Immature instars of three species of Rhodnius Stål, 1859 (Hemiptera, Reduviidae, Triatominae): morphology, morphometry, and taxonomic implications

Among the 18 genera of the Triatominae subfamily, three stand out for their diversity and epidemiological importance: Triatoma, Panstrongylus, and Rhodnius. Rhodnius includes 21 species that can transmit Trypanosoma cruzi (the etiological agent of Chagas disease, also known as American trypanosomiasis) and Trypanosoma rangeli. The Rhodnius prolixus complex comprises seven species, including Rhodnius marabaensis, Rhodnius prolixus, and Rhodnius robustus, which occur in the northern region of Brazil. Since both adults and immatures can carry T. cruzi, in this study the five nymphal instars of the three species mentioned were dorsally characterized. Using microscopy, morphometrics, and geometric morphometrics, the present work measures and describes the morphological characters of the five nymphal instars of R. marabaensis, R. prolixus, and R. robustus. The study enabled the characterization of all five nymphal instars, as well as the distinction between the three species in each of their instars. The morphological, morphometrics of the head, thorax, and abdomen and geometric morphometrics studies of the head enabled the specific distinction of these three species in all five instars.

Species belonging to Rhodnius present well-defined morphological characters that facilitate their identification in the Triatominae subfamily, but distinguishing them from one another is a complex task [2,14]. The typical morphology of Rhodnius is characterized by the position of the antennal tubercle on the apex of the head and the absence of phallosome support in the genitalia of some species [2,12,14]. Their color tends towards dark/ light brown, with spots that can be sharp [2]. Nymphs are characterized by an elongated head, antennal tubercles located in the distal one-third or one-fourth of the anteocular area, absence of ocelli, and spotted abdomen on the back. Median tubercles are located dorsally on the central longitudinal line from segment I-X [2,9,[14][15][16]. Because of the related taxonomic difficulties and its epidemiological importance, Rhodnius is widely studied, yet its phylogeny has not been clarified and requires new studies [11,17].
The tribe Rhodniini (Rhodnius + Psammolestes Bergroth, 1911) consists of a monophyletic group of two genera naturally occurring in the Neotropical region [18]. Arboreal habits are common in the genus, and most are associated with one or more palm species. Among the species studied in this work, R. robustus is found in Bolivia, Colombia, Ecuador, Peru, and Venezuela, as well as northern Brazil [2,19]. In wild environments it is generally found in a variety of palm species, its presence having also been reported in domiciles and peridomiciles [20,21]. This species is also related to food contamination and infection of forestry workers [22,23]. Rhodnius robustus is very close to R. montenegrensis, but molecular studies have confirmed the specific status of each species [24,25].
Rhodnius marabaensis, described in 2016 from the state of Pará, has a straw color, and its dorsal thorax has a trapezoidal shape limited by a straw carina. Its lobes usually show a black-spot pattern. The larger length of the second antennal segment and the keel-shaped head apex are two of the main morphological features of adults [12]. Recently, R. marabaensis had its specific status validated by transposable element analysis [25], as well as its biological cycle [26]. It is a species found in the wild with moderate epidemiological importance [12].
Rhodnius prolixus is considered the most important species in the transmission of Chagas disease in Venezuela, Colombia, and Central America [2,19]. One of the factors that contribute to this is its optimal adaptation to human dwellings. It is similar to R. robustus, which makes the separation between them more difficult [27].
Taking all these considerations into account, this study aims to characterize R. marabaensis, R. prolixus, and R. robustus both morphologically and morphometrically, making it easier to distinguish the five nymphal instars of these three species. In addition to the epidemiological importance of the five nymphal instars, as they can carry T. cruzi and T. rangeli [9], the taxonomic validity of the study of their morphological characters must also be considered. Although the epidemiological importance of R. marabaensis is still unknown on account of its recent description, R. prolixus and R. robustus are important vectors of Chagas disease in the areas where they occur. Rondônia, Brazil. Nymphs of first, second, third, fourth, and fifth instars were taken from the respective colonies on the same day they were utilized. First-instar nymphs were selected right after egg hatching. Nymphs of second, third, fourth, and fifth instars were selected immediately after ecdysis (no abdominal distension from feeding). The nymphs were only fed (on ducks), every 7 days, to pass through the instars. The morphological and morphometric studies were conducted without verifying the gender distinction of the five nymphal instars.

