Blood Parasites (Haemosporida, Trypanosomatida) in Culex pipiens: A Study and Review of Hibernating and Active Mosquitoes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors collected hibernating female C. pipiens mosquitoes during two months in the Winter 2023/24, and active C. pipiens females from 2021 -2023 using different collection methods. Parasite infection was analysed by microscopy and PCR. The authors tested for Trypanosomatides and for haemosporidian parasites. In the discussion, besides discussing their own findings, the authors present a review of previous studies on the infection status of European mosquitoes with Trypanosomatides and haemosporidian parasites.

While this study adds to the existing dataset on the prevalence of parasites in C. pipiens mosquitoes (active and hibernating), I have several concerns that need to be addressed before this study can be considered for publication.

 

Dear authors, please address the following points to improve the manuscript:

Methods:

Section 2.2: it is not clear what was done with the mosquitoes. mosquitoes were first anesthetised by cold exposure in the freezer. And then? the restriction digest analysis would have required killing of the mosquitoes and DNA extraction. as well as the dissection will have killed the mosquitoes. for what purpose anesthesize them first then?

L106 ff: Midgut preparations that were determined to be PCR positive for trypanosomatid DNA were examined microscopically - for PCR the DNA would have to be extracted from the midguts. how can they then by later analysed ny microscopy, they would have been destroyed before already. I don't understand this description

L112 ff: Hibernating mosquitoes were pooled (up to 10 individuals in one pool collected at the same locality the same day) for molecular analysis. - and how were the active females treated? also in pools? if not why the difference?

This whole section 2.2 is very cryptic and doesn't allow to understand how the ALL the samples used in this manuscript were treated and processed step by step. It has to be completely revised to be able to replicate the authors experiments.

Section 2.3: what is the "remaining samples"? specify

why were the hibernating females treated as pools but the others not? Explain or correct/add information for other samples

L120-123: provide primer sequences and reference genome on which the primers were designed

 

Results:

Why were hibernating mosquitoes collected in Dec and Jan? Explain

 

 

Figure 1: is that the combined data from 2021 -2023? Specify in figure caption. If each bar represents data from a total of 3 years, then the data should not be displayed as total numbers but as average over the 3 years and the standard deviation should be indicated.

consequently, also the parasite detections should be specified by year i.e. how much differed the parasite detections across the different years?

The authors provide a list and phylogenetic tree for Trypanomosomatides but not for haemosporidian parasites. What's the reason for that? Please add this analysis if possible.

Table 1: adding a column with the information if the parasite is has an avian or human host would make this information much faster accessible than just writing it in the text.

 

Discussion:

The discussion is in parts lengthy, and the red line is hard to follow. the authors jump between  Trypanomosomatides and haemosporidian parasites – please sort and streamline to improve readability and to enhance the clarity of the points that the authors want to make.

 

Tables 2 and 3: What does experimental research mean? Not clear, please explain in table captions

 

L296: "significant difference": where is the statistical analysis to prove statistically significant difference? Is the dataset even large enough to detect a significant difference given the low infection numbers?

 

Discuss the increasing number of findings of monoxeous trypan. in human samples, as discussed for example in Boucinha Front Cell Infect Microbiol. 2022; 12: 804707;  doi: 10.3389/fcimb.2022.804707

 

 

Major concern regarding the main question of the study, if hibernating c. pipiens can be a parasite reservoir: hibernating females were only collected in one winter. This is not a representative data set. In combination with low infection rate during summer, especially with basically absent infections in September (the generation that probably forms the hibernating mosquitoes), in my opinion there is no reliable statement possible if hibernating mosquitoes can be a reservoir or not. For that many more samples would have to be analyzed per year, and for several successive years. 

 

 

 

Supplementary material:

there were two supplemental tables (S1 and S2) provided for the review. however, these two tables are (nearly) identical to the tables 2 and 3 in the main manuscript.

Comments on the Quality of English Language

English editing is highly recommended to provide better readability of the manuscript and clarity of the language.

Author Response

Answer to reviewer

 

The authors collected hibernating female C. pipiens mosquitoes during two months in the Winter 2023/24, and active C. pipiens females from 2021 -2023 using different collection methods. Parasite infection was analysed by microscopy and PCR. The authors tested for Trypanosomatides and for haemosporidian parasites. In the discussion, besides discussing their own findings, the authors present a review of previous studies on the infection status of European mosquitoes with Trypanosomatides and haemosporidian parasites.

While this study adds to the existing dataset on the prevalence of parasites in C. pipiens mosquitoes (active and hibernating), I have several concerns that need to be addressed before this study can be considered for publication.

We are very grateful for all comments of the reviewer. We took them into account and corrected the article. Numbers of lines is provided based on PDF version of revised manuscript.

Dear authors, please address the following points to improve the manuscript:

Methods:

Section 2.2: it is not clear what was done with the mosquitoes. mosquitoes were first anesthetised by cold exposure in the freezer. And then? the restriction digest analysis would have required killing of the mosquitoes and DNA extraction. as well as the dissection will have killed the mosquitoes. for what purpose anesthesize them first then?

We used freezer for several minutes to make mosquitoes inactive / immobile, so they would not move and can be identified and dissected. The mosquito is killed by dissection (the head is removed before the salivary glands are extracted) and remnants of mosquito body are used for DNA extraction. Restriction digest analysis takes place after DNA extraction and after PCR. We wrote about the restriction digest analysis in the chapter 2.2 as this analysis is related with identification. We changed text to make methods clear. L 98-130.

