Review of Kissing Bugs (Hemiptera: Reduviidae: Triatominae) from China with Descriptions of Two New Species

Simple Summary Triatominae, commonly known as kissing bugs, are blood-feeding insects that can carry the parasite Trypanosoma cruzi to spread Chagas disease. In this study, we conducted a taxonomic review of Chinese triatomines, which involved describing two new species, Triatoma picta Zhao & Cai sp. nov. and T. atrata Zhao & Cai sp. nov. We also calculated the pairwise genetic distances of Triatoma and offered a key to species of Triatominae in China. This review may facilitate a better understanding of the diversity of Chinese kissing bugs. Abstract Triatominae, the only blood-sucking subfamily in Reduviidae, are the vectors of Chagas disease. The majority of them are distributed in the Americas, while the diversity in China has been underestimated, as only two species have been recorded. Here, we describe two new species from China, Triatoma picta Zhao & Cai sp. nov. and T. atrata Zhao & Cai sp. nov., and provide a redescription of T. sinica Hsiao, 1965, along with remarks on T. rubrofasciata (De Geer, 1773). To facilitate the identification, we include photos, especially of genitalia, as well as a distribution map and a key to Chinese triatomines. We calculated the pairwise genetic distances between 23 Triatoma species, which further supported the validity of these new species. We anticipate that our taxonomic review will be useful for identifying Chinese Triatominae.


Introduction
Triatominae, commonly known as kissing bugs, are the only hematophagous subfamily of Reduviidae [1]. They are capable of transmitting Chagas disease by carrying the parasite Trypanosoma cruzi. Chagas disease is a potentially life-threatening illness, which infects an estimated six to seven million people worldwide [2] and causes about 12,000 deaths annually [3]. Efforts to control the spread of the disease as a public health problem have been ongoing for many years, and taxonomic research on vectors may be one such effort.
Within the Triatominae subfamily, there are 154 extant species classified into eighteen genera and five tribes [4][5][6][7]. The largest and most diverse tribe is Triatomini Jeannel, 1919, which contains over half of the subfamily's species and at least six extant genera [4,8]. Among these genera, Triatoma Laporte, 1832 is the biggest and the type genus. It is also the only genus that is widely dispersed, occurring on all continents except Europe and Antarctica. In Asia, Triatoma included seven species, two of which are found in China. Triatoma rubrofasciata (De Geer, 1773) has a global distribution, while Triatoma sinica Hsiao, 1965 is an endemic species which was only collected in Nanjing, China [9]. It was first described by Hsiao, but his original description was written in Chinese and lacked a detailed description of the genitalia [10]. Few taxonomic studies about this subfamily have been conducted in China. Qian et al. reported the presence of four Triatoma species in

Specimens
All specimens were deposited in the Entomological Museum of China Agricultural University, Beijing, China (CAU). Specimens used for DNA barcoding were collected by light trapping between 2016 and 2020 (Table S1), and were preserved in 100% ethanol at-20 • C. The distribution of four species from China was mapped based on the collection sites of specimens.

Dissections and Measurements
The pygophores were removed from specimens and soaked in 100% lactic acid overnight, followed by boiling in~20% lactic acid solution for~40 min. Dissections were performed under a Motic binocular dissection microscope (Olympus SZX7), using a pair of forceps and an insect pin to separate the phallus from pygophores and stretch the endosoma. A detailed description of the dissection procedure can be found in our previous study [19]. All dissected genitalia were preserved in glycerol in plastic tubes which were pinned under the corresponding specimens. Measurements were taken using a calibrated micrometer and recorded in millimeters.

Images and Image Processing
Habitus images of the specimens were captured using a Canon EOS 7D camera equipped with a 100 mm macro lens. For obtaining detailed images of heads, pronota, and scutella, a microscope (Nikon SMZ18) equipped with a Canon EOS 7D was used. Genital images were captured using an Olympus BX51 microscope coupled with a Canon EOS 7D camera. To produce images with an extended depth of field, a focus stacking technique was applied using Helicon Focus 5.3. The species distribution map was created using ArcMap 10.8. Finally, image plates were processed and edited using Adobe Photoshop 2020.

