Detection of diverse Wolbachia 16S rRNA sequences at low titers from malaria vectors in Kayin state, Myanmar

Background : Natural Wolbachia infections in malaria mosquitoes were recently reported in Africa, and negatively correlated with the development of Plasmodium falciparum in the vectors. The occurrence and effects of Wolbachia infections outside Africa have not been described and may have been underestimated. Methods : Mosquitoes were collected by human-landing catch during May and June 2017 in ten villages in Kayin state, Myanmar. Closely related species of malaria vectors were identified with molecular assays. 16S rRNA Wolbachia DNA sequences were detected with quantitative real-time PCR. Results: Low titer of Wolbachia DNA was detected in 13/370 samples in six malaria vector species. Sequences were diverse and different from those described in the African malaria mosquitoes. Conclusion: The detection of Wolbachia DNA in malaria mosquitoes from Kayin state warrants further investigations to understand better the ecology and biology of Anopheles- Wolbachia interactions in Southeast Asia.


Introduction
Wolbachia are intracellular bacteria that infect a wide variety of arthropods and filarial nematodes. Symbiotic relationship that results from the infection have a broad range of phenotypic effects on the infected hosts, from mutualism (beneficial) to commensalism (neutral) and parasitism (harmful) 1 . In mosquitoes, Wolbachia can invade the germline and induce cytoplasmic incompatibilities between the sperm from infected males and oocytes from uninfected females 2 . Hence, mass-releases of Wolbachia-infected male mosquitoes were attempted to extinguish mosquito populations 3,4 . Cytoplasmic incompatibilities produce a fitness advantage of Wolbachia-infected over uninfected female mosquitoes, thereby driving the spread of Wolbachia-infected females in the population. In addition, Wolbachia can interfere with the development of some pathogens in the mosquito host, including dengue virus 5 , Plasmodium malaria parasites 6 and filarial nematodes 7 . Therefore, the release of Wolbachia-infected female mosquitoes is proposed for transmission-blocking of some mosquito-borne diseases 8 .
Most diversions of mosquito-Wolbachia interactions for controlling vector-borne diseases were conducted with mosquitoes artificially infected with the endosymbiont. Natural Wolbachia infections may have important effects on mosquito populations and dynamics of diseases transmission but they are less well described 9 . Wolbachia DNA was detected by PCR in 27 mosquito genera including the medically important Aedes, Armigeres, Culex, Mansonia and Stegomiya 9-18 . Interestingly, this organism was not detected in malaria mosquitoes until recent observations of naturally infected anopheline vectors in Africa 9,13,19-24 .
Only one study assessed the effects of natural Wolbachia infection on the reproductive fitness anopheline mosquitoes, namely the dominant African malaria vector Anopheles coluzzi 22 . The authors did not observe cytoplasmic incompatibilities, differences in the number of eggs laid or progeny sex ratio, but infected females laid eggs more rapidly. Two studies demonstrated the negative effects of Wolbachia infections on the development of P. falciparum 20,22 . Shaw et al. observed a negative correlation between Wolbachia infection and the development of P. falciparum in naturally blood-fed females. Gomes et al. obtained similar results on the sporozoite stage by screening large numbers of mosquitoes identified as An. gambiae sensu stricto and An. coluzzi. In addition to their field investigations, Gomes et al. infected a laboratory-adapted An. coluzzi colony with a local strain of Wolbachia, and performed artificial transmission studies with cultured gametocytes of P. falciparum strain NF54. They observed a moderate yet significant positive correlation between Wolbachia infection and oocyst development, and a negative correlation between Wolbachia infection and the number of sporozoites that subsequently invaded the salivary glands.
Natural Wolbachia infections in Southeast Asian malaria vectors have not been reported. Their potential effects on Anopheles mosquitoes and dynamics of malaria transmission are not known. The objective of this study was therefore to assess the presence of Wolbachia in malaria vector populations in Kayin state, Myanmar.

Study sites and entomological collections
Entomological surveys were conducted in May and June 2017 in ten villages in Kayin state, Myanmar ( Figure 1). Each survey consisted of five consecutive nights of collection from 06:00 pm to 06:00 am as described previously 25 . In each village, five traditional houses were selected for mosquito sampling with human-landing catches. Collectors were asked to collect every mosquitoes landing on their uncovered legs for 50 min per hour and allowed to rest for 10 min per hour. Mosquitoes were shipped to Mae Sot (Thailand) at the end of each survey.

