"Aggregates", here, perhaps refers to the 5 skandas?

Nagarjuna, belonging to the Madhyamika school of thought, argued for emptiness (even of emptiness). In doing so, he contradicted the brahmanical schools of thought, which believed in the existence of atman and brahman, and a 'permanence'. Nagarjuna on the contrary, being a mahayana buddhist, believed in 'impermanence'. This impermanence extended to that of 'being' as well, with all existence being 'momentary', and a superimposition of our cognition, of sorts.

I am confused here because it is mentioned earlier that the aggregates (I'm assuming feelings, discriminations, etc. are included in that) are nonexistent. So does selfless mean nonexistent here?

I suppose this connects to the idea of yin and yang? - They must exist simultaneously?

My takeaway from this explanation is that the crux of exiting the cycle of birth and rebirth is in a complete and total belief that it and everything it comprises is nonexistent. Upon arriving at the conclusion that is it nonexistent, there is no way to truly verify if it exists or not. It's a situation where you have decided that it is nonexistent and that action is what removes you from it altogether. So it makes sense that the person who understands all this ultimately becomes indifferent to whether it is "really existant or really nonexistant" which in action is as good as saying you believe it does not exist.

This is interesting, but what if we switched the analogy from the face/I being a reflection dependent on a mirror, to the face/I being perceived by another person. Would this logic still hold steady? How do we reconcile this with one human perceiving another? Would we then say that the I is nonexistant but the other is?

but can we take this at face value?

I found it really interesting that UI is often considered a specialized part of UX. I never realized how much overlap there is between the two, especially when it comes to user centered design, cross functional teamwork, and even using the same tools. It also makes sense now why so many job postings ask for UX/UI designers rather than separating the roles.

I am very curious to see all the ways these different facets work together to make optimally designed products and interfaces, which tools are beneficial to this collaboration and how new developments across functions could improve accessibility. Group A

User Experience (UX) is the overall feeling a person has when interacting with a product, service, or system. It involves the emotional and perceptual responses of the user upon interaction. The goal of UX is to create products that are useful, easy to use, and delightful, meeting the user's needs and enhancing their satisfaction. Some questions towards UX ask of the usability, credibility and accessibility of a product. However, UX and UI are separate fields, who altogether overlap. For example, UX designers often have the role of putting together basic wireframes and prototypes that UI designers can build off of and turn them into functional product mockups. UX and UI designers work together to ensure the best possible user experience for their product, service, or system.

I never knew UX was something and thought it was honestly just UI. But I see the difference is that UI is more for the pleasing effect for the users and that the UX is kinda the 'back-end' of UI

UX designers need to put a lot of thought process into developing the correct user experience for a target demographic.

Take input from customers and competitors, Organize the information gathered, create a sketch or design, send the design out and get it tested, then iterate and update. Send it out to a variety of people to get better, more diverse set of feedback.

It was captivating for our team to learn that it is a subset of UX. The complexion between both can be confusing for individuals. This is team Group F, Giovanni Santos, and Group Leader.

What stood out to us was information architecture. We had never heard of it prior to reading this article. It is used as a visual blueprint that outlines what each feature does and how it can help the user.

Angol megfelelőjében módosítást javasolnék, hogy érthetőbb legyen: When a business share purchase/sale takes place (possibly a capital increase or decrease occurs), a holding of the business share instrument is created, changed, or terminated.

I found this interesting that a Tsar of the Russian empire would personally go under cover and learn things from Europe. I'm sure he could have had someone else do it for him but the fact he did this by himself really shows character. I don't know much about him but considering he was called Peter the great, and did this, I think shows how much he care about his country and his people. But I could be wrong.

The text states that thew Mughal empire of India was founded with the authority of Chagatai the second son of Genghis Khan. Which is intriguing as Genghis Khan was emperor of the Mongol empire, and with his son leading in a Indian Mughal empire it shows an emphases of the prominent leadership abilities of the khan family across nations.

The "standard" is determined by people themselves. When people are comparing themselves to a "standard" they lose themselves. When people are different they struggle with fitting in which is what leads them to feel like they are impostors. If everyone looked the same or met the same standard, there would be no individuality.

I also think social media didn't force us to compare ourselves. I think our society has always done that, but social media made it much more widespread, so we are exposed to people that we compare to ourselves, who are living entirely different lives from us.

make sure head is turned to the opposite side to prevent them from rolling

how does the buzzword (so to speak..) of "latent space" figure here?

This example shows how a company used the Balanced Scorecard to test whether its strategy really worked as predicted. By tracking data across perspectives—like employee morale, customer satisfaction, and operational efficiency, it found clear chains of impact: happier employees led to happier customers, which sped up payments and boosted returns. Such evidence strengthens confidence in the strategy. The key insight is that the scorecard isn’t just about measuring results; it’s about proving or questioning the logic behind them. If those links fail to appear over time, leaders know it’s time to challenge their assumptions and possibly rethink the strategy itself, making learning and adaptation part of everyday management.

This part shows how many companies create long, impressive strategic plans that end up collecting dust, while budgets and resources are set in a completely separate process. The Balanced Scorecard closes that gap by linking strategy to budgets and daily operations, so money, time, and effort go to initiatives that truly drive long-term goals. What stands out is the realism of the critique: it’s common for plans to sound great at annual retreats but never shape day-to-day decisions. By forcing strategy and budgeting into one process, the scorecard makes strategy real, ensuring that ambitious objectives don’t just stay on paper but guide actual spending, staffing, and priorities over the year.

What stands out is how this model keeps strategy active and flexible. Instead of treating the plan as fixed, it encourages constant dialogue and learning. For complex environments—like healthcare or fast-moving industries—it’s a reminder that success depends as much on continuous alignment and adaptation as on the original strategy itself.

based on this paragraph, the Balanced Scorecard enables strategic understanding to move beyond the senior leadership team and become a shared organizational practice. By deliberately involving three levels of management, senior executives set financial and customer objectives while middle managers formulate the internal-business-process and learning-and-growth goals needed to achieve them. This broad participation enriches the strategy with operational insights, fosters stronger ownership and commitment at all levels, and ensures that day-to-day activities directly support long-term goals. In doing so, the Balanced Scorecard effectively “bottles” strategic knowledge and distributes it throughout the company, transforming strategy from an abstract plan into a widely understood and actionable framework.

It's interesting how economic changes and innovations can lead to non-economic results. I think you would also be able to do non-economic things like cleaning up their yard. This makes the whole block look better, possibly upping the property values.

The more people in a particular region, the more items will begin to cost and the benefits more items. The more costs the more people will struggle to get items and do the trades.

Certainly cuts installs by 30% or more.

something very cool that I would like to look into more

digital heritage connects to my interest in heritage and conservation, so I will look more into this

this is interesting as it is similar to what I have seen in a public history class

It is interesting to see that there is uncertainty to an all-knowing being. In other accounts, Deity is often seen as all-knowing. It is interesting to see here that the writers are uncertain if he knows or not.

O: Here it emphasizes the virtuality of plays and the participation of reader's imagination. Readers need to visualize this drama in their minds according to the author's notes or "instructions".

the difference between a play write and a book is a whole different setup, more like a script for your mind

E: The definition of Purport is: appear or claim to be or do something, especially falsely; profess. I found that the author sought to use this word, as it shows how works of literature often look like they are presenting in present tense although almost always being written in the past tense.

O: These sentences really capture what theater represents. Theater has represented adventure, challenge, and risks. And clearly shows how theater works when they write: “Present tense”.

E: contrast between the purpose of theater and the purpose of other published texts.

Q: Why does Oates think prose writers must abandon their usual “shelter of language” to succeed in theater? Couldn’t they use their strong language skills to enhance dialogues?

taxburst vs krona plots for taxonomy

Consider \(f: \mathbb{R}\rightarrow \mathbb{R}\) given by

$$ f(x) = x^2. $$

Is \(f\) injective? Surjective? Can you adjust the domain and the codomain so that \(f\) becomes bijective?

How long can cougars, or a group of cougars, last without being negatively impacted by their affected habitats and intense human development?

I wonder how long they've been separated into these two groups, and what was the main consistent factor in the differences between the clusters.

Pages hosted at hyperpost.peergos.me can be annotated via.hypothes.is

Thus anything thus shared can also b annotated without having to install a browser extension

Better still. much of the work we had been doing over the years involved sharing and collaborating on and using annotations

So we can launch our virtual launch community on the annotations margins across the web

Do check this introductory video out about Hypothesis available via.hyothes.is with annotations

https://hyp.is/vFLcpI7zEfChQQ-57btOfQ/www.youtube.com/watch?v=QCkm0lL-6lc

Rosseau was banned from France and Switzerland for his controversial writings, often criticizing the government; though he had some "modern" ideas, he still subscribes to the idea that men and women have specific gender roles. https://pittsburghquarterly.com/articles/the-trouble-with-rousseau/

If a women were to actually make a scientific discovery that could alter the way of life, because of the gender norms at the time, she would just be looked upon with jealousy and contempt rather than admiration and pride if a male were to make the scientific discovery.

In Kant's world (1724-1804) the idea that a person, male or female, would participate in actions that would go against typical gender norms, is unfathomable to Kant. Kant cannot imagine a women making contributions to science.

Women didn't have the same access to higher education that men did; many of the discoveries made by female scientists throughout history have been taken credit for by men (ex: Rosalind Franklin and the DNA double helix).

Placing Eurocentric standards upon those that are not from Europe; culture and language differences do not insinuate lesser intelligence.

Men are the only people in the family allowed to be aware or in control of finances. He is expected not to tell his wife when he has been irresponsible with their money; this takes away much of the accountability that he would face in the modern world.

What is meant specifically meant by "disguise" of this distinction? Clothing, education, or maybe career?

Energy Consumption: The text notes that PET requires 165℃ for dissolution and PP needs 135℃. How do these high temperatures compare in energy cost to traditional mechanical recycling methods, such as melt reprocessing? Would this method be more or less energy-intensive on an industrial scale?

PP within the circular economy and clarifies why its recycling predominantly relies on physical or thermal methods rather than chemical depolymerization.

I wonder how I can familiarize myself with these policies and how to navigate them in a way that works for me. As soon as I see a wall of legal jargon my eyes glaze over but I'm someone who wants to be involved in my district's policy-making.

I don't dare work in the district that I grew up in but I see them come out with policies removing Ethnic Studies classes or preventing teachers from hanging flags other than US flags (no really https://go.boarddocs.com/ca/wpusd/Board.nsf/files/DKYUET7BBF08/$file/090225%20Policy%206115.3.pdf ) and I know that I'm going to need to figure out how to navigate these situations.

I'm sorry- what? I clearly need to do more research on unions. I was under the impression that unions were recognized across the country. I'm especially surprised to see Virginia on this list; If I'm not mistaken, that state has an extensive history with unions.

If I'm not mistaken, the California Teaching Credentials travel very well within the US; other than New York I think someone with a California Credential has flexibility in the vast majority of states.

It's absolutely wild to me that the current administration actively acts against standards like this in a code of ethics that should honestly be common knowledge.

I see both sides here. On one hand, treating students differently can seem unfair. But at the same time, I get why a teacher might give a shy student more chances so they can build confidence. It’s tricky to know what’s “equal” in situations like this.

Schrödinger, Erwin. What Is Life? With Mind and Matter and Autobiographical Sketches. Cambridge University Press, 1992.

Originally published in 1944 based on lectures delivered under the auspices of the Dublin Institute for Advanced Studies at Trinity College, Dublin, in February 1943

Annotation URL: urn:x-pdf:4450b6f08de5b847d68ddef3bbe5ba47

Alternate annotation URL

historical evidence of the scientific shift from Newtonian clockwork physics into an underlying statical mechanical one

Christianity was a big religion for colonists during this time, opposing British religion. It is interesting to me that this guy wants people to explore their personal relationship with god. We haven't talked much about religion in relation to the Revolution yet so I am wondering how Christianity affects the colonists and also the leaders during the Revolution. I would assume that the leaders would try to reflect their beliefs on others but in this it seems like he wants them to just build a connection with god on their own.

In class today we talked about how the Declaration is written persuasively to convince people without privilege that they will gain equality. This revolution however contradicts that idea, allowing ideals of slavery while trying to say that slavery will be abolished. This contradiction represents how Jefferson used manipulation to gain his power, although the would never truly conform to the idea of equality. Demonstrates just how manipulating these words are, implying a lack of morality.

!

Book Review : Scientist Obscured by His Achievements - Los Angeles Times<br /> by [[Lee Dembart]] in Los Angeles Times 1988-10-04 <br /> accessed on 2025-09-11T10:29:10

proper balance between a person's life and their contributions to the world is a difficult matter

Havent seen each other in long time but deep enough connection to stay in touch

Information theorist Solomon Golomb, who directly participated in the applications of information theory to early genetics, doesn't feel that it influenced the substance of molecular biology in the 1950s and 1960s though it may have influenced the rhetoric.

Cassell includes examples of incorrect punctuation used in writing to help the reader understand what not to do during an essay. He introduced how grammar effects the voice of the writing and how to formulate it in a proper manner.

Research is not just about the information it is about who is giving or telling you this information

communication between person and Ai in which the ai operates to modify, augment, or make messages to accomplish goals

Meaning-Relating to knowledge or the degree of its validation

The process should merely be for translation and grammar norms not to rewrite but to interpret

"epistemic" PG:2 L:1 Meaning-relating to knowledge or to the degree of it's validation

Damn, that's a good point.

Is the Final Cumulative?

The group project is going to end up being the largest contributor to our final grade

4 days or 96 hours past the due date, will result in a 0 for the assignment

I am interested in learning what these principles are. What is the difference between good and bad user design?

This seems interesting and I'm excited to try out the differnt tools

The who is the people experiencing history at the time and the what epistemology is the change experienced by the poeple, but can also be construed in the production of presenting that as public history.

Is it possible that under these conditions, neurons transfer damaged mitochondria and/or other cellular material to astrocytes? It could be interesting to do neuron-specific labeling prior to co-culturing to track mitochondria.

I’m curious if you looked at CHMP2B levels by western blot in addition to immunofluorescence. Is it possible that neurons express alternate mRNA isoforms and/or proteoforms (e.g. truncated CHMP2B) that disrupt canonical ESCRT recruitment?

The associations may be based on batch differences

This is something that i believe should be noted. I think what we are seeing here is that grading is so crucial. A question I can draw from this is what as interns can we start doing now to be ready for this

I have not really looked into this before, but it seems accurate. When you continue reading under it it states "No single grading instrument can accurately report all six of the aspects above to the fullest extent. " This leads me to ask what is next

This definitely exists in our time especially when it comes to updates on social media. Too many people have fomo (fear of missing out) wether it's people hanging out or just knowing information.

The term "flow" was used to understand the structure of TV shows by analyzing.

Ads are really annoying so if it's broadcasted then there will be constant ads. Streaming apps have plans to pay for no-ads so as much as I hate spending money on subscriptions, it's worth it.

Before TV, there were books. Books would help use your own imagination. One book would usually have a beginning and end whether it was short or long. Some stories were taken from books and made into movies, but weren't always accurate.

Split the service into VAT filings with out VAT Filings. price ranges between 75-99 with out VAT filings and £100-149 with VAT filings.

this is where the most of the work lies in , if its 4-8 invoices in a month wont be a problem at all but if you have 4-5 invoices to create in a week it takes easily on an average 10mins for each invoice to check the information, create invoices, draft and send an email to client.

if we are using the software and if the bookkeeping is done by client itself, we dont really prepare VAT . We Just review the VAT reports and file the return using MTD approved software.

Are there more affixes besides that of the English language?

Are phonemes the predecessor of syllables?

When one argues "Semantics"; does that mean the meaning of what is said can be dependent on the situation? Or does that mean you can say one thing in many different ways and it will mean the same thing?

who says?

How

This loads in really weird. Are you experiencing the same? You should be able to assign this to load in first.

This idea scares me a little bit. As we talked about in class on tuesday, this is a common question to ask ourselves. how do we know that we are not in a simulation? Is there any finite proof that we can find within ourselves or others to actually prove it to be a fact that we are not. The simulation hypothesis is a really interesting idea to me. I feel like I used to call this an intrusive thought before I really understood what the idea is, but now through this reading I can really understand what the idea of questioning the reality we are living in really is.

I think this line is really interesting because of how wrapped up in the virtual world people can become. Just looking at these lines you can see how much people can become wrapped up in the virtual world and virtual reality. I think what made it so appealing at the beginning is the idea that people can escape. Escape the world that you are surrounded by and become whoever you want. A superhero, a bird that flies, a zombie a scientist and whatever else. Virtual reality gives humans the ability to experience and be whoever they want, without any consequences and dangers of reality.

establishes hierachies within the communities, has a high level of power since he can give up riches

in primitive societies, things are just things, but link to the source it came from and leads to the obligation to return them (social and spritiual)

that's no reason to change the guidance.

eLife Assessment

During the development of the unicellular eukaryote Dictyostelium discoideum, cells aggregate into mounds, which then form protrusions called tips, and the tips then become the front of migrating slugs and the top of fruiting bodies. This valuable study identifies a protein called adenosine deaminase-related growth factor (ADGF) as a key regulator of tip formation, and the authors convincingly show that ADGF catalyses the formation of ammonia from adenosine, allowing ammonia to initiate tip formation, and they then elucidate pathways upstream and downstream from ADGF. The authors discuss the intriguing possibility that mammalian ADGF may also regulate development in a similar manner.

Reviewer #1 (Public review):

Summary:

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

Strengths:

The data and statistics are excellent.

Weaknesses:

The key weakness is understanding why the cells bother to use a diffusible gas like ammonia as a signal to form a tip and continue development. The rescue of the mutant by adding ammonia gas to the entire culture indicates that ammonia conveys no positional information within the mound. By the time the cells have formed a mound, the cells have been starving for several hours, and desperately need to form a fruiting body to disperse some of themselves as spores, and thus need to form a tip no matter what. One can envision that the local ammonia concentration is possibly informing the mound that some minimal number of cells are present (assuming that the ammonia concentration is proportional to the number of cells), but probably even a miniscule fruiting body would be preferable to the cells compared to a mound. This latter idea could be easily explored by examining the fate of the adgf cells in the mound - do they all form spores? Do some form spores? Or perhaps the ADGF is secreted by only one cell type, and the resulting ammonia tells the mound that for some reason that cell type is not present in the mound, allowing some of the cells to transdifferentiate into the needed cell type. Thus elucidating if all or some cells produce ADGF would greatly strengthen this puzzling story.

Comments on revisions:

Looks better, but I think you answered my questions (listed as weaknesses in the public review) in the reply to the reviewer but not in the paper. I'd suggest carefully thinking about my questions and addressing them in the Discussion. You did however do all of the things in the paper that were listed as "Recommendations for the authors"

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

Lack of details on developmental time course of adgf activity and cell-type-specific differences in adgf expression. Absence of measurements to show that ammonia addition to the null mutant can rescue the proposed defects in cAMP signaling. No direct measurements in the dhkD mutant to show that it acts upstream of sdgf in the control of changes in cAMP signaling and tip formation.

Comments on revisions:

The revised version of the paper has improved significantly in terms of structure and clarity. The additional data on rescue of total cAMP production by ammonia (Fig. 7C) in the adgf- mutant and the 5-fold increased prespore expression of adgf RNA compared to prestalk cells (Fig 9) are useful data additions.

The link between changes in cAMP signaling (lower aca expression) and wave geometry ( concentric waves rather than spiral waves) remains speculative.

I noted that Fig 6 contains different images than the previous version (Fig 7).

The statement "Interestingly, Klebsiella pneumoniae physically separated from the Dictyostelium adgf mutants in a partitioned dish, also rescues the mound arrest phenotype suggesting a cross-kingdom interaction that drives development" in the summary is rather overdone. All experiments were performed with axenic strains (no bacteria).

as is the sentence "Remarkably, in higher vertebrates, adgf expression is elevated during gastrulation and thus adenosine deamination may be a conserved process driving organizer development in different organisms"<br /> The data supporting this in the supplementary information is hardly legible and poorly presented. What is shown is ADA expression in different tissues, not at different stages. I would suggest taking these figures out and concentrating the summary on the key mechanistic findings of the paper.

Author Response :

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

The data and statistics are excellent.

Weaknesses

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

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

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

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Recommendations for the authors):

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

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

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

Corrected

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

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

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

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

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

Corrected

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

Corrected

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

Corrected

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

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

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

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

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

Corrected.

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

Reference is included.

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

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

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

Reference is included.

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

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

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

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

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

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

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

Reviewer #2 (Recommendations for the authors):

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

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

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

The manuscript will be revised extensively.

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

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

The manuscript will be revised extensively

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

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

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

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

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

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

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

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

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

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

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

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

Figure. 2 will be moved to supplementary materials.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

eLife Assessment

This study provides a valuable examination of the social discrimination abilities of a jumping spider, Phippidus regius, based on visual cues. Behavioral essays yielded solid evidence that these spiders discriminate between familiar and unfamiliar individuals on the basis of visual cues, however the experimental support for individual recognition and long-term memory is incomplete. While the results supply evidence of discrimination, additional experiments would be needed to verify the evidence of individual recognition.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

(1) Study design: The study design is rather complicated and as a result it is difficult to interpret the results. The spiders are presented with the same individual twice in a row, called a habituation trial. Then a new individual is presented twice in a row. The first of these is a dishabituation trial and the second another habituation trial (but now habituating to a second individual). This done with three pairings and then this entire structure is repeated over three sessions. The data appear to show the strong effects of differences between habituation and dishabituation trials in the first session. The decrease in differential behavior between the so-called habituation and dishabituation trials in sessions 2 and 3 are explained as a consequence of the spiders beginning to habituate in general to all of the individuals. The claim that the spiders remember specific individuals is somewhat undercut because all of the 'dishabituation' trials in session 2 are toward spiders they already met for 14 minute previously but seemingly do not remember in session 2. In session 3 it is ambiguous what is happening because the spiders no longer differentiate between the trial types. This could be due to fatigue or familiarity. A second experiment is done to show that introducing a totally novel individual, recovers a large dishabituation response, suggesting that the lack of differences between 'habituation' and 'dishabituation' trials in session 3 is the result of general habituation to all of the spiders in the session rather than fatigue. As mentioned before, these data do support the claim that the spiders differentiate among individuals.

The data from session 1 are easy to interpret. The data from sessions 2 and 3 are harder to understand, but these are the trials in which they meet an individual again after a substantial period of separation. Other studies looking at recognition in ants and wasps (cited by the authors) have done a 4 trial design in which focal animal A meets B in the first trial, then meet C in the second trial, meets B again in the third trial, and then meets D in the last trial. In that scenario trials 1, 2 and 4 are between unfamiliar individuals and trial 3 is between potentially familiar individuals. In both the ants and wasps, high aggression is seen in species with and without recognition on trial 1, with low aggression specifically for trials with familiar individuals in species with recognition. Across different tests, species or populations that lack recognition have shown a general reduction in aggression towards all individuals that becomes progressively less aggressive over time (reminiscent of the session 2 and 3 data) while others have maintained modest levels of aggression across all individuals. The 4 session design used in those other studies provides an unambiguous interpretation of the data, while controlling for 'fatigue'. That all trials in sessions 2 and 3 are always with familiar individuals make it challenging to understand how much the spiders are habituating to each other versus having some kind of associative learning of individual identity and behavior.

The data presentation is also very complicated. How is it the case that a negative proportion of time is spent? The methods reveal that this metric is derived by comparing the time individuals spent in each region relative to the previous time they saw that individual. At the very least, data showing the distribution of distances from the wall would be much easier to interpret for the reader.

(2) "Long-term social memory": It is not entirely clear what is meant by the authors when they say 'long-term social memory', though typically long-term memory refers to a form of a memory that require protein synthesis. While the precise timing of memory formation varies across species and contexts, a general rule is that long term memory should last for > 24 hours (e.g., Dreier et al 2007 Biol Letters). The longest time that spider are apart in this trial set up is something like an hour. There is no basis to claim that spiders have long term social memory as they are never asked to remember anyone after a long time apart. The odd phrasing of the 'long-term dishabutation' trial makes it seem that it is testing a long-term memory, but it is not. The spiders have never met. The fact that they are very habituated to one set of stimuli and then respond to a new stimulus is not evidence of long-term memory. To clearly test memory (which is the part really lacking from the design), the authors would need to show that spiders - upon the first instance of re-encountering a previously encountered individual are already 'habituated' to them but not to some other individuals. The current data suggest this may be the case, but it is just very hard to interpret given the design does not directly test memory of individuals in a clear and unambiguous manner.

(3) Lack of a functional explanation and the emphasis on 'asociality': It is entirely plausible that recognition is pleitropic byproduct of the overall visual cognition abilities in the spiders. However, the discussion that discounts territoriality as a potential explanation is not well laid out. First, many species that are 'asocial' nevertheless defend territories. It is perhaps best to say such species are not group living, but they have social lives because they encounter conspecifics and need to interact with them. Indeed, there are many examples of solitary living species that show the dear enemy effect, a form of individual recognition, towards familiar territorial neighbors. The authors in this case note that territorial competition is mediated by the size of color of the chelicerae (seemingly a trait that could be used to distinguish among individuals). Apparently because previous work has suggested that territorial disputes can be mediated by a trait in the absence of familiarity has led them to discount the possibility that keeping track of the local neighbors in a potentially cannibalistic species could be a sufficient functional reason. In any event, the current evidence presented certainly does not warrant discounting that hypothesis.

Comments on Revision:

The authors have not actually addressed my points and their comments conflate discrimination with recognition. The extensive discussion about how babies are tested for discrimination tasks in their rebuttal misses the point. I believe that the data do show that the spiders discriminate between individuals but whether individuals are recognized (i.e., remembered) is less clear. The authors defend their convoluted study design, but it is overly complex and challenging to interpret the data as a result.

The main issue with the design is that they do not actually test for any kind of memory of specific individuals after a substantial time of separation. Instead they show that a new individuals is still surprising/dishabituating. That is nice evidence for discrimination but does not show memory in a clear and unambiguous way.

My comments and critique are unchanged since they didn't really change the paper. New experiments were needed and they didn't do any. Perhaps it is hard to get the spiders where they are? I don't really understand why they didn't do additional experiments as part of this revision.

Reviewer #3 (Public review):

Summary:

Jumping spiders (family Salticidae) have extraordinarily good eyesight, but little is known about how sensitive these small animals might be to the identity of other individuals that they see. Here, experiments were carried out using Phidippus regius, a salticid spider from North America. There were three steps in the experiments; first, a spider could see another spider; then its view of the other spider was blocked; and then either the same or a different individual spider came into view. Whether it was the same or a different individual that came into view in the third step had a significant effect on how close together or far apart the spiders positioned themselves. It has been demonstrated before that salticids can discriminate between familiar and unfamiliar individuals while relying on chemical cues, but this new research on P. regius provides the first experimental evidence that a spider can discriminate by sight between familiar and unfamiliar individuals.

Clark RJ, Jackson RR (1995) Araneophagic jumping spiders discriminate between the draglines of familiar and unfamiliar conspecifics. Ethology, Ecology and Evolution 7:185-190

Strengths:

This work is a useful step toward a fuller understanding of the perceptual and cognitive capacities of spiders and other animals with small nervous systems. By providing experimental evidence for a conclusion that a spider can, by sight, discriminate between familiar and unfamiliar individuals, this research will be an important milestone. We can anticipate a substantial influence on future research.

Weaknesses:

(1) The conclusions should be stated more carefully.

(2) It is not clearly the case that the experimental methods are based on 'habituation (learning to ignore; learning not to respond). Saying 'habituation' seems to imply that certain distances are instances of responding and other distances are instances of not responding but, as a reasonable alternative, we might call distance in all instances a response. However, whether all distances are responses or not is a distracting issue because being based on habituation is not a necessity.

