Baseline Characterization of the Gut Microbiota of Field and Colony Populations of Phlebotomus tobbi and Preliminary Assessment of the Anti-Leishmanial Activity of Cultivable Bacteria
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Dear colleagues,
Studies addressing the variability and richness of sand fly gut microbiota are needed, particularly because we still do not fully understand the drivers of this variability. In this context, the present manuscript represents an important contribution to the field of vector biology.
The manuscript is clearly structured, scientifically sound, and presented in a coherent and straightforward manner. In particular, the HTML-based presentation of the supplementary material was highly effective, interactive, and easy to follow. This format substantially improved the accessibility and is a helpful approach for presenting complex datasets.
Overall, I am satisfied with the relevance and quality of the findings presented in this manuscript.
Author Response
Dear colleagues,
Studies addressing the variability and richness of sand fly gut microbiota are needed, particularly because we still do not fully understand the drivers of this variability. In this context, the present manuscript represents an important contribution to the field of vector biology.
The manuscript is clearly structured, scientifically sound, and presented in a coherent and straightforward manner. In particular, the HTML-based presentation of the supplementary material was highly effective, interactive, and easy to follow. This format substantially improved the accessibility and is a helpful approach for presenting complex datasets.
Overall, I am satisfied with the relevance and quality of the findings presented in this manuscript.
We thank reviewer for valuable comments.
Reviewer 2 Report
Comments and Suggestions for Authors
The paper provides a brief descriptive study of the gut microbiota present within Phlebotomus tobbi, an important sandfly vector of Leishmania transmission in Cukurova region, Türkiye. The rationale for the studies is the suspected influence that the natural gut microbiota may have on Leishmania development and transmission, with many published studies showing both positive and negative effects. The flies were collected from an active transmission site for both cutaneous and visceral leishmaniasis. 16S rRNA amplicon sequencing was used to define the bacterial communities of the wild caught flies that were compared with colonized flies. Fourteen bacterial species were cultivable which were co-cultured with L. infantum promastigotes to compare lytic effects on the parasite. Two species showed strong leishmanicidal activity which were argued to be relevant to possible future control strategies involving paratransgenesis. Overall, the data presented appears sound and is useful as baseline information.
The main deficit of the paper is that it lacks any direct demonstration that the microbiota observed have any effect on parasite development in the vector. The authors actually indicate in the introduction that the paper fills gaps in knowledge that include the following: “(i) characterizing the bacterial communities associated with field-collected Phlebotomus tobbi populations from endemic regions of Türkiye, (ii) evaluating the influence of these bacteria on Leishmania development and survival, and (iii) testing the leishmanicidal activity of selected bacterial isolates in vitro.” There is no data that addresses the second objective. Since they have colonized flies and cultivable bacteria that show lytic effects, the addition of the bacteria to blood or sugar meals to directly observe effects on L. infantum growth and development should have been included in these studies. These sorts of experiments have been carried out by others.
The citations of prior studies relevant to the role of gut microbiota in Leishmania development are often unclear or incorrect. The referenced papers should distinguish those that have actually carried out manipulations of the gut microbiota in sandflies to show effects on Leishmania development from those that describe the microbial diversity and only infer such effects. Among the former are papers by Sant'Anna et al., 2014; Schlein et al., 1985; Louradour et al., 2017; Campolina et al., 2020; Cecilio et al., 2025.
Many papers are cited relevant to Serratia sp. and their presumed negative effects on parasite survival in the gut. Again, it needs to be specified which papers actually showed such effects. The paper by Louradour et al. showed no inhibitory effect of Serratia sp. in vivo. The feeds on Serratia actually promoted Leishmania growth in antibiotic treated flies.
The discussion is far too long given the amount of data and new information in this report.
Author Response
The paper provides a brief descriptive study of the gut microbiota present within Phlebotomus tobbi, an important sandfly vector of Leishmania transmission in Cukurova region, Türkiye. The rationale for the studies is the suspected influence that the natural gut microbiota may have on Leishmania development and transmission, with many published studies showing both positive and negative effects. The flies were collected from an active transmission site for both cutaneous and visceral leishmaniasis. 16S rRNA amplicon sequencing was used to define the bacterial communities of the wild caught flies that were compared with colonized flies. Fourteen bacterial species were cultivable which were co-cultured with L. infantum promastigotes to compare lytic effects on the parasite. Two species showed strong leishmanicidal activity which were argued to be relevant to possible future control strategies involving paratransgenesis. Overall, the data presented appears sound and is useful as baseline information.