Malaria vectors identification
Mosquitoes were immediately identified at the genus level by morphology and Anopheles specimen were stored individually at -20°C in 1.5 mL plastic tubes containing silica gel.
Anopheles were identified at the Group or Complex level using the key developed by Rattanarithikul et al. 26 . Closely related species in the Funestus, Maculatus and Leucopshyrus Groups were discriminated in a subsample of the total number of collected mosquito using allele-specific PCR assays (AS-PCR) adapted from  . Single whole mosquitoes were crushed in 200 μl of cetyl-trimethylammonium bromide solution 2% (TrisHCl pH = 8, 20mM; EDTA 10mM; NaCl, 1.4 mM; N-cetyl-N,N,N-trimethyl ammonium bromide 2%) with a TissueLyser II™ (Qiagen) set on 29 movements /second for 3 minutes. Samples were then warmed at 65°C for 5 minutes and 200 μl of chloroform were added. The aqueous phase was collected and DNA was precipitated with 200 μl of isopropanol. After centrifugation at 20,000 g for 15 minutes, the pellet was washed twice with 200 μl of 70% ethanol and suspended in 50 μl of PCR grade water 30 . The PCR mix was composed of 1X Goldstar™ DNA polymerase (Eurogentec, Seraing, Belgium) AAG GGA TAG-3' and 5'-AGC TTC GAG TGA AAC CAA TTC-3') amplified a 438 bp conserved region of the 16S rRNA genes  and W-Specf/W16S (5'-CAT ACC TAT TCG AAG GGA  TAG -3' and 5'-TTG CGG GAC TTA ACC CAA CA -3') amplified a shorter fragment of the same region (102 bp). These two sets were selected because they were previously used by other in order to detect Wolbachia in Anopheles mosquitoes 20 . Without a priori knowledge on Wolbachia DNA sequences detected in this study, the W-Specf/W-Specr primer set was selected for its ability to detect most Wolbachia strains infecting insects and to establish phylogenetic relationships among isolates 32 .
The performances of the primers W-Specf/W-Specr for the detection and quantitation of Wolbachia in mosquito samples were compared to that of the primers W-Specf/W16S as described previously 30 . Briefly, a published strain of laboratory-reared Aedes aegypti artificially infected with Wolbachia strain wMel were used as a reference material 33 . The optimal conditions for the PCR (hybridization temperature for primers annealing, and concentration of MgCl 2 and primers) were determined during a single gradient experiment in order to take into account crossinteractions between the different parameters. The range tested were 55-62°C for the hybridization temperature, 2.5-4.5 mM of MgCl 2 and 100-400 nM of each primers. The reaction conditions that gave the smallest CP (optimal conditions) were selected for all subsequent experiments. Serial-dilution experiments were then carried out in order to verify PCR efficiency (EFF) and to estimate the standard curve parameters.
All experiments were conducted with a CFX-96® (Biorad) device. Reactions were conducted in 20μl of EVAGreen qPCR Mix Plus® (Euromedex); 5μl of DNA template was used in a total reaction volume of 25μl. Specificity of the PCR was confirmed by Sanger sequencing with both W-Specf/W-Specr primers for all samples that give at least 1/3 positive reaction. Positive reaction was defined by the presence of a PCR product with the same melting temperature than the positive control at the end of the thermocycling. Macrogen (Seoul, South Korea) performed both PCR product purification and sequencing off site to avoid contamination of our facilities with post-PCR amplicons. The sequences were used for phylogenetic analysis (accession numbers MK336794 -MK336806).

Data analysis
Human-biting rate was defined as the number of collected mosquitoes divided by the corresponding number of collectionnights. Poisson confidence intervals were calculated using the epitools package version 0.5-10 in R software. Human-biting rate for sensu stricto species in the Funestus, Maculatus and Leucopshyrus Groups was estimated using the relative proportion of the species in the corresponding group.
The limit of detection of the qPCR assay (LOD) was defined as the highest dilution (lowest concentration) that gave 100% of positive reactions. The performances of the two primer sets at low concentrations of Wolbachia were also compared by scoring the proportion of positive reactions as described previously 30,34 . Crossing-point (CP) values were determined using the regression algorithm of the analysis software of the PCR device (CFX Biorad Manager version 3.01, Biorad). CP values of standard samples in the serial-dilution experiments were used to set-up the standard curve of the assay. The best fit-line and the subsequent values of the slope and y-intercept were estimated by performing least-square analysis of the linear portion of the curve (Pearson's coefficient r 2 >0.990). PCR efficiency was estimated with the formula EFF = 10 (-1/slope) -1.
For the phylogenetic analysis, chimeric PCR products were detected with the DECIPHER software version 2.0 and excluded from subsequent analysis (4/17 samples with a positive PCR result). 16S ssuRNA sequences were blasted against the National Center for Biotechnology Information nucleotide database and the most similar sequence was downloaded. Reference Rickettsiales sequences were added and alignment was performed using the DECIPHER package version 2.10 in R software. DNA sequences were converted into RNA sequences and then aligned using the AlignSeqs() function set with default parameters in order to take into account base pairing and to use single-base and double-base substitution matrices. Tamura-Nei genetic distance model and neighbor-joining tree were computed with the ape package version 5.2 of the R software. There was 373 positions in the final dataset.

Ethical considerations
This project was approved through the ethics review committee on medical research involving human beings from Myanmar, Ministry of Health and Sports, Department of Medical Research (lower Myanmar): 73/Ethics 2014. All participants provided their written consent to participate in this study.
b Nb. pos. / Nb. tested: number of positive reactions (amplification of the PCR DNA target) / total of reactions performed at a given dilution.
c Intra-assay SD : intra-assay standard deviation (SD), calculated as the average SD of the mean CP value measured for each dilution during the same experiment.
d Inter-assay SD : inter-assay standard deviation (SD), calculated as the SD of the means CP values measured during two independent experiments. e score of the proportion of positive reactions at low concentrations of Wolbachia (score was calculated on dilutions 10 -5 and 10 -6 ); an example of the calculation of the score is given here : the maximum hit for the score is 18 reactions (9 at the dilution 10 -5 , +9 at the dilution 10 -6 ), the score obtained with the primer pair W-Specf/W-Specr is 66% (12/18=(7+5)/18).
was 108 and 110% with the primer sets W-Specf/W-Specr and W-Specf/W16S respectively, and the linear dynamic spanned over six orders of magnitude (r²=0.998 and 0.999) ( Table 1). There was a one-log decrease in the LOD of the assay when using W16S as a reverse primer instead of W-Specr, and the assay scored better at low concentrations of Wolbachia (16/18 and 12/18 positive reactions respectively, χ 2 = 2.5714, P=0.109). Typical amplification and melt curves are shown in the Figure 2.
Biodiversity of Anopheles mosquitoes Four thousand seven hundreds forty-three Anopheles were collected during 500 person-nights of collection. We report the occurrence of 12 Anopheles taxa among which nine were groups of closely related or sibling species ( (Table 3). Eight unique 16S rRNA sequences were identified ( Figure 3). 16S rRNA sequences clustered with that of Wolbachia strains in the supergroups B, D and F ( Figure 4).
Crossing-point values ranged from 31.0 to 40.6 amplification cycles and Wolbachia DNA titers were generally close or below the limit of detection of the assay (only one sample gave 3/3 positive reactions) (Table 4).

