Print
Description of Triatoma mopan sp. n. from a cave in Belize (Hemiptera, Reduviidae, Triatominae)
expand article infoPatricia L. Dorn, Silvia A. Justi§|, Carolina Dale, Lori Stevens|, Cleber Galvão, Raquel Lima-Cordón|, Carlota Monroy#
‡ Loyola University New Orleans, New Orleans, United States of America
§ Smithsonian Institution Museum Support Center, Suitland, United States of America
| University of Vermont, Burlington, United States of America
¶ Instituto Oswaldo Cruz, Rio de Janeiro, Brazil
# University of San Carlos, Guatemala City, Guatemala
Open Access

Abstract

In this paper, Triatoma mopan sp. n. is described based on five males and six females collected in the Rio Frio cave, Cayo District, Belize. This species is similar to Triatoma dimidiata (Latreille), but can be distinguished by characters found on the pronotum, legs, and abdomen. Geometric morphometry and phylogenetic comparisons are also provided. Presently, the species is known only from the type locality and is a potential Chagas vector.

Keywords

Belize, Chagas disease, new species, Triatoma dimidiata, Triatoma mopan

Introduction

Species belonging to Triatominae Jeannel, 1919 (Insecta, Hemiptera, Reduviidae) are important as vectors of Chagas disease. Presently, more than 150 species within 15 genera (Justi and Galvão 2017) are recognized as valid in this subfamily. The most speciose genus is Triatoma Laporte, 1832, which includes Triatoma dimidiata (Latreille, 1811) and Triatoma infestans (Klug, 1834), historically, two of the most relevant Chagas disease vectors in Central and South America, respectively.

Because of the substantial morphological variation of T. dimidiata across its large geographic distribution (southern Mexico to Peru), this species has been split and synonymized many times since its original description (reviewed in Dorn et al. 2007). This includes morphotypes (previously considered species or subspecies) at extreme ends of the geographic distribution. In the north (Mexico, described as T. maculipennis Stål, 1859), specimens of T. dimidiata have smaller bodies, shorter heads, and larger eyes compared with specimens from the south (Colombia, once called T. capitata Usinger 1941).

Lent and Wygodzinsky (1979) examined 160 T. dimidiata specimens, including the entire distribution, and concluded that, in general, there is a clinal variation with size increasing southwards, and synonymized T. maculipennis and T. capitata with T. dimidiata; these authors also state that there are many exceptions to that rule, and commented specifically on cave specimens. Upon comparison of one “T. dimidiata” specimen from the Rio Frio cave, in Belize, with five specimens from the Lanquin cave in Guatemala, the authors conclude that they appear identical. The morphological distinctions between the cave specimens and the other specimens of T. dimidiata were regarded simply as cave adaptations.

Following the synonymizing of the species (Lent and Wygodzinsky 1979), many qualitative and quantitative phenotypic, biochemical, and molecular studies have sought to clarify the systematics of T. dimidiata. Phylogenetic studies of T. dimidiata based on DNA sequence analyses of nuclear and mitochondrial genes (Bargues et al. 2008, Monteiro et al 2003, Dorn et al. 2016) and genome-wide data (Justi et al. 2018) have shown that T. dimidiata is composed of at least three phylogenetic species (Mishler and Brandon 1987), referred to as : T. dimidiata sensu strictu (or groups 1 and 2), T. sp. affinnis dimidiata (group 3) and T. sp. aff. dimidiata – cave (group 4); the latter comprises only specimens from the Rio Frio cave, Cayo District, Belize. Interestingly, Dorn et al. (2016) included in their study specimens from both caves mentioned in Lent and Wygodzinsky (1979) and found that specimens from Lanquin are recovered within T. dimidiata s.s., whereas the ones from Rio Frio compose a distinct clade.

In this manuscript, we describe T. sp. aff. dimidiata – cave, the lineage from the Rio Frio cave, as Triatoma mopan sp. n. (Hemiptera, Reduviidae, Triatominae), a new species of the genus Triatoma.

Materials and methods

Specimen collection

We conducted field work on June 15, 2016 in the Rio Frio cave, Cayo District, Belize [(coordinates: 16.956939/-88.979675) under permits covering the research (#IA/H/1/16 (03), Institute of Archaeology), collecting (#WL/1/1/16 (33), Forest Department) and export (#WL/1/7/16 (29), Forest Department) of specimens from Belize. The sole purpose of this field work was to collect enough specimens from the Rio Frio cave Triatoma population to reliably compare this population with T. dimidiata from other localities. We collected specimens from the Rio Frio cave, Cayo, Belize because of previous results of phylogenetic studies that showed this population to be an independent lineage distinct from all other populations included under the umbrella of T. dimidiata, and to be the only phylogenetic species found in this particular cave. We collected 15 adult males and 13 adult females and more than 70 nymphs of various lifecycle stages. For this study, we focus on the adult morphology.

Morphological identification and description

Adults collected in the Rio Frio cave could not be taxonomically identified using the key for Triatoma species (Lent and Wygodzinsky 1979). Because of the morphological similarities mentioned by Lent and Wygodzinsky (1979) between T. dimidiata and the single specimen they examined from the Rio Frio cave, six T. mopan males and seven T. mopan females were compared with specimens from the Lanquin cave, Guatemala, as well as representatives of the entire distribution of T. dimidiata, including photographs of the type specimens from the extreme ends of the range, T. dimidiata capitata (from Colombia), and T. dimidiata maculipennis (from Mexico). It was not possible to directly compare with the T. dimidiata holotype, since this specimen has been lost; thus, T. dimidiata specimens used in this study were identified following the key and description in Lent and Wygodzinsky (1979). The specimens examined here were from the Triatominae collection of the Oswaldo Cruz Institute (CTIOC) in Rio de Janeiro, Brazil (Table 1) and the type specimens were from Zoologisches Museum, Berlin (T. dimidiata maculipennis) and the California Academy of Sciences, USA (T. dimidiata capitata).

