Rhodnius micki, a new species of Triatominae (Hemiptera, Reduviidae) from Bolivia

Abstract Rhodnius Stål, 1859 is the second largest genus of Triatominae after Triatoma Laporte, 1832, and includes several important Chagas vectors. Genitalia in Reduviidae are frequently used for species identification, but the current use of terminology for it is inconsistent in Triatominae. Here, Rhodnius mickisp. nov., is described from Bolivia and considered as belonging to the pictipes group based on its morphological characters and distribution. Detailed documentation of the genitalia of Rhodnius mickisp. nov. is provided with emphasis on its everted phallus, especially the endosomal sclerites, which are potentially useful as species-level diagnostic features in Rhodnius. To further verify the validity of this species, the head shapes and wing venation patterns of five species in Rhodnius are compared with morphometric analysis. After reviewing taxonomic and comparative morphology papers of assassin bugs, a vocabulary with a terminology of morphological characters, especially of external male genitalic characters, is assembled with the preferred terms and the synonyms listed. Establishing a consistent terminological framework will greatly facilitate future research on the homology of these structures across Triatominae and will ultimately contribute to our understanding of the evolution of these groups.


Introduction
Triatominae are a subfamily within Reduviidae that is known for its hematophagous feeding habit (Jansen and Roque 2010). Currently, there are 151 extant and three known fossil species assigned to 18 genera and five tribes in Triatominae (Lent and Wygodzinsky 1979;Justi and Galvão et al. 2017;Rosa et al. 2017a;Oliveira et al. 2018;Lima-Cordón et al. 2019;Nascimento et al. 2019;Poinar Jr 2019). All Triatominae possess a nearly straight labium with a flexible membranous connection between the second and third visible segments that allows upward pointing when feeding (Lent and Wygodzinsky 1979). Many species are competent vectors of Chagas disease transmitting Trypanosoma cruzi (Chagas, 1909) in their feces (Lent and Wygodzinsky 1979;Bern et al. 2011). Chagas disease is one of the ten most seriously neglected tropical diseases, which are currently estimated to affect nine million people, with more than 70 million people living under a serious risk of infection (Justi and Galvão 2017;WHO 2019).
The latest taxonomic revision of the entire genus was published approximately 40 years ago in the monograph on Triatominae by Lent and Wygodzinsky (1979) and contained descriptions of 11 of the 13 known species at that time. They regarded R. amazonicus as a synonym of R. pictipes, omitting R. dalessandroi because they were unable to examine specimens of this species. Bérenger and Pluot-Sigwalt (2002) and Rosa et al. (2017) made comparative studies between R. pictipes and R. amazonicus to prove the validity of R. amazonicus. The remaining seven species now included in Rhodnius were described after Lent and Wygodzinsky's (1979) monograph (Lent et al. 1993a;Mejia et al. 1999;Valente et al. 2001;Jurberg et al. 2009;Rosa et al. 2012;Abad-Franch et al. 2013;Souza et al. 2016). Rhodnius taquarussuensis Rosa et al., 2017a was described as a new species but is now considered a phenotypic form of R. neglectus instead of a distinct species (Nascimento et al. 2019). Bérenger and Pluot-Sigwalt (2002) published a key for the pictipes group and Galvão (2014) released a key in Portuguese which included 12 Rhodnius species.
Rhodnius is relatively easy to distinguish from other Triatominae genera because of its long head and coloration pattern but shows low non-genitalic morphological variability between species in the genus, which may account for the difficulties in species identification. The female external genitalia was described for most species of the subfamily (Lent 1948;Abalos and Wygodzinsky 1951;Sherlock and Serafim 1967), but their diagnostic importance was dismissed in papers published by Lent and Jurberg (1968, 1969, 1975 which considered them uniform and, not useful for specific identification. The resurrection of female genitalia, as an important taxonomic tool, was attributed to Rosa et al. (2010) through a detailed study by scanning electron microscopy. Subsequently, several studies corroborate the diagnostic value of female genitalia (Rosa et al. 2012(Rosa et al. , 2014(Rosa et al. , 2017bRodrigues et al. 2018). The male external genitalia are usually used for generic and specific differentiation in assassin bugs. All published species except R. barretti had been documented with the external male genitalia. However, most of these descriptions were restricted to describing or comparing the shapes of the median process of pygophore (Lent and Wygodzinsky 1979;Harry 1993;Lent et al. 1993a;Mejia et al. 1999;Valente et al. 2001;Rosa et al. 2012Rosa et al. , 2017aSouza et al. 2016). Six species (R. zeledoni, R. marabaensis, R. milesi, R. montenegrensis, R. stali, and R. colombiensis) had only detailed illustrations of non-everted phalli, thus restricting the possibility of comparison various structures on the phallosoma and endosoma, which may be helpful in species-level identifications (Lent et al. 1993a;Mejia et al. 1999;Valente et al. 2001;Rosa et al. 2012Rosa et al. , 2017bZhao et at. 2015;Souza et al. 2016). Drawings of endosomal structures that show the individual sclerites rather than the complete everted endosoma were published for only three species, R. stali, R. pictipes, and R. milesi (Lent et al. 1993a;Valente et al. 2001).
When examining the specimens of Rhodnius, two specimens from Bolivia were distinctly different from any other species found. In this study, they are named Rhodnius micki sp. nov. and described. Male genitalia are important in identifying assassin bugs, especially for Rhodnius which has low non-genitalic morphological variability between species. Therefore, special emphasis is put on their everted phallus, allowing for detailed photographic documentation of the phallus, particularly the sclerites of the endosoma. The diagnosis of the new species takes advantage of qualitative morphological features including genitalic features, and of geometric morphometric approaches to better characterize head and forewing shapes. Combining morphometric characters with distribution, we propose that this new species should be classified in the pictipes group. We also provide a synopsis of genitalic terminology applied to Triatominae and offer preferred terms to facilitate future investigations into the homology of these structures across Triatominae and even Heteroptera.

