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

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.
Author Summary Several mosquito species including Aedes aegypti transmit pathogens as adults that cause disease in humans and other vertebrates. It has also long been known that mosquitoes host bacteria in their digestive tract, which are primarily acquired during the larval stage and transstadially transmitted to adults. Our recent results indicate that axenic larvae, which lack bacteria, do not develop beyond the first instar, whereas larvae with living bacteria develop into adults. To better understand the effects of bacteria on mosquito development, we compared growth, molting and gene expression in larval Ae. aegypti that contained several species of bacteria, only one species of bacterium (Escherichia coli), or no bacteria. Results showed that larvae containing several species or only E. coli grew and molted very similarly while larvae with no bacteria grew minimally and never molted. A number of Ae. aegypti genes with roles in regulating growth were differentially expressed in larvae without bacteria when compared to larvae with bacteria. Overall, our results indicate that mosquito larvae without bacteria do not grow or molt because of defects in assimilating nutrients.