Author Response
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
This study examines the role of host blood meal source, temperature, and photoperiod on the reproductive traits of Cx. quinquefasciatus, an important vector of numerous pathogens of medical importance. The host use pattern of Cx. quinquefasciatus is interesting in that it feeds on birds during spring and shifts to feeding on mammals towards fall. Various hypotheses have been proposed to explain the seasonal shift in host use in this species but have provided limited evidence. This study examines whether the shifting of host classes from birds to mammals towards autumn offers any reproductive advantages to Cx. quinquefasciatus in terms of enhanced fecundity, fertility, and hatchability of the offspring. The authors found no evidence of this, suggesting that alternate mechanisms may drive the seasonal shift in host use in Cx. quinquefasciatus.
Strengths:
Host blood meal source, temperature, and photoperiod were all examined together.
Weaknesses:
The study was conducted in laboratory conditions with a local population of Cx. quinquefasciatus from Argentina. I'm not sure if there is any evidence for a seasonal shift in the host use pattern in Cx. quinquefasciatus populations from the southern latitudes.
We agree on the reviewers observation about the evidence on seasonal shift in the host use pattern in Cx. quinquefasciatus populations from southern latitudes. We include a paragraph in the Introduction section regarding this. Unfortunately, studies conducted in South America to understand host use by Culex mosquitoes are very limited, and there are virtually no studies on the seasonal feeding pattern. In Argentina, there is some evidence (Stein et al., 2013, Beranek, 2019) regarding the seasonal change in host use by Culex species, including Cx. quinquefasciatus, where the inclusion of mammals during the autumn has been observed. As part of a comprehensive study on characterising bridge vectors for SLE and WN viruses, our research group is currently working on the molecular identification of blood meals from engorged females to gain deeper insights into the seasonal feeding pattern of Culex mosquitoes.
While the seasonal change in host use by Culex quinquefasciatus has not been reported in Argentina so far, there has been an observed increase in reported cases of SLE virus in humans between summer and fall (Spinsanti et al., 2008). It is based on this evidence that we hypothesise there is a seasonal change in host use by Cx. quinquefasciatus, similar to what occurs in the United States. This is also considering that both countries (Argentina and the United States) have regions with similar climatic conditions (temperate climates with thermal and hydrological seasonality).
Since we work on the same species and in a similar temperate climate regimen, we assumed there is a seasonal shift in the host use by this mosquito species.
Reviewer #1 (Recommendations for the authors):
Abstract
Line 23: fed on two different hosts.
Accepted as suggested.
I think the concluding statement should be rewritten to say that immediate reproductive outcomes do not explain the shift in host use pattern of Cx. quinquefasciatus mosquitoes from birds to mammals towards autumn.
Accepted as suggested.
Introduction
No comments.
Materials and Methods
Please mention sample sizes in the text as well (n = ?) for each treatment.
Accepted as suggested.
Page 99: ......C. quinquefasciatus, since C. pipiens and its hybrids are present as well in Cordoba.
Accepted as suggested.
Results – Line 146: subsequently instead of posteriorly
Accepted all changes as suggested.
Line 148: were counted instead of was counted.
Accepted all changes as suggested.
Line 160: Subsequently instead of posteriorly
Accepted all changes as suggested.
Line 171: on fertility
Accepted all changes as suggested.
Line 174: there was an interaction effect on…
Accepted all changes as suggested.
Line 175: there were no differences in the number of eggs
Accepted all changes as suggested.
Discussion
I think the first paragraph in the discussion section is redundant and should be deleted.
The whole discussion was rewritten to be focused on our aims and results.
Line 282: this sentence needs to be rewritten.
Accepted as suggested.
Line 299: at 28{degree sign}C
Line 300: at 30{degree sign}C
Sorry, but we are not sure about your comment here. We checked. Temperatures are written as stated, 28°C and 30°C.
Line 363: I think the authors need to discuss more about the bigger question they were addressing. I think that the discussion section can be strengthened greatly by elaborating on whether there is evidence for a seasonal shift in host use pattern in Cx. quinquefasciatus in the southern latitudes. If yes, what alternate mechanisms they believe could be driving the seasonal change in host use in this species in the southern latitudes now that they show the 'deriving reproductive advantages' hypothesis to be not true for those populations.
