. Hariharan et al set out to address this with further crystallographic studies complemented with ITC and hydrogen-deuterium exchange (HDX) mass spectrometry.

They first report 4 different crystal structures of galactose derivatives to explore molecular recognition, showing that the galactose moiety itself is the main source of specificity. Interestingly, they observe a water-mediated hydrogen bonding interaction with the protein and suggest that this water molecule may be important in binding.

We appreciate the understanding of our work presented in this manuscript by this reviewer.

The results from the crystallography appear sensible, though the resolution of the data is low, with only the structure with NPG better than 3Å. However, it is a bit difficult to understand what novel information is being brought out here and what is known about the ligands. For instance, are these molecules transported by the protein or do they just bind? They measure the affinity by ITC, but draw very few conclusions about how the affinity correlates with the binding modes. Can the protein transport the trisaccharide raffinose?

The four structures with a bound sugar of different sizes aimed to identify the binding motif on both the primary substrate (sugar) and the transporter (MelB_St). Although the resolutions of the structures complexed with melibiose, raffinose, or a-MG are relatively low, the size and shape of the densities at each structure are consistent with the corresponding sugar molecules, which provide valuable data for determining the pose of the bound sugar. Additionally, there is another a-NPG-bound structure at a higher resolution of 2.7 Å. Therefore, our new data support the published binding site with the galactosyl moiety as the main interacting group. The identified water-1 in this study further confirms the orientation of C4-OH. Notably, this transporter does not recognize or transport glucosides where the orientation of C4-OH at the glucopyranosyl ring is opposite. We will provide stronger data to support the water-1.

Regarding the raffinose question, we should have clearly introduced the historical background. Bacterial disaccharide transporters have broad specificity, allowing them to work on a group of sugars with shared structural elements; for example, one sugar molecule can be transported by several transporters. As reported in the literature, the galactosides melibiose, lactose, and raffinose can be transported by both LacY and MelB of E. coli. We did not test whether MelB_St can transport the a-NPG and raffinose. To address this issue and strengthen our conclusions, we plan to conduct additional experiments to gather evidence of the translocation of these sugars by MelB_St.

The HDX also appears to be well done; however, in the manuscript as written, it is difficult to understand how this relates to the overall mechanism of the protein and the conformational changes that the protein undergoes.

Previously, we used HDX-MS to examine the conformational transition between inward- and outward-facing conformations using a conformation-specific nanobody to trap MelB_St in an inward-facing state, as structurally resolved by cryoEM single-particle analysis and published in eLife 2024. That study identified dynamic regions that may be involved in the conformational transitions; however, there was no sugar present. We also solved and published the crystal structure of the apo D59C MelB_St. The sugar-bound and apo states are virtually identical. To address the positive cooperativity of binding between the sugar and co-transport cations observed in biophysical analysis, in this study, we utilize HDX-MS to analyze the structural dynamics induced by melibiose, Na⁺, or both, focusing on the binding residues at the sugar-binding and cation-binding pockets. The results suggest that the coupling cation stabilizes sugar-binding residues at helices I and V, contributing to affinity but not specificity.

Since MelB_St favors the outward-facing conformation, and simulations on the free-energy landscape suggest that the highest affinity of the sugar-bound state is also at an outward-facing state, MelB_St in both the apo and bound states tend to remain in the outward-facing conformation. We will include a section comparing these differences. Thank you to this reviewer for the critical insight.

Reviewer #2:

This manuscript from Hariharan, Shi, Viner, and Guan present x-ray crystallographic structures of membrane protein MelB and HDX-MS analysis of ligand-induced dynamics. This work improves on the resolution of previously published structures, introduces further sugar-bound structures, and utilises HDX to explore in further depth the previously observed positive cooperatively to cotransported cation Na⁺. The work presented here builds on years of previous study and adds substantial new details into how Na⁺ binding facilitates melibiose binding and deepens the fundamental understanding of the molecular basis underlying the symport mechanism of cation-coupled transporters. However, the presentation of the data lacks clarity, and in particular, the HDX-MS data interpretation requires further explanation in both methodology and discussion.

We thank this reviewer for taking the time to read our previous articles related to this manuscript.

Comments on Crystallography and biochemical work:

(1) It is not clear what Figure 2 is comparing. The text suggests this figure is a comparison of the lower resolution structure to the structure presented in this work; however, the figure legend does not mention which is which, and both images include a modelled water molecule that was not assigned due to poor resolution previously, as stated by the authors, in the previously generated structure. This figure should be more clearly explained.

This figure shows a stereo view of a density map created in cross-eye style to demonstrate its quality. We will update this figure with a higher-resolution map, and the density for Wat-1 is clearly visible. This also addresses Reviewer-3’s comment regarding the map resolution.

(2) It is slightly unclear what the ITC measurements add to this current manuscript. The authors comment that raffinose exhibiting poor binding affinity despite having more sugar units is surprising, but it is not surprising to me. No additional interactions can be mapped to these units on their structure, and while it fits into the substrate binding cavity, the extra bulk of additional sugar units is likely to reduce affinity. In fact, from their listed ITC measurements, this appears to be the trend. Additionally, the D59C mutant utilised here in structural determination is deficient in sodium/cation binding. The reported allostery of sodium-sugar binding will likely influence the sugar binding motif as represented by these structures. This is clearly represented by the authors' own ITC work. The ITC included in this work was carried out on the WT protein in the presence of Na⁺. The authors could benefit from clarifying how this work fits with the structural work or carrying out ITC with the D59C mutant, or additionally, in the absence of sodium.

While raffinose and a-MG have been reported as substrates of MelB in E. coli, binding data are unavailable; additionally, for MelB_St, we lack data on the binding of two of the four sugars or sugar analogs. We performed a label-free binding assay using ITC to address this concern with the WT MelB_St. We will also perform the binding assay with the D59C MelB_St, since sugar binding has been structurally analyzed with this mutant, as pointed out by this reviewer. Along with other new functional results, we will prepare a new Figure 1 on functional analysis, which will also address the comment regarding extra bulk at the non-galactosyl moiety with poor affinity.

This D59C uniport mutant exhibits increased thermostability, making it a valuable tool for crystal structure determination, especially since the wild type (WT) is difficult to crystallize at high quality. Asp59 is the only site that responds to the binding of all coupling cations: Na⁺, Li⁺, or H⁺. Notably, this mutant selectively abolishes cation binding and cotransport. However, it still maintains intact sugar binding with slightly higher affinity and preserves the conformational transition, as demonstrated by an electroneutral transport reaction, the melibiose exchange, and fermentation assays with intact cells. Therefore, the structural data derived from this mutant are significant and offer important mechanistic insights into sugar transport. We will provide additional details during the revision.

Comments on HDX-MS work:

While the use of HDX-MS to deepen the understanding of ligand allostery is an elegant use of the technique, this reviewer advises the authors to refer to the Masson et al. (2019) recommendations for the HDX-MS article (https://doi.org/10.1038/s41592-019-0459-y) on how to best present this data. For example:

All authors appreciate this reviewer’s comments and suggestions, which will be incorporated into the revision.

(1) The Methodology includes a lipid removal step. Based on other included methods, I assumed that the HDX-MS was being carried out in detergent-solubilised protein samples. I therefore do not see the need for a lipid removal step that is usually included for bilayer reconstituted samples. I note that this methodology is the same as previously used for MelB. It should be clarified why this step was included, if it was in fact used, aka, further details on the sample preparation should be included.

Yes, a lipid/detergent removal step was applied in this study and in previous studies and this information was clearly described in Methods.

(2) A summary of HDX conditions and results should be given as recommended, including the mean peptide length and average redundancy per state alongside other included information such as reaction temperature, sequence coverage, etc., as prepared for previous publications from the authors, i.e., Hariharan et al., 2024.

We will update the Table S2. Thank you.

(3) Uptake plots per peptide for the HDX-MS data should be included as supporting information outside of the few examples given in Figure 6.

We will prepare the plots in supplementary information.

(4) A reference should be given to the hybrid significance testing method utilised. Additionally, as stated by Hageman and Weis (2019) (doi:10.1021/acs.analchem.9b01325), the use of P < 0.05 greatly increases the likelihood of false positive ΔD identifications. While the authors include multiple levels of significance, what they refer to as high and lower significant results, this reviewer understands that working with dynamic transporters can lead to increased data variation; a statement of why certain statistical criteria were chosen should be included, and possibly accompanied by volcano plots. The legend of Figure 6 should include what P value is meant by * and ** rather than statistically significant and highly statistically significant.

