Checklist and phenetics studies of nymphs of two species of triatomines: Triatoma lenti Sherlock & Serafim, 1967 and Triatoma sherlocki Papa, Jurberg, Carcavallo, Cerqueira, Barata, 2002 (Hemiptera: Reduviidae: Triatominae).

INTRODUCTION: Triatoma lenti and Triatoma sherlocki are endemic species of the State of Bahia, located in northeastern Brazil, where they have records of domiciliation in the human environment. In view of the epidemiological aspect and taxonomic importance of these species for the systematics of the Triatoma genus, a study was carried out with nymphs of all five instars.
METHODS: An extensive review of studies on nymphs from the subfamily Triatominae is presented. Morphology was studied using a scanning electron microscope and morphometric analyses.
RESULTS: The morphological study allowed us to characterize and discriminate species by means of scanning electron microscope of the last abdominal segment. In addition, the results show morphometric variability, with the total size of the head that best discriminates the species.
CONCLUSIONS: Studies on nymphs are fundamental to the ecosystem; however, the literature on the immature forms of certain groups is scarce, difficult to use, or nonexistent. Therefore, this study includes morphological and morphometric data of the nymphal instars of T. lenti and T. sherlocki, corroborating the specific taxonomy of these species.

INTRODUCTION

In the Americas, various species of triatomines are vectors of Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae), the etiological agent of Chagas disease . The insects of the subfamily Triatominae (Jeannel, 1919) are hematophagous and feed primarily on vertebrate blood . Even though all species of triatomines are hematophagous, species that colonize residential places or are peridomicile have increased chances of transmitting T. cruzi to humans . After the successful Triatoma infestans (Klug, 1834) control program conducted by the Brazilian National Health Foundation, other triatomines previously considered predominately sylvatic have emerged as potential vectors in several areas of Brazil , .

Presently, 157 species (including 3 fossils) within 18 genera are recognized as valid in this subfamily , , . The genus with the greatest number of species described is Triatoma Laporte, 1832, which includes members of the Triatoma brasiliensis subcomplex, Triatoma brasiliensis Neiva 1911, Triatoma brasiliensis macromelasoma Galvão 1956, Triatoma melanica Neiva & Lent, 1941, Triatoma juazeirensis Costa & Felix 2007, Triatoma sherlocki Papa, Jurberg, Carcavallo, Cerqueira, Barata, 2002, Triatoma lenti Sherlock & Serafim, 1967, T. bahiensis Sherlock & Serafim, 1967 and Triatoma petrochiae Pinto & Barreto 1925. ,

Sherlock and Serafim described T. lenti, T. pessoai, and T. bahiensis. The authors reported that T. lenti and T. pessoai were naturally infected by T. cruzi and were relatively easily maintained in the laboratory by feeding on pigeons. Currently, only T. pessoai is not considered a valid species , .

Cerqueira et al. refer to the encounter of wild triatomine, naturally infected by T. cruzi in the district of Santo Inácio, municipality of Gentio do Ouro, Bahia. Later in 1982, Cerqueira, in his doctoral dissertation, studied the biological cycle and evaluated the results of crosses of this wild triatomine with T. brasiliensis; however, it was not considered a new species. Papa et al. resumed studies of the triatomines studied by Cerqueira in 1982 and based on consistent morphological characters, such as genital structures, shorter wings, red orange spots on the connexivum and legs, inability to fly, and longer legs, concluded that it is a new species named T. sherlocki. Triatoma sherlocki was related to T. lenti by morphological characteristics, cytogenetics, molecular data, and experimental crosses, and was included as a member of the Triatoma brasiliensis complex , , , , , , .

Morphology and morphometry are tools that contribute to the knowledge of triatomines and generate useful information to establish more effective strategies for vector control . In Triatominae, biometric studies are used to characterize new species, detect populations, and define structures . For example, geometric morphometry allows the collection of information about the shape and size of organisms, which helps in systematic and phylogenetic studies , , .

