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Dear Dr. Qu, I congratulate you on the acceptance of this article for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Jennifer Vonk, a PeerJ Section Editor covering this Section #]
I noted a few remaining errors in the text. I recommend the authors carefully revise their wording and sentence construction before final acceptance.
The authors have added more information to the manuscript, which improves its clarity and transparency, particularly regarding the statistical analyses and experimental design. They clearly explained each treatment group, which wasn't clear at all before.
The authors have added information on the figures description like I suggested, which help with understanding the data.
The authors have done a good job addressing my comments and concerns, and the manuscript has been appropriately revised. I believe it will make a valuable contribution to the journal.
Dear Dr. Qu, I ask you to carefully analyze the reviewers' comments and send a new version of the article, which they can accept for publication.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
1. Line 46: some parasites can impact host survival, as the authors note later in the introduction (line 82). I would recommend changing this wording to avoid confusion.
2. Line 53: I would change “adventurous” to increased boldness, as this is more commonly used.
3. Line 62: add a space between “contexts” and the parenthesis
4. Line 143: Specify here the ambient temperature and humidity level, and the range if it fluctuates throughout the experiments.
5. Line 194-198: “Ambient temperature and humidity were maintained at stable levels representative of the natural environment, during the test, in order to prevent the test results from being influenced by the individuals themselves and by the oocysts excreted by the infected individuals, the cages were changed every day and washed with boiling water to ensure hygiene without introducing unpredictable disturbances.” : this sentence is too long and confusing. I suggest splitting into two sentences. Also, again, it would be good to specify the temperature and humidity at least once before.
6. How many animals died during the acclimation period, if any?
7. Line 209-216: the flow of reading here is disturbed by this “shopping list” of what to do with the fecal sample. I would suggest rewriting in the past tense like the rest of the methods.
8. Line 241: Specify here how many animals did not exit the refuge and were scored 120s, as this can help understand if this boldness test was effective (i.e. if a lot of individuals do not exit the shelter, this could possibly indicate that this test is not best suited for this species, and doesn't capture the whole boldness-shyness gradient).
9. Line 248: what time of the day was the RMR measured? Is there a reason it was measured only for 2 hours?
10. Line 376: Are the authors suggesting that the effects observed in the present study positive? I suggest removing “studies have focused on the negative effects of parasite infections”.
11. Line 379-380: Eimeria spp. is written twice in this sentence and correct “as common” to “a common”.
12. Line 384: add space between the and Qinghai-Tibetan
13. Line 446: change “explorations” for “exploration levels” here.
14. Line 460: I suggest using another word than attacks here.
1. Statistical analysis clarity
One main concern relates to the reporting and description of the statistical analyses. While the analyses appear conceptually sound and thoughtfully designed, several aspects require clarification to ensure transparency and reproducibility. In the main manuscript, key details about the model structure are missing. Specifically, in the description of Table 1, the authors refer to the use of “original scale approximations for Poisson models,” yet there is no prior mention of Poisson models in the manuscript. It is important to explicitly state which response variables were modelled using Poisson, Gaussian, or other model distribution within the Statistical Analysis section. In addition, Table 2 introduces variables such as sex (fixed) and ID (random) as part of the model structure; however, these variables are not described or even mention in the main text. The authors are encouraged to review the analysis section carefully and to clearly specify the structure of each model used (e.g., “Using MCMCglmm, we modelled behavioural traits [boldness, docility, exploration] with sex, treatment, and time as fixed effects, and ID as a random factor”). Providing a transparent and consistent description of all models would greatly enhance the manuscript’s methodological clarity.
2. Treatment groups
The treatment groups were a little bit ambiguous until the results section. At first, it appears that there will be an uninfected group (control group), but then, at the results section, all groups had parasites at some point in time. I’m not sure why the treatments used were chosen. One group were given the parasites (PA+); one group was given saline water (to control for the gavage effect?) (Ctrl) and one group was given an anti-parasite treatment (PA-). I would suggest giving more details about each treatment group, why they were chosen. What is the efficacity of this anti-parasite treatment? Why is there no difference between the Ctrl group and PA- group in terms of parasite counts on D5, D8 (Figure 1)? Is it possible to assess the survival of the parasites, i.e. when given the anti-parasite treatment, did the authors knew which oocysts were alive/dead from the treatment?
3. It would also be useful to clarify whether all wild-caught pikas were naturally infected with this parasite species from the start. Did the authors assess the parasite loads prior the behavioral/physiological tests, before starting the treatment? The authors mention in the methods that “healthy individuals” were captured — does “healthy” here mean parasite-free? Additionally, are other parasite species commonly found in this population?
4. Parasite count methodology
The manuscript lacks sufficient detail regarding parasite quantification. It is unclear how the abundance of Eimeria spp. was measured. From line 203-216, is this the description of how parasites were counted following the behavioral and physiological tests, at each time point? Or this is to prepare the parasite solution for gavage? or both? This is unclear.
5. Line 191: Do the authors mean that the order in which the behavioral tests were done were changed between individuals, e.g. some animals started with docility, and finished with exploration? If this is the case, I am worried about the exploration test, which needs to be a “novel environment” by definition and thus, should be the first metric measured once the pika is in the arena. Were the behavioral and physiological test done all the same day for all individuals? More information should be provided on this.
