Profiles of bacterial communities and environmental factors associated with proliferation of malaria vector mosquitoes within the Kenyan Coast

  1. Josphat Mutinda
  2. Samuel Mwakisha Mwamburi
  3. Kennedy Omondi Oduor
  4. Maurice Vincent Omolo
  5. Regina Mongina Ntabo
  6. James Muhunyu Gathiru
  7. Joseph Mwangangi
  8. James O. M. Nonoh

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Abstract

Background. Since Anopheles mosquitoes which transmit and maintain the malaria parasite breed in the outdoor environment, there is an urgent need to manage these mosquito breeding sites. In order to elaborate more on the ecological landscape of mosquito breeding sites, the bacterial community structure and their interactions with physicochemical factors in mosquito larval habitats was characterised in Kwale County (Kenya), where malaria is endemic.

Methods. The physical characteristics and water physicochemical parameters of the habitats were determined and recorded. Water samples were also collected from the identified sites for total metagenomic DNA extraction in order to characterise the bacterial communities within the breeding sites.

Results and Discussion. Sites where mosquito larvae were found were described as positive and those without mosquito larvae as negative. Electrical conductivity, total dissolved solids, salinity and ammonia were lower in the rainy season than in the dry season, which also coincided with a high proportion of positive sites. Pseudomonadota was the most common phyla recovered in all samples followed by Bacteroidota and then Actinomycetota. The presence or absence of mosquito larvae in a potential proliferation site was not related to the bacterial community structure in the sampled sites, but was positively correlated with bacterial richness and evenness.

Conclusion. Generally, the presence of Anopheles mosquito larvae was found to be positively correlated with rainy season, bacterial richness and evenness, and negatively correlated with electrical conductivity, total dissolved solids, salinity and ammonia. The findings of this study have implications for predicting the potential of environmental water samples to become mosquito proliferation sites.

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  1. Version published to 10.1099/acmi.0.000606.v4 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000606.v3 on Access Microbiology
  4. Access Microbiology

    The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. Thank you for submitting a revised version of your manuscript following the assessment of the reviewers, I have only found some minor things that I would like you to address: In the Abstract Results and Discussion section I think it would be better to skip the actual numbers in parenthesis and just highlight the main findings, for the exact details and numbers the reader can go to the main text. Figure 11 you have used the term differentially expressed, this usually refers to genes and is not the right term to use here, please amend it in the figure and the manuscript text to abundance changes/ or differential abundance of bacterial genera

  5. Version published to 10.1099/acmi.0.000606.v2 on Access Microbiology
  6. Access Microbiology

    This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. Please deposit the data underlying the work in the Society’s data repository Figshare account here: https://microbiology.figshare.com/submit. Please also cite this data in the Data Summary of the main manuscript and list it as a unique reference in the References section. When you resubmit your article, the Editorial staff will post this data publicly on Figshare and add the DOI to the Data Summary section where you have cited it. This data will be viewable on the Figshare website with a link to the preprint and vice versa, allowing for greater discovery of your work, and the unique DOI of the data means it can be cited independently.

