Differential transcriptome analysis supports Rhodnius montenegrensis and Rhodnius robustus (Hemiptera, Reduviidae, Triatominae) as distinct species

Abstract

Chagas disease is one of the main parasitic diseases found in Latin America and it is estimated that between six and seven million people are infected worldwide. Its etiologic agent, the protozoan Trypanosoma cruzi, is transmitted by triatomines, some of which from the genus Rhodnius. Twenty species are currently recognized in this genus, including some closely related species with low levels of morphological differentiation, such as Rhodnius montenegrensis and Rhodnius robustus. In order to investigate genetic differences between these two species, we generated large-scale RNA-sequencing data (consisting of four RNA-seq libraries) from the heads and salivary glands of males of R. montenegrensis and R. robustus. Transcriptome assemblies produced for each species resulted in 64,952 contigs for R. montenegrensis and 70,894 contigs for R. robustus, with N50 of approximately 2,100 for both species. SNP calling based on the more complete R. robustus assembly revealed 3,055 fixed interspecific differences and 216 transcripts with high levels of divergence which contained only fixed differences between the two species. A gene ontology enrichment analysis revealed that these highly differentiated transcripts were enriched for eight GO terms related to AP-2 adaptor complex, as well as other interesting genes that could be involved in their differentiation. The results show that R. montenegrensis and R. robustus have a substantial quantity of fixed interspecific polymorphisms, which suggests a high degree of genetic divergence between the two species and likely corroborates the species status of R. montenegrensis.

Transcriptome-based molecular systematics: Rhodnius montenegrensis (Triatominae) and its position within the Rhodnius prolixus–Rhodnius robustus cryptic–species complex

“…query sequences. The graphs present, for each of three loci (mitochondrial cytb plus nuclear rDNA ITS2, and D2- 28S ), breath-coverage (% of positions; bold vertical lines; left vertical axis) and mean depth-coverage (reads/position; bars; right vertical axis) for queries against two R. montenegrensis transcriptomes (SRX1996481 and SRX1996482; [7, 12, 13]). Red lines/bars highlight queries yielding full-breadth-coverage; queries yielding only partial-breadth-coverage are black/grey.…”

“…We used selected DNA sequences from members of the R. prolixus – R. robustus cryptic–species complex to query publicly-available transcriptome sequence read archives (SRAs) derived from UNESP colonies, namely the R. montenegrensis colony and the Peruvian ‘ R. robustus ’ colonies used as the taxonomic benchmark in the description of R. montenegrensis [7] and in later transcriptome-based comparisons [12, 13] (Table 1). We chose three loci that have been widely used to study the systematics and evolution of Rhodnius spp.…”

“…Figure 1 shows an outline of our methodological approach. We downloaded six public-domain transcriptome SRAs determined by two independent groups at UNESP and the French Centre National de la Recherche Scientifique (CNRS), respectively (see Table 1 and [12, 13, 21]). We aligned transcriptome paired-end reads to our query sequences using the Burrows-Wheeler aligner [22] with the BWA-MEM algorithm and default parameter values except for − t = 10, − B = 5, − O = 7.7, − L = 6.6, and − U = 18.…”

“…The authors concluded that, although closely related, R. montenegrensis and R. robustus are morphologically and genetically distinct [7]. These findings received further support from a comparative-transcriptomics study showing that bugs identified as R. montenegrensis and bugs from UNESP’s R. robustus colony ‘CTA 85’, had “… a substantial quantity of fixed interspecific polymorphisms …”; this was interpreted as “… suggest[ing] a high degree of genetic divergence between the two species [that] likely corroborates the species status of R. montenegrensis ” ([12], Abstract; see also [13] for details).…”

“…The striking similarity of R. montenegrensis and R. robustus cytb sequences was recently confirmed by a broader analysis [6] showing that R. montenegrensis ’ KR072682.1 is nearly identical to cytb sequences from R. robustus genotype II, one of the R. robustus cryptic taxa identified by Monteiro et al [5] in the early 2000s. While these results clearly suggest that R. montenegrensis is “… part of the variability of R. robustus II …” (caption of figure 2A in [6]), they raise the question of why morphology [7] and transcriptomics [12, 13] both discriminate R. montenegrensis from R. robustus bugs of Peruvian origin. This is even more intriguing when one considers that R. robustus II is the only R. robustus lineage known to occur in western-southwestern Amazonia [5, 6, 14–16]; R. robustus material from Peru, then, is expected to belong in genotype II and, hence, to be indistinguishable from R. montenegrensis .…”