Lack of abundant core virome in Culex mosquitoes from a temperate climate region despite a mosquito species-specific virome

ABSTRACT In arthropod-associated microbial communities, insect-specific viruses (ISVs) are prevalent yet understudied due to limited infectivity outside their natural hosts. However, ISVs might play a crucial role in regulating mosquito populations and influencing arthropod-borne virus transmission. Some studies have indicated a core virome in mosquitoes consisting of mostly ISVs. Employing single mosquito metagenomics, we comprehensively profiled the virome of native and invasive mosquito species in Belgium. This approach allowed for accurate host species determination, prevalence assessment of viruses and Wolbachia, and the identification of novel viruses. Contrary to our expectations, no abundant core virome was observed in Culex mosquitoes from Belgium. In that regard, we caution against rigidly defining mosquito core viromes and encourage nuanced interpretations of other studies. Nonetheless, our study identified 45 viruses of which 28 were novel, enriching our understanding of the mosquito virome and ISVs. We showed that the mosquito virome in this study is species-specific and less dependent on the location where mosquitoes from the same species reside. In addition, because Wolbachia has previously been observed to influence arbovirus transmission, we report the prevalence of Wolbachia in Belgian mosquitoes and the detection of several Wolbachia mobile genetic elements. The observed prevalence ranged from 83% to 92% in members from the Culex pipiens complex. IMPORTANCE Culex pipiens mosquitoes are important vectors for arboviruses like West Nile virus and Usutu virus. Virome studies on individual Culex pipiens, and on individual mosquitoes in general, have been lacking. To mitigate this, we sequenced the virome of 190 individual Culex and 8 individual Aedes japonicus mosquitoes. We report the lack of a core virome in these mosquitoes from Belgium and caution the interpretation of other studies in this light. The discovery of new viruses in this study will aid our comprehension of insect-specific viruses and the mosquito virome in general in relation to mosquito physiology and mosquito population dynamics.

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Reviewer #1 (Comments for the Author): Review of Manuscript: "Lack of abundant core virome in Culex mosquitoes from a temperate climate region despite a mosquito species-specific virome" I had the privilege of reviewing the manuscript, and I must commend the authors for their excellent work.The study delves into the intricate world of insect-specific viruses (ISVs) within arthropod-associated microbial communities, offering valuable insights into the virome of native and invasive mosquito species in Belgium.

Strengths:
1. Clarity and Conciseness: The manuscript is exceptionally well-written, with a clear and concise presentation of methods and results.The authors effectively communicate complex concepts, ensuring accessibility for a broad readership.2. Innovative Approach:The utilization of single mosquito metagenomics is a commendable approach that allows for accurate host species determination and the identification of novel viruses.This innovative methodology significantly contributes to the field.3. Novel Discoveries: The identification of 45 viruses, including 28 novel ones, enriches our understanding of the mosquito virome and ISVs.The species-specific nature of the mosquito virome, irrespective of geographical location, is a noteworthy finding.
Constructive Feedback: 1. Introduction Enhancement: I would recommend incorporating information about the World Mosquito Program (WMP) in the introduction.This addition would contextualize the study within broader mosquito control efforts and potentially enhance its relevance to a wider audience.2. Bacterial Genomes Exploration: Consider expanding the exploration of bacterial genomes beyond Wolbachia.Providing additional context and insights into other bacterial elements could further enrich the study.3. Discussion Expansion: The discussion section could be extended to delve deeper into the implications of the findings.This may include further interpretation of results, addressing study limitations, and proposing avenues for future research.Connecting the study's outcomes to practical applications in mosquito control or disease prevention would strengthen the impact of the research.

Conclusion:
In conclusion, this manuscript represents a significant contribution to the field of mosquito virology.The meticulous methodology, coupled with novel discoveries, positions this work as a valuable reference for researchers and practitioners alike.I commend the authors on their exemplary work and look forward to witnessing the continued impact of their research in the scientific community.
