Characterization of Female External Genitalia and Eggs of Four South American Species of the Triatoma Laporte, 1832 Genus (Hemiptera: Reduviidae: Triatominae)

Simple Summary We present a morphological and morphometric study with T. garciabesi, T. guasayana, T. patagonica, and T. sordida sensu stricto species within the Triatoma genus. This group of species is important for the eco-epidemic scenario of Chagas disease in the Americas; their species have morphological, biological, and behavioral similarities that make diagnosis difficult. For the first time, the description of the female external genitalia by scanning electron microscopy (SEM), a character that has helped with the delimitation of species and genera in Triatominae, is published, in addition to presenting an extensive study with eggs, covering morphology and morphometry. The study with eggs is an important tool in taxonomic studies of the subfamily. In addition to taxonomic contributions, it was possible through the descriptions to corroborate the validity of T. garciabesi and confirm the current classification of these species. Abstract Triatoma is the most diversified and one of the most important genera from an epidemiological perspective. Given the difficulty in identifying some species of the Triatoma genus, morphological, histological, and morphometric studies were performed to provide new characters that make it possible to differentiate T. garciabesi, T. guasayana, T. patagonica, and T. sordida sensu stricto, triatomines that overlap geographically and have vector potential. Through the external female genitalia, as well as morphology, morphometry, and histology of eggshells, it was possible to discriminate the four species. In addition, this study reinforces the taxonomic validity of T. garciabesi and provides new data for discussion on systematic issues of T. guasayana and T. patagonica.


Morphologic Study of External Female Genitalia and Eggshells
The images female external genitalia (n = 5) and the eggshells (n = 5) of the fo species were produced by scanning electron microscopy (SEM) according to Rosa et [25]. After death by freezing in −20 °C, the specimens were dissected from the six segment to acquire the final portion of the abdomen, with the female external genital Subsequently, the dissected structures were washed, dehydrated with an alcohol serie and oven dried for 30 min at 50° C. The female external genitalia were examined in t dorsally, posteriorly, and ventrally. The eggshells were also subjected to washin dehydration, and drying. Subsequently, they were fixed on aluminum stubs. The sampl of the female external genitalia and the eggshells were by sputtering with gold for 2 m at 10 mA, as described in Rosa et al. [25]. Subsequently, the female external genita (dorsal, posterior, and ventral views) and the eggshells (full and exochorion aspect) we examined with a Topcon SM 300 SEM at the Institute of Chemistry at Unesp, Araraquar São Paulo, Brazil. The images obtained were processed (background, contrast, brightnes in the GNU Image Manipulation Program v2.0.2 (GIMP) software free and open-sour image editor, subsequently the structures were described, and the descriptions we compared.

Histological Analysis
The eggshell histological procedures were conducted at the Laboratory Histological Processing of the Dental School, Unesp, Araraquara, São Paulo, Bra following a protocol adapted from Obara et al. [26]. Five hatched eggs of each species we

Morphologic Study of External Female Genitalia and Eggshells
The images female external genitalia (n = 5) and the eggshells (n = 5) of the four species were produced by scanning electron microscopy (SEM) according to Rosa et al. [25]. After death by freezing in −20 • C, the specimens were dissected from the sixth segment to acquire the final portion of the abdomen, with the female external genitalia. Subsequently, the dissected structures were washed, dehydrated with an alcohol series, and oven dried for 30 min at 50 • C. The female external genitalia were examined in the dorsally, posteriorly, and ventrally. The eggshells were also subjected to washing, dehydration, and drying. Subsequently, they were fixed on aluminum stubs. The samples of the female external genitalia and the eggshells were by sputtering with gold for 2 min at 10 mA, as described in Rosa et al. [25]. Subsequently, the female external genitalia (dorsal, posterior, and ventral views) and the eggshells (full and exochorion aspect) were examined with a Topcon SM 300 SEM at the Institute of Chemistry at Unesp, Araraquara, São Paulo, Brazil. The images obtained were processed (background, contrast, brightness) in the GNU Image Manipulation Program v2.0.2 (GIMP) software free and open-source image editor, subsequently the structures were described, and the descriptions were compared.

