A new species of Rhodnius from Brazil (Hemiptera, Reduviidae, Triatominae)

Abstract A colony was formed from eggs of a Rhodnius sp. female collected in Taquarussu, Mato Grosso do Sul, Brazil, and its specimens were used to describe R. taquarussuensis sp. n. This species is similar to R. neglectus, but distinct characters were observed on the head, thorax, abdomen, female external genitalia and male genitalia. Chromosomal differences between the two species were also established.

Most Rhodnius species live in palm trees, and several cases of transmission of Chagas disease have been associated with the consumption of açaí containing feces of triatomines infected by T. cruzi (Ferreira, Branquinho & Leite, 2014;Ministério da Saúde, Brasil, 2017). Apart from such cases, which occur more frequently in the northern region of the country, it is worth noting that R. neglectus was found in palm trees (species of Roystonea, Syagrus and Acrocomia] in the city of Araçatuba, São Paulo, in 2009, as well as in palm trees (Livistona australis) located in the central square of the city of Monte Alto, São Paulo, in February 2012 (Rodrigues et al. 2014, Carvalho et al. 2013.
Based on morphological, morphometric and cytogenetic characters, this paper describes R. taquarussuensis sp. n., which is similar to R. neglectus. The first collected specimen of R. taquarussuensis was a female that invaded a domicile and laid eight eggs. The colony formed from those eggs resulted in the specimens used in this description.

Morphological identification and description
On 10 November 2010 a female of Rhodnius sp. invaded a rural dwelling (22°29'07.7"S; 53°21'08.9'W) in the city of Taquarussu, Mato Grosso do Sul, Brazil, and was captured ( Fig. 1). That specimen remained alive for a few days and laid eight eggs (Fig. 2). By means of macroscopic identification and subsequent optical microscopy (OM) and using the key of Lent and Wygodzinsky (1979), a clear similarity with R. neglectus was noticed. In view of that, all characters observed and documented for Rhodnius sp. were checked for R. neglectus CTA 229, which is a colony that has been kept since June 27, 2011 at the Triatominae Insectarium of the Faculty of Pharmaceutical Sciences, UNESP, Araraquara (FCFAR/UNESP). The temperature, humidity and light cycle conditions are not controlled due to the insect's biodiversity, but these parameters are measured daily, varying the temperature between 20-35ºC and humidity 50-80%. Insects kept in colonies are fed directly on ducks every 15 days and consists of specimens from the Brazilian National and International Triatominae Taxonomy Reference Laboratory at the Oswaldo Cruz Institute in Rio de Janeiro, Brazil (LNIRTT). The colony was kindly provided by Dr. José Jurberg and Dr. Cleber Galvão, and the specimens that originated it were collected in northern Formoso, Goiás, Brazil. A colony was formed from the eight eggs laid by the R. taquarussuensis sp. n. female and identified as Araraquara Triatominae Colony (CTA) 277. The specimens of that colony were used to describe R. taquarussuensis sp. n.
Female external genitalia were observed from the dorsal, posterior, and ventral sides (Fig. 6) by SEM, according to Rosa et al. (2010). The study of the male genitalia was carried out by OM (Figs 8,9), following a technique developed by Jader de Oliveira based on Gallati (2016). The denominations used were those defined by Lent and Jurberg (1969).
The Leica MZ APO stereoscope from the Faculty of Pharmaceutical Sciences, UNESP, Araraquara, and the scanning electron microscope Topcon SM-300 located in the Department of Physical Chemistry at the Chemistry Institute, UNESP, Araraquara, were used for observation and capture of images.

Morphometric study
In the morphometric study by OM, 15 egg shells, 15 females and 15 males from the colony were measured, the same being done for R. neglectus CTA 229 (Table 1).The parameters measured were: total length, width of thorax and abdomen, length of the scutellum, three segments of the proboscis and four segments of the antenna, as well as five parameters of the head following Dujardin et al. (1999). Eggs had their length and the diameter of the opercular opening measured. The wings of R. taquarussuensis sp. n. and R. neglectus were studied by geometric morphometry using seven anatomical landmarks, according to parameters established by Gurgel- Gonçalves et al. (2008), as well as based on Rosa et al. (2012).
The observations and measurements were carried out on a Leica MZ APO stereoscope and the Motic Images Advanced System version 3.2.