Discussion
Wolbachia DNA  CP values reported in this study suggest that Wolbachia DNA titers were very low, usually close or below the limit of detection of our assay. This result is not compatible with the integration of Wolbachia DNA in the mosquito genome, which would have given much lower CP values 35 . Important precautions were taken to ensure the quality of our molecular data 36 . This was the first study on Wolbachia in our facilities. The 16S DNA sequences detected in the screened samples were different from that of the reference material, hence excluding cross-contaminations. In addition, all experiments were conducted with the real-time PCR technology (which allows amplification and detection of the PCR DNA target in a closed system) and great care was taken to perform all handlings of PCR products off site. These precautions, combined with the good laboratory practices relevant to molecular diagnosis (eg. dedicated facilities with unidirectional workflow, experiment conducted by qualified laboratory technicians and appropriate quality controls), drastically limited the risk of false positive by contamination. The risk of false positive results due to low specificity of the assay was ruled out by sequencing the PCR product in all positive samples. It is probable that some results were falsely negative due to limited sensitivity, given that most positive samples were infected at a density close of below the detection of the assay. In this study, we have shown that using the W16S as a reverse primer increases the analytical sensitivity of the qPCR assay in the optimal reaction conditions. However, in the absence of a priori data on the Wolbachia DNA sequences detected in this study, we selected the W-Specf/W-Specr primers to perform the screening because of their availability to detect a wide variety of Wolbachia infecting insects and to establish phylogenetic relationships among field isolates 32 . Molecular phylogeny based on 16S rRNA sequences revealed a high diversity of Wolbachia strains, which belonged to different lineages than those recently reported in the African malaria vectors [19][20][21][22][23][24] . Eight out of thirteen sequences reported in this study were unique. The DNA extracts were also used to assess Plasmodium infection rates in the mosquito population (data not shown), precluding multi locus sequence typing of the Wolbachia strains because there was no material remaining after the screening.
The significance of these findings regarding the biology and ecology of Wolbachia-Anopheles interactions must be interpreted cautiously as the detection of low titers of Wolbachia DNA by PCR is not unequivocal of an actual symbiosis between Wolbachia and the mosquito. The detection of Wolbachia in the supergroup D and F suggests that some DNA extracts were contaminated with Wolbachia endosymbionts of filarial nematodes rather than reflecting actual Wolbachia infections  (1-7) 1.2 (0.1-4.3) 9.6 (5.5-15.6) 6 (2.9-11) 3.6 (1.3-7.8) An. culicifacies A c 9 (5-14.8) 12.6 (7.  11.4 (6.9-17.8) 6.6 (3.3-11.8) An. introlatus An. rampae . Humanbiting rates of sensu stricto anopheline species in the Funestus, Maculatus and Leucosphyrus Groups were estimates from the relative proportion of each species in the corresponding Group assessed with molecular assays.

Conclusion
The detection Wolbachia DNA in malaria vectors from Kayin state warrants further investigations to understand better the ecology and biology of Anopheles-Wolbachia interactions in Southeast Asia.

Data availability
The data is available upon request to the Mahidol Oxford Tropical Medicine Research Unit Data Access Committee (http://www.tropmedres.ac/data-sharing) and following the Mahidol Oxford Tropical Medicine Research Unit data access policy (http://www.tropmedres.ac/_asset/file/data-sharingpolicy-v1-0.pdf).

Ewa Chrostek
Institute of Integrative Biology, University of Liverpool, Liverpool, UK Thank you for the revisions. The problematic "Wolbachia infection" has been mostly replaced, except from one place in the discussion. Also, I disagree with calling the Wolbachia DNA from previous reports an infection in the abstract and the introduction of this paper. In my opinion, this study is not defficient compared to the previous ones on Wolbachia in Anopheles. It only has the potential to be more carefully worded.
I agree with the author's interpretation of the qPCR results. However, as primer dimers contribute to the total SYBR green fluorescence in the qPCR reaction this assay cannot be quantitative. Also, only running the qPCR products on the agarose gel can reveal if the bands can indeed be distinguished by a conventional PCR. As the authors do not aim at PCR-identification or qPCR quantification of Wolbachia here this is not crucial. It is curious though, and suggests that the qPCR assay could be optimized further.
No competing interests were disclosed. Competing Interests: Reviewer Expertise: I have been applying molecular methods to study Wolbachia symbionts of insects since 2011. In 2016-2017, I was working on putative Wolbachia infections in Anopheles gambiae and Anopheles coluzzi.

© 2019 Chrostek E. This is an open access peer review report distributed under the terms of the Creative Commons
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original Attribution License work is properly cited.

