First Report of Rhodnius stali Lent, Jurberg, and Galvão 1993 (Hemiptera: Reduviidae: Triatominae) Naturally Infected by Trypanosoma cruzi in Concepción Department, Paraguay

ABSTRACT.

Most triatomine bugs inhabit nests and shelters of vertebrates, some of which are closely associated with palm trees. A few species occupy domestic and peridomestic habitats, posing a threat to human health as natural transmitters of Chagas disease. A peridomestic specimen that yielded positive results for Trypanosoma cruzi in both microscope and polymerase chain reaction tests was collected during a vector control survey in northeastern Paraguay. The specimen was identified as Rhodnius stali, marking a new record for the country. To the best of our knowledge, this is the southernmost report of this species documented to date. This record emphasizes the importance of considering this additional triatomine species in future vector and control surveillance efforts in Paraguay.

Triatominae, also known as kissing bugs, barbeiros, vinchucas, or chipos, is a mostly neotropical subfamily of Reduviidae (the family of assassin bugs and ambush bugs), which contains nearly 160 species in 18 genera.^1–3 They are all hematophagous, mostly inhabiting nests or shelters of vertebrates, which are well-known vectors of stercorarian trypanosomes, most notably the Chagas disease agent Trypanosoma cruzi (T. cruzi). A number of triatomine species belonging to genera Triatoma, Rhodnius, and Panstrongylus have been reported as transmitters of this parasite to humans. There are at least 10 species recorded from Paraguay: Psammolestes coreodes Bergroth, 1911; Panstrongylus geniculatus (Latreille, 1811); Panstrongylus guentheri Berg, 1879; Panstrongylus megistus (Burmeister, 1835); Triatoma delpontei Romaña & Abalos, 1947; Triatoma guasayana Wygodzinsky & Abalos, 1949; Triatoma guazu Lent & Wygodzinsky, 1979, which is now a junior synonym of Triatoma williami Galvão, Souza & Lima, 1965; Triatoma infestans (Klug, 1834); Triatoma platensis Neiva, 1913; and Triatoma sordida (Stål, 1859).^4–7 Although the Paraguayan National Malaria Eradication Service (SENEPA) has indicated the presence in the country of specimens belonging to the genus Rhodnius,⁸ no species identification was available thus far. This contribution reports on the presence of R. stali Lent, Jurberg & Galvão, 1993 for the first time in Paraguay based on a specimen collected by SENEPA, which is a public institution in charge of the nationwide control surveillance of vector-borne diseases.

A single adult male triatomine was collected in January 2019 by SENEPA operative personnel during an inspection of a chicken coop near a house in the San Luis neighborhood in the municipality of Belén, Concepción Department, northeastern Paraguay (23°26′05″S 57°18′11″W). The specimen, which was taxonomically unidentified upon collection, tested positive for T. cruzi through microscopic observation at 400× magnification, as described previously.⁹

The natural infection of the collected triatomine by T. cruzi was confirmed through polymerase chain reaction (PCR) analysis. The bug’s abdomen was dissected to obtain tissue for DNA extraction using the GeneJet Genomic DNA Purification kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Subsequently, a real-time PCR (RT-PCR) test was conducted in a Rotor-Gene 6000 real-time thermocycler (QIAGEN®, Hilden, Germany). A 144 bp nucleotide fragment encoding the 70 kDa heat shock protein (HSP70) was amplified by using the previously described oligonucleotide primers Fhsp70F2 (5′-GGAGAACTACGCGTACTCGATGAAG-3′) and Rhsp70C (5′-TCCTTCGACGCCTCCTGT-3′). Each PCR assay had a final volume of 20 µL, utilizing 10 µL of the mix (High-Resolution Melting PCR Master Mix®; QIAGEN), a final concentration of 50 ng/µL of the DNA template, and both primers at a final concentration of 0.7 µM. Polymerase chain reaction cycling conditions 40× consisted of initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and elongation at 72°C for 10 seconds. The PCR was immediately followed by high-resolution melting (HRM) assays (Figure 1A), with a range established between 80°C and 90°C at intervals of 2 seconds in each range of a melting curve of 0.1°C/second. HRM curves were analyzed from the raw data after normalization (Figure 1B) by using the 2.1.0 software (Rotor-Gene-6000 QIAGEN®). DNA samples from laboratory cultures of T. cruzi and Leishmania infantum served as control groups for the PCR.¹⁰

Figure 1.

Polymerase chain reaction (PCR) results. (A) Tm values were obtained with the high-resolution melt (HRM) assay for the positive controls and the sample from Rhodnius stali. (B) Normalized curve of the heat shock protein (HSP70) amplicon obtained using HRM. (C) Detection of the presence of Trypanosoma cruzi through PCR analysis in an agarose gel at 1.5%.

