Microbiota Variation Across Life Stages of European Field-Caught Anopheles atroparvus and During Laboratory Colonization: New Insights for Malaria Research

Abstract

The potential use of bacteria for developing novel vector control approaches has awakened new interests in the study of the microbiota associated with vector species. To set a baseline for future malaria research, a high-throughput sequencing of the bacterial 16S ribosomal gene V3-V4 region was used to profile the microbiota associated with late-instar larvae, newly emerged females, and wild-caught females of a sylvan Anopheles atroparvus population from a former malaria transmission area of Spain. Field-acquired microbiota was then assessed in non-blood-fed laboratory-reared females from the second, sixth, and 10th generations. Diversity analyses revealed that bacterial communities varied and clustered differently according to origin with sylvan larvae and newly emerged females distributing closer to laboratory-reared females than to their field counterparts. Inter-sample variation was mostly observed throughout the different developmental stages in the sylvan population. Larvae harbored the most diverse bacterial communities; wild-caught females, the poorest. In the transition from the sylvan environment to the first time point of laboratory breeding, a significant increase in diversity was observed, although this did decline under laboratory conditions. Despite diversity differences between wild-caught and laboratory-reared females, a substantial fraction of the bacterial communities was transferred through transstadial transmission and these persisted over 10 laboratory generations. Differentially abundant bacteria were mostly identified between breeding water and late-instar larvae, and in the transition from wild-caught to laboratory-reared females from the second generation. Our findings confirmed the key role of the breeding environment in shaping the microbiota of An. atroparvus. Gram-negative bacteria governed the microbiota of An. atroparvus with the prevalence of proteobacteria. Pantoea, Thorsellia, Serratia, Asaia, and Pseudomonas dominating the microbiota associated with wild-caught females, with the latter two governing the communities of laboratory-reared females. A core microbiota was identified with Pseudomonas and Serratia being the most abundant core genera shared by all sylvan and laboratory specimens. Overall, understanding the microbiota composition of An. atroparvus and how this varies throughout the mosquito life cycle and laboratory colonization paves the way when selecting potential bacterial candidates for use in microbiota-based intervention strategies against mosquito vectors, thereby improving our knowledge of laboratory-reared An. atroparvus mosquitoes for research purposes.

Cryopreservation of mosquito microbiota for use in microcosm experiments

“…In this study, we first developed an approach to isolate and cryopreserve microbiota from conventional larval rearing pans in the laboratory for recapitulation in experimental microcosms. High-throughput sequencing of 16S rRNA gene amplicons from water collected from four replicate rearing pans from our laboratory colony of A. aegypti revealed a bacterial community comprised of ~100 unique bacterial taxa and dominated by members of the Proteobacteria, Bacteroidetes, and Firmicutes, consistent with sequencing studies of other laboratory colonies of mosquitoes (2–4,6–16,45,47,57,60,92,97). The vast majority of this bacterial diversity was also recovered in both experimental microcosms generated using non-cryopreserved fresh material and experimental microcosms generated using cryopreserved material from the same rearing pans, regardless of stock cell density.…”

“…We next validated our isolation and cryopreservation approach using microbiota derived from water collected from a naturally occurring larval habitat in the field. As expected, the bacterial community present in this habitat was comprised of a substantially higher diversity of ~1,000 unique taxa, although these taxa were dominated by members of the same phyla (Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria) detected in laboratory rearing pans and commonly detected in field-collected mosquitoes (5,51,52,(56)(57)(58)(59)(60)(61)(62)(84)(85)(86)(87)(88)(89)(90)(91)(97)(98)(99)(100)(101)(102). Recovery of field-derived taxa in experimental microcosms generated using cryopreserved material from the same habitat was also overall much lower than what we observed in microcosms generated using cryopreserved stocks of lab-derived microbiota, although the taxa lost or significantly reduced in microcosms generated using cryopreserved material were consistent with those lost or significantly reduced in microcosms generated using fresh material-strongly suggesting that the observed shifts in alpha and beta diversity in microcosms were not the result of cryopreservation but rather the inability of many field-derived taxa to thrive under conventional mosquito rearing conditions in the laboratory.…”

“…However, the immense diversity and variability of the microbiota within and between different populations of mosquitoes in the laboratory and field have made studying mosquito-microbiome interactions–and identifying bacteria that reduce the vectorial capacity of mosquitoes–a formidable challenge (1). As in other animals, numerous factors have the potential to shape variation in mosquito microbiota and therefore variation in mosquito phenotypes, including the microbiota present in the aquatic environment in which larvae develop, environmental conditions (e.g., diet, temperature), and host genetics (2–16,51,52,57,58,60–62,84–92). However, while experiments with mosquitoes are commonly conducted using genetically identical individuals under highly controlled environmental conditions, tools to standardize the microbiota present in the larval rearing environment are comparatively limited (8,55,93–96).…”

“…13.499917 doi: bioRxiv preprint then demonstrated the utility of this approach to study the factors shaping microbiome acquisition and assembly in mosquitoes and the mechanisms by which specific microbial taxa and assemblages contribute to different mosquito traits under controlled conditions (55). However, while these results highlight the value of expanding tools to manipulate the microbiota in mosquitoes, important questions remain regarding (i) the utility of microbiome transplantation approaches to establish reproducible communities in mosquito larval rearing environments, which harbor microbial communities that are much more complex than those in adult mosquitoes (5,8,52,(56)(57)(58)(59)(60)(61)(62), and (ii) how microbiota transplantation efficacy may be shaped by long-term preservation of donor microbial communities (e.g., via cryogenic freezing), which is absolutely necessary to facilitate long-term studies and intra-and interlaboratory comparisons but may introduce additional variability via impacts on bacterial viability and recovery (63)(64)(65)(66)(67)(68).…”

Bacterial Community Diversity and Bacterial Interaction Network in Eight Mosquito Species

“…Asaia is frequently found in Aedes and Anopheles mosquitoes [ 58 , 61 ], and it can inhibit the development of Plasmodium in female mosquitoes [ 62 ]. In addition, Asaia can diminish the longevity of infected males of Anopheles stephensi [ 63 ].…”