DNA Barcoding
DNA samples were extracted from tissues (thoracic muscles or leg muscles), which followed standard methods for DNA extraction using the dNeasy Blood and tissue kit (Qiagen). The COI genes were amplified with the primers LCO 1490 (5 -GGTCAACAAATCATA AAGATATTGG-3 ) and HCO 2198 (5 -TAAACTTCAGGGTGACCAAAAAATCA-3 ) [22]. Polymerase Chain Reaction (PCR) was conducted using Premix Taq™ (Ex Taq™ Version 2.0 plus dye), with the following cycling conditions: pre-denaturation at 94 • C for 3.5 min, denaturation at 94 • C for 30 s, annealing at 55 • C for 30 s, elongation at 72 • C for 1 min, and 35 cycles and elongation at 72 • C for 8 min. Sequences were bidirectionally sequenced using the same PCR primer pairs, and the sequence data have been deposited in GenBank. Our analysis included a total of 23 COI sequences from triatomines, of which 19 sequences were obtained from GenBank. Sequence alignment was carried out using Clustal W and edited using Geneious 10. This species closely resembles T. migrans, and they both have the following characters: a general body coloration of yellowish orange with dark markings; four dark brown stripes on the posterior lobe of the pronotum; a dark rectangular marking on each segment of the connexivum. However, it can be easily distinguished from T. migrans by its relatively straight lateral edges and the conical anterolateral angles of pronotum.

Description
Coloration. Body predominantly yellowish orange with dark markings (Figure 1). Head dark brown; neck yellowish orange (Figure 2A-C). Visible labial segments mostly yellowish orange; bottom of first visible labial segment dark brown ( Figure 2B,C). First and second antennal segments dark brown, third and fourth segments yellowish ( Figure 1). Inner edge of anterolateral angle of pronotum blurry dark brown; anterior lobe of pronotum generally brown with a pair of yellowish orange petal-like markings; posterior lobe with four longitudinal dark brown stripes, and two central stripes close together in some specimens ( Figure 2D,E). Scutellum dark brown except central area and conical process yellowish orange ( Figure 2F,G). Clavus mostly dark brown, apex of clavus yellowish in some specimens; apex of corium and central irregular spot dark brown; membrane brown ( Figure 1A,D). Legs dark brown; tarsi yellowish. Each segment of connexivum with a dark brown rectangular spot. Sternites generally dark brown; spiracles with yellow narrow margins ( Figure 1C-F).

Etymology
The species name is taken from Latin, picta, in allusion to its bright yellow color.

Etymology
The species name is taken from Latin, picta, in allusion to its bright yellow color.