Several authors have used morphology and morphometry to characterize the species and correlate the known characteristics of the character, isoenzymatic and ecological, and contributed to both systematic analyses. Studies on immature instars of T. lenti and T. sherlock are scarce; therefore, we evaluated and characterized those species that make up the T. brasiliensis subcomplex, a relevant group for the ecoepidemiology of Chagas disease in the northeastern region of Brazil - , by gathering all information from the literature on the study of immature forms in Triatominae.

METHODS

Insects

We used specimens from a T. lenti ( Figure 1 ) colony collected on April 9, 2008, which were found in the county of Macaúbas (Mangabeiras and Cana Brava neighborhoods) in the state of Bahia. The specimens were collected at altitudes of 747, 755, 780, and 829 m in the peridomicile and intradomicile. On July 22, 2003, T. sherlocki ( Figure 1 ) was collected in Gentio do Outo, Santo Inácio, Bahia state, and later a colony was established in the laboratory. The specimens were kept and deposited at the Triatomine Insectario of the Faculty of Pharmaceutical Sciences, Universidade Estadual Paulista (https://www2.fcfar.unesp.br/#!/triatominae). Approved by the Ethics Committee on the Use of Animals - CEUA, CEUA/FCF/CAr: 15/2017).

FIGURE 1:

Fifth instar nymph of T. sherlocki and T. lenti. (A) Dorsal view of the fifth instar nymph of T. sherlocki. (B) Dorsal view of the fifth instar nymph of T. lenti, (C) Ventral view of the fifth instar nymph of T. sherlocki, and (D) Ventral view of the fifth instar nymph of T. lenti.

Morphological analyses

The fifth instar nymphs from T. sherlocki and T. lenti ( Figure 1 ) were cleaned using an ultrasound device. Next, the structures were dehydrated in alcohol, dried in an incubator at 45ºC for 20 min, and fixed in small aluminum cylinders with transparent glass. Sputtering metallization was then performed on the samples for 2 min at 10 mA in an Edwards sputter coater. After metallization, the samples were studied and photographed using a Topcon SM-300 scanning electron microscope (SEM), according to Rosa et al. . The images obtained were processed (background, contrast, brightness) using the GNU Image Manipulation Program v2.0.2 (GIMP) software free and open-source image editor, and the structures were described and compared.

Morphometric analyses

For the T. lenti and T. sherlocki measurements, 15 nymphs specimens in the first, second, third, fourth, and fifth instars were fixed on glass slides using a double-sided tape. Measurements were also taken to determine the thorax, abdomen, and head length, as well as interocular, ante-ocular, and postocular distance, eye diameter, and the three proboscis segments. These distances were defined by Dujardin et al. . The measurements were taken using a Leica MZ APO stereomicroscope and analyzed using the Motic Advanced 3.2 image analysis software. Descriptive statistics analyses and Welch's t-test were performed using GraphPad Prism v.5.03.

Principal component analysis

To visualize the general patterns of morphological variation in the multidimensional data obtained with the principal component analysis (PCA) of the references, a factorial map was generated using Past 3.2 .

Checklist of studies on the immature instars of triatomines

The survey of publications that study the immature instars of development had as selection criteria publications with morphology and morphometry of nymphs regardless of the methodological approach. Publications were retrieved from databases such as: National Center for Biotechnology Information- NCBI (available at https://www.ncbi.nlm.nih.gov/), Bibliography of Triatominos - BibTri (available at: https://bibtri.cepave.edu. ar/webbibtri.php), Google Academic (https://scholar.google.com.br/?hl=pt) and Scielo (https://www.scielo.org/). The keywords used for the search were: Nymphs, Triatominae, Hemiptera, Reduviidae, Morphology, Morphometry, Description, Ontogenetic, Instar, Description of nymphs, key, eggs, 1st, 2nd, 3rd, 4th, 5th and instars.

RESULTS

Through an extensive literature survey on immature forms of triatomines, we recovered 115 studies that explored the morphological aspects of nymphs; therefore, we updated the list presented by Galvão 2014 (Table 1).

TABLE 1:

Checklist of studies that include information on the nymphal instars of triatomines (Galvão, 20146, with modifications).

