I think this is a good reminder that just because two things look related in data doesn’t mean one causes the other. The candle review and COVID example is kind of funny, because it makes sense once you realize both just happen more in the fall. When there’s a lot of data, it feels easy to find patterns that aren’t actually meaningful. This part made me think we should slow down and question what the data is really showing.

This section shows that data mining can reveal surprising patterns, like predicting someone’s interests or political views from social media behavior. However, the part about spurious correlations is especially important. Just because two things move together does not mean one causes the other. The candle reviews and COVID example was funny but also a good reminder that data can be misleading. As a data science student, this makes me realize we must be careful when interpreting results and avoid jumping to conclusions too quickly.

I have noticed a lot of these on social media they like to ask you about these kinds of things, and we have to be careful because AI nowadays gives old ideas a new sense of accuracy, even though the science behind them is still wrong. It also shows how technology can reinforce bias instead of eliminating it if it’s not used

This highlights the classic problem of spurious correlation, where two trends move together without a direct causal link. For example, ice cream sales and drowning incidents are correlated because both increase in the summer, but eating ice cream does not cause drowning, seasonal factors drive both patterns.

With so much information about individuals being publicly available in the age of social media, it is very easy for people to find information about anyone — whether they are hackers who can access specific metadata or just a passing by user who observes your profile. It is interesting to observe how people’s behavior has changed over the last decade as our lives become more and more public.

In this footnote, Stowe sets the historical record straight by acknowledging the true inventor of the cotton gin, a technology that increased the demand for slave labor. In doing so, she also makes a subtle but powerful commentary on the way that American history has ignored the achievements of African American slaves. Furthermore, this footnote challenges the reader to think critically about the way that slavery was used to both oppress and exploit African American ingenuity.

I think the story does have issues but I think this line truly shows the issues with it. Of course there were people who were enslaved that were light-skin and therefore it made it easier for them to "pass" as white, but the reason this line is important is that because she is "pass-able" she doesn't have the same "accent" as her other counter-parts. Why is it that she is lighter therefore her pronunciation is better. Personally I think this encapsulates the issue at hand.

It’s so intresting that this was for a time taught as history as truth. But over time it was learned to no be true it makes me think what are we teaching now that will be proven to be not true.

Accidents do happen within history, like something happening not on purpose, but truly history does not have many accidents. Everything has causes that go into every event. But even with a true 100% accident, there are many causes that come from it. Nothing can truly happen within history without flowers of reactions that bloomed from action of the event.

This shows how early digital library design ignored user experience, making access frustrating instead of helpful, as it hsould actually be.

Besser argues that digital libraries are about new possibilities, not just things like speed or convenience.

This highlights that libraries are defined not only by what they contain, but by how they serve users and uphold values like privacy and fairness.

The author emphasizes that digital libraries aren’t just about easy access, they change how research can be done. It’s a shift from visiting a library to interacting with the material in new computational ways.

Based off of this quote, it sounds like Romeo is a very secretive and private person and doesn't like to express his feelings like how worms destroy plants before they blossom.

This paragraph defines key terms that describe how materials move and change within a watershed. It shows that matter moves through the watershed in stages. Materials must first be usable, then move, and finally become part of the ecosystem.

April 3 letter - Indian victory and accelerating catastrophe: the Pinnace capture arms/ammunition, kill the captain, and muster 200 canoes with 1000 warriors. The English are outnumbered and outgunned. This escalation shows the colony is losing control and facing near-total annihilation.

P.S. escalates the urgency - news of 26 English deaths and Indians capturing weapons means the colony's vulnerability is increasing. Indians can now use English weapons and disguise themselves as English. Frethorne frames this as life-or-death urgency requiring immediate parental intervention.

Emotional climax of the letter - Frethorne explicitly states this letter determines life or death for him. The phrase 'the Answer of this letter wilbee life or death to me' shows he sees this as his last desperate hope for parental rescue/redemption from servitude and misery.

Goodman Jackson represents human compassion in a harsh world - he treats Frethorne like a son/father, provides shelter and food. This contrast highlights both the cruelty of the system and the possibility of mercy. Jackson becomes Frethorne's lifeline and hope for redemption.

Military vulnerability and death - the colony is drastically weakened. Of the original 20 colonists and additional settlers, only a few survive. They captured two Indians but admit they are drastically outnumbered 32 to 3000, showing the precarious position of English colonists.

Food shortage is critical - workers are given only pease, water gruel, bread, and beef. The description shows extreme rationing where one small loaf must feed four men, showing colonial inability to feed settlers adequately.

Frethorne emphasizes his extreme poverty - he literally has no possessions, not even basic clothing. This illustrates the desperate conditions colonists faced in Virginia.

This quote explains that she knows that the tragedy will happen which removes suspense and the tragedy feels unavoidable. Theme of fate.

The section on microinterventions and microresistance makes me think about how public figures on social media call out subtle bias and offer support to targets of discrimination. On platforms like TikTok or Twitter, small acts like explaining why a comment hurts — even to strangers — can have a ripple effect, encouraging others to rethink their words. It connects the chapter’s leadership concepts to real-world interpersonal influences in everyday life, showing that leadership isn’t just formal authority but also everyday relational action.

This would always happen with my siblings. My older brother wanted to hug my younger brother and he would exaggerate and tell my mother that my older brother wanted to hit him and so that’s where my mom would ask my brother what happened? And he would respond with, “I just wanted to give him a hug”, but in this case they would both get in trouble. One for being exaggerated and the other one for “bothering”.

A connection that I made with this is , when we’re all young we usually get in trouble by our parents. although when for example, my family and I would visit a friends house, my parents would tell us to behave because they did not want to get on us in front of their friends. When we were in that friends house just by my mom’s look or my dads action I knew it was time for me to behave. So I connect these actions as being an unspoken rule. We see often how parents don’t even have to say nothing to their kids, but just by giving them a look, that child knows to behave. This shows how much “power” or “respected” parents can and have .

Reading this reminded me of Taylor Swift's song "Death by a Thousand Cuts" where she perfectly captures the feeling of the breaking point that microaggressions can lead to. Popular culture has the unique ability to convey lasting messages through art. Taylor Swift has undoubtedly played an integral role in this. Her music gives words to feelings we often can't put words to. While both Taylor Swift and I are part of many privileged demographics, we can also relate to microaggressions in the sense that we are women.

Simply the way our society is designed to confine women to their traditional role instead of encouraging them to thrive is an example of this. Taylor says in her song "My heart, my hips, my body, my love//Trying to find a part of me that you didn't touch." Society both subtly and unsubtly places expectations and policies on every aspect of women's lives. Like we discussed last class, just because things have gotten dramatically better for women, that does not mean they are equitable. Taylor describes the feeling that accompanies the exhaustion of this fight by saying "Gave you too much but it wasn't enough//But I'll be all right, it's just a thousand cuts." All of the small stabs women take on a daily basis might just lead to "death by a thousand cuts" if they are not addressed, and still we will persist as we bleed out.

One connection I made was interpersonal power and group dynamics that I’ve seen in real life. These sentences explain that power doesnt have to be formally put into the world to exist, but it can show up through unspoken rules and patterns. I’ve seen this in classrooms and peer groups since being in college where certain student's opinions carry more weight even when they are not professors or TA's. This connects to social hierarchies and sometimes even bias, where influence can be socially constructed rather than formally stated like how a professor and student are explicitly laid out. It helped me see that leadership responsibility has to include noticing these invisible power patterns, not just formal authority roles that are laid out obviously.

Question 1: How should a counselor address a microaggression that happened in the life of a client, while validating that the "micro" aggression may have not had a "micro" effect on the client's life?

Peer review can feel scary because you may feel uncomfortable sharing your writing at first, but remember that each writer is working toward the same goal: improving their writing and creating the best final draft possible.

It reassures you that even though sharing your writing can feel intimidating, everyone involved is working toward improving together. As the reader they can let you know if your writing is lacking something.

Las Casas shows how the Spaniards viewed the Indigenous people as worthless. This helped us understand why they were so extremely cruel and violent.

This shows that almost the entire population was wided out because of the Spanish's violence and abuse to the Native people.

Las Casas says that the Native people were peaceful. They also said that the Spaniards attacked them badly like they were wild animals.

I remember a few years back playing a video game. I forget which one, but at some point a character said something along the lines of "After everything, all you've got to show is some metal," in relation to the pursuit of gold. That's always stuck with me. Metal has value, yes, but it's not worth blood. People shouldn't die for it, and no man with an ounce of honor should kill for it. I think that Bartolomé would've thought much the same.

Although the religious focus feels strange to me, as someone who isn't religious, I also know that this is a clear sign of how deeply his compassion for these people ran. To see them as children of God in his age was radical and needed.

I can't imagine why the brutality was so common in these killings. I get that you need to send a message, but this is cruel on every level imaginable.

This is one of the most humbling facts of history. We aren't immune to the world, even when we think we've claimed it. A million deaths brought not by conquest or famine, but disease. It's a terrible, almost insulting way for a society to fall.

This basically was invented for newspapers, but had a bigger impact on the magazine area. It makes sense because magazines are less strict in terms of deadlines for certain materials. What I highlighted explains how the magazines had the outlet to express themself more for longer and detailed writing.

With everyone turning to interpretive journalism, it became a problem when the writers had to decipher real news from opinion. What they did was make two separate sections for facts and opinions. This basically tells us that both fact and opinion matter when reading texts.

Extreme case, but effects vary

Transitions in a paragraph guide readers and show how ideas connect. They are usually single words or short phrases that help the reader follow the flow of sentences and ideas.

Topic sentences should be specific and clear explain the main idea of a paragraph. In expository writing, each paragraph should focus on one idea that directly supports the thesis.

Strong primary support in writing should be specific, relevant to the thesis, and detailed. Specific examples make ideas clearer and more convincing than general ones.

An outline is a plan that organizes ideas from prewriting and helps structure paragrpah before drafting. Keeping the writing purpose in mind helps decide how ideas are grouped and ordered. Writers can organize their paper using chronological order, spatial order, or order of importance

doing things like multitasking, isnt always a good thing because people think you can work quicker that way but you cant

Teaching My Neighbor to Keep the Volume Down

• The Setup: In 2007, the author moved to an apartment where Dish Network was the only option. He upgraded to a DVR package that included an RF (Radio Frequency) remote, allowing him to control the TV from anywhere in the apartment without line-of-sight.
• The Problem: A loud neighbor moved in and also acquired Dish Network with an RF remote. Because they shared the same default frequency, the author's remote experienced interference, and he realized his remote could control the neighbor's set-top box.
• The Conflict: The neighbor frequently played his TV at high volume. The author attempted to visit the neighbor to explain the technical interference issue and build rapport, but the neighbor was rude, yelled "I'm not buying," and slammed the door.
• The Solution: Instead of reprogramming his remote to avoid interference, the author decided to "train" the neighbor. He kept the RF remote in his bedroom and established a rule: if the neighbor's volume exceeded a specific threshold (estimated level 15-20), he would use the remote to turn the neighbor's device off.
• The Result: Through "Pavlovian conditioning," the neighbor eventually learned that keeping the volume low kept the TV on, while raising it caused the TV to shut off. The author successfully conditioned the neighbor to maintain a lower volume without ever speaking to him again.

Hacker News Discussion

• Counter-Tactics: The top comment shared a similar revenge story where a user blasted System of a Down's "Chop Suey" at 4 AM to retaliate against a neighbor who watched loud reality TV late at night; the neighbor eventually complained and changed their behavior.
• Technical Warfare: Commenters discussed various gadgets for dealing with noisy neighbors, such as the "STFU" device which purportedly pipes audio back directionally, and directional ultrasound speakers.
• Pavlovian Experiments: Another user described using a fake smoke detector with an annoying buzzer triggered via Bluetooth to "train" a neighbor to stop smoking on their balcony, mirroring the blog post's conditioning theme.
• Skepticism and Meta-Commentary: Several users questioned the veracity of these "tall tales," leading to a debate about whether Hacker News is becoming more like Reddit.
• Morning vs. Night: A sub-thread debated the consideration gap between "morning people" (who make noise early) and "night people" (who make noise late), with users arguing that society unfairly favors morning noise.
• Escalation: Anecdotes ranged from passive-aggressive automated systems to extreme physical confrontations, such as a neighbor throwing a loud stereo through a window.

Document d'Information : Les Fonctions Exécutives chez l'Enfant

Résumé Exécutif

Ce document synthétise les perspectives de Julie Beaulieu, docteure en psychopédagogie, sur les fonctions exécutives chez l'enfant.

Considérées comme le « chef d'orchestre » ou la « tour de contrôle » du cerveau, ces habiletés cognitives de haut niveau, situées dans le lobe frontal, sont fondamentales pour la régulation des pensées, des émotions et des comportements.

Elles permettent à l'individu de s'adapter à son environnement et d'atteindre des objectifs.

Leur développement s'étend sur une longue période, débutant dès les premiers mois de la vie pour atteindre leur plein potentiel vers l'âge de 25 ans, avec des phases de progression accélérée durant la période préscolaire (3-7 ans) et au début de l'adolescence.

Si la maturation cérébrale est un facteur essentiel, l'environnement et la diversité des expériences vécues par l'enfant jouent un rôle crucial pour « aiguiser » ces fonctions.

Quatre composantes principales sont identifiées : la mémoire de travail, l'inhibition, la flexibilité cognitive et la planification.

Ces fonctions, bien que distinctes, sont interdépendantes et fonctionnent en synergie, à l'image d'une « toile d'araignée ».

Elles sont la fondation de nombreuses aptitudes et ont un impact direct sur la réussite éducative et le développement global de l'enfant (social, langagier, moteur).

Le soutien parental est déterminant et s'intègre dans les activités quotidiennes.

Les stratégies efficaces incluent le jeu, les interactions sociales, le questionnement (notamment le « pourquoi »), l'offre de choix, la stimulation de l'autonomie et de la créativité, ainsi que la mise en place d'un environnement sécurisant et positif.

L'objectif n'est pas d'entraîner ces fonctions comme un muscle, mais de les mobiliser à travers des expériences ludiques et variées.

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1. Définition et Nature des Fonctions Exécutives

Les fonctions exécutives sont définies comme un ensemble de processus cognitifs de haut niveau localisés dans le lobe frontal du cerveau.

Elles sont essentielles pour la réalisation de tâches complexes et pour l'adaptation de l'individu à son environnement.

Leur rôle principal est de réguler trois aspects fondamentaux du comportement humain :

• Les pensées : Organiser ses idées, raisonner, réfléchir.

• Les comportements : Contrôler ses actions, initier des tâches, s'ajuster.

• Les émotions : Gérer ses réactions émotionnelles.

Ces fonctions sont mobilisées en continu dans la vie quotidienne, dès qu'une tâche n'est pas automatique ou routinière et demande de réfléchir, raisonner, anticiper, planifier ou diriger son attention.

La Métaphore du Chef d'Orchestre

Pour illustrer leur rôle, Julie Beaulieu utilise deux analogies principales :

• La tour de contrôle : Elles permettent de se contrôler et de diriger ses actions.

• Le chef d'orchestre : C'est la métaphore la plus développée.

Les fonctions exécutives agissent comme le chef d'orchestre du cerveau, coordonnant l'ensemble des autres habiletés cognitives (les "instruments") pour produire un comportement harmonieux et adapté à un objectif précis.

Elles dirigent les pensées, les émotions et les comportements pour permettre à l'individu de s'adapter et de répondre efficacement aux exigences de son environnement.

2. Le Développement des Fonctions Exécutives

Le développement des fonctions exécutives est un processus long et progressif, influencé par la maturation biologique et les expériences environnementales.

Chronologie du Développement

• Début : Les composantes des fonctions exécutives commencent à se développer dès les premiers mois de la vie.

• Maturité : Elles atteignent leur plein potentiel au début de l'âge adulte, vers 25 ans.

• Périodes Clés : Deux périodes sont identifiées comme particulièrement propices à une progression importante :

1. L'âge préscolaire (3 à 6-7 ans).    2. Le début de l'adolescence.

Il est crucial de noter que le rythme de développement varie d'un enfant à l'autre, en fonction de facteurs individuels comme la maturation de leur propre cerveau.

Le Rôle Crucial de l'Environnement

La maturation du cerveau est une composante essentielle, mais l'environnement dans lequel l'enfant évolue est un moteur fondamental du développement.

• L'expérience : C'est principalement à travers les expériences vécues que les enfants mobilisent et développent leurs fonctions exécutives.

• L'aiguisage : Plus les fonctions sont mobilisées, plus elles s'« aiguisent », devenant « sophistiquées et spécialistes ».

• La variété : Proposer des contextes et des expériences variés permet de mobiliser un plus large éventail de fonctions exécutives et de favoriser un développement plus complet.

Le soutien offert par l'entourage (parents, éducateurs) est donc primordial.

3. Contextes Favorables au Développement

Certains contextes et situations sont particulièrement efficaces pour mobiliser et renforcer les fonctions exécutives, car ils empêchent l'enfant de recourir à des automatismes.

• Situations nouvelles : Toute situation où l'enfant n'a pas de routine établie l'oblige à s'adapter et donc à mobiliser ses fonctions exécutives.

• Tâches complexes : Les activités qui demandent plusieurs étapes, actions ou une réflexion soutenue.

• Situations inattendues : Les imprévus qui forcent une réévaluation et une adaptation.

• Résolution de problèmes : Chercher des solutions à un conflit ou à une difficulté pratique.

• Le jeu : Un contexte extrêmement favorable, en particulier :

◦ Le jeu symbolique (« faire semblant ») : Se mettre dans la peau d'un personnage.  

◦ Les jeux de société : Qui demandent de la stratégie, de la mémoire et de l'anticipation.

• Interactions sociales : Les discussions, les échanges et les conversations demandent une réflexion constante, une anticipation des réponses et une adaptation à l'interlocuteur.

• Découvertes et nature : Explorer de nouveaux environnements.

• Projets à long terme : Les projets qui s'étalent sur plusieurs jours ou semaines et nécessitent de la planification.

• Environnement stimulant et sécurisant : Un climat familial positif, où l'enfant se sent en sécurité, est plus propice à la mobilisation des fonctions exécutives.

Un stress élevé ou des émotions négatives intenses peuvent entraver leur fonctionnement.

4. Les Quatre Composantes Principales des Fonctions Exécutives

L'exposé se concentre sur quatre composantes fondamentales, qui sont interdépendantes et fonctionnent comme une « toile d'araignée ».

| Composante | Description | Exemple Clé | | --- | --- | --- | | Mémoire de travail | Capacité à retenir temporairement et à manipuler l'information pour l'utiliser dans une tâche. Elle fait le pont entre la mémoire sensorielle et la mémoire à long terme, et filtre les informations pertinentes. C'est l'aspect "actif" ou "dynamique" de la mémoire. | Le calcul mental (ex: 52 + 48). Il faut retenir les chiffres et activement les additionner pour trouver le résultat. | | Inhibition | Capacité à résister aux impulsions, aux distractions et aux habitudes pour adopter un comportement plus approprié. C'est le « frein » du cerveau qui permet de prendre un temps de recul avant d'agir et de diriger son attention. | Un jeune enfant à l'épicerie qui s'empêche de faire un commentaire à voix haute sur l'apparence physique d'une personne. | | Flexibilité cognitive | Capacité à se désengager d'une tâche ou d'une perspective pour s'adapter à une nouvelle situation, un nouveau point de vue ou de nouvelles règles. Elle permet de changer de stratégie quand la première ne fonctionne pas. | Un enfant qui accepte d'arrêter son jeu vidéo pour venir souper en famille, se désengageant cognitivement de la première activité pour se réengager dans la seconde. | | Planification | Capacité à anticiper des événements futurs, à se fixer un but et à organiser une séquence d'actions pour l'atteindre. Son développement est plus tardif, car il dépend de l'acquisition de la notion du temps. | Prévoir le trajet pour aller au parc, ou suivre les étapes d'une recette de cuisine. |

5. L'Importance Fondamentale des Fonctions Exécutives

Les fonctions exécutives sont décrites comme des compétences de base, constituant la fondation de multiples aptitudes et habiletés. Leur importance est capitale car :

• Elles soutiennent la réussite éducative à tous les niveaux (primaire, secondaire, et même post-secondaire).

• Elles ont un impact sur l'ensemble des sphères de développement de l'enfant : langagier, social, cognitif et même moteur.

• Elles sont essentielles pour fonctionner efficacement dans la vie de tous les jours et pour le bien-être futur de l'individu.

6. Stratégies de Soutien Parental pour le Développement

Les parents peuvent jouer un rôle actif dans le développement des fonctions exécutives de leur enfant à travers des actions intégrées au quotidien, sans nécessiter de matériel sophistiqué.

Approches Générales

• Développer le langage intérieur : Verbaliser à voix haute ses propres pensées pour modéliser, puis encourager l'enfant à se parler dans sa tête pour retenir des consignes ou se réguler.

• Soutenir les habiletés sociales : Favoriser les interactions sociales positives et variées.

• Offrir un soutien émotionnel : Accueillir les émotions de l'enfant avec empathie pour créer un climat de sécurité.

• Pratiquer des activités sportives et artistiques : Ces activités mobilisent l'ensemble des fonctions exécutives.

• Utiliser l'étayage et le questionnement : Accompagner l'enfant et le questionner sur ce qu'il pense, et surtout pourquoi il le pense, pour l'amener à verbaliser son raisonnement.

• Offrir des choix : Faire un choix implique de prioriser et de renoncer, ce qui mobilise la réflexion.

• Favoriser l'autonomie : Encourager l'enfant à faire les choses par lui-même.

• Tenir compte de ses intérêts : Un contexte agréable et motivant est plus propice à la mobilisation cognitive.

• Proposer des défis : Offrir des défis légèrement supérieurs à ses capacités actuelles, tout en l'accompagnant.

Stratégies Ciblées par Composante

Pour la Mémoire de Travail

• Stratégies de mémorisation : Utiliser des images mentales, la répétition (avec le langage intérieur), le regroupement d'informations (ex: retenir trois légumes), des associations ou des moyens mnémotechniques (chansons, rythmes).

• Repères visuels : Utiliser des listes, des pictogrammes pour les routines afin de se souvenir des étapes.

• Résumer et reformuler : Demander à l'enfant de redire avec ses propres mots ce qu'il a entendu ou lu.

• Réduire la charge cognitive : Éviter de surcharger l'enfant d'informations, surtout dans les moments de transition (ex: le matin avant de partir à l'école).

Pour l'Inhibition

• Prendre un temps de recul : Apprendre à l'enfant à compter jusqu'à trois avant de réagir.

• Communiquer les attentes : Avoir des attentes réalistes et les expliquer clairement à l'enfant.

• Gestion des émotions : Mettre en place des stratégies pour gérer la colère ou la tristesse (respiration, visualisation, relaxation).

• Jeux d'adresse et d'équilibre : Ces jeux demandent de contrôler ses gestes et d'éviter les mouvements impulsifs.

• Jeux de vitesse et de réflexes : Jouer à des jeux où il faut réagir rapidement mais au bon moment (ex: le jeu Dobble).

Pour la Flexibilité Cognitive

• Laisser prendre des décisions : Permettre à l'enfant de prendre des décisions par lui-même.

• Déroger aux routines : Modifier occasionnellement une routine pour l'amener à s'adapter.

• Préparer les transitions : Aviser l'enfant à l'avance d'un changement d'activité pour lui permettre de s'y préparer cognitivement.

• Stimuler la créativité : Encourager les activités créatives, les jeux de rôle et l'improvisation.

• Changer la fin des histoires : Avant de lire la fin d'un livre, demander à l'enfant d'inventer sa propre conclusion.

Pour la Planification

• Utiliser des repères de temps visuels : Calendriers, horaires de la journée ou de la fin de semaine.

• Amener à anticiper : Poser des questions comme : «

• Qu'est-ce que tu penses qu'il va se passer après ? »,
• « De quoi auras-tu besoin pour cette activité ? ».

• Créer des plans : Avant une construction en blocs LEGO, suggérer de dessiner un petit plan.

• Remettre en séquence : Après avoir lu une histoire ou vu un film, demander à l'enfant de raconter ce qui s'est passé au début, au milieu et à la fin.

Synthèse sur la Gestion de la Colère et de la Frustration chez l'Enfant

Résumé Exécutif

Ce document de synthèse présente les stratégies et les concepts clés pour aider les enfants à gérer leur colère et leur frustration, basés sur l'expertise de Madame Ly Massy, docteure en psychologie de l'éducation.

L'approche fondamentale est de considérer la colère non pas comme une émotion négative à supprimer, mais comme une émotion normale, un signal d'alarme indiquant une injustice ou un préjudice.

Elle devient problématique uniquement lorsqu'elle est mal exprimée, trop intense ou injustifiée.

