Effects of blood meal source and seasonality on reproductive traits of Culex quinquefasciatus (Diptera: Culicidae)

  1. Kevin Alen Rucci
  2. Gabriel Barco
  3. Andrea Onorato
  4. Mauricio Beranek
  5. Mariana Pueta
  6. Adrián Díaz

  • Curated by eLife

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    eLife Assessment

    This useful study provides the first assessment of potentially interactive effects of seasonality and blood source on mosquito fitness, together in one study. During revision, the manuscript has been substantively improved, providing additional solid data to support the robustness of observations. Overall, this interesting study will advance our current understanding of mosquito biology.

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Abstract

Host selection by mosquitoes is a keystone in understanding viral circulation and predicting future infection outbreaks. Culex mosquitoes frequently feed on birds during spring and early summer, shifting into mammals towards late summer and autumn. This host switch may be due to changes in mosquito fitness. The aim of this study was to assess if the interaction effect of blood meal source and seasonality may influence reproductive traits of Culex quinquefasciatus mosquitoes. For this purpose, Cx. quinquefasciatus mosquitoes were reared in simulated summer and autumn conditions and fed on two different hosts, chickens and mice, in a factorial design. Fecundity, fertility, and hatchability during two consecutive gonotrophic cycles were estimated. We found greater fecundity and fertility for mosquitoes fed upon birds than mammals. Fecundity and fertility did not vary between seasons for chicken-fed mosquitoes, whereas in autumn they decreased for mouse-fed mosquitoes. These traits decreased in the second gonotrophic cycle for mouse-fed mosquitoes, whereas they did not vary between cycles for chicken-fed mosquitoes. There was no statistically significant effect of blood meal source, seasonality or their interaction on hatchability, hence this variable was similar among treatments. Overall, these results indicate a statistically significant interaction effect of blood meal source and seasonality on fecundity and fertility. However, the pattern was opposite in relation to our hypothesis, suggesting that further studies are needed to confirm and expand our knowledge about mosquito biology and its relationship with seasonal host use shifting.

Article activity feed

  1. Version published to 10.7554/elife.89485.5 on eLife
  2. Version published to 10.7554/elife.89485 on eLife
  3. Version published to 10.7554/elife.89485.4 on eLife
  4. eLife

    eLife Assessment

    This useful study provides the first assessment of potentially interactive effects of seasonality and blood source on mosquito fitness, together in one study. During revision, the manuscript has been substantively improved, providing additional solid data to support the robustness of observations. Overall, this interesting study will advance our current understanding of mosquito biology.

  5. eLife

    Reviewer #1 (Public review):

    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.

  6. eLife

    Reviewer #2 (Public review):

    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 on 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 generalized linear mixed models to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity, fertility, and 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 from that hypothesized. The authors have done a very good job of addressing many of the reviewer's concerns, especially by adding two additional replicates.

  7. eLife

    Author response:

    The following is the authors’ response to the previous reviews

    We would like to thank you for your valuable comments and suggestions, which have greatly contributed to improving our manuscript.

    We have carefully addressed all the reviewers' suggestions, and detailed responses for each Reviewer are provided at the end of this letter. In summary:

    • The Introduction has been revised to provide a more focused discussion on results, toning down the speculative discussion on seasonal host shifts.

    • The methodology section has been clarified, particularly the power analysis, which now includes a clearer explanation. The random effects in the models have been better described to ensure transparency.

    • The Results section was reorganized to highlight the key findings more effectively.

    • The Discussion has been restructured for clarity and conciseness, ensuring the interpretation of the results is clearer and better aligned with the study objectives.

    • Minor edits throughout the manuscript were made to improve readability and accuracy.

    We hope you find this revised version of the manuscript satisfactory.

    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.

    Comments on the revision:

    Overall, the manuscript is much improved. However, the introduction and parts of the discussion that talk about addressing the question of seasonal shift in host use pattern of Cx. quin are still way too strong and must be toned down. There is no strong evidence to show this host shift in Argentinian mosquito populations. Therefore, it is just misleading. I suggest removing all this and sticking to discussing only the effects of blood meal source and seasonality on the reproductive outcomes of Cx. quin.