Distribution
China (Anhui, Hainan, Jiangxi, Yunnan); Vietnam (Ninh Binh, Nghe An.) ( Figure 4).    Triatoma atrata Zhao & Cai sp. nov. can be distinguished from other Triatoma species by the following characters: black-colored body without obvious markings on the connexivum; a pronotum more than twice as wide as the head; nearly rounded rectangular lateral sclerites of endosoma with denticles. This species is most similar to T. sinica but can be differentiated by its small eyes and the absence of markings on the connexivum.
Insects 2023, 14, x FOR PEER REVIEW 10 of 20 distinct; apex of fore and mid tibiae with spongy fossae (Figure 5C,F). Hemelytra of male approaching to middle of eighth tergum, and that of female reaching to tip of the seventh tergum ( Figure 5A,D). Genitalia. Female external genitalia: posterior edge of VII sternite sinuous; VIII gonocoxites subtriangular; IX sternite visible ( Figure 6J). Male external genitalia: transverse bridge of pygophore strongly sclerotized; median process of pygophore long and slender Genitalia. Female external genitalia: posterior edge of VII sternite sinuous; VIII gonocoxites subtriangular; IX sternite visible ( Figure 6J). Male external genitalia: transverse bridge of pygophore strongly sclerotized; median process of pygophore long and slender ( Figure 7B,C). Paramere curved with a denticle (Figure 7D-F). Width of basal plate arms equal to that of transverse bridge of basal plate; basal plate extension approximately equal to arms of basal plate in length ( Figure 7H,I). Dorsal phallothecal sclerite ellipsoid with slightly raised tip. Medial basal sclerite of phallosoma strongly sclerotized and oblong in lateral view; its tip rounded and concaved in middle of upper surface ( Figure 7H,I). Two lateral sclerites of endosoma rounded rectangular with denticles ( Figure 7G); surface of endosoma with dense denticles or wrinkles and not sclerotized; membrane in middle of dorsal surface of endosoma wrinkled and distinctly thicker than other membranous areas ( Figure 7H). Two lateral sclerites of endosoma rounded rectangular with denticles ( Figure 7G); surface of endosoma with dense denticles or wrinkles and not sclerotized; membrane in middle of dorsal surface of endosoma wrinkled and distinctly thicker than other membranous areas ( Figure 7H).

Etymology
The species name is taken from Latin, atrata, in allusion to its black color.

Diagnosis
Triatoma sinica can be distinguished from its congeners by a combination of the following characters: yellowish lateral margins and a submedian carinae of the pronotum; small and conical anterolateral angles; each segment of the connexivum being brown or dark brown with wide transverse yellow stripes on the~1/4 anterior region and~1/4 posterior region; and median-sized eyes with a width equal to the synthlipsis. This species resembles T. atrata Zhao & Cai sp. nov. and T. rubrofasciata. It can be differentiated from T. atrata Zhao & Cai sp. nov. by having yellowing markings, as noted in the diagnosis of T. atrata Zhao & Cai sp. nov. Triatoma sinica can be distinguished from T. rubrofasciata by the presence of yellow markings on the connexivum and the short first antennal segment that does not reach the apex of the maxillary plate.