Cytogenetic identification
In this study ten male specimens of R. taquarussuensis sp. n. were used for C and CMA 3 / DAPI-banding analyses and ten male specimens of R. neglectus were used for CMA 3 / DAPI-banding analyses. After being lacerated and placed on the slide, the seminiferous tubules underwent cytogenetic procedures following the C-banding (Sumner 1972) and CMA 3 /DAPI-banding protocols [Schimid 1980, with   Etymology. The name Rhodnius taquarussuensis sp. n. was chosen because this species was found in the city of Taquarussu, Mato Grosso do Sul, Brazil.
Diagnosis. Rhodnius taquarussuensis sp. n. is close to R. neglectus, their differences being the color and a variety of morphological, morphometric and cytogenetic characters (Tables 1, 2). The general color of R. taquarussuensis sp. n. is brown, whereas R. neglectus is dark brown, almost black. This difference is particularly noticeable on the hind wings. The stridulatory sulcus of R. taquarussuensis sp. n. is brown at the base and black on the sides, whereas on R. neglectus it is completely black.
On the head, differences were noticed on the vertex, genae, antennae and triangular furrow of the first segment of the rostrum. The vertex of the head of R. taquarussuensis sp. n. is quite visible, whereas on R. neglectus it is not (Fig. 3A, B, D, E). The genae of R. taquarussuensis sp. n. are longer than those of R. neglectus (Fig. 3A, D).On R. taquarussuensis sp. n. the 10 th part of the second segment of the antenna is brown; on R. neglectus, though, only the basis has that color. The triangular furrow of the first segment of the rostrum, towards the second segment, ends in a filamentous way on R. taquarussuensis sp. n. and in a rounded way on R. neglectus (Fig. 3C, F). On the thorax, differences can be found on the pronotum, wings, scutellum, prosternum, mesosternum and metasternum (Figs 4,5). The membranous portion of the hind wings is brown on R. taquarussuensis sp. n. and dark brown on R. neglectus. The scutellum ends in a rounded apex on R. taquarussuensis sp. n. and in a filamentous apex on R. neglectus (Fig. 4A, B). On R. taquarussuensis sp. n. the apex of the scutellum covers the final portion of the urotergite I process, while on R. neglectus the apex of the process of the urotergite I is perfectly visible ( Fig. 4A, B). The lines limiting the stridulatory sulcus are straight on R. taquarussuensis sp. n. and narrowed in the anterior third on R.neglectus (Fig. 5A, B). On R. taquarussuensis sp. n. the basis of the stridulatory sulcus is brown and the sides are black, whereas on R. neglectus the entire stridulatory sulcus is black. The central region of the limit between the mesosternum and the metasternum is regular and half-moon shaped on R. taquarussuensis sp. n., while on R. neglectus it is pronounced and slightly irregular (Fig. 5C, D). The beginning of the metasternum is narrow on R. taquarussuensis sp. n.   and wide on R. neglectus (Fig. 5C, D). The ventral abdomen of R. taquarussuensis sp. n. is light brown, and that of R. neglectus is dark brown (Fig. 2). The terminal portion of the paramere of the male genitalia of R. taquarussuensis sp. n. is thinner than that of R. neglectus (Fig. 9A, C). The dorsal phallothecal sclerite has a trapezoidal shape on R. taquarussuensis sp. n. and is rounded on R. neglectus (Fig. 8C, D). The external limit of the 10 th segment of the dorsal side of the female external genitalia of R. taquarussuensis sp. n. presents a concavity in the middle portion, whereas on R. neglectus that limit is straight (Fig. 6A, B). From posterior view, the limits of the 9 th segment with gonocoxite VIII are curve on R. taquarussuensis sp. n. and straight on R. neglectus, and the superior line limiting the 10 th and 9 th segments is straight on R. taquarussuensis sp. n. and curve on R. neglectus (Fig. 6C, D). In the ventral side of the female external genitalia of R. taquarussuensis sp. n. there is a concavity in the external limit with the 10 th segment that is also noticed from dorsal view; on R. neglectus that limit is a straight line. From ventral view, the external limits of the 9 th segment of the female external genitalia are curve on R. taquarussuensis sp. n. and straight on R. neglectus (Fig. 6E, F). Among the 19 characters measured, 12 showed significant differences between R. taquarussuensis sp. n. and R. neglectus in both sexes and also the eggs of both species. Two characters showed differences only between males, and five characters did not show significant differences (Tables1, 2).