We appreciate this comment and will cite this article on the hybrid significance method. We will include volcano plots for each dataset. We fully acknowledge that using a cutoff of P < 0.05 can increase the likelihood of false-positive identifications. However, given the complexity of the samples analyzed in this study, we believe that some important changes may have been excluded due to higher variability within the dataset. By applying multiple levels of statistical testing, we determined that P < 0.05 represents a suitable threshold for this study. The threshold values were marked in the residual plots and explained in the text. For Figure 6, we have revised it by showing the P value directly.

(5) Line 316 states a significant difference in seen in dynamics, how is significance measured here? There is no S.D. given in Table S4. Can the authors further comment on the potential involvement in solvent accessibility and buried helices that might influence the overall dynamics outside of their role in sugar vs sodium binding? An expected low rate of exchange suggests that dynamics are likely influenced by solvent accessibility or peptide hydrophobicity? The increased dynamics at peptides covering the Na binding site on overall more dynamic helices suggests that there is no difference between the dynamics of each site.

Table S4 was created to provide an overall view of the dynamic regions. If we understand correctly, this reviewer asked us to comment on the effect of solvent accessibility or hydrophobic regions on the overall dynamics outside the binding residues of the peptides that carry binding residues. Since the HDX rate is influenced by two linked factors: solvent accessibility and hydrogen-bonding interactions that reflect structural dynamics, poor solvent accessibility in buried regions results in low deuterium uptakes. The peptides in our dataset that include the Na⁺-binding site showed low HDX, likely due to poor solvent accessibility and structural stability. It is unclear what this reviewer meant by "increased dynamics at peptides covering the Na binding site on overall more dynamic helices." We do not observe increased dynamics in peptides covering Na⁺-binding sites.

(6) Previously stated HDX-MS results of MelB (Hariharan et al., 2024) state that the transmembrane helices are less dynamic than polypeptide termini and loops with similar distributions across all transmembrane bundles. The previous data was obtained in the presence of sodium. Does this remove the difference in dynamics in the sugar-binding helices and the cation-binding helices? Including this comparison would support the statement that the sodium-bound MelB is more stable than the Apo state, along with the lack of deprotection observed in the differential analysis.

Thanks for this suggestion. The previous datasets were collected in the presence of Na⁺. In the current study, we also have a Na-containing dataset. Both showed similar results: the multiple overlapping peptides covering the sugar-binding residues on helices I and V have higher HDX rates than those covering the Na⁺-binding residues, even when Na⁺ is present in both datasets.

(7) Have the authors considered carrying out an HDX-MS comparison between the WT and the D59C mutant? This may provide some further information on the WT structure (particularly a comparison with sugar-bound). This could be tied into a nice discussion of their structural data.

Thanks for this suggestion. Conducting the HDX-MS comparison between the WT and the D59C mutant is certainly interesting, especially given the growing amount of structural and biochemical/biophysical data available for this mutant. However, due to limited resources, we might consider doing it later.

(8) Have the authors considered utilising Li⁺ to infer how cation selectivity impacts the allostery? Do they expect similar stabilisation of a higher-affinity sugar binding state with all cations?

Thanks for this suggestion. We have demonstrated that Li⁺ also shows positive cooperativity with melibiose through ITC binding measurements. Li⁺ binds to MelB_St with higher affinity than Na⁺ but causes many different effects on MelB. It is worth investigating this thoroughly and individually. To address the second question, H⁺ is a poor coupling cation with minimal impact on melibiose binding. Since its pKa is around 6.5, only a small subpopulation of MelB_St is protonated at pH 7.5. The order of sugar-binding cooperativity is the highest with Na⁺, followed by Li⁺ and H⁺.

(9) MD of MelB suggests all transmembrane helices are reorientated during substrate translocation, yet substrate and cotransporter ligand binding only significantly impacts a small number of helices. Can the authors comment on the ensemble of states expected from each HDX experiment? The data presented here instead shows overall stabilisation of the transporter. This data can be compared to that of HDX on MFS sugar cation symporter XylE, where substrate binding induces a transition to OF state. There is no discussion of how this HDX data compares to previous MFS sugar transporter HDX. The manuscript could benefit from this comparison rather than a comparison to LacY. It is unlikely that there are universal mechanisms that can be inferred even from these model proteins. Highlighting differences instead between these transport systems provides broader insights into this protein class. Doi: 10.1021/jacs.2c06148 and 10.1038/s41467-018-06704-1.

The sugar translocation free-energy landscape simulations showed that both helix bundles move relative to the membrane plane. That analysis aimed to clarify a hypothesis in the field—that the MFS transporter can use an asymmetric mode to transition between inward- and outward-facing states. In the case of MelB, we clearly demonstrated that both domains move and each helix bundle moves as a unit, so the labeling changes were identified only in some extramembrane loops and a few highly flexible helices. Thanks for the suggestion about comparing with XylE. We will include a discussion on it.

(10) Additionally, the recent publication of SMFS data (by the authors: doi:10.1016/j.str.2022.11.011) states the following: "In the presence of either melibiose or a coupling Na⁺-cation, however, MelB increasingly populates the mechanically less stable state which shows a destabilized middle-loop C3." And "In the presence of both substrate and co-substrate, this mechanically less stable state of MelB is predominant.". It would benefit the authors to comment on these data in contrast to the HDX obtained here. Additionally, is the C3 loop covered, and does it show the destabilization suggested by these studies? HDX can provide a plethora of results that are missing from the current analysis on ligand allostery. The authors instead chose to reference CD and thermal denaturation methods as comparisons.

Thank this reviewer for reading the single-molecule force spectroscopy (SMFS) study on MelB_St. The C3 loop mentioned in this SMFS article is partially covered in the dataset Mel or Mel plus Na⁺ vs. Apo, and more coverage is in the Na⁺ vs. Apo. In either condition, no deprotection was detected. Two possible reasons the HDX data did not reflect the deprotection are: 1) The changes were too subtle and did not pass the statistical tests and 2) the longest labeling time point was still insufficient to detect the changes; much longer labeling times should be considered in future studies.

Reviewer #3:

Summary:

The melibiose permease from Salmonella enterica serovar Typhimurium (MelB_St) is a member of the Major Facilitator Superfamily (MFS). It catalyzes the symport of a galactopyranoside with Na⁺, H⁺, or Li⁺, and serves as a prototype model system for investigating cation-coupled transport mechanisms. In cation-coupled symporters, a coupling cation typically moves down its electrochemical gradient to drive the uphill transport of a primary substrate; however, the precise role and molecular contribution of the cation in substrate binding and translocation remain unclear. In a prior study, the authors showed that the binding affinity for melibiose is increased in the presence of Na⁺ by about 8-fold, but the molecular basis for the cooperative mechanism remains unclear. The objective of this study was to better understand the allosteric coupling between the Na⁺ and melibiose binding sites. To verify the sugar-recognition specific determinants, the authors solved the outward-facing crystal structures of a uniport mutant D59C with four sugar ligands containing different numbers of monosaccharide units (α-NPG, melibiose, raffinose, or α-MG). The structure with α-NPG bound has improved resolution (2.7 Å) compared to a previously published structure and to those with other sugars. These structures show that the specificity is clearly directed toward the galactosyl moiety. However, the increased affinity for α-NPG involves its hydrophobic phenyl group, positioned at 4 Å-distance from the phenyl group of Tyr26 forms a strong stacking interaction. Moreover, a water molecule bound to OH-4 in the structure with α-NPG was proposed to contribute to the sugar recognition and appears on the pathway between the two specificity-determining pockets. Next, the authors analyzed by hydrogen-to-deuterium exchange coupled to mass spectrometry (HDX-MS) the changes in structural dynamics of the transporter induced by melibiose, Na⁺, or both. The data support the conclusion that the binding of the coupling cation at a remote location stabilizes the sugar-binding residues to switch to a higher-affinity state. Therefore, the coupling cation in this symporter was proposed to be an allosteric activator.

Strengths:

(1) The manuscript is generally well written.

(2) This study builds on the authors' accumulated knowledge of the melibiose permease and integrates structural and HDX-MS analyses to better understand the communication between the sodium ion and sugar binding sites. A high sequence coverage was obtained for the HDX-MS data (86-87%), which is high for a membrane protein.

Thank this reviewer for your positive comments.

Weaknesses:

(1) I am not sure that the resolution of the structure (2.7 Å) is sufficiently high to unambiguously establish the presence of a water molecule bound to OH-4 of the α-NPG sugar. In Figure 2, the density for water 1 is not obvious to me, although it is indeed plausible that water mediates the interaction between OH4/OH6 and the residues Q372 and T373.

Thanks for your comments on the resolution. We will improve the density for the Water 1.

(2) Site-directed mutagenesis could help strengthen the conclusions of the authors. Would the mutation(s) of Q372 and/or T373 support the water hypothesis by decreasing the affinity for sugars? Mutations of Thr 121, Arg 295, combined with functional and/or HDX-MS analyses, may also help support some of the claims of the authors regarding the allosteric communication between the two substrate-binding sites.