Redescripition
Coloration. Body mostly brown to dark brown with yellowish stripes and markings ( Figure 8). Neck light brown to yellow ( Figure 9A-C). Visible labial segments yellowish except bottom area ( Figure 9B,C,E-G). First and second antennal segments dark brown ( Figure 8A-F). Anterolateral angles yellow; inner side of anterolateral angles of some specimens brown. Lateral margins of pronotum yellow; submedian carinae yellow or light brown; some specimens with a yellow longitudinal stripe on center of posterior lobe; anterior lobe of pronotum brown or dark brown with a pair of yellow or brown petal-like markings on anterior lobe; color of posterior lobe lighter than that of anterior lobe. Central region of scutellum brown, conical process of scutellum gradually yellow towards tip; outside of Y-shaped area dark brown or black ( Figure 9I-K). Bottom of clavus brown; a stripe along R+M vein yellowish, and an irregular marking on the subapical corium light yellow, most membrane pale brown ( Figure 8A,D). Legs brown to dark brown. Tarsi yellowish. Each segment of connexivum brown or dark brown with wide transverse yellow stripes on the~1/4 anterior region and~1/4 posterior region ( Figure 8A-F). Sternites generally dark brown to black; spiracles with yellow narrow margins ( Figure 8B,C,E,F). stripes on the ~1/4 anterior region and ~1/4 posterior region ( Figure 8A-F). Sternites generally dark brown to black; spiracles with yellow narrow margins ( Figure 8B-C,E-F).  Figure 9B-C,F-G). Thorax. Anterolateral angles small and conical. Surface of pronotum wrinkled, and lateral edges of pronotum straight; length of posterior lobe ~2 × as that of anterior lobe (1:2.12-2.27); posterior pronotal lobe ~1.5 ×as wide as anterior lobe (1:1.56-1.60); submedian carinae extending to half of posterior lobe; median longitudinal furrow distinct; transverse furrow shallow ( Figure 9I-K). Scutellum triangular with narrow Y-shaped ridge; conical process slender and shorter than half of whole scutellum ( Figure 9I-K). Pleura of meso-and metathoracices wrinkled  Figure 9B,C,F,G). Thorax. Anterolateral angles small and conical. Surface of pronotum wrinkled, and lateral edges of pronotum straight; length of posterior lobe~2 × as that of anterior lobe (1:2.12-2.27); posterior pronotal lobe~1.5 ×as wide as anterior lobe (1:1.56-1.60); submedian carinae extending to half of posterior lobe; median longitudinal furrow distinct; transverse furrow shallow ( Figure 9I-K). Scutellum triangular with narrow Y-shaped ridge; conical process slender and shorter than half of whole scutellum ( Figure 9I-K). Pleura of meso-and metathoracices wrinkled ( Figure 8B,E). Stridulatory sulcus narrow, length of it~5 × as long as width ( Figure 9D,H). Legs long and slender. Fore and mid femora both with two pairs of proximal denticles in most specimens ( Figure 8C,F). Hemelytra of male approaching tip of abdomen ( Figure 8A,D).
( Figure 8B,E). Stridulatory sulcus narrow, length of it ~5 × as long as width ( Figure 9D,H). Legs long and slender. Fore and mid femora both with two pairs of proximal denticles in most specimens ( Figure 8C,F). Hemelytra of male approaching tip of abdomen ( Figure  8A,D). Genitalia. Male external genitalia: Transverse bridge of pygophore strongly sclerotized and narrow; a pair of dorsal sclerites of genital opening large; median process of pygophore sharply pointed ( Figure 10A-C). Paramere curved with a denticle ( Figure  10D-F). Width of arms of basal plate equal to that of transverse bridge of basal plate (Figure 10K); basal plate extension roughly same length as arms of basal plate ( Figure 10L); dorsal phallothecal sclerite oval with raised tip ( Figure 10I); medial basal sclerite of phallosoma strongly sclerotized and oblong in lateral view, central of upper surface concaved ( Figure 10G-H). Two lateral sclerites of the endosoma sub-semicircle with denticles (Figure 10I-J); surface of endosoma granulose and not sclerotized; membrane in middle of dorsal surface of endosoma wrinkled and distinctly thicker than other membranous areas ( Figure 10K). Genitalia. Male external genitalia: Transverse bridge of pygophore strongly sclerotized and narrow; a pair of dorsal sclerites of genital opening large; median process of pygophore sharply pointed ( Figure 10A-C). Paramere curved with a denticle (Figure 10D-F). Width of arms of basal plate equal to that of transverse bridge of basal plate ( Figure 10K); basal plate extension roughly same length as arms of basal plate ( Figure 10L); dorsal phallothecal sclerite oval with raised tip ( Figure 10I); medial basal sclerite of phallosoma strongly sclerotized and oblong in lateral view, central of upper surface concaved ( Figure 10G,H). Two lateral sclerites of the endosoma sub-semicircle with denticles ( Figure 10I,J); surface of endosoma granulose and not sclerotized; membrane in middle of dorsal surface of endosoma wrinkled and distinctly thicker than other membranous areas ( Figure 10K).  The color pattern of T. rubrofasciata in China is mostly black, and the lateral margins of the whole pronotum are red and narrow. The forewing has a red strip along vein R+M, and a V-like shape spot on the subapical corium. Images of male genitalia are offered ( Figure 11). The medial basal sclerite of phallosoma is strongly sclerotized and shaped like an eagle's beak ( Figure 11H-J). The basal lateral sclerites of the endosoma form an approximate segment of a circle without denticles, and the outline is sinuous ( Figure 11G).  (Figure 11)

Remarks
The color pattern of T. rubrofasciata in China is mostly black, and the lateral margins of the whole pronotum are red and narrow. The forewing has a red strip along vein R+M, and a V-like shape spot on the subapical corium. Images of male genitalia are offered (Figure 11). The medial basal sclerite of phallosoma is strongly sclerotized and shaped like an eagle's beak ( Figure 11H-J). The basal lateral sclerites of the endosoma form an approximate segment of a circle without denticles, and the outline is sinuous ( Figure 11G).

