Description of Triatomahuehuetenanguensis sp. n., a potential Chagas disease vector (Hemiptera, Reduviidae, Triatominae)

Abstract A new species of the genus Triatoma Laporte, 1832 (Hemiptera, Reduviidae) is described based on specimens collected in the department of Huehuetenango, Guatemala. Triatomahuehuetenanguensissp. n. is closely related to T.dimidiata (Latreille, 1811), with the following main morphological differences: lighter color; smaller overall size, including head length; and width and length of the pronotum. Natural Trypanosomacruzi (Chagas, 1909) infection, coupled with its presence in domestic habitats, makes this species a potentially important vector of Trypanosomacruzi in Guatemala.


Introduction
As of 2010, more than a million cardiomyopathy cases in Latin America were caused by Chagas disease (WHO 2015) due to the parasite Trypanosoma cruzi (Chagas, 1909). This illness is mainly transmitted through the infected feces of insects of the subfamily Triatominae (Hemiptera: Reduviidae). Known colloquially as kissing bugs, the group is currently divided into five tribes and 15 genera (Justi and Galvao 2017;World Health Organization 2015;Schofield and Galvao 2009). Among these, Triatoma Laporte, 1832, Panstrongylus Berg, 1879 and Rhodnius Stål, 1859 (the first two belonging to the tribe Triatomini and the third genera belonging to Rhodniini) are the most epidemiologically relevant for Chagas transmission (WHO 2002).
The genus Triatoma is the most diverse, comprising over half of the described Triatominae species (Justi and Galvao 2017;Schofield and Galvao 2009). Triatoma dimidiata (Latreille, 1811), the most important Chagas disease vector in Central America, is in fact a species complex including at least three independently evolving lineages initially identified by sequences of nuclear (internal transcribed spacer, ITS-2) and the mitochondrial marker cytochrome b (cytb) (Bargues et al. 2008;Dorn et al. 2016), and later confirmed by phylogenetic studies using SNPs and species delimitation, obtained by a reduced representation genome genotyping by sequencing (GBS) approach (Justi et al. 2018). These studies recovered T. dimidiata as four linages: groups 1 -4, which appeared to include at least three species: groups 1 and 2 -T. dimidiata s. str., group 3 -Triatoma sp. aff dimidiata, and group 4 -Triatoma sp. aff dimidiata cave (Bargues et al. 2008, Dorn et al. 2016, Justi et al. 2018. The last of these taxa, Triatoma sp. aff dimidiata cave (= group 4) was recently described as a new species, Triatoma mopan Dorn, Justi & Dale, 2018. In this study, we formally describe Triatoma sp. aff dimidiata (group 3) based on morphological and molecular data and name it Triatoma huehuetenanguensis sp. n., after the type locality in Guatemala.

Sampling
A total of 39 Triatoma specimens was obtained between April 2015 and May 2016 through community participation in the department of Huehuetenango, Guatemala and given to personnel from the Ministry of Health of Huehuetenango who shipped them to the Applied Entomology and Parasitology Laboratory (LENAP), at San Carlos University in Guatemala City. At LENAP the specimens were preserved in 95% ethanol and 5% glycerol and stored at room temperature. Specimens were identified as T. dimidiata using the taxonomic key for the genus Triatoma published by Lent and Wygodzinsky (1979).
Three females and three males were left intact to comprise the type series used for the morphological description of the new species. DNA was extracted from the remaining 20 by Justi et al. (2018) and tested for infection with T. cruzi. Justi et al. (2018) previously recovered all 20 specimens within the same highly supported monophyletic clade named Triatoma sp. aff. dimidiata based on genome SNP phylogenies.

Morphological characterization
Since there is no known holotype for T. dimidiata (Latreille, 1811), the characterization of the new species was done following the same methodology as Dorn et al. (2018) for the description of T. mopan.
Based on (Lent and Wygodzinsky 1979), 17 morphological traits were measured from the type series and an additional 13 specimens and from 15 female and ten male Triatoma dimidiata s. str. (group 1-2) from Huehuetenango, Jutiapa and Chiquimula in Guatemala. These 25 Triatoma dimidiata s. str. specimens were preserved under the same conditions as the new species (95% ethanol and 5% glycerol). Measurements of the morphological traits were performed using a Nikon stereoscope Model SMZ-1B (see Suppl. material 1: morphological measurements). The morphological traits were: Because of unequal sample sizes for each group (T. dimidiata s. str. and the new species), an unpaired t test was used to compare the means of each of the 17 morphological traits in the two groups (JMP Pro version 13.0.0).
Insects were photographed using a Visionary Digital BK Laboratory System, a Canon 5D camera, 65 mm macro zoom lens. Photo stacks of 25-45 slices were compiled using Helicon Focus 5.3 and the image edited to balance light quality, remove background blemishes, and provide a scale on Photoshop CS6.