The authors thank this reviewer for the thoughtful suggestions. MelB_St has been subjected to Cys-scanning mutagenesis (https://doi.org/10.1016/j.jbc.2021.101090). Placing a Cys residue on the hydrogen bond-donor Q372 significantly decreased the transport initial rate, accumulation, and melibiose fermentation, with little effect on protein expression, as shown in Figure 2 of this JBC paper. Although no binding data are available, the poor initial rate of transport with a similar amount of protein expressed suggested that the binding affinity is apparently decreased, supporting the role of water-1 in the binding pocket for better binding. The T373C mutant retained most activities of the WT. We will discuss the functional characterizations of these two mutants. Thanks.

(3) The main conclusion of the authors is that the binding of the coupling cation stabilizes those dynamic sidechains in the sugar-binding pocket, leading to a high-affinity state. This is visible when comparing panels c and a from Figure S5. However, there is both increased protection (blue, near the sugar) and decreased protection in other areas (red). The latter was less commented, could the increased flexibility in these red regions facilitate the transition between inward- and outward-facing conformations?

Thanks for this important question. We will discuss the deprotected data in the conformational transition between inward-facing and outward-facing states. The two regions, loop8-9 and loop1-2, are located in the gate area on both sides of the membrane and showed increased deuterium uptakes upon binding of melibiose plus Na⁺. They are likely involved in this process.

The HDX changes induced by the different ligands were compared to the apo form (see Figure S5). It might be worth it for data presentation to also analyze the deuterium uptake difference by comparing the conditions sodium ion+melibiose vs melibiose alone. It would make the effect of Na⁺ on the structural dynamics of the melibiose-bound transporter more visible. Similarly, the deuterium uptake difference between sodium ion+melibiose vs sodium ion alone could be analyzed too, in order to plot the effect of melibiose on the Na⁺-bound transporter.

We will analyze the data as suggested by this reviewer.

(4) For non-specialists, it would be beneficial to better introduce and explain the choice of using D59C for the structural analyses.

As response to the reviewer #1 at page 3, “Asp59 is the only site that responds to the binding of all coupling cations: Na⁺, Li⁺, or H⁺. Notably, this mutant selectively abolishes cation binding and cotransport. However, it still maintains intact sugar binding with slightly higher affinity and preserves the conformational transition, as demonstrated by an electroneutral transport reaction, the melibiose exchange, and fermentation assays with intact cells. Therefore, the structural data derived from this mutant are significant and offer important mechanistic insights into sugar transport. We will provide additional details during the revision.”.

(5) In Figure 5a, deuterium changes are plotted as a function of peptide ID number. It is hardly informative without making it clearer which regions it corresponds to. Only one peptide is indicated (213-226), I would recommend indicating more of them in areas where deuterium changes are substantial.

We appreciate this comment, which will make the plots more meaningful. In the previous article published in eLife (2024), we drew boxed to mark the transmembrane regions; however, it generated much confusion, such as why some helices are very short. The revised figure will label the full length of covered positions.

(6) From prior work of the authors, melibiose binding also substantially increases the affinity of the sodium ion. Can the authors interpret this observation based on the HDX data?

This is an intriguing mechanistic question. Based on current data, we believe that the bound melibiose physically prevents the release of Na⁺ or Li⁺ from the cation-binding pocket. The cation-binding pocket and surrounding regions, including the sugar-binding residue Asp124, show low HDX, supporting this idea. Since we lack a structure with both substrates bound, figuring out the details structurally is challenging. However, we have a hypothesis about the intracellular Na⁺ release as proposed in the 2024 JBC paper (https://doi.org/10.1016/j.jbc.2024.107427). After sugar release, the rotamer change of Asp55 will help Na⁺ exit the cation pocket to the sugar pocket, and the negative membrane potential will facilitate the further movement from MelB to the cytosol. We will discuss this during the revision.

BIOmetaAnalysis

Reviewer #2 (Public review):

Summary:

Kern et al. investigated whether temporally delayed linear modeling (TDLM) can uncover sequential memory replay from a graph-learning task in human MEG during an 8-minute post-learning rest period. After failing to detect replay events, they conduct a simulation study in which they insert synthetic replay events, derived from each participant's localizer data, into a control rest period prior to learning. The simulations suggest that TDLM only reveals sequences when replay occurs at very high densities (> 80 per minute) and that individual differences in baseline sequenceness may lead to spurious and/or lackluster correlations between replay strength and behavior.

Strengths:

The approach is extremely well documented and rigorous. The authors have done an excellent job re-creating the TDLM methodology that is most commonly used, reporting the different approaches and parameters that they used, and reporting their preregistrations. The hybrid simulation study is creative and provides a new way to assess the efficacy of replay decoding methods. The authors remain measured in the scope/applicability of their conclusions, constructive in their discussion, and end with a useful set of recommendations for how to best apply TDLM in future studies. I also want to commend this work for not only presenting a null result but thoroughly exploring the conditions under which such a null result is expected. I think this paper is interesting and will be generally quite useful for the field, but I believe it also has a number of weaknesses that, if addressed, could improve it further.

Weaknesses:

The sample size is small (n=21, after exclusions), even for TDLM studies (which typically have somewhere between 25-40 participants). The authors address this somewhat through a power analysis of the relationship between replay and behavioral performance in their simulations, but this is very dependent on the assumptions of the simulation. Further, according to their own power analysis, the replay-behavior correlations are seriously underpowered (~10% power according to Figure 7C), and so if this is to be taken at face value, their own null findings on this point (Figure 3C) could therefore just reflect undersampling as opposed to methodological failure. I think this point needs to be made more clearly earlier in the manuscript. Relatedly, it would be very useful if one of the recommendations that come out of the simulations in this paper was a power analysis for detecting sequenceness in general, as I suspect that the small sample size impacts this as well, given that sequenceness effects reported in other work are often small with larger sample sizes. Further, I believe that the authors' simulations of basic sequenceness effects would themselves still suffer from having a small number of subjects, thereby impacting statistical power. Perhaps the authors can perform a similar sort of bootstrapping analysis as they perform for the correlation between replay and performance, but over sequenceness itself?

The task paradigm may introduce issues in detecting replay that are separate from TDLM. First, the localizer task involves a match/mismatch judgment and a button press during the stimulus presentation, which could add noise to classifier training separate from the semantic/visual processing of the stimulus. This localizer is similar to others that have been used in TDLM studies, but notably in other studies (e.g., Liu, Mattar et al., 2021), the stimulus is presented prior to the match/mismatch judgment. A discussion of variations in different localizers and what seems to work best for decoding would be useful to include in the recommendations section of the discussion. Second, and more seriously, I believe that the task design for training participants about the expected sequences may complicate sequence decoding. Specifically, this is because two images (a "tuple") are shown together and used for prediction, which may encourage participants to develop a single bound representation of the tuple that then predicts a third image (AB -> C rather than A -> B, B -> C). This would obviously make it difficult to i) use a classifier trained on individual images to detect sequences and ii) find evidence for the intended transition matrix using TDLM. Can the authors rule out this possibility?

Participants only modestly improved (from 76-82% accuracy) following the rest period (which the authors refer to as a consolidation period). If the authors assume that replay leads to improved performance, then this suggests there is little reason to see much task-related replay during rest in the first place. This limitation is touched on (lines 228-229), but I think it makes the lack of replay finding here less surprising. However, note that in the supplement, it is shown that the amount of forward sequenceness is marginally related to the performance difference between the last block of training and retrieval, and this is the effect I would probably predict would be most likely to appear. Obviously, my sample size concerns still hold, and this is not a significant effect based on the null hypothesis testing framework the authors employ, but I think this set of results should at least be reported in the main text. I was also wondering whether the authors could clarify how the criterion over six blocks was 80% but then the performance baseline they use from the last block is 76%? Is it just that participants must reach 80% within the six blocks *at some point* during training, but that they could dip below that again later?

Because most of the conclusions come from the simulation study, there are a few decisions about the simulations that I would like the authors to expand upon before I can fully support their interpretations. First, the authors use a state-to-state lag of 80ms and do not appear to vary this throughout the simulations - can the authors provide context for this choice? Does varying this lag matter at all for the results (i.e., does the noise structure of the data interact with this lag in any way?) Second, it seems that the approach to scaling simulated replays with performance is rather coarse. I think a more sensitive measure would be to scale sequence replays based on the participants' responses to *that* specific sequence rather than altering the frequency of all replays by overall memory performance. I think this would help to deliver on the authors' goal of simulating an "increase of replay for less stable memories" (line 246). On the other hand, I was also wondering whether it is actually necessary to use the real memory performance for each participant in these simulations - couldn't similar goals (with a better/more full sampling of the space of performance) be achieved with simulated memory performance as well, taking only the MEG data from the participant? Finally, Figure 7D shows that 70ms was used on the y-axis. Why was this the case, or is this a typo?