    Introduction and discussion have been modified, toned down and sticked to discuss the results as suggested.

    Reviewer #1 (Recommendations for the authors):

    Some more minor comments are mentioned below.

    Line 51: Because 'of' this,

    Changed as suggested.

    Line 56: specialists 'or' generalists

    Changed as suggested.

    Line 56: primarily

    Changed as suggested.

    Line 98: Because 'of' this,

    Changed 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 hostswitching 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 on 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 generalized linear mixed models to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity, fertility, and 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 from that hypothesized. The authors have done a very good job of addressing many of the reviewer's concerns, especially by adding two additional replicates. Several minor concerns remain, especially regarding unclear statements in the discussion.

    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) The methods would be improved by some additional details. For example, clarifying the number of generations for which mosquitoes were maintained in colony (which was changed from 20 to several) and whether replicates were conducted at different time points.

    Changed as suggested.

    (2) The statistical analysis requires some additional explanation. For example, you suggest that the power analysis was conducted a priori, but this was not mentioned in your first two drafts, so I wonder if it was actually conducted after the first replicate. It would be helpful to include further detail, such as how the parameters were estimated. Also, it would be helpful to clarify why replicate was included as a random effect for fecundity and fertility but as a fixed effect for hatchability. This might explain why there were no significant differences for hatchability given that you were estimating for more parameters.

    The power analysis was conducted a posteriori, as you correctly inferred. While I did not indicate that it was performed a priori, you are right in noting that this was not explicitly mentioned. As you suggested, the methodology for the power analysis has been revised to clarify any potential doubts.

    Regarding the model for hatchability, a model without a random effect variable was used, as all attempts to fit models with random effects resulted in poor validation. These points have now been clarified and explained in the corresponding section.

    (3) A number of statements in the discussion are not clear. For example, what do you mean by a mixed perspective in the first paragraph? Also, why is the expectation mentioned in the second paragraph different from the hypothesis you described in your introduction?

    Changed as suggested.

    (4) According to eLife policy, data must be made freely available (not just upon request).

    Data and code will be publicly available. The corresponding section was modified.

    Reviewer #2 (Recommendations for the authors):

    Your manuscript is much improved by the inclusion of two additional replicates! The results are much more robust when we can see that the trends that you report are replicable across 3 iterations of the experiment. Congratulations on a greatly improved study and paper! I have several minor concerns and suggestions, listed below:

    38-39: I think it is clearer to say "no statistically significant effect of season on hatchability of eggs" ... or specify if you are referring to blood or the interaction of blood and season. It isn't clear which treatment you are referring to here.

    Changed as suggested.

    54-57: This could be stated more succinctly. Instead of citing papers that deal with specific examples of patterns, I would suggest citing a review paper that defines these terms.

    Changed as suggested.

    83-84: What if another migratory bird is the preferred host in Argentina? I would state this more cautiously (e.g. "may not be applicable...").

    Changed as suggested.

    95-96: I don't understand what you mean by this. These hypotheses are specifically meant to understand mosquitoes that DO have a distinct seasonal phenology, so I'm not sure why this caveat is relevant. And naturally this hypothesis is host dependent, since it is based on specific host reproductive investments. I think that the strongest caveat to this hypothesis is simply that it hasn't been proven.

    Changed as suggested.

    97-115: This is a great paragraph! Very clear and compelling.

    Thanks for your words!

    118: Do you have an exact or estimated number of rafts collected?

    Sorry, I have not the exact number of rafts, but it was at leas more than 20-30.

    135: "over twenty" was changed to "several"; several would imply about 3 generations, so this is misleading. If the colony was actually maintained for over twenty generations, then you should keep that wording.

    Changed as suggested.

    163-164: Can you please clarify whether the replicates were conducted a separate time points?

    Changed as suggested.

    Note: the track changes did not capture all of the changes made; e.g. 163-164 should show as new text but does not.

    You are absolutely right; when I uploaded the last version, I unfortunately deleted all tracked changes and cannot recover them. In this new version, I will ensure that all minimal changes are included as tracked changes.

