Morphological approach focusing on the structure of the external genitalia of female Triatoma jatai, Triatoma costalimai, and Triatoma williami (Hemiptera: Reduviidae: Triatominae)


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Abstract Backgroud
Taxonomic identi cation of triatomines is generally performed based on aspects of their external morphology. However, the use of a multidisciplinary approach, considering morphological aspects of the external genitalia, morphometry, genetics, and phylogeography has been suggested, especially for similar and/or cryptic species. The rupestral species Triatoma jatai Gonçalves et al. 2013, Triatoma costalimai Verano & Galvão 1959, and Triatoma williami Galvão et al. 1965, which are morphologically similar, have been found with natural infection by Trypanosoma cruzi (Chagas, 1930) in wild, peridomestic, and intradomestic environments, representing a risk of new outbreaks of Chagas disease. This study presents a morphological description complementation of these species, with an emphasis on the structures of the female external genitalia, using scanning electron microscopy.

Methods
Female specimens of T. jatai and T. costalimai captured in Brazilian Municipalites of Paranã and Aurora do Tocantins were identi ed with the use of a dichotomous key for the sub-complex Matogrossensis, as well T. williami, obtained by colony of reference laboratory in triatomines. The insects were sectioned with a scalpel in the transverse direction at the height of the sixth abdominal segment. The terminal portion of the abdomen was washed, dehydrated in an alcoholic series and mounted on a metallic support on double-sided adhesive tape in the ventral or dorsal positions. Samples were then left in an oven and in a desiccator containing silica gel for drying until metallization. They were metallized with gold and examined using a scanning electron microscope at the Oswaldo Cruz/FIOCRUZ Institute Electronic Microscopy Platform.

Results
It was possible to differentiate between the species based on characteristics of the 7th, 8th, and 9th urotergites and 7th urosternite, as well as the genital plaques, gonocoxites, and gonapophyses. For the rst time, morphological differences in the thorns present on gonocoxite 8 are described in triatomines.

Conclusions
We results show that external genitalia of female it is a useful structure to differentiate T. jatai, T. costalimai and T. williami. The scanning electron microscopy contribute and corroborate, together with other tools morphological and molecular, to the differentiation of T. jatai, T. costalimai and T. williami, as well as expand the taxonomic knowledge. It may also help to understand if there is reproductive compatibility between T. jatai and T. costalimai as well possible formation of hybrids under arti cial conditions. This one is underway in order to fully contemplate all de nitions of biological species.

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Triatomines are insect vectors of the Trypanosoma cruzi (Chagas, 1909), the causative agent of Chagas disease. It is estimated that about 6 to 8 million people are infected with T. cruzi worldwide, with the highest number of cases in Latin America [1]. According to data from the Brazilian Ministry of Health, there are approximately 12 million people with chronic Chagas disease in the Americas, with 2 to 3 million in Brazil [2]. The highest number of acute cases of the disease in Brazil from 2000 to 2017 was recorded in the northern region, where two of the species analyzed in this study, Triatoma jatai Gonçalves  Morphological studies generate important data that can be used in multidisciplinary approaches that answer questions about limits applicable to species especially criptic species or very similar morphology.
The identi cation of triatomines is based on morphological characteristics [3,4]. In the 1960s, a comparative analysis of the morphology of the external genitalia of both sexes by optical microscopy started to be used as an additional tool in the taxonomy of triatomines. However, the taxonomic value of the female genitalia's morphology was questioned because it does not allow species selection in detail [5]. From 2010, scanning electron microscopy (SEM) has been used for morphological characterization of the female genitalia of several triatomine species, proving to be an important complementary methodology for determining the taxonomy of the group [6]. The rupestrian species T. jatai and T. costalimai are distributed in the Cerrado biome, found in sympatry in the municipality of Paranã, Tocantins, Brazil, and show close similarities in terms of their external morphology and classical and geometric morphometric [7]. Some also support the close genetic a nities between these two species [8,9]. The description of T. jatai was made from specimens collected in the wild environment. Even though this species has been captured in an intradomestic environment, its vectorial capacity is still unknown and there is no record of natural infection by T. cruzi [10]. Triatoma costalimai, has a geographical distribution that covers the Brazilian states of Goiás, Tocantins, Minas Gerais, and Bahia, and has also been reported in Bolivia [4,11]. This species has been found in wild, peridomestic, and intradomestic environments, in some cases with high prevalence of infection with T. cruzi [4,10,12,13] The rupestrian species Triatoma williami Galvão, Souza, Lima 1965, which is morphologically similar to T. costalimai, is found in the Cerrado Biome and Pantanal, in the states of Goiás, Mato Grosso do Sul, and Mato Grosso [4]. The rst record of a triatomine of this species with natural infection by T. cruzi was from Mato Grosso, from specimens captured in an intradomestic environment [14]. The three mentioned species, whose morphology of the female's genitals has not been studied so far, were included in Matogrossensis complex [7,15]. This study analyze the external genitalia using scanning electron microscopy (SEM), on T. jatai, T. costalimai, and T. williami, aimed to provide complementary data to the morphological characterization of this three species that could be useful for taxonomic identi cation as well as to further integrative studies to allow a better species delimitation in the Matogrossensis complex. Also the ne morphological characterization of the female genitalia could be a source of putative new characters to be used in future phylogenetic studies to allow a better comprehension of the evolutionary history of this group of vectors.