Because this is a re-analysis of a previous dataset combined with a new simulation study on that data aimed at making recommendations about how to best employ TDLM, I think the usefulness of the paper to the field could be improved in a few places. Specifically, in the discussion/recommendation section, the authors state that "yet unknown confounders" (line 295) lead to non-random fluctuations in the simulated correlations between replay detection and performance at different time lags. Because it is a particularly strong claim that there is the potential to detect sequenceness in the baseline condition where there are no ground-truth sequences, the manuscript could benefit from a more thorough exploration of the cause(s) of this bias in addition to the speculation provided in the current version. In addition, to really provide that a realistic simulation is necessary (one of the primary conclusions of the paper), it would be useful to provide a comparison to a fully synthetic simulation performed on this exact task and transition structure (in addition to the recreation of the original simulation code from the TDLM methods paper). Finally, I think the authors could do further work to determine whether some of their recommendations for improving the sensitivity of TDLM pan out in the current data - for example, they could report focusing not just on the peak decoding timepoint but incorporating other moments into classifier training.

Lastly, I would like the authors to address a point that was raised in a separate public forum by an author of the TDLM method, which is that when replays "happen during rest, they are not uniform or close". Because the simulations in this work assume regularly occurring replay events, I agree that this is an important limitation that should be incorporated into alternative simulations to ensure the lack of findings is not because of this assumption.

this things move along the curve because they are already on the X/Y axis, other factors are not

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review): 

Summary: 

In this manuscript, Singh, Wu and colleagues explore functional links between septins and the exocyst complex. The exocyst in a conserved octameric complex that mediates the tethering of secretory vesicles for exocytosis in eukaryotes. In fission yeast cells, the exocyst is necessary for cell division, where it localizes mostly at the rim of the division plane, but septins, which localize in a similar manner, are non-essential. The main findings of the work are that septins are required for the specific localization of the exocyst to the rim of the division plane, and the likely consequent localization of the glucanase Eng1 at this same location, where it is known to promote cell separation. In the absence of septins, the exocyst still localizes to the division plane but is not restricted to the rim. They also show some defects in the localization of secretory vesicles and glucan synthase cargo. They further propose that interactions between septins and exocysts are direct, as shown through Alphafold2 predictions (of unclear strength) and clean coIP experiments. 

Strengths: 

The septin, exocyst and Eng1 localization data are well supported, showing that the septin rim recruits the exocyst and (likely consequently) the Eng1 glucanase at this location. One major finding of the manuscript is that of a physical interaction between septins and exocyst subunits. Indeed, many of the coIPs supporting this discovery are very clear. 

Weaknesses: 

I am less convinced by the strength of the physical interaction of septins with the exocyst complex. Notably, one important open question is whether septins interact with the intact exocyst complex, as claimed in the text, or whether the interactions occur only with individual subunits. The two-hybrid and coIP data only show weak interactions with individual subunits, and some coIPs (for instance Sec3 and Exo70 with Spn1 and Spn4) are negative, suggesting that the exocyst complex does not remain intact in these experiments.

Given the known structure of the full exocyst complex and septin filaments (at least in S. cerevisiae), the Alphafold2 predicted structure could be used to probe whether the proposed interaction sites are compatible with full complex formation.  

We thank the reviewer for these important and insightful comments. We agree that our current data, particularly the data from yeast two-hybrid and co-immunoprecipitation (coIP) assays, primarily reveal interactions between individual septin and exocyst subunits, and do not conclusively demonstrate binding of septins to the fully assembled exocyst complex. We realize this as a key limitation and have revised the manuscript text accordingly to clarify this point.

We also appreciate the reviewer’s suggestion to use structural prediction to further assess their interaction plausibility. We have now employed the full Saccharomyces cerevisiae exocyst complex (with 4.4 Å resolution) published by the Guo group (Mei et al., 2018) to examine the interfaces of septin and the exocyst interactions, assuming that the S. pombe exocyst has the similar structure. We focused on checking all the interacting residues on the exocyst complex and septins from our AlphaFold modeling to determine whether these predicted interactions are structurally compatible. Our analysis reveals that majority subunit interactions are sterically feasible, while a few would likely require partial disassembly or flexible conformations. These new insights have been added to the revised Results and Discussion sections (Figure Supplement S4, S5 and Videos 4-7).

While we cannot fully resolve whether septins engage with the whole exocyst complex versus selected subunits, our combined data support a model that septins scaffold or spatially regulate the exocyst localization at the division site, potentially through dynamic and multivalent interactions. We now explicitly state this more cautious interpretation in the revised manuscript.

Mei, K., Li, Y., Wang, S., Shao, G., Wang, J., Ding, Y., Luo, G., Yue, P., Liu, J.-J., Wang, X. and Dong, M.-Q., Wang, H-W, Guo W. 2018. Cryo-EM structure of the exocyst complex. Nature Struct & Mol. Biol, 25(2), pp.139-146.

The effect of spn1∆ on Eng1 localization is very clear, but the effect on secretory vesicles (Ypt3, Syb1) and glucan synthase Bgs1 is less convincing. The effect is small, and it is not clear how the cells are matched for the stage of cytokinesis. 

For localizations and quantifications of Eng1, Ypt3, Syb1, and Bgs1 shown in Figures 6 and 7, cells with a closed septum (at or after the end of contractile-ring constriction) were quantified or highlighted. To quantify their fluorescence intensity at the division site using line scan, the line width used was 3 pixels. For Syb1 (Figure 6D), we quantified cells at the end of ring constriction (when Rlc1-tdTomato constricted to a dot) in the middle focal plane. The exact same lines were drawn in both Rlc1 and Syb1 channels. The center of line scan was defined as the pixel with the brightest Rlc1 value. All data were aligned by the center and plotted. For Bgs1 (Figure 7A), we quantified the cells that Rlc1 signal had disappeared from the division site. The line was drawn in the Bgs1 channel in the middle focal plane. The center of line scan was defined as the pixel with the brightest Bgs1 value.

All data were aligned by the center and plotted. These details were added to the Materials and Methods.

Reviewer #2 (Public Review): 

Summary: 

This interesting study implicates the direct interaction between two multi-subunit complexes, known as the exocyst and septin complexes, in the function of both complexes during cytokinesis in fission yeast. While previous work from several labs had implicated roles for the exocyst and septin complexes in cytokinesis and cell separation, this study describes the importance of protein:protein interaction between these complexes in mediating the functions of these complexes in cytokinesis. Previous studies in neurons had suggested interactions between septins and exocyst complexes occur but the functional importance of such interactions was not known. Moreover, in baker's yeast where both of these complexes have been extensively studied - no evidence of such an interaction has been uncovered despite numerous studies which should have detected it. Therefore while exocyst:septin interactions appear to be conserved in several systems, it appears likely that budding yeast are the exception--having lost this conserved interaction. 

Strengths: 

The strengths of this work include the rigorous analysis of the interaction using multiple methods including Co-IP of tagged but endogenously expressed proteins, 2 hybrid interaction, and Alphafold Multimer. Careful quantitative analysis of the effects of loss of function in each complex and the effects on localization and dynamics of each complex was also a strength. Taken together this work convincingly describes that these two complexes do interact and that this interaction plays an important role in post Golgi vesicle targeting during cytokinesis. 

Weaknesses: 

The authors used Alphafold Multimer to predict (largely successfully) which subunits were most likely to be involved in direct interactions between the complexes. It would be very interesting to compare this to a parallel analysis on the budding yeast septin and exocyst complexes where it is quite clear that detectable interactions between the exocyst and septins (using the same methods) do not exist. Presumably the resulting pLDDT scores will be significantly lower. These are in silico experiments and should not be difficult to carry out. 

We thank the reviewer for this insightful suggestion. To assess the specificity of the predicted interactions between septins and the exocyst complex in S. pombe, we performed a comparative AlphaFold2 analysis using some of the homologous subunits from Saccharomyces cerevisiae. We modeled two interactions between Cdc10-Sec5 and Cdc10-Sec15 (Cdc10 is the Spn2 homolog) using the same pipeline and parameters at the time when we did the modeling for S. pombe. We did not find interactions between them using the criteria we used for the fission yeast proteins in this study. These results support the notion that the predicted septin–exocyst interactions in S. pombe are not generalizable to budding yeast. Unfortunately, we did not test all other combinations at that time and the AlphaFold2 platform is not available to us now (showing system error messages when we tried recently). We thank the reviewer again for this helpful suggestion, which should strengthen the evolutionary interpretation of the septin-exocyst interactions once it is able to be systematically carried out.