Morphological study
To generate images, five specimens of each of the five instars of R. marabaensis, R. prolixus, and R. robustus were used. Images of the dorsal sides of the head, thorax, and abdomen as well as the complete images of each of the five instars from dorsal view were obtained using a Leica M205 stereoscopic microscope and Leica Application Suite X software.

Morphometric study
Fifteen specimens of first-, second-, third-, fourth-, and fifth-instar nymphs of R. marabaensis, R. prolixus, and R. robustus were measured using a Leica MZ APO stereoscopic microscope and the Motic Advanced 3.2 plus image analysis system.
The total length (TL), head length (HL), thorax length (XL), and abdomen length (AL) were measured for nymphs of all instars. Following Dujardin et al. [28], interocular (IO), anteocular (AO), and postocular (PO) distances were measured, as well as the three visible segments of the labium. The four antennal segments were also measured, according to Rosa et al. [29]. All measurements are expressed in millimeters.
The obtained data were analyzed by descriptive statistics, using t-tests for mean and standard deviation. Analysis of variance (ANOVA) and Tukey's pairwise comparison were performed to evaluate the degree of differentiation of the three species using PAST software (Additional file 1).

Geometric morphometrics of heads
Geometric morphometrics was used to evaluate variations in head shape and size using Cartesian reference coordinates. Variations among the heads of all nymphal instars of the studied species were evaluated. Fifteen heads of each instar were selected, and the images were obtained using a stereoscopic magnifying glass coupled to the Motic Advanced 3.2 plus scanning system. The coordinates of the reference points were selected according to Bookstein [30]. Four landmarks were adopted for the first and second instars and five landmarks for the other instars (Additional file 2). All of the landmarks are type 1 and were collected and processed using the modules available in the tpsDig v.1.18 software [31] and digitized using the CLIC package (https:// xyom-clic. eu/ the-clic-packa ge/). Then the file with the raw coordinates was used for a generalized Procrustes analysis (GPA). GPA is a method that allows all the information related to size, position, and orientation of previously digitized anatomical frames to be eliminated [31]. The matrix of form was held in Euclidean space to generate a set of marks known as partial warps [30]. All the additional statistical forms were performed using Procrustes residues to analyze differences in the size and shape of the heads of each nymphal instar (Additional file 3). Procrustes ANOVA (p < 0.0001) [32] is used to infer differences between species. Procrustes ANOVA is a method for quantifying relative amounts of variation at different levels. These differences in size were assessed using an isometric estimator defined as centroid size (CS) [33]. Mahalanobis distances between pairs of species were calculated for measurements of shape and significance was assessed using a non-parametric test based on permutations (bootstrap, 10,000 replications) using MorphoJ [34]. In addition to that, using distance dice from Mahalanobis, neighbor-joining trees (NJ) were recovered using PAST v.3.25 [35]. To determine the relationships between species, canonical variable analysis (CVA) was performed using MorphoJ [34]. The CVA was performed associated with a resampling method (bootstrap, 10,000 replications) to build regions of trust in relation to the median size of the species center. A factorial map of the first two canonical fathers was created using MorphoJ, version 1.0.7a [34] (Additional file 3).