(3) Besides data related to distances, other data might have been useful. For example, salticids are especially well known for the way they communicate using distinctive visual displays and, unlike distance, displaying is a discrete, unambiguous response.

(4) Methods more aligned with salticids having extraordinarily good eyesight would have useful. For example, with salticids, standardising and manipulating stimuli in experiments can be achieved by using mounts, video playback and computer-generated animation.

(5) An asocial-versus-social distinction is too imprecise, and it may have been emphasised too much. With P. regius, irrespective of whether we use the label asocial or social, the important question pertains to the frequency of encounters between the same individuals and the consequences of these encounters.

(6) Hypotheses related to not-so-strictly adaptive factors are discussed and these hypotheses are interesting, but these considerations are not necessarily incompatible with more strictly adaptive influences being relevant as well.

Comments on Revision:

The authors have responded reasonably to the comments I made. There is nothing else that I wish to add.

Author response:

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

Reviewer #1 (Public Review):

Summary:

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

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

Strengths:

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

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

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

Weaknesses:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Advantages of our paradigm: 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

We understand the reviewer's concern that data showing the distribution of distances from the wall would be much easier to interpret for the reader. We initially consider that but came to the conclusion that this approach is not straightforward. For instance, if both spiders are positioned at the very front but in different corners, the distance to the panel would be very small, but the distance between the spiders would be large. Thus, using distances from the wall could misrepresent the actual spatial distribution between the spiders.

(2) "Long-term social memory": It is not entirely clear what is meant by the authors when they say 'long-term social memory', though typically long-term memory refers to a form of a memory that requires protein synthesis.  

To address this conceptually, we used the term "long-term social memory" to describe the spiders' ability to recognize and remember individual conspecifics over multiple experimental sessions. While social memory refers to the ability of an individual to recognize other individuals within a social context, long-term memory typically involves the retention of information over extended periods. Recognizing that the term “long-term social memory” is not commonly used, we have revised the manuscript to use the more standard term “long-term memory.”

While the precise timing of memory formation varies across species and contexts, a general rule is that long-term memory should last for > 24 hours (e.g., Dreier et al 2007 Biol Letters). The longest time that spiders are apart in this trial setup is something like an hour. There is no basis to claim that spiders have long-term social memory as they are never asked to remember anyone after a long time apart.

We appreciate the reviewer’s feedback regarding the term "long-term social memory." The statement "long-term memory should last for > 24 hours" is a generalisation in discussions about memory. It oversimplifies a more complex topic. That is, long-term memory is typically distinguished from short-term memory by its persistence over time, often lasting from hours to a lifetime. However, the exact duration that qualifies memory as "long-term" varies depending on the context, model species, and type of memory. In studies involved in synaptic plasticity (LTP), the object might indeed be to look at memory that persists for at least 24 hours as a criterion for long-term memory. In studies of cellular and/or molecular mechanisms where the stabilization and consolidation of memory traces over time are key areas of interest this 24-hour interval is very common. But, defining long-term memory strictly by a 24-hour duration is by no means universally accepted nor does it apply across all fields of study.

To clarify, long-term memory is a process involving consolidation starting within minutes to hours after learning. Clearly, full consolidation can take longer, while memory persisting 24 hours is considered fully consolidated. But this does not mean that memory lasting less than 24 hours are not part of long-term memory. 

In fact, Atkinson and Shiffrin (1969) proposed that information entering short-term memory remains there for about 20 to 30 seconds before being displaced due to space limitations. During this brief interval, initial encoding processes begin transferring information to long-term memory, establishing an initial memory trace. This transfer is not indicative of full consolidation but represents the initial "laying down" of the memory trace (encoding). In our study, the focal spider’s brain forms initial memory traces of the individuals it encounters. This process continues during the period of visual separation. Upon re-encountering the same individual a few minutes later, the spider accesses the initial memory trace stored in long-term memory. This trace is fragile and not fully consolidated. The re-encounter acts as a rehearsal, reactivating specific memory traces and potentially strengthening them through additional encoding processes, allowing the spider to recognize the individual even an hour later.

According to Markowitsch (2013), initial encoding in long-term memory begins within seconds to minutes. It is also important to note that we argue for identity recognition rather than identity recall. Recognition involves correctly identifying a stimulus when it is presented again, while recall requires the volitional generation of information without an external stimulus. Thus, recall may rely on deeper forms of memory consolidation than recognition.

Is protein synthesis required for long-term memory? 

The role of protein synthesis in long-term memory has been extensively studied. According to Castellucci et al. (1978), explicit memory comprises a short-term phase that does not require protein synthesis and a long-term phase that does. Hebbian learning in its initial phase (early LTP) does not necessarily require protein synthesis. This phase involves the rapid strengthening of synapses through existing proteins and signaling pathways, such as the activation of NMDA receptors and the influx of Ca2+ ions. For the changes to persist (late LTP), protein synthesis is important. This phase involves the production of new proteins that contribute to long-term structural changes at the synapse, such as the growth of new synaptic connections or the stabilization of existing ones.

This differentiation between the early and late phases of LTP highlights that long-term memory can begin forming without immediate protein synthesis. Our study focuses on this early phase of memory encoding, which involves the initial formation of memory traces that do not yet depend on protein synthesis. 

It is however worth noting that recent research suggests that there is an early phase of protein synthesis (within minutes to hours) through the activation of immediate early genes (IEGs) and transcription factors. In this context, protein synthesis supports initial synaptic modifications. What the reviewer refers to is the consolidation phase (late phase), where continued synthesis of proteins induces structural changes at synapses, leading to the formation of new synaptic connections. In our study, it is plausible to assume that an early form of protein synthesis may contribute to stabilizing the initial memory traces during the encoding phase. However, whether or not protein synthesis occurred in our spiders is beyond the scope of this investigation and was not specifically addressed.

The critical aspect of our study is that the information transitioned from short-term memory to long-term memory during an early encoding phase, allowing recall after an hour. Due to the inherent limitations and transient nature of the short-term memory, it is implausible for spiders to retain these memory representations solely within the short-term memory for such durations. Our findings suggest that the initial encoding processes were robust enough to transfer these experiences into long-term memory, where they were stabilized and could be accessed later. 

In sum, it is important to note that long-term memory is a dynamic process, and while testing after 24 hours is a convention in some studies, this timing is arbitrary and not universally applicable to all contexts or species. The more critical consideration here is that we are dealing with a species where no prior evidence of long-term memory exists. Debating a 24-hour delay or the specifics of protein synthesis, while potentially interesting for future studies, detracts from the true significance of our findings. Our study is the first to show something akin to long-term memory representations in this species and this should remain in our focus.

Shiffrin, R. M., & Atkinson, R. C. (1969). Storage and retrieval processes in long-term memory. Psychological review, 76(2), 179. 

Markowitsch, H. J. (2013). Memory and self–Neuroscientific landscapes. International Scholarly Research Notices, 2013(1), 176027.

Castellucci, V. F., Carew, T. J., & Kandel, E. R., 1978. Cellular analysis of long-term habituation of the gill-withdrawal reflex of Aplysia californica. Science, 202(4374), 1306-1308.

The odd phrasing of the 'long-term dishabutation' trial makes it seem that it is testing a longterm memory, but it is not. The spiders have never met. The fact that they are very habituated to one set of stimuli and then respond to a new stimulus is not evidence of long-term memory. To clearly test memory (which is the part really lacking from the design), the authors would need to show that spiders - upon the first instance of re-encountering a previously encountered individual are already 'habituated' to them but not to some other individuals. The current data suggest this may be the case, but it is just very hard to interpret given the design does not directly test the memory of individuals in a clear and unambiguous manner.

While we appreciate the reviewer's feedback, we believe there may have been some misunderstanding regarding the term “long-term dishabituation.” The introduction of novel individuals at the end of Session 3 was not intended to test long-term memory by having spiders recognize these novel individuals. Instead, it aimed to investigate the nature of the habituation observed over the three sessions.

The novel individuals introduced at the end of Session 3 serve the purpose to differentiate between general habituation (a decline in response due to repeated exposure to any stimuli) and specific habituation (recognition and reduced response to previously encountered individuals). The novel spiders have never been encountered before, so the focal spiders cannot have prior representations of them. Thus, the strong dishabituation response to these novel individuals indicates that the habituation observed earlier is not due to a general fatigue effect or loss of interest but rather a specific habituation effect to the familiar individuals. By showing such strong and increased response to novel individuals, the study demonstrates that the spiders' increasingly reduced responses in Sessions 2 and 3 are not merely due to a general decrease in responsiveness but suggest cognitive habituation. This cognitive habituation implies that the spiders remember the familiar individuals (as each of them occurred three times across the three sessions), a process that relies on long-term memory. Therefore, while the novel spiders themselves are not a direct test of long-term memory, the use of these novel spiders helps us infer that the habituation observed over the three sessions is indeed due to the formation of long-term memory traces.

In other words, the organism detects and processes the novel stimulus as different from the habituated one. In our study, if a spider showed a strong dishabituation response to a novel individual introduced at the end of Session 3, it would indicate that the spider had formed specific representations of the individuals they encountered during the three sessions. These representations allow the spiders to recognise the novel individuals as different, leading to renewed interest and a stronger behavioural response. It is the absence of a prior representation for the novel spiders that triggers this dishabituation response. Since the novel spider does not match any stored representations of the previously encountered spiders, the focal spider responds more strongly.

The introduction of novel individuals at the end of Session 3 helps clarify that the increasing habituation observed in Session 2 and 3 is specific to familiar individuals, indicating cognitive habituation. This supports the presence of long-term memory processes in the spiders, as they can distinguish between previously encountered individuals and new ones. The habituationdishabituation paradigm thus effectively demonstrates the spiders' ability to form and reactivate encoded memory traces, providing clear evidence of recognition memory. 

For these reasons, we are convinced that our interpretation is accurate and hope this clarification renders the additional request for an entirely new experiment unnecessary.

(3) Lack of a functional explanation and the emphasis on 'asociality': It is entirely plausible that recognition is a pleitropic byproduct of the overall visual cognition abilities in the spiders. 

We agree with the reviewer that it is essential to consider the broader context of individual recognition and its potential adaptive significance. The possibility that recognition in jumping spiders could be a pleiotropic byproduct of their advanced visual cognition abilities is indeed a plausible explanation and has been discussed in our manuscript.

However, the discussion that discounts territoriality as a potential explanation is not well laid out. First, many species that are 'asocial' nevertheless defend territories. It is perhaps best to say such species are not group living, but they have social lives because they encounter conspecifics and need to interact with them.

The reviewer also correctly points out that many 'asocial' species still defend territories and have social interactions. Our use of the term 'asocial' was meant to indicate that jumping spiders do not live in cohesive social groups, but we acknowledge that they do have social lives in terms of interactions with conspecifics. It is more accurate to describe these spiders as non-groupliving, yet socially interactive species. A better term is “non-social” to refer to the jumping spider as a species that do not live in stable social groups and do not exhibit associated behaviours, such as cooperative behaviours. This also would imply that individuals still interact with conspecifics, especially in contexts like mating, territorial disputes or aggression. We, thus, change the term from “asocial” to “non-social” in the manuscript.  

Indeed, there are many examples of solitary living species that show the dear enemy effect, a form of individual recognition, towards familiar territorial neighbors. The authors in this case note that territorial competition is mediated by the size or color of the chelicerae (seemingly a trait that could be used to distinguish among individuals). Apparently, because previous work has suggested that territorial disputes can be mediated by a trait in the absence of familiarity has led them to discount the possibility that keeping track of the local neighbors in a potentially cannibalistic species could be a sufficient functional reason. In any event, the current evidence presented certainly does not warrant discounting that hypothesis.

The “dear enemy effect”, where solitary living species recognize and show reduced aggression towards familiar territorial neighbors, is a relevant consideration. This effect demonstrates that individual recognition can have significant functional implications even in species that are not group-living. We will elaborate on this effect in the revised manuscript to provide a more comprehensive discussion.

The reviewer mentioned that territorial disputes can be mediated by the size or color of the chelicerae, potentially serving as a feature for individual recognition. Our intention was not to discount the role of such traits but to highlight that the level of identity recognition we observed represents subordinate classification. This is different from the basic-level classification, such as distinguishing between male and female based on chelicerae colour. While we acknowledge that colour can be an important feature for identity discrimination, our findings suggest that individual recognition in jumping spiders goes beyond simple colour differentiation. 

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors investigated whether a salticid spider, Phidippus regius, recognizes other individuals of the same species. The authors placed each spider inside a container from which it could see another spider for 7 minutes, before having its view of the other spider occluded by an opaque barrier for 3 minutes. The spider was then either presented with the same individual again (habituation trial) or a different individual (dishabituation trial). The authors recorded the distance between the two spiders during each trial. In habituation trials, the spiders were predicted to spend more time further away from each other and, in dishabituation trials, the spiders were predicted to spend more time closer to each other. The results followed these predictions, and the authors then considered whether the spiders in habituation trials were generally fatigued instead of being habituated to the appearance of the other spider, which may have explained why they spent less time near the other individual. The authors presented the spiders with a different (novel) individual after a longer period of time (which they considered to be a long-term dishabituation trial), and found that the spiders switched to spending more time closer to the other individual again during this trial. This suggested that the spiders had recognized and had habituated to the individual that they had seen before and that they became dishabituated when they encountered a different individual.

We appreciate the reviewer's detailed summary of our study. The reviewer's summary accurately captures the essence of our experimental design, predictions, and findings.

Strengths:

It is interesting to consider individual recognition by Phidippus regius. Other work on individual recognition by an invertebrate has been, for instance, known for a species of social wasp, but Phidippus regius is a different animal. Importantly and more specifically, P. regius is a salticid spider, and these spiders are known to have exceptional eyesight for animals of their size, potentially making them especially suitable for studies on individual recognition. In the current study, the results from experiments were consistent with the authors' predictions, suggesting that the spiders were recognizing each other by being habituated to individuals they had encountered before and by being dishabituated to individuals they had not encountered before. This is a good start in considering individual recognition by this species.

We appreciate the reviewer's positive summary and acknowledgment of the strengths of our study. We would like to point out some more details: 

While the exceptional eyesight of salticid spiders is indeed a significant factor, our study reaches deeper in terms of processing. We do not argue at the level of sensation rather than at the level of perception. Even more, identity recognition is a higher-level perceptual process. This distinction is crucial: we are not merely examining the spiders' sensory capabilities (such as good eye sight), but rather how their brains interpret and represent what they “see”. This involves a cognitive process where the sensory input (sensation) is processed and integrated into meaningful constructs (perception) and memorised in form of representations. 

Our study also suggests that P. regius engages in “higher-level” perceptual processes. This most-likely involves complex representations of individual conspecifics, which in mammalian brains are associated with regions such as the central inferior temporal (cIT) and anterior inferior temporal (aIT) areas. We provide evidence that these spiders do not just sense visual stimuli but interpret and recognize individual identities, indicating sophisticated perceptual and cognitive abilities. In other words, the spiders do not merely respond to visual stimuli in a reflexive manner, but rather engage in sophisticated perceptual and cognitive processes that allow them to recognize and distinguish between individual identities. This indicates that the spiders are not simple Braitenberg vehicles reacting to stimuli, but are thinking organisms capable of complex mental representations. This resonates with current trends in animal cognition research, which increasingly recognize some level of consciousness and advanced cognitive abilities across a wide range of animal species. Moreover, this aligns with the growing interest and recognition of spider cognition, where research begins to provide evidence for the cognitive complexity and perceptual capabilities of these often underestimated creatures (Jackson and Cross, 2011). 

Jackson, R. R., & Cross, F. R. (2011). Spider cognition. Advances in insect physiology, 41, 115174.

Weaknesses:

The experiments in this manuscript (habituation/dishabituation trials) are a good start for considering whether individuals of a salticid species recognize each other. I am left wondering, however, what features the spiders were specifically paying attention to when recognizing each other. The authors cited Sheehan and Tibbetts (2010) who stated that "Individual recognition requires individuals to uniquely identify their social partners based on phenotypic variation." Also, recognition was considered in a paper on another salticid by Tedore and Johnsen (2013).

Tedore, C., & Johnsen, S. (2013). Pheromones exert top-down effects on visual recognition in the jumping spider Lyssomanes viridis. The Journal of Experimental Biology, 216, 1744-1756. doi: 10.1242/jeb.071118 

In this elegant study, the authors presented spiders with manipulated images to find out what features matter to these spiders when recognizing individuals.

The reviewer raises an important point regarding the specific features that Phidippus regius might be paying attention to when recognizing individual conspecifics. Our study indeed cited Sheehan and Tibbetts (2010) to highlight the importance of phenotypic variation in individual recognition. Additionally, we referenced the work by Tedore and Johnsen (2013) on visual recognition in another salticid species, which suggests that multiple sensory modalities, including visual and pheromonal cues, may be involved in the recognition process. While our current study focused on demonstrating that Phidippus regius can recognize individual conspecifics, we acknowledge that it does not specifically identify the phenotypic features involved in this recognition. 

Part of the problem with using two living individuals in experiments is that the behavior of one individual can influence the behavior of the other, and this can bias the results.  

We appreciate the reviewer's observation regarding the potential bias introduced by using two living individuals in experiments, as the behaviour of one individual can indeed influence the behaviour of the other. We shared this concern initially; however, the consistency of the data with our hypotheses suggests that this potential bias did not adversely affect the validity of our findings, rendering the concern largely illusory at least in the context of our study.

We opted for the living-individual paradigm for the following reasons:

There is a growing trend in ethological as well as animal cognition research towards more ecologically valid and biologically relevant settings, while simultaneously advancing the precision and quantification of the data collected. This is referred to as computational ethology.

This approach advocates for assessing behaviour in environments that more closely resemble natural conditions, rather than relying solely on sterile and artificial experimental setups. The rationale is that such naturalistic arenas allow animals to exhibit a broader range of behaviours and interactions, providing a more accurate reflection of their cognitive and social abilities. The challenge, however, lies in navigating the inherent tradeoff between the strict control offered by standardized procedures and the ecological validity of more naturalistic interactions.

By allowing two spiders to confront each other, we aimed to capture authentic behavioural responses while maintaining a degree of experimental standardization through the use of a controlled setup. Our approach ensures that the behaviours observed are not merely artifacts of an artificial environment but are representative of genuine social interactions. Also, to minimize potential biases arising from mutual behavioural influences, we employed a controlled and repeatable experimental environment. 

We believe that the chosen approach provides a meaningful balance (in the above-mentioned trade-off) between ecological validity and experimental rigour. By combining a standardized environment with the naturalistic interaction of real spiders, we ensured that our findings are both scientifically robust and biologically relevant.

However, this issue can be readily avoided because salticids are well known, for example, to be highly responsive to lures (e.g. dead prey glued in lifelike posture onto cork disks) and to computer animation. 

While it is true that salticid spiders are responsive to lures and computer animations, we carefully considered the most appropriate and ecologically valid approach for our study. Our aim was to capture genuine behavioural patterns in a context that closely mimics the natural encounters these spiders experience.

Additionally, creating comparable video stimuli of spiders presents its own set of challenges: Video recordings or computer animations may not fully capture the nuanced behaviours and subtle variations that occur during real-life interactions. There is also a risk that such stimuli could be perceived differently by the spiders, potentially introducing new biases or confounding factors.

Scientific progress is not made by merely relying on previously established paradigms, especially when they may not be suitable for the specific context of a study. While alternative methods like lures or computer animations can be valuable in certain situations, our approach was deliberately chosen to best capture the naturalistic and interactive aspects of spider behaviour.

These methods have already been successful and helpful for standardizing the different stimuli presented during many different experiments for many different salticid spiders, and they would be helpful for better understanding how Phidippus regius might recognize another individual on the basis of phenotypic variation. There are all sorts of ways in which a salticid might recognize another individual. Differences in face or body structure, or body size, or all of these, might have an important role in recognition, but we won't know what these are using the current methods alone. Also, I didn't see any details about whether body size was standardized in the current manuscript.

As mentioned previously, the goal of our study was to demonstrate that identity recognition occurs in spiders. This alone is of significant importance, as it challenges existing assumptions about the cognitive capabilities of small-brained animals. We did not aim at providing a proximate explanation (mechanism) for identity recognition in spiders.

The problem with what the reviewer suggested is this: As long as we do not have conclusive evidence that spiders recognize individual conspecifics, any attempt to design and manipulate stimuli would lack a solid foundation. Without understanding whether spiders have this capability, we cannot make informed decisions about which features or characteristics to manipulate in stimuli. In other words, this uncertainty means we lack a starting point for our assumptions, making it nearly impossible to create stimuli that would be useful or relevant in testing identity recognition.

Additionally, it is nearly impossible to artificially generate a stimulus set that encompasses the natural variance in features that spiders use for visual individuation. There is no guarantee that artificial stimuli, such as lures or computer animations, would capture the relevant features that spiders use in natural interactions.

In other words, the question how Phidippus regius recognizes another individual will be subject of further investigation. In this study, we focus on whether or not they individuate others.  

For another perspective, my thoughts turn to a paper by Cross et al.

Cross, F. R., Jackson, R. R., & Taylor, L. A. (2020). Influence of seeing a red face during the male-male encounters of mosquito-specialist spiders. Learning & Behavior, 48, 104-112. doi: 10.3758/s13420-020-00411-y

These authors found that males of Evarcha culicivora, another salticid species that is known to have a red face, become less responsive to their own mirror images after having their faces painted with black eyeliner than if their faces remained red. In all instances, the spiders only saw their own mirror images and never another spider, and these results cannot be interpreted on the basis of habituation/dishabituation because the spiders were not responding differently when they simply saw their mirror image again. Instead, it was specifically the change to the spider's face which resulted in a change of behavior. The findings from this paper and from Tedore and Johnsen can help give us additional perspectives that the authors might like to consider. On the whole, I would like the authors to further consider the features that P. regius might use to discern and recognize another individual.

We acknowledge that identifying the specific features used by P. regius for identity recognition is a valuable direction for future research. However, we must emphasise that without first establishing whether spiders are capable of individuating each other, it would be premature and challenging to determine the specific features they rely on for this process. A lack of response to certain features could either suggest that those features are not relevant or, more critically, that the spider does not recognize individual identities at all. Thus, our initial focus on demonstrating identity recognition is essential before delving into the specific cues or characteristics involved.

While the call for addressing the proximate causation of identity recognition in jumping spiders is valid, we need to also reiterate the significance of our findings and why they stand on their own merit:

Our study demonstrates for the first time that Phidippus regius can systematically individuate conspecifics, showing habituation within short intervals (10 minutes) and over longer intervals (1 hour). This behaviour is not due to general habituation or physical fatigue but is a result of cognitive habituation, as illustrated by the spiders' response to novel individuals introduced after repeated encounters with familiarized ones. 

What are the implications of this? Our findings indicate that these spiders possess long-term memory and form representations that can be reactivated after an hour. While this is most-likely not fully consolidated memory formation (see our reply to Reviewer 1), it represents an encoded long-term memory. This implies that small-brained animals can remember, represent, and potentially build internal mental images, which are crucial for sophisticated cognitive processing. 

Reviewer #3 (Public Review):

Summary:

Jumping spiders (family Salticidae) have extraordinarily good eyesight, but little is known about how sensitive these small animals might be to the identity of other individuals that they see. Here, experiments were carried out using Phidippus regius, a salticid spider from North America. There were three steps in the experiments; first, a spider could see another spider; then its view of the other spider was blocked; and then either the same or a different individual spider came into view. Whether it was the same or a different individual that came into view in the third step had a significant effect on how close together or far apart the spiders positioned themselves. It has been demonstrated before that salticids can discriminate between familiar and unfamiliar individuals while relying on chemical cues, but this new research on P. regius provides the first experimental evidence that a spider can discriminate by sight between familiar and unfamiliar individuals.

Clark RJ, Jackson RR (1995) Araneophagic jumping spiders discriminate between the draglines of familiar and unfamiliar conspecifics. Ethology, Ecology and Evolution 7:185-190

We appreciate the reviewer's comprehensive summary and acknowledgment of the significance of our findings.

Strengths:

This work is a useful step toward a fuller understanding of the perceptual and cognitive capacities of spiders and other animals with small nervous systems. By providing experimental evidence for a conclusion that a spider can, by sight, discriminate between familiar and unfamiliar individuals, this research will be an important milestone. We can anticipate a substantial influence on future research.

We appreciate the reviewer’s recognition of the strengths and significance of our study. We are pleased that the reviewer considers our research an important milestone. Our findings indeed suggest that even animals with relatively simple nervous systems can perform complex cognitive tasks, which has substantial implications for the broader study of animal cognition.

As pointed out by the reviewer, we also hope that our study will have a substantial influence on future research. By establishing a methodology and providing clear evidence of visual discrimination, we aim to encourage further investigations into the cognitive abilities of jumping spiders and other arthropods. Future research can build on our findings to explore the specific visual cues and mechanisms involved in individual recognition (as Reviewer 2 pointed out), as well as the ecological and evolutionary implications of these abilities.

Weaknesses:

(1) The conclusions should be stated more carefully.

We agree that clarity in our conclusions is paramount. We will revise the manuscript to ensure that our conclusions are presented with precision and appropriately reflect the data. Specifically, we will emphasize the evidence supporting our findings of visual individual recognition and clarify the limitations and scope of our conclusions to avoid any potential overstatements.

(2) It is not clearly the case that the experimental methods are based on 'habituation (learning to ignore; learning not to respond). Saying 'habituation' seems to imply that certain distances are instances of responding and other distances are instances of not responding but, as a reasonable alternative, we might call distance in all instances a response. However, whether all distances are responses or not is a distracting issue because being based on habituation is not a necessity.

We appreciate the reviewer's feedback and understand the concern regarding the use of the term 'habituation.' We agree that all distances maintained by the spiders are active responses and reflect their behavioral decisions based on perception and recognition of the other individual. We recognize that all distances are responses and interpret these as the spiders’ “active decisions”, modulated by their recognition of the same or different individuals. 

The terms 'habituation' and 'dishabituation' are used to label trial types for ease of discussion and to describe the expected behavioural modulation.

(3) Besides data related to distances, other data might have been useful. For example, salticids are especially well known for the way they communicate using distinctive visual displays and, unlike distance, displaying is a discrete, unambiguous response.

We appreciate the reviewer’s suggestion to incorporate data on visual displays, which are indeed well-known communication methods among salticids. We agree that visual displays are discrete and unambiguous responses that could provide additional insights into the spiders' recognition abilities.

Our primary focus on distance measurements was driven by the need to quantify behaviour in a continuous and scalable manner, that is, how spiders modulate their proximity based on familiarity with other individuals.

We acknowledge the potential value of including visual display measurments; however, in our study, we aimed to establish a foundational understanding of recognition behaviour through proximity measures first. Also, capturing diplays requires a different experimental paradigm, where the displays are clearly visible and analyzable. 

(4) Methods more aligned with salticids having extraordinarily good eyesight would be useful. For example, with salticids, standardising and manipulating stimuli in experiments can be achieved by using mounts, video playback, and computer-generated animation.

There is no doubt that salticids have excellent eyesight. However, our study focuses on higherlevel perceptual processes that require complex brain analysis, not just visual acuity. The goal was to investigate whether spiders can individuate and recognize conspecifics, which involves interpreting visual information and forming long-term representations.

Clearly, methods like video playback and computer animations are useful in controlled settings, where the spider is mounted, but they pose challenges for our specific research question. At this stage of research, we lack precise knowledge of which visual features are critical for individual recognition in spiders, making it difficult to design effective artificial stimuli. 