L106 ff: Midgut preparations that were determined to be PCR positive for trypanosomatid DNA were examined microscopically - for PCR the DNA would have to be extracted from the midguts. how can they then by later analysed ny microscopy, they would have been destroyed before already. I don't understand this description

We made some changes in the text. L. 115-116.

One mosquito female during investigation was divided into 1) salivary gland preparation, 2) midgut preparation, 3) all remnants after dissection were used for DNA extraction and PCR. Parasites such as haemosporidian can be found not only in certain organs (salivary glands, gut) of the mosquito body, but also in hemocoel, so extraction of salivary glands does not mean that haemosporidian parasite will not be detected in the remnants of mosquito (with removed salivary glands) using PCR as was proved also by Valkiunas et al. in https://doi.org/10.1007/s00436-013-3375-6 (for haemosporidian parasites). We used only half of a midgut for midgut preparation (for Trypanosoma parasites) and the rest of mosquito body (with removed salivary glands and half of a midgut) was used for DNA extraction and PCR. Similar method was also described by Fialova et al. in https://doi.org/10.3390/microorganisms9102101.

L112 ff: Hibernating mosquitoes were pooled (up to 10 individuals in one pool collected at the same locality the same day) for molecular analysis. - and how were the active females treated? also in pools? if not why the difference?

All females were dissected and investigated individually using microcopy. For PCR investigation hibernating females were pooled as we expected to find lower parasite prevalence in hibernating mosquitoes comparing with active females. This is very common to investigate pooled mosquitoes collected in the wild because of the low prevalence of infections, and this is described in many papers such as

https://doi.org/10.3390/microorganisms10020294

https://doi.org/10.1186/s13071-023-05965-0

DOI 10.1186/s12936-017-2035-1

https://doi.org/10.1371/journal.pone.0196052

The prevalence of investigated parasites is less than 6% even in the summer, so pooling is an efective method of investigation. This was shortly explained in the text. L 133-135.

This whole section 2.2 is very cryptic and doesn't allow to understand how the ALL the samples used in this manuscript were treated and processed step by step. It has to be completely revised to be able to replicate the authors experiments.

This section had been revised.

Section 2.3: what is the "remaining samples"?

This was corrected. L 137-138.

specify why were the hibernating females treated as pools but the others not? Explain or correct/add information for other samples

This information was added - we expected lower prevalence of parasites in hibernating mosquitoes, and this is a common method of testing mosquitoes collected in the wild for the presence of pathogens as we have already mentioned. L 133-135.

L120-123: provide primer sequences and reference genome on which the primers were designed.

Information has been provided. L 142-148.

Results:

Why were hibernating mosquitoes collected in Dec and Jan? Explain

Average daily temperature below 10°C in our country is usually from November to February and this is the hibernation time of mosquitoes. November is just the beginning of the hibernation period for Culex mosquitoes in the temperate zone of the northern hemisphere as it was also pointed in the discussion chapter. The main question was if mosquitoes infected with parasites can survive until spring. We started collecting mosquitoes in winter, from December and we believe that if we would investigate mosquitoes collected in February this would not change our results. We would like to note that towards spring the abundance of hibernating mosquitoes in cellars decreases and it is getting difficult to find them. We agree that investigation of hibernating mosquitoes collected in November would give us interesting results, as it was in November that mosquitoes infected with hemosporidian parasites were detected by Köchling et al. (we write about it in the discussion), so we plan of conducting research on this in the future together with experimental works. L 432-435.

Figure 1: is that the combined data from 2021 -2023? Specify in figure caption. If each bar represents data from a total of 3 years, then the data should not be displayed as total numbers but as average over the 3 years and the standard deviation should be indicated.

Consequently, also the parasite detections should be specified by year i.e. how much differed the parasite detections across the different years?

The figure was remade as suggested.

The authors provide a list and phylogenetic tree for Trypanomosomatides but not for haemosporidian parasites. What's the reason for that? Please add this analysis if possible.

The main purpose of providing phylogenetic tree for Trypanomosomatids was to show that based on the DNA sequences our newfound trypanosomatids are monoxenous, not dixenous and are not related with vertebrate host. This information was added. Phylogenetic trees of Plasmodium parasites are provided in many publications investigating avian haemosporidian parasites, for example phylogeny of all three genetic lineages detected in mosquitoes during our investigation is discussed in 

https://doi.org/10.1017/S0031182017000737.

Table 1: adding a column with the information if the parasite is has an avian or human host would make this information much faster accessible than just writing it in the text.

Corrected.

Discussion:

The discussion is in parts lengthy, and the red line is hard to follow. the authors jump between Trypanomosomatides and haemosporidian parasites – please sort and streamline to improve readability and to enhance the clarity of the points that the authors want to make.

We have shortened the discussion and made it easier to follow. We talked about the main findings in the beginning, later about trypanosomatids, haemosporidian parasites and feeding preference of Culex mosquitoes as this was also result from our data.

Tables 2 and 3: What does experimental research mean? Not clear, please explain in table captions

This information was provided – experimental infections of mosquitoes. L282, 379.

L296: "significant difference": where is the statistical analysis to prove statistically significant difference? Is the dataset even large enough to detect a significant difference given the low infection numbers?

Thank You for this question, we used Fisher exact test for statistical analysis and this test showed that statistically significant differences have been detected between infection rates of hibernating and active mosquitoes. L 197-198, 216-218, 239-243.