A second PCR test was conducted to confirm the presence of T. cruzi in the analyzed samples, as indicated by RT-PCR tests. Accordingly, a 188 pb segment of the repetitive 195 bp nuclear DNA sequence that is the most abundant sequence in T. cruzi was amplified using primers TCZ1 (5′-CGAGCTCTTGCCCACACGGGTGCT-3′) and TZC2 (5′-CCTCCAAGCAGCGGATAGTTCAGG-3′).⁸ PCR cycling conditions consisted of initial denaturation at 95°C for 10 minutes, followed by 35 cycles of denaturation at 95°C, annealing at 65°C, and elongation at 72°C for 1 minute. Amplified the products were run on 1.5% agarose gels¹¹ (Figure 1C).

The nucleotide fragment obtained using RT-PCR was subjected to Sanger sequencing, and the results were verified using Bioedit version 7.0 (https://thalljiscience.github.io/). Sequence data reported in this paper were deposited in GeneBank under the accession number ON653039.

The specimen was yellowish brown (although it was darkened by partial decomposition), with irregular black markings on the head, pronotum, scutellum, coxae, trochanters, and femora. The tibiae had mid and apical black rings and connexivum with H-shaped black markings on each segment. The head was approximately as long as the pronotum, and the anteocular region was 2.5 times longer than the postocular region. The genitalia, with a bifid medial process, remained sufficiently intact for identification using published identification keys.^12,13 The coloration, proportions, and genital capsule structure indicated a male specimen of R. stali Lent, Jurberg, and Galvão, 1993 (Figure 2). The specimen is now in the invertebrate’s collection of the Museo Nacional de Historia Natural del Paraguay with the catalog number MNHNP-HX 2009.

Figure 2.

External morphology of the body of specimen MNHNP-HX 2009 of Rhodnius stali. (A–C) Head and pronotum. (D) Legs. (E and F) Thorax and wings. (G and H) Abdomen and connexivum. (I–K) Pygophore. (A, E, G, I) Dorsal view; (B, J) lateral view; (C, F, H, K) ventral view. Scale bars: (A–D) 5 mm, (E–H) 5 mm, (I–K) 1 mm.

Rhodnius stali has been previously recorded in Brazil and Bolivia in localities corresponding to the biogeographical areas of Yungas (Caranavi and Palos Blancos), Southern Amazonia (Moxos, Acre), Southwestern Amazonia (Guajará-Mirim, Rondonia near Bolivia), Cerrado (Bodoquena), Pantanal (Nhecolandia), and the Cerrado–Chaco–Pantanal transition (Porto Murtinho).^12,14 It has been found both in arboreal nest of the mammal Coati (Nasua nasua), where it can even cohabit with T. sordida, in the Brazilian Pantanal,¹⁵ and in domestic (houses) and peridomestic (animal shelters and cereal stores) habitats in the Alto Beni region of Bolivia. Thus it is currently considered a species in the process of domiciliation in these areas that are marked by intense deforestation and the utilization of palm leaves as roofing material, where the principal Chagas disease vector, T. infestans, is absent; R. stali has been observed colonizing dwellings and peridomestic spaces.¹⁶ All the individuals were infected with T. cruzi in the first case, and none were infected in the second. In Bolivia, the bugs have been associated with leaves of the palm Attalea phalerata (A. phalerata) and Copernicia alba, both as building material and in the wild.^17,18 Palm trees have been tagged as one of the most important habitats for triatomines, especially Rhodniini, and particularly Rhodnius species.¹⁹

Additionally, R. stali has been found infected with Trypanosoma rangeli in Attalea palms, suggesting this tree is its ecotone. It is noteworthy that T. rangeli shares 60% of its antigens with T. cruzi, which raises concerns in areas where R. stali is prevalent.^20–22 Therefore, confirming the protozoan species associated with R. stali is imperative to accurately assess the epidemiological implications of R. stali as a vector of Trypanosomiasis.

Northeastern Paraguay, including the departments of Concepción and Amambay, belongs to the southern extension of the Cerrado biogeographical area, with transitional zones with both the Humid Chaco toward the west and the Paraná Forest to the east and south. It holds the richest palm flora of the country, with almost all reported Paraguayan species present in at least one place of the area, and approximately half of them (including A. phalerata) recorded from around the collecting area (municipality of Belén and metropolitan area of Concepción).²³ The collecting locality is placed ca. 200 km south of the nearest reported geographical record for R. stali (Porto Murtinho) in a transitional area between the Humid Chaco and the Cerrados, which was originally dominated by seasonally dry forests (cerradones), fitting well into the expected distribution of the species.²⁴

The expansion of R. stali distribution toward southern regions, along with its natural infection in its original ecotone and, as our study indicates, in peridomiciliary environments, coupled with its confirmed colonization of human dwellings in Bolivia, underscores the urgent need for entomological surveillance of this species. Such vigilance is crucial given its potential involvement in T. cruzi transmission.

The Paraguayan record of R. stali, in a peridomestic environment and positively infected by T. cruzi, implies a new Chagas disease vector in the country to be considered for future national works of vector surveillance and control. To the best of our knowledge, this is the southernmost location of this species in South America reported to date. In addition, this is the fifth triatomine species recorded in Paraguay that has been confirmed as a natural carrier of T. cruzi infection in domestic environments.⁸ This new record emphasizes the need for further studies on the occurrence, identification, and distribution of triatomine bugs in Paraguay, as the lack of information may lead to misidentifications and missing records.