Infection, Genetics and Evolution 82 (2020) 104307

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Infection, Genetics and Evolution

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Short communication petrocchiae (, , ): A Chagas disease vector of T. brasiliensis species complex associated to reptiles T ⁎ Maurício Liliosoa, , Dayane Pires-Silvaa, Fernanda von Hertwig Mascarenhas Fontesa, Jader Oliveirab, João Aristeu da Rosab, Roberto V. Vilelac, Elaine Folly-Ramosd, ⁎ Carlos Eduardo Almeidaa, a Instituto de Biologia, Universidade Estadual de Campinas – UNICAMP, b Universidade Estadual Paulista "Júlio de Mesquita Filho", FCFAR/UNESP, Brazil c Laboratório de Biologia e Parasitologia de Mamíferos Silvestres Reservatórios, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Brazil d Programa de Pós-Graduação em Ecologia e Monitoramento Ambiental, Universidade Federal da Paraíba– UFPB, Campus IV, Brazil

ARTICLE INFO ABSTRACT

Keywords: In semi-arid areas of northeastern Brazil, Chagas disease vectors of Triatoma brasiliensis species complex com- Triatomine ecology prise a monophyletic group of kissing bugs that inhabit rock outcrops. Most of them exhibit allopatric or Trophic resource parapatric distribution; the exception is T. petrocchiae, which is found in cohabitation with T. brasiliensis in rock Niche occupation outcrops. We used vertebrate mitochondrial gene sequencing applied to DNA isolated from bug midgut to vector identify the insect blood meal sources via BLAST procedure. Fourteen sylvatic from four geographic districts in the states of and Paraíba had their blood meal sources detected. While T. brasiliensis is recorded to be associated mainly (52–71%) with rodents, T. petrocchiae samples were strongly associated (86%) with reptiles of Tropidurus and Hemidactylus genera. We suggest that T. petrocchiae is the single member within this complex to be associated with reptiles, indicating a distinct niche occupation related to the trophic resources.

In semi-arid areas of northeastern Brazil, members of Triatoma mitochondrial cytochrome b gene (MT-CYB) variation. These authors brasiliensis (Hemiptera, Reduviidae, Triatominae) species complex are also stated that the latter is rare and sylvatic, contrarily to T. brasiliensis the main Chagas disease vectors (Costa et al., 2003). This group is – a species that challenges vector-control measures by infesting human composed of six species (T. bahiensis, T. juazerensis, T. lenti, T. melanica, dwellings 6–12 months after insecticide spraying (Diotaiuti et al., T. petrocchiae, and T. sherlocki) and two subspecies (T. brasiliensis bra- 2000). siliensis and T. b. macromelasoma)(Oliveira et al., 2017). For simplifi- The identification of dietary habits of insect vectors is conducted by cation, we here refer to T. b. brasiliensis,asT. brasiliensis. They differ molecular analyses of the blood ingested by the insect. Enzyme-linked significantly in terms of biology, ecology (Costa et al., 2002), mor- immunosorbent assays (ELISA) have been widely used for inferences phology (Costa et al., 1997, 2009, 2013, 2016), vector capacity (Folly- about blood meal sources in vectors (e.g., Almeida et al., 2002). Despite Ramos et al., 2016), among others. Inside this group, T. brasiliensis is the the great contribution of this technique, it finds limitations in identi- most important Chagas disease vector (Costa et al., 2003) because it fying the specifi c taxonomic status of the blood source; identifying, in combines a high rate of domiciliary infestation with a high prevalence general, only the families of the hosts. More recent techniques, based on of natural infection by – the causative agent of the the sequencing of DNA fragments from bug midgut, have emerged as an disease. Regarding the geographic distribution, most members of this alternative to solve this question, enabling searches in public genetic species complex are parapatric or allopatric (Costa et al., 2014). The databases (e.g., GenBank) for similar sequences to those obtained exception is T. petrocchiae, which we collected in cohabitation with T. through the sequencing of genes in the blood meal of insect vectors. For brasiliensis in rocky outcrops in the states of Rio Grande do Norte and T. brasiliensis, this approach was used by Almeida et al. (2016) on se- Paraíba, Brazil. Lima-Oliveira et al., 2020 reported that T. brasiliensis quencing a fragment of the MT-CYB b gene, identifying rodents (no- and T. petrocchiae exhibit similar patterns of genetic structure, based on tably the caviid rodents Galea spixii and Kerodon rupestris) as the main

⁎ Corresponding authors at: Universidade Estadual de Campinas - UNICAMP, Dept. Biologia , IB, Campus Universitário Zeferino Vaz, room 16B. Rua Monteiro Lobato, 255, Barão Geraldo, Campinas, São Paulo, Brazil. E-mail addresses: [email protected] (M. Lilioso), [email protected] (C.E. Almeida). https://doi.org/10.1016/j.meegid.2020.104307 Received 5 February 2020; Received in revised form 12 March 2020; Accepted 28 March 2020 Available online 30 March 2020 1567-1348/ © 2020 Elsevier B.V. All rights reserved. M. Lilioso, et al. Infection, Genetics and Evolution 82 (2020) 104307