Species	Approach	References
Alberprosenia goyavargasi	Description of nymphs by SEM*	Carcavallo et al. 29
Alberprosenia malheiroi	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO*, SEM)	Carcavallo et al. 30
Belminus herreri	Description and geometric morphometry of nymphs	Rocha et al. 31
Cavernicola pilosa	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Valderrama & Lizano 32
Cavernicola lenti	Description of nymphs (MO), every aspect shown by SEM	Costa et al. 33
Dipetalogaster maxima	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Jurberg et al. 34
Eratyrus mucronatus	Morphological (MO and SEM) and key	Galíndez-Girón et al. 35
Linshcosteus confumus	SEMs and description of eggs	Haridass 36
Linshcosteus costalis	SEMs and description of eggs	Haridass 36
Linshcosteus karupus	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars by SEM	Galvão et al. 37
Triatoma longipennis	Morphological (MO and SEM) and key	Galíndez-Girón et al. 35
Triatoma pallidipennis	Ontogenetic morphometrics (MO)	Rodríguez-Sánchez et al. 38
	Morphological (MO and SEM) and key	Galíndez-Girón et al. 35
Triatoma phyllosoma	Morphological (MO and SEM) and key	Galíndez-Girón et al. 35
Mepraia spinolai	Morphological (MO and SEM) and key	Galíndez-Girón et al. 35
Microtriatoma trinidadensis	Description of nymphs (MO)	Carcavallo et al. 39
		Riva et al. 40
Nesotriatoma flavida	Morphometrics	Jiménez and Fuentes, 41
Panstrongylus geniculatus	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Panstrongylus humeralis	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Panstrongylus lignarius	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Panstrongylus megistus	Morphology of spiracles 5th instar nymphs	Rosa et al. 26
	Sexual distinction between 5th instar nymphs by SEM	Rosa et al. 42
	Morphology of 5th instar nymphs by SEM	Rosa et al. 43
	Abdominal structures of 5th instar nymphs	Rosa & Barata 44
	Morphology of antennae of 1st, 2nd, 3rd, 4th, and 5th instars (SEM)	Rosa et al. 45
Paratriatoma hirsuta hirsuta	Morphology of 5th instar nymphs (MO)	Ryckman 46
	Morphological study (MO and SEM)	Galíndez-Girón et al. 35
Paratriatoma hirsuta kamiensis	Morphology of 5th instar nymphs (MO)	Ryckman 46
Paratriatoma hirsuta papagoensis	Morphology of 5th instar nymphs (MO)	Ryckman 46
Paratriatoma hirsuta pimae	Morphology of 5th instar nymphs (MO)	Ryckman 46
Paratriatoma hirsuta yumanensis	Morphology of 5th instar nymphs (MO)	Ryckman 46
Paratriatoma lecticularia	Description of nymphs (MO) and visualization of structures using SEM.	Rocha et al. 47
	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Psamolestes arthuri	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Carcavallo et al. 48
Psamolestes coreodes	Morphological (MO and SEM) and key	Galíndez-Girón et al. 35
Rhodnius brethesi	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars	Mascarenhas 50
Rhodnius dalessandroi	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Rhodnius ecuadoriensis	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Rhodnius neglectus	Morphology of spiracles 5th instar nymphs	Rosa et al. 26
	Sexual distinction between 5th instar nymphs by SEM	Rosa et al. 42
	Morphology of 5th instar nymphs by SEM	Rosa et al. 43
	Abdominal structures of 5th instar nymphs	Rosa & Barata, 44
	Morphometric of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Ponsoni et al. 49
	Morphology of antennae of 1st, 2nd, 3rd, 4th, and 5th instars (SEM)	Rosa et al. 45
Rhodnius neivai	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Rhodnius pallescens	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Rhodnius prolixus	Morphometric of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Marconato et al. 51
	Morphology of antennae of 1st, 2nd, 3rd, 4th, and 5th instars (SEM)	Rosa et al. 45
		Rosa et al. 26
	Morphology of spiracles 5th instar nymphs	Rosa et al. 42
	Sexual distinction between 5th instar nymphs by SEM	Rosa et al. 43
	Morphology of 5th instar nymphs by SEM	Rosa & Barata, 44
	Abdominal structures of 5th instar nymphs	Ponsoni et al. 49
	Morphology and key (MO and SEM)	Galíndez-Girón et al. 35
Rhodnius pictipes	Description of nymphs by MO	Lent & Valderrama 52
Triatoma arthurneivai	Nymphal instars by SEM	Rosa et al. 87
Triatoma baratai	Description of nymphs (MO) and visualization of structures using SEM	Rocha et al. 53
Triatoma barberi	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO, SEM)	Carcavallo et al. 30
Triatoma brasiliensis	Description of nymphs (MO) and visualization of structures using SEM	Jurberg et al. 54