Trypanosoma cruzi infection
Natural infection by T. cruzi was tested by PCR on genomic DNA extracted from the last three segments of the specimens' abdomen. DNA was extracted using Qiagen DNeasy blood and tissue kit, following the manufacturer's tissue protocol for the first two steps, blood protocol for subsequent steps and an additional incubation (65 °C for 10 min, followed by 95 °C for 5 min.). Primers and PCR assay conditions were used as previously described (Moser et al. 1989).

Molecular phylogenetic analysis
In order to: (a) keep the type series intact, (b) confirm that any specimens that share the same phenotype with the type series belong to Triatoma sp. aff dimidiata, and (c) width of the pronotum, width of the abdomen, (4) head length, width across eyes, length of the pronotum, ante-ocular region, post-ocular region, width of the eye, (10) synthlipsis, (11-14) each of the four antennomeres, and (15-17) each of the three labial articles.
to determine the relationship with the other groups of T. dimidiata s.l., ITS-2 and cytB were sequenced for two out of the 20 Triatoma sp. aff. dimidiata specimens studied by Justi et al. (2018). Sequencing was performed as previously described by Dorn et al. (2016). For comparison, ITS-2 and cytB sequences including representatives from all T. dimidiata s.l. groups were retrieved from GenBank (Table 1) and aligned using the algorithm Q-INS-I implemented in the online MAFFT version 7 (Katohet al. 2017) and ClustalW (Larkin et al. 2007) implemented on MEGA v. 6 (Tamura et al. 2013), respectively. Maximum likelihood phylogenies were reconstructed independently for each of the genes using PhyML v.3.1 (Guindon and Gascuel 2003), with 100 bootstrap replicates. The best fit model for each gene, according to the AIC criterion, estimated using JModeltest (Darriba et al. 2015), were model HKY+I for ITS-2 and HKY + G for cytB. Triatoma infestans (Klug, 1834) was used as the outgroup. Both phylogenies were reconstructed using the same specimens for both markers, as indicated in the original studies. Phylogenies were plotted as mirror images using the function cophyloplot, from the package ape (Paradis et al. 2004), in R (R Development Core Team, 2013). Specimens photos and clade highlights were inserted using Adobe Photoshop CC 2108.