Discuss the increasing number of findings of monoxeous trypan. in human samples, as discussed for example in Boucinha Front Cell Infect Microbiol. 2022; 12: 804707;   doi:10.3389/fcimb.2022.804707

This information has been added. L 344-348.

Major concern regarding the main question of the study, if hibernating c. pipiens can be a parasite reservoir: hibernating females were only collected in one winter. This is not a representative data set. In combination with low infection rate during summer, especially with basically absent infections in September (the generation that probably forms the hibernating mosquitoes), in my opinion there is no reliable statement possible if hibernating mosquitoes can be a reservoir or not. For that many more samples would have to be analyzed per year, and for several successive years. 

Thank You for pointing this. The prevalence of parasites in wild caught mosquitoes is usually low and depends on mosquito species (between 0.04% to 6.6% for haemosporidian parasites and from 0.005% to 22% for Trypanosoma). Investigating natural prevalence in wild caught mosquitoes we will always find low prevalences. Nevertheless, we determined that parasite prevalence in hibernating mosquitoes was statistically significantly lower than in active mosquitoes. Really, we did not detect infected hibernating mosquitoes. Data on the infection rates of hibernating mosquitoes are scarce and our results fills this data gap. We do not say that the parasite can't overwinter in a mosquito, but we say it's unlikely they can overwinter. Experimental studies are needed to determine whether the parasites fail to survive in mosquitoes or whether mosquitoes infected with parasites do not survive until spring.

Supplementary material:

there were two supplemental tables (S1 and S2) provided for the review. however, these two tables are (nearly) identical to the tables 2 and 3 in the main manuscript.

Supplementary tables have been removed. We provided another supplementary table with raw results.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The manuscript titled “Blood parasites (Haemosporida, Trypanosomatida) in Culex pipiens: a study and review of hibernating and active mosquitoes” represents a contribution to the science but also to the public health sector of Lithuania.

Before this manuscript is published there are several things that require revision.

Considering the topic focused on the parasites it is crucial to write in the introduction what is the status of these pathogens in Lithuania. Why did the author decided to test these pathogens? What are the symptoms? Who is endangered?... and so on…

In the whole manuscript genus Culex is wrongly abbreviated. It should be Cx. and not C. Please correct it.

L16 Please do not start the sentence with the number.

L30-32 Replace the citation with the original references of the authors (names given in the text).

L38 Missing citation for the Dirofilaria.

Materials and methods: in this section many details are missing considering the mosquito sampling. The authors did not give enough material to provide reproducibility of this study. Some of the questions that need to be answered are: How many times did you collect (please provide table)? How many mosquitoes per sample was collected? What was the species composition? Authors used nets but when and where? It is not enough to give name of the place. For mosquitoes micro-location is very significant. What kind of mosquito resting places did you select?

Results

The first paragraph doesn’t have a sense without giving all information about sampling and the collected samples enough information.

Figure 1. Why did you gather pathogens and number of vectors on the same graph? It cannot be comparable this way. Please improve graphical presentation of data.

 

Discussion: In the discussion authors should make connection between the place where mosquitoes were sampled and the number of positive mosquitoes. The manuscript can be significantly improved. 

Comments on the Quality of English Language

English is understandable but could be improved. 

Author Response

Dear Authors,

The manuscript titled “Blood parasites (Haemosporida, Trypanosomatida) in Culex pipiens: a study and review of hibernating and active mosquitoes” represents a contribution to the science but also to the public health sector of Lithuania.

Before this manuscript is published there are several things that require revision.

We are very grateful for all comments of the reviewer. We tried to take them into account and correct the article. Numbers of lines is provided based on PDF version of revised manuscript.

Considering the topic focused on the parasites it is crucial to write in the introduction what is the status of these pathogens in Lithuania. Why did the author decided to test these pathogens? What are the symptoms? Who is endangered?... and so on…

This information was added. L.40-45.

In the whole manuscript genus Culex is wrongly abbreviated. It should be Cx. and not C. Please correct it.

This was corrected.

L16 Please do not start the sentence with the number.

Corrected.

L30-32 Replace the citation with the original references of the authors (names given in the text).

This was corrected.

L38 Missing citation for the Dirofilaria.

Was added.

https://www.parasite-journal.org/articles/parasite/full_html/2019/01/parasite180134/parasite180134.html

and

https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-018-2845-1

Materials and methods: in this section many details are missing considering the mosquito sampling. The authors did not give enough material to provide reproducibility of this study. Some of the questions that need to be answered are: How many times did you collect (please provide table)? How many mosquitoes per sample was collected? What was the species composition?

The table with all collection of mosquitoes (with certain years, localities and numbers of collected Culex mosquitoes) is now provided as supplementary material. We investigated all Culex mosquitoes collected during warm periods collecting them periodically from May to September. This information was added. The number of investigated hibernating mosquitoes was chosen to be similar to the number of active mosquitoes. The species composition of collected mosquitoes during warm period is a subject of other publication (https://doi.org/10.1016/j.actatropica.2024.107260) and our investigation is related only with Culex mosquitoes which can be found both as active and hibernating mosquitoes. Collecting hibernating mosquitoes, we also collected a small number of Culiseta and Anopheles mosquitoes, but these mosquitoes were not target species, as we investigated only Culex mosquitoes, so they were not investigated during this research.

Authors used nets but when and where? It is not enough to give name of the place.

This information is now provided in supplementary material.

For mosquitoes micro-location is very significant. What kind of mosquito resting places did you select?