Our primary objective was to determine if spiders can individuate others. Before exploring the proximate mechanisms of how they individuate others, it was essential to establish that they have this capability. This foundational question needed to be addressed before delving into more detailed mechanistic studies.

(5) An asocial-versus-social distinction is too imprecise, and it may have been emphasised too much. With P. regius, irrespective of whether we use the label asocial or social, the important question pertains to the frequency of encounters between the same individuals and the consequences of these encounters.

Our intent was to convey that P. regius does not live in cohesive social groups but does engage in individual interactions that can have significant behavioral consequences. We will revise the manuscript to reduce the emphasis on the asocial-versus-social distinction. As discussed above, we also will change the term “asocial” to “non-social” in the manuscript.

(6) Hypotheses related to not-so-strictly adaptive factors are discussed and these hypotheses are interesting, but these considerations are not necessarily incompatible with more strictly adaptive influences being relevant as well.

We appreciate the reviewer's observation regarding the discussion of hypotheses related to notso-strictly adaptive factors. We agree that our considerations of these factors do not preclude the relevance of more strictly adaptive influences.

We will revise the manuscript to explicitly discuss how our findings can be interpreted in the context of adaptive hypotheses. This will provide a more comprehensive understanding of the evolutionary significance of individual recognition in P. regius. Modifications were made in the Discussion section.

In the following, we comment on issues not mentioned in the “public reviews” section.

Reviewer #1 (Recommendations For The Authors):

(1) I would suggest conducting experiments that actually test for recognition memory, as this seems to be a claim that the authors make. Following the ant studies by Dreier cited in this manuscript would be sufficient to test for memory. Given the relative simplicity of the measures being taken (location of spiders), this would seem like a very simple addition that would provide a much stronger and more readily interpreted dataset.

As previously explained in our detailed responses (public reviews), we believe that the current design effectively addresses the questions at hand. Our approach, using a habituationdishabituation paradigm, provides robust evidence for recognition memory within the framework of early long-term memory.

Additionally, we have explained why using the distance to the panel as a measure is not appropriate in this context. Specifically, using such a measure can misrepresent the actual interests of the spiders in each other.

While we acknowledge the merits of the ant studies by Dreier, our current design allows for a detailed understanding of the spiders' recognition capabilities over short (10 min) and slightly longer intervals (up to one hour). This is sufficient to demonstrate the presence of recognition memory without the necessity of further experiments. The observed patterns of habituation and dishabituation responses in our study clearly indicate that the spiders can distinguish between familiar and novel individuals, which supports our claims.

Given these points, we respectfully maintain that the current data and experimental design are adequate to support our findings and provide a comprehensive understanding of recognition memory in Phidippus regius.

(2) The writing is rather impenetrable. The results explain the basic finding in terms of statistical variables rather than simply stating the results. A clear and straightforward statement such as 'the spiders showed reduced interest upon habituation trials, indicating xyz' (and then citing the stats) is preferable to the introduction of results as a statistical model. The statistical model is a means of assessing the results. It is not the result. Describe the data.

We tried to improve that in the current version.

(3) Showing more straightforward data such as distance from the joint barrier would make the paper much easier to understand.

This paper has been on bioRxiv for some time and my guess is that it has ended up here because it is having trouble in review. Collecting new data that more directly test the question at hand, presenting the data in a more direct manner, and more critically evaluating your own claims will improve the paper.

While it is true that the paper has been on bioRxiv for a while, this submission marks the first instance where it has undergone peer review. Prior to this, the manuscript was submitted to other journals but was not reviewed.

We hope the explanations provided in the “public reviews” section, along with the revised manuscript, sufficiently clarify our study and its conclusions. We believe the current data robustly address the research questions, and as outlined in our detailed responses, we have critically evaluated our claims and presented the data clearly. Given these clarifications, we do not see the necessity for new experiments as the existing data adequately support our findings. We trust that these revisions and explanations will clarify any misunderstandings.

I am totally sold that the spiders are paying attention to identity at some level. The key now is to understand what that actually means in terms of recognition (i.e. memory of individuals) not just habituation.

We appreciate the reviewer’s emphasis on the distinction between habituation and memorybased individual recognition. As detailed in the preceding discussion, we have taken great care to clarify how our paradigm distinguishes simple habituation effects from true memory for individual identity. We trust that the preceding sections make clear how our findings go beyond simple habituation to establish genuine individual recognition.

Reviewer #2 (Recommendations For The Authors):

Aside from the comments in the public review, I have some additional comments that the authors may wish to consider.

Numerous times in the manuscript, the authors mentioned that recognizing individuals requires recognition memory. This seems rather obvious, and I wonder if the authors could instead be more precise about what they mean by 'recognition memory'?

Recognition memory refers to the cognitive ability to identify a previously encountered stimulus, an individual, or events as familiar. It involves both encoding and retrieval processes, allowing an organism to distinguish between novel and familiar stimuli. This form of memory is a fundamental component of cognitive functioning and is supported by neural mechanisms that, in the mammal brain, involve the hippocampus and other brain regions associated with memory processing. 

In our study, we aimed to test whether Phidippus regius recognizes conspecifics, or, in other words, utilizes recognition memory to distinguish between familiar and unfamiliar conspecifics. With the habituation - dishabituation paradigm, we assessed the spiders' ability to recognize previously encountered individuals and demonstrate memory retention over short (10 min) and extended periods (1 hour).

Encoding: In the initial trial, when a spider encounters an individual for the first time (Figure 1A, “Baseline” or “Dishabituation” for every following trial), it encodes the visual information related to that specific individual. This encoding process involves creating a memory trace of the individual's phenotypic characteristics.

Storage: During the visual separation period, this encoded information is stored in the spider's memory system. The memory trace, though initially fragile, starts to stabilize over the separation period. Whether or not this leads to some form of consolidated memory remains unaddressed. This aspect was highlighted by the first reviewer, but our focus is on the early process rather than on late processes, such as consolidation. 

Retrieval: In the subsequent trial, when the same individual is presented again, the spider retrieves the stored memory trace. If the spider recognizes the individual, its behaviour reflects habituation, indicating memory retrieval. Conversely, when a novel individual is introduced, the lack of stored memory trace triggers a different behavioural response, indicating dishabituation. This differential response demonstrates the spider's ability to distinguish between familiar and unfamiliar individuals. This differential response is also key to understanding the nature of habituation over the three sessions, as introducing novel spiders leads to a significant dishabituation response after the three sessions in Experiment 2.

In Line 39, the authors state that they used "a naturalistic experimental procedure". I would like to know how this experiment is 'naturalistic'. The authors' use of an arena does not appear naturalistic, or something the spiders would encounter in the wild.

We appreciate the reviewer's comment regarding our use of the term 'naturalistic'. We acknowledge that the experimental arena itself does not replicate the conditions found in the wild. Our approach aimed to incorporate elements of natural behaviour by allowing two spiders to freely move and interact within the controlled environment. This approach aligns with principles from computational ethology, which seeks to balance the trade-off between repeatability/standardization and observing free, naturalistic behaviour. By using this paradigm, we aimed to capture behaviours that closely resemble those exhibited in their natural habitat. This setup was chosen to balance the need for ecological validity with the requirements for standardized data collection. 

Also, and this point has been raised above, by observing the spiders' natural interactions without restraining them or using artificial stimuli like computer animations, we aimed to capture behaviours that closely resemble their natural responses to conspecifics. In contrast, we would not have any clear expectations regarding responses to arbitrarily designed artificial stimuli. This method provides a more ecologically valid assessment of the spiders' recognition abilities.

There are a few details wrong in Line 41. 'Salticidae' is a family name and shouldn't be italicized. Also, the sentence suggests that there is a spider called a 'jumping spider' in the family Salticidae, which is technically called Phidippus regius. To clarify, all spiders in the family Salticidae are known as jumping spiders, and one species of jumping spiders is called Phidippus regius.

We will correct this in the manuscript to accurately reflect the classification and terminology. Thank you for pointing out these inaccuracies.

A manuscript on individual recognition by a salticid should include citations to earlier papers that have already considered individual recognition by salticids. As well as the paper by Tedore and Johnsen (2013), the authors should be aware of the following papers.

Clark, R. J., & Jackson, R. R. (1994). Portia labiata, a cannibalistic jumping spider, discriminates between its own and foreign egg sacs. International Journal of Comparative Psychology, 7, 3843.

Clark, R. J., & Jackson, R. R. (1994). Self-recognition in a jumping spider: Portia labiata females discriminate between their own draglines and those of conspecifics. Ethology, Ecology & Evolution, 6, 371-375.

Clark, R. J., & Jackson, R. R. (1995). Araneophagic jumping spiders discriminate between the draglines of familiar and unfamiliar conspecifics. Ethology, Ecology & Evolution, 7, 185-190.

We appreciate the reviewer's suggestion to include citations to these earlier papers. We will add the recommended references to provide a comprehensive background.

In Line 203, I would not consider "interaction with human caretakers and experimenters" to be a form of behavioral enrichment. This kind of interaction has the potential to be stressful for the spiders, rather than enriching. I suggest deleting that part of the sentence.

We appreciate the reviewer's feedback and agree that interactions with human caretakers and experimenters might not always be enriching and could potentially be stressful for the spiders. We will remove that part of the sentence to better reflect the intended meaning.

Reviewer #3 (Recommendations For The Authors):

This manuscript is useful and interesting, and I predict that it will be influential, but more attention should be given to stating the objective and conclusion accurately and clearly. As I understand it, the objective was to investigate a specific hypothesis: that Phidippus regius has a capacity to identify conspecific individuals as particular individuals (i.e., individual identification). Strong evidence supporting this hypothesis being true would be especially remarkable because I am unaware of any published work having shown evidence of a spider expressing this specific perceptual capacity.

Thank you for recognizing the significance and potential influence of our manuscript. We agree that clearly stating the objective and conclusions is essential for conveying the importance of our findings. Our results provide robust evidence supporting the hypothesis that Phidippus regius can recognize and remember individual conspecifics. We will revise the manuscript to more clearly highlight the objective and our conclusions, emphasizing the novel evidence for individual identification in these spiders.

Based on reading this manuscript and based on my understanding of the meaning of 'individual identification', it seems to me that the hypothesis that P. regius has a capacity for individual identification might or might not be true, and the experiments in this manuscript cannot tell us which is the case. 

We respectfully disagree with the reviewer's assessment. Our experiments were carefully designed to test whether P. regius has the capacity for individual identification, and our results provide clear evidence supporting this hypothesis. The systematic differences in the spiders' behaviour when encountering familiar versus novel individuals indicate that they can recognize and remember specific conspecifics. We will revise the manuscript to ensure that the evidence and conclusions are stated more clearly to address any potential misunderstandings.

Determining which is the case would have required research that made better use of the literature, and displayed more critical thinking. addressed credible alternative hypotheses and adopted experimental methods that focused more strictly on individual identification. 

The distinction between whether P. regius has a capacity for individual identification is not ambiguous in our study. Our findings clearly demonstrate this capacity through systematic behavioural responses to familiar versus novel individuals. As pointed out above, the experimental procedure might be complex, but results are systematic despite this complexity. The experiments were designed to directly address the hypothesis of individual identification, and the data robustly support our conclusions. While considering alternative hypotheses is important, the results we present provide a coherent and compelling case for individual identification in P. regius. We will ensure our manuscript clearly articulates this narrative and the supporting evidence.

At the same time, I also appreciate that asking for all of that at once would be asking for too much. As I see it, this manuscript tells us about research that moves us closer to a clear focus on the details and questions that will matter in the context of considering a hypothesis that is strictly about individual identification. More importantly, I think this research reveals a perceptual capacity that is remarkable even if it is not strictly a capacity for individual identification.

We understand the desire for a more focused exploration of individual identification with paradigms more familiar to the reviewers and we acknowledge that further detailed studies could enhance our understanding of this capacity. However, our findings do indeed suggest that Phidippus regius exhibits a remarkable perceptual capacity for recognizing and remembering individual conspecifics. The systematic behavioural responses observed in our experiments strongly indicate that these spiders possess the ability for individual recognition. While our study may not have explored every potential detail (e.g. which features are most crucial for the memory matching processes), the evidence we present robustly supports the conclusion of individual identification.

We acknowledge that it is indeed valuable to follow established paradigms and build upon the frameworks that have been used successfully in similar species and studies. These paradigms provide a solid foundation for scientific inquiry and allow for comparability across different research efforts. However, it is equally important to acknowledge and explore alternative approaches. Scientific progress is driven not only by replication but also by innovation. By employing new paradigms, researchers can uncover novel insights and push the boundaries of current understanding. The paradigm we used in our study, while different from those traditionally applied to similar research, is not an invention but a well-established method in various domains. It represents an innovative application in the context of our specific research questions, offering a fresh perspective and contributing to the advancement of the field.

As I understand it, 'individual identification' means identifying another individual as being a particular individual instead of a member of a larger set (or 'class') of individuals. An 'individual' is a set containing a single individual. Interesting examples of identifying members of larger sets include discriminating between familiar and unfamiliar individuals. In the context of the specific experiments in this manuscript, familiar-unfamiliar discrimination means discriminating between recently-seen and not-so-recently-seen individuals. My impression is that the experiments in this manuscript have given us a basis for concluding that P. regius has a capacity for familiarunfamiliar (recently seen versus not so recently seen) discrimination. If this is the case, then I think this is the conclusion that should be emphasised. This would be an important conclusion.

I appreciate that, depending on how we use the words, familiar-unfamiliar discrimination might be construed as being 'individual identification'. An individual is identified as 'the individual recently seen'. As a casual way of speaking, it can be reasonable to call this 'individual identification'. The difficulty comes from the way calling this 'individual identification' can suggest something more than has been demonstrated. To navigate through this difficulty, we need an expression to use for a capacity that goes beyond familiar-unfamiliar discrimination. In the context of this manuscript about P. regius, we need expressions that will make it easy to consider two things. One of these things is a capacity for familiar-unfamiliar discrimination. The other is the capacity to identify another individual as being a particular individual.

We appreciate the reviewer's insightful comments on the distinction between familiar-unfamiliar discrimination and individual identity recognition. Our study indeed focuses on demonstrating that Phidippus regius can recognize and remember individual conspecifics, providing evidence for individual identity recognition.

Two specific behavioural hallmarks that speak against familiarity recognition:

First, the significant dishabituation response to novel individuals introduced after multiple sessions underscores the specificity of the recognition. This shows that the spiders' habituation is not general but specific to familiar individuals. 

Second, the pattern of habituation over the sessions provides further evidence: We observed the strongest systematic modulation in Session 1, a reduced modulation in Session 2, and a further diminished effect in Session 3. If the spiders were only responding based on familiarity, we would expect a more drastic decrease, resulting in a washed-out non-effect by Session 2. However, the continued, though diminishing, differentiation between habituation and dishabituation trials across sessions indicates that the spiders are not merely responding to a general sense of familiarity but are engaging in individual recognition. In other words, the spiders' ability to distinguish between familiar and novel individuals even after repeated exposures suggests that they are not just recognizing a familiar status but are identifying specific individuals.

Things people do might help clarify what this means. People have an extraordinary capacity for identifying other individuals as particular individuals. Often this is based on giving each other names. Imagine we are letting somebody see photographs and asking them to identify who they see. The answer might be, 'somebody familiar' or 'somebody I saw recently' (familiar-unfamiliar discrimination); or the question might be answered by naming a particular individual (individual identification).

We appreciate the reviewer's efforts to clarify the distinction between familiar-unfamiliar discrimination and individual recognition using human examples. However, we believe this comparison might not fully capture the complexity of individual recognition in non-human animals. 

Familiarity recognition refers to recognizing someone as having been seen or encountered before without necessarily distinguishing them from others in the same category. On the other hand, identity recognition involves recognizing a specific individual based on unique characteristics (or features). In humans, this often involves naming, but more critically, like in most animals, it involves recognizing visual, auditory, chemical or other sensory cues. In animals, including spiders, individual recognition does not involve and let alone rely on naming but on the ability to distinguish between individuals based on sensory cues and learnt associations. This is a valid and well-documented form of individual recognition across many species.

Individual recognition does not require naming or the assignment of a referential label. Animals can distinguish between specific individuals based on previously perceived and stored features and characteristics. Naming is the exception rather than the rule in the animal kingdom. Only a few species, such as humans and maybe certain cetaceans, use naming for identity recognition. This is an evolutionary rarity and not the standard mechanism for individual recognition, which primarily relies on sensory cues and learnt associations. Furthermore, the mechanism of recognition in both humans and animals involves a complex process of matching incoming sensory and perceptual information with stored memory representations. Naming is merely a tool for communication, allowing us to convey which individual we are referring to. It is not the mechanism by which recognition occurs. The core of individual recognition is this matching process, where sensory cues (visual, auditory, chemical, etc.) are compared to memory traces of previously encountered individuals. Therefore, the suggestion that individual identification necessitates naming misrepresents the actual cognitive processes involved. 

We can think of individual identification being based on more fine-grained discrimination (with this, set size = one), with familiar-unfamiliar discrimination being more coarse-grained discrimination (with this, set size can be more than one). Restricting the expression 'individual identification' to instances of having the capacity to identify another individual as being a particular individual (set size = one) is better aligned with normal usage of this expression.

Absolutely, the distinction between fine-grained and coarse-grained discrimination aligns with the concept of different category levels, such as basic and subordinate levels, put forward by Eleanor Rosch (e.g. Rosch, 1973). In the context of individual recognition, fine-grained discrimination (where set size = one) refers to the ability to identify a specific individual based on unique characteristics. This is referred to as subordinate level categorization. Coarse-grained discrimination (where set size can be more than one) refers to recognizing someone as familiar without distinguishing them from others in the same category, more similar to basic level categorization. 

Rosch, E.H. (1973). "Natural categories". Cognitive Psychology. 4 (3): 328–50.doi:10.1016/0010-0285(73)90017-0

There is a strong emphasis on an asocial-social distinction in this manuscript. It seems to me that this needs to be focused more clearly on the specific factors that would make a capacity for individual identification beneficial. In the context of this manuscript, the term 'social' may suggest too much. It seems to me that the issue that matters the most is whether individuals live in situations where important encounters occur frequently between the same individuals. Irrespective of whether other notions of the meaning of 'social' also apply, there are salticids that live in aggregated situations where they frequently have important encounters with each other. This is the case with Phidippus regius in the field in Florida, but I realize that there may not be much published information about the natural history of this salticid. Even so, there are salticids to which the word 'social' has been applied in published literature.

We appreciate the reviewer's comments on the asocial-social distinction and we agree that this terminology might need refinement. Our intent was not to categorize Phidippus regius rigidly but to explore the contextual factors influencing the benefits of individual identification. The critical factor in our study is indeed the frequency and importance of encounters between individuals, rather than a broader social structure. We will revise the manuscript to reflect this more nuanced perspective, focusing on the ecological validity of our experimental design and the adaptive significance of individual recognition in environments where repeated encounters can occur.

eLife Assessment

This important study uses data on over 56 million articles to examine the dynamics of interdisciplinarity and international collaborations in research journals. The data analytics used to quantify disciplinary and national diversity are convincing, and support the claims that journals have become more diverse in both aspects.

Reviewer #1 (Public review):

Summary:

The authors aim to explore how interdisciplinarity and internationalization-two increasingly prominent characteristics of scientific publishing-have evolved over the past century. By constructing entropy-based indices from a large-scale bibliometric dataset (OpenAlex), they examine both long-term trends and recent dynamics in these two dimensions across a selection of leading disciplinary and multidisciplinary journals. Their goal is to identify field-specific patterns and structural shifts that can inform our understanding of how science has become more globally collaborative and intellectually integrated.

Strengths and Weaknesses:

The paper's primary strength lies in its comprehensive temporal scope and use of a rich, openly available dataset covering over 56 million articles. The interdisciplinary and internationalization indices are well-founded and allow meaningful comparisons across fields and time. Moreover, the distinction between disciplinary and multidisciplinary journals adds valuable nuance. However, some methodological choices, such as the use of a 5-year sliding window to compute trend values, are insufficiently justified and under-explained. The paper also does not fully address disparities in data coverage across disciplines and time, which may affect the reliability of historical comparisons. Finally, minor issues in grammar and clarity reduce the overall polish of the manuscript.

Evaluation of Findings:

Overall, the authors have largely succeeded in achieving their stated aims. The findings-such as the sharp rise in internationalization in fields like Physics, and the divergence in interdisciplinarity trends across disciplines-are clearly presented and generally well-supported by the data. The authors effectively demonstrate that scientific journals have not followed a uniform trajectory in terms of structural evolution. However, greater clarity in trend estimation methods and better acknowledgment of dataset limitations would help to further substantiate the conclusions and enhance their generalizability.

Impact and Relevance:

This study makes a timely and meaningful contribution to the fields of scientometrics, sociology of science, and science policy. Its combination of scale, historical depth, and field-level comparison offers a useful framework for understanding changes in scientific publishing practices. The entropy-based indicators are simple yet flexible, and the use of open bibliometric data enhances reproducibility and accessibility for future research. Policymakers, journal editors, and researchers interested in publication dynamics will likely find this work informative, and its methods could be applied or extended to other structural dimensions of scholarly communication.

Reviewer #2 (Public review):

Summary:

This paper uses large-scale publication data to examine the dynamics of interdisciplinarity and international collaborations in research journals. The main finding is that interdisciplinarity and internationalism have been increasing over the past decades, especially in prestigious general science journals.

Strengths:

The paper uses a state-of-the-art large-scale publication database to examine the dynamics of interdisciplinarity and internationalism. The analyses span over a century and in major scientific fields in natural sciences, engineering, and social sciences. The study is well designed and has provided a range of robustness tests to enhance the main findings. The writing is clear and well organized.

Weaknesses:

While the research provides interesting perspectives for the reader to learn about the trends of journal preferences, I have a few points for the authors to consider that might help strengthen their work.

The first thing that comes to mind is the epistemic mechanism of the study. Why should there be a joint discussion combining internationalism and interdisciplinarity? While internationalism is the tendency to form multinational research teams to work on research projects, interdisciplinarity refers to the scope and focus of papers that draw inspiration from multiple fields. These concepts may both fall into the realm of diversity, but it remains unclear if there is any conceptual interplay that underlies the dynamics of their increase in research journals.

It is also unclear why internationalization is increasing. Although the authors have provided a few prominent examples in physics, such as CERN and LIGO, which are complex and expensive experimental facilities that demand collective efforts and investments from the global scientific community, whether some similar concerns or factors drive the growth of internationalism in other fields remains unknown. I can imagine that these concerns do not always apply in many fields, and the authors need to come up with some case studies in diverse fields with some sociological theory to support their empirical findings.

The authors use Shannon entropy as a measure of diversity for both internationalism and interdisciplinarity. However, entropy may fail to account for the uneven correlations between fields, and the range of value chances when the number of categories changes. The science of science and scientometrics community has proposed a range of diversity indicators, such as the Rao-Stirling index and its derivatives. One obvious advantage of the RS index is that it explicitly accounts for the heterogeneous connections between fields, and the value ranges from 0 to 1. Using more state-of-the-art metrics to quantify interdisciplinarity may help strengthen the data analytics.

In this section, I find this to be an example of clinician automatic thoughts that lead to distorted judgments. Relating to class, we learned that CBT uses thought records and distortions. I strongly believe that providers need to have the same practice. The alliance can split when bias enters. In class, we highlighted that it is important to repair ruptures through empathic validation.

The Process Models relate to Wave 3 on contextual therapies (mindfulness, values, flexibility). It expresses how culture should be a part of the "case conceptualization" that we learned in class.

I relate each of these models to the three waves of CBT in practice that we discussed in class lecture.

I relate the skills-based model to one of the waves in CBT practice, which is Wave 1 where it focuses on observable behaviors (skills training)

The reconciliation of black men shouldn't involve industrial slavery and civic death, but oppose it through patriotism and loyalty, even if it goes against Booker T. Washington.

AA need to peacefully fight for the rights that was given to all men by the founding fathers

Public opinion has helped protect black schools and protect AA partially in property, life, and limb. (KP)

What happened after 1750? (W)

Glad to see Washington was able to change their minds. It takes a lot to change someone's opinion from something they're dead set on believing. I wonder how long he had to convince them. (W)

(OT) Greece Enslaved by Lord Byron

Discuté avec Danny. Conserver. Il recommande de le déplacement sous les comptes à payer.

old man

exposition. one detail at a time

After reading it once analyze the text again then continue to read it one or more times again.

Pay deep attention while reading so you can get a better essential focus.

Everyone is not the same like the text talked about we are all different our brains don't work the same and we all have different ideas as well.

eLife Assessment

This important study combines imaginative and innovative experiments with a finite element modelling to demonstrate the relevance of poroelasticity in the mechanical properties of cells across physiologically relevant time and length scales. The authors present convincing evidence that cytosolic flows and pressure gradients can persist in cells with permeable membranes, generating spatially segregated influx and outflux zones. These findings are of interest to the cell biology and biophysics communities.

Reviewer #1 (Public review):

Summary:

This work investigated whether cytoplasmic poroelastic properties play an important role in cellular mechanical response over length scales and time scales relevant to cell physiology. Overall, the manuscript concludes that intracellular cytosolic flows and pressure gradients are important for cell physiology and that they act of time- and length-scales relevant to mechanotransduction and cell migration.

Strengths:

Their approach integrates both computational and experimental methods. The AFM deformation experiments combined with measuring z-position of beads is a challenging yet compelling method to determine poroelastic contributions to mechanical realization.

The work is quite interesting and will be of high value to the field of cell mechanics and mechanotransduction.

Weaknesses:

The weaknesses I noted earlier were adequately addressed in the revised version.

Reviewer #2 (Public review):

Summary:

Malboubi et al. present an experimental framework to investigate the rheological properties of the cell cytoplasm. Their findings support a model where the cytoplasm behaves as a poroelastic material governed by Darcy's law. They demonstrate that this poroelastic behavior delays the equilibration of hydrostatic pressure gradients within the cytoplasm over timescales of 1 to 10 seconds following a perturbation, likely due to fluid-solid friction within the cytoplasmic matrix. Furthermore, under sustained perturbations such as depressurization, they reveal that pressure gradients can persist for minutes, which they propose might potentially influence physiological processes like mechanotransduction or cell migration typically happening on these timescales.

Strengths:

This article holds significant value within the ongoing efforts of the cell biology and biophysics communities to quantitatively characterize the mechanical properties of cells. The experiments are innovative and thoughtfully contextualized with quantitative estimates and a finite element model that supports the authors' hypotheses.

Comments & Questions:

The authors have successfully addressed the questions and comments raised in my previous review, significantly improving the manuscript's depth. Regarding my last question on the predicted saturation of the time lag, the authors propose the interesting hypothesis that the cell cortex becomes dominant at distances beyond 30 microns and plan to test this hypothesis at a later stage.

Reviewer #3 (Public review):

Summary:

In this delightful study, the authors use local indentation of the cell surface combined with out-of-focus microscopy to measure the rates of pressure spread in the cell and to argue that the results can be explained with the poroelastic model. Osmotic shock that decreases cytoskeletal mesh size supports this notion. Experiments with water injection and water suction further support it, and also, together with a mechanical model and elegant measurements of decreasing fluorescence in the cell 'flashed' by external flow, demonstrate that the membrane is permeable, and that steady flow and pressure gradient can exist in a cell with water source/sink in different locations. Use of blebs as indicators of the internal pressure further supports the notion of differential cytoplasmic pressure.

Strengths:

The study is very imaginative, interesting, novel and important.

