Evidence for natural hybridization and novel Wolbachia strain superinfections in the Anopheles gambiae complex from Guinea

Wolbachia, a widespread bacterium which can influence mosquito-borne pathogen transmission, has recently been detected within Anopheles (An.) species that are malaria vectors in Sub-Saharan Africa. Although studies have reported Wolbachia strains in the An. gambiae complex, apparent low density and prevalence rates require confirmation. In this study, wild Anopheles mosquitoes collected from two regions of Guinea were investigated. In contrast with previous studies, RNA was extracted from adult females (n = 516) to increase the chances for the detection of actively expressed Wolbachia genes, determine Wolbachia prevalence rates and estimate relative strain densities. Molecular confirmation of mosquito species and Wolbachia multilocus sequence typing (MLST) were carried out to analyse phylogenetic relationships of mosquito hosts and newly discovered Wolbachia strains. Strains were detected in An. melas (prevalence rate of 11.6%–16/138) and hybrids between An. melas and An. gambiae sensu stricto (prevalence rate of 40.0%–6/15) from Senguelen in the Maferinyah region. Furthermore, a novel high-density strain, termed wAnsX, was found in an unclassified Anopheles species. The discovery of novel Wolbachia strains (particularly in members, and hybrids, of the An. gambiae complex) provides further candidate strains that could be used for future Wolbachia-based malaria biocontrol strategies.

Did you use a Ct threshold after which detection was considered unreliable? P4, L44-45 This reads as if the 16S PCR was done on genomic DNA rather than cDNA. Please clarify.
P4, L46-47 The CifA/CifB PCRs are not mentioned anywhere in the results or discussion. Suggest to remove the protocol here.
P5, L38 Please provide justification for using TN model of evolution.
P5, L39-40 If this means bootstrap analysis, please provide details here.
P5, L41-44 It appears the description is for the calculation of the starting tree, rather than for the ML reconstructions. Please clarify.

P5, L49ff
Please provide more details on negative and positive controls here. Did you include Wolbachiafree Anopheles specimens as negative controls? P7, L18 It appears there are 2 novel supergroup A strains and 2 belonging to supergroup B. The accession numbers for the B strains seem to be missing from Table S3 P7, L26-50 Is this level of detail really necessary? I think it is almost impossible to infer the relative abundances of two taxa in a sanger chromotogram from a mixed sample. I would suggest to either perform qPCRs for the different strains or to omit these paragraphs.
P8, L7-21 Again, I think these technical details are not very important. Suggest to consider removing this paragraph.
P9, L37-38 Please explain why singleplex appraoches would lead to reduced sensitivity for the detection of hybrids, this is not immediately apparent P10, L6-7 Unclear what is meant by this statement P10, L7-10 Sentence appears incomplete. Suggest to rephrase. P10, L21-22 Or rather, the results highlight the necessity for other types of evidence in addition to the molecular data Review form: Reviewer 2

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
Page 3, Line 32: "Interestingly, the presence of Wolbachia strains in Anopheles was inversely correlated to other bacteria species such as Asaia that are stably associated with several species". This work https://www.nature.com/articles/s41598-020-70745-0 also shed light on Wolbachiamicrobiome associations and could be added to the other works cited here.
Page 5, Line 58: It is unclear how much cDNA was used for qPCR. In the methods it is indicated 2uL, please specify the amount in ng not volume. In the results, it is stated this was normalized to ng per reaction. This should be consistent with the methods described to improve reproducibility of this study.
Wolbachia superinfection: the author have come to this conclusion by observing mixed bases in the sequenced but could not confirm the dominant strain. It is unclear why the authors have not cloned the gene/s from the samples and sequenced multiple clones to resolve which strain is the dominant one. If this is not possible for the authors to conduct this analysis now, I suggest that this approach is performed in the future.