Description. A total of 15 adult females and 15 adult males of R. taquarussuensis sp. n. and R. neglectus were measured, as well as 30 eggs shells of both species. Such measurements are detailed in Table 1.
The head of R. taquarussuensis sp. n. has a prominent brown vertex contrasting with the black sides. The clypeus is well defined. The genae are large, visible and dark brown, moving towards the anteclypeus (Figs 2A, C, 3 A, B). The limits between the genae and the clypeus are brown. The first segment of the antennae is black with mixes of brown. The articulation between the first and second segment of the antennae is brown. Roughly all the 10 th part of the beginning of the second antennal segment is brown. The second segment is mostly black. In the articulation between the second and third antennal segment there is a black ring followed by a brown one. The beginning of the third segment (around 1/3) is black and the remaining portions (2/3) are brown. The articulation between the third and fourth antennal segment is brown. The beginning of the fourth segment is black and the remaining portions are brown with mixes of black ( Fig. 2A, C).
The eyes are black and the ocelli are brown. The neck has a brown central dorsal strip flanked by two (1+1) black, narrower strips. The ventral portion of the neck between the ocelli is dark brown ( Fig. 2A, B, C, D).  The pronotum of the thorax of R. taquarussuensis sp. n. has a trapezoidal shape and is limited by a brown carina. In the antero posterior direction the pronotum has other two brown carina in the middle portion and six black strips. The three carina and the three brown strips are interspersed with the six black strips, which are larger. The collar (first portion of the pronotum) in the central part is brown and is followed by two (1+1) black glabrous areas and the two (1+1) antero lateral angles. The anterior portion of the pronotum consists of three anterior lobes which are clearly distinct from the posterior portion (hindlobe). Those three anterior lobes are limited by the carina and on each of them there are two black glabrous areas with a lengthy and irregular outline ( Fig. 2A, C). The cuticle involving the veins of the hemelytron is light brown. The corium between the veins of the coriaceous region is dark brown, whereas that of the membrane is brown (Fig. 2A, C).
The prosternum contains the stridulatory sulcus, which moves along that segment in an antero-posterior direction, having a brown color in the background and black on the sides. Two elongated tubercles limit the anterior half of the stridulatory sulcus. In the superior portion and in diagonal direction from the tubercles there are two black glabrous areas surrounded by a set of brown sensilla (Fig. 5A).
The mesosternum is limited anteriorly by the prosternum and posteriorly by the metasternum, both limits being brown. The central line dividing two dark brown elevations is also brown. Those two elevations are limited by two (1+ 1) black side glabrous areas diagonally placed. The central region of the posterior limit of the mesosternum has a half-moon shape. The metasternum is brown and resembles an isosceles triangle. Its anterior portion, i.e., its limit with the mesosternum, corresponds to the vertex of the triangle and is narrow, whereas its posterior portion, i.e., its limit with the first abdominal segment, corresponds to the basis of the triangle (Fig. 5C).