Thanks for this observation. We agree and so the Discussion section was restructured to align it with our results, as suggested.
Reviewer #2 (Public Review):
Summary:
Conceptually, this study is interesting and is the first attempt to account for the potentially interactive effects of seasonality and blood source on mosquito fitness, which the authors frame as a possible explanation for previously observed host-switching of Culex quinquefasciatus from birds to mammals in the fall. The authors hypothesize that if changes in fitness by blood source change between seasons, higher fitness in birds in the summer and on mammals in the autumn could drive observed host switching. To test this, the authors fed individuals from a colony of Cx. quinquefasciatus on chickens (bird model) and mice (mammal model) and subjected each of these two groups to two different environmental conditions reflecting the high and low temperatures and photoperiod experienced in summer and autumn in Córdoba, Argentina (aka seasonality). They measured fecundity, fertility, and hatchability over two gonotrophic cycles. The authors then used a generalized linear mixed model to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity and fertility and a null model analysis via data randomization for hatchability. The authors were trying to test their hypothesis by determining whether there was an interactive effect of season and host species on mosquito fitness. This is an interesting hypothesis; if it had been supported, it would provide support for a new mechanism driving host switching. While the authors did report an interactive impact of seasonality and host species, the directionality of the effect was the opposite of that hypothesized. While this finding is interesting and worth reporting, there are significant issues with the experimental design and the conclusions that are drawn from the results, which are described below. These issues should be addressed to make the findings trustworthy.
Strengths:
(1) Using a combination of laboratory feedings and incubators to simulate seasonal environmental conditions is a good, controlled way to assess the potentially interactive impact of host species and seasonality on the fitness of Culex quinquefasciatus in the lab.
(2) The driving hypothesis is an interesting and creative way to think about a potential driver of host switching observed in the field.
Weaknesses:
(1) There is no replication built into this study. Egg lay is a highly variable trait, even within treatments, so it is important to see replication of the effects of treatment across multiple discrete replicates. It is standard practice to replicate mosquito fitness experiments for this reason. Furthermore, the sample size was particularly small for some groups (e.g. 15 egg rafts for the second gonotrophic cycle of mice in the autumn, which was the only group for which a decrease in fecundity and fertility was detected between 1st and 2nd gonotrophic cycles). Replicates also allow investigators to change around other variables that might impact the results for unknown reasons; for example, the incubators used for fall/summer conditions can be swapped, ensuring that the observed effects are not artefacts of other differences between treatments. While most groups had robust sample sizes, I do not trust the replicability of the results without experimental replication within the study.
We agree egg lay is a variable trait and so we consider high numbers of mosquitoes and egg lay during experiments compared to our studies of the same topics. Evaluating variables such as fecundity, fertility, or other types of variables (collectively referred to as "life tables") is a challenging issue that depends on several intrinsic and extrinsic factors. Because all of this, in some experiments, sample sizes might not be very large, and in several articles, lower sample sizes could be found. For instance, in Richards et al. (2012), for Culex quinquefasciatus, during the second gonotrophic cycle, some experiments had 13 or even 6 egg rafts. For species like Aedes aegypti, the sample size for life table analysis is also usually small. As an example, Muttis et al. (2018) reported between 1 and 4 engorged females (without replicates). In addition, small sample size would be a problem if we would not have obtained any effect, which is not the case due to the fact that we were interested in finding an effect, regardless of the effect size. Because of this, we do find our sample sizes quite robust for our results.
Regarding the need to repeat the experiments in order to give more robustness to the study we also agree. However, after a review of the literature (articles cited in the original manuscript), it is apparent that similar experiments are not frequently repeated as such. Examples of this are the studies of Richards et al. (2012), Demirci et al. (2014) or Telang & Skinner (2019), which even they manipulate several cages at a time as “replicates”, they are not true replicates because they summarise and manipulate all data together, and do not repeat the experiment several times. We see these “replicates” as a way of getting a greater N.