Histological Analysis
The eggshell histological procedures were conducted at the Laboratory of Histological Processing of the Dental School, Unesp, Araraquara, São Paulo, Brazil following a protocol adapted from Obara et al. [26]. Five hatched eggs of each species were selected, and the shells were washed in acetone using ultrasound (40 kHz for 3 min) and dried at 40 • C for 30 min. The eggshells were transferred to small plastic cassettes and submitted to histological processing in a "MORSE" descaling solution (formic acid + sodium citrate) for 15 min. With the aid of a scalpel, the shells were split in half under a surgical magnifying glass. The fragments were washed in running water for 1 h, dehydrated in a graded ethanol series (2 h), diaphanized in xylene for 1 h, and embedded in liquid paraffin at 60 • C (2 h).
Subsequently, the samples were cut with a LEICA RM2145 ® microtome, and 5 µm-thick sections were stained with Harris' Hematoxylin (10 min) and Eosin (5 min). The stained sections were mounted with Permount mounting medium and observed under a Leica microscope © Leitz DM RXE with a digital camera.

Morphometric Study Morphometric Analysis of Eggshells
The total length and perimeter of the opercular opening of the eggs were measured based on classical morphometric methods [27,28]. For eggshell morphometry, 50 eggs were selected from T. garciabesi, T. guasayana, T. patagonica, and T. sordida sensu stricto. Variations were estimated by ANOVA and Tukey's post-hoc test (p < 0.05%) and were computed using GraphPad Prism (Graphpad Software v. 8.00 for Windows, San Diego, CA, USA).

Female External Genitália
The female external genitalia of T. garciabesi, T. guasayana, T. patagonica, and T. sordida sensu stricto are described for the first time. Among the species studied, the female external genitals have different characters in the three positions evaluated (dorsal, ventral, and posterior), allowing specific identification. The main differences in female external genitalia are compiled in Table 1.

. Dorsal View of Female External Genitalia
The borders (with a pair) between the segment VII and the connective are straight in T. garciabesi and T. guasayana (Figure 2A,B), in T. patagonica and T. sordida sensu stricto the lower portion is slightly curved ( Figure 2C,D).
Posterior portions (with a pair) of the VII segment at the limit with the VIII segment and connexiva end as triangular tips in T. garciabesi, T. guasayana, T. patagonica, and T. sordida sensu stricto ( Figure 2). The VIII segment has a trapezoid shape in the four species, in T. guasayana the lower part is curved in the others the angles are straight.
Segment IX has a trapezoidal shape in the four species, but in T. guasayana the region is lower and wider ( Figure 2E-H) in, T. patagonica is slightly curved on the sides ( Figure 3). Posterior portions (with a pair) of the IX segment in T. guasayana have the shape of rounded tips ( Figure 2B). The posterior portion of segment X is obtuse in T. garciabesi, T. guasayana and T. sordida sensu stricto and flat in T. patagonica ( Figure 2). in T. guasayana the lower part is curved in the others the angles are straight. Segment IX has a trapezoidal shape in the four species, but in T. guasayana the region is lower and wider ( Figure 2E-H) in, T. patagonica is slightly curved on the sides ( Figure  3). Posterior portions (with a pair) of the IX segment in T. guasayana have the shape of rounded tips ( Figure 2B). The posterior portion of segment X is obtuse in T. garciabesi, T. guasayana and T. sordida sensu stricto and flat in T. patagonica ( Figure 2).