Ewa Chrostek
Institute of Integrative Biology, University of Liverpool, Liverpool, UK In this study, the authors provide evidence for qPCR amplification of a fragment of Wolbachia 16S sequence from 13 out of 370 field-collected Southeast-Asian mosquitoes. However, a single, low level, hard to amplify (despite extensive optimisation) fragment of Wolbachia rDNA does not provide sufficient evidence for the Wolbachia infection in these malaria vectors. The environment is severely contaminated with Wolbachia as most arthropods are infected with this symbiont (Weinert . 2015 ). To show an et al infection, additional experiments are necessary, e.g. showing intracellular localization of the sequences and maternal transmission of the bacteria (see Chrostek and Gerth 2019 for further discussion). I suggest changing the wording throughout, from "natural Wolbachia infections" to "Wolbachia 16S/wSpec sequence amplification". I also recommend changing the title to: "Detection of low-density Wolbachia 16S sequences from malaria vectors in Kayin state, Myanmar".
In the results section, the authors identify the supergroups D and F Wolbachia sequences as likely environmental/parasitic contaminants (6 out of 13 positive samples). It is unclear why they do not use the same caution while interpreting data on the remaining 7 sequences from supergroup B. Figure 2 shows an interesting property of wSpec primer set. The amplification from the positive and negative samples produces similar amplification curves ( Fig. 2A, green and red lines) with very similar CPs (crossing-points), that can be distinguished from each other by the shape of their melt curved (Fig.  2B). This figure shows, that when using wSpec primer set for a PCR, negative samples produce an amplicon as well. Whether the positive and negative amplicons can be distinguished by agarose gel electrophoresis, and therefore if classical PCR with wSpec has any diagnostic value when used under this conditions, remain to be determined. The comment on this in the text would help future studies embarking on Wolbachia identification projects.

Minor comments:
The information on the origin of the laboratory-reared wMel-infected is missing. Were A. aegypti they made by the authors or are they the published strains?
The sequence of W16S primer, the amplicon sizes for both qPCR reactions and the rationale behind trying both primer sets should be added.
In the discussion, p7/28, first line: the integration into the chromosome would have produced "higher CP values", and not as it is -"lower". We thank to reviewer for her useful comments on the manuscript. The wording "natural infection" was changed in the title and throughout the manuscript as per reviewer's suggestion. In addition, part of the discussion was rewritten and suggested reference was added in order to better emphasize this study limitation in the revised version of the manuscript. The possibility of environmental contamination for all sequences including that of the supergroup B was explicitly discussed in the Discussion section. The amplicon produced in no-template controls and negative samples certainly results from the formation of primer dimers, which are unlikely to reach the size of 438 bp. Therefore, it should be possible to discriminate between primer dimers and amplification of the PCR DNA target using gel electrophoresis. Nevertheless, the reviewer is right to question the value of conventional PCR in the field of molecular diagnostic given the high risk of false positive by contamination [1]. This issue has been extensively discussed in the manuscript and we do not wish to add more details in the current version of the manuscript. Point-by-point answers to specific comments are listed below:

1.
The information on the origin of the laboratory-reared wMel-infected A. aegypti is missing.
Were they made by the authors or are they the published strains?
Were they made by the authors or are they the published strains? -infected reference samples were kindly provided by Dr. Lauren Carrington from the Wolbachia Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam. Corresponding reference [2] was added in the methods section of the revised version of the manuscript.

2.
The sequence of W16S primer, the amplicon sizes for both qPCR reactions and the rationale behind trying both primer sets should be added. The sequence of W16S primer, the amplicon sizes for both qPCR reactions and the rationale behind trying both primer sets were added in the Methods section in the revised version of the manuscript.

3.
In the discussion, p7/28, first line: the integration into the chromosome would have produced "higher CP values", and not as it is -"lower". The integration of genome into the mosquito chromosome would have resulted in a Wolbachia higher copy number of PCR DNA target in the sample (several millions versus a few dozens), and therefore in a lower CP value given the negative correlation that exists between the CP value and the logarithm of the concentration of PCR DNA target in the sample.

1.
Apfalter P, Reischl U, Hammerschlag MR. In-house nucleic acid amplification assays in research: how much quality control is needed before one can rely upon the results? J Clin

Francesco Baldini
Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, UK The second version of this manuscript provides greater clarity and detailed methodological information. Limitations of the study are also discussed. Overall the manuscript contains novel insights on natural strains in species from Myanmar, calling for further investigations on the role of Wolbachia Anopheles these endosymbionts on the biology of malaria mosquitoes.

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility? No

Are the conclusions drawn adequately supported by the results? No
No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: I believe that I have an appropriate level of expertise to assess the submitted article. I have expertise in identification of natural Wolbachia infections in natural populations of Anopheles and other vectors; I have also expertise in the methods used, mainly quantitative PCR and phylogenetic analysis. show that CP values were generated from no template controls (some below 35) and melt curve analysis was required to differentiate primer dimers from genuine target amplification. With gene qPCR I would not have expected no template control Wolbachia CPs to come up at below 35 cycles and this could be due to trying to amplify a 16S gene fragment >400 base pairs (the first report of this to my knowledge). According to Table 1, the threshold CP value for using the Wspec primer set appears to be between 30-33 cycles given only 7/9 of the 10 dilution amplified. This would be further supported by the CP of 10 (34.95 +/-1.64) not being statistically higher -5 -6 amplified. This would be further supported by the CP of 10 (34.95 +/-1.64) not being statistically higher that to the 10 dilution (34.48 +/-0.33). The authors rightly conclude that all of the samples in this study are at threshold detection (CP values >31 cycles) and the authors correctly used both melt curve analysis and sequencing to confirm this was genuine amplification. Further details of the qPCR analysis strengthens the manuscript but I'm not entirely sure how the authors estimated bacterial load. The sentence 'Given that DNA was extracted from whole mosquitoes, it was possible to estimate the bacterial load in single mosquitoes without using a calibrator to normalize the signal' needs further clarification, particularly given the title has changed to include reference to 'low density strains'. The CP values would suggest low density infections but in order to make the comparison to Mel in you would w Aedes aegypti need to account for body size and/or extraction efficiency as these factors will influence CP Wolbachia values and therefore estimating bacterial load. Lab-reared adults will likely be larger in size Aedes aegypti than wild caught species so that comparison is not possible without either normalising to host Anopheles genes or measuring the total DNA extracted. I also would suggest a better measure of prevalence rates would have resulted from using the Wspec primer set in the conventional PCR format (Werren & Windsor, 2000 ) which has been routinely used to screen mosquito populations. Figure 4 shows the phylogeny of the 16S gene and it appears that the sequences are quite Wolbachia diverse within individual species (eg. appearing to have four different strains) and the same An. maculates strains appear across multiple species (eg. and ). Although An. minimus An. baimaii Wolbachia superinfections exist in mosquito species, having the same strain of (based on 16S Wolbachia sequences) in multiple species would need confirmation from additional gene sequencing as this seems unlikely for endosymbiotic bacteria. The discussion does now contain an explanation that no Wolbachia material was left for MLST due to assessing infection rates despite having 50 μL of eluted Plasmodium DNA. Overall the reliance on only 16S sequences (some of which appear to have identical sequences across multiple species) is still problematic in my opinion and I would think a title that contains Anopheles 'genetically diverse' is inappropriate based on sequencing of only one gene fragment. Wolbachia

