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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.)

The microbiome affects important aspects of mosquito biology and differences in microbial composition can affect the outcomes of laboratory studies. To determine how the biotic and abiotic conditions in an insectary affect the composition of the bacterial microbiome of mosquitoes we reared mosquitoes from a single cohort of eggs from one genetically homogeneous inbred Aedes aegypti colony, which were split into three batches, and transferred to each of three different insectaries located within the Liverpool School of Tropical Medicine. Using three replicate trays per insectary, we assessed and compared the bacterial microbiome composition as mosquitoes developed from these eggs. We also characterised the microbiome of the mosquitoes' food sources, measured environmental conditions over time in each climate-controlled insectary, and recorded development and survival of mosquitoes. While mosquito development was overall similar between all three insectaries, we saw differences in microbiome composition between mosquitoes from each insectary. Furthermore, bacterial input via food sources, potentially followed by selective pressure of temperature stability and range, did affect the microbiome composition. At both adult and larval stages, specific members of the mosquito microbiome were associated with particular insectaries; and the insectary with less stable and cooler conditions resulted in slower pupation rate and higher diversity of the larval microbiome. Tray and cage effects were also seen in all insectaries, with different bacterial taxa implicated between insectaries. These results highlight the necessity of considering the variability and effects of different microbiome composition even in experiments carried out in a laboratory environment starting with eggs from one batch; and highlights the impact of even minor inconsistencies in rearing conditions due to variation of temperature and humidity.

Background: The endosymbiotic bacterium Wolbachia infects numerous species of insects and Wolbachia transinfection of Aedes mosquito species is now being used for biocontrol programs as Wolbachia strains can both inhibit arboviruses and invade wild mosquito populations. The discovery of novel, resident Wolbachia strains in mosquito species warrants further investigation as potential candidate strains for biocontrol strategies. Methods: We obtained mosquito specimens from diverse Culicine mosquitoes from Cameroon including ecologically diverse locations in the Central and West Regions. Wolbachia prevalence rates were assessed in addition to the environmentally acquired bacterial species Asaia in major Culicine genera. PCR-based methods were also used with phylogenetic analysis to confirm identities of host mosquito species and Wolbachia strains were classified using multi-locus sequence typing (MLST). Results : We report high Wolbachia prevalence rates for Culicine species, including in a large cohort of Aedes africanus collected from west Cameroon in which 100% of mono-specific pools were infected. Furthermore, co-infections with Asaia bacteria were observed across multiple genera, demonstrating that these two bacteria can co-exist in wild mosquito populations.  Wolbachia strain MLST and phylogenetic analysis provided evidence for diverse Wolbachia strains in 13 different mosquito species across seven different genera.  Full or partial MLST profiles were generated from resident Wolbachia strains in six Culex species ( quinquefasciatus , watti , cinerus, nigripalpus, perexiguus and rima), two Aedes species (africanus and denderensis) and in Mansonia uniformis, Catageiomyia argenteopunctata, Lutzia tigripes, Eretmapodites chrysogaster and Uranotaenia bilineata. Conclusions: Our study provides further evidence that Wolbachia is widespread within wild mosquito populations of diverse Culicine species and provides further candidate strains that could be investigated as future options for Wolbachia -based biocontrol to inhibit arbovirus transmission., Competing Interests: No competing interests were disclosed., (Copyright: © 2023 Mercant Osuna A et al.)

Introduction: Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects' biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Methods: Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase and a plasmid-based method for expressing PhiC31 when injected into early embryos. Results: Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard. Discussion: Here we have expanded the toolbox for synthetic biology in Ae. aegypti . These methods can be easily transferred into other mosquito and even insect species by identifying appropriate promoter sequences. This advances the ability to manipulate these insects for fundamental studies, and for more applied approaches for pest control., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Carabajal Paladino, Wilson, Tng, Dhokiya, Keen, Cuber, Larner, Rooney, Nicholls, Uglow, Williams, Anderson, Basu, Leftwich and Alphey.)

Wolbachia, a widespread bacterium that can reduce pathogen transmission in mosquitoes, has recently been reported to be present in Anopheles (An.) species. In wild populations of the An. gambiae complex, the primary vectors of Plasmodium malaria in Sub-Saharan Africa, Wolbachia DNA sequences at low density and infection frequencies have been detected. As the majority of studies have used highly sensitive nested PCR as the only method of detection, more robust evidence is required to determine whether Wolbachia strains are established as endosymbionts in Anopheles species. Here, we describe high-density Wolbachia infections in geographically diverse populations of An. moucheti and An. demeilloni. Fluorescent in situ hybridization localized a heavy infection in the ovaries of An. moucheti, and maternal transmission was observed. Genome sequencing of both Wolbachia strains obtained genome depths and coverages comparable to those of other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present, indicating that these strains possess the capacity to induce the cytoplasmic incompatibility phenotype, which allows Wolbachia to spread through host populations. These strains should be further investigated as candidates for use in Wolbachia biocontrol strategies in Anopheles aiming to reduce the transmission of malaria., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)