Posterior View of Female External Genitalia
In T. patagonica and T. garciabesi the border of region dividing the VIII and IX segments is convex (cs) and flat in T. guasayana and T. sordida sensu stricto ( Figure 3). In the four species the limits between the IX and X segments are open on the sides and closed in the central area ( Figure 3); moreover, in T. patagonica the region is very curved compared to the other species. The lateral borders of the IX are curved and open close to the VIII in T. garciabesi, T. guasayana, and T. sordida sensu stricto, unlike T. patagonica that close. The X segment are different between species, the border between IX and X are determinant for the differences, in T. garciabesi in T. sordida sensu stricto it is semicircular (sc) in T. gasayana it is slightly flat (fs) and in T. patagonica it is considerably curved elongated ( Figure 3).

Posterior View of Female External Genitalia
In T. patagonica and T. garciabesi the border of region dividing the VIII and IX segments is convex (cs) and flat in T. guasayana and T. sordida sensu stricto ( Figure 3). In the four species the limits between the IX and X segments are open on the sides and closed in the central area ( Figure 3); moreover, in T. patagonica the region is very curved compared to the other species. The lateral borders of the IX are curved and open close to the VIII in T. garciabesi, T. guasayana, and T. sordida sensu stricto, unlike T. patagonica that close. The X segment are different between species, the border between IX and X are determinant for the differences, in T. garciabesi in T. sordida sensu stricto it is semicircular (sc) in T. gasayana it is slightly flat (fs) and in T. patagonica it is considerably curved elongated ( Figure 3). According to the images obtained, T. sordida sensu stricto presents a greater number of bristles in relation to the others.

Ventral View of Female External Genitalia
In the four species, the border of the VII segment with Gc8 and Gp8 is concave on the sides (with a pair) and convex in the central portion ( Figure 4). Posterior portions of the connexiva of the VII segment end in pointed tips (with a pair) in T. garciabesi, T. guasayana, and T. patagonica ( Figure 4A-C) in T. sordida sensu stricto is not present ( Figure 4D). The Gc8 shape of T. guasayana and T. patagonica is triangular and in T. garciabesi and T. sordida sensu stricto it is triangular and obtuse. Gp9 is similar in all species, however in T. sordida sensu stricto it is smaller. (Figure 4). In T. garciabesi, T. patagonica and T. sordida sensu stricto the IX forms an isosceles triangle, while in T. patagonica the shape refers to a slightly obtuse equilateral triangle. ( Figure 4B). The IX is apparent in all species: T. garciabesi the segment shape is triangular and well angled, T. gasayana the segment is very visible and has the obtuse final portion, T. patagonica the region is triangular, and the outer edges are curves, and T. sordida sensu stricto the segment is triangular with many bristles, these bristles also extend throughout the central region of VII, X, and through Gc8. According to the images obtained, T. sordida sensu stricto presents a greater number of bristles in relation to the others.

Ventral View of Female External Genitalia
In the four species, the border of the VII segment with Gc8 and Gp8 is concave on the sides (with a pair) and convex in the central portion ( Figure 4). Posterior portions of the connexiva of the VII segment end in pointed tips (with a pair) in T. garciabesi, T. guasayana, and T. patagonica ( Figure 4A-C) in T. sordida sensu stricto is not present ( Figure 4D). The Gc8 shape of T. guasayana and T. patagonica is triangular and in T. garciabesi and T. sordida sensu stricto it is triangular and obtuse. Gp9 is similar in all species, however in T. sordida sensu stricto it is smaller. (Figure 4). In T. garciabesi, T. patagonica and T. sordida sensu stricto the IX forms an isosceles triangle, while in T. patagonica the shape refers to a slightly obtuse equilateral triangle. (Figure 4B). The IX is apparent in all species: T. garciabesi the segment shape is triangular and well angled, T. gasayana the segment is very visible and has the obtuse final portion, T. patagonica the region is triangular, and the outer edges are curves, and T. sordida sensu stricto the segment is triangular with many bristles, these bristles also extend throughout the central region of VII, X, and through Gc8.