Specimens used for morphological and morphometric comparison. CTIOC: Triatominae Collection of the Oswaldo Cruz Institute.

Species Specimen origin ID/voucher number Geographic Origin Sex Notes
T. sp. aff. dimidiata field A10800 Huehuetenango, Guatemala F
field A10727 Huehuetenango, Guatemala M
Triatoma dimidiata s.l. CTIOC 2838 Colombia M capitata morphotype
Colony LNIRTT* N/A Colombia F capitata morphotype
CTIOC N/A Santa Boyaca, Colombia F capitata morphotype
CTIOC 2463 Costa Rica F
CTIOC 2592 Costa Rica F
CTIOC 2587 San Jose, Costa Rica F
Colony LNIRTT* N/A Equador F
Colony LNIRTT* N/A Equador – genitalia M
CTIOC 3385 Candelaria Caves, Alta Verapaz, Guatemala M
CTIOC A6160 Lanquin Caves, Guatemala M
CTIOC 3388 Lanquin Caves, Guatemala M
CTIOC 3377 Peten M
CTIOC 3379 Peten F
CTIOC A9703 Peten F
CTIOC 8937 Mexico M maculipennis morphotype
CTIOC N/A Peru M
CTIOC 2769 N/A M
California Academy of Sciences N/A Boyacá, Colombia M capitata holotype
Triatoma gerstaeckeri CTIOC 6242 Mexico F
CTIOC N/A San Marcos, Texas, US M
CTIOC N/A Texas M
CTIOC 6239 N/A F
CTIOC 6241 N/A F
Triatoma mopan Colony 16 LNIRTT N/A Belize F colony started in 12/05/2006
Colony 147 LNIRTT N/A Belize F colony started in 12/05/2006
Field 2016BZ001 Cayo District, Rio Frio Cave, F
Field 2016BZ002 Cayo District, Rio Frio Cave, Belize F
Field 2016BZ003 Cayo District, Rio Frio Cave, Belize F
Field 2016BZ004 Cayo District, Rio Frio Cave, Belize F
Field 2016BZ005 Cayo District, Rio Frio Cave, Belize F
Field 2016BZ006 Cayo District, Rio Frio Cave, Belize F
Field 2016BZ007 Cayo District, Rio Frio Cave, Belize M
Field 2016BZ008 Cayo District, Rio Frio Cave, Belize M
Field 2016BZ009 Cayo District, Rio Frio Cave, Belize M
Field 2016BZ011 Cayo District, Rio Frio Cave, Belize M
Field 2016BZ013 Cayo District, Rio Frio Cave, Belize M

Morphological study

Character observation and measurements were made with a stereoscopic Zeiss Stemi SV11 microscope, using a graduated eyepiece micrometer, and photos were taken using a Nikon Coolpix 990 digital camera. The following characters were measured:

TL total length of the body

LOP and WOP length and width of pronotum

AOR and POR length of the ante- and post-ocular region

SYN length of the inter-ocular region or synthlipsis

HL and WOH length and width of the head

WE width of the eye

TS total length of scutellum

POS length of process of scutellum

A1 -A4 length of antennal segments

R1-R3 labial segments (Figure 1, Table 2).

The terminology and measurements used for the description were based on Lent and Wygodzinsky (1979). For comparison, these characters were also measured for specimens of T. dimidiata. For each sex separately, the two species were compared for each of the characters with a t-test using JMP® ver 13 (SAS Institute, Inc., Cary NC, USA). Significance was adjusted for multiple comparisons with the method of Benjamini and Hochberg (1995). Boxplots comparing the characters measured were also created using R (The R Development Core Team 2008) and the code and plots are provided as Suppl. material 1.

Figure 1. 

Scheme of the characters measured on the head.

Sequence information and specimens used for the phylogenetic reconstruction and calculation of genetic distances.