As was stated by the reviewer, repetition is a resource and time-consuming activity that we are not able to do. Replicating the experiment poses a significant time and resources challenge. The original experiment took over three months to complete, and it is anticipated that a similar timeframe would be necessary for each replication (6 months in total considering two more replicates). Given our existing commitments and obligations, dedicating such an extensive period solely to this would impede progress on other crucial projects and responsibilities.
Given the limitations of resources and time and the infrequent use of experimental replication in this type of studies, we performed a simulation-based analysis via a Monte Carlo approach. This approach involved generating synthetic data that mimics the expected characteristics of the original experiment and subsequently subjecting it to the same analysis routine. The main goal of this simulation was to evaluate the potential spuriousness and randomness of the results that might arise due to the experimental conditions. So, evaluating the robustness and confidence of our results and data.
(2) Considering the hypothesis is driven by the host switching observed in the field, this phenomenon is discussed very little. I do not believe Cx. quinquefasciatus host switching has been observed in Argentina, only in the northern hemisphere, so it is possible that the species could have an entirely different ecology in Argentina. It would have been helpful to conduct a blood meal analysis prior to this experiment to determine whether using an Argentinian population was appropriate to assess this question. If the Argentinian populations don't experience host switching, then an Argentinian colony would not be the appropriate colony to use to assess this question. Given that this experiment has already been conducted with this population, this possibility should at least be acknowledged in the discussion. Or if a study showing host switching in Argentina has been conducted, it would be helpful to highlight this in the introduction and discussion.
Thanks for this observation. We agree. However, we conducted the experiment beside host use data from Argentina since we used the mosquito species, and the centre region of Argentina (Córdoba) has a similar temperate weather regimen that those observed in the east coast of US.
We are aware that few studies regarding host shifting in South America are available, some such that those conducted by Stein et al. (2013) and Beranek (2019) reported a moderate host switch for Culex quinquefasciatus in Argentina. We have already performed a study about seasonal host feeding patterns for this species. However, even though there are few studies regarding host shifting, our hypothesis is based mainly in the seasonality of human cases of WNV and SLEV, a pattern that has been demonstrated for our region, see for example the study of Spinsanti et al. (2008).
We include a new paragraph in the Introduction and Discussion sections. Please see answers Reviewer #1.
(3) The impacts of certain experimental design decisions are not acknowledged in the manuscript and warrant discussion. For example, the larvae were reared under the same conditions to ensure adults of similar sizes and development timing, but this also prevents mechanisms of action that could occur as a result of seasonality experienced by mothers, eggs, and larvae.
We understand the confusion that may have arisen due to a lack of further details in the methodology. If we are not mistaken, you are referring to our oversight regarding the consideration of carry-over effects of larvae rearing that could potentially impact reproductive traits. When investigating the effects of temperature or other environmental factors on reproductive traits, it is possible to acclimate either larvae or adults. This is due to the significant phenotypic plasticity that mosquitoes exhibit throughout their entire ontogenetic cycle. In our study, we followed an approach similar to that of other authors where the adults are exposed to experimental conditions (temperature and photoperiod). For a similar approach you can refer to the studies conducted by Ferguson et al. (2018) for Cx. pipiens, Garcia Garcia & Londoño Benavides (2007) for Cx. quinquefasciatus or Christiansen-Jucht et al. (2014, 2015) for Anopheles gambiae.
(4) There are aspects of the data analysis that are not fully explained and should be further clarified. For example, there is no explanation of how the levels of categorical variables were compared.
The methodology and statistical analysis were expanded for a better understanding.
(5) The results show the opposite trend as was predicted by the authors based on observed feeding switches from birds to mammals in the autumn. However, they only state this once at the end of the discussion and never address why they might have observed the opposite trend as was hypothesized.
The discussion was restructured to focus on our results and our model.
(6) Generally speaking, the discussion has information that isn't directly related to the results and/or is too detailed in certain parts. Meanwhile, it doesn't dig into the meaning of the results or the ways in which the experimental design could have influenced results.
As mentioned above, the discussion was restructured to reflect our findings. We also included the effect that our design might have influenced our results. However, as stated above we do not fully agree that the design is inadequate for our analysis, we performed standard protocols followed by other researchers and studies in this research field.
