Triatoma vitticeps (Stal, 1859) (Hemiptera, Triatominae): A Chagas Disease Vector or a Complex of Vectors?

Abstract.

Triatoma vitticeps is a Chagas disease vector that was found infected with Trypanosoma cruzi in homes. As this species is endemic from Brazil (Bahia, Espírito Santo, Minas Gerais, and Rio de Janeiro) and no study comparing the specimens from different Brazilian states was conducted, we analyzed the genetic distance (16S rDNA, Cyt b, and COI mitochondrial genes) and the chromosomal characteristics for T. vitticeps from Minas Gerais, Rio de Janeiro, and Espírito Santo. All specimens showed the same cytogenetic characteristics. On the other hand, the different mitochondrial genes demonstrated high intraspecific variation between the genetic distances of T. vitticeps from different states ranging from 2.3% to 7.2%. Based on this, our results suggest that possibly what is characterized as T. vitticeps is a complex of cryptic species (or subspecies).

The Chagas disease is caused by the protozoan Trypanosoma cruzi (Chagas, 1909) and transmitted to humans mainly through contact with feces of insects from the Triatominae subfamily. This disease is among the main neglected diseases in Latin America and is estimated that approximately 6–7 million people are infected worldwide; in Brazil there are about 2–3 million people who are infected with T. cruzi.^1,2

The vector control is the main way to minimize the incidence of new cases of Chagas disease. In Brazil, after significant reduction of vectors, the number of cases with the acute form of Chagas disease has been reduced dramatically.^3,4 Characterizing the different species of the triatomines and the structure of the populations of these vectors can assist in the activities of vector control programs because there are species with greater or lesser degrees of importance in the transmission of T. cruzi.^4,5

Triatoma vitticeps (Stål, 1859) is an endemic species from Brazil, distributed in the states of Bahia, Espírito Santo, Minas Gerais, and Rio de Janeiro.⁵ This triatomine was initially characterized as a sylvatic species,⁶ but adults of T. vitticeps often invade houses attracted by light and form small colonies in domiciliary regions.⁷ In addition, worrying rates of domiciliary capture occurred in Rio de Janeiro^8,9 and high rates of T. cruzi infection were observed in T. vitticeps captured in homes of Espírito Santo,⁷ although it has been shown that this species does not have the habit of defecating during hematophagy.¹⁰

Souza et al.¹¹ analyzed the population dynamics of T. vitticeps in the state of Minas Gerais, by means of morphometry, isoenzymes, and random amplification of polymorphic DNA. The authors observed migration between domestic, peridomiciliary, and sylvatic habitats and gene flow between the sylvatic and domestic habitats. However, as no study comparing the specimens from different Brazilian states was conducted, we analyzed the genetic (mitochondrial markers—16S rDNA, Cyt b, and COI genes) and cytogenetic aspects (karyotype and heterochromatin) of T. vitticeps from Minas Gerais, Rio de Janeiro, and Espírito Santo.

Ten adult males of each Brazilian state were used for cytogenetic analysis. The specimens were provided by the insectariums of FCFAR/UNESP, Araraquara, São Paulo, Brazil, and of the IOC/FIOCRUZ, Rio de Janeiro, Brazil. The seminiferous tubules were torn apart, crushed, and fixed on slides in liquid nitrogen. The cytogenetic technique of C-banding¹² was then applied for the characterization of chromosome number and heterochromatin pattern and the analysis was performed using a Jenaval light microscope (Zeiss) attached to a digital camera and an Axio Vision LE 4.8 image analyzer (Copyright 2006–2009 Carl Zeiss Imaging Solutions Gmb H). For the analysis of the genetic distance between T. vitticeps coming from different states of the southeastern region of Brazil were used sequences deposited in the GenBank (Table 1), and the Tamura 3-parameter model was used to calculate genetic distances pairwise in the MEGA 6.0 software.¹³ The specimens used in cytogenetic studies were from the same populations of the material used for the molecular analysis.^14–16

Table 1.

Sequences of Triatoma vitticeps coming from different Brazilian states deposited in the GenBank

T. vitticeps	COI	Cyt b	16S
Espírito Santo	AF021219	KF826896	EU827202
Rio de Janeiro	KC249397	KC249304	KC249088
Minas Gerais	KC249395	KC249301	KC249085

All specimens analyzed showed the same cytogenetic characteristics: karyotype 2n = 24 (20A + X₁X₂X₃Y) and heterochromatin restricted to only one of the X sex chromosomes and in the Y sex chromosome (Figure 1). These results demonstrate that T. vitticeps does not present intraspecific chromosome variation as already observed for other triatomines.¹⁷

Figure 1.