Methods
Specimens were captured by a team from the Interdisciplinary Entomological Surveillance Laboratory in Diptera and Hemiptera, Instituto Oswaldo Cruz/Fiocruz, Brazil in the Brazilian municipalities of Paranã and Aurora do Tocantins, Tocantins, by active and passive search using animal bait traps [16] in 2011 (SISBIO License 43393). The specimens were identi ed using a dichotomous key for the sub-complex Matogrossensis [7] and kept at -20ºC in the laboratory. Specimens of T. williami, which have a morphology close to that of T. costalimai [3], were provided by the National and International Reference Laboratory in Triatomine Taxonomy (LNIRTT/ IOC

Results
Observation of the external genitalia of Triatoma jatai females in dorsal view, showed that the posterior edge of urotergite VII has a W shape, with a 1+1 lateral depression rising in the median region; the posterior edge of urotergite VIII is rectilinear, decaying laterally, and ending at half of the suture of the rounded apex connective; urotergite IX is trapezoidal, with bulging lateral walls and covered by thick, short, sparse hairs that end near the anal tube ( Figure 1A). Triatoma costalimai has a non-rectilinear posterior edge of urotergite VII, ending near the suture of the connective; the posterior edge of VIII is convex and ends at half of the suture of the connective with pointed apex; and IX is trapezoidal, with straight lateral walls and a 1+1 apical recess near the anal tube, covered with thick, long hairs ( Figure 1B). In T. williami the posterior edge of urotergite VII is rectilinear at the median region rising at the lateral margins towards the connexival suture; the posterior edge of VIII is rectilinear ending at the median line of the connexival suture; the IX is trapezoidal, with bulging lateral walls, presenting a 1+1 sharp depression close to the anal tube, covered by short thick hairs ( Figure 1C).
In T. jatai, ventrally, the line of the posterior edge of urosternite VIII is prominent in the median region, and the gonocoxites of the 8th segment (Gc8) are long and wide ( Figure 1D). In T. costalimai, the posterior edge line of urosternite VII is convex and Gc8 is long and narrow ( Figure 1E). In T. williami, the posterior edge of urosternite VII has the same shape as in T. jatai, but is not prominent. Gc8 is triangular and close to the median line of urosternite VII ( Figure 1F). In the median region of gonapophysis 8 (Gp8) and at the base of T. jatai Gc8, short spines were found on the apex with single, bifurcated, and trifurcated points ( Figure 1G). Similar spines were observed only in the median region of T. costalimai Gp8 ( Figure 1H). In T. williami, only short single-pointed apex spines were observed in the median region of Gp8 ( Figure 1I).
A summary of main morphological characteristics of the external genitalia female of Triatoma jatai, T. costalimai and T. williami on dorsal and ventral view are show in Table 1.

Discussion
The presence of single-pointed, bifurcated, and trifurcated spines in the median region of gonapophysis 8 in the three study species and, at the base of gonocoxite 8 in T. jatai, shows the importance of the morphological details observable by using SEM. These spines are present in the internal genitalia of Rhodnius neglectus (Lent, 1954), and also in the copulatory pouch, indicating that they may assist with spermatophore compression and sperm release [24]. However, the functions of these spines in copulation or ovulation have not been clari ed. In a recent molecular phylogeny study [25], a reorganization of Triatoma groupings was done and left the classi cation of the sister species T. jatai and T. costalimai as unde ned. However, they were included in the Triatoma pseudomaculata species group, where T. williami is also found. Although both species occur in the Cerrado Biome, the difference in phytogeography and climate pro le between the municipalities of Paranã and Aurora do Tocantins may be possible factors related to a recent process of speciation. In phenotypic terms, a difference has already been expressed in the morphology of the male genitals [5] and now also for the female genitals.
A high rate of natural infection of T. costalimai by T. cruzi in peri-and intradomestic environments has been reported [8,10,11]. Expanding understanding of characteristics that differentiate related species is important for a more accurate diagnosis, and can facilitate entomological surveillance and control of vector transmission of Chagas disease, especially in municipalities in the south and southeast Tocantins region such as Paranã and Aurora do Tocantins.

Conclusions
The morphology urotergites (7,8,9) and urosternite (7), as well as the genital plaques, gonocoxites, and gonapophyses (8) were important to differentiate the species. The results contribute and corroborate, together with other morphological and molecular studies, to the differentiation of T. jatai, T. costalimai and T. williami, as well as to the taxonomy of these species. It may also help to evaluate the reproductive compatibility between T. jatai and T. costalimai and possible formation of hybrids under arti cial conditions. This one is underway in order to fully contemplate all de nitions of biological species.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.
Availability of data and material The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.   Figure  1A) Rectilinear, decaying laterally, ending at half of the suture of the rounded apex connective ( Figure  1A) Trapezoidal, with bulging lateral walls covered by thick, short, sparse hairs that end near the anal tube ( Figure 1A) Prominent in the median region ( Figure  1D) Long and wide.
( Figure 1D) Short spines on the apex and at the base with single, bifurcated, and trifurcated points ( Figure  1G) T.