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Microbial Diversity of Adult Aedes aegypti and Water Collected from Different Mosquito Aquatic Habitats in Puerto Rico

Abstract

Mosquitoes, the major vectors of viruses like dengue, are naturally host to diverse microorganisms, which play an important role in their development, fecundity, immunity, and vector competence. The composition of their microbiota is strongly influenced by the environment, particularly their aquatic larval habitat. In this study, we used 2×300 bp 16s Illumina sequencing to compare the microbial profiles of emerging adult Aedes aegypti mosquitoes and the water collected from common types of aquatic habitat containers in Puerto Rico, which has endemic dengue transmission. We sequenced 141 mosquito and 46 water samples collected from plastic containers, septic tanks, discarded tires, underground trash cans, tree holes, or water meters. We identified 9 bacterial genera that were highly prevalent in the mosquito microbiome, and 77 for the microbiome of the aquatic habitat. The most abundant mosquito-associated bacterial OTUs were from the families Burkholderiaceae, Pseudomonadaceae, Comamonadaceae, and Xanthomonadaceae. Microbial profiles varied greatly between mosquitoes, and there were few major differences explained by container type; however, the microbiome of mosquitoes from plastic containers was more diverse and contained more unique taxa than the other groups. Container water was significantly more diverse than mosquitoes, and our data suggest that mosquitoes filter out many bacteria, with Alphaproteobacteria in particular being far more abundant in water. These findings provide novel insight into the microbiome of mosquitoes in the region and provide a platform to improve our understanding of the fundamental mosquito-microbe interactions.

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Data Availability

All raw sequence data are available through google drive (https://drive.google.com/drive/folders/17aUjQy8fwFOkepyDv0GRcHbtj5HHkaW5?usp=sharing)

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Acknowledgements

The authors wish to thank María Roubert, Jesús Estudillo, Luis Rivera, Luis Perez, and Orlando Gonzales for their assistance with field work; Betzabel Flores, Glenda Gonzales, and Gilberto Santiago for the technical support; Scot O’Dowd from Mr DNA (https://www.mrdnalab.com) for sequencing and data preparation services; and a special thanks to Luis Eduardo Martínez Villegas for the helpful discussion of the manuscript, data analysis tools, and microbial ecology. We thank the six anonymous reviewers for their helpful comments.

Code Availability

R scripts are available in the supplementary materials.

Funding

This work was funded by grants from the CDC (BAA 2017-N-18041) and USAID (AID-OAA-F-16-00096). This work has also been supported by the Bloomberg Philanthropies. CVT was supported by the Johns Hopkins Malaria Research Institutes’ postdoctoral fellowship.

Author information

Affiliations

Authors

Contributions

Conceived of or designed study: EPC, LMO, RB, and GD. Performed research: EPC and LMO. Analyzed data: EPC and CVT. Wrote the paper: EPC and GD

Corresponding author

Correspondence to G. Dimopoulos.

Ethics declarations

Ethics Approval

Not applicable

Consent to Participate

Verbal consent was solicited and obtained from home/property owners before entering and collecting mosquito and container water samples when mosquito aquatic habitats were identified on private property.

Consent for Publication

Not applicable

Conflict of Interest

The authors declare no competing interests.

Supplementary Information

Fig. S1:
figure7

Bacterial profiles of mosquitoes and breeding site water for samples from supplementary dataset A. These bar plots represent the bacterial profiles of mosquito samples collected from miscellaneous plastic containers (a), tires (b), tree holes (c), and septic tanks (d), and paired water samples collected from plastic containers (e), tires (f), tree holes (g), and septic tanks (h). Mosquito and water samples sharing the same code i.e. M1, were independent samples collected from the breeding site. Each bar represents the profile of a single mosquito or water sample. Bacterial taxa of high abundance are depicted in different colours at the Family level, or as an unclassified Family belonging to an Order (Uncl.). Common colours represent Families from the same Class. Families of lower abundance have been grouped together as ‘Other’. (PNG 397 kb)

