Microbiomes of Blood-Feeding Triatomines in the Context of Their Predatory Relatives and the Environment

Abstract

The importance of gut microbiomes has become generally recognized in vector biology. This study addresses microbiome signatures in North American Triatoma species of public health significance (vectors of Trypanosoma cruzi) linked to their blood-feeding strategy and the natural habitat. To place the Triatoma-associated microbiomes within a complex evolutionary and ecological context, we sampled sympatric Triatoma populations, related predatory reduviids, unrelated ticks, and environmental material from vertebrate nests where these arthropods reside. Along with five Triatoma species, we have characterized microbiomes of five reduviids (Stenolemoides arizonensis, Ploiaria hirticornis, Zelus longipes, and two Reduvius species), a single soft tick species, Ornithodoros turicata, and environmental microbiomes from selected sites in Arizona, Texas, Florida, and Georgia. The microbiomes of predatory reduviids lack a shared core microbiota. As in triatomines, microbiome dissimilarities among species correlate with dominance of a single bacterial taxon. These include Rickettsia, Lactobacillus, "Candidatus Midichloria," and Zymobacter, which are often accompanied by known symbiotic genera, i.e., Wolbachia, "Candidatus Lariskella," Asaia, Gilliamella, and Burkholderia. We have further identified a compositional convergence of the analyzed microbiomes in regard to the host phylogenetic distance in both blood-feeding and predatory reduviids. While the microbiomes of the two reduviid species from the Emesinae family reflect their close relationship, the microbiomes of all Triatoma species repeatedly form a distinct monophyletic cluster highlighting their phylosymbiosis. Furthermore, based on environmental microbiome profiles and blood meal analysis, we propose three epidemiologically relevant and mutually interrelated bacterial sources for Triatoma microbiomes, i.e., host abiotic environment, host skin microbiome, and pathogens circulating in host blood. IMPORTANCE This study places microbiomes of blood-feeding North American Triatoma vectors (Reduviidae) into a broader evolutionary and ecological context provided by related predatory assassin bugs (Reduviidae), another unrelated vector species (soft tick Ornithodoros turicata), and the environment these arthropods coinhabit. For both vectors, microbiome analyses suggest three interrelated sources of bacteria, i.e., the microbiome of vertebrate nests as their natural habitat, the vertebrate skin microbiome, and the pathobiome circulating in vertebrate blood. Despite an apparent influx of environment-associated bacteria into the arthropod microbiomes, Triatoma microbiomes retain their specificity, forming a distinct cluster that significantly differs from both predatory relatives and ecologically comparable ticks. Similarly, within the related predatory Reduviidae, we found the host phylogenetic distance to underlie microbiome similarities.

Keywords: Ornithodoros; Reduviidae; Triatoma; environment; microbiome.

FIG 1

UpSet graph depicting microbiome intersections among different Triatoma species. The top bar chart stands for the cumulative number of OTUs identified for each Triatoma species. The colored plot indicates how much each intersection deviates from the expected size if species membership within the analyzed data set is random. The bacterial genera shared, in 0.5 fraction, by four or more species are listed.

FIG 2

Microbiome profiles of hemolymphophagous assassin bugs. (A) Heatmap showing the distribution of the 30 most abundant bacterial taxa identified across the assassin species. (B) Shared proportion of the microbiome components among the analyzed assassins. The numbers in the parentheses stand for the absolute number of OTUs. The shared proportion of the microbiome that accounts for less than 1% is depicted only as the absolute count (C). NMDS clustering based on Bray-Curtis distances. The number in the right upper corner indicates the stress value. Two Reduvius species were merged under a single group, Reduvius sp.

FIG 3

Ornithodoros turicata microbiome profile. (A) Heatmap for the 30 most abundant genera. ND stands for the sum of the unclassified taxa at the genus level. (B) Principal-coordinate analysis based on Bray-Curtis distances. Colored shapes stand for different localities: DS, Desert Station, Tucson, AZ; Chaparral, Chaparral Wildlife Management Area, TX; LacklandAFB, Lackland Air Force Base, San Antonio, TX. (C) Bray-Curtis microbiome dissimilarities calculated among the three localities. Asterisks indicate significant differences at 95%* and 99%** confidence interval found between the diversity measures (Dunn’s Kruskal-Wallis multiple comparisons, Table S1, data sheet S17).

FIG 4

Phylogenetic inference on 16S rRNA gene sequences of Midichloriaceae OTUs associated with assassin bugs and ticks. Numbers at the nodes are bootstrap values.

FIG 5

Nest microbiome characteristics. (A) Heatmap for the 30 most abundant genera across the sampled nests. ND stands for the sum of the unclassified taxa at the genus level. (B) Venn analysis at 0.5 fraction among all sample types, i.e., ticks, kissing and assassin bugs, and nest material. The numbers in the parentheses stand for the absolute number of OTUs. The taxa in boldface were repeatedly recognized as shared microbiome members under different fractions (Table S1, data sheets S22 and S10) and thus considered of nest environmental origin. CB stands for Camp Bullis sampling site (data sheet S1).

FIG 6

Scheme for putative sources of vector microbiome based on blood meal analysis and nest microbiome profiling. Percentages next to each vector stand for successfully identified primary blood meal from which 88% matched Neotoma sp. The four taxa (Cutibacterium, Streptomyces, Pseudonocardia, and Massilia) might originate from both the host skin and the nest microhabitat.

FIG 7

(A) Schematic host phylogenetic relationships compared to the microbiome dissimilarities based on weighted UniFrac distances. (B) The individual microbiome distances are visualized using NMDS.