Decision letter (RSOS-202032.R0)
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Dear Dr Walker
The Editors assigned to your paper RSOS-202032 "Evidence for natural hybridisation and novel Wolbachia strain superinfections in the Anopheles gambiae complex from Guinea" have now received comments from reviewers and would like you to revise the paper in accordance with the reviewer comments and any comments from the Editors. Please note this decision does not guarantee eventual acceptance.
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Please note article processing charges apply to papers accepted for publication in Royal Society Open Science (https://royalsocietypublishing.org/rsos/charges). Charges will also apply to papers transferred to the journal from other Royal Society Publishing journals, as well as papers submitted as part of our collaboration with the Royal Society of Chemistry (https://royalsocietypublishing.org/rsos/chemistry). Fee waivers are available but must be requested when you submit your revision (https://royalsocietypublishing.org/rsos/waivers). This is a relatively straightforward molecular screen for Wolbachia symbionts in a collection of Anopheles mosquitoes. The main findings are that some specimens were PCR positive for Wolbachia, and that there is molecular evidence for hybridization between Anopheles melas and An. gambiae ss. The study is one of several similar recently published articles and is plagued by the same problem: the evidence for Wolbachia is entirely based on PCR, and this comes with many problems which were discussed in detail here: https://doi.org/10.1128/mBio.00784-19. Similar to previous studies, sequencing of Wolbachia loci was unsuccessful in some cases and titres of Wolbachia DNA were low; so overall the biological relevance of some of the findings is unclear. The high titre Wolbachia infections in An. melas and sp. X do appear genuine given the data. Further novel, and potentially more interesting aspects are the detection of potential hybridization events between two Anopheles species, and the molecular detection of a potentially novel Anopheles species. I think that the paper can be improved in several points, and I would like to ask the authors to consider two main issues: 1) More cautious interpretation of PCR results: I think it is misleading to claim Wolbachia detection if sequencing was unsuccessful. Samples that are PCR positive, but failed to produce a sequence should be conservatively interpreted as negative. In the case of the An. gambiae s.s samples, there was no sequence, and no amplification in the qPCR experiments. Nevertheless, Wolbachia strains in An. gambiae ss are highlighted in the abstract, and this should be removed. Using RNA extracts instead of DNA may contribute to reducing the number of false Wolbachia positives. However, this would only be efficient with DNAse treatment after RNA extractions to remove residual DNA (which is always present irrespective of the RNA extraction protocol). I couldn't find any information on this in the manuscript, so it would be good to see clarification here. Overall, the limitations should be highlighted clearly and avenues discussed for confirming the potential Wolbachia infections.
2) More concise presentation: The paper is quite long with many figures, and I believe that a more concise presentation would be beneficial. For example, is it really necessary to have separate tree figures in the main manuscript for each mitochondrial locus and the nuclear loci plus a tree from a concatenated dataset? All mitochondrial genes are essentially the same locus, and the trees don't differ a lot anyway. I would recommend to show a single tree here, and move the other figures into the supplement. The same is true for the Wolbachia loci: why so many different trees when the main goal is supergroup assignment and for that a single tree would suffice. I also recommend to remove the wsp tree entirely, as this locus is useless as phylogenetic marker. In the results section, there lengthy paragraphs about technical details that overall don't contribute much to the overall conclusions, and my suggestion would be to focus on the main findings to improve accessibility for readers. More details on this below.
Minor comments, hopefully constructive: P3, L24-28 While the limitations of these studies are mentioned in the discussion, I feel it would make sense to highlight them in the introduction as well.
P3, L59-60 Did this protocol include DNAse treatment? P4, L5-6 Did you use a Ct threshold after which detection was considered unreliable? P4, L44-45 This reads as if the 16S PCR was done on genomic DNA rather than cDNA. Please clarify.
P4, L46-47 The CifA/CifB PCRs are not mentioned anywhere in the results or discussion. Suggest to remove the protocol here.
P5, L38 Please provide justification for using TN model of evolution.
P5, L39-40 If this means bootstrap analysis, please provide details here.

P5, L41-44
It appears the description is for the calculation of the starting tree, rather than for the ML reconstructions. Please clarify.

P5, L49ff
Please provide more details on negative and positive controls here. Did you include Wolbachiafree Anopheles specimens as negative controls? P7, L18 It appears there are 2 novel supergroup A strains and 2 belonging to supergroup B. The accession numbers for the B strains seem to be missing from Table S3 P7, L26-50 Is this level of detail really necessary? I think it is almost impossible to infer the relative abundances of two taxa in a sanger chromotogram from a mixed sample. I would suggest to either perform qPCRs for the different strains or to omit these paragraphs.
P8, L7-21 Again, I think these technical details are not very important. Suggest to consider removing this paragraph.
P9, L37-38 Please explain why singleplex appraoches would lead to reduced sensitivity for the detection of hybrids, this is not immediately apparent P10, L6-7 Unclear what is meant by this statement P10, L7-10 Sentence appears incomplete. Suggest to rephrase. P10, L21-22 Or rather, the results highlight the necessity for other types of evidence in addition to the molecular data Reviewer: 2 Comments to the Author(s) Page 3, Line 32: "Interestingly, the presence of Wolbachia strains in Anopheles was inversely correlated to other bacteria species such as Asaia that are stably associated with several species". This work https://www.nature.com/articles/s41598-020-70745-0 also shed light on Wolbachiamicrobiome associations and could be added to the other works cited here.
Page 5, Line 58: It is unclear how much cDNA was used for qPCR. In the methods it is indicated 2uL, please specify the amount in ng not volume. In the results, it is stated this was normalized to ng per reaction. This should be consistent with the methods described to improve reproducibility of this study.
Wolbachia superinfection: the author have come to this conclusion by observing mixed bases in the sequenced but could not confirm the dominant strain. It is unclear why the authors have not cloned the gene/s from the samples and sequenced multiple clones to resolve which strain is the dominant one. If this is not possible for the authors to conduct this analysis now, I suggest that this approach is performed in the future.

