Transcriptome Sequencing Reveals Large-Scale Changes in Axenic Aedes aegypti Larvae

Abstract

Mosquitoes host communities of microbes in their digestive tract that consist primarily of bacteria. We previously reported that Aedes aegypti larvae colonized by a native community of bacteria and gnotobiotic larvae colonized by only Escherichia coli develop very similarly into adults, whereas axenic larvae never molt and die as first instars. In this study, we extended these findings by first comparing the growth and abundance of bacteria in conventional, gnotobiotic, and axenic larvae during the first instar. Results showed that conventional and gnotobiotic larvae exhibited no differences in growth, timing of molting, or number of bacteria in their digestive tract. Axenic larvae in contrast grew minimally and never achieved the critical size associated with molting by conventional and gnotobiotic larvae. In the second part of the study we compared patterns of gene expression in conventional, gnotobiotic and axenic larvae by conducting an RNAseq analysis of gut and nongut tissues (carcass) at 22 h post-hatching. Approximately 12% of Ae. aegypti transcripts were differentially expressed in axenic versus conventional or gnotobiotic larvae. However, this profile consisted primarily of transcripts in seven categories that included the down-regulation of select peptidases in the gut and up-regulation of several genes in the gut and carcass with roles in amino acid transport, hormonal signaling, and metabolism. Overall, our results indicate that axenic larvae exhibit alterations in gene expression consistent with defects in acquisition and assimilation of nutrients required for growth.

Axenic Aedesaegyptidevelop without live bacteria, but exhibit delayed development and reduced oviposition

“…A study of the transcriptional differences between axenic and colonized larvae may support this hypothesis. Axenic larvae (unable to develop due to a lack of a microbiome) displayed significant down-regulation of peptidase genes and an upregulation of amino acid transporters in comparison to their microbially colonized cohorts 40 . This suggests that protein and amino acid metabolism is significantly altered in axenic larvae.…”

Composition and functional roles of the gut microbiota in mosquitoes

“…aegypti larvae which leads to activation of the insulin/insulin growth factor pathway, select mitogen activated kinases and other processes with essential growth functions [61, 70] (Figure 1). Transcriptome analysis also identifies several genes with functions in digestion and nutrient acquisition as additional targets that are potentially regulated by the gut microbiota or microbiota-induced hypoxia [71]. …”

Hypoxia-induced transcription factor signaling is essential for larval growth of the mosquitoAedes aegypti

“…We recently used A. aegypti larvae fed a nutritionally complete diet and E. coli as a one-host/one-microbe model to characterize what bacteria in the gut provide that mosquito larvae require for development (19). Within each instar, mosquitoes and other insects grow by consuming nutrients until achieving a critical size, which stimulates a rise in titer of the hormone 20-hydroxyecdyone (20E) that stimulates molting (20,21). Comparing gnotobiotic larvae inoculated with wild-type E. coli with conventionally reared first instars indicated both exhibit low midgut oxygen levels during growth to critical size, which is followed by 20E release and molting to the second instar at ∼24 h posthatching (19).…”