A deeper understanding of the mechanisms underlying insecticide resistance is needed to mitigate its threat to malaria vector control. Following previously identified associations between mosquito microbiota and insecticide resistance, we demonstrate for the first time, the effects of pyrethroid exposure on the microbiota of F1 progeny of field-collected Anopheles albimanus. Larval and adult mosquitoes were exposed to the pyrethroids alphacypermethrin (only adults), permethrin, and deltamethrin. While there were no significant differences in bacterial composition between insecticide-resistant and insecticide-susceptible mosquitoes, bacterial composition between insecticide-exposed and non-exposed mosquitoes was significantly different for alphacypermethrin and permethrin exposure. Along with other bacterial taxa not identified to species, Pantoea agglomerans (a known insecticide-degrading bacterial species) and Pseudomonas fragi were more abundant in insecticide-exposed compared to non-exposed adults, demonstrating that insecticide exposure can alter mosquito bacterial communities. We also show for the first time that the cuticle surfaces of both larval and adult An. albimanus harbor more diverse bacterial communities than their internal microbial niches. Together, these findings demonstrate how insecticide pressure could be selecting for certain bacteria within mosquitoes, especially insecticide-metabolizing bacteria, thus potentially contributing to insecticide resistance.

Research on mosquito-microbe interactions may lead to new tools for mosquito and mosquitoborne disease control. To date, such research has largely utilized laboratory-reared mosquitoes that may lack the microbial diversity of wild populations. To better understand how mosquito microbiota may vary across different geographic locations and upon laboratory colonization, we characterized the microbiota of F1 progeny of wild-caught adult Anopheles albimanus from four locations in Guatemala using high throughput 16S rRNA amplicon sequencing. A total of 132 late instar larvae and 135 2-5day old, non-blood-fed virgin adult females were reared under identical laboratory conditions, pooled (3 individuals/pool) and analyzed. Larvae from mothers collected at different sites showed different microbial compositions (p=0.001; F = 9.5), but these differences were no longer present at the adult stage (p=0.12; F =1.6). This indicates that mosquitoes retain a significant portion of their field-derived microbiota throughout immature development but shed them before or during adult eclosion. This is the first time the microbiota of F1 progeny of wild-caught mosquitoes has been characterized in relation to parental collection site, and our findings provide evidence that geographically associated heterogeneity in microbiota composition persists for a single generation, but only until the end of the larval stage. These findings advance our understanding of how the mosquito microbiota is altered upon first laboratory colonization, and raises considerations for how mosquito microbiome research may be extended beyond the laboratory to field settings.

Background Research on mosquito-microbe interactions may lead to new tools for mosquito and mosquito-borne disease control. To date, such research has largely utilized laboratory-reared mosquitoes that typically lack the microbial diversity of wild populations. A logical progression in this area involves working under controlled settings using field-collected mosquitoes or, in most cases, their progeny. Thus, an understanding of how laboratory colonization affects the assemblage of mosquito microbiota would aid in advancing mosquito microbiome studies and their applications beyond laboratory settings. Methods Using high throughput 16S rRNA amplicon sequencing, the internal and cuticle surface microbiota of F1 progeny of wild-caught adult Anopheles albimanus from four locations in Guatemala were characterized. A total of 132 late instar larvae and 135 2–5 day-old, non-blood-fed virgin adult females that were reared under identical laboratory conditions, were pooled (3 individuals/pool) and analysed. Results Results showed location-associated heterogeneity in both F1 larval internal (p = 0.001; pseudo-F = 9.53) and cuticle surface (p = 0.001; pseudo-F = 8.51) microbiota, and only F1 adult cuticle surface (p = 0.001; pseudo-F = 4.5) microbiota, with a more homogenous adult internal microbiota (p = 0.12; pseudo-F = 1.6) across collection sites. Overall, ASVs assigned to Leucobacter, Thorsellia, Chryseobacterium and uncharacterized Enterobacteriaceae, dominated F1 larval internal microbiota, while Acidovorax, Paucibacter, and uncharacterized Comamonadaceae, dominated the larval cuticle surface. F1 adults comprised a less diverse microbiota compared to larvae, with ASVs assigned to the genus Asaia dominating both internal and cuticle surface microbiota, and constituting at least 70% of taxa in each microbial niche. Conclusions These results suggest that location-specific heterogeneity in filed mosquito microbiota can be transferred to F1 progeny under normal laboratory conditions, but this may not last beyond the F1 larval stage without adjustments to maintain field-derived microbiota. These findings provide the first comprehensive characterization of laboratory-colonized F1An. albimanus progeny from field-derived mothers. This provides a background for studying how parentage and environmental conditions differentially or concomitantly affect mosquito microbiome composition, and how this can be exploited in advancing mosquito microbiome studies and their applications beyond laboratory settings.

