Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Departments of Vector Biology and Tropical Disease Biology, Centre for Neglected Topical Disease, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
PLoS Negl Trop Dis. 2023 Apr 5;17(4):e0011234. doi: 10.1371/journal.pntd.0011234. eCollection 2023 Apr.
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 transmit 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.
蚊子在广泛的水生栖息地中发育，这些栖息地包含高度多样化和可变的细菌群落，这些群落塑造了幼虫和成虫的特征，包括某些蚊子物种的成年雌性将致病生物体传播给人类的能力。然而，尽管大多数蚊子研究都控制了宿主基因型和环境条件，但微生物群落的变化对蚊子表型结果的影响往往没有被考虑在内。无法对蚊子-微生物群相互作用进行可重复的实验室内和实验室间研究，也极大地限制了我们识别控制蚊媒疾病的微生物靶标的能力。在这里，我们开发了一种从黄热病蚊子埃及伊蚊的实验室和现场幼虫饲养环境中分离和冷冻保存细菌群落的方法-登革热、寨卡和基孔肯雅热病毒的主要载体。然后，我们验证了使用我们的方法来生成由标准化实验室和现场衍生的细菌群落定植的实验微宇宙。我们的研究结果总体上表明，与直接从非冷冻保存的新鲜材料中分离相比，冷冻保存对实验室和现场来源细菌的回收影响很小。我们的研究结果还表明，使用冷冻保存的库存而不是新鲜材料生成的重复微宇宙中细菌群落的重现性得到了提高。在重复的微宇宙中，群落进一步捕获了实验室和现场幼虫环境中存在的大部分总细菌多样性，尽管与未恢复的分类群相比，回收的分类群的相对丰富度在含有现场来源细菌的微宇宙中要低得多。总之，这些结果为包括由定义明确的微生物群落定植的幼虫饲养环境的蚊子研究的标准化提供了关键的下一步，它们也为蚊子-微生物相互作用的长期研究以及鉴定和操纵具有降低蚊子媒介能力潜力的分类群奠定了基础。