  7. Access Microbiology

    Comments to Author

    ACMI-D-23-00050 Metagenomic Profiles of Bacterial communities and environmental factors associated with proliferation of malaria vector mosquitoes within the Kenyan Coast SYNOPSIS AND MAJOR COMMENTS The manuscript "Metagenomic Profiles of Bacterial communities and environmental factors associated with proliferation of malaria vector mosquitoes within the Kenyan Coast" presents a broad investigation of the bacterial microbiota associated with mosquito larvae sampled at various points across the south coast of Kenya. The authors pursue this main aim by also describing a series of physicochemical parameters likely influencing the mosquito breeding sites in the selected sampling areas and making a clear association between these factors and the likely dispersal of the malaria vectors. Some highlights of the study include the detailed material and methods, the extreme care with the data obtainment and, particularly, the contextualization of the study's novelty with the underlying health challenge imposed by malaria in the region. There is an effort to pinpoint how the report might contribute to delineating future actions for mitigation and control of the neglected disease, despite the little discussion of how the taxa found in the metataxonomic analyses could direct these potential strategies. Overall, the article is well-written and has a straightforward approach to presenting data. The literature is up-to-date, despite the authors could include additional references for some of the tools and bioinformatic resources applied during their analyses. All genomic data has been properly deposited in the NCBI databases and the bioinformatic analyses were run accordingly to what is expected for a metataxonomic survey. However, the authors might need to understand that this is not a metagenomic investigation and need to replace this term with "metataxonomics" or "16S rRNA gene-based diversity analyses". In this sense, it would be interesting to retitle the work as well. In addition, most figure legends are poorly detailed and their quality and visualization might be severely improved. The Discussion is coherent and focused on the main results from the study, but the authors could extrapolate more based on the taxonomic composition seen in all samples for making claims of how their report could aid in malaria control in the area. DETAILED REVIEW Background Line 30 Restructure the first sentence, it's confusing, without a link between the Anopheles vector and the decline of malaria cases in Sub-Saharan Africa, followed by the need for innovative control strategies. Line 38 Add where Kwale County is located between parenthesis (Kenya). Line 43 Correct "genomic" with "metagenomic" or "community" to better characterize the metagenomic approach applied in this case. Lines 48-50 (and throughout all manuscript, including Figures) Correct the phyla names in accordance with the currently valid accepted nomenclature (10.1099/ijsem.0.005056): for instance, Proteobacteria → Pseudomonadota; Actinobacteria → Actinomycetota. Line 60 Write "Electricity" in lower case: "electricity". Introduction Line 93 Add a space between "environment" and "(13)". Line 107 Write "Anopheles" in italics. Lines 110-117 It lacks a proper reason why characterising these bacterial communities in the larval stages of these mosquitos is important. The biotic factor is clear while the authors managed to contextualize the big picture, but a sentence or two sentences providing how the wider comprehension of the larval microbiome could aid in designing these malaria control strategies. Material and Methods Line 121 Rewrite "figure" in upper case: "Figure". Lines 120-128 Write the geographic coordinates in degrees, minutes, and seconds. Figure 1 Add "(Kenya)" between parentheses at the end of the phrase. Line 134 Since this is the first time these Anopheles species are being cited in the manuscript text, please write them in the full form after they can be abbreviated in the text. Line 152 Correct "ml" to "mL". Line 187 Correct "ml" to "mL". Line 199 Correct "ml" to "mL". Line 203 Add a space between "escaping" and ".Once". Also, correct "ml" to "mL". Lines 206-217 Perhaps a figure to help in the comprehension of this sample distribution across geographic and source origins? Lines 218 and 231 Rewrite: "community" or "metagenomic" DNA. Line 223 and 230 Correct "ml" to "mL". Lines 234-240 So this is a metataxonomic survey and not a metagenomic one? Please, make this as clear as possible in your manuscript text. The highlight here is the fact that you've applied PacBio sequencing instead of Illumina for these 16S rRNA-derived amplicons (longer than normally used in most of the metataxonomic surveys). Line 254 Which version of the SILVA database was applied for taxonomic classification? 138? Line 256 The information between parenthesis can be removed once you specify that you have used the SILVA database v.138.1 (release date). Results Lines 299, 301, 302 and 303 The Anopheles species can be abbreviated here, once you provide it fully earlier in the text. Line 303 Write "Culex" in italics. Line 313 Add a space between "1" and "m". Line 314 Write m2 in the superscript form. Line 323 Rewrite "1360" such as "1,360". Line 336 Correct "PH": "pH" and "P-value": "p-value" in Table 1. Line 339 Rephrase this section title, better "Metataxonomic survey" or "Microbiota analyses". Line 352 Add a comma after "Fo beta-diversity analyses," and write "the" in the lowercase form. Lines 357-358 Complete this figure legend with more information. What samples? The average number of reads obtained? Line 360 Rewrite this figure legend indicating that different alpha-diversity metrics were applied for getting a glimpse of richness and diversity in studied samples. Lines 365 and 372 Correct "PCOA" to "Principal Coordinate Analysis (PCoA)" in Figure 5 and 6 legends. Line 376 Rewrite in a more direct way the section title: "Taxonomic classification". Also, update the bacterial phyla names throughout the manuscript. Line 378 Rewrite "29133": "29,133" and "1450": "1,450". Figures 8 to 11 Change the R ggplot palette colour for plotting these relative abundance graphs. The colours currently adopt make it harder for the reader to fully get the different key taxa found in all samples by each analysed taxonomic level. Figure 12 How can this figure be interpreted? Is there any way to depict the bacterial family names? Figure 13 It could be worthwhile to say in the Results text the bacterial genera that were differently abundant across the samples. It can be hard to obtain this information by solely observing Figure 13. Line 461 Add a space between "1" and "m"; the same between "10" and "m2". Don't forget to write "2" in "m2" in the superscript form. Line 523 Again, rephrase this Discussion section title in accordance with what has been previously commented on for the Results section. Line 545 Write "(4, 85)" in Times (it's currently in a different font). Lines 592-595 Adjust the font here. Line 609 Write the word "electrical" in lowercase.