Morphological description of the five nymphal instars of R. marabaensis, R. prolixus, and R. robustus by optical microscopy
First instar: the head of the nymphs has a dark-brown cuticle covering all its granular extension due to the presence of tubercles with small setae, whose color is darker than that of the cuticle. The maxillary plate and mandibular plate showed no significant differences among the species. Regarding the postocular area, the species shows Y-shaped cephalic sutures (Fig. 6a-c). On the thorax, there are tubercles with setae in the three segments, located mainly in the center. The pronotum has a trapezoidal shape and is the segment with larger external borders, followed by the metanotum and the mesonotum. The three segments are well delimited by the dividing lines, but the line separating the mesonotum from the metanotum shows a sinuous protuberance that overlaps the metanotum in about one-third of its size ( Fig. 6d-f ). The abdomen of first-instar nymphs has a lighter color in comparison with the thorax and the head. There are many tubercles with setae lighter colored than the cuticle. Connexivum with darker spots along the margin surrounding the abdomen. A lighter median longitudinal stripe is evident all over the abdomen (Fig. 6g- Second instar: the general aspects of the head of second-instar nymphs are similar to those described for first-instar nymphs. However, some differences are noticeable, such as the increase in the granulation grade and the number of setae in the three species, as well as the lighter color of the cuticle (Fig. 7a-c). Tubercles with setae are present in the three segments of the thorax, located mainly in the central portion. It is not possible to quantify the difference in size between the mesonotum and the metanotum, but, as in the first instar, the metanotum is broad on the sides and narrow in the central portion. The three segments are well delimited by the dividing lines, but the line separating the mesonotum from the metanotum has a sinuous protuberance that overlaps the metanotum in about one-third of its size ( Fig. 7d-f ). The abdomen has a median spot lighter than the cuticle in the dividing line of each of the urotergites, resembling a stripe. The connexival spots are more evident in this instar (Fig. 7g-i).
Third instar: for the three species, the maxillary plates are more rounded and extend until the end of the clypeus. Postocular cephalic sutures are also more rounded and roughly have a U-shape (Fig. 8a-c). Tubercles with setae are present in the three segments of the thorax, distributed across them. The pronotum has the shape of a trapezium and is the segment with the largest external borders, followed by the mesonotum and the metanotum. The three segments are well delimited by the dividing lines, but the line separating the mesonotum from the metanotum has a sinuous protuberance that overlaps the metanotum in about one-third of its size (Fig. 8d-f ). The prolixus; c, f, i: R. robustus; cs cephalic suture, tu tubercle, ao anteocular distance, po postocular distance, mt metanotum, ms mesonotum, pr pronotum, I-X abdominal segments three species possess 2 + 2 dark stripes across the abdomen. An increase of spots in the connexivum can also be observed. In R. marabaensis abdominal segments III and IV are the broadest. The segments widen from segment I to III and gradually narrow from segment VI onwards. A central stripe of straw color is also easily visible on the abdomen of this species, and from the sides there is another straw-colored stripe, located between two black stripes. These three stripes are arched and have the same shape as the abdomen, but they are not continuous, as they are interrupted in the intersegmental sutures. In R. prolixus and R. robustus the largest abdominal segment is the IV (Fig. 8g-i).
Fourth instar: fourth-instar nymphs present some peculiar characteristics, such as triangular mandibular plate, rounded maxillary plate surpassing the clypeus, and a higher granulation grade near the eyes (Fig. 9a-c). The three segments of the thorax have tubercles with setae. The mesonotum is the largest segment in this instar due to the presence of the first pair of wing pads. The second pair of wing pads originates from the metanotum. The three segments are well delimited by the dividing lines ( Fig. 9d-f ). In all three species the dark stripes on the abdomen are more evident, which gives the area a striped aspect. In this instar, the connexivum spots become more rounded. The central stripe on the abdomen has the same aspect as in the third instar and differentiates the three species: in R. marabaensis the three side stripes on the abdomen, a straw-colored stripe between two black ones to the right and left, are similar to what is observed in the third instar (Fig. 9g-i).
Fifth instar: in this instar, all three species also have a quite visible white stripe on the head (Fig. 10a-c). There are tubercles with setae in the three segments of the thorax. The posterior pair of wing pads can be seen overlapping, projecting from the mesonotum through the anterior pair, which in turn projects from the mesonotum (Fig. 10d-f ). It is possible to see only the central area of the metanotum as a result of this large overlapping. The anterior pair of wing pads reach the beginning of the third urotergite. In this instar, the central line of the abdomen retains the characteristics observed in the third and fourth instars. In R. marabaensis the three side stripes on the right and left (one straw and two black) observed in the third and fourth instars are still present. There is also an increase in the number of tubercles with setae ( Fig. 10g-i).
In this study, morphological differences were also observed between the three species in their five nymphal instars (Table 1).