    186 - 189: the terms should be "fixed effect" and "random effect"

    Changed as suggested.

    191: Edit: linear

    Changed as suggested.

    194: why was replicate not included as a random effect here when it was above? Also, can you please clarify "interaction effects"? Which interactions did you include?

    Changed as suggested. Explained above and in methodology. Hatchability models with random effect variable were poor fitted and validated. The interactions for hatchability were a four-way (season, blood source, cycle and replicate)

    207-208: I'm not sure what you mean by "aimed to achieve"? Weren't you doing this after you conducted the experiments, so wouldn't this be determining the power of your model (post-hoc power analysis)? Also, I think you should provide the parameter estimates that were used (e.g. effect size - did you use the effect size you estimated across the 3 replicates?).

    Changed as suggested.

    214-215: this should be reworded to acknowledge that this is estimated for the given effect size; for example, something like "This sample size was sufficient to detect the observed effect with a statistical power of 0.8" or something along those lines (unless I am misunderstanding how you conducted this test).

    Changed as suggested.

    246. Abbreviate Culex

    Changed as suggested.

    253-255: This sentence isn't clear. What do you mean by mixed? Also, the season really seemed to mainly impact the fitness of mosquitoes fed on mouse blood and here the way it is phrased seems to indicate that season has an impact on the fitness of those fed with chicken blood.

    Changed as suggested.

    258-260: You stated your hypothesis as the relative fitness shifting between seasons, but this statement about the expectation is different from your hypothesis stated earlier. Please clarify.

    You are right. Thank you for noting this. It was changed as suggested.

    263-266: I also don't understand this sentence; what does the first half of the sentence have to do with the second?

    Changed as suggested.

    269-270: This doesn't align with your observation exactly; you say first AND second are generally most productive, but you observed a drop in the second. Please clarify this.

    Changed as suggested.

    280: I suggest removing "as same as other studies"; your caveats are distinct because your experimental design was unique

    Changed as suggested.

    287: you shouldn't be looking for a "desired" effect; I suggest removing this word

    Changed as suggested.

    288: It wasn't really a priori though, since you conducted it after your first replicate (unless you didn't use the results from the first replicate you reported in the original drafts?)

    It was a posteriori. Changed as suggested.

    290: Why is 290 written here?

    It was a mistype. Deleted as suggested.

    291-298: The meaning of this section of your paragraph is not clear.

    Improve as suggested.

    304-313: This list of 3 explanations are directed at different underlying questions. Explanations 1 and 2 are alternative explanations for why host switching occurs if not due to differences in fitness. This isn't really an explanation of your results so much as alternative explanations for a previously reported phenomenon. And the third is an explanation for why you may not have observed the expected effect. I suggest restructuring this to include the fact that Argentinian quinqs may not host switch as part of your previous list of caveats. Then you can include your two alternative explanations for host switching as a possible future direction (although I would say that it is really just one explanation because "vector biology" is too broad of a statement to be testable). Also, you haven't discussed possible explanations for your actual result, which showed that mosquito fitness decreased when feeding on mouse blood in autumn conditions and in the second gonotrophic, while those that fed on chicken did not experience these changes. Why might that be?

    The discussion was restructured to include all these suggested changes. Additionally, it was also discussed some possible explanations of our results.

    315-317: This statement is vague without a direct explanation of how this will provide insight. I suggest removing or providing an explanation of how this provides insight to transmission and forecasting.

    Changed as suggested.

    319-320: According to eLife policy, all data should be publicly available. From guidelines: "Media Policy FAQs Data Availability Purpose and General Principles To maintain high standards of research reproducibility, and to promote the reuse of new findings, eLife requires all data associated with an article to be made freely and widely available. These must be in the most useful formats and according to the relevant reporting standards, unless there are compelling legal or ethical reasons to restrict access. The provision of data should comply with FAIR principles (Findable, Accessible, Interoperable, Reusable). Specifically, authors must make all original data used to support the claims of the paper, or that is required to reproduce them, available in the manuscript text, tables, figures or supplementary materials, or at a trusted digital repository (the latter is recommended). This must include all variables, treatment conditions, and observations described in the manuscript. The authors must also provide a full account of the materials and procedures used to collect, pre-process, clean, generate and analyze the data that would enable it to be independently reproduced by other researchers."