Metaphase I of Triatoma vitticeps stained by C-banding. Note the karyotype 2n = 24 (20A + X₁X₂X₃Y) and heterochromatin restricted to only one of the X sex chromosomes and in the Y sex chromosome. X = X sex chromosome; Y = Y sex chromosome. Bar: 10 μm. This figure appears in color at www.ajtmh.org.

On the other hand, the different mitochondrial genes analyzed demonstrated high intraspecific variation between the genetic distances of T. vitticeps from different states ranging from 2.3% to 7.5% (Tables 2–4). Pereira et al.¹⁸ consider that taxa with a genetic distance greater than 2% represent new species that are possibly cryptic. For Triatominae, a genetic distance of 2.48%, associated with other tools, already supports specific status (as recently observed for Triatoma bahiensis Sherlock & Serafim, 1967).¹⁹

Table 2.

Genetic distances for different populations of Triatoma vitticeps with the mitochondrial genes: COI

T. vitticeps	(1)	(2)	(3)
(1) Espírito Santo	0.000	–	–
(2) Rio de Janeiro	0.068	0.000	–
(3) Minas Gerais	0.072	0.075	0.000

Table 4.

Genetic distances for different populations of Triatoma vitticeps with the mitochondrial genes: 16S rDNA

T. vitticeps	(1)	(2)	(3)
(1) Espírito Santo	0.000	–	–
(2) Rio de Janeiro	0.031	0.000	–
(3) Minas Gerais	0.023	0.023	0.000

Table 3.

Genetic distances for different populations of Triatoma vitticeps with the mitochondrial genes: Cyt b

T. vitticeps	(1)	(2)	(3)
(1) Espírito Santo	0.000	–	–
(2) Rio de Janeiro	0.050	0.000	–
(3) Minas Gerais	0.067	0.063	0.000

Based on this, different authors evaluated the specific status of triatomines by means of genetic distance using mitochondrial genes: Alevi et al.²⁰ through the 16S gene corroborates the specific status of Triatoma pintodiasi Jurberg et al. (2013); Alevi et al.²¹ through the 16S gene also have proposed the synonimization between Nesotriatoma bruneri Usinger (1944) and Nesotriatoma flavida (Neiva, 1911); Ceretti-Junior et al.¹⁴ based on the 16S gene reported low intraspecific variation between genetic distances of triatomines (0–0.6%), except for relationships between Triatoma brasiliensis Neiva (1911) specimens (1.3%) and Triatoma sordida (Stål, 1859) specimens (1%) from different geographic populations (specimens that possibly suffered cryptic speciation)^22,23; Panzera et al.²³ using the COI gene observed a genetic distance of 5.3% between T. sordida from Brazil and from Argentina and suggest that T. sordida from Argentina is possibly a new cryptic species of T. sordida; Abad-Franch et al.²⁴ used the genetic distance of cyt b (8.7%) between Rhodnius robustus Larrousse (1927) and Rhodnius barretti to characterize that R. barretti represents a distinct entity within the “robustus lineage”; and Rua et al.²⁵ suggested that populations of Triatoma sanguisuga (Leconte, 1855) are divided into two subspecies because they have genetic distances close to five for cyt b.

Recently, T. vitticeps and Triatoma melanocephala were grouped in the monophyletic subcomplex T. vitticeps because they presented karyotypic differences when compared with the other triatomines in South America.²⁶ As observed for T. vitticeps, other populations of triatomines (e.g., T. brasiliensis) also did not present intraspecific chromosomal variations²⁷ and presented genetic distances²² that supported the description of new species. Alevi et al.²⁸ highlighted that gene evolution and chromosomal evolution are events mediated by different factors, being that the events that may lead to variations in DNA sequences are more common than those events that lead to chromosomal changes.

Thus, our results suggest that possibly what is characterized as T. vitticeps is a complex of cryptic species (or subspecies). We suggest that an extensive phylogeographic study (possibly with tools that characterize the presence of gene flow) be performed in T. vitticeps and be associated with taxonomic tools, such as experimental crosses between different populations of T. vitticeps, to evaluate the specific status of this vector of Chagas’ disease endemic to Brazil.