Fig. 1. Capture locations of Triatoma petrocchiae in the states of Paraíba and Rio Grande do Norte, Brazil. Geographic districts are in yellow (Currais Novos), blue (Patos) and green (Cajazeiras). The number and species of blood meal sources detected in T. petrocchiae for each district by molecular methods are indicated. Lizard names are in blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) blood food sources, and establishing possible links between domestic All sequences reached the best hit for searching because we assumed and sylvatic cycles of T. cruzi. These results revealed that rodents are the cut-off values of identity > 95%. The results are summarized in potential reservoirs of T. cruzi in the wild, as the same insects that fed Fig. 1. Of fourteen T. petrocchiae samples, eight (57%) fed on lizard on rodents had 52–71% prevalence of natural infection by T. cruzi. Tropidurus semitaeniatus (KR707089), three on lizard Tropidurus hispidus Active searches for insects were conducted in rocky outcrops (syl- (KU245065), two on rodent Galea spixii (GU067492), one on gecko vatic environment) throughout the states of Paraíba (PB) and Rio Hemidactylus sp. (KX902983). Both lizards of the genus Tropidurus are Grande do Norte (RN), Brazil, across a range of 240 km (East-West) and expected to co-occur in the Caatinga biome (Ribeiro and Freire, 2011). 95 km (North-South) (−06°58’48′,0“ to −06°08’49,2” latitude and -38° Although the sequence of the gecko Hemidactylus matched 97% Hemi- 35′27,6” to −36 °29’09,6″ longitude). Triatomines were collected in dactylus leschenaultii and Hemidactylus mabouia, we prefered the more the same field work, during the dry period (December to February). conservative Hemidactylus sp. However, it is worth mentioning that Sampling was distributed in three districts: Cajazeiras, PB (two sam- Hemidactylus mabouia is a lizard introduced in Brazil from Africa fre- pling spots); Patos, PB (seven sampling spots); and Currais Novos, RN quently found in the domestic environment but found also in the wild. (two sampling spots) (Fig. 1). All sampled spots were within the bio- Another reason for avoiding suggesting the specific status of this gecko geographic domain known as Caatinga, a mosaic of xerophytic, decid- is regarding the low Query cover (21%). All remaining food sources uous, semiarid thorn scrub, and forest (Leal et al., 2005). The gut detected had more than 88% of query cover. The rodent Galea spixii content of 68 T. petrocchiae from six populations was macerated in li- (Caviidae) has already been found by other authors as blood meal quid nitrogen by using a sterile crusher. The DNeasy Blood & Tissue Kit source in T. brasiliensis in the states of Rio Grande do Norte (Almeida (Qiagen) was used for DNA isolation, following the manufacturer's re- et al., 2016) and Ceará (Bezerra et al., 2018). commended protocol. We used the vertebrate-specific primers L14841/ Overall results showed that 12 of 14 (86%) T. petrocchiae captured H15149 for fragment gene amplification, as described by the authors had fed on reptiles' blood. In previous studies, authors reported that (Kocher et al., 1989). With a single exception, both strands (forward sylvatic T. brasiliensis fed mainly (53–54%) on rodents (Almeida et al., and reverse) were sequenced, which were then assembled in SEQMAN 2016; Bezerra et al., 2018), although sporadic blood meals on cold- software (Swindell and Plasterer, 1997) into contigs for edition. Only blooded (such as frogs) have been also observed for T. brasi- 14 T. petrocchiae had the fragment amplified and sequenced liensis. According to Lima-Oliveira et al. (2020), Triatoma brasiliensis is (176–372 bp, mean = 318 bp). To identify the blood meal sources, more abundant (> twice) than T. petrocchiae in all spots they were sequences were compared to those from the GenBank database using found in cohabitation. These two co-occurring triatomines are re- the BLAST program (Altschul et al., 1990). Additional information on productively isolated, according to experimental crossings (Espinola, the sampling (Municipality, State, and geographic coordinates) for each 1971). In the state of Rio Grande do Norte, Barbosa-Silva et al. (2019) blood meal source detected and details on the BLAST search (E-value, found nymphs of T. petrocchieae in domestic and peridomestic en- identity, accession codes) are available in the Supplementary File. vironments, accounting for 0.4% of the triatomines found in these

2 M. Lilioso, et al. Infection, Genetics and Evolution 82 (2020) 104307 ecotopes, whereas T. brasiliensis represented 47%. Despite the lower (Hemiptera, Reduviidae, Triatominae) genital structures and eggs of different chro- occurrence, the discovery of nymphs suggests insect colonization matic forms. Mem. Inst. Oswaldo Cruz. https://doi.org/10.1590/S0074- 02761997000400009. (OPAS, 2009), instead of sporadic invasions by winged forms. Barbosa- Costa, J., Peterson, A.T., Beard, C. Ben, 2002. Ecologic niche modeling and differentiation Silva et al. (2019) also mentioned that T. petrocchiae exhibited 1.4% of of populations of Triatoma brasiliensis neiva, 1911, the most important Chagas’ dis- T. cruzi natural infection; whereas T. brasiliensis was found by Lilioso ease vector in Northeastern Brazil (hemiptera, reduviidae, triatominae). Am. J. Trop. – Med. Hyg. 67, 516–520. https://doi.org/10.4269/ajtmh.2002.67.516. et al. (2017) having 30 40% of infection prevalence. This T. petrocchiae Costa, J., Almeida, C.E., Dotson, E.M., Lins, A., Vinhaes, M., Silveira, A.C., Beard, C. Ben, preference for reptiles may explain the lower prevalence (4%: 3/68) of 2003. The epidemiologic importance of Triatoma brasiliensis as a Chagas disease T. cruzi natural infection found by Lima-Oliveira et al. (2020) because vector in Brazil: a revision of domiciliary captures during 1993-1999. Mem. Inst. – these vertebrates are refractory to T. cruzi infection. We, therefore, Oswaldo Cruz 98, 443 449. https://doi.org/10.1590/S0074-02762003000400002. Costa, J., Peterson, A.T., Dujardin, J.P., 2009. Morphological evidence suggests homo- propose that T. petrocchiae occupies a distinct niche to survive in co- ploid hybridization as a possible mode of speciation in the Triatominae (Hemiptera, habitation with T. brasiliensis in rocky outcrops by exploring distinct Heteroptera, Reduviidae). Infect. Genet. 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