    - so you need to make your data available online; I also understand the last sentence to indicate that code should be made available.

    Data and code will be publicly available.

    Table 1: it is notable that in replicate 2, the autumn:mouse:gonotrophic cycle II fecundity and fertility are actually higher than in the summer, which is the opposite of reps 1 and 3 and the overall effect you reported from the model. This might be worth mentioning in the discussion.

    Mentioned in the discussion as suggested.

    Tables 1 and 2: shouldn't this just be 8 treatments? You included replicate as a random effect, so it isn't really a separate set of treatments.

    This table reflects the output of the whole experiment, that is why it is present the 24 expetiments.

    Figure 3: Can you please clarify if this is showing raw data?

    Changed as suggested.

    Note: grammatical copy editing would be beneficial throughout

    Grammar was improved as suggested.

  8. Version published to 10.1101/2023.06.30.546984v5 on bioRxiv
  9. Version published to 10.7554/elife.89485.3 on eLife
  10. eLife

    eLife Assessment

    This useful study provides the first assessment of potentially interactive effects of seasonality and blood source on mosquito fitness, together in one study. During revision, the manuscript has been improved, providing additional solid data to support the robustness of observations. However, the discussion still requires further refinement to present the conclusions in manner that is consistent with the data presented. Overall, this interesting study will advance our current understanding of mosquito biology.

  11. eLife

    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.

    Comments on the revision:

    Overall, the manuscript is much improved. However, the introduction and parts of the discussion that talk about addressing the question of seasonal shift in host use pattern of Cx. quin are still way too strong and must be toned down. There is no strong evidence to show this host shift in Argentinian mosquito populations. Therefore, it is just misleading. I suggest removing all this and sticking to discussing only the effects of blood meal source and seasonality on the reproductive outcomes of Cx. quin.

  12. eLife

    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 on 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 generalized linear mixed models to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity, fertility, and 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 from that hypothesized. The authors have done a very good job of addressing many of the reviewer's concerns, especially by adding two additional replicates. Several minor concerns remain, especially regarding unclear statements in the discussion.

    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) The methods would be improved by some additional details. For example, clarifying the number of generations for which mosquitoes were maintained in colony (which was changed from 20 to several) and whether replicates were conducted at different time points.
    (2) The statistical analysis requires some additional explanation. For example, you suggest that the power analysis was conducted a priori, but this was not mentioned in your first two drafts, so I wonder if it was actually conducted after the first replicate. It would be helpful to include further detail, such as how the parameters were estimated. Also, it would be helpful to clarify why replicate was included as a random effect for fecundity and fertility but as a fixed effect for hatchability. This might explain why there were no significant differences for hatchability given that you were estimating for more parameters.
    (3) A number of statements in the discussion are not clear. For example, what do you mean by a mixed perspective in the first paragraph? Also, why is the expectation mentioned in the second paragraph different from the hypothesis you described in your introduction?
    (4) According to eLife policy, data must be made freely available (not just upon request).

  13. eLife

    Author response:

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

    We have carefully addressed all the reviewers' suggestions, and detailed responses are provided at the end of this letter. In summary:

    • We conducted two additional replicates of the study to obtain more robust and reliable data.

    • The Introduction has been revised for greater clarity and conciseness.

    • The Results section was shortened and reorganized to highlight the key findings more effectively.

    • The Discussion was modified according to the reviewers' suggestions, with a focus on reorganization and conciseness.

    We hope you find this revised version of the manuscript satisfactory.

    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.

    Comments on the revision:

    Overall, I am not quite convinced about the possible shift in host use in the Argentinian populations of Cx. quinquefasciatus. The evidence from the papers that the authors cite is not strong enough to derive this conclusion. Therefore, I think that the introduction and discussion parts where they talk about host shift in Cx. quinquefasciatus should be removed completely as it misleads the readers. I suggest limiting the manuscript to talking only about the effects of blood meal source and seasonality on the reproductive outcomes of Cx. quinquefasciatus.