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility? No

Are the conclusions drawn adequately supported by the results? No
No competing interests were disclosed. Competing Interests: We thank to reviewer for his useful comments on the manuscript. Reviewer's concern on the comparison of DNA titers measured in wild and laboratory-reared Wolbachia Anopheles Aedes was addressed by removing the corresponding sentences in the Methods, Results and aegypti Discussion sections. Our data clearly demonstrate that wild are infected with very low Anopheles densities given the high CP values of the screened samples (>30 cycles) and the very Wolbachia low CP values of the reference samples (<19 cycles). The reference to "low-density Aedes aegypti infections" in the title is therefore valid, and does not overlook low densities as a specific feature of the strains detected in this study. We agree with reviewer's comment on the limitation of not having additional sequence data on other markers, and regret that it was not possible to generate such data in the current study. Although it not possible to assess accurately the phylogenic relationships between different strains only with 16S rRNA sequences, our data clearly demonstrate Wolbachia that the strains detected in this study are genetically diverse. Therefore, we wish to Wolbachia keep the title of the manuscript unchanged.
No competing interests were disclosed. Anopheles shown to potentially influence the vectorial capacity of the infected vectors. The authors state that infections were identified by using quantitative PCR approach followed Wolbachia shown to potentially influence the vectorial capacity of the infected vectors. The authors state that infections were identified by using quantitative PCR approach followed Wolbachia by sequencing of the 16S gene, although some details are missing and the methodology Wolbachia should be clarified. Phylogenetic analysis is also not completely detailed. Specific comments to the manuscript are listed below.

Introduction:
The authors state that cytoplasmic incompatibility (CI) 'enhances the "vertical" transmission of Wolbachia'. From my knowledge this is incorrect, as CI does not directly affect "vertical" transmission, but rather increases 'indirectly' the fitness of the progeny of Wolbachia infected mothers.
The authors state that 'Shaw et al. observed a negative correlation between Wolbachia infection and the development of oocysts in naturally blood-fed females'. This is not completely correct, as in P. falciparum this work the authors have quantified the prevalence of in resting blood fed females inside P. falciparum house 5 days after collection/blood feeding, without any prior knowledge on the infectious status of the mosquitoes; thereby, the stage of parasite infection (oocyst or sporozoite) was not investigated. If W-Spec primers were used, the expected product size is >400 base pairs (bp); this bp size is often too large for qPCR, as large amplicons tend to produce secondary structures during the dissociation steps, thus resulting in multiple melting peaks. If possible, it would be informative to provide more details on the optimization of this assay (in case additional reagents were added, for example) and to show the dissociation profile of the obtained amplicons, as this would enable troubleshooting of the technique if others will try to replicate the work and/or use the same methodology. Also, it should be specified how sequencing was performed, e.g. direct purification after qPCR (how?), which primer was used, etc.

Methods
In the phylogenetic analysis the authors should state what was the sequence size used to build the tree. Novel obtained sequences should also be uploaded and their unique identifier indicate in the article. Table 1-3 should be indicated (if any).

Results:
The authors state the 'we assessed species diversity, Plasmodium and Wolbachia infection rates in these Groups'. This is incorrect as Plasmodium infection rates are not shown. Table 1-3 are missing information on what each column indicates. Although these tables can be generally informative, I wonder if showing species diversity using pie-charts (for each species group) over imposed on the map in Figure 1 would provide a more direct illustration of the species composition and abundances of the Anopheles species in the study area.

Captions in
As indicated in the methods, the size of the sequence used for Figure 2 should be indicated. It would be informative to include the alignment use for the tree figure. Figure 3b shows the overall prevalence of Wolbachia in different villages without specifying the species, so I am not sure what is the purpose of illustrating the result in this way. If this is too show that some villages have higher prevalence over others this should be indicated only if statistical analysis supports it (although I doubt this is the case if species distribution is included as a variable). phylogenetic analysis. Table 1-3 should be indicated (if any). Tables 1-3 were replaced by a single table presenting human-biting rate instead of relative proportion of each mosquito species in the corresponding group. Appropriate definition of the statistics used to calculate human-biting rate are given in the Methods section.

