Re-Analysis of 16S rRNA Gene Sequence Data Sets Uncovers Disparate Laboratory-Specific Microbiomes Associated with the Yellow Fever Mosquito (Aedes aegypti).

Host-microbiome dynamics occurring in the yellow fever mosquito (Aedes aegypti) contribute to host life history traits, and particular bacterial taxa are proposed to comprise a "core" microbiota that influences host physiology. Laboratory-based studies are frequently performed to investigate these processes; however, experimental results are often presumed to be generalizable across laboratories, and few efforts have been made to independently reproduce and replicate significant findings. A recent study by Muturi et al. (FEMS Microbiol Ecol 95 (1):213, 2019) demonstrated the food source imbibed by laboratory-reared adult female mosquitoes significantly impacted the host-associated microbiota-a foundational finding in the field of mosquito biology worthy of independent evaluation. Here, we coalesce these data with two additional mosquito-derived 16S rRNA gene sequence data sets using a unifying bioinformatics pipeline to reproduce the characterization of these microbiota, test for a significant food source effect when independent samples were added to the analyses, assess whether similarly fed mosquito microbiomes were comparable across laboratories, and identify conserved bacterial taxa. Our pipeline characterized similar microbiome composition and structure from the data published previously, and a significant food source effect was detected with the addition of independent samples, increasing the robustness of this previously discovered component of mosquito biology. However, distinct microbial communities were identified from similarly fed but independently reared mosquitoes, and surveys across all samples did not identify conserved bacterial taxa. These findings demonstrated that while the main effect of the food source was supported, laboratory-specific conditions may produce inherently differential microbiomes across independent laboratory environments.

Shifts in the microbiota associated with male mosquitoes (Aedes aegypti) exposed to an obligate gut fungal symbiont (Zancudomyces culisetae).

Research characterizing arthropod-associated microbiota has revealed that microbial dynamics can have an important impact on host phenotypic traits. The influence of fungi on these interactions are emerging as targets for research, especially in organisms associated with global human health. A recent study demonstrated colonization of a widespread gut fungus (Zancudomyces culisetae) in a larval mosquito (Aedes aegypti) digestive tract affected microbiomes in larvae and newly emerged adult females (Frankel-Bricker et al. Appl Environ Microbiol, 2020. https://doi.org/10.1128/AEM.02334-19) but did not investigate these processes in males. The objective of the study presented here was to assess fungal influences on adult male mosquito microbiomes to enable a more complete assessment of fungal-bacterial-host interactions in the A. aegypti-Z. culisetae system. Sequencing of 16S rRNA gene amplicons from microbiomes harbored in adult males directly after emerging from pupae revealed larval fungal exposure significantly decreased overall microbial community diversity, altered microbiome composition and structure, and decreased within-group microbiome variation across individuals. Further, bacteria in the family Burkholderiaceae were present in high abundance in fungal-exposed males, likely contributing to the disparate microbiota between treatment groups. Comparisons between male and the female microbiomes analyzed in Frankel-Bricker et al. (2020), showed distinct shifts in bacterial communities incurred by larval exposure to fungi, potentially revealing sex-specific fungal-bacterial-host dynamics in A. aegypti. These findings highlight the complex role a gut fungus can play in influencing the microbial communities harbored in an important insect and emphasize the significance of accounting for an organism's sex when studying fungal-bacterial-host dynamics.

Variation in the Microbiota Associated with Daphnia magna Across Genotypes, Populations, and Temperature.

Studies of how the microbiome varies among individuals, populations, and abiotic conditions are critical for understanding this key component of an organism's biology and ecology. In the case of Daphnia, aquatic microcrustaceans widely used in population/community ecology and environmental science studies, understanding factors that influence microbiome shifts among individuals is useful for both basic and applied research contexts. In this study, we assess differences in the microbiome among genotypes of D. magna collected from three regions along a large latitudinal gradient (Finland, Germany, and Israel). After being reared in the lab for many years, we sought to characterize any differences in genotype- or population-specific microbial communities, and to assess whether the microbiota varied among temperatures. Our study is similar to a recent comparison of the microbial communities among D. magna genotypes raised in different temperatures published by Sullam et al. (Microb Ecol 76(2):506-517, 2017), and as such represents one of the first examples of a reproducible result in microbiome research. Like the previous study, we find evidence for a strong effect of temperature on the microbiome of D. magna, although across a much smaller temperature range representing potential near-future climates. In addition, we find evidence that the microbiomes of D. magna genotypes from different regions are distinct, even years after being brought into the laboratory. Finally, our results highlight a potentially common finding in the expanding area of microbiome research-differences among treatments are not necessarily observed in the most abundant taxonomic groups. This highlights the importance of considering sampling scheme and depth of coverage when characterizing the microbiome, as different experimental designs can significantly impact taxon-specific results, even when large-scale effects are reproduced.

Influences of a Prolific Gut Fungus (Zancudomyces culisetae) on Larval and Adult Mosquito (Aedes aegypti)-Associated Microbiota.

Adult mosquitoes inherit a bacterial community from larvae via transstadial transmission, an understudied process that may influence host-microbe interactions. Microbes contribute to important host life history traits, and analyzing transmitted microbial communities, the interrelationship between larval and adult-associated microbiota, and factors influencing host-microbe relationships provides targets for research. During its larval stage, the yellow fever mosquito (Aedes aegypti) hosts the trichomycete gut fungus Zancudomyces culisetae, and fungal colonization coincides with environmental perturbations in the digestive tract microecosystem. Natural populations are differentially exposed to fungi, thereby potentially harboring distinct microbiota and experiencing disparate host-microbe interactions. This study's objectives were to characterize larval and initial adult microbiomes, investigate variation in diversity and distribution of microbial communities across individuals, and assess whether larval fungal colonization impacted microbiomes at these developmental stages. Laboratory-based fungal infestation assays, sequencing of 16S rRNA gene amplicons, and bacterial load quantification protocols revealed that initial adult microbiomes varied in diversity and distribution. Larval fungal colonization had downstream effects on initial adult microbiomes, significantly reducing microbial community variation, shifting relative abundances of certain bacterial families, and influencing transstadial transmission outcomes of particular genera. Further, abundances of several families consistently decreased in adults relative to levels in larvae, possibly reflecting impacts of host development on specific bacterial taxa. These findings demonstrated that a prolific gut fungus impacted mosquito-associated microbiota at two developmental stages in an insect connected with global human health.IMPORTANCE Mosquitoes are widespread vectors of numerous human pathogens and harbor microbiota known to affect host phenotypic traits. However, little research has directly investigated how bacterial communities associated with larvae and adults are connected. We characterized whole-body bacterial communities in mosquito larvae preceding pupation and in newly emerged adults, and investigated whether a significant biotic factor, fungal colonization of the larval hindgut, impacted these microbiomes. Results showed that fungal colonization reduced microbial community variation across individuals and differentially impacted the outcomes of transstadial transmission for certain bacterial genera, revealing downstream effects of the fungus on initial adult microbiomes. The importance of our research is in providing a thorough comparative analysis of whole-body microbiota harbored in larvae and adults of the yellow fever mosquito (Aedes aegypti) and in demonstrating the important role a widespread gut fungus played in a host-associated microbiome.