Triatoma breyeri	Description and keys for all instars.	Rosa & Barata, 44
	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Triatoma carcavalloi	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Jurberg et al. 55
Triatoma circummaculata	Morphology of the head of 1st and 5th instar nymphs and visualization of some structures by SEM	Rosa et al. 55 , 56
Triatoma costalimai	Description and ontogenetic morphometrics of instars	Raigorodschi et al 58
Triatoma deaneorum	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Galvão &Fuentes 59
Triatoma delpontei	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
	Description and keys for all instars	Brewer et al. 60 , 61
Triatoma dimidiata	Morphological study of nymphs (MO and SEM)	Mello et al. 62
Triatoma dispar	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Triatoma eratyrusiformis	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Triatoma gerstaeckeri	Morphological study of nymphs (MO and SEM)	Galíndez-Girón et al. 35
Triatoma guasayana	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Brewer & Garay 63
Triatoma guazu	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO and SEM)	Silva et al. 64
	Comparative study of stridulatorium sulcus, bucculla and rostrum	Silva et al. 65 , 83
	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Triatoma infestans	Sexual distinction between 5th instar nymphs by SEM	Rosa et al. 26
	Morphology of 5th instar nymphs by SEM	Rosa et al. 42
	Abdominal structures of 5th stage nymphs	Rosa et al. 43
	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Galíndez-Girón et al. 35
Triatoma jurbergi	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO, MEV)	Jurberg et al. 66
	Comparative study of stridulatorium sulcus, bucculla and rostrum	Silva et al. 67
Triatoma klugi	Description of nymphs (MO) and visualization of structures using SEM.	Jurberg et al. 66 , 85
	Comparative study of the stridulatorium sulcus, buccula and rostrum of nymphs	Silva et al. 67
Triatoma lenti	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Triatoma maculata	Morphometric of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Gonçalves et al. 71
	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Triatoma matogrossensis	Abdominal structures of 5th instar nymphs	Rosa et al. 26
	Sexual distinction between 5th instar nymphs	Rosa et al. 42
	Morphology of 5th instar nymphs by SEM	Rosa et al. 43
Triatoma melanocephala	Morphometric characterization of the nymphal instars	Oliveira et al. 68 Jurberg et al. 84
Triatoma melasoma	Morphological study of nymphs (MO and SEM)	Galíndez-Girón et al. 35
Triatoma nitida	Morphology of of 1st, 2nd, 3rd, 4th, and 5th instars (SEM)	Jurberg et al. 69
Triatoma pintodiasi	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Motta & Moreira, 70
Triatoma platensis	Description of nymphs (MO) and keys	Brewer et al. 60
		Brewer & Garay, 63
Triatoma protacta	Morphology and key of 5th instar nymphs of species and subspecies	Ryckman 82
Triatoma pseudomaculata	Morphometric of 1st, 2nd, 3rd, 4th, and 5th instar (MO)	Gonçalves et al. 71
	Morphology of nymphs (MO and SEM)	Galíndez-Girón et al. 35
Triatoma ryckmani	Description of all immature instars based on MO and SEM	Rocha et al. 88
Triatoma rubrofasciata	SEM	Haridass 36
Triatoma rubrovaria	Morphology of the head of 1st and 5th instar nymphs	Rosa et al. 56
	Antenna morphometry	Rosa et al. 57
	Morphological and key (MO and SEM)	Galíndez-Girón et al. 35
Triatoma sordida	Morphometry of 1st, 2nd, 3rd, 4th, and 5th instars	Brewer et al. 60
	Description of nymphs of 1st, 2nd, 3rd, 4th, and 5th instars (MO)	Brewer et al. 61
	and keys for all instars	Brewer & Garay. 63
	Morphology and key (MO and MEV)	Galíndez-Girón et al. 35
Triatoma tibiamaculata	Abdominal structures of 5-instar nymphs	Rosa & Barata, 44
Triatoma vandae	Description of nymphs (MO) and visualization of structures using SEM.	Silva et al. 72
	Comparative study of the stridulatorium sulcus, buccula and rostrum of nymphs	Silva et al. 67
Triatoma vitticeps	Antenna morphometry and morphology	Rosa et al. 45
	Morphology compared to other Reduviidae	Weirauch 73
Triatoma williami	Description of nymphs (MO) and visualization of structures using SEM.	Silva et al. 72
	Comparative study of the stridulatorium sulcus, buccula and rostrum of nymphs	Silva et al. 67
	Morphology and key (MO and SEM)	Galíndez-Girón et al. 35
Diverse species	Some structures and key for Triatominae (MO and SEM)	Galíndez-Girón et al. 35