The three pairs of coxae are brown, except for the black glabrous areas. The trochanters of the anterior pair of legs are brown, but mixed with black glabrous areas. The middle and posterior pairs of trochanters are brown and have no glabrous areas. The three pairs of femora are black and the same color prevails in the three pairs of tibiae, except in the articulations with the femur and the spongy fossula, which are brown. The spongy fossulae are located in the first and second pairs of legs in the final portion of the tibia, alongside the articulations with the tarsi (Fig. 2A, B, C, D).
The abdomen of R. taquarussuensis sp. n. presents a brown color in the longitudinal central portion. On the sides of each segment there are (3+3) black glabrous areas, which are mixed with brown and black areas. The connexivum of the dorsal portion lies between the second and seventh segment. For each of those segments the anterior half is black and the posterior one is brown. The dorsal connexivum, also lying between the second and seventh segment, has a black color in 2/3 of the anterior portion, but that black color ends in an irregular way over the remaining 1/3, which is brown. Therefore, the black portion of the connexivum presents two edges moving towards the brown portion: one in the internal limit of the connexivum and the other in the middle portion. However, the connexivum of the second dorsal segment is black in the anterior half and brown in the posterior one, the limit between the portions having a diagonal shape. The seventh segment, on the other hand, is practically all black, except for a small brown strip located in the external posterior half. Type 1 sensilla, which prevail on the head, thorax and abdomen, have a brown color (Fig. 2B, D).
Male genitalia have the typical aspect of the genus Rhodnius. The median process of the pygophore (PrP) is short and triangular, but the base is broad and the sides are elongated with a thin edge. Parameres are hairy with a thin edge. From ventral view, the phallosome (Ph) has a broad plate whose superior region has a trapezoidal shape and occupies the middle region of the aedeagus. The support of the phallosome plate (PrPh) is broad. Conjunctival process I (PrcjI) is present and II (PrcjII) is absent. Endosomal process (En) is well-developed when seen from dorsal and ventral view (Figs 8A, C, 9B).
The dorsal side of the female external genitalia presents a concavity in the middle portion of the 10 th segment. Seen from posterior view, the limits (1+1) of the 9 th segment with gonocoxite VIII are curve, whereas the superior line limiting the 10 th and 9 th segments is straight. In the central portion of the 10 th segment of the ventral side of the female external genitalia there is another concavity that can be noticed from dorsal view. The external limits (1+1) of the 9 th segment of the female external genitalia are curve when seen from ventral view (Fig. 6A, C, E).
Egg shells of R. taquarussuensis sp. n. have a length of 1.72 mm and an opercular opening of 0.49 mm. They present lateral flattening, collar and exochorion cells, most with pentagonal or hexagonal shape (Fig. 7A, C).
Finally, although R. taquarussuensis sp. n. showed the same number of chromosomes as R. neglectus and all the tribe Rhodniini, i.e., 2n = 22 (Figure 11B), the constitutive heterochromatin pattern and the composition of the pairs of bases of DNA rich in AT and CG were completely different from R. neglectus, as the analysis of the nuclei of the initial prophases of R. taquarussuensis sp. n. has revealed a chromocenter consisting of sex chromosomes (arrow) and several heterochromatic blocks dispersed in the nucleus (Fig. 11A). The analysis of metaphase I of R. taquarussuensis sp. n. has demonstrated that this triatomine has heterochromatic blocks in both extremities of practically all the autosomes and in the Y sex chromosome (Fig. 11B), unlike what has been recently stated for many populations of R. neglectus that do not present heterochromatin in autosomes (Alevi et al. 2015a). Furthermore, R. taquarussuensis sp. n. has the X sex chromosome rich in CG (Fig. 12C), the Y rich in AT (Fig. 12D) and various  blocks rich in CG dispersed in the prophase nucleus (Fig. 12C), while R. neglectus only has the X sex chromosome rich in CG (Fig. 12A) and the Y rich in AT (Fig. 12B), which proves the genetic differences between the two Rhodnius species.

