Dynamics and diversity of bacteria associated with the disease vectors Aedes aegypti and Aedes albopictus

Aedes aegypti and Aedes albopictus develop in the same aquatic sites where they encounter microorganisms that influence their life history and capacity to transmit human arboviruses. Some bacteria such as Wolbachia are currently being considered for the control of Dengue, Chikungunya and Zika. Yet little is known about the dynamics and diversity of Aedes-associated bacteria, including larval habitat features that shape their tempo-spatial distribution. We applied large-scale 16S rRNA amplicon sequencing to 960 adults and larvae of both Ae. aegypti and Ae. albopictus mosquitoes from 59 sampling sites widely distributed across nine provinces of Panama. We find both species share a limited, yet highly variable core microbiota, reflecting high stochasticity within their oviposition habitats. Despite sharing a large proportion of microbiota, Ae. aegypti harbours higher bacterial diversity than Ae. albopictus, primarily due to rarer bacterial groups at the larval stage. We find significant differences between the bacterial communities of larvae and adult mosquitoes, and among samples from metal and ceramic containers. However, we find little support for geography, water temperature and pH as predictors of bacterial associates. We report a low incidence of natural Wolbachia infection for both Aedes and its geographical distribution. This baseline information provides a foundation for studies on the functions and interactions of Aedes-associated bacteria with consequences for bio-control within Panama.


Phylogenetic distance
Ae. aegypti larvae Ae. aegypti adults Ae. albopictus larvae Ae. albopictus adults Sequences per sample

Supplementary results based on the analysis of reverse reads Composition and structure of Aedes-associated bacterial communities
In total, 7,309,396 sequence reads of bacterial 16S rRNA gene amplicons were generated from DNA pools and individually processed mosquitoes representing all samples of immature and adult Ae. aegypti and Ae. albopictus. The mean number of reads per pool or individual sample was 40,834 reads. Due to a reduction in the quality of reads, the reverse sequence data was trimmed at 120 base pairs ( Figure S4). After quality filtering, 5,922,570 sequences were retained from 177 samples, including 75 pools (21.3 % larvae) and 20 individual adult Ae. aegypti and 62 pools (22.6 % larvae) and 20 individual adult Ae. albopictus. After rarefaction, these comprised 67 pools and 12 individuals of Ae. aegypti and 54 pools and 18 individuals of Ae. albopictus with 573 unique OTU's, averaged at 35 OTU's per individual or mosquito pool. Figure S4. Bair pair quality plot of reverse reads showing that quality is reduced consistently to below a score of 20 after 120 base pairs.
In total, the bacterial communities analysed composed 16 phyla, 33 classes, 54 orders, 73 families and 74 genera of bacteria. Due to the reduced quality and length of the reverse reads, a larger proportion of bacterial OTU's were unidentified compared to the forward reads with 4 % and 2 % unassigned bacterial OTU's compared to 0.02 % and 0.00 % for Ae. aegypti and Ae. albopictus respectively. However, the dominant bacterial Phyla remained as Proteobacteria, which totalled 89 % of the identified OTU's for both mosquito species, in addition to Bacteroidetes, Actinobacteria and Firmicutes which were found at lower relative abundances above 1%. The composition of bacterial Phyla ( Figure S5a) and Classes ( Figure  S5b

Aedes intra-and inter-species bacterial diversity
Intraspecific variability within the bacterial communities of both Aedes species was high, with average Bray-Curtis distances of 0.80 and 0.83 for Ae. aegypti and Ae. albopictus respectively. Flavobacteriales were highly variable among pools of individuals, and dominated the bacterial community composition of a small number of samples from Los Santos province including three Ae. aegypti and one Ae. albopictus ( Figure S6). The bacterial diversity of larvae was higher and had a significantly different microbial community than emergent adults in Ae. aegypti (PERMANOVA of unweighted UNIFRAC distances, pseudo-F = 7.50, P < 0.01), but not Ae. albopictus (PERMANOVA of unweighted UNIFRAC distances, pseudo-F = 1.82, P > 0.05) ( Table 1). This is likely due to a reduction in the number of identified OTU's overall for Ae. aegypti due to the lower quality and therefore shorter length of the reverse sequence data ( Figure S4).

Larval habitat features and bacterial composition
We observed no consistent differences between both the bacterial community of larvae and adult Aedes species due to larval habitats features including geographic distribution, type of container material and associated environmental variables of the water. There were few statistically significant comparisons based on PERMANOVA between oviposition sites having different container materials for adults only (Table 2). Differences were observed between the region of Panama and Bocas del Toro/Los Santos for adult Ae. aegypti as well as between Los Santos and Chiriquí/Panamá for Ae. albopictus adults, but not for larvae (Table  3). Similar to analysis based on forward reads, the water temperature and pH of the larval habitat did not strongly influence the microbiome acquired by the mosquito host, with no clear change in the microbial community with increasing values of either variable in both species.

Wolbachia occurrence and distribution
We found Wolbachia 16S rDNA positive samples in both Aedes species. This included 12 pools and four individuals of Ae. albopictus of both adults and larvae widespread across Panama and two adult Ae. aegypti from Los Santos and Panama province. The outcomes on the distribution of Wolbachia across Panama were not affected by changes in the quality of sequence reads because they were additionally supported through conventional PCR.

Conclusion
Although results based on the analysis of low-quality reverse reads largely agree with the findings and interpretations of our original submission, we lose resolution to make comparison at the intraspecific level within and between populations of Ae. aegypti and Ae. albopictus from across Panama. Therefore, we decided to exclude reverse reads from final analyses due to their lower quality.