Reviewer #2 (Comments for the Author): The manuscript by de Coninck et al. describes the diversity of viruses found in individual mosquitoes collected in different sites in Belgium.Mosquitoes belonged to three species, including two species of the Culex genus and one Aedes species.The authors screened 190 mosquito individuals in total using metagenomics.Mosquitoes were also screened for the presence of six viruses by RT-PCR.Moreover, the authors determined Wolbachia prevalence from the sequencing data obtained with metagenomics.The authors present analyses of the virus communities and phylogenies of the viruses found in their mosquito collection, including the description of new species.The main result of the manuscript, as defined by the title and the abstract, is the lack of a clear support for the existence of a core virome in the studied populations.This claim mainly derives from the limited diversity overlap in the virome of individual mosquitoes observed with metagenomics.I see two problems in the manuscript suggesting that the results do not robustly support this claim.First, assessing a core virome requires a diagnostic test allowing to robustly infer presence/absence of viruses in the samples.The approach used by the author does not seem to fully comply with this requirement.No virus was found in a large number of mosquitoes (61 %).I have not found an explanation for this result or a comparison with other studies.Few studies have analyzed individual mosquitoes with metagenomics but at least two of them (including a study from the same group) did not find such a high proportion of negative samples (references 8 and 12 in the manuscript).The low number of virus-positive samples thus suggest a sensibility problem (either due to the method or the sample quality).Secondly, there seems to be contradictory results between the metagenomics and RT-PCR approaches.The authors describe that the RT-PCR approach, an approach more sensitive than metagenomics, allows to detect six viruses in more individuals than those found with metagenomics (lines 347-349).The viruses in the RT-PCR analysis seem to have been selected based on their prevalence in the two biotypes of Culex pipiens.However, the infection rates in those biotypes obtained with RT-PCR are not clearly provided in the Results section.The number of individuals with a positive detection cannot be easily determined from Figure 5 but there seems to be a large number of individuals of each biotype infected by certain viruses (e.g., see results for Daseongdong virus).If this is the case, there seems to be some viruses that are relatively prevalent thus questioning the claim of a lack of a core virome.Another main claim of the paper is that the results show an influence of the mosquito species on the virome.The problem I see with this claim is that the sample sizes are too low to be able to do a robust analysis.The number of virus-positive individuals obtained with metagenomics is extremely low for two out of the three species (three and eight individuals for Culex torrentium and Aedes japonicus respectively).These sample sizes do not allow for a robust comparative analysis of the influence of mosquito species on the virome or on the existence of a core virome in those mosquito species.
Other comments: There are several features of the study that limit the robustness of the alpha-and beta-diversity analyses.These features include the limited number of virus-positive individuals for certain mosquito species (see comment above) and the limited number of viruses detected per virus-positive individuals (only one virus was detected in most virus-positive individuals).Those analyses are sensitive by incomplete species identification (e.g., see Beck et al., 2010 Methods in Ecology and Evolution).Moreover, there are nested factors that hamper some analyses.For example, one site provided all Aedes individuals and none from the other species (Natoye site if I have well understood Figure 1A; colors are not easily distinguishable).That is, differences between Aedes individuals and the other species may be due to the mosquito species or to specificities of the collection site.Overall, the analyses of species diversity and structure do not seem to be enough solid to allow conclusions.
The authors have used different traps that collect individuals in different life stages (e.g., gravid females versus non-gravid females).Such differences could heavily influence results.The authors do not provide data on the life stage of individuals that could help to study the influence of that factor.Lines 25-26: "Contrary to expectations..." Previous work should have led to expect the results.A study on one of the mosquito species (Culex pipiens) in the manuscript has shown that most individuals do not share a set of viruses commonly found in populations of that mosquito in different countries in Africa and Europe (Gil et al, 2023 Virus Evolution).That is, the study did not detect a core virome when comparing individuals, just like in the manuscript.
Lines 26-27.I agree with the authors that studies on the core virome should define the population level at which the core is analyzed (i.e. between individuals of a population, between populations in a country, between countries or continents, etc.).The authors clearly provide their definition of a core virome ("a select group of (insect-specific) viruses that are present in almost all mosquitoes of the same species"; lines 559-560).However, previous work has already shown that a core virome can be observed if the population scale changes.Several articles have shown that a set of viruses is found associated to populations of a given mosquito species over large sections of the mosquito geographical range, including mosquito species targeted in the manuscript (e.g., Shi et al, 2020;Gil et al, 2023;Moonen et al 2023).The fact that the authors have not observed a core virome at the studied spatial level (i.e., populations situated less than 150 km apart) does not mean that a core virome takes place at a higher level, for example between populations in different continents.The authors do not discuss this point despite a study clearly showing the influence of scale on the virome in the mosquito species with more individuals in the manuscript (Gil et al, 2023).Line 68.A quantitative analysis of publications on mosquito viromes showed that the mosquito genus with most publications is the Culex genus (Moonen et al, 2023).