Weaknesses: I have two broad critical comments:

(1) I sense that the authors are correct that the best explanation of their results is the passive poroelastic model. Yet, to be thorough, they have to try to explain the experiments with other models and show why their explanation is parsimonious. For example, one potential explanation could be some mechanosensitive mechanism that does not involve cytoplasmic flow; another could be viscoelastic cytoskeletal mesh, again not involving poroelasticity. I can imagine more possibilities. Basically, be more thorough in the critical evaluation of your results. Besides, discuss potential effect of significant heterogeneity of the cell.

(2) The study is rich in biophysics but a bit light on chemical/genetic perturbations. It could be good to use low levels of chemical inhibitors for, for example, Arp2/3, PI3K, myosin etc, and see the effect and try to interpret it. Another interesting question - how adhesive strength affects the results. A different interesting avenue - one can perturb aquaporins. Etc. At least one perturbation experiment would be good.

Comments on revisions: I am satisfied with the revisions

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

(1) Some details are not described for experimental procedures. For example, what were the pharmacological drugs dissolved in, and what vehicle control was used in experiments? How long were pharmacological drugs added to cells?

We apologise for the oversight. These details have now been added to the methods section of the manuscript as well as to the relevant figure legends.

Briefly, latrunculin was used at a final concentration of 250 nM and Y27632 at a final concentration of 50 μM. Both drugs were dissolved in DMSO. The vehicle controls were effected with the highest final concentration of DMSO of the two drugs.

The details of the drug treatments and their duration was added to the methods and to figures 6, S10, and S12.

(2) Details are missing from the Methods section and Figure captions about the number of biological and technical replicates performed for experiments. Figure 1C states the data are from 12 beads on 7 cells. Are those same 12 beads used in Figure 2C? If so, that information is missing from the Figure 2C caption. Similarly, this information should be provided in every figure caption so the reader can assess the rigor of the experiments. Furthermore, how heterogenous would the bead displacements be across different cells? The low number of beads and cells assessed makes this information difficult to determine.

We apologise for the oversight. We have now added this data to the relevant figure panels.

To gain a further understanding of the heterogeneity of bead displacements across cells, we have replotted the relevant graphs using different colours to indicate different cells. This reveals that different cells appear to behave similarly and that the behaviour appears controlled by distance to the indentation or the pipette tip rather than cell identity.

We agree with the reviewer that the number of cells examined is low. This is due to the challenging nature of the experiments that signifies that many attempts are necessary to obtain a successful measurement.

The experiments in Fig 1C are a verification of a behaviour documented in a previous publication [1]. Here, we just confirm the same behaviour and therefore we decided that only a small number of cells was needed.

The experiments in Fig 2C (that allow for a direct estimation of the cytoplasm’s hydraulic permeability) require formation of a tight seal between the glass micropipette and the cell, something known as a gigaseal in electrophysiology. The success rate of this first step is 10-30% of attempts for an experienced experimenter. The second step is forming a whole cell configuration, in which a hydraulic link is formed between the cell and the micropipette. This step has a success rate of ~ 50%. Whole cell links are very sensitive to any disturbance. After reaching the whole cell configuration, we applied relatively high pressures that occasionally resulted in loss of link between the cell and the micropipette. In summary, for the 12 successful measurements, hundreds of unsuccessful attempts were carried out.

(3) The full equation for displacement vs. time for a poroelastic material is not provided. Scaling laws are shown, but the full equation derived from the stress response of an elastic solid and viscous fluid is not shown or described.

We thank the reviewer for this comment. Based on our experiments, we found that the cytoplasm behaves as a poroelastic material. However, to understand the displacements of the cell surface in response to localised indentation, we show that we also need to take the tension of the submembranous cortex into account. In summary, the interplay between cell surface tension generated by the cortex and the poroelastic cytoplasm controls the cell behaviour. To our knowledge, no simple analytical solutions to this type problem exist.

In Fig 1, we show that the response of the cell to local indentation is biphasic with a short time-scale displacement followed by a longer time-scale one. In Figs 2 and 3, we directly characterise the kinetics of cell surface displacement in response to microinjection of fluid. These kinetics are consistent with the long time-scale displacement but not the short time-scale one. Scaling considerations led us to propose that tension in the cortex may play a role in mediating the short time-scale displacement. To verify this hypothesis, we have now added new data showing that the length-scale of an indentation created by an AFM probe depends on tension in the cortex (Fig S5).  

In a previous publication [2], we derived the temporal dynamics of cell surface displacement for a homogenous poroelastic material in response to a change in osmolarity. In the current manuscript, the composite nature of the cell (membrane, cortex, cytoplasm) needs to be taken into account as well as a realistic cell shape. Therefore, we did not attempt to provide an analytical solution for the displacement of the cell surface versus time in the current work. Instead, we turned to finite element modelling to show that our observations are qualitatively consistent with a cell that comprises a tensed submembranous actin cortex and a poroelastic cytoplasm (Fig 4). We have now added text to make this clearer for the reader.

Reviewer #2 (Public review):

Comments & Questions:

The authors state, "Next, we sought to quantitatively understand how the global cellular response to local indentation might arise from cellular poroelasticity." However, the evidence presented in the following paragraph appears more qualitative than strictly quantitative. For instance, the length scale estimate of ~7 μm is only qualitatively consistent with the observed ~10 μm, and the timescale 𝜏𝑧 ≈ 500 ms is similarly described as "qualitatively consistent" with experimental observations. Strengthening this point would benefit from more direct evidence linking the short timescale to cell surface tension. Have you tried perturbing surface tension and examining its impact on this short-timescale relaxation by modulating acto-myosin contractility with Y-27632, depolymerizing actin with Latrunculin, or applying hypo/hyperosmotic shocks?

Upon rereading our manuscript, we agree with the reviewer that some of our statements are too strong. We have now moderated these and clarified the goal of that section of the text.

The reviewer asks if we have examined the effect of various perturbations on the short time-scale displacements. In our experimental conditions, we cannot precisely measure the time-scale of the fast relaxation because its duration is comparable to the frame rate of our image acquisition. However, we examined the amplitude of the displacement of the first phase in response to sucrose treatment and we have carried out new experiments in which we treat cells with 250nM Latrunculin to partially depolymerise cellular F-actin. Neither of these treatments had an impact on the amplitude of vertical displacements (Fig. S3).

The absence of change in response to Latrunculin may be because the treatment decreases both the elasticity of the cytoplasm  and the cortical tension . As the length-scale  of the deformation of the surface scales as , the two effects of latrunculin treatment may therefore compensate one another and result in only small changes in . We have now added this data to supplementary information and comment on this in the text.   

The reviewer’s comment also made us want to determine how cortical tension affects the length-scale of the cell surface deformation created by localised microindentation. To isolate the role of the cortex from that of cell shape, we decided to examine rounded mitotic cells. In our experiments, we indented a mitotic cell expressing a membrane targeted GFP with a sharp AFM tip (Fig. S5).

In our experiments, we adjusted force to generate a 2μm depth indentation and we imaged the cell profile with confocal microscopy before and during indentation. Segmentation of this data allowed us to determine the cell surface displacement resulting from indentation and measure a length scale of deformation. In control conditions, the length scale created by deformation is on the order of 1.2μm. When we inhibited myosin contractility with blebbistatin, the length-scale of deformation decreased significantly to 0.8 μm, as expected if we decrease the surface tension γ without affecting the cytoplasmic elasticity. We have now added this data to our manuscript.

The authors demonstrate that the second relaxation timescale increases (Figure 1, Panel D) following a hyperosmotic shock, consistent with cytoplasmic matrix shrinkage, increased friction, and consequently a longer relaxation timescale. While this result aligns with expectations, is a seven-fold increase in the relaxation timescale realistic based on quantitative estimates given the extent of volume loss?

We thank the reviewer for this interesting question. Upon re-examining our data, we realised that the numerical values in the text related to the average rather than the median of our measurements. The median of the poroelastic time constant increases from ~0.4s in control conditions to 1.4s in sucrose, representing approximately a 3.5 fold increase.

Previous work showed that HeLa cell volume decreases by ~40% in response to hyperosmotic shock [3]. The fluid volume fraction in cells is ~65-75%. If we assume that the water is contained in N pores of volume , we can express the cell volume as with the volume of the solid fraction. We can rewrite .

With ∅ = 0.42  -0.6.  As  does not change in response to osmotic shock, we can rewrite the volume change to obtain the change in pore size .

The poroelastic diffusion constant scales as and the poroelastic timescale scales as . Therefore, the measured change in volume leads to a predicted increase in poroelastic diffusion time of 1.7-1.9 fold, smaller than observed in our experiments. This suggests that some intuition can be gained in a straightforward manner assuming that the cytoplasm is a homogenous porous material.

However, the reality is more complex and the hydraulic pore size is distinct from the entanglement length of the cytoskeleton mesh, as we discussed in a previous publication [4]. When the fluid fraction becomes sufficiently small, macromolecular crowding will impact diffusion further and non-linearities will arise. We have now added some of these considerations to the discussion.

If the authors' hypothesis is correct, an essential physiological parameter for the cytoplasm could be the permeability k and how it is modulated by perturbations, such as volume loss or gain. Have you explored whether the data supports the expected square dependency of permeability on hydraulic pore size, as predicted by simple homogeneity assumptions?

We thank the reviewer for this comment. As discussed above, we have explored such considerations in a previous publication (see discussion in [4]). Briefly, we find that the entanglement length of the F-actin cytoskeleton does play a role in controlling the hydraulic pore size but is distinct from it. Membrane bounded organelles could also contribute to setting the pore size. In our previous publication, we derived a scaling relationship that indicates that four different length-scales contribute to setting cellular rheology: the average filament bundle length, the size distribution of particles in the cytosol, the entanglement length of the cytoskeleton, and the hydraulic pore size. Many of these length-scales can be dynamically controlled by the cell, which gives rise to complex rheology. We have now added these considerations to our discussion.

Additionally, do you think that the observed decrease in k in mitotic cells compared to interphase cells is significant? I would have expected the opposite naively as mitotic cells tend to swell by 10-20 percent due to the mitotic overshoot at mitotic entry (see Son Journal of Cell Biology 2015 or Zlotek Journal of Cell Biology 2015).

We thank the reviewer for this interesting question. Based on the same scaling arguments as above, we would expect that a 10-20% increase in cell volume would give rise to 10-20% increase in diffusion constant. However, we also note that metaphase leads to a dramatic reorganisation of the cell interior and in particular membrane-bounded organelles. In summary, we do not know why such a decrease could take place. We now highlight this as an interesting question for further research.

Based on your results, can you estimate the pore size of the poroelastic cytoplasmic matrix? Is this estimate realistic? I wonder whether this pore size might define a threshold above which the diffusion of freely diffusing species is significantly reduced. Is your estimate consistent with nanobead diffusion experiments reported in the literature? Do you have any insights into the polymer structures that define this pore size? For example, have you investigated whether depolymerizing actin or other cytoskeletal components significantly alters the relaxation timescale?

We thank the reviewer for this comment. We cannot directly estimate the hydraulic pore size from the measurements performed in the manuscript. Indeed, while we understand the general scaling laws, the prefactors of such relationships are unknown.

We carried out experiments aiming at estimating the hydraulic pore size in previous publications [3,4] and others have shown spatial heterogeneity of the cytoplasmic pore size [5]. In our previous experiments, we examined the diffusion of PEGylated quantum dots (14nm in hydrodynamic radius). In isosmotic conditions, these diffused freely through the cell but when the cell volume was decreased by a hyperosmotic shock, they no longer moved [3,4]. This gave an estimate of the pore radius of ~15nm.

Previous work has suggested that F-actin plays a role in dictating this pore size but microtubules and intermediate filaments do not [4].

There are no quantifications in Figure 6, nor is there a direct comparison with the model. Based on your model, would you expect the velocity of bleb growth to vary depending on the distance of the bleb from the pipette due to the local depressurization? Specifically, do blebs closer to the pipette grow more slowly?

We apologise for the oversight. The quantifications are presented in Fig S10 and Fig S12. We have now modified the figure legends accordingly.

Blebs are very heterogenous in size and growth velocity within a cell and across cells in the population in normal conditions [6]. Other work has shown that bleb size is controlled by a competition between pressure driving growth and actin polymerisation arresting it[7]. Therefore, we did not attempt to determine the impact of depressurisation on bleb growth velocity or size.

In experiments in which we suddenly increased pressure in blebbing cells, we did notice a change in the rate of growth of blebs that occurred after we increased pressure (Author response image 1). However, the experiments are technically challenging and we decided not to perform more.

Author response image 1.

A. A hydraulic link is established between a blebbing cell and a pipette. At time t>0, a step increase in pressure is applied. B. Kymograph of bleb growth in a control cell (top) an in a cell subjected to a pressure increase at t=0s (bottom). Top: In control blebs, the rate of growth is slow and approximately constant over time. The black arrow shows the start of blebbing. Bottom: The black arrow shows the start of blebbing. The dashed line shows the timing of pressure application and the red arrow shows the increase in growth rate of the bleb when the pressure increase reaches the bleb. This occurs with a delay δt.

I find it interesting that during depressurization of the interphase cells, there is no observed volume change, whereas in pressurization of metaphase cells, there is a volume increase. I assume this might be a matter of timescale, as the microinjection experiments occur on short timescales, not allowing sufficient time for water to escape the cell. Do you observe the radius of the metaphase cells decreasing later on? This relaxation could potentially be used to characterize the permeability of the cell surface.

We thank the reviewer for this comment.

First, we would like to clarify that both metaphase and interphase cells increase their volume in response to microinjection. The effect is easier to quantify in metaphase cells because we assume spherical symmetry and just monitor the evolution of the radius (Fig 3). However, the displacement of the beads in interphase cells (Fig 2) clearly shows that the cell volume increases in response to microinjection. For both interphase and metaphase cells, when the injection is prolonged, the membrane eventually detaches from the cortex and large blebs form until cell lysis. In contrast to the reviewer’s intuition, we never observe a relaxation in cell volume, probably because we inject fluid faster than the cell can compensate volume change through regulatory mechanisms involving ion channels.

When we depressurise metaphase cells, we do not observe any change in volume (Fig S10). This contrasts with the increase that we observe upon pressurisation. The main difference between these two experiments is the pressure differential. During depressurisation experiments, this is the hydraulic pressure within the cell ~500Pa (Fig 6A); whereas during pressurisation experiments, this is the pressure in the micropipette, ranging from 1.4-10 kPa (Fig 3). We note in particular that, when we used the lowest pressures in our experiments, the increase in volume was very slow (see Fig 3C). Therefore, we agree with the reviewer that it is likely the magnitude of the pressure differential that explains these differences.

I am curious about the saturation of the time lag at 30 microns from the pipette in Figure 4, Panel E for the model's prediction. A saturation which is not clearly observed in the experimental data. Could you comment on the origin of this saturation and the observed discrepancy with the experiments (Figure E panel 2)? Naively, I would have expected the time lag to scale quadratically with the distance from the pipette, as predicted by a poroelastic model and the diffusion of displacement. It seems weird to me that the beads start to move together at some distance from the pipette or else I would expect that they just stop moving. What model parameters influence this saturation? Does membrane permeability contribute to this saturation?

We thank the reviewer for pointing this out. In our opinion, the saturation occurring at 30 microns arises from the geometry of the model. At the largest distance away from the micropipette, the cortex becomes dominant in the mechanical response of the cell because it represents an increasing proportion of the cellular material.

To test this hypothesis, we will rerun our finite element models with a range of cell sizes. This will be added to the manuscript at a later date.

Reviewer #3 (Public review):

Weaknesses: I have two broad critical comments:

(1) I sense that the authors are correct that the best explanation of their results is the passive poroelastic model. Yet, to be thorough, they have to try to explain the experiments with other models and show why their explanation is parsimonious. For example, one potential explanation could be some mechanosensitive mechanism that does not involve cytoplasmic flow; another could be viscoelastic cytoskeletal mesh, again not involving poroelasticity. I can imagine more possibilities. Basically, be more thorough in the critical evaluation of your results. Besides, discuss the potential effect of significant heterogeneity of the cell.

We thank the reviewer for these comments and we agree with their general premise.

Some observations could qualitatively be explained in other ways. For example, if we considered the cell as a viscoelastic material, we could define a time constant with η the viscosity and E the elasticity of the material. The increase in relaxation time with sucrose treatment could then be explained by an increase in viscosity. However, work by others has  previously shown that, in the exact same conditions as our experiment, viscoelasticity cannot account for the observations[1]. In its discussion, this study proposed poroelasticity as an alternative mechanism but did not investigate that possibility. This was consistent with our work that showed that the cytoplasm behaves as a poroelastic material and not as a viscoelastic material [4]. Therefore, we decided not to consider viscoelasticity as possibility. We now explain this reasoning better and have added a sentence about a potential role for mechanotransductory processes in the discussion.

(2) The study is rich in biophysics but a bit light on chemical/genetic perturbations. It could be good to use low levels of chemical inhibitors for, for example, Arp2/3, PI3K, myosin etc, and see the effect and try to interpret it. Another interesting question - how adhesive strength affects the results. A different interesting avenue - one can perturb aquaporins. Etc. At least one perturbation experiment would be good.

We agree with the reviewer. In our previous studies, we already examined what biological structures affect the poroelastic properties of cells [2,4]. Therefore, the most interesting aspect to examine in our current work would be perturbations to the phenomenon described in Fig 6G and, in particular, to investigate what volume regulation mechanisms enable sustained intracellular pressure gradients. However, these experiments are particularly challenging and with very low throughput. Therefore, we feel that these are out of the scope of the present report and we mention these as promising future directions.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Please add more information to Materials and methods and figure captions to more clearly share how many different cells and trials the data are coming from.

This has been done.

Please add the full equation for displacement vs. time for the poroelastic model and describe appropriately.

This cannot be done but we explain why.

Overall, the clarity of the writing in the manuscript could be improved.

This has been done.

Please increase text size in some of the figures.

This has been done.

Reviewer #2 (Recommendations for the authors):<br /> Figure 1 would benefit from some revisions for clarity. In Panel D, for the control experiment with 7 cells, why are only 3 data points shown?

This was due to the use of excel for generating the box plot. Some data points overlap. We now have used a different software.

In Panel E, there is no legend explaining the red dots in the whisker plots.

This has now been added.

Additionally, the inset in Panel D lacks a legend, and it is unclear how k was computed.

This inset panel has been removed.

Moreover, I find Figure 1, Panel C somewhat pixelated, which makes it challenging to interpret. As I am colorblind, I need to zoom in significantly to distinguish the colors, and the current resolution makes this difficult. Improving the image resolution would be helpful.

Apologies for this. We have now verified the quality of images on our submission.  

I am unsure about the method used to compute the relaxation timescale in Figure S2. If an exponential relaxation is assumed, I would expect a function of the form:

which implies that for t=t1+tau_p, the result should be d1+0.6*Delta d which does not correspond to the formula given. Have you tried fitting the data with an exponential function or using the model to extract tau_p without assuming a specific functional form?

We thank the reviewer for pointing this out. We have now added further explanation of the fitting to the figure legend.

References:

(1) Rosenbluth, M. J., Crow, A., Shaevitz, J. W. & Fletcher, D. A. Slow stress propagation in adherent cells. Biophys J 95, 6052-6059 (2008). https://doi.org/10.1529/biophysj.108.139139

(2) Esteki, M. H. et al. Poroelastic osmoregulation of living cell volume. iScience 24, 103482 (2021). https://doi.org/10.1016/j.isci.2021.103482

(3) Charras, G. T., Mitchison, T. J. & Mahadevan, L. Animal cell hydraulics. J Cell Sci 122, 3233-3241 (2009). https://doi.org/10.1242/jcs.049262

(4) Moeendarbary, E. et al. The cytoplasm of living cells behaves as a poroelastic material. Nat Mater 12, 253-261 (2013). https://doi.org/10.1038/nmat3517

(5) Luby-Phelps, K., Castle, P. E., Taylor, D. L. & Lanni, F. Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. Proc Natl Acad Sci U S A 84, 4910-4913 (1987). https://doi.org/10.1073/pnas.84.14.4910

(6) Charras, G. T., Coughlin, M., Mitchison, T. J. & Mahadevan, L. Life and times of a cellular bleb. Biophys J 94, 1836-1853 (2008). https://doi.org/10.1529/biophysj.107.113605

(7) Tinevez, J. Y. et al. Role of cortical tension in bleb growth. Proc Natl Acad Sci U S A 106, 18581-18586 (2009). https://doi.org/10.1073/pnas.0903353106

Why would you have these in the same list? They should be two different lists. It may be confusing to people what actually increases or decreases risk of transmission

(KP) This is how life was back then. Most times, AA don't even get the chance to fight back. Burke saved 2 lives that day

(KP) This was reality for most people of color back then. I believe the Jim Crow car is a metaphor for the struggles and hardships African Americans had to face back then.

eLife Assessment

In flies defective for axonal transport of mitochondria, the authors report the upregulation of one subunit, the beta subunit, of the heterotrimeric eIF2 complex via mass spectroscopy proteomics. Neuronal overexpression of eIF2β phenocopied aspects of neuronal dysfunction observed when axonal transport of mitochondria was compromised. Conversely, lowering eIF2β expression suppressed aspects of neuronal dysfunction. While these are intriguing and useful observations, technical weaknesses limit the interpretation. On balance, the evidence supporting the current claims is suggestive but incomplete, especially concerning the characterization of the eIF2 heterotrimer and the data regarding translational regulation.

Reviewer #1 (Public review):

The study presents significant findings on the role of mitochondrial depletion in axons and its impact on neuronal proteostasis. It effectively demonstrates how the loss of axonal mitochondria and elevated levels of eIF2β contribute to autophagy collapse and neuronal dysfunction. The use of Drosophila as a model organism and comprehensive proteome analysis adds robustness to the findings.

In this revision, the authors have responded thoughtfully to previous concerns. In particular, they have addressed the need for a quantitative analysis of age-dependent changes in eIF2β and eIF2α. By adding western blot data from multiple time points (7 to 63 days), they show that eIF2β levels gradually increase until middle age, then decline. In milton knockdown flies, this pattern appears shifted, supporting the idea that mitochondrial defects may accelerate aging-related molecular changes. These additions clarify the temporal dynamics of eIF2β and improve the overall interpretation.

Other updates include appropriate corrections to figures and quantification methods. The authors have also revised some of their earlier mechanistic claims, presenting a more cautious interpretation of their findings.

Overall, this work provides new insights into how mitochondrial transport defects may influence aging-related proteostasis through eIF2β. The manuscript is now more convincing, and the revisions address the main points raised earlier. I find the updated version much improved.

Reviewer #2 (Public review):

In the manuscript, the authors aimed to elucidate the molecular mechanism that explains neurodegeneration caused by the depletion of axonal mitochondria. In Drosophila, starting with siRNA depletion of milton and Miro, the authors attempted to demonstrate that the depletion of axonal mitochondria induces the defect in autophagy. From proteome analyses, the authors hypothesized that autophagy is impacted by the abundance of eIF2β and the phosphorylation of eIF2α. The authors followed up the proteome analyses by testing the effects of eIF2β overexpression and depletion on autophagy. With the results from those experiments, the authors proposed a novel role of eIF2β in proteostasis that underlies neurodegeneration derived from the depletion of axonal mitochondria, which they suggest accelerates age-dependent changes rather than increasing their magnitude.

Strong caution is necessary regarding the interpretation of translational regulation resulting from the milton KD. The effect of milton KD on translation appears subtle, if present at all, in the puromycin incorporation experiments in both the initial and revised versions. Additionally, the polysome profiling data in the revised manuscript lack the clear resolution for ribosomal subunits, monosomes, and polysomes that is typically expected in publications.

Author response:

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

Reviewer #1 (Public Review):

The authors observed a decline in autophagy and proteasome activity in the context of Milton knockdown. Through proteomic analysis, they identified an increase in the protein levels of eIF2β, subsequently pinpointing a novel interaction within eIF subunits where eIF2β contributes to the reduction of eIF2α phosphorylation levels. Furthermore, they demonstrated that overexpression of eIF2β suppresses autophagy and leads to diminished motor function. It was also shown that in a heterozygous mutant background of eIF2β, Milton knockdown could be rescued. This work represents a novel and significant contribution to the field, revealing for the first time that the loss of mitochondria from axons can lead to impaired autophagy function via eIF2β, potentially influencing the acceleration of aging.

Thank you so much for your review and comments.

Reviewer #2 (Public Review):

In the manuscript, the authors aimed to elucidate the molecular mechanism that explains neurodegeneration caused by the depletion of axonal mitochondria. In Drosophila, starting with siRNA depletion of Milton and Miro, the authors attempted to demonstrate that the depletion of axonal mitochondria induces the defect in autophagy. From proteome analyses, the authors hypothesized that autophagy is impacted by the abundance of eIF2β and the phosphorylation of eIF2α. The authors followed up the proteome analyses by testing the effects of eIF2β overexpression and depletion on autophagy. With the results from those experiments, the authors proposed a novel role of eIF2β in proteostasis that underlies neurodegeneration derived from the depletion of axonal mitochondria.

The manuscript has several weaknesses. The reader should take extra care while reading this manuscript and when acknowledging the findings and the model in this manuscript.

The defect in autophagy by the depletion of axonal mitochondria is one of the main claims in the paper. The authors should work more on describing their results of LC3-II/LC3-I ratio, as there are multiple ways to interpret the LC3 blotting for the autophagy assessment. Lysosomal defects result in the accumulation of LC3-II thus the LC3-II/LC3-I ratio gets higher. On the other hand, the defect in the early steps of autophagosome formation could result in a lower LC3-II/LC3-I ratio. From the results of the actual blotting, the LC3-I abundance is the source of the major difference for all conditions (Milton RNAi and eIF2β overexpression and depletion).

Thank you so much for your review and comments. As the reviewer pointed out, LC3-II/LC3- I ratio changes do not necessarily indicate autophagy defects. However, since p62 accumulation (Figure 2B, 2E, 3E, Figure 8C, Figure 9C), these results collectively suggest that autophagy is lowered.

As the reviewer pointed out and we described in v2, milton knockdown, eIF2β overexpression and heterozygosity increase LC3-I abundance. We do not know how these conditions increase LC3-I at this moment. We will investigate the cause of the increase in LC3-I by milton knockdown and how it contribute to impaired autophagy. We added this discussion as:

Lines 388-393; ‘Our results also suggest that milton knockdown and overexpression of eIF2β affect autophagy via increased LC3-I abundance (Figures 2 and 7), suggesting an unconventional mechanism of autophagy suppression. To our knowledge, the roles of eIF2β in aging and autophagy independent of ISR have not been reported. Our results revealed a novel function of eIF2β to maintain proteostasis during aging, while further investigation is required to elucidate underlying mechanisms.’

Another main point of the paper is the up-regulation of eIF2β by depleting the axonal mitochondria leads to the proteostasis crisis. This claim is formed by the findings from the proteome analyses. The authors should have presented their proteomic data with much thorough presentation and explanation. As in the experiment scheme shown in Figure 4A, the author did two proteome analyses: one from the 7-day-old sample and the other from the 21-day-old sample. The manuscript only shows a plot of the result from the 7-day-old sample, but that of the result from the 21-day-old sample. For the 21-day-old sample, the authors only provided data in the supplemental table, in which the abundance ratio of eIF2β from the 21-day-old sample is 0.753, meaning eIF2β is depleted in the 21-day-old sample. The authors should have explained the impact of the eIF2β depletion in the 21-day-old sample, so the reader could fully understand the authors' interpretation of the role of eIF2β on proteostasis.