Discussion
The subfamily Triatominae include 18 genera comprising 152 species, 20 of which belong to the genus Rhodnius (Galvão 2014, Mendonça et al. 2016, Souza et al. 2016). The difficulties involved in the specific identification of Rhodnius have already been noted by Neiva and Pinto (1923), as well as by Rosa et al. (2012) and Souza et al. (2016). However, even though it is difficult to specify the distinctions among the species of that genus, in the last eight years four species were described: R. zeledoni, R. montenegrensis, R. barretti, and R. marabaensis. Therefore, R. taquarussuensis sp. n. is the 21 st species of the genus and the 5 th described in the last eight years. Its similarity with R. neglectus was noticed after the capture of the first female specimen, as the most evident macroscopic characters, such as size, general aspect and connexivum, showed no differences. As a result, it was decided to base its description on the differences from R. neglectus.
In addition to the macroscopic characters, R. taquarussuensis sp. n. and R. neglectus were considered "close to" because the OM study indicated similar characters between them, including: placement of black and brown spots on the dorsal and ventral connexivum, length of the four segments of the antenna, pronotum, antero lateral angles, urotergite I process, geometric morphometry of the hind wings, median process of the pygophore and morphological characters of the eggs.
Rhodnius taquarussuensis sp. n. was considered distinct from R. neglectus on account of the observation of color, eleven morphological characters, twelve morphometric characters and cytogenetic features (Tables 1, 2). All those differences are consistent with Lent and Wygodzinsky (1979), whose descriptive key to 11 species of Rhodnius lists 13 morphological characters for specific distinction: color, tibia, legs, pronotum, head, posterior lobe of the pronotum, anterolateral angles, median process of the pygophore, connexivum, scutellum, eyes and antennae.
Regarding the color, the distinction between R. taquarussuensis sp. n. and R. neglectus was based on the general aspect, segments of the antenna, hind wings and stridulatory sulcus. The general color of R. taquarussuensis sp. n. is brown, whereas R. neglectus is dark brown, almost black, or "brown dark", as referred to by Lent and Wygodzinsky (1979).
Out of the eleven morphological characters that distinguish R. taquarussuensis sp. n. from R. neglectus, three are located on the head: dorsal vertex, genae and triangular furrow of the first segment of the proboscis. The difference related to the vertex was one of the characters used by Souza et al. (2016) to distinguish R. marabaensis from R. prolixus and R. robustus. The differences between the genae for specific characterization are being reported for the first time in this description. The ventral triangular furrow was mentioned by Rosa et al. (1999) in their study of T. rubrovaria and it was mapped by Lent and Wygodzinsky (1979), but it was not named and it is being used for the first time as a distinctive character.
In what refers to the thorax, differences on the scutellum, protothorax, mesothorax, and metathorax were noticed. The scutellum of R. taquarussuensis sp. n. and R. neglectus differs in the shape of the apex and also the position on which that apex reaches uroter-gite I, since there is no significant difference in terms of length (Table 1). The taxonomic importance of the scutellum was tackled by Obara et al. (2007), who verified the differences of that character in eight species of Triatoma. Rosa et al. (2012) and Souza et al. (2016) also used it to describe R. montenegrensis and R. marabaensis, respectively.
The differentiation of seven genera of triatomines based on the shape of the prosternal stridulatory sulcus was carried out by Lent and Wygodzinsky (1979) and also by Souza et al. (2016) in the description of R. marabaensis. The description presented herein points out color and morphological differences between R. taquarussuensis sp. n. and R. neglectus. The mesothorax and metathorax, which have been used by Souza et al. (2016) to describe a new species, were found to be different in R. taquarussuensis sp. n. and R. neglectus. Lent and Jurberg (1969) observed specific features in the characters of the male genitalia of 10 species of Rhodnius and since then that structure has been used to describe species of other genera of Triatominae, e.g., Mepraia parapatrica Frias, 2010, P. mitarakaensis Bérenger & Blanchet, 2007and T. jatai Gonçalves et al., 2013 In the case of R. taquarussuensis sp. n. and R. neglectus, the difference in the male genitalia is the shape of the phallosome. With respect to the female external genitalia, differences between R. taquarussuensis sp. n. and R. neglectus could be observed on the dorsal, posterior and ventral sides, which differ from other 13 Rhodnius species, according to Rosa et al. (2014).
The eggs of R. taquarussuensis sp. n. and R. neglectus showed differences in the measurement of their length and opercular opening, the same as R. montenegrensis and R. marabaensis on the occasion of their description. As for the morphological characters, no differences were recorded. On the other hand, it should be noted that morphological differences were found by Barata (1981) in eggs of 10 Rhodnius species, by Santos et al. (2009) in three species, by Rosa et al. (2012) in the description of R. montenegrensis and by Santos et al. (2016) when describing R. marabaensis. Dujardin et al. (1999) established the geometric morphometry of the hind wings as a distinctive character among Triatominae while studying the sexual dimorphism of R. domesticus and T. infestans. The technique proved valid, for instance, to indicate the distinction between Mepraia spinolai and M. gajardoi; T. bahiensis and T. lenti; R. colombiensis, R. ecuadoriensis and R. pallescens; five populations of T. patagonica (Campos et al. 2011, Díaz et al. 2014, Nattero et al. 2016. However, even though that technique has contributed to distinguish even very close species, it showed no significant results to distinguish R. taquarussuensis sp. n. from R. neglectus. According to Justi and Galvão (2016) (Table 3).
Cytogenetic analyses of R. taquarussuensis sp. n. made it possible to describe the karyotype (2n = 22) and observe the constitutive heterochromatin pattern in the chro- Table 3. Distinguishing characters among twelve species of the group Rhodnius prolixus.