    As mentioned in the previous revision, we agree on the reviewer observation about the lack of evidence on seasonal shift in the host use pattern in Cx. quinquefasciatus populations from Argentina. We include this topic in the discussion.

    Additionally, we also added a paragraph in the discussion section to include the limitations of our study and conclusions. One of them is the fact that our results are based on controlled conditions experiments. Future studies are needed to elucidate if the same trend is found in the field.

    Reviewer #1 (Recommendations for the authors):

    Abstract

    Line 73: shift in feeding behavior

    Accepted as suggested.

    Discussion

    Line 258: addressed that Accepted as suggested.

    Line 263: blood is nutritionally richer

    Accepted 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 on 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 model to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity, fertility, and 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 from that hypothesized. The authors have done a very good job of addressing many of the reviewer concerns, with several exception that continue to cause concern about the conclusions of the study.

    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.

    (3) The manuscript has become a lot clearer and easier to read with the revisions - thank you to the authors for working hard to make many of the suggested changes.

    Weaknesses:

    (1) The authors have decided not to follow the suggestion of conducting experimental replicates of the study. This is understandable given the significant investment of resources and time necessary, however, it leaves the study lacking support. Experimental replication is an important feature of a strong study and helps to provide confidence that the observed patterns are real and replicable. Without replication, I continue to lack confidence in the conclusions of the study.

    We included replicates as suggested.

    (2) The authors have included some additional discussion about the counterintuitive nature of their results, but the paragraph discussing this in the discussion was confusing. I believe that this should be revised. This is a key point of the paper and needs to be clear to the reader.

    Revised as suggested.

    (3) There should be more discussion of the host switching observed in the two studies conducted in Argentina referenced by the authors. Since host switching is the foundation for the hypothesis tested in this paper, it is important to fully explain what is currently known in Argentina.

    Accepted as suggested.

    (4) In some cases, the explanations of referenced papers are not entirely accurate. For example, when referencing Erram et al 2022, I think the authors misrepresented the paper's discussion regarding pre-diuresis- Erram et al. are suggesting that pre-diuresis might be the mechanism by which C. furens compensates for the lower nutritional value of avian blood, leading to no significant difference between avian/mammal blood on fecundity/fertility (rather than leading to higher fecundity on birds, as stated in this manuscript). The study performed by Erram et al. also didn't prove this phenomenon, they just suggest it as a possible mechanism to explain their results, so that should be made clear when referencing the paper.

    Changed as suggested.

    (5) In some cases, the conclusions continue to be too strongly worded for the evidence available. For example, lines 322-324: I don't think the data is sufficient to conclude that a different physiological state is induced, nor that they are required to feed on a blood source that results in higher fitness.

    Redaction was modified as suggested to tight our discussion with results.

    (6) There is limited mention of the caveat that this experiment performed with simulated seasonality that does not perfectly replicate seasonality in the field. I think this caveat should be discussed in the discussion (e.g. that humidity is held constant).

    This topic is now included in the discussion as suggested.

    Reviewer #2 (Recommendations for the authors):

    59-60: These terms should end with -phagic instead of -philic. These papers study blood feeding patterns, not preference. I understand that the Janssen papers calls it "mammalophilic" in their title, but this was an incorrect use of the term in their paper. There are some review papers that explain the difference in this terminology if it's helpful.

    Accepted as suggested.

    73: edit to "in" feeding behavior

    Accepted as suggested.

    77-78: Given that the premise of your study is based on the phenomenon of host switching, I suggest that you expand your discussion of these two papers. What did they observe? Which hosts did they switch from / to and how dramatic was the shift?

    Accepted as suggested.

    79: replace acknowledged with experienced

    Accepted as suggested.

    79-80: the way that this is written is misleading. It suggests that Spinsanti showed that seasonal variation in SLEV could be attributed to a host shift, which isn't true. This citation should come before the comma and then you should use more cautious language in the second half. E.g which MIGHT be possible to attribute to ....