Taxon Sequence ID Locality ITS-2 Cyt b
T. dimidiata 1 Sta. Theresa, Toledo DQ871354 FJ197155
10 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286693 JN585881
11 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286693 JN585881
12 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286693 JN585881
13 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286695 JN585881
14 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador KT874438 JN585881
15 Sto. Tomás, Sto. Domingo, Heredia, Costa Rica AM286693 JN585893
16 Sto. Tomás, Sto. Domingo, Heredia, Costa Rica AM286693 JN585894
17 Sto. Tomás, Sto. Domingo, Heredia, Costa Rica AM286693 JN585894
18 Angeles, San Rafael, Heredia, Costa Rica KF192843 JN585894
19 Sto. Tomás, Sto. Domingo, Heredia, Costa Rica KT874433 JN585895
2 Mérida, Yucatán, Mexico FJ197146 FJ197157
20 Colombia AM286703 KT998309
21 Colombia AM286703 KT998309
22 Colombia AM286704 KT998309
23 Colombia KF192845 KT998310
24 Lanquin, Alta Verapaz, Guatemala AM286702 KT998313
25 Lanquin, Alta Verapaz, Guatemala AM286702 KT998314
26 El Lodo Negro, San Marcos Sierra, Intibuca, Honduras AM286694 KT998315
27 El Masical, San Antonio, Copán, Honduras AM286694 KT998316
28 El Masical, San Antonio, Copán, Honduras AM286695 KT998316
29 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286693 KT998317
3 Lanquin, Alta Verapaz, Guatemala AM286694 JN585861
30 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286696 KT998318
T. dimidiata 31 El Lodo Negro, San Marcos Sierra, Intibuca, Honduras AM286695 KT998319
32 El Masical, San Antonio, Copán, Honduras AM286694 KT998320
33 El Lodo Negro, San Marcos Sierra, Intibuca, Honduras AM286693 KT998321
34 El Lodo Negro, San Marcos Sierra, Intibuca, Honduras KT874435 KT998321
35 El Lodo Negro, San Marcos Sierra, Intibuca, Honduras KT874437 KT998321
36 El Masical, San Antonio, Copán, Honduras AM286693 KT998322
37 El Masical, San Antonio, Copán, Honduras KT874436 KT998322
38 El Lodo Negro, San Marcos Sierra, Intibuca, Honduras AM286693 KT998325
39 El Lodo Negro, San Marcos Sierra, Intibuca, Honduras AM286694 KT998325
4 Lanquin, Alta Verapaz, Guatemala AM286702 JN585861
40 El Lodo Negro, San Marcos Sierra, Intibuca, Honduras AM286695 KT998325
41 El Masical, San Antonio, Copán, Honduras KT874434 KT998325
42 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286693 KT998327
43 Angeles, San Rafael, Heredia, Costa Rica AM286693 KT998328
44 Sto. Tomás, Sto. Domingo, Heredia, Costa Rica KT874432 KT998330
45 Angeles, San Rafael, Heredia, Costa Rica KF192844 KT998331
46 San Pedro Columbia, Toledo district, Belize FJ197153 FJ197154
5 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286693 JN585881
6 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286693 JN585881
7 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286693 JN585881
8 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286693 JN585881
9 Caserío la Bendición, Monte Largo, Santa Ana, El Salvador AM286693 JN585881
T. sp. aff dimidiata 1 Calla Creek, Cayo District, Belize FJ197152 FJ197156
2 Mérida, Yucatán, Mexico FJ197150 FJ197158
3 Mérida, Yucatán, Mexico FJ197147 FJ197159
4 Teya, Yucatán, Mexico KT874439 KT998296
5 Huehuetenango, Guatemala
5 Huehuetenango, Guatemala
T. mopan 1 Río Frio Cave, Cayo District, Belize KF192846 JN585883
2 Río Frio Cave, Cayo District, Belize KF192847 JN585884
Col16
T. infestans AJ576054 JN006799
T. gerstaeckeri Chihuahua JQ282707 JQ282723
T. brailovskyi Jalisco JQ282704 JQ282720
T. hegneri AM286727 JN585830

Morphometric study

Morphometric comparison of the dorsal portion of the heads was made based on eight landmarks (Figure 2). Thirteen T. mopan specimens (eleven from the field and two from a Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos – LNIRTT colony, previously regarded as T. dimidiata but found to be T. mopan based on DNA sequence (T. sp. aff. dimidiata – cave; Justi et al. 2018) were compared with all known morphotypes of T. dimidiata s.l. (T. dimidiata maculipennis: one specimen from Mexico; T. dimidiata capitata: four specimens including the holotype; Triatoma dimidiata s.l.: one each from Mexico and Peru, three from Costa Rica, two from Ecuador, six from Guatemala, including two from the Lanquin cave; T. sp. aff. dimidiata: two from Guatemala); and T. gerstaeckeri: five from the southern United States (Table 1). Triatoma gerstaeckeri was included because this species was recovered as a sister taxon to T. mopan (then identified as T. dimidiata) in a recent phylogenetic study (Justi et al. 2016). Size variation observed in colony specimens (Jaramillo et al. 2002) did not interfere with the analyses since for geometric morphometric analysis size information is removed. Additionally, colony specimens were not used for character (morphology) measurements.

Figure 2. 

Landmarks selected for the morphometric comparison. Photograph Raquel Lima.

In order to compare the head shape variation between T. mopan and closely related taxa, we used eight landmarks (clear, visible, homologous intersections between structures; Jaramillo 2011), taken for each specimen using StereoMorph (Olsen and Westneat 2015). Triatoma dimidiata specimens were grouped according to morphotype, and all comparisons were done amongst groups. Generalized Procrustes analysis was performed so shapes were directly comparable, without the effect of size and this was followed by Principal Component Analysis (PCA) and Canonical Variate Analysis (CVA). ANOVA statistics were used to compare variance between the group means for Centroid size (ANOVA) and shape (Procrustes ANOVA), with the null hypothesis that the means are not different among groups. All analyses were performed using the package MorphoJ (Klingenberg lab 2014).

Phylogeny and genetic distance

Portions of the nuclear Internal transcribed spacer 2 (ITS-2) and mitochondrial cytochrome b (cytb) gene sequences from all nominal species belonging to the dimidiata species subcomplex and species affinis (T. dimidiata, T. sp. aff. dimidiata, T. gerstaeckeri, T. brailovskyi, T. hegneri, and T. mopan; Table 2; (Justi and Galvão 2017) were used for phylogenetic reconstruction and comparison of genetic distances, with T. infestans as outgroup. Triatoma mopan specimens used for this purpose were the same as described from Rio Frio cave, Cayo, Belize in Monteiro et al. (2013), Dorn et al. (2016), Justi et al. (2018), including two specimens from the LNIRTT colony. PCR and sequencing of these genes was performed as previously described (Dorn et al. 2016).

Sequences were aligned using MAFFT version 7 (Katoh and Standley 2013) for ITS-2, with the algorithm Q-INS-I and ClustalW (Larkin et al. 2007) implemented on MEGA v. 6 (Tamura et al. 2013) for cytb. JModeltest (Darriba et al. 2015) was used to assess the best fit model of evolution under AIC criterion. Maximum likelihood phylogenies were reconstructed independently for each marker using PhyML v.3.1 (Guindon and Gascuel 2003) with 100 bootstrap replicates. The best-fit model for ITS-2 was HKY+G and for cytb, TPM2uf+G.

In order to evaluate previously reported genetic distances (Monteiro et al. 2003, Monteiro et al. 2004) and assess comparable intra- and interspecific distances with previously reported data (K2-p cytb distances < 2% for interspecific comparisons), we used the package ape (Paradis et al. 2004), in R (The R Development Core Team 2008); the code is provided as Supp. material 1 (S2).