All localities for mosquito collection were in similar habitats – mixed forests with some water bodies. And this information was added. L 90-93.

We agree that abundance of Culex mosquitoes is different in different localities, and this agree with data provided in Supplementary table. From the other hand mosquitoes can fly some distances from localities they develop, or they had bloodmeal.

Thanks to the review, we noticed that the prevalence of parasites, precisely haemosporidian, in different localities, differs from 0 to 7% (Verkiai) and even higher in Belmontas (but too few mosquitoes were investigated in Belmontas to draw conclusions). We investigated both active and hibernating mosquitoes in Verkiai (the same locality) with no infected hibernating mosquitoes detected. The prevalence of Trypanosoma parasites was not higher than 2% in all localities and no significant differences in prevalences between localities was detected during this investigation.

We think that our data are insufficient to draw broader conclusions and the relationship between location and the prevalence with parasites need to be further investigated. We think that this issue could be the subject of another investigation. We shortly discussed this. L 414-418.

Results

The first paragraph doesn’t have a sense without giving all information about sampling and the collected samples enough information.

We added information about the collecting methods, as mosquitoes were collected regularly from May till September, so information about the seasonality can be provided. L. 86-87.

Figure 1. Why did you gather pathogens and number of vectors on the same graph? It cannot be comparable this way. Please improve graphical presentation of data.

We changed the figure providing average numbers. We would like to point that the graph is provided with two vertical axes: one for number of mosquitoes and another for number of infected mosquitoes, the horizontal axes is the same. We hope that this way can be used for graphical presentation and we would like to leave this if it is possible.

Discussion: In the discussion authors should make connection between the place where mosquitoes were sampled and the number of positive mosquitoes. The manuscript can be significantly improved.

We discussed this isue, as thanks to the reviewer, we noticed that there are some differences in prevalences of parasites in investigated localities. We think that this is a point of another investigation with large amount of collected material and we tried to disscus this. L 414-418.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, while many of my comments have been clarified, I still have a few things that need correction or improvement. Especially in the methods, the descriptions are in parts still not clear or not complete enough to allow replication of the procedures.

Please find below my responses to your responses and additional issues to address:

 

Introduction:

LL36 – 38 and LL 40 – 42: almost identical (= redundant)

 

Methods:

Section 2.2: it is not clear what was done with the mosquitoes. mosquitoes were first anesthetised by cold exposure in the freezer. And then? the restriction digest analysis would have required killing of the mosquitoes and DNA extraction. as well as the dissection will have killed the mosquitoes. for what purpose anesthesize them first then?

We used freezer for several minutes to make mosquitoes inactive / immobile, so they would not move and can be identified and dissected. The mosquito is killed by dissection (the head is removed before the salivary glands are extracted) and remnants of mosquito body are used for DNA extraction. Restriction digest analysis takes place after DNA extraction and after PCR. We wrote about the restriction digest analysis in the chapter 2.2 as this analysis is related with identification. We changed text to make methods clear. L 98-130.

Response to the authors‘ corrections:

L86: „from May to September“ is double

LL 79 – 83 and LL 92-93: The description of where hibernating mosquitoes were collected is partially redundant

L108: „both slides“ – which is the second slide? You describe only one slide here for the midgut preparaitons. What is the oher one?

L110: specify SET buffer

L122: we used method based on.... the article is missing

L125: „mitochondrial DNA is affected separately“ -  mitochondrial DNA is digested differently for these two species

LL 111-113 and LL 136-141: I‘m still confused: were the active mosquitoes processed and analyzed in this study or a previous one? If it was done in a previous one and the sample processing is described there, then it should not be described here as if it was done in this study. Just refer to the previous study here.

 

 

L106 ff: Midgut preparations that were determined to be PCR positive for trypanosomatid DNA were examined microscopically - for PCR the DNA would have to be extracted from the midguts. how can they then by later analysed ny microscopy, they would have been destroyed before already. I don't understand this description

We made some changes in the text. L. 115-116. 

One mosquito female during investigation was divided into 1) salivary gland preparation, 2) midgut preparation, 3) all remnants after dissection were used for DNA extraction and PCR. Parasites such as haemosporidian can be found not only in certain organs (salivary glands, gut) of the mosquito body, but also in hemocoel, so extraction of salivary glands does not mean that haemosporidian parasite will not be detected in the remnants of mosquito (with removed salivary glands) using PCR as was proved also by Valkiunas et al. in https://doi.org/10.1007/s00436-013-3375-6 (for haemosporidian parasites). We used only half of a midgut for midgut preparation (for Trypanosoma parasites) and the rest of mosquito body (with removed salivary glands and half of a midgut) was used for DNA extraction and PCR. Similar method was also described by Fialova et al. in https://doi.org/10.3390/microorganisms9102101. 

Response to the authors‘ corrections:

That‘s clearer now, the essential part that was missing was „midgut preparations of mosquitoes“

However, there is still some unclear or imprecise phrasing (see above)

 

L112 ff: Hibernating mosquitoes were pooled (up to 10 individuals in one pool collected at the same locality the same day) for molecular analysis. - and how were the active females treated? also in pools? if not why the difference?

All females were dissected and investigated individually using microcopy. For PCR investigation hibernating females were pooled as we expected to find lower parasite prevalence in hibernating mosquitoes comparing with active females. This is very common to investigate pooled mosquitoes collected in the wild because of the low prevalence of infections, and this is described in many papers such as 

https://doi.org/10.3390/microorganisms10020294

https://doi.org/10.1186/s13071-023-05965-0

DOI 10.1186/s12936-017-2035-1

https://doi.org/10.1371/journal.pone.0196052

The prevalence of investigated parasites is less than 6% even in the summer, so pooling is an efective method of investigation. This was shortly explained in the text. L 133-135.