La gestion de ces émotions repose sur le développement de la régulation émotionnelle, une compétence qui s'articule autour de trois habiletés fondamentales :

1. L'identification des émotions et de leurs sources.

2. La modulation de l'intensité émotionnelle pour prévenir l'escalade et retrouver le calme.

3. L'expression adéquate des émotions pour résoudre les situations conflictuelles.

Le rôle des parents est central et s'articule autour de la prévention et de l'enseignement de stratégies concrètes.

La prévention consiste à instaurer un environnement sécurisant par le biais de routines prévisibles et de saines habitudes de vie, notamment une activité physique suffisante et un sommeil adéquat.

Pour l'enseignement, il est crucial d'aider l'enfant à développer un vocabulaire émotionnel, à reconnaître ses propres signaux (physiques, cognitifs, comportementaux) et à maîtriser un éventail de stratégies d'apaisement (techniques physiques, diversion de l'attention).

Le modelage parental — la manière dont les parents gèrent et expriment leurs propres émotions — est l'outil le plus puissant pour guider l'enfant.

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1. Comprendre la Colère : Une Émotion Normale

Selon Madame Ly Massy, la perception de la colère comme une émotion purement négative est une erreur. Elle doit être comprise comme un mécanisme de signalisation fondamental.

• Une Fonction d'Alarme : La colère est décrite comme "un petit bouton d'alarme" qui signale qu'une situation est perçue comme injuste, moqueuse, ou qu'elle nuit au développement personnel.

• Quand la Colère Devient Problématique : L'enjeu n'est pas de supprimer la colère, mais de gérer son expression. Elle devient problématique dans trois cas de figure :

1. Expression Inadéquate : L'enfant "explose au lieu de dire tranquillement qu'est-ce qui nous fait vivre l'émotion négative".   

2. Intensité Excessive : L'émotion est trop intense et l'enfant ne parvient pas à la contrôler.  

3. Justification Inappropriée : La colère est déclenchée par une mauvaise interprétation de la situation (par exemple, percevoir un accident comme un acte intentionnel).

2. La Régulation Émotionnelle : Un Apprentissage en Trois Temps

La régulation émotionnelle est une habileté qui se développe tout au long de la vie, mais dont les bases devraient être acquises au début du primaire. Elle se compose de trois compétences interdépendantes.

| Habileté | Description | | --- | --- | | 1\. Identification des Émotions | Comprendre l'émotion ressentie et ses sources. C'est le prérequis à toute gestion. | | 2\. Modulation des Émotions | Être capable de gérer l'intensité de l'émotion pour éviter une escalade ou pour s'apaiser après une crise et retrouver son calme. | | 3\. Expression des Émotions | Communiquer de manière adéquate ce que l'on ressent et ce qui a causé l'émotion, dans le but de résoudre la situation. |

3. Facteurs de Difficulté dans la Régulation Émotionnelle

Plusieurs caractéristiques individuelles peuvent expliquer pourquoi certains enfants ont plus de difficultés à gérer leurs émotions.

• Hypersensibilité ou Hyperréactivité : Une réaction plus intense aux émotions, aux situations et à l'environnement. Le "moindre petit élément va susciter une réaction négative".

• Seuil de Frustration Bas : L'image d'une "mèche courte" est utilisée. Il en faut très peu pour que l'enfant se sente frustré ou en colère.

• Faible Tolérance au Délai de Gratification : Une difficulté à attendre. Le besoin doit être satisfait immédiatement, sinon une réaction négative s'ensuit.

• Rigidité Mentale : Un besoin que les choses soient faites d'une manière précise ou selon leurs goûts.

Un changement dans la routine peut susciter des émotions négatives.

C'est un trait commun chez les enfants avec un trouble du spectre de l'autisme.

• Perceptions Faussées : Une tendance à interpréter les situations neutres comme hostiles.

L'enfant est en état de vigilance constant, "comme s'il était toujours en vigilance d'avoir peur d'être attaqué".

Cela peut mener à généraliser ou dramatiser les événements.

• Trauma : Un traumatisme peut accentuer tous les facteurs précédents et mener à un "surétiquetage" des émotions négatives comme de la colère, l'enfant cherchant inconsciemment à se sentir moins victime et plus combatif.

4. Stratégies Parentales : Prévention et Anticipation

La première étape pour un parent est de mettre en place des conditions pour "prévenir les pertes de contrôle" plutôt que de simplement réagir aux crises.

4.1. Un Environnement Sécurisant

• Instauration de Routines : Mettre en place des routines prévisibles, surtout pour les moments de la journée identifiés comme difficiles (le matin avant l'école, la période des devoirs, le coucher, la fin de l'effet de la médication).

La prévisibilité rend la gestion des frustrations plus facile pour l'enfant.

4.2. Saines Habitudes de Vie

• Activité Physique : Essentielle pour évacuer les tensions accumulées.

Un enfant qui ne bouge pas assez (par exemple, en s'installant directement pour les devoirs après l'école) aura plus de mal à gérer un événement frustrant.

• Sommeil : Le manque de sommeil est un des "premiers facteurs qui va nuire à la régulation émotionnelle". Les besoins varient selon l'âge :

◦ Début du primaire : Entre 11 et 13 heures.    ◦ Début du secondaire : Environ 9 heures.

4.3. Identification des Déclencheurs de Crises

Il est crucial de distinguer deux types de facteurs :

• Facteurs Précipitants (Déclencheurs) : Ce qui initie la crise. Exemples : l'envahissement de l'espace personnel, une blessure d'estime de soi, l'accumulation de stress, la faim.

En identifiant la source, on peut agir en amont (par exemple, prévoir une collation dans la voiture au retour de l'école).

• Facteurs Aggravants : Ce qui intensifie la crise. Exemples : la fatigue de fin de journée, la fin de l'effet d'une médication pour un TDAH.

5. Développer les Compétences de Régulation Émotionnelle

Le parent doit activement enseigner les trois habiletés fondamentales de la régulation, en choisissant un moment où l'enfant est calme.

5.1. Habileté 1 : Identifier les Émotions

• Enrichir le Vocabulaire : Aider l'enfant à nommer ses émotions avec précision (irrité, frustré, furieux) et à décrire ses sensations physiques ("vidé", "submergé").

• Reconnaître les Signaux : L'aider à identifier les signaux précurseurs de la colère :

◦ Signaux Corporels : Externes (tension, rougeur, froncement des sourcils) et internes (cœur qui bat vite, souffle court).   

◦ Comportements : Tendance à frapper, à se replier sur soi.  

◦ Pensées : Identifier les pensées qui nourrissent la colère ("il l'a fait exprès").

• Outils et Approches :

◦ Refléter : "Tu as l'air déçu, est-ce qu'il s'est passé quelque chose ?".  

◦ Utiliser des supports ludiques : Livres, histoires, jeux de mimes, affiches, collages, création d'un "dictionnaire des émotions".

5.2. Habileté 2 : Moduler les Émotions

• Évaluer l'Intensité : Utiliser une échelle simple comme un thermomètre des émotions (vert, jaune, orange, rouge) pour aider l'enfant à prendre conscience de son état et à choisir la bonne stratégie.

• Enseigner Deux Grandes Catégories de Stratégies :

1. Stratégies Physiques : Très efficaces pour réduire la tension physique lors d'émotions intenses.    

▪ Respiration profonde : "La plus simple et la plus efficace".     

▪ Relaxation : Relaxation musculaire, méditation de pleine conscience.    

▪ Stimulation Sensorielle : Objets lourds (couverture, toutou), se bercer, se retirer dans un endroit calme et peu stimulant (tente, garde-robe), manipuler des objets (balles de tension, "putty").  

2. Stratégies de Diversion de l'Attention : Idéales pour interrompre les pensées négatives en boucle qui nourrissent la colère.     

▪ Activités exigeant de la concentration : Compter à l'envers (de 20 à 1, ou de 200 à 0 par bonds de 4), dessiner des mandalas, jouer aux LEGO, faire une activité artistique.    

▪ Objets Visuels : Regarder des objets calmants (spirales liquides, lumières douces).

• Stratégie Cognitive : Les "Pensées Froides"

◦ Aider l'enfant à remplacer les "pensées chaudes" qui attisent la colère par des "pensées froides" qui apaisent.

Exemples : "Il ne l'a pas fait exprès", "Ça va me couler sur le dos comme sur un canard".

5.3. Habileté 3 : Exprimer les Émotions

• Le Bon Moment et le Bon Endroit : Cette étape ne peut se faire que lorsque l'enfant s'est calmé, dans un lieu propice à la discussion.

• Le "Message au Je" : Enseigner à l'enfant à structurer son expression :

1. Je me sens... (nommer l'émotion).   

2. Parce que... (expliquer la cause).  

3. J'aimerais que... (proposer une solution pour que cela ne se répète pas).

• Le Modelage Parental : "Ici encore plus, le modelage est important".

Les parents doivent eux-mêmes utiliser le "message au je" et parler de leurs propres émotions. C'est en voyant ses parents le faire que l'enfant apprendra le mieux.

6. Messages Clés et Conclusion

Madame Ly Massy conclut avec plusieurs messages fondamentaux pour les parents et les éducateurs :

• La Normalité de la Colère : Il faut cesser de diaboliser cette émotion.

• La Primauté de l'Identification : "Si on n'a pas identifié comment on se sent, on ne sera pas capable de prendre les bons moyens pour se contrôler".

• La Diversité des Stratégies : Il faut enseigner un large éventail de stratégies, car aucune n'est universellement efficace.

• Les Exutoires Sains : Proposer des moyens sains de se défouler quand l'expression verbale directe n'est pas possible (écriture, art, sport intense).

• Le Rôle du Parent : Être un modèle de calme, éviter de "mettre de l'huile sur le feu" et savoir prendre soi-même une grande respiration pour garder son calme face à l'enfant.

I think the point kyla makes here is very important. We know that it is important to examine the conent but getting engaged with the readings, taking them personal is very important and very helpful. We see kayla making that point "you read it aleast three times". reading it from different angles with very important and helpful.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

*Reviewer #1 (Evidence, reproducibility and clarity (Required): *

*Using genetics and microscopy approaches, Cabral et al. investigate how fission yeast regulates its length and width in response to osmotic, oxidative, or low glucose stress. Miller et al. have recently found that the cell cycle regulators Cdc25, Cdc13 and Cdr2 integrate information about cell volume, time and cell surface area into the cellular decision when to divide. Cabral now build on this work and test how disruption of these regulators affects cell size adaptation. They find that each stress condition shows a distinct dependence on the individual regulators, suggesting that the complex size control network enables optimized size adaptation for each condition. Overall, the manuscript is clear and the detailed methods ensure that the experiments can be replicated.

Major comments:

1.) It would be much easier to follow the authors' conclusions, if in addition to surface area to volume ratio, length and width, they would also plot cell volume at division in Figs. 1-4.*

AUTHOR RESPONSE: Due to space constraints in the main (and supplemental) figures, we focused on SA:Vol ratio together with cell length and width, which directly define cell geometry in rod-shaped fission yeast. Surface area and volume are derived from these measurements and can be misleading when considered alone, as similar surface area or volume values can arise from distinct combinations of length and width. The SA:Vol ratio therefore serves as a robust integrative metric for capturing coordinated changes in length and width that reshape cell geometry. We would be happy to include individual surface area and volume plots if requested.

2.) To me, it seems that maybe even more than upon osmotic stress, the cdc13-2x strain differs qualitatively from WT in low glucose conditions, where the increased SA-V ratio is almost completely abolished.

AUTHOR RESPONSE: We agree with the reviewer and have revised the manuscript text to point out this difference. The newly added text states: “Under low glucose, cdc13-2x cells also showed a WT-like response, decreasing length and increasing in SA:Vol ratio (Figures 3B-D). However, this SA:Vol increase was reduced compared to WT (1% vs 8.5%; Figures 1D and 3B), suggesting impaired geometric remodeling under glucose limitation.”

3.) It is not entirely clear to me why two copies of Cdc13 would qualitatively affect the responses. Shouldn't the extra copy behave similarly to the endogenous one and therefore only lead to quantitative changes? Maybe the authors can discuss this more clearly or even test a strain in which Cdc13 function is qualitatively disrupted.

AUTHOR RESPONSE: Increased Cdc13 protein concentration in cdc13-2x cells disrupts the typical time-scaling of Cdc13 protein. Consistent with this, cdc13-2x cells enter mitosis at a smaller cell size. We have modified the text to clarify this point. The new text states: “To access the role of the Cdc13 time-sensing pathway, we disrupted Cdc13 protein abundance by creating a cdc13-2x strain carrying an additional copy of cdc13 integrated at an exogenous locus. cdc13-2x cells divided at a smaller size than WT, reflecting accelerated mitotic entry upon disruption of typical time-scaling of Cdc13 protein (Figure S1A).”

4.) I don't see why the authors come to the conclusion that under osmotic stress cells would maximize cell volume. It leads to a decreased cell length, doesn't it?

AUTHOR RESPONSE: WT cells under osmotic stress do decrease in length, but this is accompanied by an increase in cell width. Because width contributes disproportionately to cell volume in rod-shaped cells, this change results in a modest but reproducible reduction in the SA:Vol ratio relative to WT cells in control medium (Figure 1D). We note that the degree of this change under osmotic stress is small (-0.4%), although statistically significant (p * Likewise, in Figure 2B, they interpret tiny changes in the SA/V. By my estimation, the difference between control and osmotic stress is only 2% (1.195/1.17), less that the wild-type case, which appears to be twice that (which is still pretty modest). The small amplitude of these changes is obscured by the fact that the graphs do not have a baseline at zero, which, as a matter of good data-presentation practice, they should.

*

AUTHOR RESPONSE: We appreciate the reviewer’s distinction between statistical and biological significance and agree that this is an important point to clarify. We now note in the revised text that changes in SA:Vol ratio under osmotic stress are numerically small and should not be overinterpreted. Our revised text now states: “Under oxidative and osmotic stress, the SA:Vol ratio decreased, indicating greater cell volume expansion relative to surface area (Figure 1D). However, we note that the reduction in SA:Vol under osmotic stress, while statistically significant, was modest in magnitude (−0.4%).”

Although small in absolute terms, even subtle geometric changes can be biologically meaningful in fission yeast due to the small size of these cells, where minor shifts in length or width translate into measurable differences in membrane area relative to cytoplasmic volume. Importantly, in Figure 2B, the key observation is not the magnitude of the change but its direction: cdc25-degron-DaMP cells exhibit a ~2% increase in SA:Vol ratio under osmotic stress, in contrast to the decrease observed in WT cells under the same condition. This opposite response reflects altered cell geometry and is supported by corresponding changes in cell length and width. We have revised the Results text to emphasize both the modest magnitude and the directional nature of these effects: “Under osmotic stress, cdc25-degron-DaMP cells exhibited a ~2% increase in SA:Vol ratio, opposite to the modest decrease observed in WT cells. This increase arose from increased cell length and reduced width (Figures 2B-D).”

Regarding data presentation, because SA:Vol ratios vary over a narrow numerical range, setting the y-axis minimum to zero would compress the data and obscure all detectable differences. Instead, we have modifed our SA:Vol ratio graphs in Fig. 1-4 to have consistent axis scaling across panels to accurately convey relative changes while maintaining visual clarity. We are happy to provide full data tables and statistical outputs upon request.

* I am also concerned about the use of manual measurement of width at a single point along the cell. This approach is very sensitive to the choice of width point and to non-cylindrical geometries, several of which are evident in the images presented. MATLAB will return the ??? as well as the length from a mask, but even better, one can more accurately calculate the surface area and volume by assuming rotational symmetry of the mask. Given that surface area and volume calculation need to be redone anyway, as discussed below, I encourage the authors to calculate them directly from the mask, instead of using the cylindrical assumption.*

AUTHOR RESPONSE: In initial experiments to calculate surface area and volume of fission yeast cells for prior work (Miller et al., 2023, Current Biology) we found that automated width measurements by MATLAB or ImageJ were inaccurate for a subset of cells leading to noisy cell surface area and volume values. Measuring cell width by hand and assuming that each cell in a given strain had the same cell radius (average of population) for calculation of cell surface area and volume gave more consistent results and recapitulated established conclusions regarding size control mechanisms.

In this previous work and the current study, abnormally skinny or wide regions of a cell were avoided when drawing a line to measure the cell width by hand. For each strain and condition, an average cell width was determined per independent experiment and used for surface area and volume calculations. Additionally, previous analysis demonstrated that this approach yields results consistent with a rotation method derived directly from cell masks, which does not assume a cylindrical cell shape (Facchetti et al., 2019, Current Biology; Miller et al., 2023, Current Biology).

To test the validity of our size measurements and confirm the robustness of our results in this study we compared the surface area and volume of cells by this rotation method. We have added this additional information to our revised methods section and also added SA:Vol ratio graphs generated from the rotation size measurement to our revised Figure S1 E-J. Importantly, both approaches used to measure cell size gave consistent results and supported the same conclusions.*

The authors also need to be more careful about their claims about size-dependent scaling. The concentration of both Cdc13 and Cdc25 scale with size (perhaps indirectly, in the case of Cdc13), but Cdr2 does not. Cdr2 activity has been proposed to scale with size, and its density at cortical nodes has been reported to scale with size, although that claim has been challenged .*

AUTHOR RESPONSE: We have modified text in the Introduction and Results to address this point. Our revised text in the introduction states: “Recent work has shown that Cdk1 activation integrates size- and time-dependent inputs: the Wee1-inhibitory kinase Cdr2 cortical node density scales with cell surface area (Pan et al., 2014; Facchetti et al., 2019); Cdc25 nuclear accumulation scales with cell volume; and cyclin Cdc13 accumulates over time in the nucleus (Miller et al., 2023) (Figure 1B).” Our revised text in the results section states: “Cdr2 functions as a cortical scaffold that regulates Wee1 activity in relation to cell size, with Cdr2 nodal density reported to scale with cell surface area, enforcing a surface area threshold for mitotic entry (Pan et al., 2014; Allard et al., 2018; Facchetti et al., 2019; Sayyad and Pollard, 2022).”*

Even taking the authors approach at face value, there are observations that do not seem to make sense, which led me to realize that the wrong formulae were used to calculate surface area and volume.

In Figure 1E,F, the KCl-treated cells get shorter and wider; surely, that should result in a lower SA/V ratio. However, as noted above, in Figure 1D, they are shown to have a similar ratio. As a sanity check, I eye-balled the numbers off of the figure (control: 14 µm x 3.6 µm and KCl: 11 µm x 3.8 µm) and calculated their surface area and volume using the formula for a capsule (i.e., a cylinder with hemispheric ends).

SA = the surface area of the two hemispheres + the surface are of the cylinder in between = 4*pi*(width/2)^2 + pi*width*(length-width), the length-width term calculates the side length of the capsule (length without the hemispheres) from the full length of the capsule (length including the hemispheres)

V = the volume of the two hemispheres + the volume of the cylinder in between = 4/3*pi*(width/2)^3 + pi*(width/2)^2*(length-width).

I got SA/V ratios of around 2, which are way off from what is presented in Figure 1D, but my calculated ratio goes down in KCl, as expected, but not as reported.

To make sure I was not doing something wrong, I was going to repeat my calculations with the formulae in Table 1, which made me realize both are incorrect. The stated formula for the cell surface area-2*pi*RL-only represents to surface area of the cylindrical side of the cells, not its hemispherical ends. And it is not even the correct formula for the surface area of the side, because that calls for L to be the length of the side (without the hemispherical ends) not the length of the cell (which includes the hemispherical ends). L here is stated to be cell length (which is what is normally measured in the field, and which is consistent with the reported length of control cells in Figure 1E being 14 µm). The formula for the volume of a capsule in the form use in Table 1 (volume of a cylinder of length L - the volume excluded from the hemispherical ends) is pi*R^2*L - (8-(4/3*pi))*R^3.

Given these problems, I think I spent too much time thinking about the rest of the paper, because all of the calculations, and perhaps their interpretations, need to be redone.*

AUTHOR RESPONSE: The surface area and volume equations for a cylinder with hemispherical ends used in our study and listed in our table are correct and widely used in other work with fission yeast cells (Navarro and Nurse, 2012; Pan et al., 2014; Facchetti et al., 2019; BayBay et al., 2020; and Miller et al., 2023). We write our equations with variables for cell length and radius because these are biologically relevant and measured parameters for fission yeast cells. Cell length (L) refers to the total tip-to-tip length of the cell, including the hemispherical ends, and radius (R) refers to half the measured cell width. We have revised the Methods section to clarify this definition and avoid ambiguity (Please see methods section “Cell geometry measurements”)

Additionally, SA or Vol calculations were performed using the length of each individual cell and the average cell radius of the population. We did not use mean cell length of the population for our calculations like the reviewer assumed in their “sanity check” above. Please see methods section “Cell geometry measurements”. We hope that these clarifications and text revisions improve transparency and reproducibility.

* Minor Points:

Strains should be identified by strain number is the text and figure legends.*

AUTHOR RESPONSE: For clarity and readability, we refer to strains by genotype in the main text and figure legends, which we believe is more informative for readers than strain numbers. All strain numbers corresponding to each genotype are provided in Table S1, ensuring traceability and reproducibility without compromising clarity in data presentation.*

In the Introduction, "Most cell control their size" should be "Most eukaryotic cell control their size".*

• *

AUTHOR RESPONSE: The text has been corrected as suggested.*

Reviewer #2 (Significance (Required)):

Nothing to add.*

*Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary This manuscript reports that fission yeast cells exhibit distinct cell size and geometry when exposed to osmotic, oxidative, or low-glucose stress. Based on quantitative measurements of cell length and width, the authors propose that different stress conditions trigger specific 'geometric adaptation' patterns, suggesting that cell size homeostasis is flexibly modulated depending on environmental cues. The study provides phenotypic evidence that multiple environmental stresses lead to distinct outcomes in the balance between cell surface area and volume, which the authors interpret as stress-specific modes of size control.

Major comments 1) The authors define the 48-hour time point as the 'long-term response', but no justification is provided for why 48 hours represents a physiologically relevant adaptation phase. It is unclear whether the size-control mode has stabilized by that time, or whether it may continue to change afterward. At minimum, the authors should provide a rationale (e.g., growth recovery dynamics, transcriptional adaptation plateau, or pilot time-course observations) to demonstrate that 48 hours corresponds to the steady-state adaptive phase rather than an arbitrarily selected time point.*

AUTHOR RESPONSE: We thank the reviewer for this important point and agree that the definition of the long-term response should be clarified. We have addressed this with new experiments and revised text. We now incorporate growth curve data and doubling time analyses for all yeast strains grown under control and stress conditions (See new Figure S3). These analyses show that following an initial transient stress-induced cell cycle delay, growth rates stabilize well before 48 hours. Notably, the slowest growth rate observed was in 1M KCl, with a doubling time of ~4 hours across all yeast strains tested. Thus, by 48 hours, cells in this condition have undergone more than 12 generations of growth, while cells in all other conditions with shorter doubling times have undergone even more divisions. So by allowing cells to grow for 48 hours prior to imaging, we are capturing cells that have resumed sustained cell cycle progression following transient stress-induced cell cycle delays. Because cell size control is tightly linked to the cell cycle, we define 48 hours as a physiologically relevant time point where cells have adapted to stress conditions.

Our revised methods now states: “Cultures were incubated at 25°C while shaking at 180 rpm for 48 h prior to imaging. This time point was chosen to ensure that cells had progressed beyond the initial transient stress response and reached a stable, condition-specific growth state, as confirmed by growth curve and doubling time analyses showing stabilization well before 48 h (Figure S3), including in the slowest growing condition (1 M KCl; doubling time ~4 h).”

* 2*）Related to the above comment, the authors propose that different stresses lead to distinct cell size adaptations, yet the rationale for the chosen stress intensities and exposure times is insufficiently described. It remains unclear whether the osmotic, oxidative, and low-glucose conditions used here induce comparable levels of cellular stress. Dose-response and time-course analyses would greatly strengthen the conclusions. Without such analyses, it is difficult to support the interpretation that geometry modulation represents a direct adaptive response.

AUTHOR RESPONSE: * *We selected the specific stress conditions based on previously published work showing that these doses elicit robust responses while preserving overall cell viability and the capacity for recovery. We note that osmotic, oxidative, and low glucose conditions perturb fundamentally different cellular systems (turgor pressure and cell wall mechanics, redox balance, and metabolism etc.) and therefore do not generate directly comparable levels of cellular stress in a quantitative sense. Our goal was not to equalize stress intensity across conditions, but to examine how cells change their geometry in response to distinct classes of stressors.

We have clarified the rationale for specific stress conditions in the revised methods: “These stress intensities were selected based on prior studies demonstrating robust cellular responses while preserving cell viability and the capacity for recovery (Fantes and Nurse, 1977, Shiozaki and Russell, 1995, Degols, et al., 1996; López-Avilés et al., 2008; Sansó et al., 2008; Satioh et al., 2015, Salat-Canela et al., 2021, Bertaux et al., 2023).”