Thank you for pointing it out. Plots of the 21-day-old proteome results was included in the main figure (Figure 4C) in v2. In this revision, we further analyzed age-dependent changes of eIF2β levels by western blotting (Figure 4G). We found that eIF2β levels increased during aging until 49-day-old then reduced at 63-day-old (Figure 4G in the revised manuscript). At the young age, eIF2β levels were higher in milton knockdown brain compared to the control , and eIF2β levels were lower in milton knockdown brains than those in the control. These results suggest that milton knockdown accelerates age-dependent changes in eIF2β. We added these results and discussion in the revised manuscript.

Lines 240-243: ‘We also investigated age-dependent changes in eIF2β by western blotting of control flies at 7-, 21-, 35-, and 49-, and 63-day-old. eIF2β levels increased during aging until 49-day-old (Figure 4G). These results suggest that upregulation of eIF2β in milton knockdown fly brain reflects early an onset of age-dependent increase of eIF2β levels.’

Lines 363-368: ‘We also found that eIF2β protein levels increase in an age-dependent manner until 49-day-old and reduces after that (Figure 4G). In the brains with neuronal knockdown of milton, eIF2β levels were higher at 7-day-old than those in control and lower at the 21-day-old (Figure 4D and Supplementary table). These results suggest that milton knockdown is likely accelerating age-dependent changes rather than increasing their magnitude.’Our new data indicate that eIF2β levels increase during aging in control flies until 49-day-old, then reduce at 63-day-old (included as Figure 4G in the revised manuscript). These age- dependent changes might explain the reduction in eIF2β levels in Milton knockdown compared to the control in middle age: higher eIF2β levels in milton knockdown flies at a young age than control and lower eIF2β levels in the middle-aged flies may reflect premature aging.

We included these sentences in the discussion section:

Lines 240-243:‘We also investigated age-dependent changes in eIF2β by western blotting of control flies at 7-, 21-, 35-, and 49-, and 63-day-old. eIF2β levels increased during aging until 49-day-old (Figure 4G). These results suggest that upregulation of eIF2β in milton knockdown fly brain reflects early an onset of age-dependent increase of eIF2β levels.’

Lines 359-371: ‘Our results suggest that the loss of axonal mitochondria is an event upstream of proteostasis collapse during aging. The number of puncta of ubiquitinated proteins was higher in milton knockdown at 14-day-old, but there was no significant difference at 30-day-old (Figure 1). Proteome analyses also showed that age-related pathways, such as immune responses, are enhanced in young flies with milton knockdown (Table 2). We also found that eIF2β protein levels increase in an age-dependent manner until 49-day-old and reduces after that (Figure 4G). In the brains with neuronal knockdown of milton, eIF2β levels were higher at 7-day-old than those in control and lower at the 21-day-old (Figure 4D and Supplementary table). These results suggest that milton knockdown is likely accelerating age-dependent changes rather than increasing their magnitude. Disruption of proteostasis is expected to contribute neurodegeneration38 , and it would be interesting to analyze the sequence of protein accumulation and axonal degeneration in milton knockdown (24,29 and Figure 1) in detail with higher time resolution.’

With our new data, we revised some of our responses to the first round of reviewer’s comments.

Reviewer #1 (Public Review):

The authors observed a decline in autophagy and proteasome activity in the context of Milton knockdown. Through proteomic analysis, they identified an increase in the protein levels of eIF2β, subsequently pinpointing a novel interaction within eIF subunits where eIF2β contributes to the reduction of eIF2α phosphorylation levels. Furthermore, they demonstrated that overexpression of eIF2β suppresses autophagy and leads to diminished motor function. It was also shown that in a heterozygous mutant background of eIF2β, Milton knockdown could be rescued. This work represents a novel and significant contribution to the field, revealing for the first time that the loss of mitochondria from axons can lead to impaired autophagy function via eIF2β, potentially influencing the acceleration of aging. To further support the authors' claims, several improvements are necessary, particularly in the methods of quantification and the points that should be demonstrated quantitatively. It is crucial to investigate the correlation between aging and the proteins eIF2β and eIF2α.

Thank you so much for your review and comments. We included analyses of protein levels of eIF2α, eIF2β, and eIF2γ at 7 days and 21 days (Figure 4D). The manuscript was revised as below;

Lines 246-249 ‘As for the other subunits of eIF2 complex, proteome analysis did not detect a significant difference in the protein levels of eIF2α and eIF2γ between milton knockdown and control flies at 7 and 21 days (Figure 4D).’

NEW TEXT: We analyzed age-dependent changes of eIF2β levels in more detail by western blotting (Figure 4G). We found that eIF2β levels increased during aging until 49-day-old then reduced at 63-day-old (Figure 4G in the revised manuscript). At the young age, eIF2β levels were higher in milton knockdown brain compared to the control , and eIF2β levels were lower in milton knockdown brains than those in the control. These results suggest that Milton knockdown accelerates age-dependent changes in eIF2β.. We added these results and discussion in the revised manuscript.

NEW TEXT: Lines 240-243: ‘We also investigated age-dependent changes in eIF2β by western blotting of control flies at 7-, 21-, 35-, and 49-, and 63-day-old. eIF2β levels increased during aging until 49-day-old (Figure 4G). These results suggest that upregulation of eIF2β in milton knockdown fly brain reflects early an onset of age-dependent increase of eIF2β levels.’

NEW TEXT: Lines 363-368: ‘We also found that eIF2β protein levels increase in an age-dependent manner until 49-day-old and reduces after that (Figure 4G). In the brains with neuronal knockdown of milton, eIF2β levels were higher at 7-day-old than those in control and lower at the 21-day-old (Figure 4D and Supplementary table). These results suggest that milton knockdown is likely accelerating age-dependent changes rather than increasing their magnitude.’

Reviewer #2 (Public Review):

In the manuscript, the authors aimed to elucidate the molecular mechanism that explains neurodegeneration caused by the depletion of axonal mitochondria. In Drosophila, starting with siRNA depletion of Milton and Miro, the authors attempted to demonstrate that the depletion of axonal mitochondria induces the defect in autophagy. From proteome analyses, the authors hypothesized that autophagy is impacted by the abundance of eIF2β and the phosphorylation of eIF2α. The authors followed up the proteome analyses by testing the effects of eIF2β overexpression and depletion on autophagy. With the results from those experiments, the authors proposed a novel role of eIF2β in proteostasis that underlies neurodegeneration derived from the depletion of axonal mitochondria.

The manuscript has several weaknesses. The reader should take extra care while reading this manuscript and when acknowledging the findings and the model in this manuscript.

The defect in autophagy by the depletion of axonal mitochondria is one of the main claims in the paper. The authors should work more on describing their results of LC3-II/LC3-I ratio, as there are multiple ways to interpret the LC3 blotting for the autophagy assessment. Lysosomal defects result in the accumulation of LC3-II thus the LC3-II/LC3-I ratio gets higher. On the other hand, the defect in the early steps of autophagosome formation could result in a lower LC3-II/LC3-I ratio. From the results of the actual blotting, the LC3-I abundance is the source of the major difference for all conditions (Milton RNAi and eIF2β overexpression and depletion). In the text, the authors simply state the observation of their LC3 blotting. The manuscript lacks an explanation of how to evaluate the LC3-II/LC3-I ratio. Also, the manuscript lacks an elaboration on what the results of the LC3 blotting indicate about the state of autophagy by the depletion of axonal mitochondria.

Thank you for pointing it out, and we apologize for an insufficient description of the result. We included quantitation of the levels of LC3-I and LC3-II in Figures 2A, 2D, 3D, 7B (Figure 6B in the previous version), and 8B (Figure 7B in the previous version). As the reviewer pointed out, LC3-II/LC3-I ratio changes do not necessarily indicate autophagy defects. However, since p62 accumulation (Figure 2B, 2E, 3E, 7C (Figure 6C in the previous version), 8C (Figure 7C in the previous version)), these results collectively suggest that autophagy is lowered. We revised the manuscript to include this discussion as below:

Lines 174-186 ‘During autophagy progression, LC3 is conjugated with phosphatidylethanolamine to form LC3-II, which localizes to isolation membranes and autophagosomes. LC3-I accumulation occurs when autophagosome formation is impaired, and LC3-II accumulation is associated with lysosomal defects31,32. p62 is an autophagy substrate, and its accumulation suggests autophagic defects31,32. We found that milton knockdown increased LC3-I, and the LC3-II/LC3-I ratio was lower in milton knockdown flies than in control flies at 14-day-old (Figure 2A). We also analyzed p62 levels in head lysates sequentially extracted using detergents with different stringencies (1% Triton X-100 and 2% SDS). Western blotting revealed that p62 levels were increased in the brains of 14-day-old of milton knockdown flies (Figure 2B). The increase in the p62 level was significant in the Triton X-100- soluble fraction but not in the SDS-soluble fraction (Figure 2B), suggesting that depletion of axonal mitochondria impairs the degradation of less-aggregated proteins.’

Line 189-190: 'At 30 day-old, LC3-I was still higher, and the LC3-II/LC3-I ratio was lower, in milton knockdown compared to the control (Figure 2D).’

Line 202-203: ‘However, in contrast with milton knockdown, Pfk knockdown did not affect the levels of LC3-I, LC3-II or the LC3-II/LC3-I ratio (Figure 3D).’

Line 279-285: ‘Neuronal overexpression of eIF2β increased LC3-II, while the LC3-II/LC3-I ratio was not significantly different (Figure 7A and B). Overexpression of eIF2β significantly increased the p62 level in the Triton X-100-soluble fraction (Figure 7C, 4-fold vs. control, p <0.005 (1% Triton X-100)) but not in the SDS-soluble fraction (Figure 7C, 2-fold vs. control, p\= 0.062 (2% SDS)), as observed in brains of milton knockdown flies (Figure 2B). These data suggest that neuronal overexpression of eIF2β accumulates autophagic substrates.’

Line 311-319: ‘Neuronal knockdown of milton causes accumulation of autophagic substrate p62 in the Triton X-100-soluble fraction (Figure 2B), and we tested if lowering eIF2β ameliorates it. We found that eIF2β heterozygosity caused a mild increase in LC3-I levels and decreases in LC3-II levels, resulting in a significantly lower LC3-II/LC3-I ratio in milton knockdown flies (Figure 8B). eIF2β heterozygosity decreased the p62 level in the Triton X- 100-soluble fraction in the brains of milton knockdown flies (Figure 8C). The p62 level in the SDS-soluble fraction, which is not sensitive to milton knockdown (Figure 2B), was not affected (Figure 8C). These results suggest that suppression of eIF2β ameliorates the impairment of autophagy caused by milton knockdown.’

Another main point of the paper is the up-regulation of eIF2β by depleting the axonal mitochondria leads to the proteostasis crisis. This claim is formed by the findings from the proteome analyses. The authors should have presented their proteomic data with much thorough presentation and explanation. As in the experiment scheme shown in Figure 4A, the author did two proteome analyses: one from the 7-day-old sample and the other from the 21-day-old sample. The manuscript only shows a plot of the result from the 7-day-old sample, but that of the result from the 21-day-old sample. For the 21-day-old sample, the authors only provided data in the supplemental table, in which the abundance ratio of eIF2β from the 21-day-old sample is 0.753, meaning eIF2β is depleted in the 21-day-old sample. The authors should have explained the impact of the eIF2β depletion in the 21-day-old sample, so the reader could fully understand the authors' interpretation of the role of eIF2β on proteostasis.

NEW TEXT: Thank you for pointing it out. We included plots of the 21-day-old proteome results as a part of the main figure (Figure 4C). As the reviewer pointed out, eIF2β protein levels are lower in milton knockdown background at the 21-day-old compared to the control. Since a reduction in the eIF2_β_ ameliorated milton knockdown-induced locomotor defects in aged flies (Figure 7D), the reduction in eIF2β observed in the 21-day-old milton knockdown flies is not likely to negatively contribute to milton knockdown-induced defects. Our new data indicate that eIF2β levels increase during aging in control flies until 49-day-old, then reduce at 63-day-old (included as Figure 4G in the revised manuscript). These age-dependent changes might explain the reduction in eIF2β levels in Milton knockdown compared to the control in middle age: higher eIF2β levels in milton knockdown flies at a young age than control and lower eIF2β levels in the middle-aged flies may reflect premature aging.

NEW TEXT: We included these sentences in the discussion section:

NEW TEXT: Lines 240-243:‘We also investigated age-dependent changes in eIF2β by western blotting of control flies at 7-, 21-, 35-, and 49-, and 63-day-old. eIF2β levels increased during aging until 49-day-old (Figure 4G). These results suggest that upregulation of eIF2β in milton knockdown fly brain reflects early an onset of age-dependent increase of eIF2β levels.’

NEW TEXT: Lines 359-371: ‘Our results suggest that the loss of axonal mitochondria is an event upstream of proteostasis collapse during aging. The number of puncta of ubiquitinated proteins was higher in milton knockdown at 14-day-old, but there was no significant difference at 30-day-old (Figure 1). Proteome analyses also showed that age-related pathways, such as immune responses, are enhanced in young flies with milton knockdown (Table 2). We also found that eIF2β protein levels increase in an age-dependent manner until 49-day-old and reduces after that (Figure 4G). In the brains with neuronal knockdown of milton, eIF2β levels were higher at 7-day-old than those in control and lower at the 21-day-old (Figure 4D and Supplementary table). These results suggest that milton knockdown is likely accelerating age-dependent changes rather than increasing their magnitude. Disruption of proteostasis is expected to contribute neurodegeneration38 , and it would be interesting to analyze the sequence of protein accumulation and axonal degeneration in milton knockdown (24,29 and Figure 1) in detail with higher time resolution.’

The manuscript consists of several weaknesses in its data and explanation regarding translation.

(1) The authors are likely misunderstanding the effect of phosphorylation of eIF2α on translation. The P-eIF2α is inhibitory for translation initiation. However, the authors seem to be mistaken that the down-regulation of P-eIF2α inhibits translation.

We are sorry for our insufficient explanation in the previous version. As the reviewer pointed out, it is well known that the phosphorylated form of eIF2α inhibits translation initiation. Neuronal knockdown of milton caused a reduction in p-eIF2α (Figure 5D and E (Figure 4J and K in the previous version)), and it also lowered translation (Figure 6 (Figure 5 in the previous version)); the relationship between these two events is currently unclear. We do not think that a reduction in the p-eIF2α suppressed translation; rather, we propose that the unbalance of expression levels of the components of eIF2 complexes negatively affects translation. We revised discussion sections to describe our interpretation more in detail as below:

Line 374-384: ‘eIF2β is a component of eIF2, which meditates translational regulation and ISR initiation. When ISR is activated, phosphorylated eIF2α suppresses global translation and induces translation of ATF4, which mediates transcription of autophagy-related genes39,40. Since ISR can positively regulate autophagy, we suspected that suppression of ISR underlies a reduction in autophagic protein degradation. We found neuronal knockdown of milton reduced phosphorylated eIF2α, suggesting that ISR is reduced (Figure 5). However, we also found that global translation was reduced (Figure 6). Increased levels of eIF2β might disrupt the eIF2 complex or alter its functions. The stoichiometric mismatch caused by an imbalance of eIF2 components may inhibit ISR induction. Supporting this model, we found that eIF2β upregulation reduced the levels of p-eIF2α (Figure 7).’We have revised the graphical abstract and removed the eIF2 complex since its role in the loss of proteostasis caused by milton knockdown has not been elucidated yet.

(2) The result of polysome profiling in Figure 4H is implausible. By 10%-25% sucrose density gradient, polysomes are not expected to be observed. The authors should have used a gradient with much denser sucrose, such as 10-50%.

Thank you for pointing it out. It was a mistake of 10-50%, and we apologize for the oversight. It was corrected (Figure 6 (Figure 5 in the previous version)).

(3) Also on the polysome profiling, as in the method section, the authors seemed to fractionate ultra-centrifuged samples from top to bottom and then measured A260 by a plate reader. In that case, the authors should have provided a line plot with individual data points, not the smoothly connected ones in the manuscript.

Thank you for pointing it out. We revised the graph (Figure 6 (Figure 5 in the previous version)).

(4) For both the results from polysome profiling and puromycin incorporation (Figure 4H and I), the difference between control siRNA and Milton siRNA are subtle, if not nonexistent. This might arise from the lack of spatial resolution in their experiment as the authors used head lysate for these data but the ratio of Phospho-eIF2α/eIF2α only changes in the axons, based on their results in Figure 4E-G. The authors could have attempted to capture the spatial resolution for the axonal translation to see the difference between control siRNA and Milton siRNA.

Thank you for your comment. We agree that it would be an interesting experiment, but it will take a considerable amount of time to analyze axonal translation with spatial resolution. We will try to include such analyses in the future. For this manuscript, we revised the discussion section to include the reviewer's suggestion as below;

Lines 355-357: ‘Further analyses to dissect the effects of milton knockdown on proteostasis and translation in the cell body and axon by experiments with spatial resolution would be needed.’

Recommendations for the authors:

From the Reviewing Editor:

As the Reviewing Editor, I have read your manuscript and the associated peer reviews. I have concerns about publishing this work in its current form. I think that your manuscript cannot claim to have found a novel function of eIF2beta because of technical uncertainties and conceptual problems that should be addressed.

Thank you so much for your review and comments. We addressed all the concerns raised by the reviewers. Point-by-point responses are listed below.

First, your manuscript is based partly on what appears to be a mistaken understanding of the mechanistic basis of the ISR. Specifically, eIF2 is a heterotrimeric complex of alpha, beta, and gamma subunits. When eIF2a is phosphorylated, the heterotrimer adopts a new conformation. This conformation directly binds and inhibits eIF2B, the decameric GEF that exchanges the GDP bound to the gamma subunit of the eIF2 complex for GTP. Unless I misunderstood your paper, you seem to propose that decreasing levels of phospho-eIF2a will inhibit translation, but this is backward from what we know about the ISR.

Thank you for your insightful comment, and we are sorry for the confusion. We did not mean to propose that decreasing levels of phospho-eIF2_a_ inhibits translation. We apologize for our insufficient explanation, which might have caused a misunderstanding (Lines 312-318 in the original version). We agree with the reviewer that ‘mismatch due to elevated eIF2-beta could change the behavior of the ISR’. We revised the text in the result section as follows:

Lines 263-268 (in the Result section) ‘Phosphorylation of eIF2α induces conformational changes in the eIF2 complex and inhibits global translation36. To analyze the effects of milton knockdown on translation, we performed polysome gradient centrifugation to examine the level of ribosome binding to mRNA. Since p-eIF2α was downregulated, we hypothesized that milton knockdown would enhance translation. However, unexpectedly, we found that milton knockdown significantly reduced the level of mRNAs associated with polysomes (Figure 6A and B).’

Lines 374-384 (in the Discussion section): ‘eIF2β is a component of eIF2, which meditates translational regulation and ISR initiation. When ISR is activated, phosphorylated eIF2α suppresses global translation and induces translation of ATF4, which mediates transcription of autophagy-related genes39,40. Since ISR can positively regulate autophagy, we suspected that suppression of ISR underlies a reduction in autophagic protein degradation. We found neuronal knockdown of milton reduced phosphorylated eIF2α, suggesting that ISR is reduced (Figure 5). However, we also found that global translation was reduced (Figure 6). Increased levels of eIF2β might disrupt the eIF2 complex or alter its functions. The stoichiometric mismatch caused by an imbalance of eIF2 components may inhibit ISR induction. Supporting this model, we found that eIF2β upregulation reduced the levels of p-eIF2α (Figure 7).’

It may be possible that a stoichiometric mismatch due to elevated eIF2-beta could change the behavior of the ISR, but your paper doesn't adequately address the expression levels of all three eIF2 subunits: alpha, beta, and gamma. The proteomic data shown in Fig 4B is unconvincing on its own because the changes in the beta subunit are subtle. The Western blot in Figure 4C suggests that the KD changes the mass or mobility of the beta subunit, and most importantly, there are no Western blots measuring the levels of eIF2a, eIF2a-phospho, or eIF2-gamma.

We appreciate the reviewer’s comment and agree that the stoichiometric mismatch due to elevated eIF2β may interfere with ISR. We found overexpression of eIF2β lowered p-eIF2 alpha (Figure S2 in V1), which supports this model. We included this data in the main figure in the revised manuscript (Figure 7D) and revised the text as below:

Lines 286-289: ‘Since milton knockdown reduced the p-eIF2α level (Figure 5E), we asked whether an increase in eIF2β affects p-eIF2α. Neuronal overexpression of eIF2β did not affect the eIF2α level but significantly decreased the p-eIF2α level (Figure 7D and E).’

Expression data of eIF2α and eIF2γ from proteomic analyses has been extracted from proteome analyses and included as a table (Figure 4D). Western blots of phospho-eIF2a (Figure S1 in V1) in the main figure (Figure 5B). The result section was revised as below;

Lines 246-249: ‘As for the other subunits of eIF2 complex, proteome analysis did not detect a significant difference in the protein levels of eIF2α and eIF2γ between milton knockdown and control flies at 7 and 21 days (Figure 4D).’

NEW TEXT: We also analyzed age-dependent changes of eIF2β by western blotting and found that eIF2β increased during aging until 49-day-old. We included this result as Figure 4G and added these sentences in the result section:

NEW TEXT: Line 240-243: ‘We also investigated age-dependent changes in eIF2β by western blotting of control flies at 7-, 21-, 35-, and 49-, and 63-day-old. eIF2β levels increased during aging until 49-day-old (Figure 4G). These results suggest that upregulation of eIF2β in milton knockdown fly brain reflects early an onset of age-dependent increase of eIF2β levels.

Reviewer #1 (Recommendations For The Authors):

L125-128: In this section, while the efficiency of Milton knockdown is referenced from a previous publication, it is necessary to also mention that the Miro knockdown has been similarly reported in the literature. Additionally, the Methods section lacks details on the Miro RNAi line used, and Table 2 does not include the genotype for Miro RNAi. This information should be included for clarity and completeness.

Thank you for pointing it out. Knockdown efficiency with this strain has been reported (Iijima- Ando et al., PLoS Genet, 2012). We revised the text to include citation and knockdown efficiency as follows:

Lines 136-147: ‘There was no significant increase in ubiquitinated proteins in milton knockdown flies at 1-day old, suggesting that the accumulation of ubiquitinated proteins caused by milton knockdown is age-dependent (Figure S1). We also analyzed the effect of the neuronal knockdown of Miro, a partner of milton, on the accumulation of ubiquitin-positive proteins. Since severe knockdown of Miro in neurons causes lethality, we used UAS-Miro RNAi strain with low knockdown efficiency, whose expression driven by elav-GAL4 caused 30% reduction of Miro mRNA in head extract24. Although there was a tendency for increased ubiquitin- positive puncta in Miro knockdown brains, the difference was not significant (Figure 1B, p>0.05 between control RNAi and Miro RNAi). These data suggest that the depletion of axonal mitochondria induced by milton knockdown leads to the accumulation of ubiquitinated proteins before neurodegeneration occurs.’

L132-L136: The current phrasing in this section suggests an increase in ubiquitinated proteins for both Milton and Miro knockdowns. However, since there is no significant difference noted for Miro, it is incorrect to state an increase in ubiquitin-positive puncta. Furthermore, combining the results of Milton knockdown to claim an increase in ubiquitinated proteins prior to neurodegeneration is misleading. At the very least, the expression here needs to be moderated to accurately reflect the findings.

Thank you for pointing it out. We revised the text as above.

L137-L141: Results in Figure 1 indicate that Milton knockdown leads to an increase in ubiquitinated proteins at 14 days, while Miro knockdown shows no difference from the control at either 14 or 30 days. Conversely, both the control and Miro exhibit an increase in ubiquitinated proteins with aging, but this trend does not seem to apply to Milton knockdown. This observation suggests that Milton KD may not affect the changes in protein quality control associated with aging. It implies that Milton's function might be more related to protein homeostasis in younger cells, or that changes due to aging might overshadow the effects of Milton knockdown. These interpretations should be included in the Results or Discussion sections for a more comprehensive analysis.

NEW TEXT: Thank you for your insightful comment. As you mentioned, the accumulation of ubiquitinated proteins significantly increases only in young flies. Age-related pathways, such as immune responses, are highlighted in young milton knockdown flies but not in the aged flies. Our new result indicates that eIF2β increases during aging in control flies (included as Figure 4G in the revised manuscript), and upregulation of eIF2β in milton knockdown is only observed at a young age. These results suggest that milton knockdown does not increase the magnitude of age-dependent changes but accelerates their onset. We revised the text to include those points as follows:

NEW TEXT: Lines 152-153: ‘These results suggest that depletion of axonal mitochondria may have more impact on proteostasis in young neurons than in old neurons.’

NEW TEXT: Lines 359-371: ‘Our results suggest that the loss of axonal mitochondria is an event upstream of proteostasis collapse during aging. The number of puncta of ubiquitinated proteins was higher in milton knockdown at 14-day-old, but there was no significant difference at 30-day- old (Figure 1). Proteome analyses also showed that age-related pathways, such as immune responses, are enhanced in young flies with milton knockdown (Table 2). We also found that eIF2β protein levels increase in an age-dependent manner until 49-day-old and reduces after that (Figure 4G). In the brains with neuronal knockdown of milton, eIF2β levels were higher at 7-day-old than those in control and lower at the 21-day-old (Figure 4 and Supplementary table). These results suggest that milton knockdown is likely accelerating age-dependent changes rather than increasing their magnitude. Disruption of proteostasis is expected to contribute neurodegeneration38 , and it would be interesting to analyze the sequence of protein accumulation and axonal degeneration in milton knockdown (24,29 and Figure 1) in detail with higher time resolution.’

L143 : Please remove the erroneously included quotation mark.

Thank you for pointing it out. We corrected it.

L145-L147:

While it is understood that Milton knockdown results in a reduction of mitochondria in axons, as reported previously and seemingly indicated in Figure 1E, this paper repeatedly refers to axonal depletion of mitochondria. Therefore, it would be beneficial to quantitatively assess the number of mitochondria in the axonal terminals located in the lamina via electron microscopy. Such quantification would robustly reinforce the argument that mitochondrial absence in axons is a consequence of Milton knockdown.

Thank you for pointing it out. We included quantitation of the number of mitochondria in the synaptic terminals (Figure 1E).

The text and figure legend was revised accordingly:

Lines 156-157: ‘As previously reported24, the number of mitochondria in presynaptic terminals decreased in milton knockdown (Figure 1E).’

The knockdown of Milton is known to reduce mitochondrial transport from an early stage, but what about swelling? By observing swelling at 1 day and 14 days, it may be possible to confirm the onset of swelling and discuss its correlation with the accumulation of ubiquitinated proteins.

Quantitation of axonal swelling has also been included (Figure 1F).

We appreciate the reviewer's comments on the correlation between the accumulation of ubiquitinated proteins and axonal swelling. Axonal swelling was not observed at 3-days-old (Iijima-Ando et al., PLoS Genetics, 2012), indicating that axonal swelling is an age-dependent event. Dense materials are found in swollen axons more often than in normal axons, suggesting a positive correlation between disruption of proteostasis and axonal damage. It would be interesting to analyze the time course of events further; however, we feel it is beyond the scope of this manuscript. We revised the text to include this discussion as:

Lines 157-160: ‘The swelling of presynaptic terminals, characterized by the enlargement and roundness, was not reported at 3-day-old24 but observed at this age with about 4% of total presynaptic terminals (Figure 1F, asterisks).’

Lines 162-167: ‘Dense materials are rarely found in age-matched control neurons, indicating that milton knockdown induces abnormal protein accumulation in the presynaptic terminals (Figure 1G and H). In milton knockdown neurons, dense materials are found in swollen presynaptic terminals more often than in presynaptic terminals without swelling, suggesting a positive correlation between the disruption of proteostasis and axonal damage (Figure 1G).’