Lines 562-564 "The "core virome" concept was originally coined after a virome study using Aedes aegypti and Culex quinquefasciatus mosquitoes captured in Guadeloupe,...".In fact, the term "core virome" had already been used in another study (Broecker et al, 2017 Gut Microbes).Moreover, the use of the term "core" was widespread in community ecology long before the paper cited by the authors.Lines 568-569.The authors do not provide a reference and there is a study that obtained results diverging from this sentence (reference 12).
Lines 577-580.There is no need to invoke the Amplification effect hypothesis.Human activity almost always leads to drops in biodiversity, including microorganism diversity.I find difficult to follow this section of the discussion.
Lines 611-612.The reason behind the limited number of bacteriophage sequences seems not the more plausible and the authors do not provide references supporting it.A previous work from the same group found a dominance of bacteriophage sequences in Culex mosquitoes (reference 8).Thus, an alternative hypothesis is a sensibility problem.
-few data on the Culex virome: to nuance: more publications on Culex viruses than the other genus (see Moonen 2023 One Health) Dear editors, Please find herewith our revised manuscript, entitled "Lack of abundant core virome in Culex mosquitoes from a temperate climate region despite a mosquito speciesspecific virome," authored by Lander De Coninck and co-authors.
Thank you for giving us the opportunity to respond to the Reviewer's comments and, hence, improve the quality of our paper.We hope this revised manuscript is now acceptable for publication in mSystems.
We responded to the comments of the reviewers as follows: Reviewer #1 (Comments for the Author): Review of Manuscript: "Lack of abundant core virome in Culex mosquitoes from a temperate climate region despite a mosquito species-specific virome" I had the privilege of reviewing the manuscript, and I must commend the authors for their excellent work.The study delves into the intricate world of insect-specific viruses (ISVs) within arthropod-associated microbial communities, offering valuable insights into the virome of native and invasive mosquito species in Belgium.

Strengths:
1. Clarity and Conciseness: The manuscript is exceptionally well-written, with a clear and concise presentation of methods and results.The authors effectively communicate complex concepts, ensuring accessibility for a broad readership.2. Innovative Approach:The utilization of single mosquito metagenomics is a commendable approach that allows for accurate host species determination and the identification of novel viruses.This innovative methodology significantly contributes to the field.3. Novel Discoveries: The identification of 45 viruses, including 28 novel ones, enriches our understanding of the mosquito virome and ISVs.The species-specific nature of the mosquito virome, irrespective of geographical location, is a noteworthy finding.
We thank reviewer #1 for their compliments and constructive feedback, our answers to their requests can be found below.
Constructive Feedback: 1. Introduction Enhancement: I would recommend incorporating information about the World Mosquito Program (WMP) in the introduction.This addition would contextualize the study within broader mosquito control efforts and potentially enhance its relevance to a wider audience.
We mentioned the WMP and its goals now on lines 77-81 in the introduction.
2. Bacterial Genomes Exploration: Consider expanding the exploration of bacterial genomes beyond Wolbachia.Providing additional context and insights into other bacterial elements could further enrich the study.
We agree that a broader view on the bacteriome would enrich the study, however our data does not easily allow this as we specifically enriched for viruses, which severaly diminishes and biases bacterial diversity.We therefore only focused on Wolbachia which is shown to influence vector competence and which we knew was naturally present in the Culex mosquito population in Belgium.
3. Discussion Expansion: The discussion section could be extended to delve deeper into the implications of the findings.This may include further interpretation of results, addressing study limitations, and proposing avenues for future research.
Connecting the study's outcomes to practical applications in mosquito control or disease prevention would strengthen the impact of the research.
We further improved our discussion based on the suggestions of both reviewers: Lines 505-514: we added a comparison with other studies.Lines 526-533: we added the low sample sizes for Aedes japonicus and Culex torrentium and the low viruses identified as a weakness for the beta diversity analyses.
Lines 545-549: Provided extra reference and comparison to a lack of core virome on the individual mosquito level

Conclusion:
In conclusion, this manuscript represents a significant contribution to the field of mosquito virology.The meticulous methodology, coupled with novel discoveries, positions this work as a valuable reference for researchers and practitioners alike.I commend the authors on their exemplary work and look forward to witnessing the continued impact of their research in the scientific community.
Reviewer #2 (Comments for the Author): The manuscript by de Coninck et al. describes the diversity of viruses found in individual mosquitoes collected in different sites in Belgium.Mosquitoes belonged to three species, including two species of the Culex genus and one Aedes species.The authors screened 190 mosquito individuals in total using metagenomics.Mosquitoes were also screened for the presence of six viruses by RT-PCR.Moreover, the authors determined Wolbachia prevalence from the sequencing data obtained with metagenomics.The authors present analyses of the virus communities and phylogenies of the viruses found in their mosquito collection, including the description of new species.
