Analysis of multiple tsetse fly populations in Uganda reveals limited diversity and species-specific gut microbiota

Abstract

The invertebrate microbiome contributes to multiple aspects of host physiology, including nutrient supplementation and immune maturation processes. We identified and compared gut microbial abundance and diversity in natural tsetse flies from Uganda using five genetically distinct populations of Glossina fuscipes fuscipes and multiple tsetse species (Glossina morsitans morsitans, G. f. fuscipes, and Glossina pallidipes) that occur in sympatry in one location. We used multiple approaches, including deep sequencing of the V4 hypervariable region of the 16S rRNA gene, 16S rRNA gene clone libraries, and bacterium-specific quantitative PCR (qPCR), to investigate the levels and patterns of gut microbial diversity from a total of 151 individuals. Our results show extremely limited diversity in field flies of different tsetse species. The obligate endosymbiont Wigglesworthia dominated all samples (>99%), but we also observed wide prevalence of low-density Sodalis (tsetse's commensal endosymbiont) infections (<0.05%). There were also several individuals (22%) with high Sodalis density, which also carried coinfections with Serratia. Albeit in low density, we noted differences in microbiota composition among the genetically distinct G. f. fuscipes flies and between different sympatric species. Interestingly, Wigglesworthia density varied in different species (10(4) to 10(6) normalized genomes), with G. f. fuscipes having the highest levels. We describe the factors that may be responsible for the reduced diversity of tsetse's gut microbiota compared to those of other insects. Additionally, we discuss the implications of Wigglesworthia and Sodalis density variations as they relate to trypanosome transmission dynamics and vector competence variations associated with different tsetse species.

FIG 1

Map of Uganda showing the locations of the tsetse sampling sites analyzed in this study. The position of Uganda on the African continent is also shown. The dotted line demarcates the separation of Trypanosoma brucei gambiense areas of endemicity in the northwest from the Trypanosoma brucei rhodesiense areas of endemicity in the southeast of Uganda. The area shaded in dark gray represents the range of Glossina fuscipes fuscipes. Light gray areas identify major bodies of water. Sampling site names are reported, and the number of tsetse flies from each site is shown. For Murchison Falls, the number of individuals assayed included samples from three different Glossina species (Glossina morsitans morsitans, n = 10; Glossina pallidipes, n = 53; G. f. fuscipes, n = 11). For all other sites, only G. f. fuscipes individuals were sampled. Maps were downloaded from “Uganda” article in South African History Online (www.sahistory.org.za).

FIG 2

16S rRNA analysis of G. f. fuscipes flies from different populations in Uganda. (A) Microbiota taxon summary for G. f. fuscipes flies from four locations. Individual taxonomy summaries were averaged for each location to represent the core microbiome for that particular sampling site. (B) We performed jackknifed principal coordinate analysis (PCoA) using the Bray-Curtis dissimilarity distance metric to uncover bacterial composition differences between locations. The Bray-Curtis dissimilarity metric is based on shared OTU counts between samples. Only principal coordinate 1 (PC1) versus PC2 is shown here; the rest of the plots, as well as uniweighted Unifrac plots, can be seen in Fig. S2 in the supplemental material. (C) Rarefaction curves for the four distinct locations and the bacteriome-removed individuals from Busime. We used the metric “Observed Species” to determine species richness in each location and Monte Carlo permutations to calculate the P value (Bonferroni corrected) to determine whether species richness differences existed between each location. Data can be seen in Table S3 in the supplemental material.

FIG 3

Quantitative PCR and 16S rRNA analysis from Murchison Falls. (A) Symbiont genome copy numbers for Wigglesworthia and Sodalis normalized to tsetse host tubulin gene based on quantitative real-time PCR analysis of individuals from Murchison Falls. Both Wigglesworthia and Sodalis densities differed in the individuals, with G. f. fuscipes flies having the highest densities of Wigglesworthia. An asterisk by a sample designation indicates the fly was PCR positive for trypanosomes (using the ITS-1 primers). Seven (2 G. m. morsitans and 5 G. pallidipes) of the total of 73 samples from Murchison Falls tested positive for the presence of trypanosomes. Values below 1.00E+02 were extrapolated from the standard curve. In sample designations, species are indicated as follows: Gff, Glossina fuscipes fuscipes; Gmm, Glossina morsitans morsitans; Gpd, Glossina pallidipes. (B) Symbiont genome copy numbers for Wigglesworthia and Sodalis normalized to tsetse host tubulin gene based on quantitative real-time PCR analysis are shown for seven whole-gut samples and for five bacteriome-removed samples. (C) 16S rRNA deep-sequencing results corresponding to the same seven whole-gut samples and five bacteriome-removed samples for which results are shown in panel B. The number inside each box corresponds to the relative abundance of the taxon in that individual. Three of the 12 G. pallidipes flies with a high density of Sodalis were found to be positive for trypanosomes; these 3 samples are indicated by asterisks.

FIG 4

Relative abundances of microbial taxa in tsetse guts and corresponding carcass tissues. Four female flies had their guts and corresponding carcasses analyzed by 16S rRNA deep-sequencing method based on quantitative real-time PCR analysis for the symbiont Sodalis. The number inside each box corresponds to the relative abundance of the taxon in that individual. An asterisk by a sample designation indicates the fly was PCR positive for trypanosomes (using the ITS-1 primers).