We thank the reviewer for the comments
The main deficit of the paper is that it lacks any direct demonstration that the microbiota observed have any effect on parasite development in the vector. The authors actually indicate in the introduction that the paper fills gaps in knowledge that include the following: “(i) characterizing the bacterial communities associated with field-collected Phlebotomus tobbi populations from endemic regions of Türkiye, (ii) evaluating the influence of these bacteria on Leishmania development and survival, and (iii) testing the leishmanicidal activity of selected bacterial isolates in vitro.” There is no data that addresses the second objective.
Correct!. We thank reviewer for this comment. It is removed and corrected.
Since they have colonized flies and cultivable bacteria that show lytic effects, the addition of the bacteria to blood or sugar meals to directly observe effects on L. infantum growth and development should have been included in these studies. These sorts of experiments have been carried out by others.
That was the plan of the project. We failed to maintain bacteria-colonized sand flies. Thus we removed those parts from the project. The limitation part is updated.
The citations of prior studies relevant to the role of gut microbiota in Leishmania development are often unclear or incorrect. The referenced papers should distinguish those that have actually carried out manipulations of the gut microbiota in sandflies to show effects on Leishmania development from those that describe the microbial diversity and only infer such effects. Among the former are papers by Sant'Anna et al., 2014; Schlein et al., 1985; Louradour et al., 2017; Campolina et al., 2020; Cecilio et al., 2025. Many papers are cited relevant to Serratia sp. and their presumed negative effects on parasite survival in the gut. Again, it needs to be specified which papers actually showed such effects. The paper by Louradour et al. showed no inhibitory effect of Serratia sp. in vivo. The feeds on Serratia actually promoted Leishmania growth in antibiotic treated flies.
We thank reviewer for the comment. Unnecessary citations are removed.
References are corrected
The discussion is far too long given the amount of data and new information in this report.
The discussion part is shortened and rephrased in some parts to make it easier for the readers.
Reviewer 3 Report
Comments and Suggestions for Authors
The manuscript titled "Comparative Analysis of Gut Microbiota in Laboratory Colony and Wild Populations of Phlebotomus tobbi and Molecular Surveillance of Leishmania Infection" addresses a relevant topic in medical entomology and vector microbiota. The integration of microbiomes, bacterial isolation, and antileishmanial activity is interesting and potentially publishable. However, in its current state, the study functions more as an exploratory preliminary characterization than as a robust functional analysis of microbiota and vector competence. Therefore, it is important to consider the following points: provide more detail on the bioinformatics section; add statistical analyses; ensure the results are interpreted correctly to avoid overinterpretation; and clarify and add controls to the Materials and Methods section. With these considerations, the manuscript could be significantly improved.
Title
Line 2-4: The authors should adjust the title, as the comparative analysis is relatively superficial, lacking robust statistical analyses of alpha and beta diversity, and without differential tests between wild and colonized populations. It would be advisable to adjust the title to something more precise and less ambitious, for example, emphasizing “baseline characterization” or “preliminary function assessment.” Regarding “molecular surveillance”, they only report negative results without a sufficiently in-depth methodological discussion of diagnostic sensitivity, expected prevalence, or statistical power.
Materials and Methods
Line 94: It is recommended to incorporate appropriate statistical analyses (associated statistical results, such as significance levels, confidence intervals, or effect sizes) for microbiomes and functional assays. The authors could include multivariate analyses commonly used in microbiome studies, such as PERMANOVA, ANOSIM, PCoA, or NMDS, to formally assess whether bacterial communities differ between biological groups.
Line 109-110: Specify if the same number of traps were used in each location and add the capture effort for each one.
Line 103-104: It is necessary to add the taxonomic references they used.
Line 120: Did the authors analyze the samples individually? The goal was to infer individual microbiota, gather intra-population information, establish real associations, and avoid favoring the dominance of certain taxa. Pooling samples significantly reduces ecological resolution and prevents the assessment of individual variability.
Line 121: How many pools were sequenced by category?
Line 123: What were the negative extraction controls and environmental controls for 16S sequencing?