The main result of the manuscript, as defined by the title and the abstract, is the lack of a clear support for the existence of a core virome in the studied populations.This claim mainly derives from the limited diversity overlap in the virome of individual mosquitoes observed with metagenomics.I see two problems in the manuscript suggesting that the results do not robustly support this claim.
We thank reviewer #2 for their critical reading of our manuscript and their insights.
Our responses can be found below: First, assessing a core virome requires a diagnostic test allowing to robustly infer presence/absence of viruses in the samples.The approach used by the author does not seem to fully comply with this requirement.No virus was found in a large number of mosquitoes (61 %).I have not found an explanation for this result or a comparison with other studies.Few studies have analyzed individual mosquitoes with metagenomics but at least two of them (including a study from the same group) did not find such a high proportion of negative samples (references 8 and 12 in the manuscript).The low number of virus-positive samples thus suggest a sensibility problem (either due to the method or the sample quality).
In agreement with reviewer #2, we were also surprised to see that we only had such a low number of virus positive samples.However, we believe this is not a result of the method and/or low sample quality: 1) Our method has been widely used to detect viruses in different sample types (human gut, insects [including mosquitoes, bees and blackflies], bats, plants, etc.).In addition, we also optimized our method in a small pilot study (not published) to recover the virus read yield for mosquitoes (eg.different homogenization speeds, concentration of viruses by centrifugation with molecular weight filters).The method used here, and in Shi et al. (2019), was still the most performant.
2) As mentioned by reviewer #2, few studies have performed single mosquito metagenomics so direct comparisons are difficult because other Culex virome studies in a temperate climate region only used pools.Pools can mask the large amount of mosquitoes without viruses.The studies we can compare to: have a markedly warmer climate (Shi et al. 2019: Guadeloupe;Batson et al. 2021: California) and for the California study, samples were also collected during a period with unusually warm temperature peaks (eg.San Diego, fall 2017: https://weatherspark.com/h/s/1816/2017/2/Historical-Weather-Fall-2017-in-San-Diego-California-United-States).In our eyes, further strengthening the hypothesis that higher temperatures could lead to more virus in these mosquitoes (mentioned on lines 569-575 of the original submission).Furthermore, a recent preprint on viromics of 2438 individual mosquitoes from China found that Culex pipiens (n=438) on average harbored 1.84 (±1.65 SD) viruses which is within the range of our study (Pan et al., 2023 bioRxiv, https://doi.org/10.1101/2023.08.28.555221).Of note, these authors did find a negative correlation between viral richness and annual mean temperature, but we have concerns regarding this result as they did not make a distinction between mosquito species, they only used the average annual temperature which might not reflect the actual temperature the mosquitoes were caught in and finally, in the low temperature range they have a lot less samples than in the high temperature range which might skew their results.
3) Reviewer #2's suggestion of low sample quality is hard to refute, but we stored our samples from collection to analysis on -80°C as usual for these kind of samples (and widely described in literature).Besides, for the Aedes mosquitoes, we did find multiple viruses in every sample suggesting that sample quality and our method were not an issue.
We added a comparison to other studies in the discussion on lines 505-514.
Secondly, there seems to be contradictory results between the metagenomics and RT-PCR approaches.The authors describe that the RT-PCR approach, an approach more sensitive than metagenomics, allows to detect six viruses in more individuals than those found with metagenomics (lines 347-349).The viruses in the RT-PCR analysis seem to have been selected based on their prevalence in the two biotypes of Culex pipiens.However, the infection rates in those biotypes obtained with RT-PCR are not clearly provided in the Results section.[We added a supplementary table with infection rates and a supplementary figure.]The number of individuals with a positive detection cannot be easily determined from Figure 5 but there seems to be a large number of individuals of each biotype infected by certain viruses (e.g., see results for Daseongdong virus).If this is the case, there seems to be some viruses that are relatively prevalent thus questioning the claim of a lack of a core virome.
