Uncovering vector, parasite, blood meal and microbiome patterns from mixed-DNA specimens of the Chagas disease vector Triatoma dimidiata

Abstract

Chagas disease, considered a neglected disease by the World Health Organization, is caused by the protozoan parasite Trypanosoma cruzi, and transmitted by >140 triatomine species across the Americas. In Central America, the main vector is Triatoma dimidiata, an opportunistic blood meal feeder inhabiting both domestic and sylvatic ecotopes. Given the diversity of interacting biological agents involved in the epidemiology of Chagas disease, having simultaneous information on the dynamics of the parasite, vector, the gut microbiome of the vector, and the blood meal source would facilitate identifying key biotic factors associated with the risk of T. cruzi transmission. In this study, we developed a RADseq-based analysis pipeline to study mixed-species DNA extracted from T. dimidiata abdomens. To evaluate the efficacy of the method across spatial scales, we used a nested spatial sampling design that spanned from individual villages within Guatemala to major biogeographic regions of Central America. Information from each biotic source was distinguished with bioinformatics tools and used to evaluate the prevalence of T. cruzi infection and predominant Discrete Typing Units (DTUs) in the region, the population genetic structure of T. dimidiata, gut microbial diversity, and the blood meal history. An average of 3.25 million reads per specimen were obtained, with approximately 1% assigned to the parasite, 20% to the vector, 11% to bacteria, and 4% to putative blood meals. Using a total of 6,405 T. cruzi SNPs, we detected nine infected vectors harboring two distinct DTUs: TcI and a second unidentified strain, possibly TcIV. Vector specimens were sufficiently variable for population genomic analyses, with a total of 25,710 T. dimidiata SNPs across all samples that were sufficient to detect geographic genetic structure at both local and regional scales. We observed a diverse microbiotic community, with significantly higher bacterial species richness in infected T. dimidiata abdomens than those that were not infected. Unifrac analysis suggests a common assemblage of bacteria associated with infection, which co-occurs with the typical gut microbial community derived from the local environment. We identified vertebrate blood meals from five T. dimidiata abdomens, including chicken, dog, duck and human; however, additional detection methods would be necessary to confidently identify blood meal sources from most specimens. Overall, our study shows this method is effective for simultaneously generating genetic data on vectors and their associated parasites, along with ecological information on feeding patterns and microbial interactions that may be followed up with complementary approaches such as PCR-based parasite detection, 18S eukaryotic and 16S bacterial barcoding.

The Role of Bacterial Symbionts in Triatomines: An Evolutionary Perspective

“…Antagonistic interactions between trypanosomes and symbiotic bacteria have been demonstrated repeatedly. For instance, T. cruzi alters the triatomine microbiota richness and beta diversity [9,234,235,237,241], and the growth media in which T. rangeli is cultured inhibits bacterial growth [223].…”

Ontogeny, species identity, and environment dominate microbiome dynamics in wild populations of kissing bugs (Triatominae)

“…In triatomines, thirteen high throughput sequencing studies published since 2015 have indicated a wide range of factors that potentially influence their microbiomes. However, it remains difficult to derive any consistent cross-species patterns since the studies utilised a wide variety of DNA templates, including pooled or individual bodies, entire abdomens, the distal part of the abdomen, whole guts, midguts, faeces, and cultured bacterial colonies [40][41][42][43][44][45][46][47][48][49][50][51][52], and were often further complicated by other variables (e.g., sex, locality, instar, T. cruzi infection status). Apart from these methodological differences, the disparity among studies could also reflect true biological characteristics of Triatominae.…”

“…Apart from these methodological differences, the disparity among studies could also reflect true biological characteristics of Triatominae. These include lengthy development times through five nymphal instars [48][49][50][51][52], complex physiology of the alimentary tract [48], and accessory feeding strategies, like haemolymphagy (feeding on arthropod haemolymph), kleptohematophagy (stealing a blood meal from another triatomine), and conspecific coprophagy (feeding on faeces) known to be employed by some triatomine species [2,[53][54][55][56].…”

Rapid detection of human blood in triatomines (kissing bugs) utilizing a lateral flow immunochromatographic assay - A pilot study

“… 2 Several techniques have been developed using DNA- and proteomics-based methodologies in an attempt to determine the blood meal sources of triatomines. 3 , 4 , 5 , 6 , 7 These procedures are sensitive and specific for identifying a blood meal source, but are not readily adaptable to field use. They require access to a laboratory with sophisticated equipment and trained personnel, are labor intensive, and are vulnerable to contamination and degradation of the blood meal.…”

“…They require access to a laboratory with sophisticated equipment and trained personnel, are labor intensive, and are vulnerable to contamination and degradation of the blood meal. 3 , 4 , 5 , 6 , 7 The Rapid Stain Identification of Human Blood (RSID™ Blood) is a lateral flow, immunochromatographic assay detecting as little as 1 µL of human blood in forensic samples within 10 min following 1-2 h of specimen preparation. We conducted a preliminary study using this assay on triatomines and their excreta fed mouse or human blood and also numerous field specimens.…”