Lines 369-371: ‘Disruption of proteostasis is expected to contribute neurodegeneration38 , and it would be interesting to analyze the sequence of protein accumulation and axonal degeneration in milton knockdown (24,29 and Figure 1) in detail with higher time resolution.’

L147-L151: Though Figures 1F and 1G provide qualitative representations, it is advisable to quantitatively assess whether dense materials significantly accumulate. Such quantitative analysis would be required to verify the accumulation of dense materials in the context of the study.

Thank you for pointing it out. We included quantitation of the number of neurons with dense material (Figure 1G). We revised the manuscript as follows:

Line 162-164: ‘Dense materials are rarely found in age-matched control neurons, indicating that milton knockdown induces abnormal protein accumulation in the presynaptic terminals (Figure 1G and H).’

Regarding Figure 1B, C:

Even though the count of puncta in the whole brain appears to be fewer than 400, the magnification of the optic lobe suggests a substantial presence of puncta. Please clarify in the Methods section what constitutes a puncta and whether the quantification in the whole brain is based on a 2D or 3D analysis. Detail the methodology used for quantification.

Thank you for your comment. We revised the method section to include more details as below:

Lines 440-443: ‘Quantitative analysis was performed using ImageJ (National Institutes of Health) with maximum projection images derived from Z-stack images acquired with same settings. Puncta was identified with mean intensity and area using ImageJ.’

What about 1-day-old specimens? Does Milton knockdown already show an increase in ubiquitinated protein accumulation at this early stage? Investigating whether ubiquitin-protein accumulation is involved in aging promotion or is already prevalent during developmental stages is a necessary experiment.

Thank you for your comment. We carried out immunostaining with an anti-ubiquitin antibody in the brains at 1-day-old. No significant difference was detected between the control and milton knockdown. This result has been included as Figure S1 in the revised manuscript. The result section was revised as below:

Line 136-139 ‘There was no significant increase in ubiquitinated proteins in milton knockdown flies at 1-day old, suggesting that the accumulation of ubiquitinated proteins caused by milton knockdown is age-dependent (Figure S1).’

For Figure 1E: In the Electron Microscopy section of the Methods, define how swollen axons were identified and describe the quantification methodology used.

Thank you for your comment. Swollen axons are, unlike normal axons, round in shape and enlarged. We revised the text as below;

Lines 157-160: ‘The swelling of presynaptic terminals, characterized by the enlargement and roundness, was not reported at 3-day-old24 but observed at this age with about 4% of total presynaptic terminals (Figure 1F, asterisks).’

Lines 689-691, Figure 1 legend: ‘Swollen presynaptic terminals (asterisks in (F)), characterized by the enlargement and higher circularity, were found more frequently in milton knockdown neurons.’

L218-L219: Throughout the text, the expression 'eIF2β is "upregulated" in response to Milton knockdown' is frequently used. However, considering the presented results, it might be more accurate to interpret that under the condition of Milton knockdown, eIF2β is not undergoing degradation but rather remains stable.

Thank you for pointing it out. We replaced ‘upregulated’ with ‘increased’ throughout the text.

L234-L235: On what basis is the conclusion drawn that there is a reduction? Given that three experiments have been conducted, it would be possible and more convincing to quantify the results to determine if there is a significant decrease.

Thank you for pointing it out. We quantified the AUC of polysome fraction and carried out a statistical analysis. There is a significant decrease in polysome in milton knockdown, and this result has been included in Figure 5B. We revised the figure and the legend accordingly.

L236: 5H-> 4H

Thank you for pointing it out, and we are sorry for the confusion. We corrected it.

L238-L239: Since there is no significant difference observed, it may not be accurate to interpret a reduction in puromycin incorporation.

Thank you for pointing it out. As described above, quantification of polysome fractions showed that milton knockdown significantly reduced polysome (Figure 6B (Figure 5B in the previous version)). We revised the manuscript as below;

Lines 267-268: ‘However, unexpectedly, we found that milton knockdown significantly reduced the level of mRNAs associated with polysomes (Figure 6A and B).’

Figure 5D and Figure 6D: Climbing assays have been conducted, but I believe experiments should also be performed to examine whether overexpression or heterozygous mutants of eIF2β induce or suppress degeneration.

Thank you for pointing it out. We analyzed the eyes with eIF2β overexpression for neurodegeneration. Although there was a tendency of elevated neurodegeneration in the retina with eIF2β overexpression, the difference between control and eIF2β overexpression did not reach statistical significance (Figure S2). This result has been included as Figure S2 in the revised manuscript, and the following sentences have been included in the text:

Lines 292-297: ‘We asked if eIF2β overexpression causes neurodegeneration, as depletion of axonal mitochondria in the photoreceptor neurons causes axon degeneration in an age- dependent manner24. eIF2β overexpression in photoreceptor neurons tends to increase neurodegeneration in aged flies, while it was not statistically significant (p>0.05, Figure S2).’

L271-L272: The results in Figure 6B are surprising. I anticipated a greater increase compared to the Milton knockdown alone. While p62 appears to be reduced, it is not clear why these results lead to the conclusion that lowering eIF2β rescues autophagic impairment. Please add a discussion section to address this point.

Thank you for pointing it out. We apologize for the unclear description of the result. Milton knockdown flies show p62 accumulation (Figure 2), and deleting one copy of eIF2beta in milton knockdown background reduced p62 accumulation (Figure 8C (Figure 7C in the previous version)). We revised the text as below:

Lines 311-319: ‘Neuronal knockdown of milton causes accumulation of autophagic substrate p62 in the Triton X-100-soluble fraction (Figure 2B), and we tested if lowering eIF2β ameliorates it. We found that eIF2β heterozygosity caused a mild increase in LC3-I levels and decreases in LC3-II levels, resulting in a significantly lower LC3-II/LC3-I ratio in milton knockdown flies (Figure 8B). eIF2β heterozygosity decreased the p62 level in the Triton X-100-soluble fraction in the brains of milton knockdown flies (Figure 8C). The p62 level in the SDS-soluble fraction, which is not sensitive to milton knockdown (Figure 2B), was not affected (Figure 8C). These results suggest that suppression of eIF2β ameliorates the impairment of autophagy caused by milton knockdown.’

L369: Please specify the source of the anti-ubiquitin antibody used.

Thank you for pointing it out. We included the antibody information in the method section.

Figure 7: While the relationship between Milton knockdown and the eIF2β and eIF2α proteins has been elucidated through the authors' efforts, I would like to see an investigation into whether eIF2β is upregulated and eIF2α phosphorylation is reduced in simply aged Drosophila. This would help us understand the correlation between aging and eIF2 protein dynamics.

Thank you for your comment. We agree that it is an important question, and we are working on it. However, we feel that it is beyond the scope of the current manuscript.

L645-L646: If the mushroom body is identified using mito-GFP, then include mito-GFP in the genotype listed in Supplementary Table 2.

We are sorry for the oversight. We corrected it in Supplementary Table 2.

Additionally, while it is presumed that the mito-GFP signal decreases in axons with Milton RNAi, how was the lobe tips area accurately selected for analysis? Please include these details along with a comprehensive description of the quantification methodology in the Methods section.

Thank you for your comment. Although the mito-GFP signal in the axon is weak in the milton knockdown neurons, it is sufficient to distinguish the mushroom body structure from the background. We revised the method section to include this information in the method section:

Line 443-447: ‘For eIF2α and p-eIF2α immunostaining, the mushroom body was detected by mitoGFP expression.’

acknowledging their concern, but also giving them a reality check, per se. The demonstrations weren't something done of a whim, for pure show, , but something that needed to be done for the good of others.

It provides your audience with you motivation, which, in the long run, can help keep them more engaged as they may also have opinions on the topic you are talking about

most of what we write is in response to something that intrigued us, and not "just because"

You need to "interact" with the other persons argument as well. if not, all you're really doing is talking at them, which can make you lose credibility, no matter how "good" your argument is

States the main goal of the book

The mastery of these moves is often what becomes this writes style and what we as readers learn to recognize. With this, we can draw connection between their works and the works of others.

Reminds me of the fact that I've learned to use the (rather old) analog registers at work so i don't really think much about what buttons im truly pressing when i ring people up, but when someone new covers for me they clearly struggle more

Considering this is one of the first terms that I have understood during this module, I understand where I am starting in this course. However, I am excited to continue learning as I am already finding this very intriguing and a new challenge for me.

eLife Assessment

This study provides valuable results on how entorhinal and hippocampal activity may support human thinking in perceptual spaces. It replicates the hexagonal symmetry of fMRI activity in the entorhinal cortex, reports novel findings on 3-fold symmetry in both behavioral performance and hippocampal fMRI activity, and links these results within a computational model. However, the methods – while potentially creative and interesting – are not fully justified or explained, and the conclusions remain incomplete. With further explanation, justification, and interpretation, this work could represent a significant step forward in understanding how cognitive maps are utilized.

Reviewer #1 (Public review):

Summary:

Zhang and colleagues examine neural representations underlying abstract navigation in the entorhinal cortex (EC) and hippocampus (HC) using fMRI. This paper replicates a previously identified hexagonal modulation of abstract navigation vectors in abstract space in EC in a novel task involving navigating in a conceptual Greeble space. In HC, the authors claim to identify a three-fold signal of the navigation angle. They also use a novel analysis technique (spectral analysis) to look at spatial patterns in these two areas and identify phase coupling between HC and EC. Finally, the authors propose an EC-HPC PhaseSync Model to understand how the EC and HC construct cognitive maps. While the wide array of techniques used is impressive and their creativity in analysis is admirable, overall, I found the paper a bit confusing and unconvincing. I recommend a significant rewrite of their paper to motivate their methods and clarify what they actually did and why. The claim of three-fold modulation in HC, while potentially highly interesting to the community, needs more background to motivate why they did the analysis in the first place, more interpretation as to why this would emerge in biology, and more care taken to consider alternative hypotheses seeped in existing models of HC function. I think this paper does have potential to be interesting and impactful, but I would like to see these issues improved first.

General comments:

(1) Some of the terminology used does not match the terminology used in previous relevant literature (e.g., sinusoidal analysis, 1D directional domain).

(2) Throughout the paper, novel methods and ideas are introduced without adequate explanation (e.g., the spectral analysis and three-fold periodicity of HC).

Reviewer #2 (Public review):

The authors report results from behavioral data, fMRI recordings, and computer simulations during a conceptual navigation task. They report 3-fold symmetry in behavioral and simulated model performance, 3-fold symmetry in hippocampal activity, and 6-fold symmetry in entorhinal activity (all as a function of movement directions in conceptual space). The analyses are thoroughly done, and the results and simulations are very interesting.

Author response:

Reviewer #1, Comment (1): Terminology

We fully acknowledge the importance of terminological consistency and will align our usage with established literature. Specifically, we will revise as follows, 

(1) Replace “sinusoidal analysis” with either “sinusoidal modulation” (Doeller et al., 2010; Bao et al., 2019; Raithel et al., 2023) or “GLM with sinusoidal (cos/sin) regressors” (Constantinescu et al., 2016). 

(2) Replace “1D directional domain” with either “angular domain of movement directions (0–360°)” or “directional modulation analysis”.

Reviewer #1, Comment (2): Spectral analysis and 3-fold periodicity

We agree that the presentation of our spectral analysis and the theoretical motivation underlying our expectation of a three-fold periodicity within hippocampal data requires further clarification.

In our revised manuscript, we will:<br /> (1) Clearly articulate the theoretical motivation for anticipating a three-fold signal, explicitly linking it to the known hexagonal grid structure encoded by the entorhinal cortex.

(2) Clarify our methodological rationale for using Fourier analysis (FFT).

a) FFT allows unbiased exploration of multiple candidate periodicities (e.g., 3–7-fold) without predefined assumptions.

b) FFT results cross-validate our sinusoidal modulation results, providing complementary evidence supporting the 6-fold periodicity in EC and 3-fold periodicity in HPC.

c) FFT uniquely facilitates analysis of periodicities in behavioral performance data, which is not feasible via standard sinusoidal GLM approaches. This consistency allows us to directly compare periodicities across neural and behavioral data.

(3) Further, we will expand our discussion to provide:

a) A deeper interpretation of potential biological bases for the observed hippocampal three-fold periodicity.

b) A careful examination of alternative explanations within existing hippocampal modeling frameworks.

Reference:

Doeller, C. F., Barry, C., & Burgess, N. (2010). Evidence for grid cells in a human memory network. Nature, 463(7281), 657-661.

Constantinescu, A. O., O'Reilly, J. X., & Behrens, T. E. J. (2016). Organizing conceptual knowledge in humans with a gridlike code. Science, 352(6292), 1464-1468.

Bao, X., Gjorgieva, E., Shanahan, L. K., Howard, J. D., Kahnt, T., & Gottfried, J. A. (2019). Grid-like neural representations support olfactory navigation of a two-dimensional odor space. Neuron, 102(5), 1066-1075.

Raithel, C. U., Miller, A. J., Epstein, R. A., Kahnt, T., & Gottfried, J. A. (2023). Recruitment of grid-like responses in human entorhinal and piriform cortices by odor landmark-based navigation. Current Biology, 33(17), 3561-3570

eLife Assessment

By combining the 'pinging' technique with fMRI-based multivariate pattern analysis, this important study provides compelling evidence for a dual-format representation of attention during the preparatory period. The findings help reconcile the debate between sensory-like and non-sensory accounts of attentional templates and shed light on how the brain flexibly deploys different forms of templates to guide attention. This work will be of broad interest to researchers in psychology, vision science, and cognitive neuroscience.

Reviewer #1 (Public review):

Summary:

The aim of the experiment reported in this paper is to examine the nature of the representation of a template of an upcoming target. To this end, participants were presented with compound gratings (consisting of tilted to the right and tilted to the left lines) and were cued to a particular orientation - red left tilt or blue right tilt (counterbalanced across participants). There two directly compared conditions: (i) no ping: where there was a cue, that was followed by a 5.5-7.5s delay, then followed by a target grating in which the cued orientation deviated from the standard 45 degrees; and (ii) ping condition in which all aspects were the same with the only difference that a ping (visual impulse presented for 100ms) was presented after the 2.5 seconds following the cue. There was also a perception task in which only the 45 degrees to the right or to the left lines were presented. It was observed that during the delay, only in the ping condition, were the authors able to decode orientation of the to be reported target using the cross-task generalization. Attention decoding, on the other hand, was decoded in both ping and non-ping conditions. It is concluded that the visual system has two different functional states associated with a template during preparation: a predominantly non-sensory representation for guidance and a latent sensory-like for prospective stimulus processing.

Strengths:

There is so much to be impressed with in this report. The writing of the manuscript is incredibly clear. The experimental design is clever and innovative. The analysis is sophisticated and also innovative -the cross-task decoding, the use of Mahalanobis distance as a function of representational similarity, the fact that the question is theoretically interesting, the excellent figures.

Comments on revisions:

I have no further comments.

Reviewer #3 (Public review):

This paper discusses how non-sensory and latent, sensory-like attentional templates are represented during attentional preparation. Using multivariate pattern analysis, they found that visual impulses can enhance the decoding generalization from perception to attention tasks in the preparatory stage in the visual cortex. Furthermore, the emergence of the sensory-like template coincided with enhanced information connectivity between V1 and frontoparietal areas and was associated with improved behavioral performance. It is an interesting paper with supporting evidence for the latent, sensory-like attentional template.

Comments on revisions:

I appreciate the authors' thoughtful revisions, which have addressed my earlier concerns. I have no further comments.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

I am impressed with the thoroughness with which the authors addressed my concerns. I don't have any further concerns and think that this paper makes an interesting and significant contribution to our understanding of VWM. I would only suggest adding citations to the newly added paragraph where the authors state "It could be argued that preparatory attention relies on the same mechanisms as working memory maintenance." They could cite work by Bettencourt and Xu, 2016; and Sheremata, Somers, and Shomstein (2018).

We thank the reviewer for the positive feedback. We have now cited the referenced work in the manuscript (Page. 19, Line 371).

Reviewer #2 (Public review):

Overall, I think that the authors' revision has addressed most, if not all, of my major concerns noted in my previous comments. The results appear convincing and I do not have additional comments.

We thank the reviewer for the positive feedback and are pleased that the revision addressed the major concerns.

Reviewer #3 (Public review):

(1) The authors addressed most of my previous concerns and provided additional data analysis. They conducted further analyses to demonstrate that the observed changes in network communication are associated with behavioral RTs, supporting the idea that the impulse-driven sensory-like template enhances informational connectivity between sensory and frontoparietal areas, and relates to behavior.

We are pleased that the revision addressed the major concerns.

(2) I would like to further clarify my previous points regarding the definition of the two types of templates and the evidence for their coexistence. The authors stated that the sensory-like template likely existed in a latent state and was reactivated by visual pings, proposing that sensory and non-sensory templates coexist. However, it remains unclear whether this reflects a dynamic switch between formats or true coexistence. If the templates are non-sensory in nature, what exactly do they represent? Are they meant to be abstract or conceptual representations, or, put simply, just "top-down attentional information"? If so, why did the generalization analysestraining classifiers on activity during the stimulus selection period and testing on preparatory activity-fail to yield significant results? While the stimulus selection period necessarily encodes both target and distractor information, it should still contain attentional information. I would appreciate more discussion from this perspective.

We thank the reviewer for the helpful clarification of previous comments. Since we addressed similar comments from Reviewer 2 (Point 2) in the previous round, our response below may appear somewhat repetitive. First, regarding whether our findings reflect a dynamic switch between non-sensory and sensory-like template, or the ‘coexistence’ of two template formats, we acknowledge that the temporal limitations of fMRI prevent us from directly testing dynamic representations. However, several aspects of our data favor the latter interpretation: (1) our key findings remained consistent in the subset of participants (N=14) who completed both No-Ping and Ping sessions in counterbalanced order. This makes it unlikely that participants systematically switched cognitive strategies (e.g., using non-sensory templates in the No-Ping session versus sensory-like templates in the Ping session) in response to the taskirrelevant, uninformative visual impulse; (2) while we agree that the temporal dynamics between the two templates remain unclear, it is difficult to imagine that orientation-specific templates observed in the Ping session emerged de novo from purely non-sensory templates and an exogenous ping. In other words, if there is no orientation information at all to begin with, how does it come into being from an orientation-less external ping? A more parsimonious explanation is that orientation information was already present in a latent format and was activated by the ping, in line with the models of “activity-silent” working memory. However, since the detailed circuit-level mechanism underlying such reactivation remain unclear, we acknowledge that this interpretation warrants direct investigation in future studies. This point is discussed in the main texts (Page 19-20, Line 389-402). 

Second, while our data cannot definitively determine the nature of the non-sensory template, we consider categorical coding a plausible candidate based on prior visual search studies. For instance, categorical attributes (e.g., left-tilted vs. right-tilted) have been shown to effectively guide attention in orientation search tasks (Wolfe et al., 1992), similar to our paradigm. Further, categorical templates are more tolerant of stimulus variability, making them well-suited to our task, which involved trial-by-trial variations in target orientation around a reference (see Page 21, Line 427- 437 for more detailed discussions).

Third, the lack of generalization from stimulus selection to preparatory attention in the Ping session may relate to the limited overlap in shared information between these two periods. Neural activity during stimulus selection encodes sensory information about both orientations, along with sensory-like attentional signals (as indicated by the attention decoding and crosstask generalization from perception task to the stimulus-selection period). In contrast, preparatory activity likely involves a dominant non-sensory template, a latent sensory-like template, and residual sensory effects from the impulse stimulus. The limited overlap in sensory-like attentional signals may therefore be insufficient to support generalization across the two periods.

Reviewer #2 ( Recommendations for the authors)

I think the central prediction of greater pattern similarity between 'attend leftward' and 'perceived leftward' in the ping session in comparison to the no-ping session (the same also holds for 'attend rightward' and 'perceived rightward' could be directly examined by a two-way ANOVA (session × the attend orientation is the same/different from the perceived orientation) for each ROI (V1 and EVC). A three-way ANOVA might complicate readers' intuitive understanding of the implications of the statistical results.

We thank the reviewer for the suggestion. Following the reviewer’s suggestion, we defined a new condition label based on orientation consistency between attended and perceived orientations: (1) same orientation: averaging “attend leftward/perceive leftward” and “attend rightward/perceive rightward”; and (2) different orientation: averaging “attend leftward/perceive rightward” and “attend rightward/perceive leftward”. A two-way mixed ANOVA (session × orientation consistency) on Mahalanobis distance revealed a main effect of orientation consistency in V1 (F(1,38) = 4.21, p = 0.047, η_p² = 0.100), indicating that activity patterns were more similar when attended and perceived orientations matched. No significant main effect of session was found (p = 0.923). Importantly, a significant interaction was found in V1 (F(1,38) = 5.00, p = 0.031, η_p² = 0.116), suggesting that visual impulse enhanced the similarity between preparatory attentional template and the perception of corresponding orientation. In EVC, the same analysis revealed only a main effect of orientation consistency (F(1,38) = 5.87, p = 0.020, η_p² = 0.134), with no significant other effects (ps > 0.240). The interaction results were consistent with those reported in the original three-way ANOVA. We have now replaced the previous analysis with the new one in the main texts (Page 11-12, Line 231-242).

eLife Assessment

The article presents important findings on the impact of climate change on odonates, integrating phenological and range shifts to broaden our understanding of biodiversity change. The study leverages extensive natural history data, offering a convincing analysis of temporal trends in phenology and range limit and their potential drivers.

Reviewer #1 (Public review):

Summary:

This study evaluates whether species can shift geographically, temporally, or both ways in response to climate change. It also teases out the relative importance of geographic context, temperature variability, and functional traits in predicting the shifts. The study system is large occurrence datasets for dragonflies and damselflies split between two time periods and two continents. Results indicate that more species exhibited both shifts than one or the other (or neither), and that geographic context and temperature variability were more influential than traits. The results have implications for future analyses (e.g. incorporating habitat availability) and for choosing winner and loser species under climate change. The results also seem to support climate vulnerability assessments for species that rely on geographic range size and geospatial climate data layers rather than more detailed information (like demographic rates, abundances, or traits) that may not be so readily available. The methodology would be useful for other taxa and study regions with strong participatory ("citizen") science and extensive occurrence data.

Strengths:

This is an organized and well written paper that builds on a popular topic and moves it forward. It has the right idea and approach, and the results are useful answers to the predictions and for conservation planning (i.e. identifying climate winners and losers). There is technical proficiency and analytical rigor driven by an understanding of the data and its limitations.

Reviewer #2 (Public review):

Summary:

This paper explores a highly interesting question regarding how species migration success relates to phenology shifts, and it finds a positive relationship. The findings are significant, and the strength of the evidence is solid. However, there are substantial issues with the writing, presentation, and analyses that need to be addressed. First, I disagree with the conclusion that species that don't migrate are "losers" - some species might not migrate simply because they have broad climatic niches and are less sensitive to climate change. Second, the results concerning species' southern range limits could provide valuable insights. These could be used to assess whether sampling bias has influenced the results. If species are truly migrating, we should observe northward shifts in their southern range limits. However, if this is an artifact of increased sampling over time, we would expect broader distributions both north and south. Finally, Figure 1 is missed panel B, which needs to be addressed.

Comments on revised version:

The revision has substantially improved the paper.

Reviewer #3 (Public review):

Summary:

In their article "Range geography and temperature variability explain cross-continental convergence in range and phenology shifts in a model insect taxon" the authors rigorously investigate the spatial and temporal trends in the occurrence of odonate species and their potential drivers. Specifically, they examine whether species shift their geographic ranges poleward or alter their phenology to cope with changing conditions. Leveraging opportunistic observations of European and North American odonates, they find that species showing significant range shifts also exhibited shifts to earlier emergence. Considering a broad range of potential predictors, their results reveal that geographical factors, but not functional traits, are associated with these shifts.

Strengths:

The article addresses an important topic in ecology and conservation that is particularly timely in the face of reports of substantial insects declines in North America and Europe over the past decades. Through data integration the authors leverage the rich natural history record for odonates, broadening the taxonomic scope of analyses of temporal trends in phenology and distribution. The combination of phenological and range shifts in one framework presents an elegant way to reconcile previous findings and informs about the drivers of biodiversity loss.

Weaknesses:

To better understand whether species shifting both their ranges and phenology are more successful, or as stated here are 'clear winners', and hence whether those that do neither are more vulnerable would require integrating population trends alongside the discussed response. The ~10% species that have not shifted their distribution or phenology might have not declined in abundance, if they have rapidly adapted to local changes in climatic conditions (i.e. they might show a plastic response). These species might be the real 'winners', while species that have recently shifted their ranges or phenology may eventually reach hard limits. The authors are discussing this limitation but might want to adapt their wording, given the potential for misinterpretation. The finding that species with more northern ranges showed lesser northward shifts would speak to the fact that some species have already reached such a geographical range limit.

Achievements and impact:

The results support broad differences in the response of odonate species to climate change, and the prediction that range geography and temperature seasonality are more important predictors of these changes than functional traits. Simultaneously addressing range and phenological shifts highlights that most species exhibit coupled responses but also identifies a significant portion of species that do not respond in these ways that are of critical conservation concern. These results are important for improving forecasts of species' responses to climate change and identifying species of particularly conservation concern. Although not exhaustive regarding abundance trends, the study presents an important step towards a general framework for investigating the drivers of multifaceted species responses.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #2 (Recommendations for the authors):

Line 364-370: This paragraph is not very clear to me.

Thank you for pointing this out, we agree our point could have been made clearer. We have clarified as follows:

“The geographic positions of species’ ranges determine the local pressures and environmental factors to which they are exposed (MacLean and Beissinger, 2017; Pacifici et al., 2020), potentially masking or confounding the effects of traits that evolved under conditions determined by range geography (Schuetz et al., 2019). This process could cause trait-related trends to differ across levels of biological organization (Srivastava et al., 2021), from local populations (where traits might be critical) to biogeographical extents (where traits might be unrelated to range or phenological shifts; Grewe et al., 2013; Gutiérrez and Wilson, 2021; Sunday et al., 2015; Zografou et al., 2021).” (Lines 370-377).

Reviewer #3 (Recommendations for the authors):

L313: '...higher population growth' compared to what? Does this mean that species shifting to earlier emergence really show higher population growth over time?

Thank you for this suggestion, we have clarified as follows: “Earlier seasonal timing allows species to stay within their climatic limits and maintain population growth rates (Macgregor et al., 2019), although earlier emergence could expose individuals to early season weather extremes (McCauley et al., 2018).” (Lines 316-319).

L336: Same here. Please refer to your comparative counterpart in such statements. Does 'plasticity may enable higher population growth' mean higher than for species shifting range or phenology or higher compared to the previous level for a given species. In many cases it seems you are referring to an overall baseline, so that the 'higher' means 'lesser decline'. Wouldn't plasticity maintain population growth at similar levels as before? The current wording suggests that plasticity results in species exceeding their previous population growth. Please rephrase.

We agree it was confusing with no comparative counterpart, so we changed the sentence as follows: “Adaptive evolution and plasticity may enable high population growth rates in newly-colonized areas (Angert et al., 2020; Usui et al., 2023), but this possibility can only be directly tested with long term population trend data.” (Lines 341-343).

L307: The term 'universal winners' appears too strong and not well justified given the lack of the crucial third dimension of response. In fact, changes in phenology are less indicative than abundance trends. Combined with range shifts they would tell a story of success or failing, while phenological shifts would rather help to understand how species adapted. I am not saying the insight cannot stand alone, but it is important to adapt the wording in this regard.