Morphological characteristics

The morphological characteristics of the two species are presented in Figure 2. According to the genital morphology of fifth instar nymphs, the ninth ventral abdominal segment is wider in T. lenti than in T. sherlocki, as well as the presence of a hole in the posterior portion of this segment in T. lenti and its absence in T. shelocki. The ninth ventral abdominal segment of fifth instar nymphs shows parallel grooves in the posterior region, which are most evident in T. lenti, while their presence in T. sherlocki is poorly visible. The eighth segment was trapezoidal in T. sherlocki and oval in T. lenti. The laterals were irregular at the apex. It was found that the ninth segment had few sensilla, as well as segments 7, 8, and 10. The tenth segment was curved ventrally in the posterior portion. Sexual dimorphisms of the nymphs are characterized by the size of the ninth segment ventrally, in which females have a narrow (Figure 2 A, B) while males have a wide ninth segment (Figure 2 C, D).

FIGURE 2:

Ventral view of the terminal segments of the male and female fifth instar nymph of T. sherlock and T. lenti. (A) Female of T. sherlocki, (B) Female of T. lenti, (C) Male of T. sherlocki, (D) Male of T. lenti. X, IX, VIII: tenth, ninth, eighth ventral abdominal segment.

Morphometric characteristics

Morphometric characteristics of the two species are presented in Table 2. The averages of the total length of heads in T. sherlocki and T. lenti were 1.34 and 1.42 mm, 1.84 and 1.83 mm, 2.65 and 2.93 mm, 3.61 and 3.86 mm, and 4.56 and 4.53 mm in the first, second, third, fourth, and fifth instar, respectively.

TABLE 2:

Mean and standard deviation results of head (including Outer distance between the eyes, Inner distance between the eyes, Postocular distance, Ante-Ocular distance), thorax and abdomen measurements of T. lenti and T. sherlocki nymphs.