Based on Figure 5, an argument could be made to question the lack of a core virome as some viruses (DV, CPV and XCV) seem to be present in a large number of individuals.However, based on the following arguments, we would like to better explain our arguments, for NOT claiming the presence of an "abundant" core virome.First of all, samples with a genome copy number of 10,000 or less were positive for the virus but their Ct was higher than the lowest concentration of our standard curve, making the genome copy determination not entirely accurate (we mentioned this on lines 355-358 of the original manuscript).Certainly for DV only a small number of samples had copy numbers above this threshold, DV is therefore not very abundant when it is present and this is mentioned in the title of the manuscript: "Lack of abundant core virome…".Secondly, primers were designed in the RdRP gene (across multiple sequences from the different samples), this gene might be more conserved across viral species.Therefore, there is also the possibility that we detected similar, but different, viral species with our RT-qPCR.Finally, our most compelling argument to not call it a core virome comes from the observation that these viruses are quite locally present (see new supplementary tables and figure below).DV is mostly present in Leuven and Bertem (<5km apart) with a few samples in Eupen and Maasmechelen, CPV is almost exclusively present in Vrasene (1 sample in Maasmechelen).XCV on the other hand is present in almost all locations, but the infection rates for Cx.pipiens molestus and Cx.pipiens pipiens are respectively 4.26% and 14.96%.All this information leads us to conclude that there is a lack of support for an (abundant) core virome.
We added this reasoning in the Results section on lines 331-338.
Another main claim of the paper is that the results show an influence of the mosquito species on the virome.The problem I see with this claim is that the sample sizes are too low to be able to do a robust analysis.The number of virus-positive individuals obtained with metagenomics is extremely low for two out of the three species (three and eight individuals for Culex torrentium and Aedes japonicus respectively).These sample sizes do not allow for a robust comparative analysis of the influence of mosquito species on the virome or on the existence of a core virome in those mosquito species.
We agree that for Culex torrentium and Aedes japonicus virus-positive sample counts are low, however it is already known from literature that viromes seem to be species-specific.Certainly differences between Aedes and Culex have been shown before (Shi et al. 2019;Thongspirong et al., 2021, Sci. Rep.;Li et al., 2023, Microbiology Spectrum;Wang et al. 2024, Microbiology Spectrum).
We mention this now as a weakness of the study on lines 526-528.

Culex torrentium
Other comments: There are several features of the study that limit the robustness of the alpha-and beta-diversity analyses.These features include the limited number of virus-positive individuals for certain mosquito species (see comment above) and the limited number of viruses detected per virus-positive individuals (only one virus was detected in most virus-positive individuals).Those analyses are sensitive by incomplete species identification (e.g., see Beck et al., 2010 Methods in Ecology and Evolution).
We acknowledge the concern of reviewer #2 about the issue of undersampling and although we cannot exclude the possibility that we have missed to identify viruses, we believe that our method is still sensitive enough to do these types of analyses (see also answers above).However, the low number of viruses per individual warrants a cautious interpretation of the NMDS and PCoA plots (added this as a weakness on lines 531-533).In addition, these analyses are also sensitive to overestimation of (virus) species.Therefore, we have chosen to be more stringent in our identification of viruses to not include genome fragments.
Moreover, there are nested factors that hamper some analyses.For example, one site provided all Aedes individuals and none from the other species (Natoye site if I have well understood Figure 1A; colors are not easily distinguishable) [Colors have been made more divergent in all figures].That is, differences between Aedes individuals and the other species may be due to the mosquito species or to specificities of the collection site.Overall, the analyses of species diversity and structure do not seem to be enough solid to allow conclusions.
We agree and we added this as a weakness in the discussion on lines 526-528.But other studies have observed similar differences between Aedes and Culex, and between different mosquito genera in general (Shi et al. 2019;Thongspirong et al., 2021, Sci. Rep.;Li et al., 2023, Microbiology Spectrum;Wang et al. 2024, Microbiology Spectrum as a few examples).
The authors have used different traps that collect individuals in different life stages (e.g., gravid females versus non-gravid females).Such differences could heavily influence results.The authors do not provide data on the life stage of individuals that could help to study the influence of that factor.Unfortunately, we do not have the information anymore on the gravidness of the mosquitoes (the samples were homogenized before assessing).However, we added the trap type as an extra explaining variable into the PERMANOVA analysis.This showed that trap type (and by assumption gravidness, if we consider mosquitoes caught in gravid traps to be gravid and mosquitoes caught by the other traps to be non-gravid) only had a minor influence on the virome composition (4% vs 25% for mosquito species, and 10% for collection location).Lines 25-26: "Contrary to expectations..." Previous work should have led to expect the results.A study on one of the mosquito species (Culex pipiens) in the manuscript has shown that most individuals do not share a set of viruses commonly found in populations of that mosquito in different countries in Africa and Europe (Gil et al,