Nowak Karol H, Hartop Emily, Prus-Frankowska Monika, Buczek Mateusz, Kolasa Michał R, Roslin Tomas, Ovaskainen Otso, Łukasik Piotr
Institute of Environmental Sciences, Faculty of Biology, Jagiellonian University, Kraków, Poland.
Doctoral School, PhD Programme in Biology, Faculty of Biology, Jagiellonian University, Kraków, Poland.
Microbiome. 2025 Aug 12;13(1):186. doi: 10.1186/s40168-025-02169-9.
Symbiotic microorganisms can profoundly impact insect biology, including their life history traits, population dynamics, and evolutionary trajectories. However, microbiota remain poorly understood in natural insect communities, especially in 'dark taxa'-hyperdiverse yet understudied clades.
Here, we implemented a novel multi-target amplicon sequencing approach to study microbiota in complex, species-rich communities. It combines four methodological innovations: (1) To establish a host taxonomic framework, we sequenced amplicons of the host marker gene (COI) and reconstructed barcodes alongside microbiota characterisation using 16S-V4 rRNA bacterial gene amplicons. (2) To assess microbiota abundance, we incorporated spike-in-based quantification. (3) To improve the phylogenetic resolution for the dominant endosymbiont, Wolbachia, we analysed bycatch data from the COI amplicon sequencing. (4) To investigate the primary drivers of host-microbe associations in massive multi-dimensional datasets, we performed Hierarchical Modelling of Species Communities (HMSC). Applying this approach to 1842 wild-caught scuttle flies (Diptera: Phoridae) from northern Sweden, we organised them into 480 genotypes and 186 species and gained unprecedented insights into their microbiota. We found orders-of-magnitude differences in bacterial abundance and massive within-population variation in microbiota composition. Patterns and drivers differed among microbial functional categories: the distribution and abundance of facultative endosymbionts (Wolbachia, Rickettsia, Spiroplasma) were shaped by host species, genotype, and sex. In contrast, many other bacterial taxa were broadly distributed across species and sites.
This study highlights facultative endosymbionts as key players in insect microbiota and reveals striking variations in distributional patterns of microbial clades. It also demonstrates the power of integrative sequencing approaches in uncovering the ecological complexity and significance of symbiotic microorganisms in multi-species natural communities. Video Abstract.
共生微生物可深刻影响昆虫生物学，包括其生活史特征、种群动态和进化轨迹。然而，在自然昆虫群落中，微生物群仍知之甚少，尤其是在“黑暗类群”——高度多样化但研究不足的进化枝中。
在此，我们采用了一种新颖的多靶点扩增子测序方法来研究复杂、物种丰富的群落中的微生物群。该方法结合了四项方法创新：（1）为建立宿主分类框架，我们对宿主标记基因（COI）的扩增子进行测序，并在使用16S-V4 rRNA细菌基因扩增子进行微生物群特征分析的同时重建条形码。（2）为评估微生物群丰度，我们纳入了基于内参的定量方法。（3）为提高对主要内共生菌沃尔巴克氏体的系统发育分辨率，我们分析了COI扩增子测序的副产物数据。（4）为研究大规模多维数据集中宿主-微生物关联的主要驱动因素，我们进行了物种群落层次建模（HMSC）。将该方法应用于从瑞典北部野生捕获的1842只蚤蝇（双翅目：蚤蝇科），我们将它们分为480个基因型和186个物种，并对其微生物群有了前所未有的深入了解。我们发现细菌丰度存在数量级差异，且微生物群组成在种群内部存在巨大变异。不同微生物功能类别的模式和驱动因素有所不同：兼性内共生菌（沃尔巴克氏体、立克次氏体、螺原体）的分布和丰度受宿主物种、基因型和性别的影响。相比之下，许多其他细菌类群在物种和地点之间广泛分布。
本研究强调兼性内共生菌是昆虫微生物群中的关键参与者，并揭示了微生物进化枝分布模式的显著差异。它还展示了综合测序方法在揭示多物种自然群落中共生微生物的生态复杂性和重要性方面的强大作用。视频摘要。