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  8. Access Microbiology

    Comments to Author

    1. Methodological rigour, reproducibility and availability of underlying data This research covers important questions surrounding Anopheles proliferation. Research in this area is needed and welcomed. The methodology used in this manuscript is scientifically sound and appropriate for the study. The collection of the data has been collected in a thorough and suitable manor which enables the research aims to be met. However, there are some issues with the data analysis and presentation of the results which would need to be corrected prior to acceptance. All sequence data has been deposited to NCBI and accession numbers are provided within the manuscript. The raw data used for the other elements of this study have not been provided. Whilst the manuscript includes summary data the raw data has not been provided in either supplementary information nor deposited on a relevant database. Authors should consider including this data or a statement to explain that the data is available upon request. 2. Presentation of results Most of the figures are well presented however some of the results requires more explanation and editing to the presentation to ensure that the reader is able to interpret the information presented. Please see more specific comments below. Lines 289-296 appear to be explaining that there is no significant difference in the percentage of negative and positive sites during the dry season. However it is also important to investigate whether the proportion of positive sites is different in the dry and rainy seasons. From the numbers presented it is clear that the proportion of positive sites is higher in the rainy season (as would be expected) but no stats for this seems to have been presented. You should test for a significant difference in positive site proportion between the dry and rainy season. Lines 304-305 states that the results suggest that more habitats were suitable for breeding Anopheles than Culex. It would be important to understand how this fits in to the mosquito abundance in this area. Could it not just be that there are more Anopheles than Culex. Some discussion on this, probably best placed within the discussion, would benefit the interpretation of these results. Figure 2A displays the % of positive and negative sites in the dry season, rainy season, and overall. A stacked barchart is more appropriate for displaying proportions and would make it easier to interpret whether the proportion is different in the different seasons. I'm also not sure that you should have error bars on these plots. Has the percentage positive and negative not been calculated across all sample sites to give overall percentages? How were the error bars for figure 2 calculated? They seem to be very similar across different bars, are they correct? For figure 2C the error bars are putting the relative abundance above 100 and below 0, error bars should not go below 0 or above 100. There also appear to be error bars for some of the characteristics (type, vegetation) where there is no actual bar. For readability I would recommend Figure 2C being in its own figure. This would allow you to increase readability of the figure and flow of the main text. Stacked bar charts might also work better here to reduce the number of individual bars and increase readability. Please check the data in figure 2C. For example, shouldn't the % of artificial negative sites and natural negative sites add to 100%? They don't appear to, and there are similar issues throughout this figure. Figure legends require more detail added throughout to ensure figures can be interpreted and are stand alone. Where statistical analysis has been conducted that should be included in the legend to highlight significant difference (or no differences). Table 1 is well presented. It could benefit from including asterisks to indicate where differences are significant. Have the physiochemical parameters in table 1 also been tested for significance between the positive and negative sites. If so, please include the results. If not, please complete this analysis as it is important to the study. Identifying which physicochemical characters differ between positive and negative sites would provide an important insight into the factors associated with mosquito oviposition choice (as discussed in your introduction). The refraction curve (figure 3) is well presented and explained in the main text (lines 341-343). I wonder if it would be more appropriate in the methods rather than results. Diversity of bacterial communities results section (lines 140-355) is well and explained and accompanied by well presented figures. The number of reads generated, before and after QC trimming (lines 377 - 381) might be better presented in a table. Figures 7-11 presenting the results of the taxonomic assignment are very well presented. However in the results section they are just listed and not specifically referred to in the main text of the results section. Please ensure that the main text refers directly to relevant figures pointing out key message in each figure. This would make it easier for the reader to interpret these results. It is useful to know that more bacterial families in positive sites compared to negative sites (as explained in lines 389-391) however figure 12 does not include labelling to enable the reader to understand which families specifically are found in each site. Figure should be edited to include family names. Lines 391 - 393 states that 19 genera were more significantly more abundant in positive sites. However, no p value has been provided. Has this been tested statistically? If not please avoid using the term significant. It would be useful to present this analysis for all genera (not just the significant 19) as supplementary information. The size of the points on figure 13 could be reduced to improve readability. Please explain what is meant by N/A on figure 13 in the figure legend. 3. How the style and organization of the paper communicates and represents key findings The paper has all relevant sections, and the structure and style are effective at communicating the key findings. There are places where the results/discussion require further clarification, and this has been indicated by my specific comments throughout the review. The abstract is very long and could be condensed to more concisely convey the key findings of the study. The background section of the abstract for example could be significantly cut down. The results section could also be cut down, just focusing on key results rather than each individual result. This would make the key take home message more clear in the abstract. 