Morphology of Eggs
Through SEM and light microscopy, it was possible to describe the architecture of the ultrastructure and the general appearance of the eggs of the four species. In general, the eggs analyzed are ellipsoidal in the four species studied ( Figure 5). The four species show longitudinal chamfers that are very apparent in T. guasayana, T. patagonica, and T. sodida and less apparent in T. garciabesi ( Figure 5).
The surface of exochoria in SEM is different between species. In general, the predominant shapes are pentagons, hexagons, and heptagons. In T. garciabesi, T. guasayana and T. sordida sensu stricto the shapes of the exochorion surface are varied without a defined pattern, while in T. patagonica the shapes are hexagonal. The profile of the limiting lines is different between species, it can be seen that in T. garciabesi and T. guasayana the lines are deep and define a quilted appearance for the exochorial structures, while in T. patagonica and T. sordida sensu stricto the lines are shallow and offer a flat appearance for exochorion Insects 2021, 12, 537 7 of 14 ( Figure 6). In the four species, numerous perforations can be observed scattered throughout the exochorion (Figure 6).

Morphology of Eggs
Through SEM and light microscopy, it was possible to describe the architecture of the ultrastructure and the general appearance of the eggs of the four species. In general, the eggs analyzed are ellipsoidal in the four species studied ( Figure 5). The four species show longitudinal chamfers that are very apparent in T. guasayana, T. patagonica, and T. sodida and less apparent in T. garciabesi ( Figure 5).
The surface of exochoria in SEM is different between species. In general, the predominant shapes are pentagons, hexagons, and heptagons. In T. garciabesi, T. guasayana and T. sordida sensu stricto the shapes of the exochorion surface are varied without a defined pattern, while in T. patagonica the shapes are hexagonal. The profile of the limiting lines is different between species, it can be seen that in T. garciabesi and T. guasayana the lines are deep and define a quilted appearance for the exochorial structures, while in T. patagonica and T. sordida sensu stricto the lines are shallow and offer a flat appearance for exochorion ( Figure 6). In the four species, numerous perforations can be observed scattered throughout the exochorion ( Figure 6).

Histological Analysis of Eggs
The egg sections of the four species were analyzed using optical microscopy ( Figure  7). Photomicrographs make it possible to identify four layers of tissue. The internal tissues: soft endochorion and the stiff endochorion ( Figure 7) and the external tissues, the stiff exochorion and the soft exochorion ( Figure 7).

Histological Analysis of Eggs
The egg sections of the four species were analyzed using optical microscopy ( Figure 7). Photomicrographs make it possible to identify four layers of tissue. The internal tissues: soft endochorion and the stiff endochorion ( Figure 7) and the external tissues, the stiff exochorion and the soft exochorion ( Figure 7). Different textures can be observed between the studied tissues: the endochoria of T. garciabesi and T. patagonica (internal part) is smooth ( Figure 7A,C), while in T. sordida sensu stricto and T. guasayana is wavy ( Figure 7B,D). The profile of the egg exochoria is wavy in the four species, probably because of the hexagonal and pentagonal ornamentation of the exochoria. In the photomicrographs it is possible to perceive the differences in the thickness of the tissues: T. garciabesi and T. patagonica are thin and relationship and T. guasayana and T. sordida sensu stricto thick. There are also differences in the thickness of the stiff exochorion, in guasayana and T. sordida are thick in T. garciabesi and T. patagonica are less apparent.

Morphometry of Eggs
The two measured parameters show the metric variability between eggs of the four studied species (Figure 8). Regarding the total length of the eggs, T. patagonica presents the largest average size with 1.77 ± 0.05 mm followed by T. sordida sensu stricto 1.72 ± 0.05 mm. T. garciabesi with 1.70 ± 0.09 mm and T. guasayana 1.61 ± 0.09 mm have a lower mean total length of the eggs. T. garciabesi and T. sordida sensu stricto have statistically similar total egg length (Table 2). Therefore, the relation according to the egg length is: T. patagonica > T. sodida sensu stricto > T. garciabesi > T. guasayana (Figure 8). exochoria. In the photomicrographs it is possible to perceive the differences in the thickness of the tissues: T. garciabesi and T. patagonica are thin and relationship and T. guasayana and T. sordida sensu stricto thick. There are also differences in the thickness of the stiff exochorion, in guasayana and T. sordida are thick in T. garciabesi and T. patagonica are less apparent.