Taxonomy

Family Reduviidae Latreille, 1807

Subfamily Triatominae Jeannel, 1919

Genus Triatoma Laporte, 1832

Triatoma mopan Dorn, Justi & Dale, 2018, sp. n.

Figure 6

Material

Holotype Male. BELIZE: Cayo: Rio Frio Cave, coordinates: 16.956939/-88.979675, 15 June 2016, Dorn, Justi, Stevens, Monroy, CTIOC, FIOCRUZ. Paratypes. Five males and six females, Cayo: Rio Frio Cave, coordinates: 16.956939/-88.979675, 15 June 2016, Dorn, Justi, Stevens, Monroy (CTIOC; FIOCRUZ [four males and five females]; National Museum of Natural History, Smithsonian Institution [one male and one female])

Diagnosis

Triatoma mopan has an overall vestiture similar to T. dimidiata, generally with a more slender body. It is believed to have been wrongly identified as T. dimidiata by Lent and Wygodzinsky (1979). Upon close examination, T. mopan can be easily distinguished from T. dimidiata specimens by the consistently pale-yellow hemelytra, and uniformly dark brown to black head and pronotum with scarce pale setae, absent in T. dimidiata. Triatoma mopan has the pronotum with a straight latitudinal depression dividing it in half and the anterior lobe of the pronotum rugose without any distinguishable tubercles (all the examined morphotypes of T. dimidiata present such tubercles) (Figure 3). In addition, the fore-femora with 1+1 apical, small denticles, 2 +1 subapical denticles in both males and females; and mid-femora with 1+1 apical, small denticles, 2 +2 asymmetrical subapical larger denticles on males and 2 +2 larger, asymmetrical subapical denticles on females. In contrast, T. dimidiata presents 1+1 small subapical denticles on both fore- and mid-femora in both sexes (Figure 4). Abdominal spiracles are close or adjacent to the connexival suture and surrounded by spot slightly darker then the tegument in T. mopan, but never adjacent and always surrounded by a well-defined dark spot in T. dimidiata (Figure 5).

Figure 3. 

Drawings of the pronotum of T. mopan and distinct T. dimidiata morphotypes. Note that for T. dimidiata the anterior lobe shows discal and lateral tubercles, that are absent in T. mopan, which also presents a shorter and rounder anterolateral angle. Drawings by Carolina Dale.

Figure 4. 

Comparison between the fore and mid femora of T. mopan and T. dimidiata from Jutiapa, Guatemala. Drawings: Carolina Dale. Photographs Silvia Justi.

Figure 5. 

Comparison between the position and color of the abdominal spiracles from T. mopan and T. dimidiata from Jutiapa, Guatemala. Drawings by Carolina Dale. Photographs Silvia Justi and Raquel Lima.

Description

Length of male 26.6–30.1 mm., of female 32.1–38 mm.; width of pronotum of male 6.2–6.3 mm., of female 6–7.4 mm (Table 4). Overall color dark brown or black, with connexivum and corium pale yellow. Pilosity pale yellow, short and scarce (Figure 6).

Figure 6. 

Overall vestiture of T. mopan male and female. Scalebar 10 mm. Photographs Raquel Lima.

Head distinctively rugose dorsally (Figure 7), averaging twice as long as width across the eyes (1:0.39–0.46) and slightly longer than the pronotum (1:1.09–1.23). Short, scarce, pale yellow pilosity present in both dorsal and ventral portions (Figure 7). Anteocular region from 2.8 to 3.2 times as long as post ocular (1:0.31–0.35), post ocular region with sides almost straight, subparallel and not rounded. Eyes in lateral view surpassing the level of the ventral surface of the head. Ratio of the width of eye to synthlipsis: 1:1.81–2.46. Ocelli very small, with diameter averaging about half the distance from their anterior border to the posterior margin of the eye. Antenniferous tubercles subcylindrical, situated slightly posterior of middle of anteocular region of head; first antennal segment attaining to the level of or surpassing the apex of clypeus; second segment with many strong setae. Antennal segments presenting a darker to lighter coloration from the first to the fourth segment, going from dark brown (first segment), reddish brown (second segment) to pale yellow (third and fourth segments). Ratio of antennal segments: 1:2.71–3.4:2.5–2.6:1.15. Labium slender; first visible segment extending almost to the base of the antenniferous tubercle in males and to the level of apex of antenniferous tubercle in females; second visible segment extending to the anterior portion of the thorax, almost reaching the stridulatory sulcus in males, and attaining level of posterior border of head on females; third visible segment attaining almost to the posterior portion of the stridulatory sulcus in males, and to the anterior half of the stridulatory sulcus on females (Figure 7). Ratio of visible labium segments: 1:1.79–2.15:0.44–0.69. Color dark brown (first visible segment) to light reddish brown (third visible segment). Setae pale yellow, with length and density increasing from the first visible through the third visible segment, being the longest and denser on the third segment (Figure 7).

Figure 7. 

Comparison between the dorsal and ventral portions of the heads from T. mopan and T. dimidiata from Jutiapa, Guatemala males and females. Photographs: Silvia Justi and Raquel Lima.

Neck dark, with 1+1 lateral light brown to yellowish spots. Pronotum uniformly dark brown to black, with a distinctive depression forming a straight latitudinal line from the neck to the posterior portion of the pronotum (Figure 8). Anterior lobe rugose, without distinctive tubercles (Figures 3 and 8). Anterolateral angles, short, rounded, in some specimens subconical. Scutellum shallowly rugose, with central median depression heart shaped, apical process about as long as body of scutellum, subcylindrical, slightly downwardly bent at apex. Hemelytra usually surpassing the apex of abdomen but leaving female genital segments exposed. Basal portion of clavus dark brown, apex pale yellow. Corium pale yellow, with the extreme apex black, and with a dark central spot of variable size. Membrane almost as pale as the corium. Legs uniformly dark. Forelegs with 1+1 apical small denticles 2 +1 subapical denticles on both males and females. Middle legs with 1+1 apical small denticles 2 +2 asymmetrical subapical denticles on males and 2 +2 asymmetrical subapical denticles on females (Figure 4).