4. Literature analysis or discussion When discussing the presence of Anopheles and Culex reported in this study (lines 427 - 439) it is important to give some information on known distribution/occurrence of these mosquitoes in the local area. What might the co-occurrence of both Anopheles and Culex mean for vector control programs? Can you provide any information on relationships between Anopheles and Culex. There are studies which have investigated predation between the two genera as well as the effect on life history traits etc. You have reported co-occurrence of the two genera here, what does this mean for vector control? There is an in-depth discussion on breeding site characteristics in relation to Anopheles but your study also found Culex. Culex should also be discussed in this context. It may be beneficial to add a brief mention of the potential for Culex to be vectors, especially for west nile virus. Physiochemical parameters are described to be high (line 481). High in relation to what? Could you give some context to this or provide a reference to explain why you classify these to be high in your study? The statement that Anopheles mosquitoes have become less selective to environmental parameters (lines 489-491) is not supported by the results of this study. You explain that there was a difference in temperature between positive and negative sites which does suggest that temperature affects larval abundance however the temperature in the dry and rainy season presented in table 1 are very similar (31.74 and 32.06). Could it not just be that the temperature did not fluctuate much between the different seasons/sites in this study? A discussion on optimal temperatures for mosquito development would be helpful here. Could it just be that the temperatures in this study were always within the optimal temperatures for mosquito development? Limitations of the study are not well addressed in the discussion, please ensure that you have addressed the limitations thoroughly. More in depth discussion on the significance of this study would improve the manuscript. How does increasing the knowledge of bacterial communities associated with mosquito larval presence feed into vector control. What are the applications of this (and other similar) research? When discussing bacterial control agents (line 594-597) it would be beneficial to include some specific examples of this and how successful they have been. The discussion makes no reference to the absence of other malaria vectors in this study. There are other species in this area, why were they not found in your study? 5. Any other relevant comments In the abstract it is not clear what is meant by positive and negative sites (this is made clear in the manuscript but abstract should be stand alone). It would be helpful to give some examples of what the optimal conditions for larval survival and growth discussed in lines 80 - 88 include. The introduction should mention the malaria vaccine as this is a key development in malaria control. However vector control is still important. The introduction mentions various vector control strategies but needs to more explicitly explain why environmental management to reduce oviposition sites is a preferred method (cheaper, no resistance, no environmental effects etc.). This helps to justify why understanding of larval ecology is important to vector control. The introduction needs a bit more background information on the situation in the study area specifically, this would help to justify why this area has been chosen. The map (figure 1) could benefit from differentiation between each site to make it clearer, for example using numbering or different colours for each site. The structure of the methods has meant that in places details are repeated, could this be improved to improve flow? The methods should include when the sampling took place. This is especially relevant given the discussion of variation in rainy seasons explained in lines 129-135. This is provided later in the methodology (lines 155-156) but would be helpful to the reader if provided with discussion on rainy seasons (lines 129-135). It is also important to provide differentiation in when the dry and rainy season collections took place, as these are presented separately in the results. Study design and sample size section could be clearer. Was there a total of 35 proliferation sites samples across all sample sites? If so how many from each site? Were all mosquitoes identified to species level or some just to genus? Would be helpful to specify this in lines 205-207. The methodology gives a very detailed and concisely explained description of the genomic DNA extraction process. I find the labelling of the water samples in lines 210 - 217 to be a little confusing, as the labels don't really indicate anything about the samples. This also means that during the results the reader needs to continuously flip back to the methods to understand what is being presented. This makes the presentation of the metagenomic results a little difficult to follow. Could the labelling be changed to something more intuitive and related to the samples themselves for example P for positive sites? More details on the positive and negative controls used for sequencing is needed (lines 238-240). Could you provide a list of the 17 isolates included in the positive control in supplementary information? What exactly is the negative control? The quality control parameters given in lines 248 should be explained in plain terms rather than providing the code as it is not clear exactly what each parameter is. Could you provide the conditions used for successful 16S rRNA amplification (lines 234-236)? There is an inconsistency with the number of sites samples, in the methods it states 35 sites were sampled, in the results 65. Please clarify this and correct in the manuscript. Line 450, typo - farmers come pastoralists. There is an inconsistency with the formatting of genus names in the manuscript, some are italicised, and others are not.

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  9. Version published to 10.1099/acmi.0.000606.v1 on Access Microbiology