Fig. S2:
figure8

Bacterial profiles of mosquitoes and breeding site water for samples from supplementary dataset B. These bar plots represent the bacterial profiles of mosquito samples collected from large plastic buckets (a), and septic tanks (b), and water sample collected from large plastic buckets (c), and septic tanks (d). Mosquito and water samples sharing the same code i.e. P5, were independent samples collected from the breeding site. Each bar represents the profile of a single mosquito or 1-2 water samples. Bacterial taxa of high abundance are depicted in different colours at the Family level, or as an unclassified Family belonging to an Order (Uncl.). Common colours represent Families from the same Class. Families of lower abundance have been grouped together as ‘Other’. (PNG 380 kb)

Fig. S3:
figure9

NMDS ordination plots of mosquito and water samples for s supplementary dataset A. Ordination plots for mosquito (k = 2, stress = 0.21873) (a), and water samples (k = 2, stress = 0.16743) (b) split by breeding site type. A further panel (c) compares all water and mosquito samples (k = 3, stress = 0.16419). Plots were generated through non-metric multidimensional scaling (NMDS) using the metaMDS() function. Each dot depicts one sample, with samples from the same breeding site type sharing colours. Samples that are closer together in space have more similar bacterial profiles. (PNG 123 kb)

Fig. S4:
figure10

Venn diagrams comparing the bacteria present in samples from different breeding site types. Two-way Venn diagrams describe the number of bacterial genera that were identified in mosquito and water samples within the main data set. The region of overlap indicates the number of genera found in both water and mosquito samples. The other regions indicate unique bacterial genera associated with either sample type. Sample comparisons as follows: all mosquitoes and all breeding site water (a), plastics mosquitoes and their breeding site water (b), septics mosquitoes and their breeding site water (c), tire mosquitoes and their breeding site water (d), trash can mosquitoes and their breeding site water (e), tree hole mosquitoes and their breeding site water (f), water meter mosquitoes and their breeding site water (g). Panel (h) depicts the total number of genera identified in mosquitoes from each of the different breeding sites. (PNG 321 kb)

Fig. S5:
figure11

Family level bacterial profiles of mosquito samples from Qiime2 pipeline data. Bar plots represent the bacterial profiles of adult female Ae. aegypti mosquitoes collected within 24 hours post-emergence from six different breeding site types: large plastic buckets (a), septic tanks (b), discarded tires (c), inground trash cans (d), tree holes (e), and water meter pits (f). Each bar represents the profile of a single mosquito sample. Bacterial taxa of high abundance are depicted in different colours at the Family level, or as an unclassified Family (Uncl.) belonging to a bacterial Order. Common colours represent Families from the same Class. Families of lower abundance have been grouped together as ‘Other’. Graphs are based on Qiime2 pipeline data. Dashed blue lines separate samples collected from different breeding sites. (PNG 430 kb)

Fig. S6:
figure12

Family level bacterial profiles of breeding site water samples from Qiime2 pipeline data. Bar plots represent the bacterial profiles of breeding site water samples collected from large plastic buckets (a), septic tanks (b), discarded tires (c), inground trash cans (d), tree holes (e), and water meter pits (f). Each bar represents the profile of 1-2 water samples. Bacterial taxa of high abundance are depicted in different colours at the Family level, or as an unclassified Family belonging to an Order (Uncl.). Common colours represent Families from the same Class. Families of lower abundance have been grouped together as ‘Other’. Graphs are based on Qiime2 pipeline data. (PNG 306 kb)

Fig. S7:
figure13

NMDS ordination plots of mosquito and water samples from Qiime2 pipeline data. Ordination plots for mosquito (k = 4, stress = 0.12496) (a) and water (k = 2, stress = 0.09972) (b) samples generated through non-metric multidimensional scaling (NMDS) using the metaMDS() function. Each dot depicts one sample, with samples from the same breeding site type sharing colours. Samples that are closer together in space have more similar bacterial profiles. Plots are based on Qiime2 pipeline data. (PNG 148 kb)

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Caragata, E.P., Otero, L.M., Tikhe, C.V. et al. Microbial Diversity of Adult Aedes aegypti and Water Collected from Different Mosquito Aquatic Habitats in Puerto Rico. Microb Ecol (2021). https://doi.org/10.1007/s00248-021-01743-6

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Keywords

  • Aedes aegypti
  • Microbiome
  • Dengue
  • bacteria
  • 16s sequencing
  • Aquatic container habitat