This whole section 2.2 is very cryptic and doesn't allow to understand how the ALL the samples used in this manuscript were treated and processed step by step. It has to be completely revised to be able to replicate the authors experiments.

This section had been revised.

Response to the authors‘ corrections:

Thank you for clarifying some of the issues. There are still some left though (see above)

 

 

Section 2.3: what is the "remaining samples"? 

This was corrected. L 137-138.

specify why were the hibernating females treated as pools but the others not? Explain or correct/add information for other samples

This information was added - we expected lower prevalence of parasites in hibernating mosquitoes, and this is a common method of testing mosquitoes collected in the wild for the presence of pathogens as we have already mentioned. L 133-135.

L120-123: provide primer sequences and reference genome on which the primers were designed.

Information has been provided. L 142-148.

 

 

Results:

Why were hibernating mosquitoes collected in Dec and Jan? Explain

Average daily temperature below 10°C in our country is usually from November to February and this is the hibernation time of mosquitoes. November is just the beginning of the hibernation period for Culex mosquitoes in the temperate zone of the northern hemisphere as it was also pointed in the discussion chapter. The main question was if mosquitoes infected with parasites can survive until spring. We started collecting mosquitoes in winter, from December and we believe that if we would investigate mosquitoes collected in February this would not change our results. We would like to note that towards spring the abundance of hibernating mosquitoes in cellars decreases and it is getting difficult to find them. We agree that investigation of hibernating mosquitoes collected in November would give us interesting results, as it was in November that mosquitoes infected with hemosporidian parasites were detected by Köchling et al. (we write about it in the discussion), so we plan of conducting research on this in the future together with experimental works. L 432-435.

 

Figure 1: is that the combined data from 2021 -2023? Specify in figure caption. If each bar represents data from a total of 3 years, then the data should not be displayed as total numbers but as average over the 3 years and the standard deviation should be indicated.

Consequently, also the parasite detections should be specified by year i.e. how much differed the parasite detections across the different years?

The figure was remade as suggested.

Response to the authors‘ corrections:

This is scientifically much more valuable now

 

The authors provide a list and phylogenetic tree for Trypanomosomatides but not for haemosporidian parasites. What's the reason for that? Please add this analysis if possible.

The main purpose of providing phylogenetic tree for Trypanomosomatids was to show that based on the DNA sequences our newfound trypanosomatids are monoxenous, not dixenous and are not related with vertebrate host. This information was added. Phylogenetic trees of Plasmodium parasites are provided in many publications investigating avian haemosporidian parasites, for example phylogeny of all three genetic lineages detected in mosquitoes during our investigation is discussed in  

https://doi.org/10.1017/S0031182017000737. 

Table 1: adding a column with the information if the parasite is has an avian or human host would make this information much faster accessible than just writing it in the text.

Corrected.

 

 

Additional things to address in the results:

LL 209 – 211: “All detected by PCR Trypanosoma species were obtained microscopically in midgut preparations of investigated mosquitoes (Fig. 2), but we did not detect T. theileri in preparations of mosquito midguts” – Well, then it was not all, because T. theileri was not detected, correct?

Moreover it should be: All Trypanosoma species detected by PCR…

 

Discussion:

The discussion is in parts lengthy, and the red line is hard to follow. the authors jump between Trypanomosomatides and haemosporidian parasites – please sort and streamline to improve readability and to enhance the clarity of the points that the authors want to make.

We have shortened the discussion and made it easier to follow. We talked about the main findings in the beginning, later about trypanosomatids, haemosporidian parasites and feeding preference of Culex mosquitoes as this was also result from our data.

Tables 2 and 3: What does experimental research mean? Not clear, please explain in table captions

This information was provided – experimental infections of mosquitoes. L282, 379.

L296: "significant difference": where is the statistical analysis to prove statistically significant difference? Is the dataset even large enough to detect a significant difference given the low infection numbers?

Thank You for this question, we used Fisher exact test for statistical analysis and this test showed that statistically significant differences have been detected between infection rates of hibernating and active mosquitoes. L 197-198, 216-218, 239-243.

Discuss the increasing number of findings of monoxeous trypan. in human samples, as discussed for example in Boucinha Front Cell Infect Microbiol. 2022; 12: 804707;   doi:10.3389/fcimb.2022.804707

This information has been added. L 344-348.

Major concern regarding the main question of the study, if hibernating c. pipiens can be a parasite reservoir: hibernating females were only collected in one winter. This is not a representative data set. In combination with low infection rate during summer, especially with basically absent infections in September (the generation that probably forms the hibernating mosquitoes), in my opinion there is no reliable statement possible if hibernating mosquitoes can be a reservoir or not. For that many more samples would have to be analyzed per year, and for several successive years. 

Thank You for pointing this. The prevalence of parasites in wild caught mosquitoes is usually low and depends on mosquito species (between 0.04% to 6.6% for haemosporidian parasites and from 0.005% to 22% for Trypanosoma). Investigating natural prevalence in wild caught mosquitoes we will always find low prevalences. Nevertheless, we determined that parasite prevalence in hibernating mosquitoes was statistically significantly lower than in active mosquitoes. Really, we did not detect infected hibernating mosquitoes. Data on the infection rates of hibernating mosquitoes are scarce and our results fills this data gap. We do not say that the parasite can't overwinter in a mosquito, but we say it's unlikely they can overwinter. Experimental studies are needed to determine whether the parasites fail to survive in mosquitoes or whether mosquitoes infected with parasites do not survive until spring.