A deeper understanding of the mechanisms underlying insecticide resistance is needed to mitigate its threat to malaria vector control. Building upon our earlier identified associations between mosquito microbiota and insecticide resistance, we demonstrate for the first time, type-specific effects of pyrethroid exposure on internal and cuticle surface bacteria in adult progeny of field-collected Anopheles albimanus. In contrast, larval cuticle surface—but not internal—bacteria were affected by pyrethroid exposure. Being over five-folds more abundant in pyrethroid resistant adults, as compared to susceptible or non-insecticide-exposed mosquitoes, Klebsiella (alphacypermethrin), Pantoea and Asaia (permethrin) were identified as potential markers of pyrethroid resistance in An. albimanus. We also show for the first time that An. albimanus larvae and adult cuticles harbor more diverse bacterial communities than their internal microbial niches. Our findings indicate insecticide selection pressures on mosquito microbiota, and support the hypothesis of an undescribed microbe-mediated mechanism of insecticide metabolism in mosquitoes.

BackgroundDespite the progress to eliminate malaria in Central America, focalized transmission persists, and insecticide resistance is on the rise in the primary vector, Anopheles albimanus. Many of the new control methods being developed depend on the release of a large number of male mosquitoes that must be sorted prior to release. However, An. albimanus manual pupal-sex-sorting is not feasible, and therefore, we explored the use of RNA interference (RNAi) targeting genes with a sex-biased expression for female elimination. Here, we evaluated the effect of feeding larvae with dsRNA for a female-biased orthologue of the flight muscle actin gene.ResultsTwo sex-biased actin forms were identified in An. albimanus. Gene expression analysis showed a >40-fold higher expression of the AALB015469 transcript in female pupae (p = 0.0048) and adults (p = 0.0078) when compared to males. Tissue-specific analysis also suggests this female-biased actin can be an orthologue of the flight muscle actin of Aedes aegypti. At the same time, the AALB015481 transcript showed a >40-fold higher expression in male pupae and adults when compared to females, with no detectable expression in flight muscle. The potential effects of oral-induced RNAi for the female-biased actin were evaluated. Larvae were fed a diet containing either dsRNA for the female-biased actin 3'-UTR alone or for the UTR with an adjoining portion of the C-terminal coding region. A significant number of flightless females resulted from feedings with 3' UTR alone (10.50 ± 5.92 %, p < 0.05) or with the coding region (6.00 ± 2.16 %, p < 0.01). Treatment with the 3' UTR alone resulted in a significant number of flightless males (8.25 ± 3.10 %, p < 0.01). Both diets produced significant mortality in both female and male adults (p < 0.0001). ConclusionsFeeding of An. albimanus larvae with dsRNA targeting the female-biased flight muscle actin orthologue impairs flight in both sexes and affects the overall survival of female and male mosquitoes. Providing dsRNA in the larval diet shows promise as a method for screening other differentially expressed genes as potential targets for female elimination in mosquito breeding facilities.