Figure 8. 

Comparison between the pronotum morphology of T. mopan and T. dimidiata from Jutiapa, Guatemala males and females. Photographs: Silvia Justi.

Abdomen ventrally convex, minutely striate transversally, with scarce yellow pilosity. Mostly dark brown, with yellow spots extending from the connexival suture (Figure 9). Spiracles close or adjacent to connexival suture, usually surrounded by a spot slightly darker than tegument (Figure 9). Connexival segments piceous or black on anterior third to half across entire width, almost always as an extension of the piceous portion of the abdomen, pale posteriorly.

Figure 9. 

Comparison between ventral views of from T. mopan and T. dimidiata from Jutiapa, Guatemala males and females. Photographs: Silvia Justi and Raquel Lima.

External genitalia dark brown to black, almost round, with setae darker than the rest of the tegument in males; triangular, with long reddish setae on females (Figure 9).

Distribution

To date, the species is only known from the type locality.

Type-locality

Rio Frio cave, Cayo District, Belize, coordinates 16.956939/-88.979675.

Etymology

The specific epithet mopan was chosen to honor the indigenous Mopan people, one of the Mayan groups historically and presently in this area of Belize and Guatemala, and comprises the lineage previously referred to as T. sp. aff. dimidiata (Group 4 – cave; Monteiro et al. 2013, Stevens et al. 2014, Dorn et al. 2016, Justi et al. 2018).

Host-parasite data

Specimens of T. mopan sp. n. collected prior to its description, in the same Rio Frio cave, were found to be infected with Trypanosoma cruzi (Monroy, unpublished data). The type series was not investigated for parasite infection in order to preserve the integrity of the samples.

Earlier studies completed by our research group, identifying blood sources on specimens of the then undescribed T. mopan collected in the same Rio Frio cave indicate that this species feeds on human, pig, goat or sheep, rat, mouse, duck, bat, opossum, and synanthropic and wild vertebrates (Stevens et al. 2014).

Bionomics

Found in caves, in cracks in rocks near water, in humid environment.

Results

Morphological and morphometric study

Character measures used for the description were compared separately for males and females between T. mopan and the morphotypes of T. dimidiata (Table 3). Barplots showing the mean and standard deviation for each of the significantly different characters are shown on Figure 10, and the bar plots for all the characters are shown in the Suppl. material 1. After the correction for multiple comparisons five of the 18 characters varied between species for females and six of 17 for males (because of missing third and fourth antennae segments, not all 19 characters were measured for T. dimidiata (see Table 3). Four characters were significantly different between species for both females and males (HL, POR, R3 and SYN. For females, the two species also differed for the R1 and R2. For males, A1 and AOR differed between species (Table 3).

Character measurements (mm) of T. mopan and T. dimidiata specimens and significance of t-test results for the comparison between the sexes of both species. Key: asterisk (*) significant value based on Benjamini-Hochberg multiple comparison False Discovery Rate, FDR = 0.05. n.s. non-significant value, N/A – not available.

Character T. mopan females T. dimidiata females T. mopan males T. dimidiata males
N Mean (mm) Min–Max (mm) N Mean (mm) Range (mm) P-value N Mean (mm) Range (mm) N Mean (mm) Range (mm) P-value
A1 6 1.63 1.406–1.781 6 1.492 1.200–1.750 n.s. 5 1.538 1.406–1.625 6 1.223 1.000–1.563 < 0.02*
A2 6 4.719 4.500–5.281 5 4.2 3.400–4.800 n.s. 5 4.769 4.125–5.313 4 4.255 4.000–4.719 n.s.
A3 3 4.365 4.219–4.531 3 3.567 3.100–3.800 n.s. 5 4.194 3.688–4.469 0 N/A N/A N/A
A4 1 1.844 1.844–1.844 3 2.617 1.750–3.100 N/A 5 2.725 0.000–4.188 0 N/A N/A N/A
AOR 6 3.781 3.688–4.063 3 2.567 1.800–3.250 n.s. 5 3.513 3.313–3.813 7 2.734 2.250–3.500 < 0.002*
BOS 6 1.823 1.563–2.063 3 1.717 1.550–1.950 n.s. 5 1.525 1.188–2.000 7 1.857 1.500–2.063 n.s.
HL 6 6.192 5.923–6.538 6 5.383 4.650–5.900 < 0.005* 5 5.569 5.308–5.846 7 4.758 4.308–5.615 < 0.005*
LOP 6 5.295 5.000–6.000 6 4.767 3.950–5.400 n.s. 5 4.723 4.308–5.077 7 4.805 3.950–7.000 n.s.
POR 6 1.281 1.188–1.375 6 0.942 0.800–1.050 < 0.0001* 5 1.15 1.125–1.188 7 0.929 0.750–1.188 < 0.005*
POS 6 1.458 1.000–1.688 3 1.55 1.200–1.750 n.s. 5 1.425 0.813–1.625 7 1.307 1.063–1.625 n.s.
R1 6 2.133 2.000–2.250 6 1.808 1.650–2.000 < 0.002 5 1.87 1.750–2.050 6 1.8 1.650–2.150 n.s.
R2 6 4.133 3.750–4.450 6 3.417 2.900–3.850 < 0.002 5 3.83 3.750–3.950 6 3.292 2.950–4.000 n.s.
R3 6 1.202 1.000–1.610 6 0.967 0.850–1.050 < 0.05 n.s. 5 1.2 1.150–1.250 6 0.933 0.700–1.150 < 0.01*
S 6 1.308 1.231–1.538 6 1.033 0.900–1.250 < 0.005* 5 1.169 1.077–1.231 7 0.95 0.850–1.231 < 0.005*
TL 6 34.58 32.170–38.000 6 33.12 28.830–34.200 n.s. 5 28.73 26.670–30.170 7 29.98 23.830–35.500 n.s.
TS 6 3.281 2.750–3.500 3 3.15 2.500–3.650 n.s. 5 2.95 2.750–3.188 7 3.164 2.750–3.688 n.s.
WAE 6 2.487 2.385–2.692 6 2.492 2.200–2.800 n.s. 5 2.323 2.231–2.462 7 2.415 2.154–2.900 n.s.
WE 6 0.599 0.500–0.625 6 0.733 0.600–0.900 n.s. 5 0.594 0.563–0.625 7 0.654 0.500–0.800 n.s.
WOP 6 6.526 6.077–7.385 6 6.633 2.800–8.000 n.s. 5 6.308 6.231–6.385 7 5.352 3.100–7.385 n.s.