Response to the authors‘ corrections:

Thank you for the explanation. I am still concerned, though, about basing the statement, that hibernating mosquitoes are unlikely reservoirs, on animals collected only during one winter season (even though the total number of analyzed active and hibernating mosquitoes was similar). Checking animals only in one winter does not provide representative data (the numbers for the active females are also based on three years, not only one). Therefore, the authors should at least include the cautionary note in the discussion, that their conclusions on hibernating mosquitoes are based on the animals collected in a single winter. 

 

 

Additional things to address in the discussion:´

LL 237 – 238: “No infected with investigated parasites mosquitoes were found among hibernating ones.”

That’s not true. L 192: 2 hibernating mosquitoes with trypanosomatids were detected.

Moreover, please check the grammar of the sentence

 

LL287 – 289: “Monoxenous trypanosomatids are generally considered non-pathogenic, however there is some evidence suggesting negative effects on insect fitness, although this has been investigated in only a few insect species” - Please provide the corresponding reference(s)

 

L 315: “infections seem to be the highest during the rains” – this should be: infections seem to be the highest during the raining periods

 

L347: "We cannot ignore the significant difference between the prevalence of parasites in mosquitoes collected during warm and cold periods.” -  I don’t understand what the authors want to say with this statement in this context

 

The discussion is more streamlined now and easier to read. But please revise the new sentences from L 339 until L 347 carefully for language, grammar and missing words.

 

L 367: “the prevalence of haemosporidian parasites differed between study sites and was high in Verkiai (7 %) during warm period" – are you sure you don’t mean Kairenai? Based on the supplemental table there were twice more positive mosquitoes found there than in Verkiai.

Comments on the Quality of English Language

The language should be carefully checked for missing articles throughout the manuscript, for grammar and sometimes also missing words or word order in the sentences.

Author Response

Dear authors, while many of my comments have been clarified, I still have a few things that need correction or improvement. Especially in the methods, the descriptions are in parts still not clear or not complete enough to allow replication of the procedures.

Please find below my responses to your responses and additional issues to address:

Thank You for Your comments and suggestions.

Introduction:

LL36 – 38 and LL 40 – 42: almost identical (= redundant)

The sentence was deleted and changed. L 40-41.

 

Methods:

Section 2.2: it is not clear what was done with the mosquitoes. mosquitoes were first anesthetised by cold exposure in the freezer. And then? the restriction digest analysis would have required killing of the mosquitoes and DNA extraction. as well as the dissection will have killed the mosquitoes. for what purpose anesthesize them first then?

We used freezer for several minutes to make mosquitoes inactive / immobile, so they would not move and can be identified and dissected. The mosquito is killed by dissection (the head is removed before the salivary glands are extracted) and remnants of mosquito body are used for DNA extraction. Restriction digest analysis takes place after DNA extraction and after PCR. We wrote about the restriction digest analysis in the chapter 2.2 as this analysis is related with identification. We changed text to make methods clear. L 98-130.

Response to the authors‘ corrections:

L86: „from May to September“ is double

Corrected. L88.

LL 79 – 83 and LL 92-93: The description of where hibernating mosquitoes were collected is partially redundant

Information has been shorten. L 80-85.

L108: „both slides“ – which is the second slide? You describe only one slide here for the midgut preparaitons. What is the oher one?

The sentence was corrected. L 110.

L110: specify SET buffer

This information was added. L 112-113.

L122: we used method based on.... the article is missing

The article was [27]. We corrected the sentence to be more clear. L 124-125.

L125: „mitochondrial DNA is affected separately“ -  mitochondrial DNA is digested differently for these two species

Corrected.

LL 111-113 and LL 136-141: I‘m still confused: were the active mosquitoes processed and analyzed in this study or a previous one? If it was done in a previous one and the sample processing is described there, then it should not be described here as if it was done in this study. Just refer to the previous study here.

Thank You for this comment. Only haemosporidian parasites in active mosquitoes were investigated before as described in [24]. We screened hibernating mosquitoes for the presence of haemosporidian parasites and trypanosomatids using both PCR and microscopy, and we investigated active mosquitoes for the presence of trypanosomatids using PCR and microscopy. We have shortened a bit details of the description of DNA extraction from active mosquitoes. L 137-141.

To emphasize that, the study methods were similar for both groups of mosquitoes (hibernating and active), we have left a brief information of the study methods (DNA extraction) to facilitate the reader's understanding. We investigated overwintering mosquitoes for both groups of parasites, so we would like to keep the primers used for both haemosporidian and Trypanosoma parasites.

L106 ff: Midgut preparations that were determined to be PCR positive for trypanosomatid DNA were examined microscopically - for PCR the DNA would have to be extracted from the midguts. how can they then by later analysed ny microscopy, they would have been destroyed before already. I don't understand this description

We made some changes in the text. L. 115-116. 