    Accepted as suggested.

    80-82: this is not convincing. Even if the Robin isn't in Argentina, Argentina does have migrating birds, so couldn't this be the case for other species of birds? Do any of the birds observed in previous blood meal analyses in Argentina migrate? If so, couldn't this hypothesis indeed play a role?

    A paragraph about this topic was added to the discussion as suggested.

    90: hypotheses for what? The fall peak in cases? Or host switching?

    Changed to be clearer.

    98: where was this mentioned before? I think "as mentioned before" can be removed.

    Accepted as suggested.

    101: edit to "whether an interaction effect exists"

    Accepted as suggested.

    104: edit to "We hypothesize that..."

    Accepted as suggested.

    106: reported host USE changes, not host PREFERENCE changes, right?

    All the terminology was change to host pattern and not preference to avoid confusion.

    200: Briefly reading Carsey and Harden, it looks like the methodology was developed for social science. Is there anything you can cite to show this applied to other types of data? If not, I think this requires more explanation in your MS.

    This was removed as replicates were included.

    237-239: I think it is best not to make a definitive statement about greater/higher if it isn't statistically significant; I suggest modifying the sentences to state that the differences you are listing were not significantly different up front rather than at the end, otherwise if people aren't reading carefully, they may get the wrong impression.

    Accepted as suggested.

    245: you only use the term MS-I once before and I forgot what it meant since it wasn't repeated, so I had to search back through with command-F. I suggest writing this out rather than using the acronym.

    Accepted as suggested.

    249: edit to: "an interaction exists between the effect of..."

    Accepted as suggested.

    253-254: greater compared to what?

    Change for clearness. 258-260: edit for grammar

    Accepted as suggested.

    260-262: edit for grammar; e.g. "However, this assumption lacks solid evidence; there is a scarcity of studies regarding nutritional quality of avian blood and its impact on mosquito fitness."

    Accepted as suggested.

    263: edit: blood is nutritionally...

    Accepted as suggested.

    264-267: This doesn't sound like an accurate interpretation of what the paper suggests regarding pre-diuresis in their discussion - they are suggesting that pre-diuresis might be the mechanism by which C. furens compensates for the lower nutritional value of avian blood, leading to no significant difference between avian/mammal blood on fecundity/fertility. They also don't show this, they just suggest it as a possible mechanism to explain their results.

    This topic was removed given the restructuring of discussion.

    253-269: You should tie this paragraph back to your results to explicitly compare/contrast your findings with the previous literature.

    Accepted as suggested.

    270-282: This paragraph would be a good place to explain the caveat of working in the laboratory - for example, humidity was the same across the two seasons which I'm guessing isn't the case in the field in Argentina. You can discuss what aspects of laboratory season simulation do not accurately replicate field conditions and how this can impact your findings. You said in your response to the reviewers that you weren't interested in measuring other variables (which is fair, and not expected!), but the beauty of the discussion section is to be able to think about how your experimental design might impact your results - one possibility is that your season simulation may not have produced the results produced by true seasonal shifts.

    Accepted as suggested.

    279-281: You say your experiment was conducted within the optimal range, which would suggest that both summer and autumn were within that range, but then you only talk about summer as optimal in the following sentence.

    Changed for clearness.

    281-282: You should clarify this sentence - state what the interaction has an effect on.

    Accepted as suggested.

    283-291: I appreciate that your discussion now acknowledges the small sample size and the questions that remain unanswered due to the results being opposite to that of the hypothesis, but this paragraph lacks some details and in places doesn't make sense.

    I think you need to emphasize which groups had small sample size and which conclusions that might impact. I also think you need to explain why the sample size was substantially smaller for some groups (e.g. did they refuse to feed on the mouse in the autumn?). I appreciate that sample sizes are hard to keep high across many groups and two gonotrophic periods, but unfortunately, that is why fitness experiments are so hard to do and by their nature, take a long time. I understand that other papers have even lower sample size, but I was not asked to review those papers and would have had the same critique of them. I don't believe that creating simulated data via a Monte Carlo approach can make up for generating real data. As I understand it from your explanation, you are parametrizing the Monte Carlo simulations with your original data, which was small to begin with for autumn mouse. Using this simulation doesn't seem like a satisfactory replacement for an experimental replicate in my opinion. I maintain that at least a second replicate is necessary to see whether the patterns that you have observed hold.