Triatoma mopan character measures for males, females and the holotype.

Males (mm) Females (mm) Holotype (mm)
Min. Mean Max. sd Min. Mean Max. sd
A1 1.41 1.538 1.63 0.086 1.41 1.63 1.78 0.138 1.59
A2 4.13 4.77 5.31 0.483 4.5 4.718 5.28 0.293 5.19
A3 3.69 4.194 4.47 0.303 4.22 4.363 4.53 NA 4.31
A4 2.75 2.952 3.19 0.197 2.75 3.282 3.5 0.279 3.13
AOR 3.31 3.512 3.81 0.189 3.69 3.782 4.06 0.145 3.56
BOS 1.19 1.526 2 -0.332 1.56 1.823 2.06 0.191 1.5
HL 5.31 5.57 5.85 0.261 5.92 6.193 6.54 0.217 5.85
LOP 4.31 4.724 5.08 0.305 5 5.295 6 0.371 4.77
POR 1.13 1.154 1.19 0.033 1.19 1.282 1.38 0.076 1.19
POS 0.81 1.426 1.63 -0.349 1 1.46 1.69 0.28 1.63
R1 1.75 1.87 2.05 0.130 2 2.133 2.25 0.103 2.05
R2 3.75 3.83 3.95 0.084 3.75 4.133 4.45 0.225 3.85
R3 1.15 1.2 1.25 0.05 1 1.202 1.61 0.211 1.25
S 1.08 1.168 1.23 0.063 1.23 1.308 1.54 0.120 1.15
TL 26.67 28.74 30.17 1.677 32.17 34.58 38 2.056 29.67
TS 2.84 3.408 4.19 NA 1.84 1.84 1.84 NA 3.13
WAE 2.23 2.324 2.46 0.084 2.38 2.485 2.69 0.117 2.31
WE 0.56 0.592 0.63 0.025 0.5 0.602 0.63 0.052 0.59
WOP 6.23 6.306 6.38 0.075 6.08 6.525 7.38 0.458 6.38
Figure 10. 

Barplot of the character measures that were significantly different for at least one comparison between male and females of T. mopan and T. dimidiata (see Table 3).

Head shape comparison between T. mopan specimens and T. dimidiata and T. gerstaeckeri revealed a unique separate cluster comprising T. mopan for both PCA and CVA (Figure 11). ANOVA results led to the rejection of the null hypothesis (p<0.0001), that is, our results indicate that the variance between the means of the groups are different. PCA results show that most of the variation is from landmarks corresponding to the antennal tubercle and the anterior portion of the eye (Figure 12).

Figure 11. 

PCA and CVA of the Morphometric comparison of the heads of T. mopan, T. gerstaeckeri, Triatoma from Lanquin and T. dimidiata different morphotypes. Note that both T. mopan and Triatoma from Lanquin form clusters separated from the other species.

Figure 12. 

PCA thin plate showing that landmarks 1 and 2 are the most variable within the compared specimens.

Phylogenetic reconstruction and genetic distances

Both ML phylogenies recovered T. mopan as an independently evolving lineage, with the highest bootstrap support recovered for each phylogeny. Triatoma mopan was recovered as a sister taxon to T. dimidiata upon ITS-2 phylogenetic reconstruction and as sister to T. gerstaeckeri when cytb was used for the reconstruction (Figure 13).

Figure 13. 

ML phylogenies reconstructed using ITS-2 (right) and cytb (left) sequences. Numbers above branches represent bootstrap support > 50. T. mopan clade is highlighted in purple.

Pairwise Kimura 2-parameter genetic distances revealed T. mopan to diverge at least 2% from the other lineages when distances were calculated for ITS-2; while for cytb the minimum pairwise distance between T. mopan and the closest examined species increases to about 10% (Figure 14).

Figure 14. 

Plot of the K2-p distances calculated pairwise between T. mopan and all the other specimens used for the phylogenetic reconstruction. Dotted line indicates 2% divergence.

Discussion

Phylogenetic studies of the diverse T. dimidiata have long shown the taxa to be composed of at least three independently evolving lineages (Dorn et al. 2007, 2016, Bargues et al. 2008, Herrera-Aguilar et al. 2009, Monteiro et al. 2013, Justi et al. 2018). One such lineage, the Triatoma from the Rio Frio cave in Belize, was first observed to be a separate phylogenetic species by Monteiro et al. 2013, and referred to as T. dimidiata group 4. Later, more comprehensive studies confirmed the specific phylogenetic status of the lineage referred to as T. sp. aff. dimidiata – cave, pending the formal description of the new species (Dorn et al. 2016, Justi et al. 2018). In this study, we describe the Triatoma lineage from the Rio Frio cave, in Belize and and name it Triatoma mopan, after the Mopan people of that area.

We have compared T. mopan with T. dimidiata using different systematic approaches: classic morphology, geometric morphometric and molecular phylogeny, and the results agree in that these are separate species. Diagnostic characters were observed on the pronotum, legs and abdomen and PCA and CVA results place both in separate clusters (Figures 3, 4, 5, 11).