Distribution map
Reported confirmed distributions of T. huehuetenanguensis sp. n. and specimens previously identified as Triatoma sp. aff. dimidiata, by molecular means were compiled (Table 2, Fig. 1) and GPS coordinates and altitudes were inferred using Google Maps. Localities were plotted on a map of Central America, using the packages plyr (Wickham 2011), raster (Hijmans 2012) and maps (Becker 2017)   Differential diagnosis. Specimens of T. huehuetenanguensis are classified as T. dimidiata following the key published by Lent and Wygodzinsky (1979). On closer examination, T. huehuetenanguensis differs from T. dimidiata in the following diagnos- tic characters: overall color of connexivum, color of head pilosity, ocelli, setae in the second antennomere, anterolateral angles, labial articles joints, setae in the abdomen, spiracles, and female and male terminalia.
In contrast to the connexivum and corium color of T. dimidiata (pale yellow to orange yellow), T. huehuetenanguensis is brown, with connexivum and corium from yellow to pale yellow. The ventral color in T. huehuetenanguensis is light yellow, while in T. dimidiata it is piceous or black (Fig. 2). The setae around the abdomen are less dense in T. huehuetenanguensis when compared to T. dimidiata. In T. huehuetenanguensis, the spiracles are adjacent to the connexival suture, while in T. dimidiata, the spiracles are close but not adjacent to the connexival suture. In addition, spiracles are surrounded by a dark spot in T. dimidiata, while the spot is absent in T. huehuetenanguensis (Fig. 5). The first antennomere in T. huehuetenanguensis does not reach the apex of the head, whereas in T. dimidiata, it does. The setae in the second antennomere of T. huehuetenanguensis are not as dense as in T. dimidiata (Fig. 3). Anterolateral angles are laterally oriented in T. huehuetenanguensis, while in T. dimidiata they are anterolaterally oriented. The three labial articles of T. huehuetenanguensis are light colored while in T. dimidiata they are dark, similar to the dark body color. The joint of each of the labial articles are pale yellow only in T. huehuetenanguensis (Fig. 3). The collar is relatively thicker in T. huehuetenanguensis compared with T. dimidiata. The scutellum is rugose in both, T. dimidiata and T. huehuetenanguensis. However, the central scutellum area in T. huehuetenanguensis is more depressed as compared with T. dimidiata (Fig. 4). Legs in T. huehuetenanguensis with 1 + 1 subapical denticles, sometimes with a very small apical denticle on fore and/or mid-femora. Females sometimes with only one subapical denticle or a callosity, slightly lighter than the tegument on its proximal side. Fore and mid-femora of both males and females with lighter ventral subapical band, ranging from almost imperceptible to yellow.
The terminalia in males of T. huehuetenanguensis is almost square-shaped and darker than the rest of the tegument, presenting sparse dark pilosity, while in T. dimidiata it is ovoid and dark, presenting abundant dark pilosity. Posterior margin of urosternite VIII convex on T. dimidiata and almost straight in T. huehuetenanguensis. Posterior margin of urosternite IX slightly sinuous and not exceeding the abdomen on T. huehuetenanguensis, convex and exceeding the abdomen on T. dimidiata. Female terminalia in both species is triangle-shaped with very dark and dense pilosity. However, in T. huehuetenanguensis it is pale and very pointed while, in T. dimidiata it has rounded apex and is dark colored. Posterior margin of sternite VII sinuous on T. dimidiata and very sinuous in T. huehuetenanguensis; gonocoxite VIII (Gc8) pointed on T. huehuetenanguensis and rounded on T. dimidiata; gonapophysis VIII (Gp8) is wider than long in T. huehuetenanguensis compared to T. dimidiata. Gonocoxite IX (Gc9) strongly expanded exceeding the abdomen in T. huehuetenanguensis compared to T. dimidiata (Fig. 6).
Description. Overall color brown, connexivum, and corium yellow to light yellow. Pilosity short, distinctively yellow, covering whole body, except male and female terminalia, where pilosity is brown.
Total length, male 22.5-26.5 mm, female 22.2-29.3 mm; pronotum width, male 4.9-6.2 mm, females 4.9-6.4 mm; pronotum length, male 3.7-4.2 mm, female 3.4-4.5 mm (Table 3). Head dark brown with scarce yellow pilosity and overall smooth surface; central band with very shallow rugosity. Head length, male 4.1-4.7 mm, female 4.0-4.8 mm. Ocelli large, lighter than tegument, placed on a pronounced tubercle. Antenniferous tubercles subcylindrical, very short, situated in the middle of the anteocular region; first antennomere not attaining apex of head. Second antennomere lighter than first, with long setae. Ratio of antennomeres I-IV 1:3.9-4.4:3-4:2.5. Apex of clypeus distinctively lighter than rest of head. Labium (Fig. 3) slender, first article reaching level of apex of antenniferous tubercle; second article exceeding posterior border of head, attaining neck; third article light brown, with interarticular areas light yellow (Fig. 3). Ratio of labial articles 1: 1.6:0.5. Most labial setae short, not very numerous on first and second article. Third labial article reaching first third of stridulatory sulcus on males, half on females. Neck dark brown, with very smooth surface, and a pair of lateral yellowish spots.
Pronotum brown, with humerus blunt and pointed, usually lighter in color. Anterolateral angles short, almost round, laterally oriented, almost perpendicular to neck. Submedian carinae very pronounced, with tubercle aspect. Scutellum triangular, shallowly rugose, with the central area distinctly depressed, apical process sometimes lighter in color. (Fig. 4).
Hemelytra not reaching apex of the abdomen, darker at membrane, with scarce light yellow pilosity, dark brown spots around the intersection of the claval suture and Abdomen ventrally convex, shortly pilose, yellow to light yellow (Fig. 3). Spiracles adjacent, but not touching connexival suture, not very pronounced, concolorous with rest of tegument. Connexival segments with a piceous or black spot covering the entire width of the anterior third, yellow posteriorly (Fig. 5).
Males terminalia almost square-shaped, darker than the rest of the body, with scarce dark pilosity. The posterior margin of urosternite VIII almost straight. Posterior margin of urosternite IX slightly sinuous and not exceeding the abdomen. Female external terminalia triangle-shaped, pale, with very dark, dense pilosity (Fig. 6). Posterior margin of sternite VII very sinuous; gonocoxite VIII (Gc8) pointed; gonapophysis VIII (Gp8) is wider than long. Gonocoxite IX (Gc9) strongly expanded exceeding the abdomen in T. huehuetenanguensis (Fig. 6). Distribution. Holotype and paratypes specimens of T. huehuetenanguensis were obtained by community participation and reported to be found in domestic environments, near to tropical forest. Huehuetenango is at the northwest of Guatemala and is characterized by pine forest. The altitude ranges from 300 to >3,000 m above sea level. Other localities (Table 1 and Fig. 1) were inferred from specimens collected for previously published molecular phylogenetics studies (Bargues et al. 2008, Dorn et al. 2016, Justi et al. 2018.
Host-parasite data. 18 out of the 20 specimens tested were found to be infected with Trypanosoma cruzi.
Discussion. The ecological diversity within the subfamily Triatominae (>150 species) and its wide distribution through the Americas, and particularly Latin America, have made it difficult to control vector-borne transmission of Chagas disease (WHO 2002). Triatoma dimidiata is one of the main vector taxa involved in Chagas transmission in Latin America, specifically the main vector for Central America and a secondary vector in Mexico and Colombia. Its broad geographic range and phylogenetic diversity have posed taxonomic challenges for many years (Dorn et al. 2007). Therefore, understanding the taxonomy, phylogenetic and ecological diversity of the T. dimidiata complex is important for understanding T. cruzi transmission.   Here we are presenting three lines of evidence that support T. huehuetenanguensis as a distinct species: morphological, nuclear genetic (ITS-2) and mitochondrial genetic (cytB). The morphological characters included in the taxonomic key for Triatoma species (Lent and Wygodzinsky 1979) for T. dimidiata encompass T. huehuetenanguensis, specifically overall size and measurements of the head and eyes. However, on closer examination, macroscopic differences, including those summarized in Table 4, reveal T. huehuetenanguensis as morphologically different. Color differentiation on connexivum, pilosity, ocelli, labial articles joints, and female and male terminalia separate T. huehuetenanguensis from T. dimidiata. Differentiation based on the color pattern of the connexivum and other body regions was used in the description of T. brailovskyi (Martinez, Carcavallo & Pelaez, 1984), T. gomeznunezi (Martinez, Carcavallo & Jurberg, 1994) and most recently Triatoma mopan (Dorn et al., 2018). This latter study attributed the diminished pigmentation of T. mopan as the result to the cave environment. In our case, where both, T. huehuetenanguensis and T. dimidiata are found in sympatry and in domestic environments, it would be Adjacent, but not touching the connexival suture. Same color as the tegument.