Morphometric study of the five nymphal instars of R. marabaensis, R. prolixus, and R. robustus
With the acquired data it was possible to calculate the mean for each parameter and species, and then compare them to evaluate the degree to which the three Rhodnius species differ.
In the first and second instars, none of the parameters were statistically significant to evaluate the degree of the differences among the three Rhodnius species. As for the third instar, the parameter of the third segment of the antenna (F (2,42) 23.12, P = 1.693) was significant ( Table 2). In the fourth instar, only the postocular distance stands out (F (2,42) 13.64, P = 2.718) ( Table 3). Lastly, on the fifth instar, just the second segment of the antenna (F (2,42) 36.32, P = 6.965) ( Table 2) made it possible to evaluate the degree of the difference between R. marabaensis, R. prolixus, and R. robustus. By ontogenetic geometric morphometry of the heads of nymphs, it was possible to describe the differences in shape and size of the five instars of R. robustus, R. prolixus, and R. marabaensis. CS measures show variability in the head size of the species. Furthermore, by the isometric measurement of the CS, the size gain among immature shapes can be clearly seen (Fig. 11). Analysis of the CS shows that differences among the size means are significant (p < 0.0001, supplementary material). Rhodnius robustus and R. prolixus have larger means than R. marabaensis (Fig. 11). Differences can also be explained as a percentage of the total variance among groups in the eigenvalues (auto values), the percentages being 89% for the first instar, 83% for the second, 98% for the third, 93% for the fourth, and 90% for the fifth. Mahalanobis distance was used as a metric estimator. The estimator considers the variations and correlations among groups defined a priori and enables pairwise comparison. Mahalanobis distances were significant among the pairs of the assessed species (p < 0.001, supplementary material). Dendrograms were built based on the values recovered for Mahalanobis distances and neighbor joining (NJ). The topology is identical for all instars (Fig. 12). It was possible to delimit the proximity between R. prolixus and R. marabaensis (Fig. 12). Procrustes ANOVA test also recovered significant values, showing shape differences among the species (p < 0.0001, supplementary material).
The projection of the three species in the space defined by canonical axes 1 (CVA1) and 2 (CVA2) provides a description of the specified groups in the set of multivariate data. The analyses of the canonical variables resulted Fig. 9 Dorsal view of fourth-instar nymphs. Head, thorax, and abdomen. a, d, g: R. marabaensis; b, e, h: R. prolixus; c, f, i: R. robustus; ne neck, ju mandibular plate, ge maxillary plate, cl clypeus, ao anteocular distance, po postocular distance, mt metanotum, ms mesonotum, pr pronotum, I-X abdominal segments in 10 variables and explain 100% of the discrimination among the species (Fig. 13). The first two variables (CVA1 and CVA2) generated the following percentages: 85.2% and 22.49% for the first instar; 47.76% and 21.48% for the second; 97.1% and 3% for the third; 92.8% and 3% for the fourth; and 85.2% and 22.49% for the fifth (Fig. 13). The grouping in the space of the canonical axis shows an overlapping relationship between R. prolixus, R. robustus, and R. marabaensis in the first and fourth instar; however, the separation of populations in the second, third, and fifth instars is clear. R. marabaensis is the species that was best separated in the CVA analysis.

