Influences of a Prolific Gut Fungus (Zancudomyces culisetae) on Larval and Adult Mosquito (Aedes aegypti)-Associated Microbiota

Abstract

Adult mosquitoes inherit a bacterial community from larvae via transstadial transmission, an understudied process that may influence host-microbe interactions. Microbes contribute to important host life history traits, and analyzing transmitted microbial communities, the interrelationship between larval and adult-associated microbiota, and factors influencing host-microbe relationships provides targets for research. During its larval stage, the yellow fever mosquito (Aedes aegypti) hosts the trichomycete gut fungus Zancudomyces culisetae, and fungal colonization coincides with environmental perturbations in the digestive tract microecosystem. Natural populations are differentially exposed to fungi, thereby potentially harboring distinct microbiota and experiencing disparate host-microbe interactions. This study's objectives were to characterize larval and initial adult microbiomes, investigate variation in diversity and distribution of microbial communities across individuals, and assess whether larval fungal colonization impacted microbiomes at these developmental stages. Laboratory-based fungal infestation assays, sequencing of 16S rRNA gene amplicons, and bacterial load quantification protocols revealed that initial adult microbiomes varied in diversity and distribution. Larval fungal colonization had downstream effects on initial adult microbiomes, significantly reducing microbial community variation, shifting relative abundances of certain bacterial families, and influencing transstadial transmission outcomes of particular genera. Further, abundances of several families consistently decreased in adults relative to levels in larvae, possibly reflecting impacts of host development on specific bacterial taxa. These findings demonstrated that a prolific gut fungus impacted mosquito-associated microbiota at two developmental stages in an insect connected with global human health.IMPORTANCE Mosquitoes are widespread vectors of numerous human pathogens and harbor microbiota known to affect host phenotypic traits. However, little research has directly investigated how bacterial communities associated with larvae and adults are connected. We characterized whole-body bacterial communities in mosquito larvae preceding pupation and in newly emerged adults, and investigated whether a significant biotic factor, fungal colonization of the larval hindgut, impacted these microbiomes. Results showed that fungal colonization reduced microbial community variation across individuals and differentially impacted the outcomes of transstadial transmission for certain bacterial genera, revealing downstream effects of the fungus on initial adult microbiomes. The importance of our research is in providing a thorough comparative analysis of whole-body microbiota harbored in larvae and adults of the yellow fever mosquito (Aedes aegypti) and in demonstrating the important role a widespread gut fungus played in a host-associated microbiome.

Keywords: Aedes aegypti; Zancudomyces culisetae; gut fungi; metagenomics; microbial ecology; microbiome; mosquitoes; mycology; transstadial transmission.

FIG 1

Zancudomyces culisetae colonization of a dissected fourth-instar larval mosquito digestive tract (magnified view of area boxed in inset image, bottom right) visualized with phase-contrast microscopy (×100 magnification). Significant regions are labeled as follows: i, midgut; ii, hindgut. The black arrow indicates mature fungal hyphae. The inset shows a larva with the head removed and midgut and hindgut exposed.

FIG 2

Box plots of microbiome alpha diversity measures across developmental stages and treatments. (a) Comparative analyses of Simpson diversity index values show a significant increase in variation of measures across developmental stages for nonfungal mosquitoes and treatments for newly emerged adults. (b) Comparative analyses of Shannon diversity index values show a significant increase in diversity across developmental stages for fungal mosquitoes, an increase in variation of measures across developmental stages for nonfungal mosquitoes, and treatments for newly emerged adults (n = 87). Upper and lower limits of boxes represent quartiles around the mean, and horizontal lines within boxes represent median values. Significant differences of CV values (CV ***, P < 0.001) were calculated with asymptotic and modified signed-likelihood ratio tests. Significant differences of mean alpha diversity measures were calculated with a linear mixed model (same letter, no significant difference; different letter and asterisk, P < 0.05).

FIG 3

Analyses of unweighted UniFrac distance for larval microbiomes across treatments. (a) Nonmetric multidimensional scaling plot based on unweighted UniFrac distance. (b) Box plots of within-group variation of unweighted UniFrac distance, represented by the distance of each sample’s value from the group’s centroid value, show that nonfungal larvae have higher within-group variation for the metric (n = 38). Upper and lower limits of boxes represent quartiles around the mean, and horizontal lines within boxes represent median values. Significant differences of within-group variation across treatments were calculated with a permutational statistical test for the homogeneity of group dispersions (*, P < 0.05).

FIG 4

Nonmetric multidimensional scaling plots of beta diversity measures across developmental stages for each treatment group. (a and b) Bray-Curtis dissimilarity and unweighted UniFrac distance for nonfungal mosquitoes (n = 36), respectively. (c and d) Bray-Curtis dissimilarity and unweighted UniFrac distance for fungal mosquitoes (n = 31), respectively.

FIG 5

Mean relative abundances of the 15 most abundant families shared across developmental stages of nonfungal (n = 36) (a) and fungal (n = 31) (b) mosquitoes.

FIG 6

Log₂(fold change) values for genera from families identified to significantly differ in mean relative abundances across developmental stages of nonfungal (blue) and fungal (red) mosquitoes. Only genera present at greater than 1% relative abundances in either developmental stage within each treatment group are shown. Positive values indicate an increase while negative values indicate a decrease in the abundance of each genus in adults relative to levels in larvae.

FIG 7

Relative abundances of genera from families identified to significantly change in mean relative abundances across developmental stages of nonfungal (blue) and fungal (red) mosquito larvae (a) and adults (b). Only genera present at greater than 1% relative abundances in either developmental stage within each treatment group are shown.

FIG 8

Bar plots of mean read counts calibrated using spike-in calibration to microbial load (SCML) across developmental stages for each treatment group. Comparative analyses show larger bacterial loads in larvae than in adults for both groups. Error bars indicate standard error (n = 42). Statistical differences were calculated with a linear mixed model (*, P < 0.05).