Discussion
The discovery of new distribution localities and previously unknown species has long been of interest to researchers, as it sheds light on their diversity and evolution. Traditionally, species identification and classification have relied on morphological characteristics, such as color pattern, head shape, and genital structure. However, with the help of recent advances in molecular techniques, researchers can potentially obtain more information and tackle intricate problems. In this study, we combined both morphological and molecular approaches to identify and describe or redescribe three Triatominae species.
The newly described Triatoma picta Zhao & Cai sp. nov. is similar to T. migrans in color pattern, but there are noticeable differences between these two species [8]. Notably, T. picta Zhao & Cai sp. nov. is uniformly dark brown on the dorsal surface of the head, while T. migrans features a yellowish stripe in the middle. Additionally, the forewing bottom area of T. picta Zhao & Cai sp. nov. is yellowish, whereas T. migrans' is dark brown. The anterolateral angles of T. migrans are much longer than those of T. picta Zhao & Cai sp. nov. Moreover, T. migrans is generally larger than T. picta Zhao & Cai sp. nov. in size, although a few exceptions existed. Barcoding analysis reveals that the genetic divergence between T. picta Zhao & Cai sp. nov. and T. migrans is significant, with a value of 0.17, which exceeds the species delimitation empirical threshold, 0.02 [13]. The genetic distances between T. picta Zhao & Cai sp. nov. and other Triatoma species are also distinct, with values much higher than 0.02.
Among the Old-World Triatominae species, T. atrata Zhao & Cai sp. nov. stands out due to its unique characteristics. Unlike other species, T. atrata Zhao & Cai sp. nov. is generally dark and lacks distinct markings on the connexivum. Regarding morphological structure, T. sinica is the species that most resembles T. atrata Zhao & Cai sp. nov., while there are notable differences between them. Triatoma sinica has much larger eyes, with its width equal to the synthlipsis, and T. atrata Zhao & Cai sp. nov. has relatively sharper humeral angles. Moreover, there is a difference in the shape of the basal lateral sclerites of endosoma, with T. sinica having sub-semicircle sclerites and T. atrata Zhao & Cai sp. nov. having slightly angular ones. The stridulatory sulcus of T. atrata Zhao & Cai sp. nov. is wider, and its width-to-length ratio is greater than that of T. sinica. The two species also have distinct color patterns. Triatoma atrata Zhao & Cai sp. nov. is generally black, and its light yellowish markings are not distinct, while T. sinica is brown to dark brown with yellowish stripes and markings. The observed genetic divergences between T. atrata Zhao & Cai sp. nov. and the other 22 Triatoma species are found to be significantly higher than the empirical threshold, 0.02 [13]. Regrettably, we are unable to calculate the genetic distance between T. sinica and T. atrata Zhao & Cai sp. nov due to the unavailability of ethanol-preserved specimens and our inability to extract DNA from dry specimens of T. sinica.
Our examination of specimens allowed us to identify three suspected T. sinica specimens. Although we did not examine the type specimens, we believe that one of the examined species was collected from the same locality as the type specimens, based on its collection information. Furthermore, we observed that two of the specimens collected in Taiwan were slightly darker than those from Nanjing. After dissecting their genitalia, we discovered that they both have an oblong medial basal sclerite of the phallosoma and lateral sclerites of the endosoma in the form of sub-semicircles with denticles, which are identical to those of specimen from Nanjing. Therefore, we concluded that T. sinica was found in a new distribution locality in Taiwan, China.
By combining morphological and molecular methods, we were able to gain a more comprehensive understanding of these species, and this highlights the importance of using multiple approaches to accurately identify and classify Triatominae species.