Discussion
A striking feature of Triatominae is that males, females, and nymphs of all instars can transmit T. cruzi if infected [36,37]. In several field collections carried out between 1989 and 2021 by Rosa et al. [unpublished data], a higher prevalence of nymphs than adults was observed. Thus, studies regarding the specific identification of nymphal instars become necessary, which is the objective of this study. Therefore, studies about nymphal instars not only have taxonomic and phylogenetic interest, but also have epidemiological importance. Specifically, about the genus Rhodnius, the following works can be mentioned: Mascarenhas [38], which studied the five instars of R. brethesi; Ponsoni et al. [39] and Marconato et al. [40], which carried out a biometric study of nymphs of R. neglectus Lent, 1954    Morphological characters are useful tools for taxonomic and systematic studies in Triatominae, in addition to being useful for epidemiological surveillance. The morphological analyses showed the separation of the three species by characters observed on the head, thorax, and abdomen shape. This made it possible to separate them in all five nymphal instars and to characterize for the first time the development instars of R. marabaensis. In their   chapter about nymphal instars, Lent and Wygodzinsky [2] mentioned that R. prolixus and R. robustus do not have sub median tubercles or aggregations of granules along the midline, but such characters were observed in all five nymphal instars of those species, as well as in R. marabaensis. Rosa et al. [37], studying first-and secondinstar nymphs of Triatoma wygodzinskyi Lent, 1951, distinguished the two instars by morphological characters of the thorax. Thus, by scanning electronic microscopy, they observed the absence of collar, glabrous areas, and tubercles in the first instar of T. wygodzinskyi, which were present in the second instar. Nevertheless, the differentiation among third, fourth, and fifth instars of R. marabaensis, R. prolixus, and R. robustus was made using the same characters observed by Rosa et al. [37] in nymphs of the previously mentioned instars of T. wygodzinskyi, i.e., the formation and conformation of the two pairs of wing pads located on the thorax. Although the ventral surface was not described, it is very likely that in future studies, new differential characters between the three species will be added to those already described in this work.
In this study, the results of the morphometry of characters from the abdomen, antenna, head, labium, and thorax showed little discrimination between the three species. In general, the compared averages are small or not significant, and the morphometric study is not suitable for identification. However, R. marabaensis had its nymphs characterized morphometrically and morphologically for the first time.
The relative length of the four antennal segments in R. marabaensis showed the same pattern for the first three instars, another for the fourth instar, and a third pattern for the fifth instar, whereas R. prolixus and R. robustus showed the same pattern for the first and second instars, another for the third and fourth instars, and a third one for the fifth instar. Santos [41], measuring R. colombiensis, R. ecuadoriensis, and R. stali, found two patterns of relative length for antennal segments of the five nymphal instars. For R. milesi, the author found three patterns, one for the first and second instars, another for the fourth and fifth instars, and a third one for the third instar, hence different patterns from the ones observed in R. marabaensis, R. prolixus, and R. robustus.
Rosa et al. [42] carried out a morphometric study of the four antennal segments of nymphs of the five instars and adults of Panstrongylus megistus (Burmeister, 1835),   [42]. However, in relation to the relative length of the four antennal segments, it is not possible to differentiate the studied species. The different results were described for R. colombiensis, R. ecuadoriensis, R. milesi, R. stali [41], and R. neglectus [42], T. rubrovaria [29], P. megistus, and T. vitticeps [42]. Furthermore, our data show that R. prolixus and R. robustus are like R. neglectus [42] and can be distinguished from R. colombiensis, R. ecuadoriensis, R. milesi, and R. stali [41] as well as T. rubrovaria [29], P. megistus, and T. vitticeps [42] for this characteristic. Geometric morphometry enables the evaluation of the variation in shape in relation to causal effects [43]. The technique allows us to quantify biological forms and discuss the evolution of phenetic patterns [34]. The technique is used in paleontological, anthropological, ecological, zoological, and botanical studies [30,34]. In triatomines, geometric morphometry is used to assess the shape and size variables of hemelytra [44,45], heads [13,46], and eggs [47]. It is also used for ontogenetic studies [48][49][50].
Recently two subcomplexes of the genus Triatoma Laporte, 1832 were studied using geometric morphometrics, which indicated the potential of the technique to study specimens that are phylogenetically close [44,46]. Geometric morphometrics allowed the differences in head shape and size of the five nymphal instars to be described. In relation to the CS, all values obtained were significant and enabled the differentiation of the three species in the five nymphal instars. Variation was observed among the instars, but considering the general aspect, R. robustus was easily characterized by the geometric profile of the heads of nymphs. The second and fourth instar showed less discrimination potential, i.e., only approximated size means were recovered.
The metric estimator of Mahalanobis distance was used to recover NJ dendrograms, where it is possible to visualize that in all evaluated instars R. robustus is distant, whilst R. prolixus and R. marabaensis form a single clade. However, CVA ellipses showed that in the first and second instars, R. marabaensis and R. robustus remain close, while groups are clearly separated in the third, fourth, and fifth instars. Regarding the shape, the values of the Procrustes ANOVA test revealed differences among the cephalic capsules, enabling discrimination. It was shown that the multivariate morphometric technique is more efficient for discriminating against the studied species when confronted with linear morphometric data.

Conclusion
In this study, the morphological and morphometric differences in three Rhodnius species were evaluated. New data were also provided for R. marabaensis. Furthermore, it was shown that the morphology of the head (third, fourth, and fifth), thorax (second and fifth instar), and abdomen (first, second, third, and fifth instar) are useful in discriminating the studied species. Through morphometric analysis of the head, it was verified that the postocular distance of the fourth instar and the lengths of the antennal segments of the third and fifth instars distinguish the three species. Lastly, geometric morphometry proved to be useful for these species. The size and shape variables clearly show the differences between R. marabaensis, R. prolixus, and R. robustus.