A cryopreservation method to recover laboratory- and field-derived bacterial communities from mosquito larval habitats

Mosquitoes develop in a wide range of aquatic habitats containing highly diverse and variable bacterial communities that shape both larval and adult traits, including the capacity of adult females of some mosquito species to vector disease-causing organisms to humans. However, while most mosquito studies control for host genotype and environmental conditions, the impact of microbiota variation on phenotypic outcomes of mosquitoes is often unaccounted for. The inability to conduct reproducible intra- and inter-laboratory studies of mosquito-microbiota interactions has also greatly limited our ability to identify microbial targets for mosquito-borne disease control. Here, we developed an approach to isolate and cryopreserve bacterial communities derived from lab- and field-based larval-rearing environments of the yellow fever mosquito Aedes aegypti–a primary vector of dengue, Zika, and chikungunya viruses. We then validated the use of our approach to generate experimental microcosms colonized by standardized lab- and field-derived bacterial communities. Our results overall reveal minimal effects of cryopreservation on the recovery of both lab- and field-derived bacteria when directly compared with isolation from non-cryopreserved fresh material. Our results also reveal improved reproducibility of bacterial communities in replicate microcosms generated using cryopreserved stocks over fresh material. Communities in replicate microcosms further captured the majority of total bacterial diversity present in both lab- and field-based larval environments, although the relative richness of recovered taxa as compared to non-recovered taxa was substantially lower in microcosms containing field-derived bacteria. Altogether, these results provide a critical next step toward the standardization of mosquito studies to include larval-rearing environments colonized by defined microbial communities. They also lay the foundation for long-term studies of mosquito-microbe interactions and the identification and manipulation of taxa with potential to reduce mosquito vectorial capacity.

蚊子可在多种水生栖息地中生长发育，这些栖息地产育着高度多样且动态变化的细菌群落，共同调控蚊子的幼虫与成虫性状，其中包括部分蚊种的雌性成虫向人类传播致病病原体的能力。然而，尽管绝大多数蚊子相关研究均对宿主基因型与环境条件进行了控制，但菌群变异对蚊子表型结果的影响往往未被纳入考量。无法开展可重复的蚊子-菌群互作实验室内部及跨实验室研究，同样极大限制了我们筛选蚊媒疾病防控相关微生物靶点的能力。本研究开发了一种可分离并冷冻保存细菌群落的方法，该方法的样本来源于实验室及野外环境下的黄热病蚊子埃及伊蚊（Aedes aegypti）的幼虫饲养环境，而埃及伊蚊是登革热、寨卡及基孔肯雅病毒的主要传播媒介。随后，我们验证了利用该方法构建由标准化实验室来源及野外来源细菌群落定殖的实验微群落的可行性。整体而言，我们的研究结果显示，与直接从未冷冻保存的新鲜样本中分离的细菌相比，冷冻保存对实验室来源及野外来源细菌的回收效果均影响极小。此外，相较于使用新鲜样本构建的重复微群落，使用冷冻保存菌种构建的重复微群落中细菌群落的可重复性得到了提升。含野外来源细菌的重复微群落中，回收类群相较于未回收类群的相对丰富度显著更低，但此类重复微群落仍覆盖了实验室及野外幼虫饲养环境中绝大多数的细菌总多样性。综上，本研究结果为蚊子研究的标准化工作迈出了关键的下一步，该标准化工作将纳入由明确微生物群落定殖的幼虫饲养环境。同时，本研究也为蚊子-微生物互作的长期研究，以及筛选并改造具备降低蚊子媒介效能潜力的类群奠定了基础。