Thank you for this comment, we have clarified the text as follows: “These results suggest that some species may have an advantage with respect to climate change: they demonstrate the flexibility to respond both temporally and spatially to the onset of rapid climate change.” (Lines 310-313).

We also softened language around winners and losers on line 388: “It remains unclear if range and phenology shifts relate to trends in abundance, but our results suggest that there may be ‘winners’ and ‘losers’ under climate change (Figure 4).” (Lines 387-388).

L326-240: I agree with line 330 that abundance trends are needed to clarify the situation of species shifting or not shifting ranges and phenology. However, this abstract should clarify that this is particularly important to understand whether non shifting species are really the 'losers'. If these species show adapted evolution or plasticity, we would expect they do not decline in abundance. Even without shifts in range or phenology they would be the 'ultimate winners' as you call it.

Thank you for this comment, we agree that abundance trends are necessary to understand potential winners and losers. We have made this addition to the abstract as follows: “Species shifting in both space and time may be more resilient to extreme conditions, although further work integrating abundance data is needed.” (Lines 16-18).

eLife assessment

This paper provides valuable insights into the consequences of hydrogen sulfide (H2S) exposure on the behavior and physiology of the nematode C. elegans. While solid evidence supports most of the paper's findings, the evidence that H2S is detected by the nervous system to mediate behavioral avoidance is incomplete. The paper provides a wide range of intriguing observations that could serve as a foundation for future work to synthesize these disparate results or provide insight into the mechanisms of H2S detection in C. elegans.

Reviewer #3 (Public review):

Summary:

The manuscript explores behavioral responses of C. elegans to hydrogen sulfide, which is known to exert remarkable effects on animal physiology in a range of contexts. The possibility of genetic and precise neuronal dissection of responses to H2S motivates the study of responses in C. elegans. The revised manuscript does not seem to have significantly addressed what was lacking in the initial version.

The authors have added further characterization of possible ASJ sensing of H2S by calcium imaging but ASJ does not appear to be directly involved. Genetic and parallel analysis of O2 and CO2 responsive pathways do not reveal further insights regarding potential mechanisms underlying H2S sensing. Gene expression analysis extends prior work. Finally, the authors have examined how H2S-evoked locomotory behavioral responses are affected in mutants with altered stress and detoxification response to H2S, most notably hif-1 and egl-9. These data, while examining locomotion, are more suggestive that observed effects on animal locomotion are secondary to altered organismal toxicity as opposed to specific behavioral responedse

Overall, the manuscript provides a wide range of intriguing observations, but mechanistic insight or a synthesis of disparate data is lacking.

Reviewer #4 (Public review):

Summary:

The authors establish a behavioral paradigm for avoidance of H2S and conduct a large candidate screen to identify genetic requirements. They follow up by genetically dissecting a large number of implicated pathways - insulin, TGF-beta, oxygen/HIF-1, and mitochondrial ROS, which have varied effects on H2S avoidance. They additionally assay whole-animal gene expression changes induced by varying concentrations and durations of H2S exposure.

Strengths:

The implicated pathways are tested extensively through mutants of multiple pathway molecules. The authors address previous reviewer concerns by directly testing the ability of ASJ to respond to H2S via calcium imaging. This allows the authors to revise their previous conclusion and determine that ASJ does not directly respond to H2S and likely does not initiate the behavioral response.

Weaknesses:

Despite the authors focus on acute perception of H2S, I don't think the experiments tell us much about perception. I think they indicate pathways that modulate the behavior when disrupted, especially because most manipulations used broadly affect physiology on long timescales. For instance, genetic manipulation of ASJ signaling, oxygen sensing, HIF-1 signaling, mitochondrial function, as well as starvation are all expected to constitutively alter animal physiology, which could indirectly modulate responses to H2S. The authors rule out effects on general locomotion in some cases, but other physiological changes could relatively specifically modulate the H2S response without being involved in its perception.

I am actually not convinced that H2S is directly perceived by the C. elegans nervous system at all. As far as I can tell, the avoidance behavior could be a response to H2S-induced tissue damage rather than the gas itself.

Author response:

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

Reviewer #1 (Public Review): 

Summary: 

This paper sets out to achieve a deeper understanding of the effects of hydrogen sulfide on C. elegans behavior and physiology, with a focus on behavior, detection mechanism(s), physiological responses, and detoxification mechanisms.

Strengths: 

The paper takes full advantage of the experimental tractability of C. elegans, with thorough, welldesigned genetic analyses. 

Some evidence suggests that H<SUB>2</SUB>S may be directly detected by the ASJ sensory neurons.  The paper provides interesting and convincing evidence for complex interactions between responses to different gaseous stimuli, particularly an antagonistic role between H<SUB>2</SUB>S and O2 detection/response.  Intriguing roles for mitochondria and iron homeostasis are identified, opening the door to future studies to better understand the roles of these components and processes. 

We thank the reviewer for the supportive comments.

Weaknesses: 

The claim that worms' behavioral responses to H<SUB>2</SUB>S are mediated by direct detection is incompletely supported. While a role for the chemosensory neuron ASJ is implicated, it remains unclear whether this reflects direct detection. Other possibilities, including indirect effects of ASJ and the guanylyl cyclase daf-11 on O2 responses, are also consistent with the authors' data. 

We thank the reviewer for the insightful comment and agree that the role of ASJ neurons in H<SUB>2</SUB>S detection was not clear. We included new experiments and revised our text to make it clearer.

Since our initial analyses suggest a role of ASJ neurons in H<SUB>2</SUB>S-evoked locomotory responses (Figure 2F and G), We thought that this would offer us a starting point to dissect the neuronal circuit involved in H<SUB>2</SUB>S responses. Expression of the tetanus toxin catalytic domain in ASJ, which blocks neurosecretion, inhibited H<SUB>2</SUB>S-evoked locomotory speed responses (Figure 2H), suggesting that neurosecretion from ASJ promotes H<SUB>2</SUB>S-evocked response (Lines 162–165). We then performed calcium imaging of ASJ neurons in response to H<SUB>2</SUB>S exposure. However, while we observed CO<SUB>2</SUB>-evoked calcium transients in ASJ using GCaMP6s, we did not detect any calcium response to H<SUB>2</SUB>S, under several conditions, including animals on food, off food, and with different H<SUB>2</SUB>S concentrations and exposure times (Figure2—Figure supplement 2E and F) (Lines 166–170). Since signaling from ASJ neurons regulates developmental programs that modify sensory functions in C. elegans (Murakami et al., 2001), the involvement of ASJ neurons is not specific to H<SUB>2</SUB>S and ASJ neurons are unlikely to serve as the primary H<SUB>2</SUB>S sensor (Discussed in Line 449–458). Therefore, the exact sensory neuron, circuit and molecular triggers mediating acute H<SUB>2</SUB>S avoidance remain to be elucidated.

Our subsequent investigation on mitochondrial components suggests that a burst of mitochondrial ROS production may be the trigger for H<SUB>2</SUB>S avoidance, as transient exposure to rotenone substantially increases baseline locomotory speed (Figure 7E) (Line 391–396). However, to initiate avoidance behavior to H<SUB>2</SUB>S, mitochondrial ROS could potentially target multiple neurons and cellular machineries, making it challenging to pinpoint specific sites of action. Nevertheless, we agree that further dissection of the neural circuits and mitochondrial signaling in H<SUB>2</SUB>S avoidance will be important and should be explored in future studies.

The role of H<SUB>2</SUB>S-mediated damage in behavioral responses, particularly when detoxification pathways are disrupted, remains unclear. 

We thank the reviewer for the insightful comment and fully agree with the concern raised. The same issue was also noted by the other reviewers. We agree that decreased locomotory responses in H<SUB>2</SUB>S-sensitized animals can arise from distinct causes, either systemic toxicity or behavioral adaptation, and distinguishing between these is critical. We have included new experiments and revised the text to clarify this issue.

Our data suggest that increased initial omega turns and a rapid loss of locomotion in hif-1 and detoxification-defective mutants including sqrd-1 and ethe-1 likely reflect an enhanced sensitivity to H<SUB>2</SUB>S toxicity due to their failure to induce appropriate adaptative responses (Figure 5D–F, Figure 5J–L, Figure 5—Figure supplement 1F–P).  Supporting this, hif-1 mutants become less responsive to unrelated stimuli (near-UV light) after 30 minutes of H<SUB>2</SUB>S exposure (Figure 5I).

In contrast, egl-9 and SOD-deficient animals show reduced initial omega-turn and reduced speed responses (Figure 5B, Figure 7G, Figure 5—Figure supplement 1A and B, and Figure 7—Figure supplement 1F and G), although both egl-9 and sod mutants respond normally to the other stimuli prior or after H<SUB>2</SUB>S exposure (Figure 5I, Figure 5—Figure supplement 1C, and Figure 7—Figure supplement 1H). Since disrupting egl-9 stabilizes HIF-1 and upregulates the expression of numerous genes involved in cellular defense against H<SUB>2</SUB>S toxicity, the enhanced detoxification capacity in egl-9 mutants likely increases animals’ tolerance to H<SUB>2</SUB>S, thereby reducing avoidance to otherwise toxic H<SUB>2</SUB>S levels. Similarly, persistently high ROS in SOD deficient animals activates a variety of stress-responsive signaling pathways, including HIF-1, NRF2/SKN-1 and DAF-16/ FOXO signaling (Lennicke & Cocheme, 2021; Patten et al., 2010), facilitating cellular adaptation to redox stress and reducing animals’ responsiveness to toxic H<SUB>2</SUB>S levels. Taken together, these findings support the view that reduced locomotory speed during H<SUB>2</SUB>S exposure can arise from distinct mechanisms: early systemic toxicity in hif-1 and detoxificationdefective mutants, versus enhanced cellular adaptation in egl-9 and SOD mutants. We have integrated the relevant information across the result section and discussed this in Lines 485-536. 

The findings of the paper are somewhat disjointed, such that a clear picture of the relationships between H<SUB>2</SUB>S detection, detoxification mechanisms, mitochondria, and iron does not emerge from these studies. Most importantly, the relative roles of H<SUB>2</SUB>S detection and integration, vs. general and acute mitochondrial crisis, in generating behavioral responses are not convincingly resolved.  

We thank the reviewer for this comment and agree that our presentation did not fully connect different findings into a cohesive picture. To address this, we have acquired new data, and revised the abstract, results and discussion sections to clarify two phases of H<SUB>2</SUB>S-evoked responses: an initial avoidance behavior upon H<SUB>2</SUB>S exposure, followed by a later phase of adaption and detoxification when the escape is not successful.

In brief, we began with the basic characterization of H<SUB>2</SUB>S-induced locomotory speed response, followed by a candidate gene screen to identify key molecules and pathways involved in initial speed response to H<SUB>2</SUB>S. Subsequently, we focused on three major intersecting pathways that contributed to the acute behavioral response to H<SUB>2</SUB>S. These include cGMP signaling, which led to the identification of ASJ neurons; nutrient-sensitive pathways that modulate behavioral responses to both H<SUB>2</SUB>S and CO2; and O2sensing signaling, whose activation inhibits responses to H<SUB>2</SUB>S. However, the molecules and neurons in these pathways, including ASJ, likely play modulatory roles and are unlikely to serve as the primary H<SUB>2</SUB>S sensors. Our subsequent analysis, however, suggests that mitochondria play a critical role in triggering avoidance behavior upon H<SUB>2</SUB>S exposure. Brief treatment with rotenone, a potent inducer of ROS, led to marked increase in locomotory speed (Figure 7E). This suggests the possibility that a burst of ROS production triggered toxic levels of H<SUB>2</SUB>S (Jia et al., 2020) may initiate the avoidance behavior.

When the initial avoidance fails, H<SUB>2</SUB>S detoxification programs are induced as a long-term survival strategy. The induction of detoxification programs appears to enhance tolerance to H<SUB>2</SUB>S exposure and contributes to the gradual decrease of locomotory speed in H<SUB>2</SUB>S. We now provide a clearer image of how different pathways modulate H<SUB>2</SUB>S detoxification and adaptation (see our responses to other comments). Briefly, mutants defective in detoxification, such as hif-1 and other detoxification-defective mutants, showed stronger initial omega-turn response and a rapid loss of locomotion. This loss of locomotion is likely caused by early cellular toxicity as the mutants failed to respond to other unrelated stimuli (nearUV light) after 30 minutes of H<SUB>2</SUB>S exposure (Figure 5I). Likewise, smf-3 mutants and BP-treated animals were hypersensitive to H<SUB>2</SUB>S (Figure 6D and E, and Figure 6—Figure supplement 1G and I), likely due to impaired H<SUB>2</SUB>S detoxification under low iron conditions, as iron is a co-factor required for the activity of the H<SUB>2</SUB>S detoxification enzyme ETHE-1 (Figure 5K and Figure 5—Figure supplement 1E).

In contrast, reduced locomotion and response in other contexts such as egl-9 mutants and SODdeficient animals reflect H<SUB>2</SUB>S-induced adaptive mechanism rather than toxicity as they remain responsive to the other stimuli after H<SUB>2</SUB>S exposure. Since disrupting egl-9 stabilizes HIF-1 and upregulates the expression of numerous genes involved in cellular defense against H<SUB>2</SUB>S toxicity, the enhanced detoxification capacity in egl-9 mutants likely increases animals’ tolerance to H<SUB>2</SUB>S, thereby reducing avoidance to otherwise toxic H<SUB>2</SUB>S levels. Similarly, persistently high ROS in SOD deficient animals activates a variety of stress-responsive signaling pathways, including HIF-1, NRF2/SKN-1 and DAF-16/ FOXO signaling (Lennicke & Cocheme, 2021; Patten et al., 2010), facilitating cellular adaptation to redox stress and reducing animals’ responsiveness to toxic H<SUB>2</SUB>S levels. Therefore, different animals decline their locomotory speed to the effects of H<SUB>2</SUB>S through distinct mechanisms. We have integrated the relevant information across the result section and discussed this in Lines 485-536.

Reviewer #2 (Public Review): 

Summary: 

H<SUB>2</SUB>S is a gas that is toxic to many animals and causes avoidance in animals such as C. elegans. The authors show that H<SUB>2</SUB>S increases the frequency of turning and the speed of locomotion. The response was shown to be modulated by a number of neurons and signaling pathways as well as by ambient oxygen concentrations. The long-term adaptation involved gene expression changes that may be related to iron homeostasis as well as the homeostasis of mitochondria. 

Strengths: 

Overall, the authors provide many pieces that will be important for solving how H<SUB>2</SUB>S signals through neuronal circuits to change gene expression and physiological programs. The experiments rely mostly on a behavioral assay that measures the increase of locomotion speed upon exposure to H<SUB>2</SUB>S. This assay is then combined with manipulations of environmental factors, different wild-type strains, and mutants. The mutants analyzed were obtained as candidates from the literature and from transcriptional profiling that the authors carried out in worms that were exposed to H<SUB>2</SUB>S. These studies imply several genetic signaling pathways, some neurons, and metabolism-related factors in the response to H<SUB>2</SUB>S. Hence the data provided should be useful for the field.  

We thank the reviewer for the supportive comments.

Weaknesses: 

On the other hand, many important aspects of the underlying mechanisms remain unsolved and the reader is left with many loose ends. For example, it is not clear how H<SUB>2</SUB>S is actually sensed, how sensory neurons are activated and signal to downstream circuits, and what the role of ciliated and RMG neurons is in this circuit. It remains unclear how signals lead to gene expression and physiological changes such as metabolic rewiring. Solving all this would clearly be beyond the scope of a single manuscript. Yet, the manuscript also does not focus on understanding one of these central aspects and rather is all over the place, which makes it harder to understand for readouts that are not in this core field. Multiple additional methods and approaches exist to dig deeper into these mechanisms in the future, such as neuronal calcium imaging, optogenetics, and metabolic analysis. To generate a story that will be interesting to a broad readership substantial additional experimentation would be required. Further, in the current manuscript, it is often difficult to understand the rationales of the experiments, why they were carried out, and how to place them into a context. This could be improved in terms of documentation, narration/explanation, and visualization.  

We thank the reviewer for the comment, which has also been raised by the other reviewers. We agree that our initial submission was poorly presented. We also acknowledge the fact that some aspects, such as detailed neural circuit and sensory transduction, still remain unresolved. We have now included additional experiments and revised the manuscript to clarify the logic of our experiments, provided better context for our findings, and improved both the narrative flow and data visualization to make the manuscript more accessible to readers. We now provide a clearer image of how different pathways interact to modulate the initial avoidance response, and the H<SUB>2</SUB>S detoxification and behavioral habituation during prolonged H<SUB>2</SUB>S exposure. The following response is similar to the one for reviewer #1.

In brief, we began with the basic characterization of H<SUB>2</SUB>S-induced locomotory speed response, followed by a candidate gene screen to identify key molecules and pathways involved in initial speed response to H<SUB>2</SUB>S. Subsequently, we focused on three major intersecting pathways that contributed to the acute behavioral response to H<SUB>2</SUB>S. These include cGMP signaling, which led to the identification of ASJ neurons; nutrient-sensitive pathways that modulate behavioral responses to both H<SUB>2</SUB>S and CO2; and O2sensing signaling, whose activation inhibits responses to H<SUB>2</SUB>S. However, the molecules and neurons in these pathways, including ASJ, likely play modulatory roles and are unlikely to serve as the primary H<SUB>2</SUB>S sensors. Our subsequent analysis, however, suggests that mitochondria play a critical role in triggering avoidance behavior upon H<SUB>2</SUB>S exposure. Brief treatment with rotenone, a potent inducer of ROS, led to marked increase in locomotory speed (Figure 7E). This suggests the possibility that a burst of ROS production triggered toxic levels of H<SUB>2</SUB>S (Jia et al., 2020) may initiate the avoidance behavior.

When the initial avoidance fails, H<SUB>2</SUB>S detoxification programs are induced as a long-term survival strategy. The induction of detoxification programs appears to enhance tolerance to H<SUB>2</SUB>S exposure and contributes to the gradual decrease of locomotory speed in H<SUB>2</SUB>S. We now provide a clearer image of how different pathways modulate H<SUB>2</SUB>S detoxification and adaptation (see our responses to other comments). Briefly, mutants defective in detoxification, such as hif-1 and other detoxification-defective mutants, showed stronger initial omega-turn response and a rapid loss of locomotion. This loss of locomotion is likely caused by early cellular toxicity as the mutants failed to respond to other unrelated stimuli (nearUV light) after 30 minutes of H<SUB>2</SUB>S exposure (Figure 5I). Likewise, smf-3 mutants and BP-treated animals were hypersensitive to H<SUB>2</SUB>S (Figure 6D and E, and Figure 6—Figure supplement 1G and I), likely due to impaired H<SUB>2</SUB>S detoxification under low iron conditions, as iron is a co-factor required for the activity of the H<SUB>2</SUB>S detoxification enzyme ETHE-1 (Figure 5K and Figure 5—Figure supplement 1E).

In contrast, reduced locomotion and response in other contexts such as egl-9 mutants and SODdeficient animals reflect H<SUB>2</SUB>S-induced adaptive mechanism rather than toxicity as they remain responsive to the other stimuli after H<SUB>2</SUB>S exposure. Since disrupting egl-9 stabilizes HIF-1 and upregulates the expression of numerous genes involved in cellular defense against H<SUB>2</SUB>S toxicity, the enhanced detoxification capacity in egl-9 mutants likely increases animals’ tolerance to H<SUB>2</SUB>S, thereby reducing avoidance to otherwise toxic H<SUB>2</SUB>S levels. Similarly, persistently high ROS in SOD deficient animals activates a variety of stress-responsive signaling pathways, including HIF-1, NRF2/SKN-1 and DAF-16/ FOXO signaling (Lennicke & Cocheme, 2021; Patten et al., 2010), facilitating cellular adaptation to redox stress and reducing animals’ responsiveness to toxic H<SUB>2</SUB>S levels. Therefore, different animals decline their locomotory speed to the effects of H<SUB>2</SUB>S through distinct mechanisms. We have integrated the relevant information across the result section and discussed this in Lines 485-536.

Reviewer #3 (Public Review): 

Summary: 

The manuscript explores the behavioral responses of C. elegans to hydrogen sulfide, which is known to exert remarkable effects on animal physiology in a range of contexts. The possibility of genetic and precise neuronal dissection of responses to H<SUB>2</SUB>S motivates the study of responses in C. elegans. The manuscript is well-written in communicating the complex physiology around C. elegans behavioral responses to H<SUB>2</SUB>S and in appropriately citing prior and related relevant work. 

There are three parts to the manuscript.

In the first, an immediate behavioral response-increased locomotory rate-upon exposure to H<SUB>2</SUB>S is characterized. The experimental conditions are critical, and data are obtained from exposure of animals to 150ppm H<SUB>2</SUB>S at 7% O2. The authors provide evidence that this is a chemosensory response to H<SUB>2</SUB>S, showing a requirement for genes encoding components of the cilia apparatus and implicating a role for tax-4 and daf-11. Neuron-specific rescue in the ASJ neurons suggests the ASJ neurons contribute to the response to H<SUB>2</SUB>S. One caveat is that previous work has shown that the dauer-constitutive phenotype of daf-11 mutants can be suppressed by ASJ ablation, suggesting that there may be pervasive changes in animal nervous system signaling that are ASJ-dependent in daf-11 mutants, which may indirectly alter chemosensory responses to H<SUB>2</SUB>S. More direct methods to assess whether ASJ senses H<SUB>2</SUB>S, e.g. using calcium imaging, would better assess a direct role for the ASJ neurons in a behavioral response to H<SUB>2</SUB>S. The authors also point out interesting parallels between the response to H<SUB>2</SUB>S and CO2 though provide some genetic data separating the two responses. Importantly, the authors note that when aerotaxis (O2sensing and movement) in the presence of bacterial food is intact, as in npr-1 215F animals, the response to H<SUB>2</SUB>S is abrogated. Mutation in gcy-35 in the npr-1 215F background restores the H<SUB>2</SUB>S chemosensory response. 

There is a second part of the paper that conducts transcriptional profiling of the response to H<SUB>2</SUB>S that corroborates and extends prior work in this area. 

The final part of the paper is the most intriguing, but for me, also the most problematic. The authors examine how H<SUB>2</SUB>S-evoked locomotory behavioral responses are affected in mutants defective in the stress and detoxification response to H<SUB>2</SUB>S, most notably hif-1. Prior genetic studies have established the pathways leading to HIF-1 activation/stabilization, as well as potential downstream mechanisms. The authors conduct logical genetic analysis to complement studies of the hif-1 mutant and in part motivated by their transcriptional profiling studies, examine the role of iron sequestration/free iron in the locomotory response to H<SUB>2</SUB>S, and further speculate on how the behavior of mutants defective in mitochondrial function might be affected by exposure to H<SUB>2</SUB>S. 

In some regard, this part of the manuscript is interesting because the analysis begins to connect how the behavior of an animal to a toxic compound is affected by mutations that affect sensitivity to the toxic compound. However, what is unclear is what is being studied at this point. In the context, of noting that H<SUB>2</SUB>S at 150ppm is known to be lethal, its addition to mutants clearly sensitized to its effects would be anticipated to have pervasive effects on animal physiology and nervous system function. The authors note that the continued increased locomotion of wild-type animals upon H<SUB>2</SUB>S exposure might be due to the byproducts of detoxification or the detrimental effects of H<SUB>2</SUB>S. The latter explanation seems much more likely, in which case what one may be observing is the effects of general animal sickness, or even a bit more specifically, neuronal dysfunction in the presence of a toxic compound, on locomotion. As such, what is unclear is what conclusions can be taken away from this part of the work.  

Strengths: 

(1) Characterization of a motor behavior response to H<SUB>2</SUB>S 

(2) Transcriptional profiling of the response to H<SUB>2</SUB>S corroborating prior work.  

We thank the reviewer for the supportive comments.

Weaknesses: 

Unclear significance and experimental challenges regarding the study of locomotory responses to animals sensitized to the toxic effects of H<SUB>2</SUB>S under exposure to H<SUB>2</SUB>S. 

We thank the reviewer for the comment, which has also been raised by the other reviewers. We agree that our initial submission left several important questions open, and we acknowledge the fact that some aspects, such as detailed neural circuit and sensory transduction, still remain unresolved. Nevertheless, we acquired new data and revised our text, aiming to clarify the distinct mechanisms underlying the reduced locomotion in different mutants during prolonged H<SUB>2</SUB>S exposure.

Our data suggest that increased initial omega turns and a rapid loss of locomotion in hif-1 and detoxification-defective mutants including sqrd-1 and ethe-1 likely reflect an enhanced sensitivity to H<SUB>2</SUB>S toxicity due to their failure to induce appropriate adaptative responses (Figure 5D–F, Figure 5J–L, Figure 5—Figure supplement 1F–P).  Supporting this, hif-1 mutants become less responsive to unrelated stimuli (near-UV light) after 30 minutes of H<SUB>2</SUB>S exposure (Figure 5I).

In contrast, egl-9 and SOD-deficient animals show reduced initial reorientation and reduced speed responses (Figure 5B, Figure 7G, Figure 5—Figure supplement 1A and B, and Figure 7—Figure supplement 1F and G), although both egl-9 and sod mutants respond normally to the other stimuli prior or after H<SUB>2</SUB>S exposure (Figure 5I, Figure 5—Figure supplement 1C, and Figure 7—Figure supplement 1H). Since disrupting egl-9 stabilizes HIF-1 and upregulates the expression of numerous genes involved in cellular defense against H<SUB>2</SUB>S toxicity, the enhanced detoxification capacity in egl-9 mutants likely increases animals’ tolerance to H<SUB>2</SUB>S, thereby reducing avoidance to otherwise toxic H<SUB>2</SUB>S levels. Similarly, constant high ROS in SOD deficient animals activates a variety of stress-responsive signaling pathways, including HIF-1, NRF2/SKN-1 and DAF-16/ FOXO signaling (Lennicke & Cocheme, 2021; Patten et al., 2010), facilitating cellular adaptation to redox stress and reducing animals’ responsiveness to toxic H<SUB>2</SUB>S levels. Taken together, these findings support the view that reduced locomotory speed during H<SUB>2</SUB>S exposure can arise from distinct mechanisms: early systemic toxicity in hif-1 and detoxification-defective mutants, versus enhanced cellular adaptation in egl-9 and SOD mutants. We have integrated the relevant information across the result section and discussed this in Lines 485-536.

Reviewer #1 (Recommendations For The Authors): 

To better substantiate a role for H<SUB>2</SUB>S detection, it would be useful for the authors to image Ca responses to H<SUB>2</SUB>S in ASJ in WT and unc-13, and to rule out the possibility that the requirement for daf-11 in ASJ reflects a role in O2 rather than H<SUB>2</SUB>S detection. 

We thank the reviewer for this comment. As suggested, we performed calcium imaging of ASJ neurons using GCaMP6s. As previously described, 3% CO<SUB>2</SUB> evoked a calcium transient in ASJ (Figure 2—figure supplement 2F). To investigate whether H<SUB>2</SUB>S evoked a calcium transient in ASJ neurons, we tested several conditions, including animals on food or off food, with different H<SUB>2</SUB>S concentrations (~75 or ~150ppm), and different exposure time (4 or 8 mins). However, we did not detect a calcium response to H<SUB>2</SUB>S in ASJ under any of the conditions tested (Figure2—figure supplement 2E) (Lines 166–168). Given that neuronspecific rescue of daf-11 or tax-4 mutants pointed to a role of ASJ neurons in promoting H<SUB>2</SUB>S responses, we sought to determine how ASJ neurons were involved. Expression of the tetanus toxin catalytic domain in ASJ neurons, which blocks neurosecretion, inhibited H<SUB>2</SUB>S-evoked locomotory speed responses (Figure 2H), similar to the phenotypes observed in daf-11 and daf-7 mutants (Figure 2C and D) (Lines 162–165). These results confirm that ASJ activity and neurosecretion contribute to the H<SUB>2</SUB>S responses, although ASJ is unlikely to serve as the primary H<SUB>2</SUB>S sensor. One potential explanation is that DAF-7 released by ASJ controls the starvation program, which in turn modulates the animal’s response to H<SUB>2</SUB>S. We also discussed this in Lines 449–458.