Parameters	Triatoma sherlocki					Triatoma lenti
	1st instar	2nd instar	3rd instar	4th instar	5th instar	1st instar	2nd instar	3rd instar	4th instar	5th instar
Total lenght of head (mm)	1,34 ± 0,03	1,84 ± 0,05	2,65 ± 0,06	3,61 ± 0,17	4,56 ± 0,14	1,42 ± 0,08	1,83 ± 0,05	2,93 ± 0,06	3,86 ± 0,12	4,39 ± 0,16
Outer distance between the eyes (OE) (mm)	0,25 ± 0,01	0,39 ± 0,03	0,53 ± 0,03	0,69 ± 0,04	0,94 ± 0,04	0,24 ± 0,01	0,36 ± 0,03	0,53 ± 0,04	0,70 ± 0,03	0,93 ± 0,03
Inner distance between the eyes (IE) (mm)	0,51 ± 0,01	0,66 ± 0,02	0,92 ± 0,03	1,23 ± 0,07	1,54 ± 0,05	0,55 ± 0,02	0,68 ± 0,02	1,01 ± 0,03	1,29 ± 0,03	1,69 ± 0,07
Postocular distance (PO) (mm)	0,24 ± 0,02	0,31 ± 0,03	0,41 ± 0,02	0,53 ± 0,04	0,60 ± 0,04	0,28 ± 0,03	0,30 ± 0,02	0,48 ± 0,03	0,60 ± 0,04	0,88 ± 0,04
Ante-Ocular distance (AO) (mm)	0,83 ± 0,02	1,20 ± 0,04	1,81 ± 0,04	2,50 ± 0,01	3,21 ± 0,12	0,89 ± 0,04	1,22 ± 0,04	1,95 ± 0,05	2,66 ± 0,07	3,65 ± 0,15
Total thorax lenght (mm)	0,98 ± 0,03	1,43 ± 0,05	2,08 ± 0,06	2,93 ± 0,15	4,68 ± 0,18	0,99 ± 0,05	1,43 ± 0,05	2,02 ± 0,05	3,26 ± 0,11	5,29 ± 0,15
Total abdomen lenght (mm)	1,75 ± 0,11	3,17 ± 0,24	5,24 ± 0,23	6,68 ± 0,42	11,29 ± 0,39	1,57 ± 0,10	3,04 ± 0,13	5,60 ± 0,22	6,33 ± 0,33	11,14 ± 0,75
Proboscis 1st segment (1S) (mm)	0,29 ± 0,03	0,43 ± 0,02	0,58 ± 0,04	0,84 ± 0,04	1,18 ± 0,07	0,29 ± 0,03	0,40 ± 0,02	0,6 ± 0,04	0,85 ± 0,03	1,19 ± 0,06
Proboscis 2nd segment (2S) (mm)	0,65 ± 0,02	1,0 ± 0,04	1,35 ± 0,04	1,85 ± 0,07	2,50 ± 0,08	0,64 ± 0,02	0,98 ± 0,03	1,48 ± 0,03	1,94 ± 0,06	2,58 ± 0,09
Proboscis 3rd segment (3S) (mm)	0,35 ± 0,02	0,54 ± 0,01	0,69± 0,02	0,93 ± 0,03	1,26 ± 0,05	0,36 ± 0,02	0,52 ± 0,02	0,72 ± 0,02	0,97 ± 0,02	1,22 ± 0,06

The averages of the total length of thoraxes in T. sherlocki and T. lenti were 0.98 and 0.99 mm, 1.43 and 1.43 mm, 2.08 and 2.02 mm, 2.93 and 3.26 mm, and 4.68 and 5.29 mm in the first, second, third, fourth, and fifth instar, respectively.

The averages of the total length of abdomens in T. sherlocki and T. lenti were 1.75 and 1.57 mm, 3.17 and 3.04 mm, 5.24 and 5.60 mm, 6.68 and 6.63 mm, and 11.29 and 11.14 mm in the first, second, third, fourth, and fifth instar, respectively. The mean lengths of the abdomen were larger than those of the head, which were larger than those of the thorax in the first stage nymphs in both T. lenti and T. sherlocki. As in the first stage nymphs, the average abdominal length in second instar nymphs was longer than those of the head and the thorax nymphs for both species.

In T. lenti and T. sherlocki, the highest measurement observed was the total length of the abdomen that was longer than the head as well as the thorax. The average lengths of the abdomen were higher than those of the head as well as those of the thorax for T. lenti and T. sherlocki. Abdomen and eye diameter measurements showed no significant difference between T. lenti and T. sherlocki (Table 2. p<0.001). Analyzing the fifth instar nymphs of the two species, we found that the abdomen was the largest segment, and unlike the other nymphal instars, the thorax was larger than the head in the fifth stage nymphs of both species.

The PO, IE, and AO followed an ascending order: first instar > second instar > third instar > fourth instar > fifth instar, for both species (Table 2). Among these parameters, the largest length was the distance before the eye, and the smallest was the diameter of the eyes and the distance between both T. lenti and T. sherlocki.