* 3) The authors describe stress-induced size changes as an 'adaptive' response. While this is an appealing hypothesis, the presented data do not demonstrate that the change in cell size itself confers a fitness advantage. Evidence showing that blocking the size change reduces stress survival-or that the altered size improves growth recovery- would be required to support this claim. Without such data, the use of the term 'geometric adaptation' seems overstated.*

AUTHOR RESPONSE: We have revised the text to remove the term “adaptive” and now describe stress-induced size changes in descriptive terms. As discussed further in response to Comment 4, new growth curve and doubling time analyses show that defects in surface area or volume expansion do not uniformly impair growth or survival over the stress exposure examined here, reinforcing the decision to avoid fitness-based language.*

4) The authors conclude that mutants exhibit no major defects in growth or viability during 48-hour stress exposure based on comparable septation index values (Fig. S2). However, septation index alone does not fully capture growth performance or cell-cycle progression and is not sufficient to support claims regarding fitness or robustness of proliferation. If the authors intend to make statements about 'growth', 'viability', or 'cell-cycle progression', additional quantitative measures (e.g., growth curves, doubling time, colony-forming units, or microcolony growth measurements) would be necessary. Alternatively, the claims should be toned down to align with the measurements currently provided.*

AUTHOR RESPONSE: We have addressed this concern with new experiments and revised text. In addition to septation index measurements (now analyzed using chi-square tests of proportions; Figure S2), we performed growth curve experiments and doubling time analyses for all genotypes under control and stress conditions (new Figure S3). These additional data show that growth rates are largely comparable across genotypes in control, oxidative, and low-glucose conditions, with more pronounced genotype-dependent differences emerging under osmotic stress. Defects in surface area or volume expansion did not uniformly correspond to impaired population growth, indicating that geometric remodeling is not strictly required for proliferation over the 48-hour stress exposure examined here. We have refined our conclusion to emphasize that defects in surface area or volume expansion do not uniformly impair growth or survival. See revised Results text under the heading “Defects in surface area or volume expansion do not uniformly compromise growth or survival”.*

5) Related to the above comment, the manuscript does not adequately rule out the possibility that the decreased division size simply results from slower growth or delayed cell-cycle progression rather than a shift in the size-control mechanism. Measurements and normalizations of growth rate are required; without them, the interpretation remains speculative.*

AUTHOR RESPONSE: We agree that changes in growth rate or altered cell cycle timing are important to consider. We have revised our text: “Changes in growth rate or cell cycle progression under stress may influence division size by altering mitotic regulator accumulation. Future studies measuring mitotic regulator dynamics alongside growth rates will be needed to distinguish direct changes in size control mechanisms from growth- or timing-dependent effects.”

* 6) Regarding the phenotypes of wee1-2x cells, it is interesting that they increase the SA:Vol ratio under all stress conditions and show phenotypes distinct from cdr2Δ cells. From these observations, the authors claims that Cdr2 and Wee1 function as a surface-area-sensing module that complements the volume-sensing and time-sensing pathways to maintain geometric homeostasis. To support this interpretation, the authors could consider additional experiments, such as analyzing cdr2Δ + wee1-2x cells under the same stress conditions. Such data would test whether increased Wee1 can rescue or modify the cdr2Δ phenotype, providing functional evidence for the proposed Cdr2-Wee1-Cdk1 regulatory relationship. Measurements of cell length, width, SA:Vol ratio, and, if feasible, Cdk1 activity markers in the strain would greatly strengthen the mechanistic claims.*

AUTHOR RESPONSE: We thank the reviewer for this insightful suggestion. While analysis of a cdr2Δ wee1-2x strain could provide additional mechanistic detail, such experiments address a distinct question beyond the scope of our current study, which focuses on how cell geometry changes under different stress conditions in cells with perturbed surface area-, volume-, or time-sensing pathways. Our conclusions regarding a surface area-sensing role for Cdr2-Wee1 signaling are based on previous studies (Pan et al., 2014; Facchetti et al., 2019; Miller et al., 2023) and the cell geometry phenotypes we observe of cdr2Δ and wee1-2x cells under stress conditions. *

Minor comments 1) The manuscript focuses on adaptation through changes in the surface-to-volume ratio; however, only the ratio is shown. Presenting the underlying values of surface area and volume would clarify which geometric parameter primary contributes to the observed changes.*

AUTHOR RESPONSE: Please see our response to Reviewer 1 major comment 1.*

*2) Statistical analysis for Fig.S2 should be provided.

AUTHOR RESPONSE: We have completed this. See revised Figure S2 and methods.*

3) The paper by Kellog and Levin 2022 is missing from the reference list.*

AUTHOR RESPONSE: Thank you for catching this. This reference has now been added. *

**Referees cross-commenting**

After reading the other reviewer's reports, I recognize that focal points differ, but they appear sequential rather than contradictory.

Reviewer 2 raises concerns regarding the surface area/volume calculations, which-if incorrect-would influence many of the quantitative conclusions. I agree that confirming the validity of these calculations (and recalculating if necessary) should be the top priority before evaluating the biological interpretations.

Reviewer 1 raises more mechanistic biological questions. These are certainly important, but in my view they depend on the robustness of the quantitative analysis highlighted by Reviewer 2.

Therefore, I regard the reports as complementary rather than conflicting. Once the analytical issue pointed out by Reviewer 2 is resolved, the field will be in a better position to assess the significance of the mechanistic points raised by Reviewer 1 (as well as those in my own report).

Reviewer #3 (Significance (Required)):

General assessment One of the major strengths of this manuscript is its quantitative, side-by-side comparison of multiple environmental stresses under a unified experimental and analytical framework. The authors provide well-controlled morphometric measurements, allowing direct comparison of geometry changes that would otherwise be difficult to evaluate across studies. The observation that different stress types generate distinct geometric outcomes is particularly intriguing and has the potential to stimulate new conceptual thinking in the field of size control. However, the strength of the conceptual conclusion is currently limited by several aspects of the experimental design and interpretation. In particular, it remains unclear whether the observed geometry changes represent active adaptive responses rather than non-specific consequences of prolonged or string stress exposure. Demonstrating whether geometry remodeling provides a fitness advantage, clarifying whether the changes reach a steady-state rather than reflecting slow drift over time, or identifying upstream stress pathways that govern the response would substantially strengthen the conceptual advance. Even if additional mechanistic or fitness-related data cannot be added, refining the interpretation so that it remains aligned with the present evidence will enhance the clarity, and impact of the study.

Advance Previous study - including the 2023 publication by the James B. Moseley group - established that fission yeast integrates distinct size-control pathways related to surface area, volume, and time under normal growth conditions. The present manuscript extends this line of work to stressed environments and argues that each stress condition elicits a distinct size-control pattern. To our knowledge, a systematic comparison of cell geometry across multiple stress types in the context of size-control pathways has not been reported, and this represents a potentially valuable conceptual advance. The advance is primarily phenomenological and conceptual rather than mechanistic: the work presents new correlation between stress types and geometry but does not yet elucidate the pathways governing these responses or demonstrate a functional advantage. With additional evidence - or with qualifiers ensuring that claims match the current data - the study could make an important contribution to understanding how cells integrate environmental cues into size-control strategies.

Audience Although the primary audience consists of researchers in the fields of cell growth, cell-cycle control, and stress responses in yeast, the conceptual contribution may interest broader fields such as growth homeostasis, metabolic adaptation, and pathological cell size changes in higher eukaryotes. Beyond yeast biology, the modular view of size regulation proposed here may inspire new investigations in stem cell biology, cancer research, and biotechnology where environmental adaptation and cell size are closely linked.

Expertise: nuclear morphology; cell morphology; cell growth; cell cycle; cytoskeleton*

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #3

Evidence, reproducibility and clarity

Summary

This manuscript reports that fission yeast cells exhibit distinct cell size and geometry when exposed to osmotic, oxidative, or low-glucose stress. Based on quantitative measurements of cell length and width, the authors propose that different stress conditions trigger specific 'geometric adaptation' patterns, suggesting that cell size homeostasis is flexibly modulated depending on environmental cues. The study provides phenotypic evidence that multiple environmental stresses lead to distinct outcomes in the balance between cell surface area and volume, which the authors interpret as stress-specific modes of size control.

Major comments

1) The authors define the 48-hour time point as the 'long-term response', but no justification is provided for why 48 hours represents a physiologically relevant adaptation phase. It is unclear whether the size-control mode has stabilized by that time, or whether it may continue to change afterward. At minimum, the authors should provide a rationale (e.g., growth recovery dynamics, transcriptional adaptation plateau, or pilot time-course observations) to demonstrate that 48 hours corresponds to the steady-state adaptive phase rather than an arbitrarily selected time point.

2）Related to the above comment, the authors propose that different stresses lead to distinct cell size adaptations, yet the rationale for the chosen stress intensities and exposure times is insufficiently described. It remains unclear whether the osmotic, oxidative, and low-glucose conditions used here induce comparable levels of cellular stress. Dose-response and time-course analyses would greatly strengthen the conclusions. Without such analyses, it is difficult to support the interpretation that geometry modulation represents a direct adaptive response.

3) The authors describe stress-induced size changes as an 'adaptive' response. While this is an appealing hypothesis, the presented data do not demonstrate that the change in cell size itself confers a fitness advantage. Evidence showing that blocking the size change reduces stress survival-or that the altered size improves growth recovery- would be required to support this claim. Without such data, the use of the term 'geometric adaptation' seems overstated.

4) The authors conclude that mutants exhibit no major defects in growth or viability during 48-hour stress exposure based on comparable septation index values (Fig. S2). However, septation index alone does not fully capture growth performance or cell-cycle progression and is not sufficient to support claims regarding fitness or robustness of proliferation. If the authors intend to make statements about 'growth', 'viability', or 'cell-cycle progression', additional quantitative measures (e.g., growth curves, doubling time, colony-forming units, or microcolony growth measurements) would be necessary. Alternatively, the claims should be toned down to align with the measurements currently provided.

5) Related to the above comment, the manuscript does not adequately rule out the possibility that the decreased division size simply results from slower growth or delayed cell-cycle progression rather than a shift in the size-control mechanism. Measurements and normalizations of growth rate are required; without them, the interpretation remains speculative.

6) Regarding the phenotypes of wee1-2x cells, it is interesting that they increase the SA:Vol ratio under all stress conditions and show phenotypes distinct from cdr2Δ cells. From these observations, the authors claims that Cdr2 and Wee1 function as a surface-area-sensing module that complements the volume-sensing and time-sensing pathways to maintain geometric homeostasis. To support this interpretation, the authors could consider additional experiments, such as analyzing cdr2Δ + wee1-2x cells under the same stress conditions. Such data would test whether increased Wee1 can rescue or modify the cdr2Δ phenotype, providing functional evidence for the proposed Cdr2-Wee1-Cdk1 regulatory relationship. Measurements of cell length, width, SA:Vol ratio, and, if feasible, Cdk1 activity markers in the strain would greatly strengthen the mechanistic claims.

Minor comments

1) The manuscript focuses on adaptation through changes in the surface-to-volume ratio; however, only the ratio is shown. Presenting the underlying values of surface area and volume would clarify which geometric parameter primary contributes to the observed changes.

2) Statistical analysis for Fig.S2 should be provided.

3) The paper by Kellog and Levin 2022 is missing from the reference list.

Referees cross-commenting

After reading the other reviewer's reports, I recognize that focal points differ, but they appear sequential rather than contradictory.

Reviewer 2 raises concerns regarding the surface area/volume calculations, which-if incorrect-would influence many of the quantitative conclusions. I agree that confirming the validity of these calculations (and recalculating if necessary) should be the top priority before evaluating the biological interpretations.

Reviewer 1 raises more mechanistic biological questions. These are certainly important, but in my view they depend on the robustness of the quantitative analysis highlighted by Reviewer 2.

Therefore, I regard the reports as complementary rather than conflicting. Once the analytical issue pointed out by Reviewer 2 is resolved, the field will be in a better position to assess the significance of the mechanistic points raised by Reviewer 1 (as well as those in my own report).

Significance

General assessment

One of the major strengths of this manuscript is its quantitative, side-by-side comparison of multiple environmental stresses under a unified experimental and analytical framework. The authors provide well-controlled morphometric measurements, allowing direct comparison of geometry changes that would otherwise be difficult to evaluate across studies. The observation that different stress types generate distinct geometric outcomes is particularly intriguing and has the potential to stimulate new conceptual thinking in the field of size control. However, the strength of the conceptual conclusion is currently limited by several aspects of the experimental design and interpretation. In particular, it remains unclear whether the observed geometry changes represent active adaptive responses rather than non-specific consequences of prolonged or string stress exposure. Demonstrating whether geometry remodeling provides a fitness advantage, clarifying whether the changes reach a steady-state rather than reflecting slow drift over time, or identifying upstream stress pathways that govern the response would substantially strengthen the conceptual advance. Even if additional mechanistic or fitness-related data cannot be added, refining the interpretation so that it remains aligned with the present evidence will enhance the clarity, and impact of the study.

Advance

Previous study - including the 2023 publication by the James B. Moseley group - established that fission yeast integrates distinct size-control pathways related to surface area, volume, and time under normal growth conditions. The present manuscript extends this line of work to stressed environments and argues that each stress condition elicits a distinct size-control pattern. To our knowledge, a systematic comparison of cell geometry across multiple stress types in the context of size-control pathways has not been reported, and this represents a potentially valuable conceptual advance. The advance is primarily phenomenological and conceptual rather than mechanistic: the work presents new correlation between stress types and geometry but does not yet elucidate the pathways governing these responses or demonstrate a functional advantage. With additional evidence - or with qualifiers ensuring that claims match the current data - the study could make an important contribution to understanding how cells integrate environmental cues into size-control strategies.

Audience

Although the primary audience consists of researchers in the fields of cell growth, cell-cycle control, and stress responses in yeast, the conceptual contribution may interest broader fields such as growth homeostasis, metabolic adaptation, and pathological cell size changes in higher eukaryotes. Beyond yeast biology, the modular view of size regulation proposed here may inspire new investigations in stem cell biology, cancer research, and biotechnology where environmental adaptation and cell size are closely linked.

Expertise: nuclear morphology; cell morphology; cell growth; cell cycle; cytoskeleton.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Cabral et al. present a analysis of the effects of environmental stress of cellular geometry in the fission yeast S. pombe. The stresses they study-oxidative, osmotic and nutritional-have previously been shown to affect cell size in fission yeast. Here, the authors do a more sophisticated analysis, measuring surface area as well as volume (for which length had previously been used as a proxy, assuming fission yeast cells are cylinders of constant width). In addition, they investigate the effect of mutations in three cell-cycle control proteins that have been proposed to regulate cell geometry: Cdc13, Cdc25 and Cdr2. It is an interesting study that could provide insight into cell-size control and environmental-stress response in fission yeast. However, I have serious concerns about the analysis of the data. In fact, as I was writing up my concerns, I noticed that the formulae in Table 1 for surface area and volume are incorrect, so the whole paper appears to require reanalysis.

One general problem is that the authors seem to confuse statistical significance with biological significance. They claim that both oxidative and osmotic stress cause a reduction in SA/V ratio. For oxidative stress, the difference is evident, but the control and KCl-treated cells look to have indistinguishable distributions. Perhaps there is a significant statistical difference between the, but I am skeptical. (I would ask for the data table to try out the stats myself, but given the revelation below that the number will all need to be recalculated, that point is moot). In any case, the difference is certainly not biologically significant.

Likewise, in Figure 2B, they interpret tiny changes in the SA/V. By my estimation, the difference between control and osmotic stress is only 2% (1.195/1.17), less that the wild-type case, which appears to be twice that (which is still pretty modest). The small amplitude of these changes is obscured by the fact that the graphs do not have a baseline at zero, which, as a matter of good data-presentation practice, they should.

I am also concerned about the use of manual measurement of width at a single point along the cell. This approach is very sensitive to the choice of width point and to non-cylindrical geometries, several of which are evident in the images presented. MATLAB will return the ??? as well as the length from a mask, but even better, one can more accurately calculate the surface area and volume by assuming rotational symmetry of the mask. Given that surface area and volume calculation need to be redone anyway, as discussed below, I encourage the authors to calculate them directly from the mask, instead of using the cylindrical assumption.

The authors also need to be more careful about their claims about size-dependent scaling. The concentration of both Cdc13 and Cdc25 scale with size (perhaps indirectly, in the case of Cdc13), but Cdr2 does not. Cdr2 activity has been proposed to scale with size, and its density at cortical nodes has been reported to scale with size, although that claim has been challenged <https://pubmed.ncbi.nlm.nih.gov/36093997>.

Even taking the authors approach at face value, there are observations that do not seem to make sense, which led me to realize that the wrong formulae were used to calculate surface area and volume.

In Figure 1E,F, the KCl-treated cells get shorter and wider; surely, that should result in a lower SA/V ratio. However, as noted above, in Figure 1D, they are shown to have a similar ratio. As a sanity check, I eye-balled the numbers off of the figure (control: 14 µm x 3.6 µm and KCl: 11 µm x 3.8 µm) and calculated their surface area and volume using the formula for a capsule (i.e., a cylinder with hemispheric ends).

SA = the surface area of the two hemispheres + the surface are of the cylinder in between = 4pi(width/2)^2 + piwidth(length-width), the length-width term calculates the side length of the capsule (length without the hemispheres) from the full length of the capsule (length including the hemispheres)

V = the volume of the two hemispheres + the volume of the cylinder in between = 4/3pi(width/2)^3 + pi(width/2)^2(length-width).

I got SA/V ratios of around 2, which are way off from what is presented in Figure 1D, but my calculated ratio goes down in KCl, as expected, but not as reported.

To make sure I was not doing something wrong, I was going to repeat my calculations with the formulae in Table 1, which made me realize both are incorrect. The stated formula for the cell surface area-2piRL-only represents to surface area of the cylindrical side of the cells, not its hemispherical ends. And it is not even the correct formula for the surface area of the side, because that calls for L to be the length of the side (without the hemispherical ends) not the length of the cell (which includes the hemispherical ends). L here is stated to be cell length (which is what is normally measured in the field, and which is consistent with the reported length of control cells in Figure 1E being 14 µm). The formula for the volume of a capsule in the form use in Table 1 (volume of a cylinder of length L - the volume excluded from the hemispherical ends) is piR^2L - (8-(4/3pi))R^3.

Given these problems, I think I spent too much time thinking about the rest of the paper, because all of the calculations, and perhaps their interpretations, need to be redone.

Minor Points:

Strains should be identified by strain number is the text and figure legends.

In the Introduction, "Most cell control their size" should be "Most eukaryotic cell control their size".

Significance

Nothing to add.

The consistency is also impressive given the fact that when the causal arrow is reversed and one considers research that investigates the influence of repressive behavior on dissent (exploring one of the core aspects of collective action theory regarding the importance of costs), the results are highly inconsistent.

This is the part that confuses researchers. We know that dissent causes repression, but we don't know if repression actually stops dissent. Sometimes people get scared and go home (negative impact), but other times it just makes them angrier and leads to more protests (positive impact). It’s a puzzle because the government keeps using repression even though they aren't totally sure it works.

shows how a person can work with good time rather than another person

explains how a person can set times but needs to learn how to work through it

I like how this text included the specific death toll because it really shows the full impact that Covid-19 had on the population, economy, and even families. I also found it interesting that they named these numbers but pointed out the problem with the count of direct Covid deaths (statistics) and the difference between that to excess mortality.

This definition focuses on more than just biological causes but the social patterns of the diseases and impairments. This means that epidemiologist focus more one who is getting sick, where are they getting sick at, and what are the conditions that this person is going through. Based on the video we watched in class, "Rohrman on Social Epidemiology" they focus on the upstream of diseases rather than trying to rapidly heal one person at a time, they look at the situation as a social situation.

Please someone correct me if I'm wrong, but it seems that because Black people are not receiving adequate care when they are sick, it causes them to be sicker, and as a result, it affects studies that determine how likely someone is to be sick with a certain disease or infection This leads to even more racist misconceptions, for example that Black people contract more illnesses than white people. The structural racism in the U.S. is not only present in every corner of our society, it is also cyclical and almost self sustaining. This is why naming it and intentionally taking action wherever possible is so important.

This is wild but shows the lengths people will go to use data in such a negative way.

Scary

I think this a good direction toward finding a solution. But, how often is this used?

I also think that it's important to have more representation in our healthcare systems to bring more of this information to light.

I had no idea but it's interesting to see how algorithms are used for everyday rights such as healthcare.

This section made me realize how much data social media platforms collect, even beyond what we intentionally share. I used to think they only stored basic info like my name or email, but they also track behaviors like what I click on, how long I look at posts, and even where I go online. It feels a little uncomfortable because many of these things happen without us noticing. It shows that our online actions can reveal a lot about us, not just what we directly say. This makes me think we should be more careful about privacy and what platforms are allowed to collect.

This section clearly shows how much data social media platforms collect, often beyond what users knowingly provide. I was especially surprised by the idea that platforms can collect data about non-users through photos or contact lists. It makes it clear that participation in social media data systems isn’t always a choice, which raises serious concerns about privacy and consent.

This is interesting because people often share this data, which gives platforms detailed insight into others' identities and behaviors. That information can be used to personalize experiences, but also raises privacy and data-use concerns.

What stood out to me is how much of this data is collected passively rather than intentionally shared. Actions like pausing on a post or scrolling speed feel almost subconscious, yet they can reveal a lot about a user’s interests or emotional state. This raises an ethical question about whether users truly understand how much they are communicating through behavior alone, not just through what they choose to post.

This reminds me of growing concerns about data privacy on social media, especially when platforms collect information not only from users but also from non-users. It raises an ethical question about whether people need to get consent before posting content that includes others, like friends or family. This is especially important for children, whose digital lives are often shared by their parents before they are old enough to understand or express concern.

Data collection has a variety of gains and drawbacks for both parties, the platform and the users. But it is important to maintain ethics when collecting data by asking questions such as what are the legal limitations on how much data can be collected and how that data can be used.

I know for a fact google knows all the info about me, gender age shopping preferences ect., but since I am under 18 it claims to not store that information. That is something that I find hard to believe, given that I do experience targeting ads and marketing on my google account. I am not really comfortable nor uncomfortable about them storing information about me, anyway, because most sites do it anyway.

This section shows how powerful—and dangerous—data mining can be when patterns are taken out of context. The examples make it clear that just because data lines up does not mean it reveals a true cause, especially with spurious correlations. It highlights how easily data can be used to support misleading or biased conclusions, which is especially concerning when these inferences affect real people’s identities and social outcomes.

I found it interesting that APIs create a filtered version of reality for researchers and developers. When we analyze Reddit data through the API, it’s easy to forget that we are only seeing what the platform allows us to see, not the full picture of user behavior. This makes me wonder how data mining conclusions might change if hidden data—like deleted interactions or internal metrics—were included.

I want to inform my boss that due to illness I will not be able to attend work in the morning. I also want to point out in this email that I have done some leg work but cannot make for certain that everythign will be ready and set up for the meeting.

This emphasizes that trolling and data poisoning aren't necessarily a bad thing; we see the word "poison" and instantly think of negative connotations like illness, fatigue, and death. However, it can often be used in a positive light, as in this example, where they used it to fight back against a massive corporation that chose greed over humanity. The same can be said of the ICE applications recently: people began applying to ICE just to deny the position, stalling DHS and wasting the time they would have used to hire, but then undertrain, overpay, and undermine immigration security.

I had previously known that intentionally poisoning datasets was possible, and that unintentionally doing so was also possible, but I wasn't aware that something like a TikTok video was able to make that big of an impact. It's interesting to see how one influencer's video and her audience were able to make such a big impact and effectively ruin most of the researcher's data- considering her audience is mainly women in their 20s.

This statement highlights a crucial point: when data quality is poor to a certain extent, continuing the analysis is not "working harder," but rather "more dangerous." This is because biases and missing data can systematically push the model or conclusions in the wrong direction, and it's difficult to detect this from the results alone. In such situations, the most important step is to conduct a data audit: checking sample representativeness, missing data mechanisms, and whether abnormal distributions are explainable. If necessary, one must acknowledge that "this dataset cannot answer this question," rather than forcing a conclusion.

This example really blurs the line between harm and protest. Poisoning data is usually framed as something unethical, but here it’s being used as a tactic to support workers and push back against corporate power. It made me think about how the intent behind data poisoning matters a lot for how we judge it ethically, even if the technical action is the same.

Yeah, that’s such a good example of “poisoning” that isn’t even malicious — it’s just the internet doing internet things. A dataset can get totally skewed if one group floods it, because suddenly the results stop representing the wider population the researchers were trying to study.

When finding the best solution to a problem, it is so important to not just settle with the first option. You can try something and believe it helps you, but it is also important that you try other things too, before deciding that’s the only way. I think this can also relate to deciding where you want to go to school. Comparing other peoples experiencing and ratings of different schools can help you find the right one for you, rather than just liking the rating on one and sticking with that one.