Specimens
Type specimens and an additional male specimen of R. robustus Larrousse, 1927 are deposited in The Natural History Museum (NHMUK), London, United Kingdom.
Specimens of R. stali, R. pictipes, R. pallescens, and R. ecuadoriensis which were used for the geometric analysis came from colonies reared at Fundação Oswaldo Cruz (FIOCRUZ) in Brazil and were deposited at Fundação Oswaldo Cruz (FIOCRUZ).

Dissections and measurements
After softening the abdomens of dried specimens with wet tissue, the pygophores were removed and soaked in 100% lactic acid overnight ( Fig. 1). They were then boiled in 20% lactic acid solution for ~ 30 minutes to remove muscles (Fig. 2). Dissections were carried out in the lactic acid under a Motic binocular dissection microscope. At this point, the endosoma was gently stretched with a pair of forceps (Ideal-Tek SS.SA) and insect pins (0#). The tip of the pins should be blunt (Fig. 3). At first, we inserted the insect pin along the membrane of the endosoma from the opening where the endosoma is everting out, and then gently agitated the pin along the membrane from one side to the other to make the phallosoma loose and make the endosoma move towards the tip of phallosoma, so that the opening is big enough and the forceps would enable to touch the sclerites of endosoma without breaking the membrane (Figs 4, 5). Forceps were used to grasp the sclerite and to stretch the endosoma (Fig. 6). After taking the photographs and other procedures, the dissected genitalia were preserved in glycerol in plastic tubes which were pinned under the corresponding specimens. Measurements were made using a calibrated micrometer and given in millimeters.
Figures 1-6. Process of dissection 1 soaking genitalia in 100% lactic acid overnight 2 boiling genitalia in 20% lactic acid solution for ~ 30 minutes 3-6 dissecting genitalia under microscope with forceps and blunted insect pin 4, 5 inserting the insect pin along the membrane of the endosoma and agitate the pin 6 using forceps to stretch the endosoma.

Terminology
Because of the inconsistent use of terminology in Triatominae, after reviewing many taxonomic and comparative morphology papers of assassin bugs, the terminology adopted in this paper are listed in Table 1. It includes the preferred terms, definition of terms, previously used terms, and references.

Images and image processing
Habitus images were obtained using a Canon EOS 7D and 60mm macro lens. Detail images of heads, pronota, and wings were obtained using a Microscope (Nikon SMZ18) with a Canon EOS 600D. Genital images were taken using an Olympus BX51 with a Canon EOS 450D. Images were stacked using the EOS Utility 2, and Helicon focus 5.3. Photographs were edited with Adobe Photoshop CS4, including adjustment of background color and cropping without modifying any characters. All the images were taken in the laboratory by the authors. The plate of male genitalia is that of the paratype. Table 1. Terminology used in this study with synonyms from the literature.

