Microbiome reduction prevents lipid accumulation during early diapause in the northern house mosquito, Culex pipiens pipiens

Abstract

The mosquito microbiome is critical to multiple facets of their biology, including larval development and disease transmission. For mosquitoes that reside in temperate regions, periods of diapause are critical to overwintering survival, but how the microbiome impacts this state is unknown. In this study, we compared the midgut microbial communities of diapausing and non-diapausing Culex pipiens and assessed how a reduced midgut microbiome influences diapause preparation. High community variability was found within and between non-diapausing and diapausing individuals, but no specific diapause-based microbiome was noted. Emergence of adult, diapausing mosquitoes under sterile conditions generated low bacterial load (LBL) lines with nearly a 1000-fold reduction in bacteria levels. This reduction in bacterial content resulted in significantly lower survival of diapausing females after two weeks, indicating acquisition of the microbiome in adult females is critical for survival throughout diapause. LBL diapausing females had high carbohydrate levels, but did not accumulate lipid reserves, suggesting an inability to process ingested sugars necessary for diapause-associated lipid accumulation. Expression patterns of select genes associated with mosquito lipid metabolism during diapause showed no significant differences between LBL and control lines, suggesting transcriptional changes may not underlie impaired lipid accumulation. Overall, a diverse, adult-acquired microbiome is critical for diapause in C. pipiens to process sugar reserves and accumulate lipids that are necessary to survive prolonged overwintering.

Keywords: Culex pipiens; Dormancy; Lipid levels; Metabolism; Microbiome.

Figure 1.. Midgut family composition of diapausing and nondiapausing C. pipiens.

Relative abundances of bacterial families associated with each sample (N=10 per group). Midgut microbiome familial distribution for diapausing (D) and nondiapausing (ND) C. pipiens. Families within a single phylum are assigned shades of the same color. No significant differences in diversity or composition were identified (MRPP, D=0.258; NMS, R^2=0.415, 0.288, 0.201)

Figure 2.

Comparison of CFU counts and ovarian follicle lengths by treatment group. A. Colony forming units (CFU) (df=5, D=diapause, N=nondiapause). B. Primary ovarian follicle length of diapausing and ND LBL, reconstituted, and untreated mosquitoes (df=5 N=5–7, 3 technical replicates per sample, Table S1). All diapausing groups retained ovarian follicles under 0.08mm suggesting that all successfully entered diapause whereas all ND groups had primary ovarian follicles above 0.1mm suggesting that treatment does not impact diapause or ND status in C. pipiens.

Figure 3.. Survival Assessment.

Survival was assessed for groups of 75 untreated, LBL and reconstituted mosquitoes (df=2, N=7). Reconstituted mosquitoes displayed significantly lower survivorship than untreated mosquitoes (p=0.006) though significantly lower than LBL mosquitoes (p=0.001).

Figure 4.

Mass and nutritional reserves for untreated, LBL and reconstituted mosquitoes. A. Dry mass (g) of individual mosquitoes in each treatment, normalized to mean untreated weight for visualization, LBL mosquitoes weighed significantly less than their untreated (p=0.0004) and reconstituted (p=0.05) counterparts (df=2, N=144). B. Percent lipid in relation to total dry mass. LBL mosquitoes accumulated significantly less lipids than reconstituted (p=0.009) or untreated mosquitoes (p=0.008) (df=2, N=8). C. Glycogen in relation to dry mass. No significant differences were noted between the groups. D. Total carbohydrates in relation to dry mass. LBL mosquitoes had significantly more total carbohydrates present than mosquitoes from the untreated (p=0.02) or reconstituted groups (p=0.04) (df=2, N=8).

Figure 5.

Expression of genes important for nutrient mobilization via qPCR. A. Fatty acid synthase-1 (df=2, N=5, 3 technical replicates, f=0.525). B. Fatty acid binding protein (df=2, N=5, 3 technical replicates, f=0.865). C. Dihydrolipoamide S-acetyltransferase (df=2, N=5, 3 technical replicates, f=0.504). None of the genes investigated were significantly different between groups possibly due to variable success of LBL and reconstitution treatments.