I believe it is most important to take all means of advice into account. In this example, this individual has already done some research of their own, when they hear about someone else’s experience with said car. It is important to weigh these options and decide if its better to rely on the ratings or someone close to you.

u/loveucrispina at https://www.reddit.com/r/typewriters/comments/1qtbwq3/comment/o365fib/?context=1

This is wild but also great setup for elite storytelling which the native Americans seem to have

Pleading for life with the bear

This can be compared to the African American soldiers that fought for the Confederates (or maybe even fought at all) during the Civil War.

Industrial policy and government-led planning is an important change from previous governments. Not only regulating but setting goals. There are budget constraints that could be detrimental for a rapid implementation of a clean energy transition.

(I remember we only need to do one post, I'm doing an additional one because I had thoughts I wanted to express) It seems to me that the early focuses on truth, impartiality, purity, etc. especially as they relate to the history of a nation are indicative of a sort of imperialist, or just broadly nationalist approach to record keeping. The concept of a pure truth that is muddied by human inadequacies seems tied to other scientific movements of the time like eugenics. It feels like a weird comparison to make, but you see the common thread of logic drawn from evolutionary theory and various industrial philosophies: Humans are flawed, thus we must erase the fallible parts of humanity so that there is only the most rational and advantageous parts left. It's interesting to see how much archival theory was dominated by a similar approach until the material impracticality forced a change in perspective.

I'm interested by this approach to archivists as a distinct community, separate from an outside world whose records they keep. I get how it works for this paper on a rhetorical level, to help talk about the field and its aims in the same way that we talk about the communities it serves, but I think it ignores the fact that archivists are also human beings that exist within other communities. You can't decontextualize an archivist from the society around them simply because they're an archivist. I don't think this is necessarily what the author is arguing, but setting up the discussion of future possibilities for the profession by framing archivists as distinct from the people they serve limits how archivists can envision themselves. If we want an actually effective paradigm or framework to consider archival work, we should try to move away from seeing The Archive as an apparatus that exists separately from the information it stores.

This is a good representation of being a great communicator. Listening to understand and not just respond not only builds firm communication but opens a person up to vulnerability and safety in communication when it comes to all topics.

UNDERLYING point of contention is issue of LAND OWNERSHIP -> Perhaps now in the news because Afrikaners are concerned about requisitions

Ramaphosa admin passed a law that would allow govt yo exporpriate private property for "public purposes" , most often with compensation -> similar to other expropriation laws arounf the world (Canada?) -> So far no land, contrary to Trump's claims, has been expropriated w/o compensation SINCE 1994.

And in fact this is kind of the key issue here -> notes that Black South Africans were disspossessed of farmland and continue to own only around 30% of all agricultural land. Whites own the remaining 70%

Basically going to explain article and then explain that this actually matter of controlling history. Farm murders have been around for a long time. V, C describe how they are used by Afrikaners to avoid historical culpability for crimes of Apartheid -> to avoid trials for direct violence, in some cases, but also to avoid broader questions about continued benefitting from the racial divide

Drop fact that 70% of wealth is still owned by white South Africans.

Likewise, by asserting this is a genocide, are also thereby asserting their own definition as an ethnic group -> shores up unstable identity.

I know that data mining is inevitable, however I'm still concerning about the safety of my personal information. As long as we go online surfing we will definitely leave online footprint, that would be seen by social media company, network carrier, etc. Something real happened to me, when I registered an account of an application, my cellphone number got leaked and been sold to fraud company that kept phone calls me and text me that really causing some troubles to my daily life

Reviewer #3 (Public review):

Summary:

In their study McDermott et al. investigate the neurocomputational mechanism underlying sensory prediction errors. They contrast two accounts: representational sharpening and dampening. Representational sharpening suggests that predictions increase the fidelity of the neural representations of expected inputs, while representational dampening suggests the opposite (decreased fidelity for expected stimuli). The authors performed decoding analyses on EEG data, showing that first expected stimuli could be better decoded (sharpening), followed by a reversal during later response windows where unexpected inputs could be better decoded (dampening). These results are interpreted in the context of opposing process theory (OPT), which suggests that such a reversal would support perception to be both veridical (i.e., initial sharpening to increase the accuracy of perception) and informative (i.e., later dampening to highlight surprising, but informative inputs).

Strengths:

The topic of the present study is of significant relevance for the field of predictive processing. The experimental paradigm used by McDermott et al. is well designed, allowing the authors to avoid common confounds in investigating predictions, such as stimulus familiarity and adaptation. The introduction provides a well written summary of the main arguments for the two accounts of interest (sharpening and dampening), as well as OPT. Overall, the manuscript serves as a good overview of the current state of the field.

Weaknesses:

In my opinion the study has a few weaknesses. Some method choices appear arbitrary (e.g., binning). Additionally, not all results are necessarily predicted by OPT. Finally, results are challenging to reconcile with previous studies. For example, while I agree with the authors that stimulus familiarity is a clear difference compared to previous designs, without a convincing explanation why this would produce the observed pattern of results, I find the account somewhat unsatisfying.

Author response:

The following is the authors’ response to the previous reviews

Reviewer 1

Minor

The main substance of my previous comment I suppose targeted a deeper issue - namely whether such a result is reflecting a resolution to a 'neural prediction' puzzle or a 'perceptual prediction' puzzle. Of course, these results tell us a great deal about a potential resolution for how dampening and sharpening might co-exist in the brain - but in the absence of corresponding perceptual effects (or a lack of correlation between neural and perceptual variables - as outlined in this revision) I do wonder if any claims about implications for perception might need moderation or caveating. To be honest, I don't think the authors *need* to make any more changes along these lines for this paper to be acceptable - it is more an issue they might wish to consider themselves when contextualizing their findings.

Thank you for the thoughtful comment. We have now added a caveat to the relevant section of the discussion to make it clearer that we are discussing neural results, not perceptual results (p.20, lines 378-379).

I am also happy with the changes that the authors have made justifying which claims can and cannot made based on a statistical decoding test against 'chance' in a single condition using t-tests. I was perhaps a little unclear when I spoke about 'comparisons against 0' in my original review, when the key issue (as the authors have intuited!) is about comparisons against 'chance' (where e.g., 0% decoding above chance is the same thing as 'chance'!). The authors are of course correct in the amendment they have made on p.29 to make clear this is a 'fixed effects analysis' - though I still worry this could be a little cryptic for the average reader. I am not suggesting that the authors run more analyses, or revise any conclusions, but I think it would be more transparent if a note was added along the lines of "while the fixed effects approach (one-sample t-test) enables us to establish whether some consistent informative patterns are detectable in these particular subjects, the results from our paired t-tests support inference to the wider population".

This sentence has been added for increased transparency (p. 27, lines 544-547).

Reviewer 3

Major

(1) In the previous round of comments, I noted that: "I am not fully convinced that Figures 3A/B and the associated results support the idea that early learning stages result in dampening and later stages in sharpening. The inference made requires, in my opinion, not only a significant effect in one-time bin and the absence of an effect in other bins. Instead to reliably make this inference one would need a contrast showing a difference in decoding accuracy between bins, or ideally an analysis not contingent on seemingly arbitrary binning of data, but a decrease (or increase) in the slope of the decoding accuracy across trials. Moreover, the decoding analyses seem to be at the edge of SNR, hence making any interpretation that depends on the absence of an effect in some bins yet more problematic and implausible". The authors responded: "we fitted a logarithmic model to quantify the change of the decoding benefit over trials, then found the trial index for which the change of the logarithmic fit was < 0.1%. Given the results of this analysis and to ensure a sufficient number of trials, we focused our further analyses on bins 1-2". However, I do not see how this new analysis addresses the concern that the conclusion highlights differences in decoding performance between bins 1 and 2, yet no contrast between these bins are performed. While I appreciate the addition of the new model, in my current understanding it does not solve the problem I raised. I still believe that if the authors wish to conclude that an effect differs between two bins they must contrast these directly and/or use a different appropriate analysis approach.

Relatedly, the logarithmic model fitting and how it justifies the focus on analysis bin 1-2 needs to be explained better, especially the rationale of the analysis, the choice of parameters (e.g., why logarithmic, why change of logarithmic fit < 0.1% as criterion, etc), and why certain inferences follow from this analysis. Also, the reporting of the associated results seems rather sparse in the current iteration of the manuscript.

We thank the reviewer for this important point. Following your suggestion, we conducted additional post-hoc tests directly comparing the first and second bins. We found significant differences between bins in the invalid trials, but not the valid trials, suggesting that sharpening/dampening effects are condition specific. This is discussed in the manuscript on p.14, lines 268-271; p.15, 280-284; p.20, lines 382-386.

A logarithmic analysis was chosen as learning is usually found to be a nonlinear process; learning effects occur rapidly before stabilising relatively early, as seen in Fig. 2D. This is consistent with other research which found that logarithmic fits efficiently describe learning curves in statistical learning (Kang et al., 2023; Siegelman et al., 2018; Choi et al., 2020). By utilising a change of logarithmic fit at <0.1% as a criterion, it is ensured that virtually zero learning took place after that point, allowing us to focus our analysis on learning effects as they developed and providing a more accurate model of representational change. This is explained in the manuscript on p.13, lines 250-251; p.27-28, lines 557-563.

(2) A critical point the authors raise is that they investigate the buildup of expectations during training. They go on to show that the dampening effect disappears quickly, concluding: "the decoding benefit of invalid predictions [...] disappeared after approximately 15 minutes (or 50 trials per condition)". Maybe the authors can correct me, but my best understanding is as follows: Each bin has 50 trials per condition. The 2:1 condition has 4 leading images, this would mean ~12 trials per leading stimulus, 25% of which are unexpected, so ~9 expected trials per pair. Bin 1 represents the first time the participants see the associations. Therefore, the conclusion is that participants learn the associations so rapidly that ~9 expected trials per pair suffice to not only learn the expectations (in a probabilistic context) but learn them sufficiently well such that they result in a significant decoding difference in that same bin. If so, this would seem surprisingly fast, given that participants learn by means of incidental statistical learning (i.e. they were not informed about the statistical regularities). I acknowledge that we do not know how quickly the dampening/sharpening effects develop, however surprising results should be accompanied with a critical evaluation and exceptionally strong evidence (see point 1). Consider for example the following alternative account to explain these results. Category pairs were fixed across and within participants,i.e. the same leading image categories always predicted the same trailing image categories for all participants. Some category pairings will necessarily result in a larger representational overlap (i.e., visual similarity, etc.) and hence differences in decoding accuracy due to adaptation and related effects. For example, house  barn will result in a different decoding performance compared to coffee cup  barn, simply due to the larger visual and semantic similarity between house and barn compared to coffee cup and barn. These effects should occur upon first stimulus presentation, independent of statistical learning, and may attenuate over time e.g., due to increasing familiarity with the categories (i.e., an overall attenuation leading to smaller between condition differences) or pairs.

We apologise for the confusion, there are 50 expected trials per bin per condition. The trial breakdown is as follows. Each participant completed 1728 trials, split equally across 3 mappings (two 2:1 maps and one 1:2 map), giving 1152 trials in the 2:1 mapping. Stimuli were expected in 75% of trials (864), leaving 216 per bin, and 54 per leading image in each bin. We have clarified this in the script (p.14, line 267; p.15, line 280). This is in line with similar studies in the field (e.g. Han et al., 2019).

(3) In response to my previous comment, why the authors think their study may have found different results compared to multiple previous studies (e.g. Han et al., 2019; Kumar et al., 2017; Meyer and Olson, 2011), particularly the sharpening to dampening switch, the authors emphasize the use of non-repeated stimuli (no repetition suppression and no familiarity confound) in their design. However, I fail to see how familiarity or RS could account for the absence of

sharpening/dampening inversion in previous studies.

First, if the authors argument is about stimulus novelty and familiarity as described by Feuerriegel et al., 2021, I believe this point does not apply to the cited studies. Feuerriegel et al., 2021 note: "Relative stimulus novelty can be an important confound in situations where expected stimulus identities are presented often within an experiment, but neutral or surprising stimuli are presented only rarely", which indeed is a critical confound. However, none of the studies (Han et al., 2019; Richter et al., 2018; Kumar et al., 2017; Meyer and Olson, 2011) contained this confound, because all stimuli served as expected and unexpected stimuli, with the expectation status solely determined by the preceding cue. Thus, participants were equally familiar with the images across expectation conditions.

Second, for a similar reason the authors argument for RS accounting for the different results does not hold either in my opinion. Again, as Feuerriegel et al. 2021 correctly point out: "Adaptation-related effects can mimic ES when the expected stimuli are a repetition of the last-seen stimulus or have been encountered more recently than stimuli in neutral expectation conditions." However, it is critical to consider the precise design of previous studies. Taking again the example of Han et al., 2019; Kumar et al., 2017; Meyer and Olson, 2011. To my knowledge none of these studies contained manipulations that would result in a more frequent or recent repetition of any specific stimulus in the expected compared to unexpected condition. The crucial manipulation in all these previous studies is not that a single stimulus or stimulus feature (which could be subject to familiarity or RS) determines the expectation status, but rather the transitional probability (i.e. cue-stimulus pairing) of a particular stimulus given the cue. Therefore, unless I am missing something critical, simple RS seems unlikely to differ between expectation condition in the previous studies and hence seems implausible to account for differences in results compared to the current study.

Moreover, studies cited by the authors (e.g. Todorovic & de Lange, 2012) showed that RS and ES are separable in time, again making me wonder how avoiding stimulus repetition should account for the difference in the present study compared to previous ones. I am happy to be corrected in my understanding, but with the currently provided arguments by the authors I do not see how RS and familiarity can account for the discrepancy in results.

The reviewer is correct in that the studies cited (Han et al., 2019; Kumar et al., 2017; Meyer and Olson, 2011) ensure that participants are equally familiar with the images across expectation conditions. Where the present study differs is that participants are not familiar with individual exemplars at all. Han et al., 2019 used a pool of 30 individual images, and subjects underwent exposure sessions lasting two hours each daily for 34 days prior to testing. Kumar et al., 2017 used a pool of 12 images with subjects being exposed to each sequential pair 816 times over the course of the training period. Meyer & Olsen, 2011 used pure tones at five different pitch levels. While familiarity of stimuli across conditions was controlled for in these studies in the sense that familiarity was constant across conditions, novelty was not controlled for. The present study uses a pool of ~3500 images, which are unrepeated across trials.

Feuerriegel et al., 2021 also points out: “There are also effects of adaptation that are dependent on the recent stimulation history extending beyond the last encountered stimulus and long-lag repetition effects that occur when the first and second presentation of a stimulus is separated by tens or even hundreds of intervening images”. Bearing this in mind, and given the very small pool of stimuli being used by Han et al., 2019; Kumar et al., 2017; Meyer and Olson, 2011, it stands to reason that these studies may still have built-in but unaccounted for effects relating to the repetition of exemplars. Thus, our avoidance of those possible confounds, in addition to foregoing any prior training, may elicit differing results. Furthermore, as pointed out by Walsh et al. 2020, methodological heterogeneity (such as subject training) can produce contrasting results as PP makes divergent predictions regarding the properties of prediction error given different permutations of variables such as training, transitional probabilities, and conditional probabilities. In our case, the use of differing methodology was intentional. These issues have been discussed in more detail on p.5, lines 112-115; p.19, lines 368-377; p.20, lines 378-379).

Minor

(1) The authors note in their reply to my previous questions that: "As mentioned above, we opted to target our ERP analyses on Oz due to controversies in the literature regarding univariate effects of ES (Feuerriegel et al., 2021)". This might be a lack of understanding on my side, but how are concerns about the reliability of ES, as outlined by Feuerriegel et al. (2021), an argument for restricting analyses to 1 EEG channel (Oz)? Could one not argue equally well that precisely because of these concerns we should be less selective and instead average across multiple (occipital) channels to improve the reliability of results?

The reviewer is correct in suggesting that a cluster of occipital electrodes may be more reliable than reporting one single electrode. We have amended the analysis to examine electrodes Oz, O1, and O2 (p.9, lines 187-188; p.11, lines 197-201).

(2) The authors provide a github link for the dataset and code. However, I doubt that github is a suitable location to share EEG data (which at present I also cannot find linked in the github repo). Do the authors plan to share the EEG data and if so where?

Thank you for bringing this to my attention. EEG data has now been uploaded at osf.io/x7ydf and linked to the github repository (p.28, lines 569-570).

(3) The figure text could benefit from additional information; e.g. Fig.1C and Fig.3 do not clarify what the asterisk indicates; p < ? with or without multiple comparison correction?

Thank you for pointing out this oversight, the figure texts have been amended (p. 9, line 168; p.16, line 289).

Song arrangements are a common complaint (some feel overly simplified), but the structured lesson progression is more methodical than that of pure gamification apps.

At roughly $200/year, it is the most expensive option here, but still a fraction of the cost of weekly private lessons.

Unlike many learning apps, there is no AI feedback or automated play-along scoring. Instead, Pianote offers an experience that feels more like attending an online piano school.

In my testing, single-note passages were accurately registered about 85-90% of the time on an acoustic piano, but chord recognition was less reliable in noisy rooms

The curriculum follows a method-book progression (right hand, left hand, both hands theory), but unlike a static book, it waits when you slow down and highlights errors so you can self-correct. This approach helps kids develop true musical literacy—such as reading notes, understanding rhythm, and honing technique—rather than depending on gamification rewards, giving young learners skills that transfer to real-world playing.

Akin to McLuhan's tetrad :)

Note the impact shall be different depending on the game! In social games, take VR chat, or Second Lind, perhaps even The Sims, this is much more prominent. These aren't examined. Games like Minecraft allow more than parametric customisation, they have mods and skins... and in this sense, ethnographies on game worlds, will be exemplatory, but also limited by these constraints. Sure, white is a terrible default... but we shouldn't ask indie devs to add perfect customisation settings when their games lack basic accessibility features like high contrast or text read-aloud.

eLife Assessment

Muetter et al. provide an important argument that luminescence is a reliable, high-throughput alternative to colony-forming units (CFU) for super-MIC investigations, particularly when the quantity of interest is biomass. By examining 20 antimicrobials spanning 11 classes, the work shows that discrepancies between CFU and luminescence are often biological (filamentation, Viable But Not Culturable). The work provides a compelling view of how these three common measurements (luminescence, optical density, and CFU) relate to one another across a range of drug treatments, although testing on clinical isolates could be of further benefit.

Reviewer #1 (Public review):

Summary:

This study examines how luminescence can be used to measure bacterial population dynamics during antimicrobial treatment by comparing it directly with optical density and colony counts. The authors aim to determine when luminescence reflects changes in population size and when it instead captures metabolic or physiological states induced by drug exposure. By generating parallel datasets under controlled conditions, the work provides a detailed view of how these three common measurements relate to one another across a range of drug treatments.

Strengths:

The study is technically strong and thoughtfully designed. Measuring luminescence, optical density, and colony counts from the same cultures allows the authors to make clear and informative comparisons between methods. The data are compelling, and the analyses highlight both agreements and divergences in a way that is easy to interpret. The manuscript also succeeds in showing why these divergences arise. For example, the observation that filamentation and metabolic shifts can sustain luminescence even when colony counts drop provides valuable information on how different readouts capture distinct aspects of bacterial physiology. The writing is clear, the figures are effective, and the work will be useful for researchers who need high-throughput approaches to quantify microbial population dynamics experimentally.

Weaknesses:

The study also exposes some inherent limitations of luminescence-based measurements. Because luminescence depends on metabolic activity, it can remain high when cells are damaged or unable to resume growth, and it can fall quickly when drugs disrupt energy production, even if cells remain physically intact. These properties complicate interpretation in conditions that induce strong stress responses or heterogeneous survival states. In addition, the use of drug-free plates for colony counts may overestimate survival when filamented or stressed cells recover once the antibiotic is removed, making differences between luminescence and colony counts harder to attribute to killing alone. Finally, while the authors discuss luminescence in the context of clinically relevant concentration ranges, the current implementation relies on engineered laboratory strains and does not directly demonstrate applicability to clinical isolates. These limitations do not detract from the technical value of the work but should be kept in mind by readers who wish to apply the method more broadly.

Reviewer #2 (Public review):

Summary:

This preprint proposes luxCDABE-based luminescence as a high-throughput alternative (or complement) to CFU time-kill assays for estimating antimicrobial rates of population change at super-MIC concentrations, by comparing luminescence- and CFU-derived rates across 20 antimicrobials (22 assays) and attributing divergences primarily to filamentation (luminescence closer to biomass/volume than cell number) and changes in culturability/carryover (CFU undercounting viable cells).

Strengths:

The authors do not merely report discrepancies; they experimentally validate the biological causes. Specifically, they successfully attribute the slower decline of luminescence in certain drugs to bacterial filamentation (maintaining biomass despite halted division) and the rapid decline of CFU in others to loss of culturability or carryover effects.

The inclusion of 20 antimicrobials spanning 11 classes provides a robust dataset that allows for broad categorization of drug-specific assay behaviors.

The study critically exposes flaws in the "gold standard" CFU method, specifically regarding antimicrobial carryover (demonstrated with pexiganan) and the potential for CFU to overestimate cell death in the presence of VBNC (viable but non-culturable) states induced by drugs like ciprofloxacin.

The use of chromosomal integration for the lux operon to minimize plasmid copy-number effects and the validation of linearity between light intensity and cell density establish a solid technical foundation.

Weaknesses:

The study is conducted exclusively using Escherichia coli. While E. coli is a standard model organism, the paper claims to evaluate luminescence as a generalizable high-throughput tool. Many of the discrepancies observed are driven by filamentation. However, distinct morphological responses occur in other critical pathogens (e.g., Staphylococcus aureus does not filament in the same way).

The authors propose that luminescence data can be corrected using microscopy-derived volume data to better align with CFU counts. The primary appeal of luminescence is high-throughput efficiency. If a researcher must perform time-lapse microscopy to calculate cell volume changes to "correct" their luminescence data, the high-throughput advantage is lost.

The paper argues that for ciprofloxacin, CFU underestimates viability because cells remain intact and impermeable to propidium iodide. While the cells are metabolically active and membrane-intact, if they cannot divide to form a colony (even after drug removal/dilution), their clinical relevance as "living" pathogens is debatable.

Some other comments:

The use of a population dynamical model to simulate filamentation effects is excellent. The finding that light intensity tracks volume ($\psi_V$) better than cell number ($\psi_B$) is a key theoretical contribution.

The model assumes linear elongation. The authors should briefly comment on whether this holds true for the specific drug mechanisms tested (e.g., PBP inhibition vs. DNA gyrase inhibition).

The use of bootstrapping to estimate rate distributions is appropriate and robust.

Conclusion:

Muetter et al. provide a compelling argument that luminescence is a reliable, high-throughput alternative to CFU for super-MIC investigations, particularly when the quantity of interest is biomass. The paper effectively warns researchers that discrepancies between CFU and luminescence are often biological (filamentation, VBNC) rather than methodological failures.

eLife Assessment

This valuable study examined how sensory adaptation supports visual perception in the presence of noise. The authors used a combination of human psychophysics, electroencephalography (EEG), and deep neural networks to show that adaptation to noise can improve perception. The results are solid but are, at present, weakened by a number of concerns, including some related to the experimental design and some regarding the interpretation of the results in terms of particular mechanisms. With these concerns adequately addressed, the study and conclusions would be likely to be of broad interest to the neuroscience community.

Reviewer #1 (Public review):

The authors sought to investigate the role of adaptation in supporting object recognition. In particular, the extent to which adaptation to noise improves subsequent recognition of objects embedded in the same or similar noise, and how this interacts with target contrast. The authors approach this question using a combination of psychophysics, electroencephalography, and deep neural networks. They find better behavioural performance and multivariate decoding of stimuli preceded by noise, suggesting a beneficial effect of adaptation to noise. The neural network analysis seeks to provide a deeper explanation of the results by comparing how well different adaptation mechanisms capture the empirical behavioural results. The results show that models incorporating intrinsic adaptation mechanisms, such as additive suppression and divisive normalisation, capture the behavioural results better than those that incorporate recurrent interactions. The study has the potential to provide interesting insights into adaptation, but there are alternative (arguably more parsimonious) explanations for the results that have not been refuted (or even recognised) in the manuscript. If these confounds can be compellingly addressed, then I expect the results would be of interest to a broad range of readers.

The study uses a multi-modal approach, which provides a rich characterisation of the phenomenon. The methods are described clearly, and the accompanying code and data are made publicly available. The comparison between univariate and multivariate analyses is interesting, and the application of neural networks to distinguish between different models of adaptation seems quite promising.

There are several concerning confounding factors that need to be addressed before the results can be meaningfully interpreted. In particular, differences in behavioural accuracy may be explained by a simple change detection mechanism in the "same noise" condition, and temporal cuing by the "adaptor" stimulus may explain differences in reaction time. Similarly, interference between event-related potentials may explain the univariate EEG results, and biased decoder training may explain the multivariate results. Thus, it is currently unclear if any of the results reflect adaptation.