The paper would be significantly strengthened by testing the possibility (as the authors acknowledge in lines 348-52) that disruption of detoxification mechanisms reduces sustained behavioral responses to H<SUB>2</SUB>S because of physiological damage. Authors use acute exposure to high O2 for this purpose earlier in the paper, but not to probe the consequences of loss of hif-1 and detoxification factors.  

We thank the reviewer for the valuable suggestion. As the reviewer highlighted, we attributed the brief locomotory speed responses to H<SUB>2</SUB>S observed in hif-1 mutants to the lack of detoxification response, leading to the rapid intoxication of the animals. Several lines of evidence support this conclusion. First, we observed that hif-1 and the detoxification mutants displayed a stronger initial reorientation response (omega turns) and a more rapid decline in speed and reversals compared to wild type (Figure 5 D–F). Second, to test if hif-1 mutants were indeed more susceptible to H<SUB>2</SUB>S toxicity, we exposed WT and hif-1 animals to H<SUB>2</SUB>S for 30 mins and subsequently tested their ability to respond to near-UV light. Unlike WT animals, the speed response to near-UV light was inhibited in hif-1 mutants (Figure 5I), suggesting that exposure to H<SUB>2</SUB>S for 30 min causes a stronger toxicity in animals deficient of HIF-1 signaling. Third, hif-1 and detoxification mutants displayed a sustained high speed in response to 1% O<SUB>2</SUB> , suggesting the specific impairment of H<SUB>2</SUB>S response. The data were presented in Lines 318–347, and were further discussed this in Lines 485–508.

To better understand whether mitochondrial damage has a role in H<SUB>2</SUB>S-evoked behavior, it might be useful for the authors to determine whether general ROS response pathways are important for H<SUB>2</SUB>S behavioral responses.

We thank the reviewer for this insightful comment. As suggested, we investigated whether ROS detoxification pathways contribute to H<SUB>2</SUB>S-evoked locomotory speed responses by analyzing mutants in the superoxide dismutase (SOD) family. These experiments, together with other observations, suggest that mitochondrial ROS play a dual role in H<SUB>2</SUB>S-evoked locomotion. The relevant results were presented in Lines 401–425, and were further discussed in Lines 509–536.

First, we found that increased mitochondrial ROS formation, either induced pharmacologically by rotenone or genetically in mitochondrial electron transport chain (ETC) mutants (Ishii et al., 2013; Ochi et al., 2016; Ramsay & Singer, 1992; Yang & Hekimi, 2010; Zorov, Juhaszova, & Sollott, 2014), suppressed the behavioral response to toxic H<SUB>2</SUB>S (Figure 7A–E). This indicates that mitochondrial ROS plays a significant role in H<SUB>2</SUB>S-evoked responses. One likely explanation is that high ROS formation may dampen the H<SUB>2</SUB>S-triggered ROS spike, or may impair other H<SUB>2</SUB>S signaling processes required to initiate avoidance. Second, consistent with previous reports (Onukwufor et al., 2022), we observed that shortterm rotenone exposure (<1 hour) significantly increased baseline locomotory speed. Given that toxic H<SUB>2</SUB>S levels promote ROS formation (Jia et al., 2020), our findings suggest that acute mitochondrial ROS production by toxic levels of H<SUB>2</SUB>S exposure may serve as a trigger for the avoidance response.

In contrast, animals with sustained mitochondrial ROS production do not have an increased baseline locomotory speed. This effect was observed after 2 hours of rotenone exposure, in mitochondrial ETC mutants, and in animals lacking all SOD enzymes (Figures 7A–K). A likely explanation for the reduced basal locomotory speed during sustained mitochondrial ROS production is the activation of ROSresponsive signaling pathways including HIF-1, NRF2/SKN-1, and DAF-16/FOXO (Lennicke & Cocheme, 2021; Patten, Germain, Kelly, & Slack, 2010), which may promote adaptation to prolonged oxidative stress (Figure 7H). Notably, unlike hif-1 mutants, SOD-deficient animals remained as responsive as WT to other stimuli after 30 minutes of H<SUB>2</SUB>S exposure (Figure 7—figure supplement 1H), indicating that elevated ROS levels do not compromise overall viability or the ability to detoxify H<SUB>2</SUB>S.

Taken together, these results support a model in which mitochondrial ROS exerts a biphasic effect on H<SUB>2</SUB>S-induced avoidance. It enhances detection and avoidance under acute stress but contributes to locomotory suppression when ROS levels remain elevated chronically.

Reviewer #2 (Recommendations For The Authors):

The way the manuscript is presented could be improved without much effort by rewriting/editing. For the reader, it is hard at present to understand the rationales of the experiments, why they were carried out, and how to place them into a context. This could be improved on three levels:

(1) Documentation 

(2) Narration/Explanation 

(3) Visualization 

(1) Documentation

Not all of the results in the text are well documented. The results should be described with more details in the written text and improved documentation and quantification of the results. Example: 

Turning behavior is mentioned as an important aspect of the response to H<SUB>2</SUB>S. There is no citation given but this effect is not well documented. The authors image the animals and could provide video footage of the effect, could quantify eg turning/pirouettes, and provide the data. At the moment the manuscript largely relies on measuring the increase in speed, but the reader is left wondering what other behavioral effects occur and how this is altered in all of the mutant and other conditions tested. Just quantifying speed reduces the readout and seems like an oversimplification to characterize the behavioral response.  

We are grateful for this comment. We now provide a video footage of the H<SUB>2</SUB>S effects (Figure 1—Video 1). As suggested, we analyzed the recordings to extract reorientation (omega-turns) and reversals. These analyses are now included in the Supplemental file 1 with representative panels displayed in Figure 5 and supplements to Figures 2, 3, 5, 6 and 7. Even though the mutant effects on omega-turns were often subtle, and reversal responses showed considerable variability (likely due to differences in population density, food availability, or animals’ physiological state prior to the assay), this analysis has proven valuable for distinguishing mutants that exhibit adaptation from those that display hypersensitivity to H<SUB>2</SUB>S toxicity. For instance, although both SOD-deficient and BP-treated animals failed to increase their locomotory speed in H<SUB>2</SUB>S (Figure 6E and Figure 7G), they exhibited distinct omega-turn responses (Figure 6—figure supplement 1I and Figure 7—figure supplement 1F), suggesting that different mechanisms likely underlie the locomotory defects of these two animals. We have integrated the omega-turn and reversal data into the text and discussed under relevant contexts.

(2) Narration/Description.

Generally, the description of the results part is very brief and it is often not clear why a certain experiment was carried out and how. Surely it is possible to check the methods but this interrupts the flow of reading and it would be easier for the reader to be guided through the results with more information what the initial motivation for an experiment is, what the general experimental outline is, and what specific experiments are carried out. 

We apologize for the lack of clarity and logical structure in the initial submission. In the revised manuscript, we have thoroughly revised the text to improve its organization and readability.

Examples: 

Line 97ff: The authors performed a candidate screen yet it is not described why which genes were chosen. Are there also pathways that were tested that turned out to not be involved? 

We thank the reviewer for the suggestion. To address this, we have added a new section, explaining the rationale for selecting genes and pathways in our candidate screen. Briefly, we focused on genes known or predicted to be involved in sensory responses to gaseous stimuli in C. elegans and mammals, including globins and guanylate cyclases (21% O<SUB>2</SUB> sensing), potassium channels (acute hypoxia), and nutrientsensitive pathways (CO<SUB>2</SUB> responses). We also included mutants defective in sensory signal transduction and neurotransmission. In addition, mitochondrial mutants were analyzed because mitochondria play a central role in H<SUB>2</SUB>S detoxification. The pathways that contributed to the acute H<SUB>2</SUB>S response included cGMP, insulin, and TGF-β signaling, as well as mitochondrial components. In contrast, globins, potassium channels, and biogenic amine signaling did not appear to play significant roles under our assay conditions. The results of the candidate screen are described in Lines 106–138 and summarized in Supplementary File 1.

line 262ff: the paragraph starts with explaining ferritin genes that are important for iron control but the reader does not yet know why. Then it is explained that a ferritin gene is DE in the H<SUB>2</SUB>S transcriptomes. then a motivation to look into the labile iron pool is described. Why not first explain what genes are strongly regulated and why they are selected based on their DE? Then explain what is known about these genes and pathways, and then motivate a set of experiments. 

We agree with the reviewer that our initial description could have been more logically organized. We reframed this section to first present the RNA-seq data, followed by an explanation of their known biological functions and the motivation for the subsequent experiments (Lines 350–357).

nhr-49 appears suddenly in the results part and it is not clear why it was tested and how the result links. Is nhr-49 a key transcription factor that is activated by H<SUB>2</SUB>S sensory or physiological response, and does it control the signaling or protective changes induced by H<SUB>2</SUB>S?  

We thank the reviewer for the comment. As suggested, we revised the text to present the information more clearly. In our candidate gene screen, a set of mutants exhibiting reduced speed responses to H<SUB>2</SUB>S has previously been shown to be defective in response to CO<SUB>2</SUB> stimulation (Hallem & Sternberg, 2008). These included animals deficient in nutrient-sensitive pathways, including insulin, TGF-beta, and NHR49, which were reported by Sternberg’s lab to exhibit dampened responses to CO<SUB>2</SUB> (Hallem & Sternberg, 2008) (Lines 173–179). We also included a simply cartoon to further illustrate this (Figure 3C).

The nuclear hormone receptor NHR-49 has been implicated in a variety of stress responses, including starvation (Van Gilst, Hadjivassiliou, & Yamamoto, 2005), bacterial pathogen (Naim et al., 2021; Wani et al., 2021), and hypoxia (Doering et al., 2022). The nhr-49 mutants exhibited a rapid decline in locomotory speed during H<SUB>2</SUB>S exposure, implicating a role in sustaining high speed in the presence of H<SUB>2</SUB>S. Furthermore, we observed that fmo-2, a well-characterized target gene of NHR-49, was significantly upregulated after 1 hour of exposure to 50 and 150 ppm H<SUB>2</SUB>S (Supplementary file 2), suggesting that NHR-49 signaling is rapidly activated by H<SUB>2</SUB>S exposure. Exactly how NHR-49 contributes to H<SUB>2</SUB>S response requires further investigation.

(3) Visualization 

Adding a model/cartoon summary that describes the pathways tested and their interaction would be helpful in some of the figures for the reader to keep an overview of the pathways that were tested. Also, a final summary cartoon that integrates all the puzzle pieces into one larger picture would be helpful. Such a final cartoon overview could also point to the key open questions of the underlying mechanisms. 

We thank the reviewer for this suggestion. We have added a series of models/cartoons to illustrate the different pathways and their interactions. These include starvation regulatory mechanisms (Figure 3C), 21% O<SUB>2</SUB> sensing mechanisms (Figure 3G), HIF-1 signaling and detoxification (Figure 5—figure supplement 1E), HIF-1 signaling and the regulation of labile iron (Figure 6H), as well as ROS signaling and regulation (Figure 7L). To help interpretation and to elaborate on these models, we have also included explanatory sentences in the corresponding figure legends.

Other comments: 

Introduction and line 93: The authors mention that 50 ppm H<SUB>2</SUB>S has beneficial effects on lifespan yet does not have a detectable phenotype." Are there any concentrations of H<SUB>2</SUB>S that cause attraction of C. elegans and what is the preferred range if it exists? Could this be measured in an H<SUB>2</SUB>S gradient? 

We thank the reviewer for the insightful comment. We performed an H<SUB>2</SUB>S gradient assay, which suggests that wild type animals are attracted toward low concentrations of H<SUB>2</SUB>S around 40 ppm (Figure 1G and H) (Lines 95–104). These results suggest that H<SUB>2</SUB>S acts as a strong repellent for C. elegans at high concentrations but as an attractant at low levels. This dual role may be ecologically relevant, as wild C. elegans lives in complex and dynamic environments where H<SUB>2</SUB>S levels likely fluctuate over short distances (Adams, Farwell, Pack, & Bamesberger, 1979; Budde & Roth, 2011; Morra & Dick, 1991; Patange, Breen, Arsuffi, & Ruvkun, 2025; Rodriguez-Kabana, Jordan, & Hollis, 1965; Romanelli-Cedrez, Vairoletti, & Salinas, 2024).

Line 146: "Local H<SUB>2</SUB>S concentrations could also be significantly higher in decomposing substances where wild C. elegans thrives" please provide a citation.

As suggested, we included a set of references that have described the H<SUB>2</SUB>S enrichment in the natural environment in early field studies (Adams et al., 1979; Morra & Dick, 1991; Rodriguez-Kabana et al., 1965), as well as references that have discussed and implied this in C. elegans studies (Budde & Roth, 2011; Patange et al., 2025; Romanelli-Cedrez et al., 2024). They can be found in the introduction (Lines 59–62) and in the result (Lines 197–199).

Line 311 "Wild C. elegans isolates thrive in the decomposing matters, where the local concentrations of O2 are low while the levels of CO2 and H<SUB>2</SUB>S could be high. These animals have adapted their behavior in such an environment, displaying increased sensitivity to high O2 exposure but dampened responses to CO2." Please provide citations for these statements.  

As suggested, we cited the relevant articles or books describing the variation of O<SUB>2</SUB> and CO<SUB>2</SUB> levels in the decomposing matters including several C. elegans papers that mentioned this in Lines 197–199 (Bretscher, Busch, & de Bono, 2008; Gea, Barrena, Artola, & Sanchez, 2004; Hallem & Sternberg, 2008; Oshins, Michel, Louis, Richard, & Rynk, 2022), and the above-mentioned articles for H<SUB>2</SUB>S (Adams et al., 1979; Budde & Roth, 2011; Morra & Dick, 1991; Patange et al., 2025; Rodriguez-Kabana et al., 1965; Romanelli-Cedrez et al., 2024).

For C. elegans’ sensitivity to O2 and CO2, these articles were cited in Lines 201–203 (Beets et al., 2020; Bretscher et al., 2008; Carrillo, Guillermin, Rengarajan, Okubo, & Hallem, 2013; Hallem & Sternberg, 2008; Kodama-Namba et al., 2013; McGrath et al., 2009).

Reviewer #3 (Recommendations For The Authors): 

More work could be conducted establishing the neuronal circuitry involved in the initial, tractable response to H<SUB>2</SUB>S. 

We thank the reviewer for the insightful comment. Since our initial analyses suggest a role of ASJ neurons in H<SUB>2</SUB>S-evoked locomotory responses (Figure 2F and G), We thought that this would offer us an entry point to dissect the neuronal circuit involved in H<SUB>2</SUB>S responses. Expression of the tetanus toxin catalytic domain in ASJ, which blocks neurosecretion, inhibited H<SUB>2</SUB>S evoked locomotory responses (Figure 2H), suggesting that neurosecretion from ASJ promotes the speed response to H<SUB>2</SUB>S (Lines 162– 165). We then performed calcium imaging of ASJ neurons in response to H<SUB>2</SUB>S exposure. However, while we observed CO<SUB>2</SUB> -evoked calcium transients in ASJ using GCaMP6s, we did not detect any calcium response to H<SUB>2</SUB>S, under several conditions, including animals on food, off food, and with different H<SUB>2</SUB>S concentrations and exposure times (Figure2—Figure supplement 2E and 2F) (Lines 166–168). Since signaling from ASJ neurons regulates developmental programs that modify sensory functions in C. elegans, including CO<SUB>2</SUB> and O<SUB>2</SUB> responses (Murakami, Koga, & Ohshima, 2001), the involvement of ASJ neurons is not specific to H<SUB>2</SUB>S responses and ASJ neurons are unlikely to serve as a primary H<SUB>2</SUB>S sensor (Discussed in Line 449–458). Therefore, the exact sensory neuron, circuit and molecular triggers mediating acute H<SUB>2</SUB>S avoidance behavior remain to be elucidated.

Our subsequent investigation on mitochondrial components suggests that a burst of mitochondrial ROS production may be the trigger for H<SUB>2</SUB>S avoidance, as transient exposure to rotenone substantially increases baseline locomotory activity (Figure 7E) (Line 391–396). However, mitochondrial ROS could potentially target multiple neurons and cellular machineries to initiate avoidance behavior to H<SUB>2</SUB>S, making it challenging to pinpoint specific sites of action. Nevertheless, we agree that further dissection of the neural circuits and mitochondrial signaling in H<SUB>2</SUB>S avoidance will be important and should be explored in future studies. We discussed this in Lines 509–536. 

I am not sure how to overcome the challenges involved in reaching conclusions from the decreased locomotory responses of animals that are sensitized to the effects of H<SUB>2</SUB>S. Perhaps this conundrum could be discussed in more detail in the text. 

We thank the reviewer for this important comment. We agree that decreased locomotory speed during H<SUB>2</SUB>S exposure can arise from distinct causes, either systemic toxicity or adaptation, and distinguishing between these is critical. We have included new experiments and revised the text to clarify this issue.

Our data suggest that increased initial omega turns and a rapid loss of locomotion in hif-1 and detoxification-defective mutants including sqrd-1 and ethe-1 likely reflect an enhanced sensitivity to H<SUB>2</SUB>S toxicity due to their failure to induce appropriate adaptative responses (Figure 5D–F, Figure 5J–L, Figure 5—Figure supplement 1F–P).  Supporting this, hif-1 mutants become less responsive to unrelated stimuli (near-UV light) after 30 minutes of H<SUB>2</SUB>S exposure (Figure 5I).

In contrast, egl-9 and SOD-deficient animals show reduced initial reorientation and reduced speed responses (Figure 5B, Figure 7G, Figure 5—Figure supplement 1A and B, and Figure 7—Figure supplement 1F and G), although both egl-9 and sod mutants respond normally to the other stimuli prior or after H<SUB>2</SUB>S exposure (Figure 5I, Figure 5—Figure supplement 1C, and Figure 7—Figure supplement 1H). Since disrupting egl-9 stabilizes HIF-1 and upregulates the expression of numerous genes involved in cellular defense against H<SUB>2</SUB>S toxicity, the enhanced detoxification capacity in egl-9 mutants likely increases animals’ tolerance to H<SUB>2</SUB>S, thereby reducing avoidance to otherwise toxic H<SUB>2</SUB>S levels. Similarly, persistently high ROS in SOD deficient animals activates a variety of stress-responsive signaling pathways, including HIF-1, NRF2/SKN-1 and DAF-16/ FOXO signaling (Lennicke & Cocheme, 2021; Patten et al., 2010), facilitating cellular adaptation to redox stress and reducing animals’ responsiveness to toxic H<SUB>2</SUB>S levels. Taken together, these findings support the view that reduced locomotory speed during H<SUB>2</SUB>S exposure can arise from distinct mechanisms: early systemic toxicity in hif-1 and detoxificationdefective mutants, versus enhanced cellular adaptation in egl-9 and SOD mutants. We have integrated the relevant information across the result section and discussed this in Lines 485–536. 

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"Policies Supporting Renewable in the Industry Sector" redirects to the home page instead of the section!

I'm surprised the Chinese gov published this, maybe to their audience and residents it makes them look strong. I appreciate the use of attribution as this number might be hard to fact check and could be larger than they state.

If H. declined to comment, I wonder how they legally navigated what they could share especially if this source and his wife are so high profile. I know with anonymous sourcing there is a small glimmer of truth regarding the source's proximity to the story that can be shared that is hopefully not traceable. I just wonder if this is traceable, we know H. owns a vineyard in Bordeaux and his wife is Zhao Wei.

I know they interviewed subjects in over 100 countries, I wonder why they would open with a subject that declined to comment and could only use initial. I understand this story is deeply sensitive for many involved, maybe this was the strongest lede they had

I find it fascinating thqat both Ma and H. were vulnerable to attacks from the chinese government, and they tasked Ma with persuading H. There could be an elaborate and powerful system of puppetry built on blackmail and threats I'm not yet aware of.

I appreciate the honesty, I value the pursuit of scientific study but dont appreciate when others don't acknowledge it can be pretentious

I find it interesting the author dubbed himself one of the prototypes, that is usually a level of recognition reserved for another professional to determine

I think this is very important, where journalists level up skills to meet the evolving modern moment with tech, cultural understanding and improved fact checking they are still dedicated to a timeless pursuit

1. مشکل خودت میتونه مشکل بقیه هم باشه.
2. سوار ترندها بشو و در راستای اونها پروداکت بساز. (چطور ترند رو تشخیص بدیم و از هایپ افتراقش بدیم؟ هر ترندی؟ یا یه سری ترند خاص؟)

As long as your account is registered with an email address, and as long as you create revenue through Substack and its contracted sub-processors (such as Stripe), you are not anonymous from parties with authority to audit such systems. In other words, adopting an anonymous identity on Substack only serves to hide your identity, and therefore your claim to authority, from users of the site.

if knowledge is not sought as a product without the path the learning that lead to its creation

everything would be innovated t the point of creation

obvioulsy with some explicit intent

but if the intent itself was comPlexualized

suitable mappings could be constructed

But that would require human intellect

All we need to do is stop the practice of putting man in the loop of th machine

and put the machine where it belongs in human loops

Bu tthat would be just difficult to won and control so we wont go there

How interesting

if you get your comPlexes right intelligence will take care of itself

The very fact that we do not create content (text or otherwise) that does not record in an explicit manner all the relevant pertinent named associative complexes

is the problem

amyone considred search by "semantic" what a bad term

antinomity

That's the true source of true creativity

I am interested in Contextual Retrieval

or rather ConPlexual retieval because it is not conText matters

but associative comPlexes that can be wowen around/throug/with them with a bit of extra work

Something that requires human attention and human intentions and readiness to ariculate, formulate the structure of meaning that can be formulated around 1s learning as part of that process

https://bafybeicyhws72tr4xdgjcpyb4cflrkx5254gqqrakyjymvsbizi6jausma.ipfs.dweb.link?filename=Introducing%20Contextual%20Retrieval%20%EF%BC%BC%20Anthropic.html

for pages that are important I tend to save them with SingleFile Chrome extension

and Pin then using Pianata.cloud on IPFS and add them to my locally running IPFS Desktop too

Soeven if the page gets changd out of recognition or vanish, which does happen most of the time I still have the page i spent time with available in the future

eLife Assessment

This study presents significant and novel insights into the roles of zinc in mammalian meiosis/fertilization events. These findings are useful to our understanding of these processes. The evidence presented is solid, with experiments being well-designed, carefully described, and interpreted with appropriate rigor. The main limitation of lack of mechanistic insight needs to be acknowledged.

Reviewer #1 (Public review):

The revised manuscript addresses several reviewer concerns, and the study continues to provide useful insights into how ZIP10 regulates zinc homeostasis and zinc sparks during fertilization in mice. The authors have improved the clarity of the figures, shifted emphasis in the abstract more clearly to ZIP10, and added brief discussion of ZIP6/ZIP10 interactions and ZIP10's role in zinc spark-calcium oscillation decoupling. However, some critical issues remain only partially addressed.

(1) Oocyte health confound: The use of Gdf9-Cre deletes ZIP10 during oocyte growth, meaning observed defects could result from earlier disruptions in zinc signaling rather than solely from the absence of zinc sparks at fertilization. The authors acknowledge this and propose transcriptome profiling as a future direction. However, since mRNA levels often do not accurately reflect protein levels and activity in oocytes, transcriptomics may not be particularly informative in this context. Proteomic approaches that directly assess the molecular effects of ZIP10 loss seem more promising. Although current sensitivity limitations make proteomics from small oocyte samples challenging, ongoing improvements in this area may soon allow for more detailed mechanistic insights.

(2) ZIP6 context and focus: The authors clarified the abstract to emphasize ZIP10, enhancing narrative clarity. This revision is appropriate and appreciated.

(3) Follicular development effects: The biological consequences of ZIP6 and ZIP10 knockout during folliculogenesis are still unknown. The authors now say these effects will be studied in the future, but this still leaves a major mechanistic gap unaddressed in the current version.

(4) Zinc spark imaging and probe limitations: The addition of calcium imaging enhances the clarity of Figure 3. However, zinc fluorescence remains inadequate, and the authors depend solely on FluoZin-3AM, a dye known for artifacts and limited ability to detect subcellular labile zinc. The suggestion that C57BL/6J mice may differ from CD1 in vesicle appearance is plausible but does not fully address concerns about probe specificity and resolution. As the authors acknowledge, future studies with more selective probes would increase confidence in both the spatial and quantitative analysis of zinc dynamics.

(5) Mechanistic insight remains limited: The revised discussion now recognizes the lack of detailed mechanistic understanding but does not significantly expand on potential signaling pathways or downstream targets of ZIP10. The descriptive data are useful, but the inability to pinpoint how ZIP10 mediates zinc spark regulation remains a key limitation. Again, proteomic profiling would probably be more informative than transcriptomic analysis for identifying ZIP10-dependent pathways once technical barriers to low-input proteomics are overcome.

Overall, the authors have reasonably revised and clarified key points raised by reviewers, and the manuscript now reads more clearly. However, the main limitation, lack of mechanistic insight and the inability to distinguish between developmental and fertilization-stage roles of ZIP10, remains unresolved. These should be explicitly acknowledged when framing the conclusions.

Author response:

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

Reviewer #1 (Public review):

The authors investigated the role of the zinc transporter ZIP10 in regulating zinc sparks during fertilization in mice. By utilizing oocyte-specific Zip6 and Zip10 conditional knockout mice, the authors effectively demonstrate the importance of ZIP10 in zinc homeostasis, zinc spark generation, and early embryonic development. The study is overall useful as it identifies ZIP10 as an important component of oocyte processes that support embryo development, thus opening the door for further investigations. While the study provides solid evidence for the requirement of ZIP10 in the regulation of zinc sparks and zinc homeostasis, it falls short of revealing the underlying mechanism of how ZIP10 exerts this important function.

This report is the first to clarify the role of the zinc transporters ZIP10 expressed in oocytes, which was previously unknown, and does not focus on the detailed mechanism. As you pointed out, we believe that the mechanism will also be important information in the field of fertilization and embryogenesis research, and we believe that it is necessary to consider this issue in the future.

(1) The zinc transporters the authors are knocking out are expressed in mouse oocytes through follicular development, and the Gdf9-cre driver used means these oocytes were grown in the absence of appropriate Zinc signaling. Thus, it would be difficult to assert that the lack of fertilization associated with zinc sparks is solely responsible for the failure of embryo development. Spindle morphology and other meiotic parameters do not necessarily report oocyte health, so normalcy of these features may not be a strong argument when it comes to metabolic issues.

As you rightly observe, the results of this study do not entirely exclude the possibility of oocyte health in the absence of adequate zinc homeostasis during oocyte growth. However, evidence has been presented demonstrating that spindle formation does occur in Zip10^d/d mouse oocytes (Fig.2 C), that fertilization occurs despite the absence of zinc spark (Fig.3 and Fig. 4A), and that some embryos develop to blastocysts (Fig. 4 B). We believe that future studies should evaluate the transcriptome profile of Zip10^d/ mouse oocytes.

(2) While comparing ZIP6 and ZIP10 in the abstract provides context, focusing more on ZIP10 would improve reader comprehension, as ZIP10 is the primary focus of the study. Emphasizing the specific role of ZIP10 will help the reader grasp the core findings more clearly.