Definition Previously used terms References
Phallus (P) Intromittent organ inside the pygophore Phallus (Ph) (P) Lent and Jurberg 1984;Lent and Jurberg 1987;Lent et al. 1993a, b;Mejia et al. 1999;Carcavallo et al. 2001;Valente et al. 2001;Cai and Tomokuni 2003;Ishikawa et al. 2007;Sandoval et al. 2007;Weirauch 2008;Jurberg et al. 2009;Frías-Lasserre 2010;Forero et al. 2010;Forero and Weirauch 2012;Rosa et al. 2012;  Processes of the conjunctiva (PrCj) Lent et al. 1993b Processes of the conjunctiva 3 (PrCj 3) Mejia et al. 1999 Morphometrics In total, 42 specimens of five species, R. ecuadoriensis (ten specimens), R. pallescens (ten specimens), R. pictipes (ten specimens), R. stali (ten specimens), and R. micki sp. nov. (two specimens), were used in the analysis. and nine anatomical landmarks were extracted respectively on the heads and forewings. Thirteen landmarks of head (type II points, which combine geometric and biological or histological descriptions) (Gurgel- Gonçalves et al. 2008;Oliveira et al. 2017), and nine landmarks of wings (type I points, which homology comes from unique patterns in biological form) (Gurgel- Gonçalves et al. 2008;Feliciangeli et al. 2007;Costa et al. 2009;Oliveira et al. 2017) were extracted based on the landmarks used in previous works. These landmarks were digitized with tpsUtil 1.46 (Rohlf 2010) and tpsdig2 v.2.16 (Rohlf 2008). To quantify the shape variation related with the shape dimensions, the digitized data were analyzed using morphoJ 1.06d (Klingenberg 2011). Variability in the shape space was assessed using a Principal Component Analysis (PCA). To better visualize the shape variation, thin plate spline visualization was used to get the average shapes of these characters. Diagnosis. General coloration dark brown. Head relatively short, only slightly longer than the pronotum. Eyes small, width of the eye shorter than the synthlipsis. Central area of the anterior lobe of the pronotum conspicuously dark and its humeral angle of the posterior lobe relatively sharply curved. Legs brown. The median process of the pygophore long and bifid on the tip. The medial basal sclerite of the phallosoma with two straight and flat projections. One distal dorsal sclerite of the endosoma bifurcated, and its tip rounded and curved slightly inward.

Taxonomy
Description. Coloration. Body generally dark brown. Head with light median longitudinal stripe extending from the apex of clypeus to the posterior portion of ocelli; eyes blackish; middle of third segment and posterior half of forth segment yellow; a pair of black stripes on the dorsal surface of neck, half of lateral side and ventral side dark. Pronotum with a pair of submedian carinae and lateral margin yellow; concave areas on anterior lobe, especially the central area darkened; posterior lobe dark with scattered irregular small yellow spots. Scutellum dark with a yellow "Y"-shaped ridge; the tip of scutellar process white. Hemelytra generally brown and mottled; corium with small lightly colored spots; membrane with narrowly rimmed pale-yellow veins, area between veins with scattered light color spots. Legs mottled with yellow spots; tarsi yellowish (Fig. 7). Connexivum dark and mottled with yellow spots, posterior one fourth of every segment almost yellow; ventral surface of the abdomen yellowish with scattered irregular dark brown spot; sternites light brown to black, with irregular dark brown spots, center of sternite II and a pair of sublateral elliptical spots of each segment dark (Fig. 9); spiracles with a brown narrowly margin (Fig. 8).
Etymology. The species epithet is named in honor of Mr. Mick Webb (NHMUK), who had helped us in many ways in the study of Hemiptera.
Measurements  Additional material. Rhodnius robustus Larrousse, 1927(1♂, Brazil: Belém, Instituto Evandro Chagas, reared in lab, 20.II.1992 tively short head, only slightly longer than the pronotum, and a defined transverse sulcus on their pronotum. On the other hand, Rhodnius ecuadoriensis, R. pallescens, and R. micki sp. nov. do not have dark rings on the tibiae which is a significant diagnostic character of Rhodnius. Based on the morphometrics of the head and the particular coloration of the legs, we compared before mentioned four species to R. micki sp. nov. For head shape analysis (Fig. 27), the contribution of the first principal (PC1) component accounted for 81.79% of the total variation, whereas the second principal component (PC2) accounted for 6.26%. In the factorial map, five species were separated. The type specimens of R. micki sp. nov. were far away from the others. The thin plate spline visualization showed that the fifth and tenth landmarks located on the anterior margin of eye contributed most to the shape difference among these species. The size of the eye and the length of the anteocular and postocular regions might be the most significant differences among them. For wing vein analysis (Fig. 28), the contribution of the first principal (PC1) component accounted for 58.46% of the total variation and the second principal component (PC2) accounted for 22.21%. The points of R. micki sp. nov. were also distinct from those of the other four species, and these four species were separated from each other too. The thin plate spline visualization showed that the seventh landmark contributed most to the shape difference among these species. It implied that the position of the intersection of the Cu and An1 veins may be the most variable among them.