The first, second, and third proboscis segment lengths were in the following order: first segment > third segment > second (Table 2). In fifth stage nymphs, the second and third segments showed significant differences in their length for both species. In both cases, it was observed that the second segment was larger than the third and this was larger than the first for all nymphal instars (Table 2). After measuring and performing statistical analysis on the three segments of the proboscis in first stage nymphs, it was observed that only the second segment showed a significant difference, while the first and third segments did not show significant differences between T. lenti and T. sherlocki (Table 3).

TABLE 3:

Comparative statistical analysis of T. lenti and T. sherlocki by Welch's t-test for nymph measurements.

T. lenti x T. sherlocki		1st instar		2nd instar		3rd instar		4th instar		5th instar
		p value	significance	p value	significance	p value	significance	p value	significance	p value	significance
Head	Total lenght	0,0057	**	0,6471	NS	<0,0001	***	0,0001	***	0,0087	**
	OE	0,0517	*	0,0025	**	0,6927	NS	0,2272	NS	0,05686	*
	IE	<0,0001	***	0,0187	*	<0,0001	***	0,0099	*	<0,0001	***
	PO	0,0012	**	0,2312	NS	<0,0001	***	0,0002	**	<0,0001	***
	AO	<0,0001	***	0,2621	NS	<0,0001	***	0,0003	**	<0,0001	***
Proboscis	1S	0,7318	NS	0,0003	**	0,1862	NS	0,3192	NS	0,1506	NS
	2S	0,0338	*	0,0066	**	<0,0001	***	0,0003	**	0,0099	*
	3S	0,3836	NS	0,0076	**	0,0006	**	0,0058	*	0,0396	*
Total thorax lenght		0,4811	NS	0,7834	NS	0,0267	*	<0,0001	***	<0,001	**
Total abdômen lenght		<0,0001	***	0,0923	NS	0,0003	**	0,0186	*	0,4947	NS

Outside distance between the eyes (OE); Inner distance between the eyes (IE); Postocular distance (PO); Ante-Ocular distance (AO), and NS, not significant.

Comparisons between proboscis segments and head and abdomen lengths of the two species are presented in Table 3. In the second instar nymphs, the three segments of the proboscis revealed measurements that showed significant differences, according to the statistical analysis, for the two species. In the third and fourth instar nymphs, measurements of the second and third proboscis segments showed a significant difference between T. lenti and T. sherlocki.

In the first instar nymphs, statistical analyses revealed significant differences in total head and abdominal length measurements. Thorax measurements were not different between the two species. Regarding the measurements of head parameters of the first stage nymphs, the distance between the anterior, postocular, interocular, and eye diameters were significantly different when comparing T. lenti and T. sherlocki. Statistical analysis showed significant differences only for interocular distance and eye diameter in second instar nymphs.

Measurements of the thorax, abdomen, ante-ocular distance, postocular distance, and total head length revealed no significant differences between the two species (Table 3). For the third instar nymphs, all measurements except for the eye diameter and first proboscis segment measurements, showed significant differences between both species, (Table 3). For the fourth instar nymphs, all parameters showed statistically significant differences, except for the eye diameter and first segment of the proboscis, as was also observed for the third instar nymphs (Table 3). The measurements of total head length, ante-ocular distance, postocular distance, and interocular and thorax distance of fifth instar nymphs were significantly different between the two species.

The main components (PCA1 and PCA2) are presented through biplot graphics showing the morphometric variability between T. lenti and T. sherlocki. The total size of the head was responsible for greater discrimination between the studied specimens. Alternatively, PC1 and PC2 were 99.569% and 0.431% for the first stage nymphs ( Supp. Figure 1), 99.966% and 0.034% for the second stage (Supp. Figure 2), 99.937% and 0.062% for the third stage (Supp. Figure 3), 99.791% and 0.208% for the fourth (Supp. Figure 4), and 99.84 and 0.15% for the fifth (>Supp. Figure 5).

DISCUSSION

Studies on immature forms of triatomines are relevant to taxonomy and provide important information for the understanding of several biological aspects of these vectors. In this study, a list of works with immature forms were presented and a morphological characterization of five nymphal instars of T. lenti and T. sherlocki, species that are closely related phylogenetically , , were described.