Line 125: The authors can add more data on the sequencing methodology, such as sequencing platform, number of reads per sample, coverage, quality filtering, bioinformatics pipeline, OTUs vs ASVs, allocation criteria, identity thresholds, chimera removal, and batch effect controls.
Line 127: The authors could specify the exact final concentration of bacteria, MOI, whether supernatants or live bacteria were used, and normalization between isolates.
Line 129: The functional analysis of leishmanicidal activity using the XTT assay only evaluated promastigotes, making it useful only as a preliminary approximation. However, the functional assessment remains quite weak, as it lacks analysis of intracellular amastigotes, macrophage infection, and interaction within the vector.
Results
Line 161: In Table 1, is it advisable to specify which district the sandflies belong to, Imamopu and Kozan districts?
Line 169: How many Ph. tobbi midguts were analyzed?
Line 171: How many females collected in the field were analyzed?
Line 177: How many females from the colony were analyzed?
Line 178: How many males from the colony were analyzed?
Line 179: What type of alpha and beta diversity analysis was performed, and what indices were considered? This is not mentioned in the materials and methods section.
Line 183: In Table 2, in addition to the percentages, it is necessary to add the raw data of the number of specimens analyzed.
Line 192: The authors could add statistical tests to determine whether the observed differences between bacterial isolates are significant, as they only report average mortality percentages and standard deviations. This limitation reduces the study's ability to distinguish between biologically relevant patterns and variations attributable to experimental chance.
Line 193: This word in italics, L. infantum
Line 196: Although the 32% mortality rate for Serratia liquefaciens is relatively low, it is interesting but far from representing a highly potent effect. Therefore, the discussion should adhere to these results and not exaggerate the biological significance of the finding.
Line 207: The authors could clarify whether it is a direct bacterial culture or of bacterial supernatants.
Discussion
Line 227: Check the word "pmparasite"
Line 244-245: Review grammatically this sentence: "Such reasons like controlled diets and environmental factors"
Line 316: The discussion about the impact of COVID-19 lockdowns on the absence of Leishmania is highly speculative and weakly supported. There is no longitudinal entomological or local epidemiological data to support the conclusion that vector-host contact decreased, transmission dynamics changed, or vector prevalence was reduced. Furthermore, it occupies too much space and diverts attention from scientific discussion.
Conclusions
Line 373: The authors claim that the study is the “first global characterization”; however, the study is regional, the sampling is limited, the effective microbiome size is not very large, and multiple similar studies exist in other sandflies. Therefore, it is recommended to be more conservative, using terms such as “first characterization in Turkey”, “baseline characterization in endemic populations” or “regional microbiota assessment.”
Lines 340, 346, 359, 369: Due to the results and scientific evidence, the authors should use these words with caution: “critical ecological baseline”, “promising candidates”, “ideal ecological setting”, “few scalable innovations available”.
Author Response
The manuscript titled "Comparative Analysis of Gut Microbiota in Laboratory Colony and Wild Populations of Phlebotomus tobbi and Molecular Surveillance of Leishmania Infection" addresses a relevant topic in medical entomology and vector microbiota. The integration of microbiomes, bacterial isolation, and antileishmanial activity is interesting and potentially publishable. However, in its current state, the study functions more as an exploratory preliminary characterization than as a robust functional analysis of microbiota and vector competence. Therefore, it is important to consider the following points: provide more detail on the bioinformatics section; add statistical analyses; ensure the results are interpreted correctly to avoid overinterpretation; and clarify and add controls to the Materials and Methods section. With these considerations, the manuscript could be significantly improved.
We thank reviewer for valuable comments. By following the suggestions of the reviewer, me and my colleagues revised the manuscript.
Title
Line 2-4: The authors should adjust the title, as the comparative analysis is relatively superficial, lacking robust statistical analyses of alpha and beta diversity, and without differential tests between wild and colonized populations. It would be advisable to adjust the title to something more precise and less ambitious, for example, emphasizing “baseline characterization” or “preliminary function assessment.” Regarding “molecular surveillance”, they only report negative results without a sufficiently in-depth methodological discussion of diagnostic sensitivity, expected prevalence, or statistical power.