Reviewer #3 (Public Review): 

Septins in several systems are thought to guide the location of exocytosis, and they have been found to interact with the exocyst vesicle-tethering complex in some cells. However, it is not known whether such interactions are direct or indirect. Moreover, septin-exocyst physical associations were not detected in several other systems, including yeasts, making it unclear whether such interactions reflect a conserved septin-exocytosis link or whether they may missed if they depend on septin polymerization or association into higher-order structures. Singh et. al., set out to define whether and how septins influence the exocyst during S. pombe cytokinesis. Based on three lines of evidence, the authors conclude that septins directly bind to exocyst subunits to regulate localization of the exocyst and vesicle secretion during cytokinesis. The conclusions are consistent with the data presented, but some interpretations need to be clarified and extended: 

(1) The first line of evidence examines septin and exocyst localization during cytokinesis in wild-type and septin-mutant or exocyst-mutant yeast. Quantitative imaging convincingly shows that the detailed localization of the exocyst at the division site is perturbed in septin mutants, and that this is accompanied by modest accumulation of vesicles and vesicle cargos. Whether that is sufficient to explain the increased thickness of the division septum in septin mutants remains unclear.

The modest accumulation of vesicles and vesicle cargos at the division site is one of the reasons for the increased thickness of the division septum in septin mutants. It is more likely that the misplaced exocyst can still tether vesicles along the division plane (less likely at the rim) without septins. Due to the lack of the glucanase Eng1 at the rim of the division plane in septin mutants, daughter-cell separation is delayed and then cells continue to thicken the septum. We have added these points to the Discussion.

(2) The second line of evidence involves a comprehensive Alphafold2 analysis of potential pair-wise interactions between septin and exocyst subunits. This identifies several putative interactions in silico, but it is unclear whether the identified interaction surfaces would be available in the full septin or exocyst complexes.  

We thank the reviewer for raising this important point. We fully agree that a key limitation of pairwise AlphaFold predictions is that they do not account for the higher-order structural context of multimeric protein complexes, such as septin hetero-oligomers or the assembled exocyst complex. As a result, some of the predicted interfaces could indeed be conformationally restricted in the native state.

To address this concern, we predicted the S. pombe exocyst and septin structures using AlphaFold3. We mapped predicted contact residues onto the predicted structure. Most predicted interfaces (86% for the exocyst and 86-96% for septins) appear to be located on accessible surfaces in the assembled complexes (Figure supplement S4, S5, videos 4 - video 7), suggesting that these interactions are sterically plausible. We have added this important caveat to the text of the revised manuscript highlighting the interface accessibility within the assembled complexes. We appreciate the reviewer’s insight, which helped us strengthen the interpretation and limitations of the AlphaFold-based analysis.

(3) The third line of evidence uses co-immunoprecipitation and yeast two hybrid assays to show that several physical interactions predicted by Alphafold2 can be detected, leading the authors to conclude that they have identified direct interactions. However, both methods leave open the possibility that the interactions are indirect and mediated by other proteins in the fission yeast extract (co-IP) or budding yeast cell (two-hybrid). 

We thank the reviewer for this important clarification. We agree that coimmunoprecipitation (co-IP) and yeast two-hybrid (Y2H) assays cannot conclusively distinguish between direct and indirect interactions. As the reviewer points out, co-IPs may reflect associations mediated by bridging proteins within the fission yeast extract, and Y2H readouts can be influenced by fusion context or endogenous host proteins. In our manuscript, we have now revised the relevant statements in the Results and Discussion sections to clarify that the observed associations are consistent with direct interactions predicted by AlphaFold2, but cannot alone establish direct binding. We have also tempered our terminology—substituting phrases such as “direct interaction” with “physical association consistent with direct binding,” where appropriate.

(4) Based on prior studies it would be expected that the large majority of both septins and exocyst subunits are present in cells and extracts as stoichiometric complexes. Thus, one would expect any septin-exocyst interaction to yield associations detectable with multiple subunits, yet co-IPs were not detected in some combinations. It is therefore unclear whether the interactions reflect associations between fully-formed functional complexes or perhaps between transient folding intermediates. 

We thank the reviewer for this thoughtful observation. We agree that both septins and exocyst subunits are generally understood to exist in cells as stable, stoichiometric complexes, and that interactions between fully assembled complexes might be expected to yield co-immunoprecipitation signals involving multiple subunits from each complex. However, it was also found that >50% of septins Spn1 and Spn4 are in the cytoplasm even during cytokinesis when the septin double rings are formed (Table 1 of Wu and Pollard, Science 2005, PMID: 16224022). Thus, it is possible that there are pools of free septin and exocyst subunits in the cytoplasm, which were detected in our Co-IP assays. 

In our experiments, we observed selective co-IP signals between certain septin and exocyst subunits, while other combinations did not yield detectable interactions. We believe these findings could reflect several other possibilities besides the possible interactions among the free subunits in the cytoplasm:

(1) Some interactions may only be strong enough between specific subunits at exposed interfaces under the Co-IP conditions, rather than through wholesome complex–complex interactions;

(2) The detergent and/or salt conditions used in our co-IPs may disrupt labile complex interfaces or partially dissociate multimeric assemblies.

To address this concern, we now include in the Discussion a paragraph highlighting the possibility that some of the observed interactions may not reflect binding between fully assembled, functional complexes. Notably, most detected interactions pairs are consistent with the AlphaFold predictions, which suggest specific subunit interfaces may be responsible for mediating contact. While we cannot fully resolve whether septins engage with the whole exocyst complex versus selected subunits, our combined data supports a model that septins scaffold or spatially regulate the exocyst localization at the division site, potentially through dynamic and multivalent interactions. We now explicitly state this more cautious interpretation in the revised manuscript. Future biochemical studies using native complex purifications, cross-linking mass spectrometry, or in vitro reconstitution with fully assembled septin and exocyst complexes, or in vivo FRET assays will be essential to clarify whether the interactions we observe occur between intact assemblies or intermediate forms.

Reviewer #1 (Recommendations for the Authors): 

A major finding from the manuscript is the description of physical interaction of septin subunits with exocyst subunits. The analysis starts from Alphafold2 predictions, shown in Figures 3 and S3. However, some of the most useful metrics of Alphafold, the PAE plot and the pTM and ipTM values, are not provided. It is thus very difficult to estimate the value of the predicted structures (which are also obscured by all side chains). The power of a predicted structure is that it suggests binding interfaces, which is not explored here. At the very least, it would not be difficult to examine whether the proposed binding interfaces are free in the septin filaments and octameric exocyst complex. 

Please also see response to reviewer #1 (Public Review).

We thank the reviewer for these very helpful suggestions. We agree that inclusion of AlphaFold2 model confidence metrics—specifically the Predicted Aligned Error (PAE) plots, as well as pTM and ipTM values—is essential for evaluating the reliability of the predicted septin–exocyst interfaces.

In the revised manuscript, we have now included the PAE plots (Figure 3 and Supplementary S3) and summarizes the pTM scores for each predicted septin–exocyst subunit pair. We also provide a short description of these metrics in the figure legend to help guide interpretation. The old Alphafold2 version (alphafold2advanced) that we used doesn’t give iPTM score, so are not included. However, according to our methodology, we only counted the interacting residues which have pLDDT scores >50%, predicting the resulting iPTM score should not be very weak.

In addition, we have updated Figures 3 and S3 to show simplified ribbon diagrams of the interface regions, with side chains hidden by default and selectively displayed only at predicted interaction hotspots. This improves structural clarity and makes the interface regions easier to interpret. We mentioned in the Discussion that the preliminary studies show that the predicted interacting interfaces of Sec15 and Sec5 with septin subunits are accessible for interaction in the whole exocyst complex. The new Figure Supplement S4 and S5 and Videos 4-7 now show the interface residues of both the exocyst and septins that are involved in the interactions.

Two further points on the interaction: 

The 2H interaction data is not very convincing. The insets showing beta-gal assays do not look very different from the negative control (compare for instance in panel 4E the Sec15BD alone, last column, with the Sec15-BD in combination with Spn4-AD, third column: roughly same color), which suggests it is mostly driven by autoactivation of Sec15-BD. Providing growth information in addition to beta-gal may be helpful. 

We appreciate the reviewer’s close evaluation of the yeast two-hybrid (Y2H) assay data, and we agree that the signals observed in the Spn4–Sec15 combination is indeed weak. Unfortunately, we did not perform growth assays. However, we would like to clarify that this is consistent with the nature of the interactions that we are investigating. The interaction between individual septin and exocyst subunits is not strong and/or transient as supported by the weak interactions by Co-IP experiments. Given the exocyst only tethers/docks vesicles on the plasma membrane for tens of seconds before vesicle fusion, the multivalent interactions between septins and the exocyst should be very dynamic and not be too strong. 