Triatoma lenti and T. sherlocki have reproductive compatibility with other members of the species T. brasiliensis subcomplex , which are frequently found in dwellings and infected with T. cruzi; therefore, they are potential vectors of Chagas disease , . Costa et al. conducted a comparative morphological analysis of the external genital structures and eggs of T. brasiliensis to differentiate chromatic forms. Gonçalves et al. used classic and geometric morphometry as a tool to distinguish T. jatai from other species. Mendonça et al. , used morphological, morphometric, molecular, and cytogenetic approaches as well as experimental crosses to revalidate the specific status of T. bahiensis and differentiate it from T. lenti. Combining morphometric and molecular approaches has provided important clues about the T. brasiliensis complex, which includes the species and subspecies T. lenti, T. petrocchiae, T. b. brasiliensis, T. b. macromelasoma, T. juazeirensis, T. sherlocki, T. melanica, and T. bahiensis .

In the present study, using SEM images, morphological differences were observed in the ninth ventral abdominal segment of female and male nymphs of the fifth instar. Comparing the morphology of the ninth ventral abdominal segment of male and female nymphs in the fifth instar of the species T. melanocephala Neiva & Pinto, 1923, T. brasiliensis, T. infestans, T. matogrossensis Leite and Barbosa, 1953, T. tibiamaculata (Pinto, 1926), T. lenti, and T. sherlocki, it can be seen that these seven species differ by this character , , . This indicates that the shape and size of the ninth abdominal segment in fifth instar nymphs may be taxonomically valid.

The measurements of the head, thorax, and abdomen served to better characterize and distinguish T. lenti and T. sherlocki across their evolutionary instars, as well as in the comparative analysis of nymphal instars of other species of the Triatoma genus. The combination of morphometric and morphological approaches provides important clues about the delimitation of the complex , , . Oliveira et al. morphometrically analyzed the species of the T. brasiliensis complex and showed that the variations in the shape of the head were statistically significant. The wings showed sexual dimorphism in shape, while the heads were not dimorphic as expected.

In this study, as in all other nymphal instars, we found that the largest measurement among the head measurements was the anocular distance and the smallest was the postocular distance, In the morphometry, all parameters in the first instar, except the average eye diameter and the first and third proboscis segments, were significantly different between T. lenti and T. sherlocki. Measurements of interocular distance, eye diameter, and the three segments of the proboscis revealed significant differences between the second instar nymphs of T. lenti and T. sherlocki. The third and fourth instar nymphs showed significant differences in the measurements of the abdomen, head, thorax, ante-ocular, interocular, postocular, and second and third proboscis segments. In the fifth instar, measurements of thorax length, head length, ante-ocular, interocular, postocular, and second and third proboscis segments showed significant differences in taxonomic differentiation between T. lenti and T. sherlocki. In all nymphal instars, the total length measurement ratio were in the following order: abdomen > head > thorax. In Triatoma melanocephala Neiva & Pinto, 1923, the nymphal instars presented the following length pattern: in the first instar, thorax > abdomen > head; in the second instar, abdomen > head > thorax; and in the third, fourth, and fifth instars, abdomen > thorax > head .

In all nymphal instars of T. lenti and T. sherlocki, it was observed that the second segment of the proboscis was larger than the third which was larger than the first segment. In T. melanocephala nymphs, it was found that specimens in the first three nymphs presented the same length order (2 > 3 > 1), while those in the fourth and fifth instars, along with the adults, possessed mouthpart segments of the same order (2 > 1 > 3) . The main components (PCA1 and PCA2) illustrated the differences between the studied parameters and showed that the total size of the head is or that it discriminates against T. lenti and T. sherlocki.

Studies on nymphs are crucial for the systematic development of certain groups. However, the literature on immature forms of certain groups is scarce, difficult to use, or nonexistent . Epidemiological studies and control measures require precise taxonomic determination of T. brasiliensis subcomplex , , . Therefore, this study provides morphological and morphometric data on the nymphal instars of T. lenti and T. sherlocki, corroborating the specific taxonomy of these species.