    The performing of a power analysis and addition of more replicates tried to solve the issue of sample size. More about this critic is added in the discussion. The simulation approach was totally removed.

    Regarding the directionality of the interaction effect, I think this warrants more discussion. Lines 287-291 don't make sense to me. You suggest that feeding on birds in the autumn may confer a reproductive advantage when conditions are more challenging. But then why wouldn't they preferentially feed on birds in the autumn, rather than mammals? I suggest rewriting this paragraph to make it clearer.

    Accepted as suggested.

    297: earlier mentioned treatments? Do you mean compared to the first gonotrophic cycle? This isn't clear.

    Changed for clearness.

    302-303: Did you clarify whether you are allowed to reference unpublished data in eLife?

    This was removed to follow the guidelines of eLife.

    316-317: "it becomes apparent" sounds awkward, I suggest rewording and also explaining how this conclusion was made.

    Accepted as suggested.

    322-324: I think that this statement is too strongly worded. I don't think your data is sufficient to conclude that a different physiological state is induced, nor that they are required to feed on a blood source that results in higher fitness. Please modify this and make your conclusions more cautious and closely linked to what you actually demonstrated.

    Accepted as suggested.

    325: change will perform to would have

    Accepted as suggested.

    326: add to the sentence: "and vice versa in the summer"

    Accepted as suggested.

    330: possible explanations, not explaining scenarios.

    Accepted as suggested.

    517: I think you should repeat the abbreviation definitions in the caption to make it easier for readers, otherwise they have to flip back and forth which can be difficult depending on formatting.

    Accepted as suggested.

    In general, I think that your captions need more information. I think the best captions explain the figure relatively thoroughly such that the reader can look at the figure and caption and understand without reading the paper in depth. (e.g. the statistical test used).

    Data availability: The eLife author instructions do say that data must be made available, so there should be a statement on data availability in your MS. I also suggest you make the code available.

    Accepted as suggested.

  14. Version published to 10.1101/2023.06.30.546984v4 on bioRxiv
  15. Version published to 10.7554/elife.89485.2 on eLife
  16. eLife

    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.

    1. 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.

  17. eLife

    eLife assessment

    This useful study provides the first assessment of the potentially interactive effects of seasonality and blood source on mosquito fitness, together in one study. However, the experimental approach is incomplete because it is limited without replication of the experiments and because of the small sample sizes for some groups. The work will be of interest to those studying mosquito biology.

  18. eLife

    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.

    Comments on the revision:

    Overall, I am not quite convinced about the possible shift in host use in the Argentinian populations of Cx. quinquefasciatus. The evidence from the papers that the authors cite is not strong enough to derive this conclusion. Therefore, I think that the introduction and discussion parts where they talk about host shift in Cx. quinquefasciatus should be removed completely as it misleads the readers. I suggest limiting the manuscript to talking only about the effects of blood meal source and seasonality on the reproductive outcomes of Cx. quinquefasciatus.

  19. eLife

    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 on 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 model to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity, fertility, and 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 from that hypothesized. The authors have done a very good job of addressing many of the reviewer concerns, with several exception that continue to cause concern about the conclusions of the study.

    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.
    (3) The manuscript has become a lot clearer and easier to read with the revisions - thank you to the authors for working hard to make many of the suggested changes.