My main concerns relate to how adaptation is induced and how differences between conditions are interpreted. The adaptation period is only 1.5 s. Although brief adaptors (~1 s) can produce stimulus history effects, it is unclear whether these reflect the same mechanisms as those observed with standard, longer adaptation durations (e.g., 10-30 s). Prior EEG work on visual adaptation using longer adaptors has shown that feature-specific effects emerge very early (<100 ms) after test onset in both univariate and multivariate responses (Rideaux et al., 2023, PNAS). In contrast, the present study finds no difference between same and different adaptor conditions until much later (>300 ms). These later effects likely reflect cognitive processes such as template matching or decision-making, rather than sensory adaptation. Although early differences appear between blank and adaptor conditions, these could be explained by interactions between ERPs elicited by adaptor onset/offset and those elicited by the test stimulus; therefore, they cannot be attributed to adaptation. This contradicts the statement in the Discussion that "Our EEG measurements show clear evidence of repetition suppression, in the form of reduced responses to the repeated noise pattern early in time."

A second concern is the brief inter-stimulus interval. The adaptor is shown for 1.5 s, followed by only a 134 ms blank before the target. When the "adaptor" and test noise are identical, improved performance could simply arise from detecting the pixels that change, namely, those forming the target number. Such change detection does not require adaptation; even simple motion detector units would suffice. If the blank period were longer-beyond the temporal window of motion detectors-then improved performance would more convincingly reflect adaptation. Given the very short blank, however, a more parsimonious explanation for the behavioural effect in the same-noise condition is that change detection mechanisms isolate the target.

Differences between the blank and adaptor conditions may also be explained by temporal cueing. In the noise conditions, the noise reliably signals the upcoming target time, whereas the blank condition provides no such cue. Given the variable inter-trial interval and the brief target presentation, this temporal cue would strongly facilitate target perception. This account is consistent with the reaction time results: both adaptor conditions produce faster reaction times than the blank condition, but do not differ from each other.

The decoding analyses are also difficult to interpret, given the training-testing protocol. All trials from the three main conditions (blank, same, different) were used to train the classifier, and then held-out trials - all from one condition-were decoded. Because ERPs in the adaptor conditions differ substantially from those in the blank condition, and because there are twice as many adaptor trials, the classifier is biased toward patterns from the adaptor conditions and will naturally perform worse on blank trials. To compare decoding accuracy meaningfully across conditions, the classifier should be trained on a separate unbiased dataset (e.g., the "clean" data), or each condition should be trained and tested separately using cross-fold validation.

Reviewer #2 (Public review):

Summary:

Neurons adapt to prolonged or repeated sensory inputs. One function of such adaptation may be to save resources to avoid representing the same inputs over and over again. However, it has been hypothesized that adaptation could additionally help improve the representation of sensory stimuli, especially during difficult recognition scenarios. This study sheds light on this question and provides behavioral evidence for such enhancement. The behavioral results are interesting and compelling. The paper also includes scalp electroencephalographic (EEG) data, which are noisy but point toward similar conclusions. The authors finally implement a deep convolutional neural network (DCNN) with adaptation mechanisms, which nicely capture human behavior.

Strengths:

(1) The authors introduce an interesting hypothesis about the role of adaptation in visual recognition.

(2) The authors present interesting and compelling behavioral data consistent with the hypothesis.

(3) The authors introduce a computational model that can capture mechanisms that can lead to adaptation, enhancing visual recognition.

Weaknesses:

(1) The main weakness is the scalp EEG data. As detailed below, the results are minimal at best and do not contribute to understanding the mechanisms of adaptation. The paper would be stronger without the EEG data.

(2) I wonder whether the hypothesis also holds with real-world objects in natural scenes, beyond the confines of MNIST digits.

Reviewer #3 (Public review):

Summary:

Brands and colleagues investigate how temporal adaptation can aid object recognition, and what neural computations may underlie these effects. They employed a previously published experimental paradigm to study how adaptation to temporally constant distractor input facilitates the recognition of a newly appearing target object. Specifically, they studied how this effect is modulated by the contrast of the target object.

They found that adaptation enhances the recognition of high-contrast objects more than that of low-contrast objects. This behavioral effect was mirrored by a larger effect of adaptation on the response to the high-contrast objects in relatively higher visual areas.

To investigate what neural computations can support this interaction, they implement several candidate neural mechanisms in a deep convolutional neural network: additive suppression, divisive suppression, and lateral recurrence. The authors conclude that divisive and additive suppression, which are intrinsic to the neuron, best explain the interaction between contrast and adaptation in the human data. They further show that these mechanisms, and divisive suppression in particular, show increased robustness to spatial shifts of the adaptor stimulus, hinting and potential perceptual benefits.

Strengths:

(1) Overall, this is a well-written paper, supported by thorough analyses and illustrated with clear, well-designed figures that effectively show overall trends as well as data variance. The authors tell a compelling story while responsibly steering away from overreaching conclusions.

(2) What makes this paper stand out is its comprehensive approach to understanding the behavioral benefit of neural adaptation and its mechanistic underpinnings. The authors effectively achieve this through integrating new behavioral and neural data with simulations using neural network models.

(3) The findings convincingly demonstrate that neuronally intrinsic adaptation mechanisms are sufficient to explain the observed interaction between temporal adaptation, contrast, and object recognition. Furthermore, the paper highlights that these intrinsic mechanisms offer superior robustness compared to learned lateral recurrence mechanisms, which, while being more expressive, can also be more brittle.

Weaknesses:

While the results and conclusion are well supported, there were a few major points that need clarification for me.

(1) Divisive normalization

I was confused by the author's classification of divisive normalization as a neuronally intrinsic mechanism, that is, one that operates within a single neuron, independent of interactions with other neurons.

My understanding is that divisive normalization, as originally proposed by Heeger in the early nineties, describes a mechanism where neurons integrate pooled activity from neighboring cells to mutually inhibit one another. In this form, divisive normalization is fundamentally an interneuronal mechanism involving recurrence. Adding to the confusion, the authors highlight in the introduction their interest in divisive normalization for its relation to stimulus contrast, a relation likely linked to neuronal pooling.

However, my reading of the methods section (Equations 6 and 7) suggests the authors implemented only a temporal feedback component, leaving out the pooling across neurons (Equation 5). This distinction should be disambiguated early in the paper. I recommend choosing a less ambiguous term than "divisive normalization". Even "temporal divisive normalization" is still ambiguous, as lateral neuronal interactions are also inherently temporal.

(2) Parietal electrodes

The paper's adapter-specific effects are centered around the P9/P10 electrodes, which the authors identify as "parietal." However, it is unclear to me which part of the cortex drives these electrodes, particularly whether it is actually the parietal cortex. I am no expert in EEG, but based on the topomaps in Figures 4 and 5, it appears that these electrodes cover more posterior occipito-temporal regions rather than truly parietal regions. Given the central role of P9/P10 to the main findings, the paper would be significantly improved for non-EEG readers by clarifying which cortical regions are covered by these electrodes.

(3) Interpretation of non-significant statistical results

In some places, the authors attach relatively strong claims to non-significant statistical results. For example, in Figure 5D, they claim that there is no effect of contrast on occipital electrodes, based on a non-significant p-value. P-values do not quantify evidence for the null hypothesis, so the authors should be careful with such claims. In fact, Figure 5D shows such a clear negative slope, with variance comparable to Figure 5A, that I am surprised that the p-value for the slope of Figure 5D was in fact so large. A similar issue arises in the discussion for Figure 6, where the authors claim that the effect of contrast is adapter-specific. However, this claim is based on the observation that is significant for same-noise trials, but not for different-noise or blank trials. To statistically substantiate the claims that there is an adapter-specific effect, the authors should directly compare the slope for same-noise trials with the slope for different-noise/blank trials.

(4) The match between behavior and models

The authors' claim that models with intrinsic adaptation better match the interaction between contrast and temporal adaptation observed in human behavior is not fully substantiated. This conclusion appears to be based on a qualitative assessment of Figure 8, which, in my view, does not unambiguously rule out an interaction for lateral recurrence. Furthermore, a potential confounding factor is the ceiling effect that limits higher accuracy values. Indeed, conditions where the interaction was not/less (i.e., shorter time sequences and lateral inhibition) are also the conditions where accuracy values are closer to this ceiling, which may mask a potential interaction.

Reviewer #1 (Public review):

Summary:

Extracellular electrophysiology datasets are growing in both number and size, and recordings with thousands of sites per animal are now commonplace. Analyzing these datasets to extract the activity of single neurons (spike sorting) is challenging: signal-to-noise is low, the analysis is computationally expensive, and small changes in analysis parameters and code can alter the output. The authors address the problem of volume by packaging the well-characterized SpikeInterface pipeline in a framework that can distribute individual sorting jobs across many workers in a compute cluster or cloud environment. Reproducibility is ensured by running containerized versions of the processing components.

The authors apply the pipeline in two important examples. The first is a thorough study comparing the performance of two widely used spike-sorting algorithms (Kilosort 2.5 and Kilosort 4). They use hybrid datasets created by injecting measured spike waveforms (templates) into existing recordings, adjusting those waveforms according to the measured drift in the recording. These hybrid ground truth datasets preserve the complex noise and background of the original recording. Similar to the original Kilosort 4 paper, which uses a different method for creating ground truth datasets that include drift, the authors find Kilosort 4 significantly outperforms Kilosort 2.5. The second example measures the impact of compression of raw data on spike sorting with Kilosort 4, showing that accuracy, precision, and recall of the ground truth units are not significantly impacted even by lossy compression. As important as the individual results, these studies provide good models for measuring the impact of particular processing steps on the output of spike sorting.

Strengths:

The pipeline uses the Nextflow framework, which makes it adaptable to different job schedulers and environments. The high-level documentation is useful, and the GitHub code is well organized. The two example studies are thorough and well-designed, and address important questions in the analysis of extracellular electrophysiology data.

Weaknesses:

The pipeline is very complete, but also complex. Workflows - the optimal artifact removal, best curation for data from a particular brain area or species - will vary according to experiment. Therefore, a discussion of the adaptability of the pipeline in the "Limitations" section would be helpful for readers.

Reviewer #3 (Public review):

Summary:

The authors provide a highly valuable and thoroughly documented pipeline to accelerate the processing and spike sorting of high-density electrophysiology data, particularly from Neuropixels probes. The scale of data collection is increasing across the field, and processing times and data storage are growing concerns. This pipeline provides parallelization and benchmarking of performance after data compression that helps address these concerns. The authors also use their pipeline to benchmark different spike sorting algorithms, providing useful evidence that Kilosort4 performs the best out of the tested options. This work, and the ability to implement this pipeline with minimal effort to standardize and speed up data processing across the field, will be of great interest to many researchers in systems neuroscience.

Strengths:

The paper is very well written and clear in most places. The accompanying GitHub and ReadTheDocs are well organized and thorough. The authors provide many benchmarking metrics to support their claims, and it is clear that the pipeline has been very thoroughly tested and optimized by users at the Allen Institute for Neural Dynamics. The pipeline incorporates existing software and platforms that have also been thoroughly tested (such as SpikeInterface), so the authors are not reinventing the wheel, but rather putting together the best of many worlds. This is a great contribution to the field, and it is clear that the authors have put a lot of thought into making the pipeline as accessible as possible.

Weaknesses:

There are no major weaknesses. I have only a handful of very minor questions and suggestions that could clarify/generalize aspects of the pipeline or make the text more understandable to non-specialists.

(1) Could the authors please expand on the statement on line 274, that processing their test dataset serially "on a single GPU-capable cloud workstation... would take approximately 75 hours and cost over 90 USD." How were these values calculated? I was a bit surprised that this is a >4-fold slow-down from their pipeline, but only increases the cost by ~1.35x, if I understood correctly. More context on why this is, and maybe some context on what a g4dn.4xlarge is compared to the other instances, might help readers who are less familiar with AWS and cloud computing.

(2) One of the most commonly used preprocessing pipelines for Neuropixels data is the CatGT/ecephys pipeline from the developers of SpikeGLX at Janelia. It may be worth commenting very briefly, either in the preprocessing section or in the discussion, on how the preprocessing steps available in this pipeline compare to the steps available in CatGT. For example, is "destriping" similar to the "-gfix" option in catGT to remove high-amplitude artifacts?

(3) Why are there duplicate units (line 194), and how often is this an issue? I understand that this is likely more of a spike sorter issue than an issue with this pipeline, but 1-2 sentences elaborating why might be helpful for readers.

(4) It seems from the parameter files on GitHub that the cluster curation parameters are customizable - correct? If so, it may be worth explicitly saying so in the curation section of the text, as the presented recipe will not always be appropriate. A presence ratio of >0.8 could be particularly problematic for some recordings, for example, if a cell is only active during a specific part of the behavior, that may be a feature of the experiment, or the animal could be transitioning between sleep and wake states, in which different units may become active at different times.

(5) The axis labels in Figures 3d-e are too small to see, and Figure 3d would benefit from a brief description of what is shown.

(6) What is the difference between "neural" and "passing QC" in Figure 4?

(7) I understand the current paper is focused on spike data, so there may not be an answer to this, but I am curious about the NP2.0 probes that save data in wideband. Does the lossy compression negatively affect the LFP data? Is software filtering applied for the spike band before or after compression?

Reviewer #2 (Public review):

This manuscript presents an impressive and novel investigation of organizational principles governing brain activity at both global and local scales during naturalistic viewing paradigms. The proposed multi-scale nested structure offers valuable new insights into functional brain states and their dynamics. Importantly, investigation of global brain states in the context of a naturalistic viewing context represents an important and timely contribution that addresses unresolved issues about global signals and anticorrelations in resting-state fMRI. This manuscript presents a novel investigation of organizational principles governing brain activity at both global and local scales during naturalistic viewing paradigms. The authors demonstrate that brain activity during naturalistic viewing is dominated by two anti-correlated states that toggle between each other with a third transitional state mediating between them. The successful replication across three independent datasets (StudyForrest, NarrattenTion, and CamCAN) is a particular strength. The successful replication across three independent datasets (StudyForrest, NarrattenTion, and CamCAN) is a particular strength, and I appreciate the authors' careful documentation of both convergent and divergent findings across these samples.

Overall, this manuscript makes important contributions to our understanding of large-scale brain organization during naturalistic cognition. The multi-scale framework and robust replication across datasets are notable strengths. Addressing the concerns raised below will substantially strengthen the impact and interpretability of this work.

(1) Network Definition and Specificity

(a) The authors adopt an overly broad characterization of the Default Mode Network (DMN). The statement that "areas most active in the default mode state... consist of the precuneus, angular gyrus, large parts of the superior and middle temporal cortex, large parts of the somatomotor areas, frontal operculi, insula, parts of the prefrontal cortex and limbic areas" includes regions typically assigned to other networks. The insula is canonically considered a core node of the Salience Network/Ventral Attention Network (VAN), not the DMN. Also, not clear which limbic areas? The DMN findings reported need to be critically reassessed in this context.

(b) Given the proposed role of state switching in your framework, a detailed analysis of salience network nodes (particularly insula and dorsal ACC) would be highly informative.

(c) While you report transition-related signals in the visual and auditory cortex, the involvement of insular and frontal control systems in state transitions remains unaddressed.

(d) My recommendation is to provide a more anatomically precise characterization of network involvement, particularly distinguishing DMN from salience/VAN regions, and analyze the specific role of salience network nodes in mediating state transitions.

(2) Distinguishing Top-Down from Stimulus-Driven Effects

(a) The finding that "the superior parietal lobe (SPL) and the frontal eye fields (FEF) show the greatest overlap between their local ROI state switches and the global state switches" raises an important question: To what extent are these effects driven by overt changes in visual gaze or attention shifts triggered by stimulus features versus internally-generated state changes?

(b) Similarly, the observation that DAN areas show the highest overlap with global state changes in StudyForrest and NarrattenTion, while VAN shows the highest overlap in CamCAN, lacks sufficient anatomical detail regarding which specific nodes are involved. This information would help clarify whether insular regions and other VAN components play distinct roles in state switching.

(c) It will be important to (i) discuss potential confounds from eye movements and stimulus-driven attention shifts; (ii) provide detailed anatomical breakdowns of network nodes involved in state transitions, particularly for VAN; (iii) if eye-tracking data or any other relevant stimulus-related data are available, include analyses examining relationships between these measures and state transitions.

(3) Physiological Interpretation of the "Down" State

The linkage between the "Down" state and the Default Mode State (DMS) is intriguing but requires deeper physiological grounding. Recent work by Epp et al. (Nature Neuroscience, 2025) demonstrates that decreased BOLD signal in DMN regions does not necessarily indicate reduced metabolic activity and can reflect neurovascular coupling modes with specific metabolic profiles. It would be useful to discuss whether your "Down" state might represent a particular neurovascular coupling mode with distinct metabolic demands rather than simply reduced neural activity. Alternatively, your analytical approach might be insensitive to or unconfounded by such neurovascular uncoupling. This discussion would substantially enrich the biological interpretation of the DMS versus TPS dual mechanism framework.

(4) Statistical Validation of Bimodality Detection

The method of selecting bimodal timepoints using the Dip test followed by sign-alignment is novel and creative. However, this filter-then-align procedure could potentially introduce circularity by imposing the anticorrelated structure the authors aim to detect. It would be important to implement validation analyses to confirm that anticorrelation is an intrinsic property rather than a methodological artifact. Approaches include leave-one-subject-out cross-validation, unsupervised dimensionality reduction (e.g., PCA) applied independently to verify the anticorrelated structure, and split-half reliability analysis. Such validation would significantly strengthen the statistical foundation of findings.

(5) Quantifying Hyperalignment Contribution

The appendix notes that non-hyperaligned data show a coarser structure, but the specific contribution of hyperalignment to your findings requires more thorough quantification. Please provide a systematic comparison of results with and without hyperalignment, demonstrating that similar (even if weaker) anatomical correspondence exists in native subject space. This would establish that the mesoscale organizational principles you identify are not artifacts of the alignment procedure but reflect genuine neurobiological organization. Consider presenting correlation coefficients or overlap metrics quantifying the similarity of state structures before and after hyperalignment.

(6) Functional Characterization of the Unimodal State

The observation that the brain spends approximately 34% of its time in a "Unimodal State" is presented primarily as a transition period. This is an interesting observation. However, it would be useful to characterize the functional connectivity profile of the unimodal state. Specifically, investigate whether it represents a distinct functional state with its own characteristic connectivity pattern. More detailed analysis would provide a more complete picture of temporal brain dynamics during naturalistic viewing and could yield new perspectives on how the brain reorganizes between stable states.

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Reply to the reviewers

We sincerely appreciate the feedback, attention to detail and timeliness of the referees for our manuscript. Below, we provide a point-by-point response to all comments from the referees, detailing the changes we have already made, and those that are in progress. Referee's comments will appear in bolded text, while our responses will be unbolded. Any text quoted directly from the manuscript will be italicised and contained within "quotation marks". Additionally, we have grouped all comments into four categories (structural changes, minor text changes, experimental changes, figure changes), comments are numbered 1-n in each of these categories. Please note: this response to reviewer's comments included some images that cannot be embedded in this text-only section.

1. General Statements

We appreciate the overall highly positive and enthusiastic comments from all reviewers, who clearly appreciated the technical difficulty of this study, and noted amongst other things that this study represents" a major contribution to the future advancement of oocyst-sporozoite biology" and the development of the segmentation score for oocysts as a "major advance[ment]". We apologise for the omission of line numbers on the document sent to reviewers, we removed these for the bioRxiv submission without considering that this PDF would be transferred across to Review Commons.

We have responded to all reviewers comments through a variety of text changes, experimental inclusions, or direct query response. Significant changes to the manuscript since initial submission are as follows:

1. Refinement of rhoptry biogenesis model: Reviewers requested more detail around the content of the AORs, which we had previously suggested were a vehicle for rhoptry biogenesis as we saw they carried the rhoptry neck protein RON4. To address this, we first attempted to address this using antibodies against rhoptry bulb proteins but were unsuccessful. We then developed a * berghei* line where there rhoptry bulb protein RhopH3 was GFP-tagged. Using this parasite line, we observed that the earliest rhoptry-like structure, which we had previously interpreted as an AOR contained RhopH3. By contrast, RhopH3 was absent from AORs. Reflecting these observations we have renamed this initial structure the 'pre-rhoptry' and suggested a model for rhoptry biogenesis where rhoptry neck cargo are trafficked via the AOR but rhoptry bulb cargo are trafficked by small vesicles that move along the rootlet fibre (previously observed by EM).
2. Measurement of rhoptry neck vs bulb: While not directly suggested by the reviewers, we have also included an analysis that estimates the proportion of the sporozoite rhoptry that represents the rhoptry neck. By contrast to merozoites, which we show are overwhelmingly represented by the rhoptry bulb, the vast majority of the sporozoite rhoptry represents the rhoptry neck.
3. Measurement of subpellicular microtubules: One reviewer asked if we could measure the length of subpellicular microtubules where we had previously observed that they were longer on one side of the sporozoite than the other. We have now provided absolute and relative (% sporozoite length) length measurements for these subpellicular microtubules and also calculated the proportion of the microtubule that is polyglutamylated.
4. More detailed analysis of RON11cKD rhoptries: Multiple comments suggested a more detailed analysis of the rhoptries that were formed/not formed in RON11cKD We have included an updated analysis that shows the relative position of these rhoptries in sporozoites.

2. Point-by-point description of the revisions

Reviewer #1

Minor text changes (Reviewer #1)

1. __Text on page 12 could be condensed to highlight the new data of ron4 staining of the AOR. __

We agree with the reviewer that it is a reasonable suggestion. After obtaining additional data on the contents of the AOR (as described in General Statements #1), this section has been significantly rewritten to highlight these findings. 2.

__Add reference on page 3 after 'disrupted parasites' __

This sentence has been rewritten slightly with some references included and now reads: "Most data on these processes comes from electron microscopy studies 6-8, with relatively few functional reports on gene deleted or disrupted parasites9-11. 3.

__Change 'the basal complex at the leading edge' - this seems counterintuitive __

This change has been made. 4.

__Change 'mechanisms underlying SG are poorly' - what mechanisms? of invasion or infection? __

This was supposed to read "SG invasion" and has now been fixed. 5.

__On page 4: 'handful of proteins' __

This error has been corrected. 6.

__What are the 'three microtubule spindle structures'? __

The three microtubule spindle structures: hemispindle, mitotic spindle, and interpolar spindle are now listed explicitly in the text. 7.

__On page 5: 'little is known' - please describe what is known, also in other stages. At the end of the paper I would like to know what is the key difference to rhoptry function in other stages? __

The following sentence already detailed that we had recently used U-ExM to visualise rhoptry biogenesis in blood-stage parasites, but the following two sentences have been added to provide extra detail on these findings: "In that study, we defined the timing of rhoptry biogenesis showing that it begun prior to cytokinesis and completed approximate coincident with the final round of mitosis. Additionally, we observed that rhoptry duplication and inheritance was coupled with centriolar plaque duplication and nuclear fission." 8.

__change 'rhoptries golgi-derived, made de novo' __

This has been fixed. 9.

__change 'new understand to' __

This change has been made 10.

__'rhoptry malformations' seem to be similar in sporozoites and merozoites. Is that surprising/new? __

We assume this is in reference to mention of "rhoptry malformations" in the abstract. In the RON11 merozoite study (PMID:39292724) the authors noted no gross rhoptry malformations, only that one was not formed/missing. The abstract sentence has been changed to the following to better reflect this nuance: "*We show that stage-specific disruption of RON11 leads to a formation of sporozoites that only contain half the number of rhoptries of controls like in merozoites, however unlike in merozoites the majority of rhoptries appear grossly malformed."

* 11.

__What is known about crossing the basal lamina. Where rhoptries thought to be involved in this process? Or is it proteins on the surface or in other secretory organelles? __

We are unaware of any studies that specifically look at sporozoites crossing the SG basal lamina. A review, although now ~15 years old stated that "No information is available as to how the sporozoites traverse the basal lamina" (PMID:19608457) and we don't know any more information since then. To try and better define our understanding of rhoptry secretion during SG invasion, we have added the following sentence:

"It is currently unclear precisely when during these steps of SG invasion rhoptry proteins are required, but rhoptry secretion is thought to begin before in the haemolymph before SG invasion16." 12.

__On page change/specify: 'wide range of parasite structures' __

The structures observed have been listed: centriolar plaque, rhoptry, apical polar rings, rootlet fibre, basal complex, apicoplast. 13.

__On page 7: is Airyscan2 a particular method or a specific microscope? __

Airyscan2 is a detector setup on Zeiss LSM microscopes, this was already detailed in the materials and methods sections, but figure legends have been clarified to read: "...imaged by an LSM900 microscopy with an Airyscan2 detector". 14.

__how large is RON11? __

RON11 is 112 kDa in * berghei*, as noted in the text. 15.

__There is no causal link between ookinete invasion and oocyst developmental asynchrony __

We have deleted the sentence that implied that ookinete invasion was responsible for oocyst asynchrony. This section now simply states that "Development of each oocyst within a midgut is asynchronous..." 16.