Female external terminalia
Triangle-shaped with rounded apex Triangle-shaped with pointed apex

Male external terminalia
Ovoid shape Square-shaped Limit of the 8 th urosternite is curved Limit of the 8 th urosternite is straight † T. dimidiata features are based on Lent and Wygodzinsky (1979) and T. dimidiata s. str. (group 1-2) specimens from Jutiapa, Guatemala.
interesting to determine the process that caused such color differentiation between these two species. Another important morphological character is the location and pigmentation of the spiracles. These structures in T. huehuetenanguensis are adjacent to the connexival suture, whereas in T. dimidiata, they are close but not adjacent to the connexival suture. Differentiation based on the location and color of the respiratory spiracles was reported in the description of Rhodnius montenegrensis (Rosa et al. 2012) and most recently in the description of Triatoma mopan (Dorn et al., 2018).
The phylogenetic molecular analysis from the nuclear ITS-2 and the mitochondrial cytB gene, recovered a single monophyletic clade with high support (Fig. 7), and low genetic divergence (<3.1% for cytb and < 0.6% for ITS-2) indicating that Triatoma dimidiata auct., non (Latreille, 1811), Triatoma sp. aff. dimidiata (Bargues et al. 2008), Triatoma sp. aff dimidiata group 3 (Monteiro et al. 2013, Dorn et al. 2016, Justi et al. 2018 and T. huehuetenanguensis are the same species (see Suppl. material 3: cytB maximum likelihood phylogeny and Suppl. material 4: ITS-2 maximum likelihood phylogeny). Previously published molecular phylogenies (Bargues et al. 2008, Monteiro et al. 2013, Dorn et al. 2016, Justi et al. 2018) have thoroughly described the ITS-2 and cybB diversity within and among the four groups that comprise T. dimidiata s. l. This allowed us to compare these two regions in two of the specimens used by Justi et al. (2018), and verify that they fall within the monophyletic clade. Two Triatominae species were differentiated based on molecular analysis of the mitochondrial gene (Cytb), R. montenegrensis (Da Rosa et al. 2012) and Rhodnius marabaensis (Souza et al. 2016).
As supported by our genetic data, we suggest the inclusion of T. huehuetenanguensis in the subcomplex T. dimidiata. Based on Justi and Galvao (2017), this subcomplex Table 5. Distinguishing features between the species of T. dimidiata subcomplex based on Justi and Galvão (2017). This reference was used as the original description is not very detailed, and Lent and Wygodzinsky (1979) provide a much more detailed description of the morphology upon inspection of 160 specimens.