Statistical analysis used in
The authors state the 'we assessed species diversity, Plasmodium and Wolbachia infection rates in these Groups'. This is incorrect as Plasmodium infection rates are not shown. The inaccurate statement was removed from the revised version of the manuscript.
Captions in Table 1-3 are missing information on what each column indicates. Although these tables can be generally informative, I wonder if showing species diversity using pie-charts (for each species group) over imposed on the map in Figure 1 would provide a more direct illustration of the species composition and abundances of the Anopheles species in the study area. Table 1-3 were removed from the manuscript and replaced by a single table showing human-biting rate estimates collated by village and species. Given the number of study villages and diversity of mosquitoes, we do not think that figuring multiple pie-charts on the map will improve the Anopheles readability of the data.
As indicated in the methods, the size of the sequence used for Figure 2 should be indicated. It would be informative to include the alignment use for the tree figure. The sequence alignment was added to the revised version of the manuscript and the number of positions in the final dataset used to build the tree was added to the figure legend of the revised version of the manuscript.

Figure 3b shows the overall prevalence of Wolbachia in different villages without specifying the species, so I am not sure what is the purpose of illustrating the result in this way. If this is too show that some villages have higher prevalence over others this should be indicated only if statistical analysis supports it (although I doubt this is the case if species distribution is included as a variable).
The reviewer is right to question the relevance of our prevalence data. Given the low sample size and the diversity of strains and species, prevalence data were removed Wolbachia Anopheles from the revised version of the manuscript.
The authors state that 'Crossing-point values ranged from 31.0 to 40.6 amplification cycles. Infected specimens were generally infected at a density close or below the limit of detection of the assay (only one sample gave 3/3 positive reactions).' More details should be given regarding the rational for inclusion (or exclusion) of an infected/amplified sample; it is not clear to me if 'reactions' refers to technical replicates in the same qPCR run or in different qPCR assays. This should be described with more details. It would also be informative to normalize the quantity of the amplified Wolbachia 16S using a mosquito housekeeping gene, for example. Indeed, as 'density [was] close or below the limit of detection of the assay' normalization would provide information on the likelihood of false negatives in samples, as you would expect if the total DNA is very low (for example due to inefficient DNA extraction). It is common that at low parasite concentration, only some replicates give a positive result because the distribution of the DNA template in the reaction tube follow a Poisson distribution (Sterkers, Varlet-Marie et al. 2010, Stahlberg and Kubista 2014, Chaumeau, Andolina et al. 2016). This observation does not challenge the validity of our results. A clear statement that some Wolbachia infected sample have probably been missed because bacterial density observed in are Anopheles close or below the limit of detection of the assay that give 95% positive reaction. Misleading interpretations on the prevalence of infection were removed given the small sample size Wolbachia and the possibility of false negative. laboratory population (not virus inhibition). Furthermore, including references 5 & 6 in the context of pathogen blocking is not appropriate given this was work which was proposing to use cytoplasmic incompatibility to reduce mosquito populations Culex and artificial -infected mosquito lines were only established in the mid 2000s. Wolbachia The paragraph describing natural infections in mosquitoes also needs further references . Wolbachia The final paragraph in the introduction presents the fact that Natural infections in Southeast Wolbachia Asian malaria vectors have not been reported. However, the authors should reference the studies in which screening of Anopheles species for Wolbachia was undertaken despite finding no evidence of natural infections .

Methods
The primers used for Wolbachia detection W-Specf (CATACC TATTCGAAGGGATAG) and W-Specr (AGCTTCGAGTGAA ACCAATTC) produce a product size of 438 bp and this (to my knowledge) would not be possible or has not been reported using a qrtPCR format. The authors also reference Gomes et al.
which used a different reverse primer (5′-TTGCGGGACTTAACCCAACA-3′) that results in amplification of a smaller fragment of the 16S rRNA gene for qrtPCR. The accession numbers MK336794 -MK336806 refer to sequences with >400 bases indicating W-Specf/W-Specr was used. The authors need to clarify if W-Specf/W-Specr was used on a qrtPCR format or if both were used independently and report the differences in prevalence rates using these two primer sets.

Results
The inclusion for analysis of only what would be considered 'primary malaria vectors' needs more explanation if the authors overall aim was to provide evidence for natural infections in Wolbachia species given the mosquitoes were not screened for infection.

Anopheles Plasmodium
Tables 1-3 provide a breakdown of the species composition collected at the different villages but I think it would be more informative to have all the different species grouped according to villages. Currently it's difficult to determine mosquito species prevalence on a village level.
The statistics used in tables 1-3 don't appear to be explained either in the manuscript methods or in the table legends. For example, I am assuming 'n/N' means the species/total number collected but again this would be much easier to understand if species were grouped by villages.

Wolbachia infections
With reference to my previous point raised in the methods, which 16S PCR primer set and format was used to determine the prevalence rates and to generate sequences for Figure 2? The authors provide the overall prevalence rate (13/370) and then have Figure 3 to show the individual species. Figure 3a I don't feel is needed because plotting 1/11 (PSE) and 1/12 (DIR) seems unnecessary and could be in a table that incorporates prevalence rates by species and village. Having an overall village prevalence rate (Fig 3b) has little biological relevance given you have variable Anopheles species containing what appears to be different Wolbachia strains based on 16S analysis.
A major limitation of the phylogenetic analysis (and even the prevalence rates) is only using a single 1,2,3 4 5 A major limitation of the phylogenetic analysis (and even the prevalence rates) is only using a single gene (16S) but I appreciate that Cp values ranging from 31-40.6 are at the limit of detection.

