A nuclear single-nucleotide polymorphism (SNP) potentially useful for the separation of Rhodnius prolixus from members of the Rhodnius robustus cryptic species complex (Hemiptera: Reduviidae)

Abstract

The design and application of rational strategies that rely on accurate species identification are pivotal for effective vector control. When morphological identification of the target vector species is impractical, the use of molecular markers is required. Here we describe a non-coding, single-copy nuclear DNA fragment that contains a single-nucleotide polymorphism (SNP) with the potential to distinguish the important domestic Chagas disease vector, Rhodnius prolixus, from members of the four sylvatic Rhodnius robustus cryptic species complex. A total of 96 primer pairs obtained from whole genome shotgun sequencing of the R. prolixus genome (12,626 random reads) were tested on 43 R. prolixus and R. robustus s.l. samples. One of the seven amplicons selected (AmpG) presented a SNP, potentially diagnostic for R. prolixus, on the 280th site. The diagnostic nature of this SNP was then confirmed based on the analysis of 154 R. prolixus and R. robustus s.l. samples representing the widest possible geographic coverage. The results of a 60% majority-rule Bayesian consensus tree and a median-joining network constructed based on the genetic variability observed reveal the paraphyletic nature of the R. robustus species complex, with respect to R. prolixus. The AmpG region is located in the fourth intron of the Transmembrane protein 165 gene, which seems to be in the R. prolixus X chromosome. Other possible chromosomal locations of the AmpG region in the R. prolixus genome are also presented and discussed.

Figure 1. Geographical distribution of sampled specimens of R. prolixus and R. robustus s.l.

Each location is numbered according to Table 1. The colors indicate the range of R. prolixus and the four members of the R. robustus cryptic species complex (R. robustus I-IV).

Figure 2. Phylogenetic relationship between Rhodnius prolixus and R. robustus I-IV haplotypes

Species are indicated by different colors. (A) Non-rooted 60% majority-rule Bayesian consensus tree obtained from all 154 AmpG sequences of R. prolixus and R. robustus. Posterior probability values higher than 0.9 are indicated near the nodes. Shared genotypes between different R. robustus species is represented by more than one circle in a single terminal branch. Note that R. robustus I specimens (red circles) are more closely related to R. prolixus (blue circles) than to the other R. robustus species, evidencing its paraphyletic assemblage. (B) Median-Joining network with Maximum-Parsimony post-processing. Each node (or circle) represents a unique haplotype. The size of each node is proportional to the number of specimens that shared the same haplotype, and the branch size is proportional to the number of mutational steps. Note that the distance (measured by branch sizes) between R. prolixus and R. robustus I is smaller than between R. robustus I and R. robustus II-IV, confirming their sister-species status. The R. robustus paraphyly is shown by dot and dashed circles, which highlight the R. prolixus + R. robustus I group, and the Amazonian R. robustus (II-IV), respectively.

Figure 3. AmpG neighborhood

Rhodnius prolixus contig GL563069 (region from 1110523-1123517) that contains Transmembrane protein 165 (TP165). Exons were shown as gray rectangles with black outlines linked together. AmpG region (1116877-1117340) is inside the fourth gene intron. Outcast_TE and RT_TE are in the fifth intron and represent an outcast class I transposable element and its reverse transcriptase (A). Rhodnius prolixus contig GL563069 (region from 925000 to 1319500) contains 14 genes and four transposons. AmpG region is shown as a black vertical dashed line. Genes were represented as gray arrows, and EST-supported genes as gray arrows with black outlines. Gene 8 had fully manual curation and EST support, and was represented as a gray outlined arrow. (1- ephrin; 2- ribonuclease; 3- src tyrosine kinase; 4- odorant receptor; 5- sarcoplasmic Ca-binding protein; 6- small Glu-rich tetratricopeptide repeat-containing protein alpha; 7- X11Lbeta; 8- transmembrane protein 165; 9- interleukin 16; 10- WNT4 protein; 11- UNC112 related protein; 12- NADH dehydrogenase; 13- KN motif and ankyrin repeat domain-containing protein; 14- band 4.1-like protein 4). Transposable elements were represented as black arrows (a- hAT – class II; b- outcast – class I; c- mariner – class II; d- L1 – class I) (B). Homologous genes were identified at the protein level using reciprocal best hit strategy (Tatusov et al., 1997). Numbers mean chromosome number. X= chromosome; X. nf= homologs not found; nm= homologs not mapped; *= not homologous, only a similar gene (C).