One mosquito female during investigation was divided into 1) salivary gland preparation, 2) midgut preparation, 3) all remnants after dissection were used for DNA extraction and PCR. Parasites such as haemosporidian can be found not only in certain organs (salivary glands, gut) of the mosquito body, but also in hemocoel, so extraction of salivary glands does not mean that haemosporidian parasite will not be detected in the remnants of mosquito (with removed salivary glands) using PCR as was proved also by Valkiunas et al. in https://doi.org/10.1007/s00436-013-3375-6 (for haemosporidian parasites). We used only half of a midgut for midgut preparation (for Trypanosoma parasites) and the rest of mosquito body (with removed salivary glands and half of a midgut) was used for DNA extraction and PCR. Similar method was also described by Fialova et al. in https://doi.org/10.3390/microorganisms9102101. 

Response to the authors‘ corrections:

That‘s clearer now, the essential part that was missing was „midgut preparations of mosquitoes“

However, there is still some unclear or imprecise phrasing (see above)

We have taken comments into consideration.

L112 ff: Hibernating mosquitoes were pooled (up to 10 individuals in one pool collected at the same locality the same day) for molecular analysis. - and how were the active females treated? also in pools? if not why the difference?

All females were dissected and investigated individually using microcopy. For PCR investigation hibernating females were pooled as we expected to find lower parasite prevalence in hibernating mosquitoes comparing with active females. This is very common to investigate pooled mosquitoes collected in the wild because of the low prevalence of infections, and this is described in many papers such as 

https://doi.org/10.3390/microorganisms10020294

https://doi.org/10.1186/s13071-023-05965-0

DOI 10.1186/s12936-017-2035-1

https://doi.org/10.1371/journal.pone.0196052

The prevalence of investigated parasites is less than 6% even in the summer, so pooling is an efective method of investigation. This was shortly explained in the text. L 133-135.

This whole section 2.2 is very cryptic and doesn't allow to understand how the ALL the samples used in this manuscript were treated and processed step by step. It has to be completely revised to be able to replicate the authors experiments.

This section had been revised.

Response to the authors‘ corrections:

Thank you for clarifying some of the issues. There are still some left though (see above)

We considered the comments.

 

Section 2.3: what is the "remaining samples"? 

This was corrected. L 137-138.

specify why were the hibernating females treated as pools but the others not? Explain or correct/add information for other samples

This information was added - we expected lower prevalence of parasites in hibernating mosquitoes, and this is a common method of testing mosquitoes collected in the wild for the presence of pathogens as we have already mentioned. L 133-135.

L120-123: provide primer sequences and reference genome on which the primers were designed.

Information has been provided. L 142-148.

 

Results:

Why were hibernating mosquitoes collected in Dec and Jan? Explain

Average daily temperature below 10°C in our country is usually from November to February and this is the hibernation time of mosquitoes. November is just the beginning of the hibernation period for Culex mosquitoes in the temperate zone of the northern hemisphere as it was also pointed in the discussion chapter. The main question was if mosquitoes infected with parasites can survive until spring. We started collecting mosquitoes in winter, from December and we believe that if we would investigate mosquitoes collected in February this would not change our results. We would like to note that towards spring the abundance of hibernating mosquitoes in cellars decreases and it is getting difficult to find them. We agree that investigation of hibernating mosquitoes collected in November would give us interesting results, as it was in November that mosquitoes infected with hemosporidian parasites were detected by Köchling et al. (we write about it in the discussion), so we plan of conducting research on this in the future together with experimental works. L 432-435.

 

Figure 1: is that the combined data from 2021 -2023? Specify in figure caption. If each bar represents data from a total of 3 years, then the data should not be displayed as total numbers but as average over the 3 years and the standard deviation should be indicated.

Consequently, also the parasite detections should be specified by year i.e. how much differed the parasite detections across the different years?

The figure was remade as suggested.

Response to the authors‘ corrections:

This is scientifically much more valuable now

Thank You.

The authors provide a list and phylogenetic tree for Trypanomosomatides but not for haemosporidian parasites. What's the reason for that? Please add this analysis if possible.

The main purpose of providing phylogenetic tree for Trypanomosomatids was to show that based on the DNA sequences our newfound trypanosomatids are monoxenous, not dixenous and are not related with vertebrate host. This information was added. Phylogenetic trees of Plasmodium parasites are provided in many publications investigating avian haemosporidian parasites, for example phylogeny of all three genetic lineages detected in mosquitoes during our investigation is discussed in  

https://doi.org/10.1017/S0031182017000737. 

Table 1: adding a column with the information if the parasite is has an avian or human host would make this information much faster accessible than just writing it in the text.

Corrected.

 

Additional things to address in the results:

LL 209 – 211: “All detected by PCR Trypanosoma species were obtained microscopically in midgut preparations of investigated mosquitoes (Fig. 2), but we did not detect T. theileri in preparations of mosquito midguts” – Well, then it was not all, because T. theileri was not detected, correct?

Moreover it should be: All Trypanosoma species detected by PCR…

The sentence has been corrected. L 211-213.

 

Discussion:

The discussion is in parts lengthy, and the red line is hard to follow. the authors jump between Trypanomosomatides and haemosporidian parasites – please sort and streamline to improve readability and to enhance the clarity of the points that the authors want to make.

We have shortened the discussion and made it easier to follow. We talked about the main findings in the beginning, later about trypanosomatids, haemosporidian parasites and feeding preference of Culex mosquitoes as this was also result from our data.

Tables 2 and 3: What does experimental research mean? Not clear, please explain in table captions

This information was provided – experimental infections of mosquitoes. L282, 379.

L296: "significant difference": where is the statistical analysis to prove statistically significant difference? Is the dataset even large enough to detect a significant difference given the low infection numbers?