Morphometry of Eggs
The two measured parameters show the metric variability between eggs of the four studied species (Figure 8). Regarding the total length of the eggs, T. patagonica presents the largest average size with 1.77 ± 0.05 mm followed by T. sordida sensu stricto 1.72 ± 0.05 mm. T. garciabesi with 1.70 ± 0.09 mm and T. guasayana 1.61 ± 0.09 mm have a lower mean total length of the eggs. T. garciabesi and T. sordida sensu stricto have statistically similar total egg length (Table 2). Therefore, the relation according to the egg length is: T. patagonica > T. sodida sensu stricto > T. garciabesi > T. guasayana (Figure 8). The perimeter of the opening of the operculum showed greater capacity to discriminate eggs among the four species. The largest mean size of the perimeter of the opening   The perimeter of the opening of the operculum showed greater capacity to discriminate eggs among the four species. The largest mean size of the perimeter of the opening of the operculum was T. sordida sensu stricto 1.83 ± 0.10 mm, followed by T. patagonica with 1.5 5 ± 0.07 mm, the averages of T. garciabesi and T. patagonica were lower 1.47 ± 0.10 mm and 1.42 ± 0.08 mm, respectively ( Table 2). The morphometric relationship of the perimeter opening of the operculum from the largest to the smallest is: T. sordida sensu stricto > T. patagonica > T. garciabesi > T. guasayana.
The variables of length and perimeter of the operculum forma the estimates for the levels of significance. The four species show significant differences between the averages. However, T. garciabesi and T. sordida sensu stricto do not show differences in length averages (Table 3). Table 3. Results of Turkey's post-hoc test for length (bold) opening of the perimeter of the operculum (italic).