We thank the reviewer for this valuable suggestion. We agree that the original title may imply a more comprehensive comparative microbiome analysis than was actually performed. Since our study was primarily designed as a baseline characterization of the gut bacterial composition of Phlebotomus tobbi and a preliminary assessment of the anti-leishmanial activity of cultivable isolates, we have revised the title accordingly. We have also toned down the emphasis on molecular surveillance, as no Leishmania infections were detected during the study period. New title is : Baseline Characterization of the Gut Microbiota of Field and Colony Populations of Phlebotomus tobbi and Preliminary Assessment of the Anti-Leishmanial Activity of Cultivable Bacteria
Materials and Methods
Line 94: It is recommended to incorporate appropriate statistical analyses (associated statistical results, such as significance levels, confidence intervals, or effect sizes) for microbiomes and functional assays. The authors could include multivariate analyses commonly used in microbiome studies, such as PERMANOVA, ANOSIM, PCoA, or NMDS, to formally assess whether bacterial communities differ between biological groups.
We thank the reviewer for this important comment. We agree that alpha diversity, beta diversity, PERMANOVA, ANOSIM, PCoA, and NMDS analyses are commonly used in microbiome studies. However, due to the pooled sampling design and the limited number of pooled sequencing samples generated in this exploratory study, robust statistical comparisons of community structure could not be performed without risking overinterpretation of the data. We have therefore clarified this limitation in both the Materials and Methods and Discussion sections and explicitly stated that our microbiome analyses should be considered descriptive rather than inferential.
Line 109-110: Specify if the same number of traps were used in each location and add the capture effort for each one.
We thank the reviewer for pointing this out. Additional information regarding trapping effort has been added. The number of CDC light traps used at each sampling site and the total trapping effort per location and sampling period are now provided in the Materials and Methods section.
Line 103-104: It is necessary to add the taxonomic references they used.
We thank the reviewer for this suggestion. The taxonomic identification references used for sand fly species determination have now been added to the Materials and Methods section.
Line 120: Did the authors analyze the samples individually? The goal was to infer individual microbiota, gather intra-population information, establish real associations, and avoid favoring the dominance of certain taxa. Pooling samples significantly reduces ecological resolution and prevents the assessment of individual variability.
We appreciate this important observation. The objective of the sequencing component was not to infer individual microbiomes but rather to obtain an overview of dominant bacterial taxa associated with different biological groups. Pooling was adopted to reduce sequencing costs and obtain sufficient DNA yield from small midgut samples. We fully acknowledge that this approach limits the assessment of individual-level variation and have now explicitly stated this limitation in the manuscript.
Line 121: How many pools were sequenced by category?
We thank the reviewer for identifying this omission. The number of pools sequenced in each biological category has now been added to the Materials and Methods section and summarized in the Results section.
Line 123: What were the negative extraction controls and environmental controls for 16S sequencing?
We thank the reviewer for this important methodological comment. Information regarding extraction blanks and negative controls has been added to the revised manuscript. Sterile extraction controls were processed alongside biological samples and no detectable bacterial amplification was observed in these controls.
Line 125: The authors can add more data on the sequencing methodology, such as sequencing platform, number of reads per sample, coverage, quality filtering, bioinformatics pipeline, OTUs vs ASVs, allocation criteria, identity thresholds, chimera removal, and batch effect controls.
We thank the reviewer for this suggestion. Additional details regarding sequencing methodology have been incorporated, including sequencing platform, target region, quality filtering procedures, taxonomic assignment workflow, and sequence processing steps provided by the commercial sequencing facility (Illumina MiSeq).
Line 127: The authors could specify the exact final concentration of bacteria, MOI, whether supernatants or live bacteria were used, and normalization between isolates.
We thank the reviewer for pointing this out. The final bacterial concentrations, co-incubation conditions, and normalization procedures used for the XTT assay have now been clarified in the Materials and Methods section.
Line 129: The functional analysis of leishmanicidal activity using the XTT assay only evaluated promastigotes, making it useful only as a preliminary approximation. However, the functional assessment remains quite weak, as it lacks analysis of intracellular amastigotes, macrophage infection, and interaction within the vector.
We agree with the reviewer that the XTT assay performed on promastigotes provides only a preliminary indication of anti-leishmanial activity. We have therefore revised the Discussion section to clearly acknowledge that no intracellular amastigote, macrophage infection, or vector-based experiments were conducted and that our findings should be interpreted as preliminary functional observations.
Results
Line 161: In Table 1, is it advisable to specify which district the sandflies belong to, Imamopu and Kozan districts?