Wolbachia
The authors should provide these 16S Cp values to allow the reader to see the variation both between technical and biological replicates.
Could the authors also provide the rationale for concluding that samples were positive where not all technical replicates produced positive amplification given 'only one sample gave 3/3 positive reactions'? How do these results fit with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines ?
Could the authors not have used another qPCR assay based on a second gene that targets a Wolbachia broad range of strains ?
The density comparison to laboratory-reared Aedes aegypti artificially-infected with the wMel Wolbachia strain is not particularly informative for several reasons. Firstly, quantifying Wolbachia density without accounting for mosquito body size and/or DNA extraction efficiency is problematic. Secondly, the wMel is a strain that naturally infects so a better comparison would be to natural Drosophila melanogaster infections in mosquito species (such as Pip in or even the w Culex quinquefasciatus wAlbA/wAlbB strains in ). Therefore, I would question the inclusion of this density data given Mel in Aedes albopictus w Ae. is an artificial infection. aegypti

Discussion
The sentence 'It was not possible to study more in detail the phylogeny of strain detected in Wolbachia this study by multi locus sequence typing because of the lack of DNA extracts after the screening' needs clarification. Do the authors mean that they were unable to amplify any of the MLST genes? Wolbachia Did they try using degenerate primer protocols or nested PCR given the qPCR data would indicate low density infections?
The statement "This is consistent with previous attempts to quantify in naturally infected Wolbachia malaria vectors" is incorrect and refers to some (An. gambiae complex) but not all species analysed in Sub-Saharan Africa . The authors should expand this discussion as the low density infections presented in this study are comparable to those strains detected in mosquitoes from Sub-Saharan An. gambiae Africa. Some of these studies have only resulted in 16S gene amplification and sequencing resulting in conflicting phylogenetics which appear incompatible with the traditional criteria for vertically transmitted endosymbionts (reviewed in reference 9 ). The authors should provide some further discussion points on whether their results only amplifying 16S could have resulted from either 1) integration into the mosquito genome or 2) some form of contamination. However, additional gene analysis would allow Wolbachia more confidence in these detected strains given the high 16S qPCR Cp values are at the limit of detection. . 2018. bioRxiv Publisher Full Text

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly We thank to the reviewer for his useful feedback on the manuscript. More details on the qPCR assay used to detect in this study were provided in the revised version of the Wolbachia manuscript, including the raw quantitative data as per reviewer's suggestion. We also agree that additional genotyping would have been an important added value to the manuscript. Wolbachia However, there was not enough DNA material to attempt additional genotyping of the Wolbachia strains detected in this study given that mosquito samples were also screened for Plasmodium infection and identified at the species level with molecular assays.
Response to point specific comments are listed below: The introduction needs significant improvement in the referencing. For example, the sentence 'In addition, Wolbachia can interfere with the development of some pathogens, including dengue virus' contains a reference to a publication that only describes Wolbachia establishment and invasion in an Aedes aegypti laboratory population (not virus inhibition). Furthermore, including references 5 & 6 in the context of pathogen blocking is not appropriate given this was work which was proposing to use cytoplasmic incompatibility to reduce Culex mosquito populations and artificial Wolbachia-infected mosquito lines were only established in the mid 2000s. The paragraph describing natural Wolbachia infections in mosquitoes also needs further references. The final paragraph in the introduction presents the fact that Natural Wolbachia infections in Southeast Asian malaria vectors have not been reported. However, the authors should reference the studies in which screening of Anopheles species for Wolbachia was undertaken despite finding no evidence of natural infections. The referencing of the introduction was improved as per reviewer's suggestions.
The primers used for Wolbachia detection W-Specf (CATACC TATTCGAAGGGATAG) and W-Specr (AGCTTCGAGTGAA ACCAATTC) produce a product size of 438 bp and this (to my knowledge) would not be possible or has not been reported using a qrtPCR format. The authors also reference Gomes et al. which used a different reverse primer (5′-TTGCGGGACTTAACCCAACA-3′) that results in amplification of a smaller fragment of the 16S rRNA gene for qrtPCR. The accession numbers MK336794 -MK336806 refer to sequences with >400 bases indicating W-Specf/W-Specr was used. The authors need to clarify if W-Specf/W-Specr was used on a qrtPCR format or if both were used independently and report the differences in prevalence rates using these two primer sets. Amplification of fragments much longer than 438 bp with real-time PCR technology has been reported previously (Rothfuss, Gasser et al. 2010). Without knowledge on the DNA a priori sequences of the strains detected in this study, the primer W-Specf and W-Specr were Wolbachia chosen for their ability to detect most strains infecting insects and to establish Wolbachia phylogenic relationship among isolates (Werren and Windsor 2000). The results of additional assay optimization and serial dilution experiments with the W-Specf/W16S primers used by were added to the revised version of the manuscript, although we did not use these Gomes et al. primers for the screening of in field mosquito samples.

Wolbachia
The inclusion for analysis of only what would be considered 'primary malaria vectors' needs more explanation if the authors overall aim was to provide evidence for natural infections in species given the mosquitoes were Wolbachia Anopheles not screened for infection.