    Weaknesses:

    (1) The authors have decided not to follow the suggestion of conducting experimental replicates of the study. This is understandable given the significant investment of resources and time necessary, however, it leaves the study lacking support. Experimental replication is an important feature of a strong study and helps to provide confidence that the observed patterns are real and replicable. Without replication, I continue to lack confidence in the conclusions of the study.
    (2) The authors have included some additional discussion about the counterintuitive nature of their results, but the paragraph discussing this in the discussion was confusing. I believe that this should be revised. This is a key point of the paper and needs to be clear to the reader.
    (3) There should be more discussion of the host switching observed in the two studies conducted in Argentina referenced by the authors. Since host switching is the foundation for the hypothesis tested in this paper, it is important to fully explain what is currently known in Argentina.
    (4) In some cases, the explanations of referenced papers are not entirely accurate. For example, when referencing Erram et al 2022, I think the authors misrepresented the paper's discussion regarding pre-diuresis- Erram et al. are suggesting that pre-diuresis might be the mechanism by which C. furens compensates for the lower nutritional value of avian blood, leading to no significant difference between avian/mammal blood on fecundity/fertility (rather than leading to higher fecundity on birds, as stated in this manuscript). The study performed by Erram et al. also didn't prove this phenomenon, they just suggest it as a possible mechanism to explain their results, so that should be made clear when referencing the paper.
    (5) In some cases, the conclusions continue to be too strongly worded for the evidence available. For example, lines 322-324: I don't think the data is sufficient to conclude that a different physiological state is induced, nor that they are required to feed on a blood source that results in higher fitness.
    (6) There is limited mention of the caveat that this experiment performed with simulated seasonality that does not perfectly replicate seasonality in the field. I think this caveat should be discussed in the discussion (e.g. that humidity is held constant).

  20. Version published to 10.1101/2023.06.30.546984v3 on bioRxiv
  21. Version published to 10.7554/elife.89485.1 on eLife
  22. eLife

    Author Response

    Reviewer #1 (Public Review):

    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.

    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 pattern of host use. In Argentina, there is some evidence (Stein et al., 2013; Beranek, 2018) regarding the seasonal change in host use by Culex species, including Culex quinquefasciatus, where the inclusion of mammals during the autumn has been observed. As part of a comprehensive study on characterizing 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 host use by 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 autumn (Spinsanti et al., 2008). It is based on this evidence that we hypothesize there is a seasonal change in host use by Culex 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).

    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.

    We will restructure our discussion to align it with our results, as suggested.

    Grammar and writing

    The manuscript will be grammatically revised.

    Reviewer #2 (Public Review):

    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 artifacts 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 of 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). 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 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 it 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 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 repetition in this type of studies, we suggest performing a simulation-based analysis. This approach involves 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 will be to evaluate the potential spuriousness and randomness of the results that might arise due to the experimental conditions. We will introduce this simulation-based analysis in the next revised version of the manuscript.

    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.

    We are aware that few studies regarding host shifting in South America are available, some such those conducted by Stein et al. (2013) and Beranek (2018) reported a moderate host switch for Culex quinquefasciatus in Argentina. We have already performed a study about seasonal host feeding patterns for this species. As you suggested, we could mention it in the discussion to highlight our partial findings. 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).

    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 and Christiansen-Jucht et al. (2014, 2015) for Anopheles gambiae.

    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 will discuss into our hypotheses and theoretical model to explain this seasonal shift in host use.

    Grammar and writing

    The manuscript will be grammatically revised by a professional translator.

  23. eLife

    eLife assessment

    This useful study presents convincing evidence that blood meal source and season affect Culex quinquefasciatus mosquito reproduction. Its unique focus on the interactive effects of both factors on mosquito fitness is of considerable relevance for the field, but the work suffers from inadequate experimental design - no replication, a population mismatch with the hypothesis region, and small sample sizes, limitations that were not sufficiently acknowledged in the discussion. The work will be of interest to those studying malaria and vector-borne diseases.

  24. eLife

    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.

  25. eLife

    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 artifacts 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.
    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.
    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.
    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.
    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.
    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.
    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.
    8. Throughout the manuscript, there are grammatical errors that make it difficult to understand certain sentences, especially for the results.

    This study is driven by an interesting question and has the potential to be a valuable contribution to the literature.

  26. Version published to 10.1101/2023.06.30.546984v2 on bioRxiv
  31. Version published to 10.1101/2023.06.30.546984v1 on bioRxiv