(7) Beyond the issue of lack of replication limiting trust in the conclusions in general, there is one conclusion reached at the end of the discussion that would not be supported, even if additional replicates are conducted. The results do not show that physiological changes in mosquitoes trigger the selection of new hosts. Host selection is never measured, so this claim cannot be made. The results don't even suggest that fitness might trigger selection because the results show that physiological changes are in the opposite direction as what would be hypothesized to produce observed host switches. Similarly, the last sentence of the abstract is not supported by
the results.
We agree with this observation. However, we did not evaluate the impact of fitness on host selection in this study. Instead, we aimed to investigate the potential influence of seasonality on mosquito fitness as a potential trigger for a shift in host selection. We agree that we have incorrectly used the term “host selection” when we should actually be discussing “host use change”. Our results indicate a seasonal alteration in mosquito fitness in response to temperature and photoperiod changes. Building upon this observation, we re-discussed our hypothesis and theoretical model to explain this seasonal shift in host use.
(8) Throughout the manuscript, there are grammatical errors that make it difficult to understand certain sentences, especially for the results.
All English grammar and writing of the manuscript was revised and corrected to be easily understood.
This study is driven by an interesting question and has the potential to be a valuable contribution to the literature.
Reviewer #2 (Recommendations for The Authors):
I hope that the authors will consider the suggested revisions and experimental replication to improve the quality of the study and paper.
This study tests a very interesting hypothesis. I understand that additional replicates are difficult to conduct, but I do believe that fitness studies absolutely require experimental replicates. Unless you are able to replicate the observed effects, I personally would not trust the results of this study. I hope that you will consider conducting replicates so that this important question can be answered in a more robust manner. Below, I expand upon some additional points in the public review and also provide more specific suggestions. I provided some copy-editing feedback, but was not able to point out all grammatical mistakes. I suggest that you use ChatGPT to help you edit the English. For example, you can feed ChatGPT your MS and ask it to bold the grammatical errors or you can ask it to edit grammatical errors and bold the sections that were edited. I understand that writing in a second language is very difficult (from personal experience!), so I view ChatGPT as a great tool to help even the playing field for publishing. Below are line item suggestions. Apologies that wording is curt, I was trying to be efficient in writing.
20-21: I suggest that you emphasize that you are investigating the interactive effect.
Accepted as suggested.
22: they weren't "reared" (from larvae) in different conditions, they were "maintained" as adults
Accepted as suggested.
26-27: increased/decreased is a bit misleading since you did not evaluate these groups sequentially in time. It might be more accurate to describe it as less than/greater than. Also, if you say increased/decreased or less than/greater than, you should always say what you are comparing to. The same applies throughout the MS.
Accepted as suggested.
29-30: "finding the" is not correct here; could be "with the lowest..."
Accepted as suggested.
34-36: I do not think that your results suggest this, even if you were to replicate the results of this experiment. You haven't shown metabolic changes.
We understand the point. Accepted as suggested.
42-44: "one of the main responsible" should be "one of the main species responsible..."
Accepted as suggested.
48: I think that "host preference" is better than selection here; -philic denotes preference
Accepted as suggested.
50: "Moreover" isn't the correct transition word here
Accepted as suggested.
57: "could" isn't correct here; consider saying "... species sometimes feed primarily on mammal hosts, including humans, in certain situations."
Accepted as suggested.
58: Different isn't correct word here
Accepted as suggested.
60: delete "feeding"
Accepted as suggested.
66-68: I am not familiar with any blood meal analysis studies in the southern hemisphere that show host switching for Culex species between summer and autumn. If this hasn't been shown, then this critique of the host migration hypothesis doesn't make sense.
There are some studies pointing this out (Stein et al., 2013, Beranek 2019), and unpublished data from us). However, our hypothesis has supported by epidemiological data observed in human population which indicate a seasonal activity pattern. It was explained in depth in the Introduction section.
68: ensures is not the right word; I suggest "suggests"
Accepted as suggested.
68-70: this explanation isn't clear to me; please revise
It will be revised. Accepted as suggested.
70: change cares to care
Accepted as suggested.
76-77: can you explain how they were not supported by the data for the benefit of those who are not familiar with these papers please?
Accepted as suggested.