As evidenced by our Co-IP experiments and multivalent interactions predicted by Alphafold2, the interaction between Spn4 and Sec15 is detectable but weak, suggesting that this may be a low-affinity or transient interaction. Given that Y2H assays have known limitations in detecting such low-affinity interactions—especially those that depend on conformational context or are not optimal in the yeast nucleus—it is perhaps not surprising that the X-gal color development is subtle. These limitations of the Y2H system have been well-documented (e.g., Braun et al., 2009; Vidal & Fields, 2014), particularly for interactions with affinities in the micromolar range or those requiring conformational specificity. Therefore, the weak signal observed is in line with expectations for a lowaffinity, transient interaction such as between Spn4 and Sec15.

Vidal, M. and Fields, S., 2014. The yeast two-hybrid assay: still finding connections after 25 years. Nature methods, 11(12), pp.1203-1206.

Braun, P., Tasan, M., Dreze, M., Barrios-Rodiles, M., Lemmens, I., Yu, H., Sahalie, J.M., Murray, R.R., Roncari, L., De Smet, A.S. and Venkatesan, K., 2009. An experimentally derived confidence score for binary protein-protein interactions. Nature methods, 6(1), pp.91-97.

In the coIP experiments, I am confused by the presence of tubulin signal in some of the IPs. For instance, in Fig 4B, but not 4D, where the same Sec15-GFP is immunoprecipitated. There is also a signal in 4C but not 4A. This needs to be clarified. 

The presence of tubulin in some immunoprecipitates is not unexpected, particularly in experiments involving cytoskeleton-associated proteins such as septins and exocyst subunits. The occasional presence of tubulin in our co-IP samples is consistent with well-documented reports showing tubulin as a frequent non-specific co-purifying protein, particularly under native lysis conditions used to preserve large complexes (Vega and Hsu, 2003; Gavin et al., 2006; Mellacheruvu et al., 2013; Hein et al., 2015). The CRAPome database and quantitative interactomics studies highlight tubulin as one of the most common background proteins in affinity-based workflows. Importantly, tubulin was used as a loading control but not as a marker for interaction in our study, and its variable presence does not reflect a specific interaction with Sec15-GFP or other bait proteins, and we have clarified this point in the revised figure legend.

Gavin, A.C., Aloy, P., Grandi, P., Krause, R., Boesche, M., Marzioch, M., Rau, C., Jensen, L.J., Bastuck, S., Dümpelfeld, B. and Edelmann, A., 2006. Proteome survey reveals modularity of the yeast cell machinery. Nature, 440(7084), pp.631-636.

Mellacheruvu, D., Wright, Z., Couzens, A.L., Lambert, J.P., St-Denis, N.A., Li, T., Miteva, Y.V., Hauri, S., Sardiu, M.E., Low, T.Y. and Halim, V.A., 2013. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data. Nature methods, 10(8), pp.730736.

Hein, M.Y., Hubner, N.C., Poser, I., Cox, J., Nagaraj, N., Toyoda, Y., Gak, I.A., Weisswange, I., Mansfeld, J., Buchholz, F. and Hyman, A.A., 2015. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell, 163(3), pp.712-723.

Vega, I.E., Hsu, S.C. 2003. The septin protein Nedd5 associates with both the exocyst complex and microtubules and disruption of its GTPase activity promotes aberrant neurite sprouting in PC12 cells. Neuroreport, 14, pp.31-37.

Regarding the localization of Ypt3 and Syb1 in WT and spn1∆ in Figure 6C-D and Bgs1 in Figure 7A, it would help to add a contractile ring marker to be able to match the timing of cytokinesis between WT and mutants and ensure that cells of same stage are compared (and add some quantification for Ypt3). In fact, in Figure 7A, next to the cells being pointed at, there are very similar localizations of Bgs1 in WT and spn1∆ at the rim of the ingressing septum, which makes me wonder how the quantified cells were chosen. 

For localizations and quantifications of Eng1, Ypt3, Syb1, and Bgs1 shown in Figures 6 and 7, cells with a closed septum (at or after the end of contractile-ring constriction) were quantified or highlighted. To quantify their fluorescence intensity at the division site using line scan, the line width used was 3 pixels. For Syb1 (Figure 6D), we quantified cells at the end of ring constriction (when Rlc1-tdTomato constricted to a dot) in the middle focal plane. The exact same lines were drawn in both Rlc1 and Syb1 channels. The center of line scan was defined as the pixel with the brightest Rlc1 value. All data were aligned by the center and plotted. For Bgs1 (Figure 7A), we quantified the cells that Rlc1 signal had disappeared from the division site. The line was drawn in the Bgs1 channel in the middle focal plane. The center of line scan was defined as the pixel with the brightest Bgs1 value. All data were aligned by the center and plotted. These details were added to the Materials and Methods.

Finally, the manuscript would benefit from some figure reorganization/compaction. Unless work on the binding interfaces is added, Figure 3 and S3 could be removed and summarized by providing the pTM and ipTM values of the predicted interactions. Figure 5 could be combined with Figure 2, as it is essentially a repeat with additional exocyst subunits. 

Because the binding interfaces are added, we keep the original Figures 3 and S3. The experiments in Figure 5 could not be performed before the interaction tests between septins and the exocyst. Thus, to aid the flow of the story, we keep Figures 2 and 5 separated.

Minor comments: 

The last sentence of the first paragraph of the results does not make much sense at this point of the paper. After the first paragraph, there is no evidence that colocalization would be required for proper function.  

We agree that the sentence in question may have overstated the functional implications of colocalization too early in the Results section, before presenting supporting evidence. Our intention was to introduce the hypothesis that spatial proximity between septins and exocyst subunits may be relevant for their coordination during cytokinesis, which we examine in later figures. We have revised the sentence to more accurately reflect the observational nature of the data at this stage in the manuscript as below:

"These observations suggest the spatial proximity between septins and the exocyst during certain stage of cytokinesis, raising the possibility of their functional coordination, which we would further investigate below."

What is the indicated n in Figure 6B? Number of cells? 

Yes, the n in Figure 6B refers to the thin sections of electron microscopy quantified in the analysis. We have now updated the figure legend to explicitly state this for clarity.

The causal inference made between the alteration of Exocyst localization in septin mutants and the thicker septum is possible, but by no means certain. It should be phrased more cautiously. 

We agree that our original phrasing may have overstated the causal relationship between altered exocyst localization in septin mutants and septum thickening. Our data supports a correlation between these phenotypes, but additional experiments would be required to establish direct causality.

To reflect this, we have revised the relevant sentence in the Discussion to read:

“The modest accumulation of vesicles and vesicle cargos at the division site is one of the reasons for the increased thickness of the division septum in septin mutants. It is more likely that the misplaced exocyst can still tether vesicles along the division plane without septins. Due to the lack of the glucanase Eng1 at the rim of the division plane in septin mutants, daughter-cell separation is delayed and then cells continue to thicken the septum.”

Reviewer #2 (Recommendations for the Authors): 

(1) In the display of the AlphaFold Model for the interactions (Figure 3 and Supplemental Figure 3) it is difficult to identify which subunits are where. Residue numbers and subunits should be labeled and only side chains important for the interactions should be present in the model. 

We appreciate this valuable suggestion. We agree that clearer visual labeling is essential for interpreting the predicted interactions and have revised Figures 3 and S3 accordingly to improve readability and emphasize key structural features.

Specifically, we have:

• Labeled each subunit with its name and color-coded consistently across panels.

•  Annotated key interface residues with residue numbers directly in the figure.

• Removed non-interacting side chains to declutter the model and highlight only those involved in predicted interactions as well as expanded the figure legend for explanation.

(2) In Table 1 the column label "Genetic Interaction at 25C" is confusing when synthetic growth defects are shown with a "plus". Rather this column could be labeled "Growth of double mutants at 25C" and then designate the relative growth rate observed at 25C as in Table 2. Designating a negative effect on growth with a plus is confusing. 

Thanks for the thoughtful suggestions. We have made the suggested changes by deleting the last column so that Tables 1 and 2 are consistent.

(3) In Figure 4, why is tubulin being co-immunoprecipitated in two of the four anti-GFP IPs? Are the IPs dirty and if so why does it vary between the four experiments? If they are dirty can the non-specific tubulin be removed by additional washes with IP buffer or conversely is it necessary to do minimal washes in order to detect the exocyst-septin interaction by coIP? A comment on this would be helpful. 

