Comprehensive characterization of internal and cuticle surface microbiota of laboratory-reared F1 Anopheles albimanus originating from different sites

Abstract

Background: Research on mosquito-microbe interactions may lead to new tools for mosquito and mosquito-borne disease control. To date, such research has largely utilized laboratory-reared mosquitoes that typically lack the microbial diversity of wild populations. A logical progression in this area involves working under controlled settings using field-collected mosquitoes or, in most cases, their progeny. Thus, an understanding of how laboratory colonization affects the assemblage of mosquito microbiota would aid in advancing mosquito microbiome studies and their applications beyond laboratory settings.

Methods: Using high throughput 16S rRNA amplicon sequencing, the internal and cuticle surface microbiota of F₁ progeny of wild-caught adult Anopheles albimanus from four locations in Guatemala were characterized. A total of 132 late instar larvae and 135 2-5 day-old, non-blood-fed virgin adult females that were reared under identical laboratory conditions, were pooled (3 individuals/pool) and analysed.

Results: Results showed location-associated heterogeneity in both F₁ larval internal (p = 0.001; pseudo-F = 9.53) and cuticle surface (p = 0.001; pseudo-F = 8.51) microbiota, and only F₁ adult cuticle surface (p = 0.001; pseudo-F = 4.5) microbiota, with a more homogenous adult internal microbiota (p = 0.12; pseudo-F = 1.6) across collection sites. Overall, ASVs assigned to Leucobacter, Thorsellia, Chryseobacterium and uncharacterized Enterobacteriaceae, dominated F₁ larval internal microbiota, while Acidovorax, Paucibacter, and uncharacterized Comamonadaceae, dominated the larval cuticle surface. F₁ adults comprised a less diverse microbiota compared to larvae, with ASVs assigned to the genus Asaia dominating both internal and cuticle surface microbiota, and constituting at least 70% of taxa in each microbial niche.

Conclusions: These results suggest that location-specific heterogeneity in filed mosquito microbiota can be transferred to F₁ progeny under normal laboratory conditions, but this may not last beyond the F₁ larval stage without adjustments to maintain field-derived microbiota. These findings provide the first comprehensive characterization of laboratory-colonized F₁ An. albimanus progeny from field-derived mothers. This provides a background for studying how parentage and environmental conditions differentially or concomitantly affect mosquito microbiome composition, and how this can be exploited in advancing mosquito microbiome studies and their applications beyond laboratory settings.

Keywords: 16S rRNA gene amplicon sequencing; Anopheles albimanus; Laboratory colonization; Mosquito microbiome; Mosquito microbiota; Next generation sequencing.

Fig. 1

Map showing the maternal origins of F₁ Anopheles albimanus. F₁ laboratory-colonized mosquitoes were derived from gravid and/or blood fed females collected from each location. Mosquito icons show the maternal origin of the F₁ life stages studied

Fig. 2

Principal coordinate analysis (PCoA) ordinations of internal and cuticle surface microbiota from F₁ laboratory-colonized An. albimanus. The PCoA plots are based on Bray–Curtis distances between the microbiota of mosquitoes with differing collection sites. Each point on the plot represents the microbial composition of a pool of three individuals, and mosquito pools are color-coded by their origin. For larvae, the first two principal component (PC) axes captured 50% (internal) and 48% (cuticle surface) of the variance in the data, with both internal and cuticle surface microbiota clustering distinctly by collection site. For adults, the first two PC axes captured 59% (internal) and 47% (cuticle surface) of the variance in the data, with cuticle surface but not internal microbiota clustering distinctly by collection site. PERMANOVA statistics are presented at the bottom of each plot

Fig. 3

Number of unique and shared microbial taxa between microbial niches (A) and number of unique and shared microbial taxa between collection sites (B). The number of taxa shown in the Venn diagram represent bacterial taxa except in the cuticle surface microbiota of adults from Las Cruces 4, where two archaeal taxa were present. n = pools of mosquito samples analyzed per location or microbial niche (3 individuals/pool)

Fig. 4

Frequency of ASVs from the internal (a) and cuticle surface (b) microbiota of laboratory-colonized An. albimanus F₁ larvae originating from different collection sites. ASVs were annotated to the genus level or the lowest possible taxonomic level (in square brackets) and are clustered by the average nearest-neighbors chain algorithm. Only taxonomically annotated ASVs with frequencies ≥ 2000 are presented. n = pools of mosquito samples analyzed per location (3 individuals/pool)

Fig. 4

Frequency of ASVs from the internal (a) and cuticle surface (b) microbiota of laboratory-colonized An. albimanus F₁ larvae originating from different collection sites. ASVs were annotated to the genus level or the lowest possible taxonomic level (in square brackets) and are clustered by the average nearest-neighbors chain algorithm. Only taxonomically annotated ASVs with frequencies ≥ 2000 are presented. n = pools of mosquito samples analyzed per location (3 individuals/pool)

Fig. 5

Volcano plots of differentially abundant bacterial taxa in F₁ laboratory colonized An. albimanus larvae originating from three different collection sites. The plots show results of analysis of composition of microbiomes (ANCOM) tests for differentially abundant microbial taxa between collection site, with an effect size set to log F ≥ 20, and a cut-off of differential abundance set to W ≥ 20 (i.e., a taxon was differentially abundant across collection sites if the ratio of its abundance to those of at least 20 other taxa—25% of all included taxa—differed significantly across sites). Differentially abundant taxa are highlighted (blue shaded area), and the taxa names and locations in which they were most abundant are presented in the adjoining tables

Fig. 6

Frequency of ASVs from the internal and cuticle surface microbiota of laboratory-colonized F₁ adult An. albimanus originating from different locations. ASVs were annotated to the genus level or the lowest possible taxonomic level (in square brackets) and are clustered by the average nearest-neighbors chain algorithm. Only taxonomically annotated ASVs with frequencies ≥ 1000 are presented. n = pools of mosquito samples analyzed per location (3 individuals/pool)