Species
T

Discussion
In the subfamily Triatominae, the Triatoma genus currently has the largest number of species, with its complexes and subcomplexes [9] clearly being the most diverse of the subfamily [6]. Discussions about species of Triatoma deserve attention, especially in view of the eco-epidemic scenario in which are inserted [29]. In this study, we present new taxonomic characters for four species of the genus: T. garciabesi, T. guasayana, T. patagonica, and T. sordida sensu stricto, in addition, the female external genitalia and histology of eggs are presented with new diagnostic characters.
Although the recent phylogenies [7] show a distancing of T. guasayana and T. patagonica from T. garciabesi and T. sordida sensu stricto, these species have similar morphology and biology, in addition they overlap geographically by several South American regions where they have vectorial potential and consequently deserve attention [10,11,13]. Triatoma garciabesi has been described from specimens captured in central Argentina [30] but synonymized with T. sordida by Lent and Wygodzinsky [17]. Through morphological characters of the head, male genitalia, izoenzymes, and cytogenetics, the species was revalidated in 1998 [13]. In the present study, the results allowed to differentiate T. garciabesi from the other studied species, despite the morphological similarities shared mainly with T. sordida sensu stricto, the results corroborate the revalidation. We demonstrate that it is possible to discriminate the species by the characters of the external female genitalia and by the morphometry of the eggs, however the morphology of the eggs presents similarities that make the diagnosis difficult. We demonstrate that it is possible to differentiate the species based on the morphological differences of the female external genitalia evaluated in dorsal, posterior, and ventral view. The dorsal and ventral position has a greater number of characters useful for discriminating these triatomines. In the dorsal position, IX and X have different shapes, being similar in T. garciabesi and T. sordida stricto sensu. In the ventral view, Gc8 are determinants for taxonomy of the studied species. The characterization of the female external genitalia by SEM in Triatominae allows diagnosis at the level of gender [31] and species, in this case assisting in the diagnosis of nearby taxons [2,25,32]. The female external genitalia were used in a study with 10 species of the T. brasiliensis subcomplex [9] corroborating the diagnosis of the studied species, in addition to helping in inferring the group's systematic relationships [32].
Eggs of triatomines possess characteristics that are useful in taxonomic studies [27,33]. In this study, the morphological and morphometric data of the eggs proved to be useful to discriminate the four species studied. The results show that the eggs of T. patagonica are the ones that show the greatest morphological and morphometric differences, Triatoma guasayana presents eggs of shorter length when compared to the studied species in addition to morphological differences. Triatoma garciabesi and T. sordida sensu stricto have morphology and morphometric similarities in eggs, however the histological profile is different.
By comparing the results of this study with previous publications, it was possible to determine morphological relationships between the eggs of the species studied with others of Triatoma. The characteristics described for eggs of T. guasayana and T. patagonica allow to show that they present morphological similarities with eggs of species of the T. rubrovaria subcomplex. The results show that the eggs of T. patagonica are the ones that present the greatest morphological and morphometric differences, the elongated profile is similar to the published descriptions for eggs of the species Triatoma rubrovaria (Blan-chard, 1846) and Triatoma klugi Jurbergi, Lent and Galvão, 2001 [34,35]. The approximate average length of the T. guasayana eggs are like those described for Triatoma circummaculata (Stål, 1859) and Triatoma carcavalloi Jurberg, Rocha and Lent, 1998, species of the T. rubrovaria subcomplex [34,36]. It was also possible to show that the morphology of T. sordida sensu stircto eggs is similar to the morphology of T. matogrossensis Leite e Barbosa, 1953; T. vandae Carcavallo et al., 2002 and T. jurbergi Carcavallo, Galvão and Lent, 1998 [36-38]. Triatoma matogrossensis, T. vandae, and T. jurbergi were recently inserted together with the T. sordida subcomplex [7,8].
The proximity of T. guasayana and T. patagonica it was first discussed by Abalos and Wygodzinsky [14] classifying both as allopatric species. Usinger et al. [39] suggest the proximity between T. guasayana and T. patagonica to T. sordida. At present we show that the studied species present morphological and morphometric differences that can corroborate the hypotheses of Pita et al. [8] and Belintani et al. [7].
The evaluation of the eggs exochoria by SEM reveals an ornamentation with homogeneous geometric shapes, a common characteristic of the Triatoma genus as demonstrated by Barata [27]. In Triatomines, eggs have been explored in different approaches and have been shown to be an excellent taxonomic character [34,[36][37][38]. The distinct sublayers of the chorion were first described by Beament [40] used eggs of Rhodnius prolixus Stål, 1859. Beament [40] makes observations about the composition of each layer and the process of formation of the geometric ornamentation present in the chorion. Barata [33] reiterate the histological descriptions by observing eggshells of ten Rhodnius Stål, 1859 species. Recently, the technique was used by Obara et al. [26] where it was possible to characterize the histological profile of eggs of six species of the Triatoma. In this study, it was possible to characterize the histological sections of the chorion of eggs of the four species for the first time. The photomicrographs of the histological sections did not allow the structures to be observed in great detail; however, it was possible to observe important characteristics, such as the four layers that form the egg chorion and their differences.

Conclusions
Bearing in mind that the four species studied are important for the current scenario of Chagas disease in South America, this study shows that the morphology and especially the morphometry of eggs are useful characters for differentiation between the studied species. Moreover, the female external genitalia by SEM are useful for taxonomy of these triatomines, especially visualized in the dorsal and ventral position. Finally, useful characters for the diagnosis and specific discrimination between T.garciabesi, T. guasayana, T. patagonica and T. sordida sensu stricto are presented, in addition, relevant systematic information is presented, confirming the current classification of these species.