The presence of tubulin in some immunoprecipitates is not unexpected, particularly in experiments involving cytoskeleton-associated proteins such as septins and exocyst subunits. The occasional presence of tubulin in our co-IP samples is consistent with welldocumented reports showing tubulin as a frequent non-specific co-purifying protein, particularly under native lysis conditions used to preserve large complexes (Vega and Hsu, 2003; Gavin et al., 2006; Mellacheruvu et al., 2013; Hein et al., 2015). The CRAPome database and quantitative interactomics studies highlight tubulin as one of the most common background proteins in affinity-based workflows. Importantly, tubulin was used as a loading control but not marker for interaction in our study, and its variable presence does not reflect a specific interaction with Sec15-GFP or other bait proteins, and we have clarified this point in the revised figure legend.

Gavin, A.C., Aloy, P., Grandi, P., Krause, R., Boesche, M., Marzioch, M., Rau, C., Jensen, L.J., Bastuck, S., Dümpelfeld, B. and Edelmann, A., 2006. Proteome survey reveals modularity of the yeast cell machinery. Nature, 440(7084), pp.631-636.

Mellacheruvu, D., Wright, Z., Couzens, A.L., Lambert, J.P., St-Denis, N.A., Li, T., Miteva, Y.V., Hauri, S., Sardiu, M.E., Low, T.Y. and Halim, V.A., 2013. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data. Nature methods, 10(8), pp.730736.

Hein, M.Y., Hubner, N.C., Poser, I., Cox, J., Nagaraj, N., Toyoda, Y., Gak, I.A., Weisswange, I., Mansfeld, J., Buchholz, F. and Hyman, A.A., 2015. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell, 163(3), pp.712-723.

Vega, I.E., Hsu, S.C. 2003. The septin protein Nedd5 associates with both the exocyst complex and microtubules and disruption of its GTPase activity promotes aberrant neurite sprouting in PC12 cells. Neuroreport, 14, pp.31-37. 

In response to the second part of reviewer’s comment, we washed the pulldown product for 5 times each time with 1 ml IP buffer at 4ºC. We used this standard protocol for all the Co-IP experiments to detect the interaction between different septin-exocyst subunits. So, we are not sure if and how more washes or more stringent buffer conditions can interfere with detection of the interactions.

Reviewer #3 (Recommendations for the Authors): 

In addition to the issues noted in the public review, there were some confusing findings and references to previous literature that merit further consideration or discussion: 

• The current gold standard for validating Alphafold predictions involves making targeted mutants suggested by the structural predictions. The absence of any such validation weakens the conclusions significantly. 

We agree that the targeted mutagenesis based on AlphaFold2-predicted interaction interfaces represents a powerful approach to experimentally validate the in silico models. While we did not pursue structure-guided mutagenesis in this study, our goal was to identify putative interactions between septin and exocyst subunits as a foundation for future functional work. Our current conclusions are intentionally limited to proposing putative interfaces, supported by co-immunoprecipitation and genetic interaction data.

We recognize that direct validation of specific contact residues would significantly strengthen the model. Accordingly, we have revised the Discussion to explicitly state this limitation and to note that structure-based mutagenesis will be an important next step to test the functional relevance of predicted interactions. We have added the following statement:

“Future studies are needed to refine the residues involved in the interactions because the predicted interacting residues from AlphaFold are too numerous. However, it is encouraging that most of the predicted interacting residues are clustered in several surface patches. Experimental validation through targeted mutagenesis is an important next step.”

• Much of the writing appears to imply that differences in mutant phenotypes indicate differences in septin (or exocyst) subunit behaviors/functions. However, my reading of the work in budding yeast is that such differences reflect the partial functionality that can be conferred by aberrant partial septin complexes that assemble and may polymerize in mutants lacking different subunits. In this view, which is supported by data showing that essentially all septins are in stoichiometric octameric complexes in cells, the wild-type functions are all mediated by the full complex. Similarly, the separate exocyst subunit localizations based on tagged Sec3 (Finger et al) were not supported by later work from the Brennwald lab with untagged Sec3, and the idea that different exocyst subunits may function separately from the full complex has very limited support in yeast. I would suggest that the text be edited to better reflect the literature, or that different views be better justified. 

Thanks for the suggestions. We have revised the text accordingly.

• The comprehensive set of Alphafold2 predictions is a major strength of the paper, but it is unclear to this reader whether the multiple predicted interactions truly reflect multivalent multimode interactions or whether many (most?) predictions would not be consistent with interactions between full complexes and may not indicate physiological interactions. Better discussion of these issues is needed to interpret the findings. 

We appreciate the reviewer’s suggestion to use structural prediction to further assess interaction plausibility. We have now employed the full Saccharomyces cerevisiae exocyst complex (with 4.4 Å resolution) published by the Guo group to examine the interfaces of septins and the exocyst interactions, assuming that the S. pombe exocyst has the similar structure. We mapped predicted contact residues onto the predicted structure. Most predicted interfaces (86% for the exocyst and 86-96% for septins) appear to be located on accessible surfaces in the assembled complexes (Figure supplement S4, S5, videos 4 - video 7), suggesting that these interactions are sterically plausible. We have added this important caveat to the text of the revised manuscript highlighting the interface accessibility within the assembled complexes. We appreciate the reviewer’s insight, which helped us strengthen the interpretation and limitations of the AlphaFold-based analysis.

• Some but not all co-IP blots appear to show tubulin (negative control) coming down with the GFP pull-downs. Why is that, and what does it imply for the reliability of the co-IP protocol? 

The presence of tubulin in some immunoprecipitates is not unexpected, particularly in experiments involving cytoskeleton-associated proteins such as septins and exocyst subunits. The occasional presence of tubulin in our co-IP samples is consistent with welldocumented reports showing tubulin as a frequent non-specific co-purifying protein, particularly under native lysis conditions used to preserve large complexes (Vega and Hsu, 2003; Gavin et al., 2006; Mellacheruvu et al., 2013; Hein et al., 2015). The CRAPome database and quantitative interactomics studies highlight tubulin as one of the most common background proteins in affinity-based workflows. Importantly, tubulin was used as a loading control but not a marker for interaction in our study, and its variable presence does not reflect a specific interaction with Sec15-GFP or other bait proteins, and we have clarified this point in the revised figure legend.

Gavin, A.C., Aloy, P., Grandi, P., Krause, R., Boesche, M., Marzioch, M., Rau, C., Jensen, L.J., Bastuck, S., Dümpelfeld, B. and Edelmann, A., 2006. Proteome survey reveals modularity of the yeast cell machinery. Nature, 440(7084), pp.631-636.

Mellacheruvu, D., Wright, Z., Couzens, A.L., Lambert, J.P., St-Denis, N.A., Li, T., Miteva, Y.V., Hauri, S., Sardiu, M.E., Low, T.Y. and Halim, V.A., 2013. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data. Nature methods, 10(8), pp.730736.

Hein, M.Y., Hubner, N.C., Poser, I., Cox, J., Nagaraj, N., Toyoda, Y., Gak, I.A., Weisswange, I., Mansfeld, J., Buchholz, F. and Hyman, A.A., 2015. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell, 163(3), pp.712-723.

Vega, I.E., Hsu, S.C. 2003. The septin protein Nedd5 associates with both the exocyst complex and microtubules and disruption of its GTPase activity promotes aberrant neurite sprouting in PC12 cells. Neuroreport, 14, pp.31-37.

• Why were two different protocols used for different yeast-two-hybrid analyses? 

The purpose of using two protocols was to test which protocol is more reliable and sensitive.

• The different genetic interactions between septin and exocyst mutants when combined with TRAPP-II mutants merits further discussion: might the difference reflect relocation of exocyst from rim to center in septin mutants versus inactivation of exocyst in exocyst mutants? 

We appreciate this insightful comment and agree that this distinction is likely meaningful. The reviewer correctly notes that septin mutants may not abolish exocyst function but rather cause its spatial mislocalization: from the rim to the center of the division site, whereas the exocyst mutants likely result in partial or complete loss of vesicle tethering activity at the plasma membrane.

To address this important nuance, we have expanded the Discussion as follows:

“The genetic interactions between mutations in the exocyst and septins when combined with TRAPP-II mutants may reflect fundamentally different consequences for compromising the exocyst function (Tables 1 and 2). In septin mutants, the exocyst complex still localizes to the division site but is mispositioned from the rim to the center of the division plane. This mislocalization allows partial retention of exocyst function, leading to very mild synthetic or additive defects when combined with compromised TRAPP-II trafficking and tethering. In contrast, in exocyst subunit mutants, the exocyst becomes partial or non-functional, resulting in a more severe loss of exocyst activity. These differing consequences could explain the qualitative differences in genetic interactions observed with TRAPP-II mutants (Tables 1 and 2). Thus, septins and the exocyst also work in different genetic pathways for certain functions in fission yeast cytokinesis.”

• The vesicle accumulation in septin mutants was quite modest. Does that imply that most vesicles are still fusing in the septum? Further discussion would be beneficial to understand what the authors think this means. 