The morphological comparison of T. mopan with the description of T. dimidiata (Lent and Wygodzinsky 1979) and photos of the holotypes of T. dimidiata capitata and T. dimidiata maculipennis (Figure 15), along with the molecular phylogenetic results published previously (Dorn et al. 2007, 2009, 2016, Bargues et al. 2008, Monteiro et al. 2013, Justi et al. 2018) show the uniqueness of the Rio Frio species. All diagnostic characters and the description of the species, allowed us to observe a combination of characters unique to T. mopan.

Figure 15. 

Triatoma dimidiata capitata (Photo: Rachel Diaz-Bastin, California Academy of Sciences), T. mopan (Photo: Raquel Lima-Cordón), and T. dimidiata maculipennis (Photo: © Schurian / MfN.berlin) holotypes. Photographs are not to scale.

Lent and Wygodzinsky (1979) observed 160 specimens of T. dimidiata and concluded that the morphological variation observed within the taxa displays a clinal pattern with size increasing southwards. Amongst those, they describe five specimens from the Lanquin cave, in Guatemala, highlighting differences in the length and ratio of the antennae, and mentioning characteristics related to the cave environment, such as diminished pigmentation and eye and ocelli size when compared with non-cave populations of T. dimidiata. The authors also stated that the single specimen examined from the Rio Frio cave “is identical phenotypically with the specimens from Lanquin cave”. In light of this statement, we compared T. mopan with the Lent and Wygodzinsky (1979) description and to two specimens from the Lanquin cave population and noticed distinctive characteristics in the head (Table 5), wing, and abdomen coloration pattern (Figure 16) that clearly separate T. mopan and the Lanquin population of Triatoma. Likely convergent evolution due to the cave environment is observed in the diminished pigmentation and relative smaller eye and ocelli sizes and the absence of tubercles on the anterior lobe of the pronotum. Even though Lent and Wygodzinsky stated that these are T. dimidiata populations from caves, it is not possible to taxonomically place these Lanquin and Rio Frio cave specimens using their key to the species of Triatoma (combination of characters described on dychotomy 39 does not correspond to either population).

Character comparison between T. mopan, T. dimidiata, and Triatoma from Lanquin cave, based on the description of Lent and Wygodzinsky (1979) for the latter.

T. mopan Triatoma Lanquin T. dimidiata
Antenna first antennal segment attaining to the level of or surpassing the apex of clypeus first antennal segment surpassing the apex of clypeus first antennal segment attaining to the level of the apex of clypeus
POR/AOR 2.8–3.2 4 2.5–3
Ratio antennae segments 1:2.7–3.4:2.5-2.6:1.15 1:2.5:2.2:2.3 1:3.2–3.8:2.5:2.2.
Eyes surpassing the level of the ventral surface of the head not surpassing the level of the ventral surface of the head attaining to the level of the ventral surface of the head
Figure 16. 

Comparison between Triatoma from Lanquin and T. mopan. Photographs to scale. Photo: Carolina Dale.

The geometric morphometric comparison of the head shape (PCA and CVA) also placed the Lanquin specimens closer to T. dimidiata (but separate from) than to T. mopan, which forms a unique separate cluster (Figure 11). Similar results were previously found, placing the Lanquin population in a separate cluster from the T. dimidiata populations (Bustamante et al. 2004). Additionally, previous molecular phylogeny results have shown that the Triatoma specimens collected in the Lanquin cave fall within T. dimidiata s.s (Monteiro et al. 2013, Dorn et al. 2016), while the ones collected in the Rio Frio cave comprise an independently evolving lineage. These results were confirmed by the broader phylogenetic recontruction and comparison of genetic distances performed for this study (Figures 13, 14). The widely distinct morphology of the Lanquin cave specimens, combined with these phylogenetic results show the need for a deeper study of this population to better understand its evolution and taxonomy.

The comparison of the morphology of T. mopan with T. dimidiata shows a clear trend in cave adaptation evolution. Besides the diminished overal pigmentation of the specimens, T. mopan exhibits much denser sensillae on the visible labial segments (Figure 7), which are significantly longer than in T. dimidiata, probably to compensate for the reduced visual cues in such an environment.

In combination, the morphological characters with molecular phylogeney and geometric morphometry of the head show that T. mopan is an independently evolving lineage, separate from T. dimidiata. The comparison with the types of T. dimidiata maculipennis, T. dimidiata capitata and the description given by Lent and Wygodzinsky (1979) for T. dimidiata, in the absence of the type, make it clear that T. mopan is a separate species, not previously formally described as any morphotype or subspecies of T. dimidiata.

Acknowledgements

We are grateful for prior work by Heather Axen, Bethany Richards, Nicholas de la Rúa, and Dulce Bustamante that suggested T. mopan was a separate species. We also thank Leticia Nery and Renata Cardoso, from the Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, FIOCRUZ for help with the collection specimens used in this study; Lisa Chamberland for the help with the photos; Dr. Jurgern Deckert and Bernhard Schurian from the Museum für Naturkunde Berlin for the photos of the types of T. dimidiata maculipennis; and Rachel Diaz-Bastin from the California Academy of Sciences for the photos of the type of T. dimidiata capitata. We also thank Thomas Henry, curator of the Heteroptera collection at the National Museum of Natural History, Smithsonian Institution for the invaluable comments on this manuscript.

Funding was provided by grant R03AI26268/1-2 from the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH); 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 CNPq Research Productivity grant 305055/2016-0. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the funding organizations.

