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The microbiome influences critical aspects of mosquito biology and variations in microbial composition can impact the outcomes of laboratory studies. To investigate how biotic and abiotic conditions in an insectary affect the composition of the mosquito microbiome, a single cohort of Aedes aegypti eggs was divided into three batches and transferred to three different climate-controlled insectaries within the Liverpool School of Tropical Medicine. The bacterial microbiome composition was compared as mosquitoes developed, the microbiome of the mosquitoes' food sources was characterised, environmental conditions over time in each insectary were measured, and mosquito development and survival were recorded. While developmental success was similar across all three insectaries, differences in microbiome composition were observed between mosquitoes from each insectary. Environmental conditions and bacterial input via food sources varied between insectaries, potentially contributing to the observed differences in microbiome composition. At both adult and larval stages, specific members of the mosquito microbiome were associated with particular insectaries; the insectary with less stable and cooler conditions resulted in a slower pupation rate and higher diversity of the larval microbiome. These findings underscore that even minor inconsistencies in rearing conditions can affect the composition of the mosquito microbiome, which may influence experimental outcomes., (© 2025 The Author(s). Environmental Microbiology published by John Wiley & Sons Ltd.)

Mosquitoes transmit medically important human pathogens, including viruses like dengue virus and parasites such as Plasmodium spp., the causative agent of malaria. Mosquito microbiomes are critically important for the ability of mosquitoes to transmit disease-causing agents. However, while large collections of bacterial isolates and genomic data exist for vertebrate microbiomes, the vast majority of work in mosquitoes to date is based on 16S rRNA gene amplicon data that provides limited taxonomic resolution and no functional information. To address this gap and facilitate future studies using experimental microbiome manipulations, we generated a bacterial Mosquito-Associated Isolate Collection (MosAIC) consisting of 392 bacterial isolates with extensive metadata and high-quality draft genome assemblies that are publicly available, both isolates and sequence data, for use by the scientific community. MosAIC encompasses 142 species spanning 29 bacterial families, with members of the Enterobacteriaceae comprising 40% of the collection. Phylogenomic analysis of 3 genera, Enterobacter, Serratia, and Elizabethkingia, reveal lineages of mosquito-associated bacteria isolated from different mosquito species in multiple laboratories. Investigation into species' pangenomes further reveals clusters of genes specific to these lineages, which are of interest for future work to test for functions connected to mosquito host association. Altogether, we describe the generation of a physical collection of mosquito-associated bacterial isolates, their genomic data, and analyses of selected groups in context of genome data from closely related isolates, providing a unique, highly valuable resource for research on bacterial colonisation and adaptation within mosquito hosts. Future efforts will expand the collection to include broader geographic and host species representation, especially from individuals collected from field populations, as well as other mosquito-associated microbes, including fungi, archaea, and protozoa., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Foo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)