87-89: I suggest the following wording: "In the autumn, we expect a greater number of eggs (fecundity) and larvae (fertility) in mosquitoes after feeding on a mammal host compared to an avian host, and the opposite relationship in the summer."
Accepted as suggested.
99: edit for grammar
Accepted as suggested.
102: suggest: "...offered a blood meal from a restrained chicken twice a month"
Accepted as suggested.
107: powder
Accepted as suggested.
108: inbred? Is this the term you meant to use?
Changed as suggested.
109: "several" cannot be used to describe 20 generations; suggest using "over twenty generations"; also, it would be good to acknowledge in your discussion that lab adaptation could force evolution, especially since mosquitoes are kept at constant temperatures and fed with certain hosts (with easy access) in the lab. Also, it would be good to know when the experiments were conducted to know the lapse of time between the creation of the colony and the experiments.
Accepted as suggested.
110-111: Does humidity vary between summer and fall in Córdoba? If so, I suggest acknowledging in the discussion that if humidity differences are involved in a potential interaction between host species and seasonality, then this would not have been captured by your experimental design.
Several variables change during seasons. We were interested in capturing the effects of temperature and photoperiod, since humidity is a variable difficult to control.
113-116: I suggest combining into one sentence to make more concise.
Accepted as suggested.
135: You might be obscuring the true impact of seasonality by rearing the larvae under the same conditions. There may be signals that mothers/eggs/larvae receive that influence their behavior (e.g. I believe this is the case for diapause), so this limitation should also be acknowledged. I understand why you decided to do this to control for development time and size, but it is something that should be considered in the discussion.
As it was explained above, Cx. quinquefasciatus do not suffer diapause in our country. Maintaining mosquitoes from adults was an approach selected by us based on other studies.
138: edit: "with cotton pads soaked in... on plastic..."; what is plastic glass? Do you mean plastic dishes?
Accepted as suggested.
141: here and throughout paragraph, full should be "fully"
Accepted as suggested.
144: located should be "placed"
Accepted as suggested.
147: suggest editing to "at which point, they were fixed with 1 mL of 96% ethanol and the number of L1 larvae per raft was counted."
Accepted as suggested.
154-155: edit for grammar
Accepted as suggested.
157: Your GLM explanation doesn't say anything about how you made pairwise comparisons between your levels; did you use emmeans?
This revised version includes a more detailed methodology and statistical analysis. Accepted as suggested.
158-160: I don't understand why you took this approach - it seems strange to me to use this analysis, but I am not familiar with it, so it might be that I lack the knowledge to be able to adequately evaluate. Please provide more explanation so that readers can better understand this analysis. A citation for this kind of application of the analysis would be helpful.
It was changed to be in accordance with the remaining analyses.
173: replace neither with either
Accepted as suggested.
174: this applies throughout; edit to : "An interaction effect was observed..."
Accepted as suggested.
175: "it was not found" is grammatically incorrect; instead : "We did not find ..." or "no differences in... were detected", etc
Accepted as suggested.
183: "it was detected" is grammatically incorrect
Accepted as suggested.
185-186: "being this treatment... in terms of fitness": I do not understand what this means. Please rephrase
Accepted as suggested.
170-199: you should provide the effect sizes and p values in text and/or in the figure for the pairwise comparisons
Accepted as suggested.
193-196. These two sentences are confusing and I am not sure what you mean, especially in the first sentence.
It was rewritten. Accepted as suggested.
Figure 1: This figure is great and easy to read and interpret! Thank you for the comment!
218-219: it is important to state which mosquito species you are referring to here.
Accepted as suggested.
226-227: you definitely should acknowledge the small sample size here.
Considered.
227: "it was observed" should be "We observed" or "A greater hatching rate.... was observed."
Accepted as suggested.
228-229: is the result really comparable even though you took very different approaches to the analysis for these outcomes?
Changed to be comparable.
230-278: the discussion of these hypotheses is too long and detailed, especially since the comparison of mouse vs chicken wasn't your main question; you really wanted to understand this in the context of seasonality. I suggest cutting this down a lot and making room to dig into your results more, and also to discuss the potential impacts of your experimental design/limitations on the results.
Discussion was changed to focus on our results and model. Accepted as suggested.
281: Hoffman is an old citation; I suggest you cite a modern review.
