Bacterial Communities of Lab and Field Northern House Mosquitoes (Diptera: Culicidae) Throughout Diapause

Abstract

Diapause is a hormonally driven response which is triggered by environmental cues that signal impending adverse conditions and prompts metabolic, developmental, and behavioral changes to allow survival until the return of favorable conditions. Microbial symbionts have been shown to influence the metabolism, development, and behavior of their host organisms, all of which are common diapause-associated characteristics. Surveys of bacterial components in relation to diapause have been examined in few systems, of which the species are usually inactive during dormancy, such as eggs or pupae. This is specifically intriguing as adult female diapause in Culex pipiens (Diptera: Culicidae) can last between 4 and 7 mo and females remain mobile within their hibernacula. Furthermore, it is unknown how microbiota changes associated with prolonged dormancy are different between the lab and field for insect systems. This study aims to characterize how the microbiota of C. pipiens changes throughout diapause under both field and lab settings when provided identical food and water resources. Based on these studies, C. pipiens microbiota shifts as diapause progresses and there are considerable differences between field and lab individuals even when provided the same carbohydrate and water sources. Specific bacterial communities have more association with different periods of diapause, field and lab rearing conditions, and nutritional reserve levels. These studies highlight that diapausing mosquito microbiota studies ideally should occur in field mesocosms and at multiple locations, to increase applicability to wild C. pipiens as prolonged exposure to artificial rearing conditions could impact metrics related to diapause-microbiome interactions. Additionally, these findings suggest that it would be worthwhile to establish if the microbiota shift during diapause impacts host physiology and whether this shift is critical to diapause success.

Keywords: Culex pipiens; diapause; microbiome.

Fig. 1.

Whole body microbial composition at the phylum level of diapausing C. pipiens in lab and field conditions at different time points. Relative abundances of bacterial phyla associated with each sample (N = 3–6 per group). Midgut microbiota phyla distribution for field (F) and lab (L) samples at 2 wk (2wks), 2 mo (2M), 3 mo (3M), 4 mo (4M), and diapause break (DB). Data were assessed through two different pipeline/reference datasets: (A) SeqMatic-Greenegenes and (B) QIIME/SILVA. Both methods returned similar results at the phylum level (Supp Data S3 [online only]). See online version for color figure.

Fig. 2.

Midgut family composition of diapausing C. pipiens at the family level of diapausing C. pipiens in lab and field conditions at different time points. Relative abundances of bacterial families associated with each sample (N = 3–6 per group). Midgut microbiota familial distribution for field (F) and lab (L) samples at 2 wk (2wks), 2 mo (2M), 3 mo (3M), 4 mo (4M), and diapause break (DB). Families within a single phylum are assigned shades of the same color. Data were assessed through two different pipeline/reference datasets: (A) SeqMatic-Greenegenes and (B) QIIME/SILVA. Both methods returned relatively similar results at the phylum level (Supp Data S3 [online only]). See online version for color figure.

Fig. 3.

Alpha diversity metrics of diapausing C. pipiens in lab and field conditions at different time points. (A) Chao diversity and (B) Shannon diversity. Despite a general downward trend as diapause progressed, no significant differences were seen between groups (see Supp Data S1 [online only] for statistics). See online version for color figure.

Fig. 4.

Comparison of bacterial communities between field and lab mosquitoes and across diapause. Nonmetric multidimensional scaling analysis (NMDS) of proportional OTU abundance data using the Bray–Curtis dissimilarity matrix. The symbols indicate field and lab and coloration is based on duration within diapause. Large symbols represent mean of all samples. Small symbols represent each individual sample. See online version for color figure.

Fig. 5.

WGCNA reveals OTUs associated with diapausing C. pipiens in lab and field conditions at different time points and nutritional reserve levels. (A) OTU dendrogram of co-expressed OTU modules. (B) Heat map depicts the OTU modules positively (red) or negatively (blue) correlated with specific treatments. (C) Summary of group type, taxa, and highly expressed tax associated with each module color. The associated taxa listed were the three most abundant families associated with each module while highly expressed is taxa information for OTUs that had an average read total over 1000. Not all OTUs within a specific taxon were similarly expressed. See Supp Data S4 (online only) for all OTU-module associations. * denotes significance at 0.05, ** denotes significance at 0.01, and *** denotes significance at 0.001. See online version for color figure.