We thank the reviewer for this important point. We agree that the modest vesicle accumulation observed in septin mutants suggests that a significant proportion of vesicles continue to successfully fuse at the division site, even in the absence of fully functional septin structures.

We now discuss this in greater detail in the revised manuscript:

“The relatively modest vesicle accumulation in septin mutants suggests that septins are not absolutely required for vesicle tethering or fusion per se at the division site. Instead, septins primarily function to spatially organize the targeting sites of exocyst-directed vesicles by stabilizing the localization of the exocyst at the rim of the cleavage furrow. In septin mutants, mislocalization of the exocyst reduces the spatial precision of membrane insertion but still permits vesicle tethering and fusion, albeit in a less controlled manner. Thus, septins likely play a modulatory rather than essential role in exocytic vesicle delivery during cytokinesis. This interpretation aligns with our localization and genetic interaction data, which indicates that septins act as scaffolds to optimize secretion geometry, rather than as core components of the fusion machinery.”

• It was unclear to this reader why relocation of some exocyst complexes from the rim to the center of the septal region would lead to dramatic thickening of the septum. Further discussion would be beneficial to understand what the authors think this means. 

The modest accumulation of vesicles and vesicle cargos at the division site is one of the reasons for the increased thickness of the division septum in septin mutants. It is more likely that the misplaced exocyst can still tether vesicles along the division plane without septins. Because of the lack of glucanase Eng1 at the rim of the division plane in septin mutants, daughter-cell separation is delayed and then cells continue to thicken the septum. We have added these points to the Discussion.

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encontre esta informacion, favor de checar si es de utilidad

📜 Timeline comparado Marco Antonio (83–30 a.C.)

83 a.C. → Nace en Roma.

41 a.C. → Conoce a Cleopatra en Tarso; empieza la relación.

36–31 a.C. → Gobierno conjunto con Cleopatra en Oriente, hijos en común.

31 a.C. → Derrota en Accio frente a Octavio.

30 a.C. → Se suicida en Alejandría tras la caída de Cleopatra. 👉 Ejemplo clásico de cómo el amor debilitó al gobernante.

Tito (39–81 d.C.)

39 d.C. → Nace en Roma, hijo de Vespasiano.

66 d.C. → Conquista Jerusalén; conoce a Berenice.

70 d.C. → Regresa a Roma, relación abierta con Berenice.

79 d.C. → Sube al trono como emperador. Presiones políticas → debe enviarla lejos.

81 d.C. → Muere joven, sin volver a verla. 👉 Amó a Berenice, pero el poder lo obligó a sacrificarla.

Adriano (76–138 d.C.)

76 d.C. → Nace en Itálica (Hispania).

100 d.C. → Se casa con Vibia Sabina (sobrina nieta de Trajano), matrimonio conflictivo.

117 d.C. → Sube al trono tras la muerte de Trajano (y la presión de Plotina).

123 d.C. → Viaja por Grecia; conoce a Antínoo (su gran amor).

130 d.C. → Muerte de Antínoo en el Nilo (misteriosa: ¿accidente, suicidio, sacrificio?). Adriano lo deifica y funda la ciudad de Antinoópolis.

138 d.C. → Muere en Baiae. 👉 A diferencia de Tito y Marco Antonio, Adriano no perdió el poder por amor, pero lo sublimó en culto y memoria (Antínoo convertido en dios).

1. The graph is looking at the evolution across 5 linguistic groups (French, Spanish, Germanic, and Arabic) for the amount of those who it is their official language. The y-axis denotes number of people in millions, while the x-axis denotes years from 1965 to projected values of 1965. these later values display French as exponentially growing while other language plateua or steadily rise at a slower rate.

okay, so Bliss definitely thinks very highly of science; he regards it well over the idea of culture and communit,y I believe? I

La PTH nunca va a interactuar directamente sobre los osteoclastos, porque los osteoclastos no tienen receptores para PTH. Únicamente van a interactuar con osteoblastos y los osteoblastos van a sintetizar lo que necesitan los osteoclastos para empezar a realizar su función.

Un compilador es un programa que toma el código de alto nivel, como lo es Java y este lo traduce a lenguaje de computadora que el sistema operativo puede procesar directamente.

Segmentation?

Reviewer #1 (Public review):

This is my first review of this manuscript. The authors included previous reviews for a different journal with a length of 90 and 39 pages; I did not review this reply in my assessment of the paper itself. Influenza prediction is not my area of expertise.

A major concern is that the model is trained in the midst of the COVID-19 pandemic and its associated restrictions and validated on 2023 data. The situation before, during, and after COVID is fluid, and one may not be representative of the other. The situation in 2023 may also not have been normal and reflective of 2024 onward, both in terms of the amount of testing (and positives) and measures taken to prevent the spread of these types of infections. A further worry is that the retrospective prospective split occurred in October 2020, right in the first year of COVID, so it will be impossible to compare both cohorts to assess whether grouping them is sensible.

The outcome of interest is the number of confirmed influenza cases. This is not only a function of weather, but also of the amount of testing. The amount of testing is also a function of historical patterns. This poses the real risk that the model confirms historical opinions through increased testing in those higher-risk periods. Of course, the models could also be run to see how meteorological factors affect testing and the percentage of positive tests. The results only deal with the number of positive (only the overall number of tests is noted briefly), which means there is no way to assess how reasonable and/or variable these other measures are. This is especially concerning as there was massive testing for respiratory viruses during COVID in many places, possibly including China.

(1) Although the authors note a correlation between influenza and the weather factors. The authors do not discuss some of the high correlations between weather factors (e.g., solar radiation and UV index). Because of the many weather factors, those plots are hard to parse.

(2) The authors do not actually compare the results of both methods and what the LSTM adds.

Minor comments:

(3) The methods are long and meandering. They could be cleaned up and shortened. E.g., there is no need for 30 lines on PCR testing; the study area should come before the study design. The authors discuss similar elements in multiple places; this whole section can be shortened considerably without affecting the content.

(4) How reliable is the "Our Word in Data" website for subnational coverage of restrictions? Some of the authors are from Putian and should be able to confirm the accuracy for both studied areas.

(5) Figure 2A is hard to parse; it would make more sense to plot these as line plots (y=count, x=month).

This small paragraph stood out to me because of the high statistical number of older adult who experience agism. This shows that agism is happening much more than we realize and not in good ways. The most common form is internalized which means that older people are even thinking of this of themselves.

This part of the data was surprise but not to me. It shows that women experienced more ageism than man have. I wonder why is this? Is this because many assume that men "do more" for them selfs and can be more stubborn. This also makes he wander if women are just more open to talk about it then men as well. I assume that men have a harder time being honst about agesim than possibly women.

This sentence is very interesting to me, because it says that only 7.2% of the group rated their mental health as fair/poor, but 32.3% say that they have depressive symptoms. This suggests that many participants experiencing depressive symptoms may not interpret or label their overall mental health as being “poor.” This tells me that the elderly population either does not want to discuss mental health issues, or is not properly informed about mental health.

I liked how this section was incorporated since there is not one specific reason for everyday ageism. I also like how it shows the difference in how the use of social media can change someone. This can help people caring for this population to understand factors that have gone into everyday ageism.

Ok, somehow you like to stick to this finite definition of the population. In the next sentence, however, you say (correctly) that each possible sample must have a non-zero probability. In real life: you could select a sample plot at any point in the total forest area. In a finite population view this is not the case (and somehow you are limited to quadratic cells as plots, which is not in line with what you explain later)?? Why is it so important to define the population as finite here? Imagine we conduct SRS by random selection of x,y coordinates in the area (and install plots there). Then we are outside of your population concept (the population of possible plots is infinite).

should look like. We believe in diversity.

7ww w.si deways.earth

Main benefits

• Exchange and commerce
• Cross-organization dispute resolution
• International recognition
• Member hosting and benefits across organizations
• Values alignment / adherence to standards

DAOs vouched

Enlever les majuscules

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La herramienta 3D de ixina permite diseñar una cocina en unos pocos clics, incluso aunque no tengas experiencia previa con este tipo de herramientas online. Su interfaz intuitiva te va guiando paso a paso. Te ayuda a tener en cuenta aspectos fundamentales como la ergonomía: la altura ideal de las encimeras según tu estatura, la accesibilidad de los muebles altos o la ubicación óptima de los electrodomésticos respecto a las zonas de trabajo de la cocina. Cada paso está diseñado para que el resultado sea funcional, estético y perfectamente adecuado a tus necesidades.

Consultar el catélogo online y dejarme guiar para crear mi proyecto

Here \(p\) is the logical exclusive or (XOR) function. It is usually denoted

$$ p(x, y) = x \oplus y. $$

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