Imamoglu and Kozan districts are not separated from each other’s. Those two regions are the main vector sampling districts and almost identical in terms of ecological parameters. Thus, two different districts were analyzed together.
Line 169: How many Ph. tobbi midguts were analyzed?
We thank the reviewer for noting this omission. The exact numbers of analyzed midguts are present in the text for each biological group have and also added to the Results section and corresponding tables.
Line 171: How many females collected in the field were analyzed?
We thank the reviewer for noting this omission. The exact numbers of analyzed midguts and specimens in each biological group have now been added to the Results section and corresponding tables.
Line 177: How many females from the colony were analyzed?
We thank the reviewer for noting this omission. The exact numbers of analyzed midguts and specimens in each biological group have now been added to the Results section and corresponding tables.
Line 178: How many males from the colony were analyzed?
We thank the reviewer for noting this omission. The exact numbers of analyzed midguts and specimens in each biological group have now been added to the Results section and corresponding tables.
Line 179: What type of alpha and beta diversity analysis was performed, and what indices were considered? This is not mentioned in the materials and methods section.
We appreciate this suggestion. Raw sample numbers corresponding to the relative abundance values have now been added to Table 2 where applicable.
Line 183: In Table 2, in addition to the percentages, it is necessary to add the raw data of the number of specimens analyzed.
Alpha and Beta diversity data with all raw data are presented in Supp. File 1
Line 192: The authors could add statistical tests to determine whether the observed differences between bacterial isolates are significant, as they only report average mortality percentages and standard deviations. This limitation reduces the study's ability to distinguish between biologically relevant patterns and variations attributable to experimental chance.
Line 193: This word in italics, L. infantum
Corrected
Line 196: Although the 32% mortality rate for Serratia liquefaciens is relatively low, it is interesting but far from representing a highly potent effect. Therefore, the discussion should adhere to these results and not exaggerate the biological significance of the finding.
We agree with the reviewer that the observed mortality rate should not be interpreted as a highly potent anti-leishmanial effect. The Discussion section has therefore been revised to present these findings more cautiously and to emphasize that the observed activity is moderate and preliminary in nature. Also XTT replicates file is added to Supp. Files
Line 207: The authors could clarify whether it is a direct bacterial culture or of bacterial supernatants.
We thank the reviewer for identifying this. We have clarified that the experiments were performed using live bacterial cultures rather than cell-free supernatants
Discussion
Line 227: Check the word "pmparasite"
Thanks. Corrected.
Line 244-245: Review grammatically this sentence: "Such reasons like controlled diets and environmental factors"
The sentence removed.
Line 316: The discussion about the impact of COVID-19 lockdowns on the absence of Leishmania is highly speculative and weakly supported. There is no longitudinal entomological or local epidemiological data to support the conclusion that vector-host contact decreased, transmission dynamics changed, or vector prevalence was reduced. Furthermore, it occupies too much space and diverts attention from scientific discussion.
We thank the reviewer for this valuable observation. We agree that the discussion linking the absence of Leishmania detection to COVID-19-related restrictions was speculative and not directly supported by data collected in this study. Therefore, Covid 19 part is removed.
Conclusions
Line 373: The authors claim that the study is the “first global characterization”; however, the study is regional, the sampling is limited, the effective microbiome size is not very large, and multiple similar studies exist in other sandflies. Therefore, it is recommended to be more conservative, using terms such as “first characterization in Turkey”, “baseline characterization in endemic populations” or “regional microbiota assessment.”
We agree with the reviewer that the term “first global characterization” overstates the scope of the study. The statement has been revised to more accurately reflect the regional nature of the work.
Lines 340, 346, 359, 369: Due to the results and scientific evidence, the authors should use these words with caution: “critical ecological baseline”, “promising candidates”, “ideal ecological setting”, “few scalable innovations available”.
We thank the reviewer for this suggestion. We have carefully revised the manuscript and replaced potentially overstated expressions such as “critical ecological baseline”, “promising candidates”, “ideal ecological setting”, and “few scalable innovations available” with more balanced and evidence-based wording.
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors
none
Reviewer 3 Report
Comments and Suggestions for Authors
The authors responded appropriately to the observations, addressing the points raised in the title, methodology, results, and discussion. They acknowledged the study's limitations, adjusted the scope of their conclusions, and added important methodological details. Overall, the responses are satisfactory, and the manuscript, with the corrections made, can be accepted.
