Limited influence of the microbiome on the transcriptional profile of female Aedes aegypti mosquitoes

Abstract

The microbiome is an assemblage of microorganisms living in association with a multicellular host. Numerous studies have identified a role for the microbiome in host physiology, development, immunity, and behaviour. The generation of axenic (germ-free) and gnotobiotic model systems has been vital to dissecting the role of the microbiome in host biology. We have previously reported the generation of axenic Aedes aegypti mosquitoes, the primary vector of several human pathogenic viruses, including dengue virus and Zika virus. In order to better understand the influence of the microbiome on mosquitoes, we examined the transcriptomes of axenic and conventionally reared Ae. aegypti before and after a blood meal. Our results suggest that the microbiome has a much lower effect on the mosquito's gene expression than previously thought with only 170 genes influenced by the axenic state, while in contrast, blood meal status influenced 809 genes. The pattern of expression influenced by the microbiome is consistent with transient changes similar to infection rather than sweeping physiological changes. While the microbiome does seem to affect some pathways such as immune function and metabolism, our data suggest the microbiome is primarily serving a nutritional role in development with only minor effects in the adult.

Figure 1

Canonical Analysis of Principal Coordinates (CAP) analysis of transcript abundance by mosquito pool. Constrained Analysis of Principal Coordinates (CAP) ordination for the transcriptomes of the mosquito pools sequenced, based on Jaccard dissimilarity index. CAP axes 1 and 2 explain 65.9 and 15.3% of the variance, respectively. Explanatory variables are indicated by arrows, while diet is indicated by the colours, tissue type by the shape and treatment by the internal dot colour.

Figure 2

MA plots of axenic versus conventionally reared mosquito genes. The beta value in Sleuth indicates the estimated fold change for each gene. Each dot is representative of one gene; grey dots represent no significant differences between axenic and conventionally raised groups. Red dots represent genes that were either enriched (above the x-axis) or depleted (below the x-axis) and were considered biologically significant in abundance. The four treatment groups investigated were midguts blood-fed (MBF) (a), midguts sugar-fed (MSF) (b), carcasses blood-fed (CBF) (c) and carcasses sugar-fed (CSF) (d).

Figure 3

Upset plot of biologically significant genes expressed in the four treatment groups investigated. The plot shows the number of shared genes between the four treatment groups. The set size is the total number of biologically significant genes identified. The interaction size shows how many genes are found either only in one treatment group or in multiple treatment groups. The four treatment groups investigated were midguts blood-fed (MBF), midguts sugar-fed (MSF), carcasses blood-fed (CBF), and carcasses sugar-fed (CSF).

Figure 4

Hierarchical classification of the biologically significant GO terms identified. The treemap shows the comparative abundance of Level 2 GO terms by treatment type, midguts blood-fed (MBF) (a), midguts sugar-fed (MSF) (b), carcasses blood-fed (CBF) (c) and carcasses sugar-fed (CSF) (d).

Figure 5

The relative expression of the six genes used to validate transcriptome results. Stars indicate statistical significance; *** < 0.0001 and * < 0.01. Gene codes used are all accepted standard abbreviations and are found in Table 2.

Figure 6

MA plots of significantly expressed mosquito genes during a blood or sugar meal. The beta value in Sleuth indicates the estimated fold change for each gene. Each dot is representative of one gene; grey dots represent no significant differences between axenic and conventionally raised groups. Red dots represent genes that were either enriched (above the x-axis) or depleted (below the x-axis) and were considered biologically significant. The two groups investigated were midguts conventionally raised (a), and midguts axenically raised (b).