__First sentence of page 24 appears to contradict what is written in results____ I don't understand the first two sentences in the paragraph titled Comparison between Plasmodium spp __

This sentence was worded confusingly, making it appear contradictory when that was not the intention. The sentence has been changed to more clearly support what is written in the discussion and now reads: "Our extensive analysis only found one additional ultrastructural difference between Plasmodium spp."

__On page 25 or before the vast number of electron microscopy studies should be discussed and compared with the authors new data. __

It is not entirely clear which new data should be specifically discussed based on this comment. However, we have added a new paragraph that broadly compares MoTissU-ExM and our findings with other imaging methods previously used on mosquito-stage malaria parasites:

"*Comparison of MoTissU-ExM and other imaging modalities

Prior to the development of MoTissU-ExM, imaging of mosquito-stage malaria parasites in situ had been performed using electron microscopy7,8,11,28, conventional immunofluorescence assays (IFA)10, and live-cell microscopy25. MoTissU-ExM offers significant advantages over electron microscopy techniques, especially volume electron microscopy, in terms of accessibility, throughput, and detection of multiple targets. While we have benchmarked many of our observations against previous electron microscopy studies, the intracellular detail that can be observed by MoTissU-ExM is not as clear as electron microscopy. For example, previous electron microscopy studies have observed Golgi-derived vesicles trafficking along the rootlet fibre8 and distinguished the apical polar rings44; both of which we could not observe using MoTissU-ExM. Compared to conventional IFA, MoTissU-ExM dramatically improves the number and detail of parasite structures/organelles that can be visualised while maintaining the flexibility of target detection. By contrast, it can be difficult or impossible to reliably quantify fluorescence intensity in samples prepared by expansion microscopy, something that is routine for conventional IFA. For studying temporally complex processes, live-cell microscopy is the 'gold-standard' and there are some processes that fundamentally cannot be studied or observed in fixed cells. We attempt to increase the utility of MoTissU-ExM in discerning temporal relationships through the development of the segmentation score but note that this cannot be applied to the majority of oocyst development. Collectively, MoTissU-ExM offers some benefits over these previously applied techniques but does not replace them and instead serves as a novel and complementary tool in studying the cell biology of mosquito-stage malaria parasites.**"

*

__First sentence on page 27: there are many studies on parasite proteins involved in salivary gland invasion that could be mentioned/discussed. __

The sentence in question is "To the best of our knowledge, the ability of sporozoites to cross the basal lamina and accumulate in the SG intercellular space has never previously been reported."

This sentence has now been changed to read as follows: "While numerous studies have characterized proteins whose disruption inhibited SG invasion9,10,15,59-63, to the best of our knowledge the ability of sporozoites to cross the basal lamina and accumulate in the SG intercellular space has never previously been reported ."

__On page 10 I suggest to qualify the statement 'oocyst development has typcially been inferred by'. There seem a few studies that show that size doesn't reflect maturation. __

In our opinion, this statement is already qualified in the following sentence which reads: "Recent studies have shown that while oocysts increase in size initially, their size eventually plateaus (11 days pot infection (dpi) in P. falciparum4)."

__On page 16 the authors state that different rhoptries might have different function. This is an interesting hypothesis/result that could be mentioned in the abstract. __

The abstract already contains the following statement: "...and provide the first evidence that rhoptry pairs are specialised for different invasion events." We see this as an equivalent statement.

Experimental changes (Reviewer #1)

1. On page 19: do the parasites with the RON11 knockout only have the cytoplasmic or only the apical rhoptries?

The answer to this is not completely clear. We have added the following data to Figures 6 and 8 where we quantify the proportion of rhoptries that are either apical or cytoplasmic: In both wildtype parasites and RON11ctrl parasites, oocyst spz rhoptries are roughly 50:50 apical:cytoplasmic (with a small but consistent majority apical), while almost all rhoptries are found at the apical end (>90%) in SG spz. Presumably, after the initial apical rhoptries are 'used up' during SG invasion, the rhoptries that were previously cytoplasmic take their place. In RON11cKD the ratio of apical:cytoplasmic rhoptries is fairly similar to control oocyst spz. In RON11cKD SG spz, the proportion of cytoplasmic rhoptries decreases but not to the same extent as in wildtype or RON11Ctrl. From this, we infer that the two rhoptries that are lost/not made in RON11cKD sporozoites are likely a combination of both the apical and cytoplasmic rhoptries we find in control sporozoites.

__in panel G: Are the dense granules not micronemes? What are the dark lines? Rhoptries?? __

We have labelled all of Figure 1 more clearly to point out that the 'dark lines' are indeed rhoptries. Additionally, we have renamed the 'protein-dense granules' to 'protein-rich granules', as it seems we are suggesting that these structures are dense granules the secretory organelle. At this stage we simply do not know what all of these granules are. The observation that some but not all of these granules contain CSP (Supplementary Figure 2) suggests that they may represent heterogenous structures. It is indeed possible that some are micronemes, however, we think it is unlikely that they are all micronemes for a number of reasons: (1) micronemes are not nearly this protein dense in other Plasmodium lifecycle stages, (2) some of them carry CSP which has not been demonstrated to be micronemal, (3) very few of these granules are present in SG sporozoites, which would be unexpected because microneme secretion is required for hepatocyte invasion.

__Figure 2 seems to add little extra compared to the following figures and could in my view go to the supplement. __

We agree that Figure 2b adds little and so have moved that to Supplementary Figure 2, but think that the relative ease at which it can be distinguished if sporozoites are in the secretory cavity or SG epithelial cell is a key observation because of the difficulty in doing this by conventional IFA.

__On page 8 the authors mention a second layer of CSP but do not further investigate it. It is likely hard to investigate this further but to just let it stand as it is seems unsatisfactory, considering that CSP is the malaria vaccine. What happens if you add anti-CSP antibodies? I would suggest to shorten the opening paragraphs of this paper and to focus on the rhoptries. This could be done be toning down the text on all aspects that are not rhoptries and point to the open question some of the observations such as the CSP layers raise for future studies. __

When writing the manuscript, we were unsure whether to include this data at all as it is a purely incidental finding. We had no intention of investigating CSP specifically, but anti-CSP antibodies were included in most of the salivary gland imaging experiments so we could more easily find sporozoites. Given the tremendous importance of CSP to the field, we figured that these observations were potentially important enough that they should be reported in the literature even though they are not something we have the intention or resources to investigate subsequently. Additionally, after consultation with other microscopists we think there is a reasonable chance that this double-layer effect could be a product of chemical fixation. To account for this, we have qualified the paragraph on CSP with this sentence:

"We cannot determine if there is any functional significance of this second CSP layer and considering that it has not been observed previously it may well represent an artefact of chemical (paraformaldehyde) fixation."

__Maybe include more detail of the differences between species on rhoptry structure into Figure 4. I would encourage to move the Data on rhoptries in Figure S6 to the main text ie to Figure 4. __

We have moved the images of developing rhoptries in * falciparum *(previously Figure S6a and b) into figure 4, which now looks as follows:

Figure S8 (previously S6c) now consists only of the MG spz rhoptry quantification

Manuscript structural changes (Reviewer #1)

1. Abstract: don't focus on technique but on the questions you tried to answer (ie rewrite or delete the 3rd and 4th sentence)

2. 'range of cell biology processes' - I understand the paper that the key discovery concerns rhoptry biogenesis and function, so focus on that, all other aspects appear rather peripheral.

3. 'Much of this study focuses on the secretory organelles': I would suggest to rewrite the intro to focus solely on those, which yield interesting findings.

4. Page 11: I am tempted to suggest the authors start their study with Figure 3 and add panel A from Figure 2 to it. This leads directly to their nice work on rhoptries. Other features reported in Figures 1 and 2 are comparatively less exciting and could be moved to the supplement or reported in a separate study.____ Page 23: I suggest to delete the first sentence and focus on the functional aspects and the discoveries.

5. __Maybe add a conclusion section rather than a future application section, which reads as if you want to promoted the use of ultrastructure expansion microscopy. To my taste the technological advance is a bit overplayed considering the many applications of this techniques over the last years, especially in parasitology, where it seems widely used. In any case, please delete 'extraordinarily' __

Response to Reviewer#1 manuscript structural changes 1-5: This reviewer considers the findings related to rhoptry biology as the most significant aspect of the study and suggests rewriting the manuscript to emphasize these findings specifically. Doing so might make the key findings easier to interpret. However, in our view, this approach could misrepresent how the study originated and what we see as the most important outcomes. We did not develop MoTissU-ExM specifically to investigate rhoptry biology. Instead, this technique was created independently of any particular biological question, and once established, we asked what questions it could answer, using rhoptry biology as a proof of concept. Given the authors' previous work and available resources, we chose to focus on rhoptry biology. Since this was driven by basic research rather than a specific hypothesis, it's important to acknowledge this in the manuscript. While we agree that the findings related to rhoptry biology are valuable, we believe that highlighting the technique's ability to observe organelles, structures, and phenotypes with unprecedented ease and detail is more important than emphasizing the rhoptry findings alone. For these reasons, we have decided not to restructure the manuscript as suggested.

Reviewer #2

Minor text changes (Reviewer #2)

1. __The 'image Z-depth' value indicated in the figures is ambiguous. It is not clear whether this refers to the distance from the coverslip surface or the starting point of the z-stack image acquisition. A precise definition of this parameter would be beneficial. __

In the legend of Figure 1, the image Z-depth has been clarified as "sum distance of Z-slices in max intensity projection". 2.

__Paragraph 3 of the introduction - line 7, "handful or proteins" should be handful of proteins __

This has been corrected. 3.

__Paragraph 5 of the introduction - line 7, "also able to observed" should be observe __

This has been changed. 4.

__In the final paragraph of the introduction - line 1, "leverage this new understand" should be understanding __

This has been fixed. 5.

__The first paragraph of the discussion summary contains an incomplete sentence on line 7, "PbRON11ctrl-infected SGs." __

This has been removed. 6.

__The second paragraph of the discussion - line 10, "until cytokinesis beings" should be begins __

This mistake has been corrected. 7.

__One minor point that author suggest that oocyst diameter is not appropriate for the development of sporozoite develop. This is not so true as oocyst diameter tells between cell division and cell growth so it is important parameter especially where the proliferation with oocyst does not take place but the growth of oocyst takes place. __

We agree that this was not highlighted enough in the text. The final sentence of the results section about this now reads:

"While diameter is a useful readout for oocyst development in the early stages of its growth, this suggests that diameter is a poor readout for oocyst development once sporozoite formation has begun and highlights the usefulness of the segmentation score as an alternative.", and the final sentence of the discussion section about this now reads "Considering that oocyst size does not plateau until cytokinesis begins4, measuring oocyst diameter may represent a useful biological clock specifically when investigating the early stages of oocyst development." 8.

__How is the apical polarity different to merozoite as some conoid genes are present in ookinete and sporozoite but not in merozoite. __

Our hypothesis is that apical polarity is established by the positioning and attachment of the centriolar plaque to the parasite plasma membrane in both forming merozoites and sporozoites. While the apical polar ring proteins are obviously present at the apical end, and have important functions, we think that they themselves are unlikely to regulate polarity establishment directly. Additionally, it seems that the apical polar rings are visible in forming sporozoites far before the comparable stages of merozoite formation. An important note here is that at this point, this is largely inferences based on observational differences and there is relatively little functional data on proteins that regulate polarity establishment at any stage of the Plasmodium 9.

__Therefore, I think that electron microscopy remains essential for the observation of such ultra-fine structures __

We have added a paragraph in the discussion that provides a more clear comparison between MoTissU-ExM and other imaging modalities previously applied on mosquito-stage parasites (see response to Reviewer#1 (Minor text changes) comment #17). 10.

__The author have not mentioned that sometimes the stage oocyst development is also dependent on the age of mosquito and it vary between different mosquito gut even if the blood feed is done on same day. __

In our opinion this can be inferred through the more general statement that "development of each oocyst within a midgut is asynchronous..."

Figure changes (Reviewer #2)

1. __Fig 3B: stage 2 and 6 does not show the DNA cyan, it would-be good show the sate of DNA at that particular stage, especially at stage 2 when APR is visible. And box the segment in the parent picture whose subset is enlarged below it. __

We completely agree with the reviewer that the stage 2 image would benefit from the addition of a DNA stain. Many of the images in Figure 3b were done on samples that did not have a DNA stain and so in these * yoelii samples we did not find examples of all segmentation scores with the DNA stain. Examples of segmentation score 2 and 6 for P. berghei, and 6 for P. falciparum* can be found with DNA stains in Figure S8. 2.

__For clarity, it would be helpful to add indicators for the centriolar plaques in Figure 1b, as their locations are not immediately obvious. __

The CPs in Figure 1a and 1b have been circled on the NHS ester only panel for clarity. +

__Regarding Figure 1c, the authors state that 'the rootlet fiber is visible'. However, such a structure cannot be confirmed from the provided NHS ester image. Can the authors present a clearer image where the rootlet fibre is more distinct? Furthermore, please provide the basis for identifying this structure as a rootlet fiber based on the NHS ester observation alone. __

The image in Figure 1c has been replaced with one that more clearly shows the rootlet fibre.

Based on electron microscopy studies, the rootlet fibre has been defined as a protein dense structure that connects the centriolar plaque to the apical polar rings (PMID: 17908361). Through NHS ester and tubulin staining, we could identify the apical polar rings and centriolar plaque as sites on the apical end of the parasite and nucleus that microtubules are nucleated from. There is a protein dense fibre that connects these two structures. Based on the fact that the protein density of this structure was previously considered sufficient for its identification by electron microscopy, we consider its visualisation by NHS ester staining sufficient for its identification by U-ExM.

__Fig 1B - could the tubulin image in the hemispindle panel be made brighter? __

The tubulin staining in this panel was not saturated, and so this change has been made.

__Fig 4A - the green text in the first image panel is not visible. Also, the cyan text in the 3rd image in Fig 1A is also difficult to see. There's a few places where this is the case __

We have made all microscopy labels legible at least when printed in A4/Letter size.

__Fig 6A - how do the authors know ron11 expression is reduced by 99%? Did they test this themselves or rely on data from the lab that gifted them the construct? Also please provide mention the number of oocyst and sporozoites were observed. __

The way Figure 6a was previously designed and described was an oversight, that wrongly suggested we had quantified a >99% reduction in *ron11 * The 99% reduction has been removed from Figure 6a and the corresponding part of the figure legend has been rewritten to emphasise that this was previously established:

"(a) Schematic showing previously established Ron11Ctrl and Ron11cKD parasite lines where ron11 expression was reduced by >99%9."

As to the second part of the question, we did not independently test either protein or RNA level expression of RON11, but we were gifted the clonal parasite lines established by Prof. Ishino's lab in PMID: 31247198 not just the genetic constructs.

__Fig 6E - are the data point colours the wrong way round on this graph? Just looking at the graph it looks as though the RON11cKD has more rhoptries than the control which does not match what is said in the text. __

Thank you for pointing out this mistake, the colours have now been corrected.

__Fig S8C, PbRON11 ctrl, pie chart shows 89.7 % spz are present in the secretory cavity while the text shows 100 %, 35/35 __

The text saying 100% (35/35) only considered salivary glands that were infected (ie. Uninfected SGs were removed from the count. The two sentences that report this data have been clarified to reflect this better:

"Of *PbRON11ctrl SGs that were infected (35/39), 100% (35/35) contained sporozoites in the secretory cavity (Figure S8c). Conversely of infected PbRON11cKD SGs (59/82), only 24% (14/59) contained sporozoites within the secretory cavity (Figure S9d)."

*

__Fig S9D shows that RON11 ckd contains 17.1% sporozoites in secretory cavity while the text says 24%. __

Please see the response to Reviewer#2 Figure Changes Comment #8 where this was addressed.

Experimental changes (Reviewer #2)

1. __Why do the congruent rhoptries have similar lengths to each other, while the dimorphic rhoptries have different lengths? Is this morphological difference related to the function of these rhoptries? __

We hypothesise that this morphological difference arises because the congruent rhoptries are 'used' during SG invasion, while the dimorphic rhoptries are utilized during hepatocyte invasion. It is not straightforward to test this functionally at this point, as no protein is known to have differential localization between the two. Additionally, RON11 is likely directly involved in both SG and hepatocyte invasion through a secreted portion of the protein (as seen in RBC invasion). Therefore, RON11cKD sporozoites may have combined defects, meaning we cannot assume any defect is solely due to the absence of two rhoptries. Determining this functionally is of high interest to our research groups and remains an area of ongoing study, but it is beyond the scope of this study. 2.

Would it be possible to show whether RON11 localises to the dimorphic rhoptries, the congruent rhoptries, or both, by using expansion microscopy and a parasite line that expresses RON11 tagged with GFP or a peptide tag?

__ __We do not have access to a parasite line that expresses a tagged copy of RON11, or anti-PbRON11 antibodies. Based on previously published localisation data, however, it seems likely that RON11 localises to both sets of rhoptries. Below are excerpts from Figure 1c of PMID: 31247198, where RON11 (in green) seems to have a more basally-extended localisation in midgut (MG) sporozoites than in salivary gland (SG) sporozoites. From this we infer that in the MG sporozoite you're seeing RON11 in both pairs of rhoptries, but only the one remaining pair in the SG sporozoite.

__The knockdown of RON11 disrupts the rhoptry structure, making the dimorphic and congruent rhoptries indistinguishable. Does this suggest that RON11 is important for the formation of both types of rhoptries? I believe that it would be crucial to confirm whether RON11 localises to all rhoptries or is restricted to specific rhoptries for a more precise discussion of RON11's function. __

Based on our analysis, it does indeed seem that RON11 is important for both types of rhoptries as when RON11 isn't expressed sporozoites still have both apical and cytoplasmic rhoptries (ie. Not just one pair is lost; see Reviewer #1 Experimental changes comment #1).

__The authors state that 64% of RON11cKD SG sporozoites contained no rhoptries at all. Does this mean RON11cKD SG sporozoites used up all rhoptries corresponding to the dimorphic and congruent pairs during SG invasion? If so, this contradicts your claims that sporozoites are 'leaving the dimorphic rhoptries for hepatocyte invasion' and that 'rhoptry pairs are specialized for different invasion events'. If that is not the case, does it mean that RON11cKD sporozoites failed to form the rhoptries corresponding to the dimorphic pair? A more detailed discussion would be needed on this point and, as I mentioned above, on the specific role of RON11 in the formation of each rhoptry pair. __

We do not agree that this constitutes a contradiction; instead, more nuance is needed to fully explain the phenotype. As shown in the new graph added in response to Reviewer#1 Figure changes comment #1 in RON11cKD oocyst sporozoites, 64% of all rhoptries are located at the apical end. Our hypothesis is that these rhoptries are used for SG invasion and, therefore, would not be present in RON11cKD SG sporozoites. Consequently, the fact that 64% of RON11cKD sporozoites lack rhoptries is exactly what we would expect. Essentially, we predict three slightly different 'pathways' for RON11cKD sporozoites: If they had 2 apical rhoptries in the oocyst, we predict they would have zero rhoptries in the SG. If they had 2 cytoplasmic rhoptries in the oocyst, we predict they would have two rhoptries in the SG. If they had one apical and one cytoplasmic rhoptry in the oocyst, we predict they would have one rhoptry in the SG. In any case, we expect the apical rhoptries to be 'used up,' which appears to be supported by the data.

__Out of pure curiosity, is it possible to measure the length and number of subpellicular microtubules in the sporozoites observed in this study using expansion microscopy? __

We have performed an analysis of subpellicular microtubules which is now included as Supplementary Figure 2. We could not always distinguish every SPMT from each other and so have not quantified SPMT number. We have, however, quantified their absolute length on both the 'long side' and 'short side', their relative length (as % sporozoite length) and the degree to which they are polyglutamylated.

A description of this analysis is now found in the results section as follows: "*We quantified the length and degree of polyglutamylation of SPMTs on the 'long side' and 'short side' of the sporozoite (Figure S2). 'Short side' SPMTs were on average 33% shorter (mean = 3.6 µm {plus minus}SD 1.0 µm) than 'long side' SPMTs (mean = 5.3 µm {plus minus}SD 1.5 µm) and extended 17.4% less of the total sporozoite length. While 'short side' SPMTs were significantly shorter, a greater proportion of their length (87.9% {plus minus}SD 11.2%) was polyglutamylated compared to 'long side' SPMTs (69.4% {plus minus}SD 13.8%)." *

Supplementary Figure 2: Analysis of sporozoite subpellicular microtubules. Isolated P. yoelii salivary gland sporozoites were prepared by U-ExM and stained with anti-tubulin (microtubules) and anti-PolyE (polyglutamylated SPMTs) antibodies. SPMTs were defined as being on either the 'long side' (nucleus distant from plasma membrane) or 'short side' (nucleus close to plasma membrane) of the sporozoite as depicted in Figure 1f. (a) SPMT length along with (b) SPMT length as a proportion of sporozoite length were both measured. (c) Additionally, the proportion of the SPMT that was polyglutamylated was measured. Analysis comprises 25 SPMTs (11 long side, 14 short side) from 6 SG sporozoites. ** = p The following section has also been added to the methods to describe this analysis: * "Subpellicular microtubule measurement

• To measure subpellicular microtubule length and polyglutamylation maximum intensity projections were made of sporozoites stained with NHS Ester, anti-tubulin and anti-PolyE antibodies, and SYTOX Deep Red. The side where the nucleus was closest to the parasite plasma membrane was defined as the 'short side', while the side where the nucleus was furthest from the parasite plasma membrane was defined as the 'long side'. Subpellicular microtubules were then measured using a spline contour from the apical end of the sporozoite to the basal-most end of the microtubule with fluorescence intensity across the contour plotted (Zeiss ZEN 3.8). Sporozoite length was defined as the distance from the sporozoite apical polar rings to the basal complex, measuring through the centre of the cytoplasm. The percentage of the subpellicular microtubule that was polyglutamylated was determined by assessing when along the subpellicular microtubule contour the anti-PolyE fluorescence intensity last dropped below a pre-defined threshold."

*

__In addition to the previous point, in the text accompanying Figure 7a, the authors claim that "64% of PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries". Could this data be used to infer which rhoptry pair are missing from the RON11cKD oocyst sporozoites? Can it be inferred that the 64% of salivary gland sporozoites that had no rhoptries in fact had 2 congruent rhoptries in the oocyst sporozoite stage and that these have been discharged already? __

Please see the response to Reviewer #2 Experimental Changes Comment #4.

__Is it possible that the dimorphic rhoptries are simply precursors to the congruent rhoptries? Could it be that after the congruent rhoptries are used for SG invasion, new congruent rhoptries are formed from the dimorphic ones and are then used for the next invasion?____ Would it be possible to investigate this by isolating sporozoites some time after they have invaded the SG and performing expansion microscopy? This would allow you to confirm whether the dimorphic rhoptries truly remain in the same form, or if new congruent rhoptries have been formed, or if there have been any other changes to the morphology of the dimorphic rhoptries. __

In theory, it is possible that the dimorphic rhoptries are precursors to the uniform rhoptries, specifically how the larger one of the two in the dimorphic pair might be a precursor. Maybe the smaller one is, but we have no evidence to suggest that this rhoptry lengthens after SG invasion. We are interested in isolating sporozoites from SGs to add a temporal perspective, but currently, this isn't feasible. When sporozoites are isolated from SGs, they are collected at all stages of invasion. Additionally, we don't know how long each step of SG invasion takes, so a time-based method might not be effective either. We are developing an assay to better determine the timing of events during SG invasion with MoTissU-ExM, but this is beyond the scope of this study.

__In the section titled "Presence of PbRON11cKD sporozoites in the SG intercellular space", the authors state that "the majority of PbRON11cKD-infected mosquitoes contained some sporozoites in their SGs, but these sporozoites were rarely inside either the SG epithelial cell or secretory cavity". - this is suggestive of an invasion defect as the authors suggest. Could the authors collect these sporozoites and see if liver hepatocyte infection can be established by the mutant sporozoites? They previously speculate that the two different types of rhoptries (congruent and dimorphic) may be specific to the two invasion events (salivary gland epithelial cell and liver cell infection). __

It has already been shown that RON11cKD sporozoites fail hepatocyte invasion (PMID: 31247198), even when isolated from the haemolymph and so it seems very unlikely that they would be invasive following SG isolation. As mentioned in the discussion, RON11 in merozoites has a 'dual-function' where it is partially secreted during merozoite invasion in addition to its rhoptry biogenesis functions. Assuming this is also the case in sporozoites, using the RON11cKD parasite line we cannot differentiate these two functions and therefore cannot ascribe invasion defects purely to issues with rhoptry biogenesis. In order to answer this question functionally, we would need to identify a protein that only has roles in rhoptry biogenesis and not invasion directly.

Reviewer #3

Minor text changes (Reviewer #3)

1. __Page 3 last paragraph: ...the molecular mechanisms underlying SG (invasion?) are poorly understood. __

This has been corrected 2.