Thank You for this question, we used Fisher exact test for statistical analysis and this test showed that statistically significant differences have been detected between infection rates of hibernating and active mosquitoes. L 197-198, 216-218, 239-243.

Discuss the increasing number of findings of monoxeous trypan. in human samples, as discussed for example in Boucinha Front Cell Infect Microbiol. 2022; 12: 804707;   doi:10.3389/fcimb.2022.804707

This information has been added. L 344-348.

Major concern regarding the main question of the study, if hibernating c. pipiens can be a parasite reservoir: hibernating females were only collected in one winter. This is not a representative data set. In combination with low infection rate during summer, especially with basically absent infections in September (the generation that probably forms the hibernating mosquitoes), in my opinion there is no reliable statement possible if hibernating mosquitoes can be a reservoir or not. For that many more samples would have to be analyzed per year, and for several successive years. 

Thank You for pointing this. The prevalence of parasites in wild caught mosquitoes is usually low and depends on mosquito species (between 0.04% to 6.6% for haemosporidian parasites and from 0.005% to 22% for Trypanosoma). Investigating natural prevalence in wild caught mosquitoes we will always find low prevalences. Nevertheless, we determined that parasite prevalence in hibernating mosquitoes was statistically significantly lower than in active mosquitoes. Really, we did not detect infected hibernating mosquitoes. Data on the infection rates of hibernating mosquitoes are scarce and our results fills this data gap. We do not say that the parasite can't overwinter in a mosquito, but we say it's unlikely they can overwinter. Experimental studies are needed to determine whether the parasites fail to survive in mosquitoes or whether mosquitoes infected with parasites do not survive until spring.

Response to the authors‘ corrections:

Thank you for the explanation. I am still concerned, though, about basing the statement, that hibernating mosquitoes are unlikely reservoirs, on animals collected only during one winter season (even though the total number of analyzed active and hibernating mosquitoes was similar). Checking animals only in one winter does not provide representative data (the numbers for the active females are also based on three years, not only one). Therefore, the authors should at least include the cautionary note in the discussion, that their conclusions on hibernating mosquitoes are based on the animals collected in a single winter. 

We added the sentence in the end of discussion. L 360-362.

 

Additional things to address in the discussion:´

LL 237 – 238: “No infected with investigated parasites mosquitoes were found among hibernating ones.”

That’s not true. L 192: 2 hibernating mosquitoes with trypanosomatids were detected.

Moreover, please check the grammar of the sentence

Sentences have been corrected: Haemosporidian and Trypanosoma parasites were not detected in hibernating mosquitoes…. L 242-243.

LL287 – 289: “Monoxenous trypanosomatids are generally considered non-pathogenic, however there is some evidence suggesting negative effects on insect fitness, although this has been investigated in only a few insect species” - Please provide the corresponding reference(s)

References have been added. L 283.

L 315: “infections seem to be the highest during the rains” – this should be: infections seem to be the highest during the raining periods

Corrected. L 302-303.

L347: "We cannot ignore the significant difference between the prevalence of parasites in mosquitoes collected during warm and cold periods.” -  I don’t understand what the authors want to say with this statement in this context

The sentence was changed. L 335-336.

The discussion is more streamlined now and easier to read. But please revise the new sentences from L 339 until L 347 carefully for language, grammar and missing words.

The text was corrected. L 330-341.

L 367: “the prevalence of haemosporidian parasites differed between study sites and was high in Verkiai (7 %) during warm period" – are you sure you don’t mean Kairenai? Based on the supplemental table there were twice more positive mosquitoes found there than in Verkiai.

Yes, it was detected 9 positive mosquitoes in Kairenai and only 4 positive mosquitoes in Verkiai. But we caught and investigated more mosquitoes in Kairenai with the infection prevalence being 2.2% and less mosquitoes were investigated from Verkiai with the prevalence being 7.0%.

Comments on the Quality of English Language

The language should be carefully checked for missing articles throughout the manuscript, for grammar and sometimes also missing words or word order in the sentences.

We checked all the manuscript once more and made some small changes.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

L40 Correct to – mosquito or mosquitoes

L44 Instead of – in our country give the name of country

Your table in the supplement is very good. I recommend to add it in the main text and not as supplementary material. The table requires revision: The authors should put: 2021 and then all months in this year. After that 2022 and then all months and so on…The first column should be a year, then months, then area, method, positives… and the authors should calculate the percentage of positives from the total collected for each of parasite.

 

This sentence is given as a response of the authors  - we also collected a small number of Culiseta and Anopheles mosquitoes, but these mosquitoes were not target species, as we investigated only Culex mosquitoes … should be modified and added to the results. 

Comments on the Quality of English Language

English is fine. 

Author Response

Thank You for Your comments and suggestions.

L40 Correct to – mosquito or mosquitoes

Corrected.

L44 Instead of – in our country give the name of country

Corrected.

Your table in the supplement is very good. I recommend to add it in the main text and not as supplementary material. The table requires revision: The authors should put: 2021 and then all months in this year. After that 2022 and then all months and so on…The first column should be a year, then months, then area, method, positives… and the authors should calculate the percentage of positives from the total collected for each of parasite.

The table has been corrected as suggested. This table provides row data and is long, so we would like to leave it as a supplementary table.

This sentence is given as a response of the authors  - we also collected a small number of Culiseta and Anopheles mosquitoes, but these mosquitoes were not target species, as we investigated only Culex mosquitoes … should be modified and added to the results. 

This information has been added to the Results, first paragraph.

Author Response File: Author Response.docx