References

  • Bargues MD, Klisiowicz DR, Gonzalez-Candelas F, Ramsey JM, Monroy C, Ponce C, Salazar-Schettino PM, Panzera F, Abad-Franch F, Sousa OE, Schofield CJ, Dujardin JP, Guhl F, Mas-Coma S (2008) Phylogeography and genetic variation of Triatoma dimidiata, the main Chagas disease vector in Central America, and its position within the genus Triatoma. PLoS neglected tropical diseases 2: e233. https://doi.org/10.1371/journal.pntd.0000233
  • Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society 57: 289–300.
  • Bustamante DM, Monroy C, Menes M, Rodas A, Salazar-Schettino PM, Rojas G, Pinto N, Guhl F, Dujardin JP (2004) Metric Variation Among Geographic Populations of the Chagas Vector Triatoma dimidiata (Hemiptera: Reduviidae: Triatominae) and Related Species. Journal of Medical Entomology 41: 296–301. https://doi.org/10.1603/0022-2585-41.3.296
  • Dorn PL, Calderon C, Melgar S, Moguel B, Solorzano E, Dumonteil E, Rodas A, de la Rua N, Garnica R, Monroy C (2009) Two distinct Triatoma dimidiata (Latreille, 1811) taxa are found in sympatry in Guatemala and Mexico. PLoS neglected tropical diseases 3: e393. https://doi.org/10.1371/journal.pntd.0000393
  • Dorn PL, de la Rúa NM, Axen H, Smith N, Richards BR, Charabati J, Suarez J, Woods A, Pessoa R, Monroy C, Kilpatrick CW, Stevens L (2016) Hypothesis testing clarifies the systematics of the main Central American Chagas disease vector, Triatoma dimidiata (Latreille, 1811), across its geographic range. Infection, Genetics and Evolution 44: 431–443. https://doi.org/10.1016/j.meegid.2016.07.046
  • Dorn PL, Monroy C, Curtis A (2007) Triatoma dimidiata (Latreille, 1811): A review of its diversity across its geographic range and the relationship among populations. Infection, Genetics and Evolution 7: 343–352. https://doi.org/10.1016/j.meegid.2006.10.001
  • Herrera-Aguilar M, Be-Barragán LA, Ramirez-Sierra MJ, Tripet F, Dorn P, Dumonteil E (2009) Identification of a large hybrid zone between sympatric sibling species of Triatoma dimidiata in the Yucatan peninsula, Mexico, and its epidemiological importance. Infection, Genetics and Evolution 9: 1345–1351. https://doi.org/10.1016/j.meegid.2009.09.009
  • Jaramillo ON, Castillo D, Wolff EM (2002) Geometric morphometric differences between Panstrongylus geniculatus from field and laboratory. Memorias do Instituto Oswaldo Cruz 97: 667–673. https://doi.org/10.1590/S0074-02762002000500015
  • Justi SA, Cahan S, Stevens L, Monroy C, Lima-cordón R, Dorn PL (2018) Vectors of diversity : Genome wide diversity across the geographic range of the Chagas disease vector Triatoma dimidiata sensu lato (Hemiptera: Reduviidae) . Molecular Phylogenetics and Evolution 120: 144–150. https://doi.org/10.1016/j.ympev.2017.12.016
  • Justi SA, Galvão C, Schrago CG (2016) Geological Changes of the Americas and their Influence on the Diversification of the Neotropical Kissing Bugs (Hemiptera: Reduviidae: Triatominae). PLOS Neglected Tropical Diseases 10: e0004527. https://doi.org/10.1371/journal.pntd.0004527
  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30: 772–780. https://doi.org/10.1093/molbev/mst010
  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics (Oxford, England) 23: 2947–8. https://doi.org/10.1093/bioinformatics/btm404
  • Lent H, Wygodzinsky P (1979) Revision of the Triatominae (Hemiptera: reduviidae), and their significance as vectors of Chagas Disease. Bulletin of the American Museum of Natural History 163: 123–520.
  • Monteiro FA, Peretolchina T, Lazoski C, Harris K, Dotson EM, Abad-Franch F, Tamayo E, Pennington PM, Monroy C, Cordon-Rosales C, Salazar-Schettino PM, Gómez-Palacio AM, Grijalva MJ, Beard CB, Marcet PL (2013) Phylogeographic Pattern and Extensive Mitochondrial DNA Divergence Disclose a Species Complex within the Chagas Disease Vector Triatoma dimidiata. PLoS Neglected Tropical Diseases 8: e70974. https://doi.org/10.1371/journal.pone.0070974
  • Monteiro FA, Barrett TV, Fitzpatrick S, Cordon-Rosales C, Feliciangeli D, Beard CB (2003) Molecular phylogeography of the Amazonian Chagas disease vectors Rhodnius prolixus and R. robustus. Molecular ecology 12: 997–1006. https://doi.org/10.1046/j.1365-294X.2003.01802.x
  • Monteiro FA, Donnelly MJ, Beard CB, Costa J (2004) Nested clade and phylogeographic analyses of the Chagas disease vector Triatoma brasiliensis in Northeast Brazil. Molecular phylogenetics and evolution 32: 46–56. https://doi.org/10.1016/j.ympev.2003.12.011
  • Olsen AM, Westneat MW (2015) StereoMorph: an R package for the collection of 3D landmarks and curves using a stereo camera set-up. Methods in Ecology and Evolution 6: 351–356. https://doi.org/10.1111/2041-210X.12326
  • Stevens L, Monroy MC, Rodas AG, Dorn PL (2014) Hunting, Swimming, and Worshiping: Human Cultural Practices Illuminate the Blood Meal Sources of Cave Dwelling Chagas Vectors (Triatoma dimidiata) in Guatemala and Belize. PLoS Neglected Tropical Diseases 8. https://doi.org/10.1371/journal.pntd.0003047
  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30: 2725–2729. https://doi.org/10.1093/molbev/mst197
  • The R Development Core Team (2008) 2.6.2 R: A Language and Environment for Statistical Computing. 2547 pp.
  • Usinger (1941) Notes and descriptions of neotropicalTriatominae (Hemiptera, Reduviidae). Pan Pacific Entomology 17: 49–57.