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Decision letter (RSOS-202032.R1) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. We would like to thank the reviewer for a very constructive and useful review that has improved our manuscript. Our responses to reviewer 2 are shown as follows in bold and changes to the manuscript are highlighted in yellow: Reviewer: 2 Comments to the Author(s) Page 3, Line 32: "Interestingly, the presence of Wolbachia strains in Anopheles was inversely correlated to other bacteria species such as Asaia that are stably associated with several species". This work https://www.nature.com/articles/s41598-020-70745-0 also shed light on Wolbachia-microbiome associations and could be added to the other works cited here. Thank you for this suggestion and we have added this reference.
Page 5, Line 58: It is unclear how much cDNA was used for qPCR. In the methods it is indicated 2uL, please specify the amount in ng not volume. In the results, it is stated this was normalized to ng per reaction. This should be consistent with the methods described to improve reproducibility of this study. Apologies and thank you for spotting this mistake. As we used 'RNA' in our onestep qRT-PCR assays, we have corrected this error (replaced 'DNA' with 'RNA'). Earlier in this paragraph we have the details that we used 2.0 ng/uL RNA in each reaction.
Wolbachia superinfection: the author have come to this conclusion by observing mixed bases in the sequenced but could not confirm the dominant strain. It is unclear why the authors have not cloned the gene/s from the samples and sequenced multiple clones to resolve which strain is the dominant one. If this is not possible for the authors to conduct this analysis now, I suggest that this approach is performed in the future Thank you for this useful suggestion, unfortunately it wasn't possible to attempt this but we agree this would be very useful for further work and therefore have included a sentence within the discussion regarding further work for greater characterisation of the superinfection strains.
We would like to thank the reviewer for a very constructive and useful review that has improved our manuscript. Our responses to reviewer 1 are shown as follows in bold and changes to the manuscript are highlighted in yellow: Reviewer: 1 Comments to the Author(s) This is a relatively straightforward molecular screen for Wolbachia symbionts in a collection of Anopheles mosquitoes. The main findings are that some specimens were PCR positive for Wolbachia, and that there is molecular evidence for hybridization between Anopheles melas and An. gambiae ss. The study is one of several similar recently published articles and is plagued by the same problem: the evidence for Wolbachia is entirely based on PCR, and this comes with many problems which were discussed in detail here: https://doi.org/10.1128/mBio.00784-19. Similar to previous studies, sequencing of Wolbachia loci was unsuccessful in some cases and titres of Wolbachia DNA were low; so overall the biological relevance of some of the findings is unclear. The high titre Wolbachia infections in An. melas and sp. X do appear genuine given the data. Further novel, and potentially more interesting aspects are the detection of potential hybridization events between two Anopheles species, and the molecular detection of a potentially novel Anopheles species. I think that the paper can be improved in several points, and I would like to ask the authors to consider two main issues: 1) More cautious interpretation of PCR results: I think it is misleading to claim Wolbachia detection if sequencing was unsuccessful. Samples that are PCR positive, but failed to produce a sequence should be conservatively interpreted as negative. In the case of the An. gambiae s.s samples, there was no sequence, and no amplification in the qPCR experiments. Nevertheless, Wolbachia strains in An. gambiae ss are highlighted in the abstract, and this should be removed. We agree that for An. gambiae ss without amplification in qPCR experiments and failed sequencing attempts we should be more conservative and interpret as negative. We have adjusted our summary (abstract) accordingly as we agree that detection of Wolbachia DNA sequences from these samples should not be presented in the abstract as a major result of this manuscript.
Using RNA extracts instead of DNA may contribute to reducing the number of false Wolbachia positives. However, this would only be efficient with DNAse treatment after RNA extractions to remove residual DNA (which is always present irrespective of the RNA extraction protocol). I couldn't find any information on this in the manuscript, so it would be good to see clarification here. Overall, the limitations should be highlighted clearly and avenues discussed for confirming the potential Wolbachia infections. Although we did not include a DNase treatment the Qiagen RNeasy 96 kit is optimised for RNA extraction. However, we completely agree that this does not fully prevent the possibility of dsDNA from being present and amplified in our one step RT PCR assays (in which a normalised RNA amount is added to the reaction). We have modified our existing discussion on this for clarity by adding the following sentences: "Although RNA extraction kits are optimised for RNA and we measured high levels of total RNA using a Fluorometer, there is a small possibility that our amplification and sequencing could result from co-extracted Wolbachia gDNA. Detection and sequencing of Wolbachia gDNA has been used previously in numerous studies to characterise strain phylogenies [25][26][27][28][29]. However, to provide greater confidence on expression of Wolbachia genes in future work, a DNase treatment could be undertaken to ensure amplification is resulting from only cDNA.''