Plasmodium
The vector status of species in the Thailand-Myanmar border area has been Anopheles 5 The vector status of species in the Thailand-Myanmar border area has been Anopheles determined previously (Somboon, Aramrattana et al. 1998, Chaumeau, Fustec et al. 2018 Tables 1-3 provide a breakdown of the species composition collected at the different villages but I think it would be more informative to have all the different species grouped according to villages. Currently it's difficult to determine mosquito species prevalence on a village level. The reviewer is right to question the relevance of presenting specific diversity as a proportion of a given species in the corresponding group. In the revised manuscript, human-biting rates were reported is a single table instead of the relative proportions.
The statistics used in tables 1-3 don't appear to be explained either in the manuscript methods or in the table legends. For example, I am assuming 'n/N' means the species/total number collected but again this would be much easier to understand if species were grouped by villages. In the revised version of the manuscript, table 1-3 were merged in a single table and appropriate description of the statistics used in this table were added to the Methods section.
With reference to my previous point raised in the methods, which 16S PCR primer set and format was used to determine the prevalence rates and to generate sequences for Figure 2? The primer W-Specf and W-Specr were used in a real-team PCR format for both estimation of the prevalence rates and phylogenetic analysis as described in the Methods section. The authors provide the overall prevalence rate (13/370) and then have Figure 3 to show the individual species. Figure 3a I don't feel is needed because plotting 1/11 (PSE) and 1/12 (DIR) seems unnecessary and could be in a table that incorporates prevalence rates by species and village. Having an overall village prevalence rate (Fig 3b) has little biological relevance given you have variable Anopheles species containing what appears to be different Wolbachia strains based on 16S analysis. The reviewer is right to question the biological significance of plotting prevalence estimates per species and per village. In the revised manuscript, the screening results collated by village and species are presented in a table, and the two plots were removed.
A major limitation of the phylogenetic analysis (and even the prevalence rates) is only using a single gene (16S) but I appreciate that Cp values ranging Wolbachia from 31-40.6 are at the limit of detection. The authors should provide these 16S Cp values to allow the reader to see the variation both between technical and biological replicates. We agree with the reviewer the analyzing only 16S ssuRNA genes is a limitation of our study. Raw quantitative data were added to the revised version of the manuscript as per reviewer suggestion.
Could the authors also provide the rationale for concluding that samples were positive where not all technical replicates produced positive amplification given 'only one sample gave 3/3 positive reactions'? How do these results fit with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines ?
It is common that at low parasite concentration, only some replicates give a positive result because It is common that at low parasite concentration, only some replicates give a positive result because the distribution of the DNA template in the reaction tube follow a Poisson distribution (Sterkers, Varlet-Marie et al. 2010, Stahlberg and Kubista 2014, Chaumeau, Andolina et al. 2016). This observation does not challenge the validity of our results. A clear statement that some Wolbachia infected sample have probably been missed because bacterial density observed in are Anopheles close or below the limit of detection of the assay that give 95% positive reaction. Misleading interpretations on the prevalence of infection were removed given the small sample size Wolbachia and the possibility of false negative.
Could the authors not have used another qPCR assay based on a Wolbachia second gene that targets a broad range of strains.
In the absence of knowledge on the strains infecting samples, the a priori Wolbachia Anopheles W-Specf/ W-Specr primers were chosen for their ability to detect a broad range of strains infecting insects (Werren and Windsor 2000). There was not enough DNA material remaining after the screening with the W-Specf/ W-Specr primers to use another assay. However, the specificity of the PCR was confirmed in all positive sample by Sanger sequencing of the PCR product.
The density comparison to laboratory-reared Aedes aegypti artificially-infected with the wMel Wolbachia strain is not particularly informative for several reasons. Firstly, quantifying Wolbachia density without accounting for mosquito body size and/or DNA extraction efficiency is problematic. Secondly, the wMel is a strain that naturally infects so a better comparison would be to Drosophila melanogaster natural infections in mosquito species (such as Pip in or w Culex quinquefasciatus even the in ). Therefore, I would question wAlbA/wAlbB strains Aedes albopictus the inclusion of this density data given Mel in is an artificial infection. w Ae. aegypti We agree with the reviewer that there is little biological relevance in comparing the density of infection in artificially infected and naturally infected . We Wolbachia Aedes aegypti Anopheles think that presenting those quantitative data is an added value to support that natural Wolbachia infection in this study actually occur at low density rather than resulting from of a low assay sensitivity. We believe that a calibrator to normalize the signal is not necessary as DNA was extracted from whole mosquitoes and bacterial loads expressed as an (arbitrary) number of bacteria per mosquito rather the a number of bacteria per weight-unit of mosquito body (Varlet-Marie, Sterkers et al. 2014, Chaumeau, Andolina et al. 2016. The sentence 'It was not possible to study more in detail the phylogeny of strain detected in this study by multi locus sequence typing because of Wolbachia the lack of DNA extracts after the screening' needs clarification. Do the authors mean that they were unable to amplify any of the MLST genes? Did they Wolbachia try using degenerate primer protocols or nested PCR given the qPCR data would indicate low density infections? The methodology used for detection in this study was described into detail in the Wolbachia Methods section and we did not attempt additional experiments on than that described Wolbachia in the manuscript. In addition to detection and molecular identification of the mosquito Wolbachia species, samples were also screened for infection (data not shown). There was no Plasmodium DNA material to perform additional experiment after the screening with the W-Specf/ W-Specr primers.
The statement "This is consistent with previous attempts to quantify in Wolbachia naturally infected malaria vectors" is incorrect and refers to some (An. gambiae complex) but not all species analysed in Sub-Saharan Africa . The authors should expand this discussion as the low density infections presented in this study are comparable to those strains detected in mosquitoes from Sub-Saharan An. gambiae Africa. Some of these studies have only resulted in 16S gene amplification and sequencing resulting in conflicting phylogenetics which appear incompatible with 8 sequencing resulting in conflicting phylogenetics which appear incompatible with the traditional criteria for vertically transmitted endosymbionts (reviewed in reference 9 ). The authors should provide some further discussion points on whether their results only amplifying 16S could have resulted from either 1) integration into the mosquito genome or 2) some form of contamination. However, additional gene analysis would allow more confidence in these detected Wolbachia strains given the high 16S qPCR Cp values are at the limit of detection. We thank to the reviewer for his useful feedback on the interpretation of our quantitative data. Suggested edits and references were added in the revised version of the manuscript.