Species
Reference Features T. dimidiata Lent and Wygodzinsky (1979)* First antennae segment attaining level of apex of clypeus; anterolateral angles anterolaterally directed; central area of the scutellum not depressed; spiracles close but not adjacent to connexival suture, connexivum dark. T. hegneri Lent and Wygodzinsky (1979) Labium very short; abdomen flattened below with spiracles distant from connexival suture; venter and connexivum uniformly dark. T. brailovskyi Martinez et al. (1984) Overall size small with very large eyes and ocelli, pronotum with an evident keel at the border, anterolateral angles short and subconical, fore and mid femora with 1 + 1 subapical denticles. T. gomeznunezi Martinez et al. (1994) Antenniferous tubercle laterally covered with long setae and dorsally glabrous; neck polished and entirely black; corium dark brown with two basal and distal yellowish spots; venter convex but longitudinally flattened; venter black. T. mopan Dorn et al. (2018) Pronotum without discal tubercles and presenting a straight latitudinal depression dividing it in half, fore-femora with 1+1 apical, small denticles, 2 +1 subapical denticles in both males and females; and 1+1 apical, small denticles, 2 +2 asymmetrical subapical larger denticles on males and 2 +2 larger, asymmetrical subapical denticles on females, and spiracles close adjacent to connexival suture, surrounded by a spot slightly darker then the tegument T. huehuetenanguensis This study Short yellow pilosity in the whole body except in the genitalia; connections between each segment of the labium are very visible and light-yellow colored; color of venter light yellow.
is comprised by: T. dimidiata, T. hegneri, T. brailovskyi and T. gomeznunezi, and more recently T. mopan has been added (Dorn et al. 2018). The most relevant differences between these six species are summarized in Table 5. Relevant Triatominae species for human T. cruzi transmission are those that have evolved to live close to humans and have been found to be infected T. cruzi (WHO 2002). Triatoma huehuetenanguensis was collected in both peridomestic and intradomestic environments. The high natural infection with T. cruzi (> 90% of the specimens) suggests it is a potentially important vector and its role in human Chagas disease should be further evaluated.
of the Ministry of Health of Huehuetenango, especially to Mirna Sosa for their help in field collections. The reviewers who helped greatly improve this manuscript.
This manuscript was prepared in part while SAJ held a Postdoctoral fellowship from the National Science Foundation (NSF) grant BCS-1216193 as part of the joint NSF-NIH-USDA (United States Department of Agriculture) Ecology and Evolution of Infectious Diseases program; and, in part as a National Research Council Research Associate Awardee at the Walter Reed Army Institute of Research. The material published reflects the views of the authors and should not be construed to represent those of the Department of the Army, the Department of Defense or the Department of Agriculture.
Financial Support: This work was supported with a subsidy of the Ecohealth Initiative Program of the Center of Investigations for the Development of Canada (IDRC; http://www.idrc.ca/ecohealth) (Subsidy no 106531) to Carlota Monroy; a grant from the World Health Organization (Tropical Disease Research-World Health Organization grant, TDR -WHO ID# A10249) awarded to CM, by National Science Foundation (NSF) grant BCS-1216193 as part of the joint NSF-NIH-USDA (United States Department of Agriculture) Ecology and Evolution of Infectious Diseases program to CM, PD and LS and grant R03AI26268/1-2 from the National Institute of Allergy and Infectious Disease (NIAID) of the National Institutes of Health (NIH) to LS.