6. I would suggest specifying what software you are working with and the version of R in the beginning of this paragraph rather than the end. Also, for the distribution of residuals; I would suggest reporting this before explaining the models.
7. Was the distribution of residuals inspected prior to the LMMs for the repeatability models? Also, I find it a little bit confusing: why use the rptR package to measure repeatability, if it was done later with the MCMCglmm package?
1. Figure 2: what does the black horizontal bar represent? Is it the contrast between the groups, showing if there is a significant difference between them? It seems like we would expect a significant difference in exploration at D8 for the Ctrl group giving its much higher exploration levels? Also, for the inset plots showing the relationship between the trait and number of parasites, what are the points? Is this for all the groups (PA+, Ctrl and PA-) all together, for all the time point (5d, 8d, 18d)? Is it raw data or from a specific model? Specify in the figure description what the points represent please. For the bar plot showing the behaviors, are these raw data or predicted values from the MCMCglmm models?
2. Figure 3: same comment as before for the inset plots. I suggest adding more details regarding what data is used (raw or predicted by the models) here for transparency and less confusion.
3. Line 322: “overall, with an increase in Eimeria spp., pikas became more exploratory.” However, we see a big increase in exploration on D8 for the Ctrl group, which coincides with a decrease in the number of parasites on D8 for this group. Is there any comparison between the days? For e.g. do we know the contrast between the same group in their exploration at D5 versus D8? Or the models only compared the traits on the same day?
4. Raw data are available; however, the authors should include a meta-data sheet that describes all variables.
This study by Wang et al. explores the relationship between parasite infection, personality, and physiology in pikas. The paper is well-structured and examines these relationships across different time points, providing valuable insight into the timing of infection and its impact on host behaviour and physiology. I believe this is an important line of research, and the study represents a meaningful contribution to the literature and should be considered for publication once the comments are addressed.
The study of the features of the evolutionary relationships between parasites and their hosts is of great importance for the stability of ecosystems. Therefore, these studies on determining the influence of single-celled parasites of the digestive system Eimeria spp. on the physiology and behavior of a small mammal (Ochotona curzoniae) are of considerable interest to specialists in the field of ecology, parasitology, veterinary medicine and medicine. The authors have reviewed a large amount of scientific literature on the topic of the research. The presented hypothesis consists of three points, which are fully analyzed in the "Discussion" section.
The purpose of the work is clearly stated. The authors have provided sufficient information on the current state of research in this area. They have also identified issues that have not yet been resolved. All research has been conducted at a high technical level and in accordance with current ethical standards in this field.
The conclusions are clearly formulated. Although in the "Conclusions" section it would be desirable to briefly provide a generalization regarding the previously proposed points of the hypothesis.
This scientific work is quite interesting, although it has some comments and questions:
1. It is not entirely clear what treatment the animals of the three groups received. Were the feces of animals of all three groups previously examined for the presence of Eimeria oocysts? Were the animals naturally infected with Eimeria before the experiment? Were the animals of the second and third groups infected with Eimeria, which were given saline and an anticoccidial drug, respectively? It would probably be better to explain this in more detail in the “Materials and Methods” section.
2. The “Materials and Methods” section states that the experiment was divided into four stages, i.e. 5, 8 and 18 days. That is, the study was carried out, as is clear, on days 5, 8 and 18. Maybe the authors meant three stages, according to the indicated days?
3. The “Materials and Methods” section contains a subsection “Eimeria spp. and anthelmintic agents”, although anticoccidial drugs were used to treat the animals, since protozoa do not belong to helminths.
4. The subsection “Eimeria spp. and anthelmintic agents” would be desirable to be divided into items that relate separately to the collection, cultivation of Eimeria in laboratory conditions and testing for the presence of Eimeria, their counting by the McMaster method.
5. It is not clear why, according to the McMaster method, 9 ml of water was added to 1 ml of the filtered mixture with oocysts and then placed in counting chambers. If, according to the method, water is not added to the filtrate. The filtrate is immediately placed in counting chambers. Or, the feces are first washed with water and then filtered (Zajac and Conboy, 2012).
6. In the subsection “Statistical analysis” of the section “Materials and methods”, the term “anthelmintics” is also used, although anticoccidial drugs were used.
7. If water and saline promote the excretion of metabolic waste and parasites (Worthington et al., 2013; Zakeri et al., 2018; Ahmed, 2023; Halliez & Buret, 2015), why does this suggest, as the authors point out in the Discussion section, that as the intake of saline via gavage decreased, the number of Eimeria gradually decreased?
8. Why was the number of Eimeria spp. in the PA- (anticoccidial drugs) group higher than in the Ctrl group on D5? And how exactly did environmental factors (the density of parasites in the habitat and the nutritional status of the host, as indicated by the authors) influence this result, if the animals of all three groups were placed in the same conditions?
9. At the end of the subsection “Variations in the Eimeria spp. numbers” of the “Discussion” section, the authors conclude “Thus, the Eimeria spp. was higher in the PA- group than in the PA+ group on D18”. That is, in the group of animals that were given anticoccidial drugs at the end of the experiment, the number of Eimeria spp. was higher than in the other group? Then what is the point of treating animals with anticoccidial drugs at all, if without treatment with these drugs the number of Eimeria spp. in sick animals becomes lower?
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