Department of Parasitology, Faculty of Science, University of South Bohemia, Ceske Budejovice, Czechia.
Institute of Parasitology, Biology Centre of ASCR, Ceske Budejovice, Czechia.
Front Cell Infect Microbiol. 2020 Mar 17;10:86. doi: 10.3389/fcimb.2020.00086. eCollection 2020.
Symbiotic bacteria affect competence for pathogen transmission in insect vectors, including mosquitoes. However, knowledge on mosquito-microbiome-pathogen interactions remains limited, largely due to methodological reasons. The current, cost-effective practice of sample pooling used in mosquito surveillance and epidemiology prevents correlation of individual traits (i.e., microbiome profile) and infection status. Moreover, many mosquito studies employ laboratory-reared colonies that do not necessarily reflect the natural microbiome composition and variation in wild populations. As a consequence, epidemiological and microbiome studies in mosquitoes are to some extent uncoupled, and the interactions among pathogens, microbiomes, and natural mosquito populations remain poorly understood. This study focuses on the effect the pooling practice poses on mosquito microbiome profiles, and tests different approaches to find an optimized low-cost methodology for extensive sampling while allowing for accurate, individual-level microbiome studies. We tested the effect of pooling by comparing wild-caught, individually processed mosquitoes with pooled samples. With individual mosquitoes, we also tested two methodological aspects that directly affect the cost and feasibility of broad-scale molecular studies: sample preservation and tissue dissection. Pooling affected both alpha- and beta-diversity measures of the microbiome, highlighting the importance of using individual samples when possible. Both RNA and DNA yields were higher when using inexpensive reagents such as NAP (nucleic acid preservation) buffer or absolute ethanol, without freezing for short-term storage. Microbiome alpha- and beta-diversity did not show overall significant differences between the tested treatments compared to the controls (freshly extracted samples or dissected guts). However, the use of standardized protocols is highly recommended to avoid methodological bias in the data.
共生细菌会影响昆虫媒介（包括蚊子）传播病原体的能力。然而，由于方法学原因，蚊子-微生物组-病原体相互作用的知识仍然有限。目前，在蚊子监测和流行病学中使用的具有成本效益的样本混合实践，阻止了个体特征（即微生物组特征）和感染状态的相关性。此外，许多蚊子研究采用的是实验室饲养的群体，这些群体不一定反映出自然微生物组的组成和野生种群的变异。因此，蚊子的流行病学和微生物组研究在某种程度上是脱节的，病原体、微生物组和自然蚊子种群之间的相互作用仍然知之甚少。本研究重点研究了混合实践对蚊子微生物组特征的影响，并测试了不同的方法，以找到一种优化的低成本方法，用于广泛的采样，同时允许进行准确的个体水平微生物组研究。我们通过比较野外捕获的、单独处理的蚊子和混合样本，测试了混合的效果。对于单独的蚊子，我们还测试了两个直接影响广泛分子研究成本和可行性的方法学方面：样本保存和组织解剖。混合处理既影响了微生物组的 alpha 多样性，也影响了 beta 多样性，这突出了在可能的情况下使用个体样本的重要性。使用价格低廉的试剂，如 NAP（核酸保存）缓冲液或绝对乙醇，而不是短期储存的冷冻，可以提高 RNA 和 DNA 的产量。与对照组（新鲜提取的样本或解剖的肠道）相比，测试处理与控制处理之间的微生物组 alpha 和 beta 多样性没有总体上的显著差异。然而，强烈建议使用标准化的方案，以避免数据中的方法学偏差。