Accepted as suggested. We deleted it due to the re-writing of the manuscript.
282: "It can be recognise".. I am not sure what you are trying to say here
Accepted as suggested.
- After the first time you write a species name, you can abbreviate the genus in all future mentions unless it is at the beginning of a sentence.
Accepted as suggested.
303-305: Revise this sentence. E.g "Fewer studies are available regarding photoperiod and show mixed results; Mogi (1992) found that mid and long day lengths induced greater fecundity while Costanzo et al. (2015) did not find differences in fecundity by day length."
Accepted as suggested.
315-316: typically, unpublished data shouldn't be referenced; I'm not sure if eLife has a policy on this.
We will check this with eLife guidelines. However, since the lack of evidence on this pattern we consider important to include this unpublished data.
316: Aegypti should be lowercase
Accepted as suggested.
328-330: This sentence is redundant with the first sentence of the paragraph
Accepted as suggested.
321-336: You never reintroduced your hypothesis in your discussion. I suggest that you center your whole discussion more directly around the hypothesis that motivated the study. If you decide not to restructure your discussion, you should at least reintroduce your hypothesis here and discuss how your results do not support the hypothesis.
Accepted as suggested.
337-348: This paragraph is a bit confusing as you jump between fertility and hatchability
Accepted as suggested.
353: is viral transmission the right word to use here? I think you might mean bridge vector transmission to humans specifically?
Accepted as suggested.
357: you say "neither" but never define which traits you are referring to
Accepted as suggested.
361: I suggest "two variables previously analyzed separately..."
Accepted as suggested.
General: There is no statement about the availability of data; it is eLife policy to require all data to be publicly available. Also, it would be helpful to share your code to help understand how you conducted pairwise comparisons, etc.
In the submission it was not mentioned anything about data availability. However, all data and scripts will be uploaded with the VOR if it is required.
Recommendations for the authors:
I found your study interesting and potentially promising. However, there are some fundamental problems with the study design and the hypothesis, including:
<(1) Seasonality simulation - Seasonality is strongly associated with time, so it is unusual to simulate seasonal factors without accounting for time. The actual factors associated with seasonal change in reproductive output may be neither a difference in host blood meal nor temperature and photoperiod. It is therefore, odd to reduce seasonality to a difference in photoperiod and temperature in summer and autumn without even mentioning the time of year when the experiment was carried (except for the mention of February as the time the stock samples were collected from the wild).
The temperature and photoperiod settings are established according to a representative day in both autumn and summer. To determine these settings, we utilized climate data spanning a 3-year period (2020-2022), encompassing the most frequently occurring temperatures and day lengths. The weather conditions remained notably consistent throughout this time frame, which is why the specific year was not mentioned. Moreover, including the year in laboratory experiment details is uncommon, as evident in various papers. This practice can be corroborated by referring to multiple sources (cited in the original manuscript). We mention this in the new version.
(2) Hypothesis - While the hypothesis alludes to the 'reason' for seasonal host shift, the prediction is on the outcome of the interaction between blood meal type and season.
It might be nicer to frame your hypothesis to be consistent with the aim, which is, testing the partial contributions of blood meal type, versus photoperiod and temperature to seasonal change in the reproductive output of Culex quinquefasciatus. A hypothesis like that can be accompanied by alternative predictions according to the expected individual and interactive effects of both factors.
It was rewritten in the revised version to be consistent with our predictions and findings.
Blood meal type, temperature, and photoperiod are all components of seasonality, so the strength of the study is its potential to decouple the effect of blood meal type from that of temperature and photoperiod on the seasonal reproductive output of Culex quinquefasciatus by comparing the two blood meal types under simulated summer and winter conditions. Ideally, this should have been over a natural summer and winter because a natural time difference captures the effect of other seasonal factors other than temperature and photoperiod.
Furthermore, the hypothesis stemmed from field observations, while the study itself was conducted under laboratory conditions using a local population of Culex quinquefasciatus from Argentina. It remains uncertain whether there is supporting evidence for a seasonal shift in host usage in Culex quinquefasciatus from the stock population. Discussing the field observations within the stock population would provide valuable insights.
It was considered in the new version.