__The term "APR" does not refer to a tubulin structure per se, but rather to the proteinaceous structure to which tubulin anchors. Are there any specific APR markers that can be used in Figure 1C? If not, I recommend avoiding the use of "APR" in this context. __

The text does not state that the APR is a tubulin structure. Given that it is a proteinaceous structure, we visualise the APRs through protein density (NHS Ester). It has been standard for decades to define APRs by protein density using electron microscopy, and it has previously been sufficient in Plasmodium using expansion microscopy (PMIDs: 41542479, 33705377) so it is unclear why it should not be done so in this study. 3.

__I politely disagree with the bold statements ‚ Little is known about cell biology of sporozoite formation.....from electron microscopy studies now decades old' (p.3, 2nd paragraph); ‚To date, only a handful of (instead of ‚or') proteins have been implicated in SG invasion' (p. 4, 1st paragraph). These claims may overlook existing studies; a more thorough review of the literature is recommended. __

This study includes at least 50 references from papers broadly related to sporozoite biology, covering publications from every decade since the 1970s. The most recent review that discusses salivary gland invasion cites 11 proteins involved in SG invasion. We have replaced "handful" with a more precise term, as it is not the best adjective, but it is hardly an exaggeration.

Figure changes (Reviewer #3)

1. __The hypothesis that Plasmodium utilizes two distinct rhoptry pairs for invading the salivary gland and liver cells is intriguing but remains clearly speculative. Are the "cytoplasmic pair" and "docked pair" composed of the same secretory proteins? Are the paired rhoptries identical? How does the parasite determine which pair to use for salivary gland versus liver cell invasion? Is there any experimental evidence showing that the second pair is activated upon successful liver cell invasion? Without such data this hypothesis seems rather premature. __

We are unaware of any direct protein localisation evidence suggesting that the rhoptry pairs may carry different cargo. However, only a few proteins have been localised in a way that would allow us to determine if they are associated with distinct rhoptry pairs, so this possibility cannot be ruled out either. It seems unlikely that the parasite 'selects' a specific pair, as rhoptries are typically always found at the apical end. What appears more plausible is that the "docked pair" forms first and immediately occupies the apical docking site, preventing the cytoplasmic pair from docking there. Regarding any evidence that the second pair is activated during liver cell invasion, it has been well documented over decades that rhoptries are involved in hepatocyte invasion. If the dimorphic rhoptries are the only ones present in the parasite during hepatocyte invasion, then they must be used for this process. 2.

__The quality of the "Roolet fibre" image is not good and resembles background noise from PolyE staining. Additional or alternative images should be provided to convincingly demonstrate that PolyE staining indeed visualizes the Roolet fibre. It is puzzling that the structure is visible with PolyE staining but not with tubulin staining. __

This is a logical misinterpretation based on the image provided in Figure 1c. Our intention was not to imply that PolyE staining enables us to see the rootlet fibre but that PolyE and tubulin allow us to see the APR to which the rootlet fibre is connected. There is some PolyE staining that likely corresponds to the early SPMTs that in 1c appears to run along the rootlet fibre but this is a product of the max-intensity projection. Please see Reviwer#2 Figure Changes Comment #3 for the updated Figure 1c. 3.

__More arrows should be added to Figures 6b and 6c to guide readers and improve clarity. __

We have added arrows to Figure 6b and 6c which point out what we have defined as normal and aberrant rhoptries more clearly. These panels now look like this: 4.

__Figure 2a zoomed image of P. yoelii infected SG is different than the highligted square. __

We agree that the highlighted square and the zoomed area appear different, but this is due to the differing amounts of light captured by the objectives used in these two panels. The entire SG panel was captured with a 5x objective, while the zoomed panel was captured with a 63x objective. Because of this difference, the plane of focus of the zoomed area is hard to distinguish in the whole SG image. The zoomed image is on the 'top' of the SG (closest to the coverslip), while most of the signal you see in the whole SG image comes from the 'middle' of the SG. To demonstrate this more clearly, we have provided the exact region of interest shown in the 63x image alongside a 5x image and an additional 20x image, all of which are clearly superimposable.__

__ 5.

__Figure 3 legend: "P. yoelii infected midguts harvested on day 15" should be corrected. More general, yes, "...development of each oocyst within a single midgut is asynchronous." but it is still required to provide the dissection days. __

We are unsure what the suggested change here is. We do not know what is wrong with the statement about day 15 post infection, that is when these midguts were dissected. __ Experimental Changes (Reviewer #3)__

1. __The proposed role of AOR in rhoptry biogenesis appears highly speculative. It is unclear how the authors conclude that "AORs carry rhoptry cargo" solely based on the presence of RON4 within the structure. Inclusion of additional markers to characterize the content of AOR and rhoptries will be essential to substantiate the hypothesis that this enigmatic structure supports rhoptry biogenesis. __

It is important to note that the hypothesis that AORs, or rhoptry anlagen, carry rhoptry cargo and serve as vehicles of rhoptry biogenesis was proposed long before this study (PMID: 17908361). In that study, it was assumed that structures now called AORs or rhoptry anlagen were developing rhoptries. Although often visualised by EM and presumed to carry rhoptry cargo (PMID: 33600048, 26565797, 25438048), it was only more recently that AORs became the subject of dedicated investigation (PMID: 31805442), where the authors stated that "...AORs could be immature rhoptr[ies]...". Our observation that AORs contain the rhoptry protein RON4, which is not known to localize to any other organelle, we therefore consider sufficient to conclude that AORs carry rhoptry cargo and are thus vehicles for rhoptry biogenesis. 2.

__The study of RON11 appears to be a continuation of previous work by a collaborator in the same group. However, neither this study nor the previous one adequately addresses the evolutionary context or structural characteristics of RON11. Notably, the presence of an EF-hand motif is an important feature, especially considering the critical role of calcium signaling in parasite stage conversion. Given the absence of a clear ortholog, it would be interesting to know whether other Apicomplexan parasites harbor rhoptry proteins with transmembrane domains and EF-hand motifs, and if these proteins might respond similarly to calcium stimulation. Investigating mutations within the EF-hand domain could provide valuable functional insights into RON11. __

We are unsure what suggests that RON11 lacks a clear orthologue. RON11 is conserved across all apicomplexans and is also present in Vitrella brassicaformis (OrthoMCL orthogroup: OG7_0028843). A phylogenetic comparison of RON11 across apicomplexans has previously been performed (PMID: 31247198), and this study provides a structural prediction of PbRON11 with the dual EF-hand domains annotated (Supplementary Figure 9). 3.

__The study cannot directly confirm that membrane fusion occurs between rhoptries and AORs. __

This is already stated verbatim in the results "Our data cannot directly confirm that membrane fusion occurs between rhoptries and AORs..." 4.

__It is unclear what leads to the formation of the aberrant rhoptries observed in RON11cKD sporozoites. Since mosquitoes were not screened for infection prior to salivary gland dissection, The defect reports and revisited of RON11 knockdown does not aid in interpreting rhoptry pair specialization, as there was no consistent trend as to which rhoptry pair was missing in RON11cKD oocyst sporozoites. The notion that RON11cKD parasites likely have ‚combinatorial defects that effect both rhoptry biogenesis and invasion' poses challenges to understand the molecular role(s) of RON11 on biogenesis versus invasion. Of note, RON11 also plays a role in merozoite invasion. __

We are unclear about the comment or suggestion here, as the claims that RON11cKD does not help interpret rhoptry pair specialization, and that these parasites have combined defects, are both directly stated in the manuscript. 5.

__Do all SG PbRON11cKD sporozoites lose their reduced number of rhoptries during SG invasion as in Figure 7a (no rhoptries)? __

Not all RON11cKD SG sporozoites 'use up' their rhoptries during SG invasion. This is quantified in both Figure 7a and the text, which states: "64% of *PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries."

* 6.

Different mosquito species/strains are used for P. yoelii, P. berghei, and P. falciparum. Does it effect oocyst sizes/stages? Is it ok to compare?

__ __We agree that a direct comparison between for example * yoelii and P. berghei *oocyst size would be inappropriate, however Figure 3c and Supplementary Figure 4 are not direct comparisons between two species, but a summation of all oocysts measured in this study to indicate that the trends we observe transcend parasite/mosquito species differences. Our study was not set up with the experimental power to determine if mosquito host species alter oocyst size. 7.

__While I acknowledge that UExM has significantly advanced resolution capabilities in parasite studies, the value of standard microscopy technique should not be overlooked. Particularly, when discussing the function of RON11, relevant IFA and electron microscopy (EM) images should be included to support claims about RON11's role in rhoptry biogenesis. This would complement the UExM data and substantially strengthen the conclusions. Importantly, UExM can sometimes produce unexpected localization patterns due to the denaturation process, which warrants caution. __

The purpose of this study is not to discredit, undermine, or supersede other imaging techniques. It is simply to use U-ExM to answer biological questions that cannot or have not been answered using other techniques. Please refer to Reviewer # 1 Minor text changes comment#17 to see the new paragraph "Comparison of MoTissU-ExM and other imaging modalities" that addresses this

Both conventional IFA and immunoEM have already been performed on RON11 in sporozoites before (PMID: 31247198). When assessing defects caused by RON11 knockdown, conventional IFA isn't especially helpful because it doesn't allow visualization of individual rhoptries. Thin-section TEM also doesn't provide the whole-cell view needed to draw these kinds of conclusions. Volume EM could likely support these observations, but we don't have access to or expertise in this technique, and we believe it is beyond the scope of this study. It's also important to note that for the defect we observe-missing or abnormal rhoptries-the visualization with NHS ester isn't significantly different from what would be seen with EM-based techniques, where rhoptries are easily identified based on their protein density.

The statement that "UExM can sometimes produce unexpected localisation patterns due to the denaturation process..." is partially correct but lacks important nuance in this context. Based on our extensive experience with U-ExM, there are two main reasons why the localisation of a single protein may look different when comparing U-ExM and traditional IFA images. First, denaturation: in conventional IFAs, antibodies need to recognize conformational epitopes to bind to their target, whereas in U-ExM, antibodies must recognize linear epitopes. This doesn't mean the target protein's localisation changes, only that the antibody's ability to recognize it does. Second, antibody complexes seem unable to freely diffuse out of the gel, which can result in highly fluorescent signals not related to the target protein appearing in the image, as we have previously reported (PMID: 36993603). Importantly, neither of these factors applies to our phenotypic analysis of RON11 knockdown. All phenotypes described are based solely on NHS Ester (total protein) staining, so the considerations about changes in the localisation of individual proteins are not relevant.

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Referee #3

Evidence, reproducibility and clarity

Overall, the manuscript is well-written and -structured. However, I would like to raise several major points for consideration:

1. While I acknowledge that UExM has significantly advanced resolution capabilities in parasite studies, the value of standard microscopy technique should not be overlooked. Particularly, when discussing the function of RON11, relevant IFA and electron microscopy (EM) images should be included to support claims about RON11's role in rhoptry biogenesis. This would complement the UExM data and substantially strengthen the conclusions. Importantly, UExM can sometimes produce unexpected localization patterns due to the denaturation process, which warrants caution.
2. The proposed role of AOR in rhoptry biogenesis appears highly speculative. It is unclear how the authors conclude that "AORs carry rhoptry cargo" solely based on the presence of RON4 within the structure. Inclusion of additional markers to characterize the content of AOR and rhoptries will be essential to substantiate the hypothesis that this enigmatic structure supports rhoptry biogenesis.
3. The hypothesis that Plasmodium utilizes two distinct rhoptry pairs for invading the salivary gland and liver cells is intriguing but remains clearly speculative. Are the "cytoplasmic pair" and "docked pair" composed of the same secretory proteins? Are the paired rhoptries identical? How does the parasite determine which pair to use for salivary gland versus liver cell invasion? Is there any experimental evidence showing that the second pair is activated upon successful liver cell invasion? Without such data this hypothesis seems rather premature.
4. The study of RON11 appears to be a continuation of previous work by a collaborator in the same group. However, neither this study nor the previous one adequately addresses the evolutionary context or structural characteristics of RON11. Notably, the presence of an EF-hand motif is an important feature, especially considering the critical role of calcium signaling in parasite stage conversion. Given the absence of a clear ortholog, it would be interesting to know whether other Apicomplexan parasites harbor rhoptry proteins with transmembrane domains and EF-hand motifs, and if these proteins might respond similarly to calcium stimulation. Investigating mutations within the EF-hand domain could provide valuable functional insights into RON11.
5. The study cannot directly confirm that membrane fusion occurs between rhoptries and AORs.
6. It is unclear what leads to the formation of the aberrant rhoptries observed in RON11cKD sporozoites. Since mosquitoes were not screened for infection prior to salivary gland dissection, The defect reports and revisited of RON11 knockdown does not aid in interpreting rhoptry pair specialization, as there was no consistent trend as to which rhoptry pair was missing in RON11cKD oocyst sporozoites. The notion that RON11cKD parasites likely have ‚combinatorial defects that effect both rhoptry biogenesis and invasion' poses challenges to understand the molecular role(s) of RON11 on biogenesis versus invasion. Of note, RON11 also plays a role in merozoite invasion. I like the introduction of a segmentation score to Plasmodium oocyst maturation.

Minor comments:

1. The term "APR" does not refer to a tubulin structure per se, but rather to the proteinaceous structure to which tubulin anchors. Are there any specific APR markers that can be used in Figure 1C? If not, I recommend avoiding the use of "APR" in this context.
2. The quality of the "Roolet fibre" image is not good and resembles background noise from PolyE staining. Additional or alternative images should be provided to convincingly demonstrate that PolyE staining indeed visualizes the Roolet fibre. It is puzzling that the structure is visible with PolyE staining but not with tubulin staining.
3. Figure 2a zoomed image of P. yoelii infected SG is different than the highligted square.
4. Figure 3 legend: "P. yoelii infected midguts harvested on day 15" should be corrected. More general, yes, "...development of each oocyst within a single midgut is asynchronous." but it is still required to provide the dissection days.
5. More arrows should be added to Figures 6b and 6c to guide readers and improve clarity.
6. Do all SG PbRON11cKD sporozoites lose their reduced number of rhoptries during SG invasion as in Figure 7a (no rhoptries)?
7. Different mosquito species/strains are used for P. yoelii, P. berghei, and P. falciparum. Does it effect oocyst sizes/stages? Is it ok to compare?
8. I politely disagree with the bold statements ‚ Little is known about cell biology of sporozoite formation.....from electron microscopy studies now decades old' (p.3, 2nd paragraph); ‚To date, only a handful of (instead of ‚or') proteins have been implicated in SG invasion' (p. 4, 1st paragraph). These claims may overlook existing studies; a more thorough review of the literature is recommended.
9. Page 3 last paragraph: ...the molecular mechanisms underlying SG (invasion?) are poorly understood.

Significance

In this study, the authors explore Ultrastructure Expansion Microscopy (U-ExM) in Plasmodium-infected mosquito tissue with the aim to enhance the visualization of parasite ultrastructure. For this purpose, they revisit sporogony, the maturation of sporozoites inside oocysts, and sporozoite invasion of salivary glands, which has been studied both by cell biological methods and experimental genetics over four decades. They focus their analysis on the biogenesis and function of key secretory organelles, termed rhoptries, which are central to parasite invasion and, again, have been studied extensively.

This study is a follow-up of a previous study by the same authors (Ref. 19). In the former study the authors showed that U-ExM allows to visualize subcellular structures in sporozoites, including the nucleus, rhoptries, Golgi, apical polar rings (APR), and basal complex, as well as midgut-associated oocysts with developing sporozoites. Here, the authors claim a new finding by stating that sporozoites possess two distinct rhoptry pairs. Supposedly, only one pair is utilized during salivary gland invasion. The authors suggest specialization of rhoptries for different cell invasion events. The authors also revisit a RON11 knock-down parasite line, which was previously shown to be deficient in salivary gland invasion, host cell attachment, gliding locomotion, and liver invasion (Ref. 14).

I find it difficult to estimate the significance. Obviously, attention will be limited to Plasmodium researchers only, as this study is descriptive and revisits a well-studied aspect of the Plasmodium life cycle in the Anopheles vector.

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Referee #2

Evidence, reproducibility and clarity

The manuscript by Liffner et al have used the modified expansion microscopy as they term Mosquito Tissue Ultrastructure Expansion Microscopy (MoTissU-ExM) to study a cell biology of temporal development of malaria parasite sporozoite biogenesis within mosquito host. They employed three different malaria parasite models Plasmodium yoelii, P.beghei and P falciparum and infected them in mosquito host.

The application of MoTissU-ExM to infected mosquito tissues is a significant technical advance, enabling visualizations previously only achievable with electron microscopy.

The major conclusion and advances are as following

• The establishment of a "segmentation score" as a great tool for staging asynchronous oocyst development.
• The location of Centriolar plaques, rootlet and other structures which are difficult to analyse
• The first detailed timeline for sporozoite rhoptry biogenesis.
• Clear quantification showing that sporozoites possess four rhoptries and utilise two during salivary gland (SG) invasion.
• A characterization of the RON11 knockout phenotype, linking it to defects in rhoptry biogenesis and a specific block in SG epithelial cell invasion. The following points are intended to further strengthen the paper for publication.

Points for Revision

1. For clarity, it would be helpful to add indicators for the centriolar plaques in Figure 1b, as their locations are not immediately obvious.
2. The 'image Z-depth' value indicated in the figures is ambiguous. It is not clear whether this refers to the distance from the coverslip surface or the starting point of the z-stack image acquisition. A precise definition of this parameter would be beneficial.
3. Regarding Figure 1c, the authors state that 'the rootlet fiber is visible'. However, such a structure cannot be confirmed from the provided NHS ester image. Can the authors present a clearer image where the rootlet fibre is more distinct? Furthermore, please provide the basis for identifying this structure as a rootlet fiber based on the NHS ester observation alone.
4. Why do the congruent rhoptries have similar lengths to each other, while the dimorphic rhoptries have different lengths? Is this morphological difference related to the function of these rhoptries?
5. Would it be possible to show whether RON11 localises to the dimorphic rhoptries, the congruent rhoptries, or both, by using expansion microscopy and a parasite line that expresses RON11 tagged with GFP or a peptide tag?
6. The knockdown of RON11 disrupts the rhoptry structure, making the dimorphic and congruent rhoptries indistinguishable. Does this suggest that RON11 is important for the formation of both types of rhoptries? I believe that it would be crucial to confirm whether RON11 localises to all rhoptries or is restricted to specific rhoptries for a more precise discussion of RON11's function.
7. The authors state that 64% of RON11cKD SG sporozoites contained no rhoptries at all. Does this mean RON11cKD SG sporozoites used up all rhoptries corresponding to the dimorphic and congruent pairs during SG invasion? If so, this contradicts your claims that sporozoites are 'leaving the dimorphic rhoptries for hepatocyte invasion' and that 'rhoptry pairs are specialized for different invasion events'. If that is not the case, does it mean that RON11cKD sporozoites failed to form the rhoptries corresponding to the dimorphic pair? A more detailed discussion would be needed on this point and, as I mentioned above, on the specific role of RON11 in the formation of each rhoptry pair.
8. Out of pure curiosity, is it possible to measure the length and number of subpellicular microtubules in the sporozoites observed in this study using expansion microscopy?
9. Is it possible that the dimorphic rhoptries are simply precursors to the congruent rhoptries? Could it be that after the congruent rhoptries are used for SG invasion, new congruent rhoptries are formed from the dimorphic ones and are then used for the next invasion? Would it be possible to investigate this by isolating sporozoites some time after they have invaded the SG and performing expansion microscopy? This would allow you to confirm whether the dimorphic rhoptries truly remain in the same form, or if new congruent rhoptries have been formed, or if there have been any other changes to the morphology of the dimorphic rhoptries.
10. In addition to the previous point, in the text accompanying Figure 7a, the authors claim that "64% of PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries". Could this data be used to infer which rhoptry pair are missing from the RON11cKD oocyst sporozoites? Can it be inferred that the 64% of salivary gland sporozoites that had no rhoptries in fact had 2 congruent rhoptries in the oocyst sporozoite stage and that these have been discharged already?
11. In the section titled "Presence of PbRON11cKD sporozoites in the SG intercellular space", the authors state that "the majority of PbRON11cKD-infected mosquitoes contained some sporozoites in their SGs, but these sporozoites were rarely inside either the SG epithelial cell or secretory cavity". - this is suggestive of an invasion defect as the authors suggest. Could the authors collect these sporozoites and see if liver hepatocyte infection can be established by the mutant sporozoites? They previously speculate that the two different types of rhoptries (congruent and dimorphic) may be specific to the two invasion events (salivary gland epithelial cell and liver cell infection).

There are a few typing errors in the document:

1. Paragraph 3 of the introduction - line 7, "handful or proteins" should be handful of proteins
2. Paragraph 5 of the introduction - line 7, "also able to observed" should be observe
3. In the final paragraph of the introduction - line 1, "leverage this new understand" should be understanding
4. The first paragraph of the discussion summary contains an incomplete sentence on line 7, "PbRON11ctrl-infected SGs."
5. The second paragraph of the discussion - line 10, "until cytokinesis beings" should be begins

Some suggestions for figures

Fig 1B - could the tubulin image in the hemispindle panel be made brighter?

Fig 3B: stage 2 and 6 does not show the DNA cyan, it would-be good show the sate of DNA at that particular stage, especially at stage 2 when APR is visible. And box the segment in the parent picture whose subset is enlarged below it.

Fig 4A - the green text in the first image panel is not visible. Also, the cyan text in the 3rd image in Fig 1A is also difficult to see. There's a few places where this is the case

Fig 6A - how do the authors know ron11 expression is reduced by 99%? Did they test this themselves or rely on data from the lab that gifted them the construct? Also please provide mention the number of oocyst and sporozoites were observed.

Fig 6E - are the data point colours the wrong way round on this graph? Just looking at the graph it looks as though the RON11cKD has more rhoptries than the control which does not match what is said in the text.

Fig S8C, PbRON11 ctrl, pie chart shows 89.7 % spz are present in the secretory cavity while the text shows 100 %, 35/35

Fig S9D shows that RON11 ckd contains 17.1% sporozoites in secretory cavity while the text says 24%.

Some point to discuss

1.One minor point that author suggest that oocyst diameter is not appropriate for the development of sporozoite develop. This is not so true as oocyst diameter tells between cell division and cell growth so it is important parameter especially where the proliferation with oocyst does not take place but the growth of oocyst takes place.<br /> 2. The author have not mentioned that sometimes the stage oocyst development is also dependent on the age of mosquito and it vary between different mosquito gut even if the blood feed is done on same day. 3. How is the apical polarity different to merozoite as some conoid genes are present in ookinete and sporozoite but not in merozoite.

Significance

The following aspects are important:

This is novel and more cell biology approach to study the challenging stage of malaria parasite within mosquito. By using MoTissU-ExM, the authors have enabled the three-dimensional observation of ultrastructures of oocyst-sporozoite development that were previously difficult to observe with conventional electron microscopy alone. This includes the developmental process and entire ultrastructure of oocysts and sporozoites, and even the tissue architecture of the mosquito salivary gland and its epithelia cells.

Advances:

By observing sporozoites formation within the oocyst and the overall ultrastructure of the sporozoite with MoTissU-ExM, the authors have provided detailed descriptions of the complete structure and three-dimensional spatial relationships of the rhoptries, rootlet fibre, nucleus, and other organelles. Furthermore, their detailed localisation analysis of sporozoites within the salivary gland is also a great achievement. Considering that such observations were technically and laboriously very difficult with conventional electron microscopy, enabling these analyses with higher efficiency and relatively lower difficulty represents a major contribution to the future advancement of oocyst-sporozoite biology. The development of the 'segmentation score' for sporozoite formation within the oocyst is another major advance. I think this will enable detailed descriptions of structural changes at each developmental stage and of the molecular mechanisms involved in the development of oocysts-sporozoites This has its advantages if antibodies can be used and somewhat reduces the need for immuno-EM. Secondly, in terms of sporozoite rhoptry biology, the Schrevel et al Parasitology 2007 seems to only focus on oocyst sporozoite rhoptries as they say that the sporozoites have 4 rhoptries. This study on the other hand also looks at salivary gland sporozoites and shows that there are potentially important differences between the two - namely the reduction from 4 rhoptries to two. This also leads to further questions about the different types of rhoptries in oocyst sporozoites and whether they're adapted to invasion of different cell types (sal gland epithelial cells or liver hepatocytes)

Limitation

It would be that expansion microscopy alone still has its limits when it comes to observing ultra-fine structures. For example, visualising the small vesicular structures that Schrevel et al. observed in detail with electron microscopy, or seeing ultra-high resolution details such as the fusion of membrane structures and their interactions with structures like the rootlet fibre and microtubules. Therefore, I think that electron microscopy remains essential for the observation of such ultra-fine structures The real impact of this work is mostly cell biologist working with malaria parasite and more in mosquito stages. But the approaches can be applied to any material from any species where temporal dynamics need to be studied with tissue related structures and where UExM can be applied. I am parasite cell biologist working with parasites stages within mosquito vector host.