Comparison with other species
It is relatively easy to distinguish this species from other Rhodnius species because of its relatively sharply curved humeral angles and unique color pattern. Rhodnius stali and R. pictipes are similar to R. micki sp. nov. because their heads are all relatively short, only slightly longer than their pronota, and their pronota have a defined transverse sulcus. However, the tibiae of R. micki sp. nov. are uniformly dark brown, the humeral angle is sharply curved (Fig. 10), and the third antennal segment is black, whereas the other two both have a distinct dark ring on each tibia, only the anterior half of the third antennal segment is black, and the humeral angle is broadly rounded (Fig. 11). Rhodnius ecuadoriensis and R. pallescens do not have any tibial rings. Rhodnius ecuadoriensis is smaller than R. micki sp. nov., and the head of R. pallescens is obviously longer than the pronotum. Rhodnius micki sp. nov. is darker and its submedian carinae on the posterior lobe are not obvious; the posterior quarter of every connexival segment on the dorsal side is yellow. Differences between R. micki sp. nov. and the other species in the male genitalia are significant. The median processes of the pygophore of R. micki sp. nov., R. stali, and R. pictipes are bifid, but the former one is bifid at its tip, with small projections, whereas the median processes of the pygophores of R. stali and R. pictipes are bifid (Lent et al. 1993a) at the base with long projections, and those of R. ecuadoriensis and R. pallescens are not bifid (Lent and Wygodzinsky 1979;Mejia et al. 1999). The parameres of R. micki sp. nov. are narrower than those of R. stali and R. pictipes (Lent et al. 1993). The medial basal sclerite of its phallosoma (MBSPh) is bifid with two flat and straight arms; other Rhodnius species do not have a medial basal sclerite or it is not bifid (Y. Zhao unpublished data). The distal ventral sclerite of the endosoma (DVSEn) of R. micki sp. nov. is smaller and less sclerotized than those in R. stali and R. pictipes (Lent et al. 1993a), and the distal dorsal sclerite (DDSEn) is bifurcated deeply, curved inward, and more heavily sclerotized than R. ecuadoriensis and R. pallescens (our unpublished data). Therefore, Genitalic structures, especially distal ventral sclerite of the endosoma (DVSEn) and distal dorsal sclerite of the endosoma (DDSEn), can provide more information to fully compare the species of Rhodnius. According to geometric morphological analysis, R. micki sp. nov. is relatively isolated on the factorial map, which suggests that the R. micki sp. nov. is also distinguished from those species relatively easily based on the shapes of the head and wing.

Species group assignment
Rhodnius micki sp nov. is known from Santa Cruz, Bolivia, where some species of pictipes group and prolixus group, i.e., R. stali, R. pictipes, and R. robustus are distributed (Chávez 2006;Schofield and Galvão 2009;Justi et al. 2010;Soto-Vivas et al. 2018). Rhodnius stali and R. pictipes, which are the most similar species to R. micki sp. nov. based on the non-genitalic characters mentioned above, both belong to the pictipes group. With respect to genitalic characters, they are also similar because they all have a single distal dorsal sclerite on the endosoma (Lent et al. 1993a). Based on our observations (unpublished), species in the prolixus group, such as R. robustus, have two symmetrical sclerites located in the same position, and the shape of the ventral sclerite of endosoma is triangle (Figs 25, 26). Therefore, we infer that R. micki sp. nov. should be included in the pictipes group based on distribution and genitalic characters.

Terminology of morphological characters
Historically, the terminology of Triatominae, especially male genitalic terms, has developed at least partially in isolation from that of Reduviidae. A plethora of terms have been used for homologous genitalic structures, and in some cases different structures have used the same name. This inconsistency results in incompatible and sometimes misleading terminology for taxonomic descriptions and diagnoses. For example, some researchers have variously used the terms aedeagus, phallus, phallosoma, conjunctiva and phallothecal plate when describing the apical apart of the intromittent organ, and the sclerotized plate beneath the basal plate (Lent and Wygodzinsky 1979;Mejia et al. 1999;Valente et al. 2001;Jurberg et al. 2009;Rosa et al. 2012;Gil-Santana and Galvão 2013;Souza et al. 2016;Oliveira et al. 2018). To avoid ambiguity and achieve consistency with the description of other assassin bugs, we adopt the following terms in this study. Male genitalia consist of pygophore, parameres, and phallus. The articulatory apparatus is composed of basal plate, basal plate bridge, and basal plate extension. The dorsal phallothecal sclerite (DPS) is regarded as the dorsal part of phallosoma. To clarify each sclerite's position, we rename these sclerites with adjectives describing their position, while being as consistent as possible with previous terms. We adopt medial basal sclerite of phallosoma to denote the sclerite on the dorsal side of phallosoma. Two pairs of sclerites on the lateral and ventral sides of phallosoma are called lateral flatlike prolongation of phallosoma (LFPPh) and ventral sclerite of phallosoma (VSPh) respectively. Sclerites at the tip of the endosoma are renamed distal dorsal sclerite of endosoma (DDSEn) and distal ventral sclerite of endosoma (DVSEn). All the preferred terms and synonyms are shown in Table 1.