R. barretti
The third antennal segment appears to be relatively shorter. The scutellar process is narrowly pointed. Abad-Franch et al. 2013

R. dalessandroi
Antenniferous tubercle slightly pilose and with triangular glabrous depression in the upper region. Semicircular spot on the posterior end of the neck.

Carcavallo and Barreto 1976
R. domesticus Head comparatively long, distinctly longer that pronotum. Process of pygophore rectangular.

R. marabaensis
The second antennal segment is 10.3 times larger than the first. The scutellum is larger and includes two prominent internal lateral carinea.

R. milesi
The male genitalia presents a second process of the phallosoma. Divergent antennal tubercle with an apical denticle.

R. montenegrensis
Anterior wings with well-demarcated veins, notable the Sc by a yellow tonality. Abdomen presents yellow spots interposed with dark ones over the ventral abdomen lengthwise.

R. nasutus
Overall color light reddish brown, trochantera not contrasting conspicuously with femora. Median process of pygophore wide at base.

R. neglectus
Overall color dark brown, trochantera very light colored. Median process of pygophore narrow at base.

Lent and Wygodzinsky 1979
R. neivai Pronotum entirely dark brown or black, including the carine. Connexivum blackish, with very small reddish spots.

R. prolixus
Anteocular region slightly over three times as long as postocular. Distance between eyes dorsally larger than width of eyes in dorsal view.

R.robustus
Anteocular region about four times as long as postocular. Specimens distance between eyes dorsally smaller than, or equal to, width of eye in dorsal view.
Head with a prominent brown vertex contrasting with the black sides. The phallosome (Ph) has a broad plate whose superior region has a trapezoidal shape and occupies the middle region of the aedeagus.
This work *group R. prolixus according to Justi and Galvão 2016. mosomes (extremities of most autosomes), which are rich in CG. All the species in the tribe Rhodniini have 22 chromosomes (Alevi et al. 2013(Alevi et al. , 2015b. On the other hand, out of the 14 species of the genus Rhodnius whose chromosomes have been studied in the literature, only four show heterochromatic blocks in the autosomes, namely R. colombiensis, R. nasu tus, R. pallescens and R. pictipes (Dujardin et al. 2002). R. neglectus, which is a similar species for R. taquarussuensis sp. n., does not show heterochromatic blocks in the autosomes (Dujardin et al. 2002;Panzera et al. 2012;Alevi et al. 2015a).
Although the evolutionary process in triatomine is disruptive (Dujardin et al. 2009) and intraspecific chromosome variation has been described for R. ecuadoriensis (Pita et al. 2013), R. pallescens (Gómez-Palacio et al. 2008), P. geniculatus (Crossa-Pérez et al. 2002), T. dimidiata (Panzera et al. 2006) T. infestans (Panzera et al. 2004(Panzera et al. , 2012 and T. sordida (Panzera et al. 1997), generally the distribution of species is associated with different countries [for exemple, R. ecuadoriensis from Peru and Ecuador (Pita et al. 2013) and T. sordida from Brazil and Argentina (Panzera et al. 1997)] or different regions [for example, R. pallescens from North and West regions from Colombia (Gómez-Palacio et al. 2008) and T. infestans from Andean group and Non-Andean group (Panzera et al. 2004(Panzera et al. , 2012]. However, a population study was previously performed with R. neglectus (endemic species of Brazil) coming from different Brazilian states (Alevi et al. 2015a) and the authors pointed out that there is no intraspecific chromosome variation for this species. This fact and the morphological data described sustain the specific status of R. taquarussuensis sp. n., since the gain and loss of heterochromatin in the autosomes of Rhodnius are adaptive processes that can be linked to speciation processes, as recently noted for the group pallescens (Alevi et al. 2015c). Support and Evaluation of Graduate Education (CAPES), grant process number 23038-005285/2011-2012; Brazilian National Council for Scientific Technological Development (CNPq process number 142284/2015-7).