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Referee #1

Evidence, reproducibility and clarity

In this paper the authors use ultrastructure expansion microscopy to investigate the mosquito stages of the malaria parasite, specifically the stage called oocyst and the process of sporozoite development. They report a number of observations of which the ones concerning rhoptries are the most interesting. There are four of these organelles in the first form of sporozoites in the oocyst and only two in the mature form in the salivary gland. Using a gene knockout of a protein that was reported to be important for rhoprty formation in merozoites, the parasites invading into human red blood cells, they found that fewer rhoptries are formed also in sporozoites and that these cannot enter into the salivary gland cells any more. The presented data are in my view conclusive and no additional experiments are needed for this work to be published. The described experiments should be readily reproducible and have a high statistical power. The text is mostly clearly written but could be improved to make it more concise and more precise and to avoid overstatements. Some references could be added. It would have helped to have line numbers in the manuscript. My suggestions are as following:

Abstract: don't focus on technique but on the questions you tried to answer (ie rewrite or delete the 3rd and 4th sentence)

Add reference on page 3 after 'disrupted parasites' Change 'the basal compelx at the leading edge' - this seems counterintuitive Change 'mechanisms underlying SG are poorly' - what mechanisms? of invasion or infection? On page 4: 'handful of proteins' 'range of cell biology processes' - I understand the paper that the key discovery concerns rhoptry biogenesis and function, so focus on that, all other aspects appear rather peripheral. what are the 'three microtubule spindle structures'? 'Much of this study focuses on the secretory organelles': I would suggest to rewrite the intro to focus solely on those, which yield interesting findings. On page 5: 'little is known' - please describe what is known, also in other stages. At the end of the paper I would like to know what is the key difference to rhoptry function in other stages? change 'rhoptries golgi-derived, made de novo' change 'new understand to' 'rhoptry malformations' seem to be similar in sporozoites and merozoites. Is that surprising/new? What is known about crossing the basal lamina. Where rhoptries thought to be involved in this process? Or is it proteins on the surface or in other secretory organelles? On page change/specify: 'wide range of parasite structures' On page 7: is Airyscan2 a particular method or a specific microscope? what are the dark lines in panel E? in panel G: Are the dense granules not micronemes? What are the dark lines? Rhoptries?? On page 8 the authors mention a second layer of CSP but do not further investigate it. It is likely hard to investigate this further but to just let it stand as it is seems unsatisfactory, considering that CSP is the malaria vaccine. What happens if you add anti-CSP antibodies? I would suggest to shorten the opening paragraphs of this paper and to focus on the rhoptries. This could be done be toning down the text on all aspects that are not rhoptries and point to the open question some of the observations such as the CSP layers raise for future studies. Figure 2 seems to add little extra compared to the following figures and could in my view go to the supplement. On page 10 I suggest to qualify the statement 'oocyst development has typcially been inferred by'. There seem a few studies that show that size doesn't reflect maturation. Page 11: I am tempted to suggest the authors start their study with Figure 3 and add panel A from Figure 2 to it. This leads directly to their nice work on rhoptries. Other features reported in Figures 1 and 2 are comparatively less exciting and could be moved to the supplement or reported in a separate study. Text on page 12 could be condensed to highlight the new data of ron4 staining of the AOR. Maybe include more detail of the differences between species on rhoptry structure into Figure 4. I would encourage to move the Data on rhoptries in Figure S6 to the main text ie to Figure 4. On page 16 the authors state that different rhoptries might have different function. This is an interesting hyopthesis/result that could be mentioned in the abstract. how large is RON11? On page 19: do the parasites with the RON11 knockout only have the cytoplasmic or only the apical rhoptries? Page 23: I suggest to delete the first sentence and focus on the functional aspects and the discoveries. There is no causal link between ookinete invasion and oocyst developmental asynchrony First sentence of page 24 appears to contradict what is written in results I don't understand the first two sentences in the paragraph titled Comparison between Plasmodium spp On page 25 or before the vast number of electron microscopy studies should be discussed and compared with the authors new data. First sentence on page 27: there are many studies on parasite proteins involved in salivary gland invasion that could be mentioned/discussed. Maybe add a conclusion section rather than a future application section, which reads as if you want to promoted the use of ultrastructure expansion microscopy. To my taste the technological advance is a bit overplayed considering the many applications of this techniques over the last years, especially in parasitology, where it seems widely used. In any case, please delete 'extraordinarily'

Significance

This interesting study investigates the development of malaria parasites in the mosquito using ultrastructure expansion microscopy adapted to mosquito tissue. It provides new and beautiful views of the process of sporozoite formation. The authors discovered that four secretory vesicles called rhoptires are formed in the sporozoites with two pairs being important for distinct functions, one pair functions during invasion of the salivary glands of the mosquito and the other in liver infection, although the latter is not shown but inferred from prior data.

This study will thus be of interest to scientists investigating malaria parasites in the mosquito as well as to scientist working on vesicle secretion and invasion in these parasites.

The authors use a previously generated parasite line that lack a protein to investigate its function in rhoptry biogenesis and find that its absence leads to fewer rhoptries which impacts the capacity of the parasite to enter into salivary gland cells. This is a nice functional addition to an otherwise largely descriptive study, but mimics largely the previously reported results from the blood stages. It is not clear to this reviewer how much the study advances the field over the many previous electron microscopy studies. This could be better elaborated in the text.

Strength of the study: beautiful microscopy, new insights into rhoptry formation and function, new technique to study malaria parasites in the mosquito

Weakness of the study: Some loose ends in the description of spindles and CSP layers, text could be more focussed on the key advancements reported

Reviewer #1 (Public review):

Summary:

Renard, Ukrow et al. applied their recently published computational pipeline (CHROMAS) to the skin of Euprymna berryi and Sepia officinalis to track the dynamics of cephalopod chromatophore expansion. By segmenting each chromatophore into radial slices and analyzing the co-expansion of slices across regions of the skin, they inferred the motor control underlying chromatophore groups.

Strengths:

The authors demonstrate that most motor units of cephalopod skin include a subregion of multiple chromatophores, creating "virtual chromatophores" in between the fixed chromatophores. This is an interesting concept that challenges prevailing models of chromatophore organization, and raises interesting possibilities for how chromatophore arrays may be patterned during development.

This study introduces new analyses of cephalopod skin that will be valuable for the quantitative study of cephalopod behavior.

Weaknesses:

The authors chose to image spontaneous skin changes in sedated animals, rather than visually-evoked skin changes in awake, freely-moving animals. Spontaneous chromatophore changes tend to be small shimmers of expansion and contraction, rather than obvious, sizable expansions. This may make it more challenging to distinguish truly co-occurring expansions from background activity. The authors don't provide any raw data (videos) of the skin, so it is difficult to independently assess the robustness of the inferred chromatophore groupings.

The patch-clamp experiments in E. berryi are used to test the validity of their approach for inferring motor units. The stimulations evoke expansions of sub-regions of each chromatophore, creating "virtual chromatophores" as predicted from the behavioral analysis. However, the authors were not able to predict these specific motor units from behavioral analysis before confirming them with patch-clamp, limiting the strength of the validation. It would be informative to quantify the results of the patch-clamp experiments - are the inferred motor units of similar sizes to those predicted from behavior?

The authors report testing multiple experimental conditions (e.g., age, size, behavioral stimuli, sedation, head-fixation, and lighting), but only a small subset of these data are presented. It is difficult to determine which conditions were used for which experiments, and the manuscript would benefit from pooling data from multiple experiments to draw general conclusions about the motor control of cephalopod skin.

The authors use a different clustering algorithm for E. berryi and S. officinalis, but do not discuss why different clustering approaches were required for the two species.

Impact:

The authors use their computational pipeline to generate a number of interesting predictions about chromatophore control, including motor unit size, their spatial distribution within the skin, and the independent control of subregions within individual chromatophores by putatively distinct motor neurons. While these observations are interesting, the current data do not yet fully support them.

The CHROMAS tool is likely to be valuable to the field, given the need for quantitative frameworks in cephalopod biology. The predictions outlined here provide a useful foundation for future experimental investigation.

Reviewer #2 (Public review):

Summary:

Overall, this is an excellent paper, making use of a newly developed system for monitoring the behaviour of chromatophores in the skin of (mostly) free-swimming bobtail squid and European cuttlefish. The manuscript is very well-written, clearly presented and very well-structured. The central finding, that individual chromatophores are connected to multiple motor neurones, is not new. Novelty instead comes from the ability to measure the actuation of chromatophore sections across wide areas of skin in free-swimming animals, showing the diversity of local motor units and reinforcing the notion that individual chromatophores are not necessarily the individual units of colour change, but rather local motor units that cover multiple neighbour and near-neighbour chromatophore muscles. This is an excellent finding and one that will shape our understanding of the neural control of cephalopod skin colour.

Strengths:

The methodological approach to collecting large amounts of data about local variations in the expansion of sections of chromatophores is exciting, and the analysis pipeline for clustering sections of chromatophores whose spontaneous activity correlated over time is powerful and exciting.

Weaknesses:

Some minor edits and typographical errors need correcting. I also had some concerns that the preparation for the electrophysiological section of the manuscript complies with the journal's ethical requirements, so I would urge that this be carefully checked.

Reviewer #3 (Public review):

Summary:

This study uses high-resolution videography and a custom computer-vision pipeline to dissect the motor control of cephalopod chromatophores in Euprymna berryi and Sepia officinalis. By quantifying anisotropic chromatophore deformations and applying dimensionality reduction methods, the authors infer that individual chromatophores can be a part of multiple motor units. Clustering analyses reveal putative motor units that often span multiple chromatophores, with diverse and overlapping geometries. Chromatophore expansion dynamics are faster and more stereotyped than relaxation, consistent with active neural contraction followed by passive recoil. Together, the results show that chromatophores function not as uniform pixels but as fractionated, coordinately controlled elements that enable flexible pattern generation

Strengths:

The authors present compelling, direct evidence that a). chromatophore deformations are anisotropic, and indirect evidence that b) individual chromatophores can be split across multiple putative motor units. This evidence is provided through data collected over large spatial scales, but also at a sub-chromatophore resolution. This combination of scale and resolution is not possible using traditional neuroanatomical and physiological approaches alone.

The authors also develop a new non-invasive, image analysis approach to extract information about chromatophore deformation across large spatial scales on the organism's body. In principle, this approach is applicable across species and may allow for further comparative characterization of chromatophore motor control. It is therefore a promising new tool and useful resource for the community.

Weaknesses:

An important weakness of the work is that the methods the authors develop can only be applied during resting, spontaneous 'flickering' activity of chromatophores. The inability to reliably apply their technique during any kind of realistic camouflage is a large limitation, as it means this method cannot be used to study the dynamics of motor control during realistic camouflage behaviors.

Another weakness of this paper is the rather limited electrophysiological validation of the computational findings. The authors present only one electrophysiology experiment in E. berryi, the species that they used only for 'methodological development' and not for detailed characterization. A complementary electrophysiological experiment in S. officinalis, or some visualization of neuron morphology confirming that motor neurons do indeed project to multiple chromatophores, would strengthen the generalizability of their computational analysis. This would be particularly pertinent to validate the author's claim that some motor units contain chromatophores that are quite distant from one another on the animal.

Overall, the authors' technical contributions and method development are an important advance. This work serves as an excellent proof of concept that their method can extract useful information about chromatophore motor control. Further validation of their method is needed to fully trust the fine-scale conclusions drawn about the distribution and composition of multi-innervated chromatophores. Furthermore, the authors raise many interesting ideas about developmental constraints on circuit wiring and potential adaptive significance of multi-innervated chromatophores for certain features of camouflage patterning. Their method may be able to help resolve some of these questions in the future if it is refined and applied across developmental stages, regions of the animal, and across species

Low Band (Sub-1 GHz) - Covers a wide area, including cities, suburbs, and rural areas. - Good for IoT devices and deep indoor coverage. - Speed is lower: up to ~100 MB/s.

Mid Band (1–6 GHz) - Balances speed and coverage. - Includes frequencies like 3.3–3.8 GHz, 1800 MHz, 2.3 GHz, 2.6 GHz. - Wireless speed is 5–10 times faster than 4G. - This is usually the first spectrum sold in auctions.

High Band (Above 6 GHz, e.g., 26 GHz) - Extremely fast and low-latency (up to ~1 GB/s). - Covers short distances only. - Enables advanced services like vehicle-to-vehicle communication and remote surgery.

In short: low band = wide coverage, mid band = good mix, high band = super fast but short range

Of course, there are many interests to dislodge, but we should believe in enough good faith to move forwards.

Social media data is used to maximize user engagement and profit, primarily through targeted advertising, but this same system can be exploited to manipulate vulnerable populations and undermine democratic processes.

1. As someone who's on social media pretty often, I can 100% attest to the fact that any sort of interaction with an ad or sponsored post will cause your timeline to be filled with other ads relating to a similar product, if not the same one. Sometimes ads do get my attention and I look at the item to see the price, and when I return to my homepage I manage to see the same post 3 or 4 times in the same hour. I also do recognize that gambling advertisements are often shown to children, which I think should 100% monitored, considering children are both not able to grasp the concept of money at their age, and they're children, the idea that it's legal to show these ads to children when they're using electronics is beyond me.****

Assuming cloud based models. Why? You could drop up to 200 month on a VPS and be really self sufficient, but probably wouldn't need a VPS that heavy?

clawdbot as additional attack surface

again this is arriving at database queries you already had, but through a way more convoluted way. When 'reasoning' is deterministic then you don't need a probabilistic layer at all, no?

knowledge graphs represent semantic context and relationships / constraints. K-graphs are a 1980s thing, I know we added them into the reference architecture for systems of digital twins I cowrote. But have no understanding more recent than the 1990s. - [ ] spend #30mins collecting current state of the art on #knowledgegraphs #pkm

criticism of retrieval augmented generatio (RAG): fails in cross domain settings, finds similar text not relations between facts or meaning

This part really played with my emotions because at first I thought her father was supportive, but later I realized he only saw college as a tool for her to find someone to marry instead of getting an education.

In this paragraph, Cisneros expresses how her identity and background shape who she is. When she lists the 3 different ways to describe herself, she talks about her gender, culture, and social class.

Blessing and a curse all in the same. However, it depends on how one utilizes it. Afterall, we are the only ones in control of our own actions.

All of them depend on what I am learning. #1 -3 I've learned about being motivated and interested in what I'm learning which is one of the major aspects of my learning. A preferred learning style is just what it says, how one PREFERS to learn something. I agree that a preference on learning something shouldn't be categorized in a box, however it should just be looked at a a preference by that individual. 4- Somewhat, I believe that teachers are a big part, but the most significant aspect is me.

Did this using qwen3-code, but then Claude Code just keeps spinning, even with the smallest prompt. Not sure about the cause (first load of model??)

It didn't occur to me that AI could be addictive but I can totally see why it would be.

I feel like AI is amplifying an issue that has been around for awhile but now we will see it on a much more dramatic scale as AI evolves and more and more people use it.

It seems that the hidden curriculum is not helpful to a ne student, I get that it exists, but should it? Can it be avoided?

I find the idea of essentialism especially important because it reveals how stereotypes often disguise themselves as “cultural knowledge.” Saying “they’re just like that culturally” can sound neutral, but it can actually deny people agency and adaptability.

Observation: Bossuet warns kings that they will be judged by God for how they use their power

Interpretation: This shows that although kings had power, they were still expected to rule with their power responsibly escpecially with the religious standards they were being held up to.

Connection: This connects to the tertiary sourse, which explains that absolutist ideology still made a moral and ethical responsibility, even though political authority was the main rule.

Consequence: When abusing the power that they have, there are consequences that can take place if rulers failed to live up to moral and religious expecations.

Observation: Bossuet says that the king is sacred and that harming or going against him is considered a crime.

Interpretation: This shows that people who beleived in divine rights from their God saw opposing the king as morally and religiously wrong, but not politically illegal.

Connection: I think this connects to the tertiary source, which says that absolutist rulers justified their authority by saying that it was directly in line with God, in order to prevent people from rebelling.

Cause: The belief was that the king was sacred and helped cause obedience throughout the population and caused people to less likely act out against authority.

Observation: Bossuet says that the Royal power is absolute and that the king doesn't answer to anyone on earth

Interpretation: This shows that people who may have supported absolutionism believed the king should not be limited by institutions like parliaments or assemblies, because his authority came from God, so that should be who is asked upon.

Connection: This connects to the tertiary source, which says that French absolutism places all political authority in the hands of the monarch.

Context: When you understand the contect of 17th century France it helps understand and interpret how these arguments were played out, since some monarchs were trying to justify centralized power and prevent changed to their rule.

Teaching the opportunity is good, but teaching the lesson is better.

Definitely a weak spot for me. I always have my phone with me for music usually but i easily get distracted and take too many breaks. If i could focus longer i would stay more consistent with the material at hand.

what happened to me this afternoon, because of a silly mistake I made on an assignment to miss a point.

The fiscal crisis is a recurring pattern in capitalist economies like NYC. Similar breakdowns will continue to happen when economic expansion is continuously prioritized. Instead of addressing the causes of a fiscal crisis, service and budget are cut, and the government divests in poor communities. Only the government can choose to prioritize social needs over market desires, and only then will this cycle become less popular.

Is increased sufficiency brought upon by technology an advancement of society? What defines societies attachment to humanity? Can society outpace humanity?

These notions attach "intrinsic value" to our perception of our selves, and humanity in general. Advances in technology =/= an advancement of humanity, maybe in some aspects but overall I do not believe so.

These problems have existed, and still exist today, but in a much more complex manner. The normalization of depersonalized traits and alienation from one another is the scariest threat to humanity at this time with the increasing prevalence of technology stripping away our fundamental human values.

But what happens when people think that the models that they use are benign? What happens when people "assume best intentions."

yes!!!! However, they don't always work out. I personally love when stats can back up something, like stealing bases, but I know that although stealing bases is statistically not a smart choice, it adds drama to the game. The same can be said with boxing, but when we lose the integrity of sport, it just becomes numbers and playing in the favor of whomever get to benefit from those numbers

like an unspoken "truth" that is observed by many and not questions because it is in "good faith"

Something that I never thought about...but makes sense.

Dissapointing

Type of data that is not necessarily manipulated, but created due to the inevitable surroundings of historically marginalized youth.

Ridiculous and tiresome to even go through all these options. A little glimpse into her mind as a mathematician

I think it's important to do all this but perhaps may not turn out to be the ideal "model"

can the same be said for any sport? I think of the times where underdogs win. I watch a lot of volleyball and never thought A&M would win the national championship. But their game throughout the season became increasingly better. Were player statistics getting to the level of a #1 team?

Sports data amazes me and I see how coaches make so much. Not only do they have to be experts in the game, but they also have to constantly monitor trends on game days and make critical decisions, especially during high stakes games.

It shouldn't be this hard to seek explanation. How are decision makers able to back data when they cant explain or justify it?

hard to read but true

I think this clearly explains how math is used to influence bad economic decisions by individuals.

This final stanza now reflects how in this generation we live in, generation before may try to cut the legs we stand us but to question themselves, they act like they know everything similar to the white men from the previous poem but we got to question them in what they do, not on what we do.

This wording, I'm trying to understand what it means like I think I got an idea but need to finish this reading to fully understand

This an intresting opener, it so far sounds like they believe in something but others who I'm guessing due to the conenction to the other poem, they don't believe them due to a certain bias or other reason.

I would drop this paragraph down in a new visual section. Centre the heading, and add 3 columns underneath with each idea.

5f defines digestion as a form of translation that assesses your own thoughts, ideas, and observations. This tells me I need to explain how the sources I use help me understand my film community more. I will ask questions such as "How does this theory of ... explain what I saw in the communities Reddit page?" This will hopefully help me to go beyond summary.

To prove this point, Charles Darwin examined the island of Galapagos, specifically the finches of said island. According to a documentary that I saw for an anthropology class, over the course of many years, Darwin returned to the island and noted the changes in the plumes and beaks of the finches as generations of them went by; from this, he proved his theory of evolution. This section of the chapter mentions 3 names in particular of prominent people who disproved, in one way or another, the ideology behind Christianity on a biological level. Aside from these 3, who all influenced this challenge to Christian ideology? Were there others who may have influenced any of these 3, such as someone whose work individually would not propose much of an opposition, but who drove forth this level of thinking nonetheless?

From my prior knowledge, the majority of Issac Newton’s work was publicly stated to be based on his desire to understand heaven better; he allegedly referred to himself as a theologian, not a scientist. This was referenced in the previous paragraph, although not in much depth. For his work to have been part of the foundation in sciences that caused the separation of science and religion is fascinating, considering how he was religious, although not to the extent he portrayed himself openly.

how data travels through the internet. It uses a protocol called packet switching and imagine you're sending an email to your friend and it's a long email. Your email application is going to hand that off to a protocol that's going to break that message into fixed size, relatively small packets. So, those are P1, P2, P3. Here, those packets are going to be sent through the internet to the destination. So, this is the source your computer, let's say, and this is your friends. Can take different routes through the network when they arrive at their destination. Of course, they have to be gathered together there and then then reassembled, and there s no guarantee they re going to arrive in the order P1, P2, P3, but they have to be reassembled in that order in order for you to read them. Include email packet switching example

The internet uses a basic end-to-end architecture. Messages are sent from, say, a host A to host B, but messages have to travel through one or more routers, depending on where these two hosts are are located, and hosts are simply computers that are connected to the internet, so that could mean your laptop or your smartphone or tablet and hosts are given IP addresses or Internet. Hosts are reachable either by means of their IP address or by their domain name. For example, the host trincall.edu, the main computer for Trinity College, has this domain name and this IP address: 157.252.10.123. We're going to learn a lot more about how that works in a subsequent lesson. Routers compared to hosts are dedicated computers that transmit data between networks within the internet. Include diagram of hosts and routers

This highlights again about something to do with a authority figures which the white men seem to represent but their oppressive figures of authority.

But even with all her defense is still doesn't make difference to others no matter how many tears cried, if your are not like the white men then it just feels like a waste of time and resources to even try to speak up to others.

This is just a bombshell and it all makes sense now. Girls face alot stuff but weren't believed for it, this supported by the earlier lines. Others only believe it because someone who many people favor such as an author, John Krakauer, they now treat the issue like it's a specactle, a new genre of writting, or just pretend like they always known and try to assume some sort of high ground. While the girl who was trying to say that the whole time is still left forgotten, like this is not her issue and one she isn't making up but actively suffered in.

I'm assuming these are the type of sentences she being fed by others which could moslty refer to the old white men which is even supported at the end of the lines. But one more intresting thing is that she metions "taking up the most space".

Were continuing from the reality check, were they are questioning her confidence since before they were talking about "speak it like you mean it". But she pointing out that she has a confidence and it's not like the words they would understand would make her speak up any louder than she is already.

Now I'm getting the picture that, this is like a reality check, in her shoes nobody takes her seriosuly or believe she really believes her own stuff due to her words and tone but can also be because she a girl.

Countercultures like the 1960s hippie movement serve a vital function by questioning assumptions that mainstream culture has stopped examining. When ways of living become "just how things are done," they often persist not because they're optimal but because they're familiar. Countercultures act as a kind of cultural R&D lab, testing alternative approaches to fundamental questions about meaning, community, and how to live. Countercultures also provide a "pressure valve" for people who genuinely don't fit the dominant mold. Not everyone thrives in the prevailing structure, and having alternative spaces and communities can be psychologically essential for those who experience the mainstream as alienating or oppressive.

These terms explain the important part of writing a narrative. a writer must help the reader understand who is involved where and when events happen and organize the story in a clear way. Good narrative mix scenes, which show important moments with details and dialogue, and summaries, which mean something.

we can use this guideline to write a great narrative.

all these tools will help you write a great narrative.

it is more important to capture the emotional truth of an experience than to remember every exact detail, they should say the narratives just the way they are.

This narrative highlights how communication and honesty shape family relationships.

the writer can always understand a person who is starting to write like and maybe understand what I am trying to say through my feelings.

is Always great to have other recourses or people to ask about great narratives or ask any related questions on the reading or how to simply make a great narrative as well.

Being able to communicate clearly and convey what you want the reader to understand is important, as it allows the reader to interpret your story properly.

At times my opinion is less could be more in a situation but still sticking to strong points in the story leaving out details that don't pertain to the story especially if they are not true and just being used as fillers in the story.

This would definitely catch the readers attention and tune their emotions into the text as it did mine.

A more in depth visual explanation going beyond the writer themselves while leaving open opportunities for the reader to relate. Quoting text "Here’s an example, I used to spend summers at my grandmother’s house in New Jersey–snore. Who cares, right?" leaves no room for the reader to relate unless they have a grandma in Jersey. For example, "Spending summers with grandma was something that will never be forgotten" could open up more possibilities for the reader to connect dots in their mind with similar feelings about their own summer experiences with their grandma.

Creative writing is used to reach an audience soliciting an emotional response to the writers writing. Forming a connection between readers and writer.