https://orcid.org/0000-0001-8333-3764
Figures
Abstract
Triatomines are hematophagous insects of great epidemiological importance, since they are vectors of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. Triatoma brasiliensis complex is a monophyletic group formed by two subspecies and six species: T. b. brasiliensis, T. b. macromelasoma, T. bahiensis, T. juazeirensis, T. lenti, T. melanica, T. petrocchiae and T. sherlocki. The specific status of several species grouped in the T. brasiliensis complex was confirmed from experimental crossing and analysis of reproductive barriers. Thus, we perform interspecific experimental crosses between T. lenti and other species and subspecies of the T. brasiliensis complex and perform morphological analysis of the gonads and cytogenetic analysis in the homeologous chromosomes of the hybrids of first generation (F1). Besides that, we rescue all the literature data associated with the study of reproductive barriers in this monophyletic complex of species and subspecies. For all crosses performed between T. b. brasiliensis, T. b. macromelasoma, T. juazeirensis and T. melanica with T. lenti, interspecific copulas occurred (showing absence of mechanical isolation), hybrids were obtained, none of the male hybrids presented the phenomenon of gonadal dysgenesis and 100% pairing between the chromosomes homeologous of the hybrids was observed. Thus, we demonstrate that there are no pre-zygotic reproductive barriers installed between T. lenti and the species and subspecies of the T. brasiliensis complex. In addition, we demonstrate that the hybrids obtained between these crosses have high genomic compatibility and the absence of gonadal dysgenesis. These results point to reproductive compatibility between T. lenti and species and subspecies of the T. brasiliensis complex (confirming its inclusion in the complex) and lead us to suggest a possible recent diversification of the taxa of this monophyletic group.
Citation: Pinotti H, de Oliveira J, Ravazi A, Madeira FF, dos Reis YV, de Oliveira ABB, et al. (2021) Revisiting the hybridization processes in the Triatoma brasiliensis complex (Hemiptera, Triatominae): Interspecific genomic compatibility point to a possible recent diversification of the species grouped in this monophyletic complex. PLoS ONE 16(10): e0257992. https://doi.org/10.1371/journal.pone.0257992
Editor: Brian W. Davis, Texas A&M University College of Veterinary Medicine, UNITED STATES
Received: August 20, 2020; Accepted: September 15, 2021; Published: October 15, 2021
Copyright: © 2021 Pinotti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript.
Funding: This work was carried out with the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil – Financing Code 001 (HP, JO and FFM), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil (Process number 2013/19764-0) (KCCA) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil (AR, YVR, ABBO, MTVAO and JAR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Triatomines are hematophagous insects of great epidemiological importance, since they are vectors of the protozoan Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae), the etiological agent of Chagas disease [1]. This disease is neglected, has no cure in the chronic phase, affects about eight million people and puts at risk of infection, approximately, 25 million people worldwide [1]. Cases of cure from the use of the drugs Benznidazole or Nifurtimox are reported only when the diagnosis is made in the acute phase of the infection (usually asymptomatic) [1]. Thus, the control of vector populations is considered the main way to minimize the incidence of new chagasic cases [1], being the studies related to triatomines of extreme importance for public health, since they can generate subsidies to assist these programs in the prophylaxis of Chagas disease.
There are currently 157 species of triatomines, grouped into 18 genera and five tribes [2–5]. Based mainly on morphological characters and/or geographic distribution, triatomines were grouped into complexes and subcomplexes [6–10]. Although these groupings are not valid according to the International Code of Zoological Nomenclature [11], Justi et al. [12] emphasize that complexes and subcomplexes of species must form natural groups (monophyletic groups), which led to several changes in the initial groupings [12–19].
Triatoma brasiliensis complex is a group of species and subspecies that has its center of dispersion in the semi-arid region of Northeast Brazil [20]. This grouping of species and subspecies was initially proposed by Lucena [7] as a systematic arrangement composed mainly of taxa found in the Northeast region of Brazil: T. b. brasiliensis Neiva, 1911, T. petrocchiae Pinto & Barreto, 1925, T. lenti Sherlock & Serafim, 1967, T. pessoai Sherlock and Serafim, 1967 (which was later synonymous with T. lenti [8]), T. bahiensis Sherlock & Serafim, 1967 (which was later synonymous with T. lenti [8]), T. b. melanica Neiva & Lent, 1941 (which was later synonymous with T. b. brasiliensis [8]) and T. b. macromelasoma Galvão, 1956 (which was also synonymous with T. b. brasiliensis [8]). Multidisciplinary studies based on morphology [21–23], biology [24], crossing experiments [18, 25–29], ecology [30, 31], isoenzymes [32], dispersal abilities [33], cytogenetics [34–38] and phylogenetics [22, 28, 39–41] allowed to demonstrate that T. b. macromelasoma and T. b. melanica were different taxa from T. b. brasiliensis (T. b. macromelasoma was redescribed and revalidated [42] and the status of T. b. melanica was raised to T. melanica [43]), allowed to describe the distinct population of T. brasiliensis from Juazeiro, Bahia, Brazil as T. juazeirensis Costa & Félix, 2007 [44], as well as allowed to revalidate the specific status of T. bahiensis [28], demonstrating that the T. brasiliensis complex is a monophyletic group formed by two subspecies and six species: T. b. brasiliensis, T. b. macromelasoma, T. bahiensis, T. juazeirensis, T. lenti, T. melanica, T. petrocchiae and T. sherlocki Papa et al. 2002 [22, 28, 39–41, 45].
Triatoma b. brasiliensis is one of the main Chagas disease vectors in the Northeast region of Brazil [46], as it is directly related to the semi-arid climate of this region [47–50]. This subspecies was reported in five Brazilian states: Maranhão, Piauí, Ceará, Rio Grande do Norte and Paraíba [31]. Until 2007, T. juazeirensis was considered as a population of T. b. brasiliensis collected in Juazeiro, Bahia, Brazil [44], being this species distributed in the states of Bahia and Pernambuco [31, 41]. In addition, T. melanica, until 2006, was considered as a subspecies of T. b. brasiliensis [43] endemic to the state of Minas Gerais and south of Bahia [31, 51]. Triatoma petrocchiae is a species morphologically related to T. b. brasiliensis [7] that is distributed in the states of Bahia, Ceará, Paraíba, Pernambuco and Rio Grande do Norte [31, 51]. Triatoma lenti and T. bahiensis were described, in 1967, as melanic forms of T. b. brasiliensis [52]. For 37 years, T. bahiensis was considered synonymous with T. lenti [8] and, recently, specimens were collected in the state of Bahia and the species was revalidated [28]. Triatoma lenti was notified in two Brazilian states, namely, Bahia and Goiás [31, 51, 53]. Triatoma sherlocki is an endemic species from Bahia [50, 54]. Finally, the subspecies T. b. macromelasoma, described based on melanic forms of T. b. brasiliensis found at home in the state of Pernambuco [31, 51], was redescribed and revalidated by Costa et al. [42].
Evolutionary studies, based on interspecific experimental crossings contribute to the taxonomy and systematics of triatomines (considering the biological concept of species proposed by Mayr [55, 56] and Dobzhansky [57]) [6, 18, 58]. The specific status of species grouped in the T. brasiliensis complex (such as T. petrocchiae [59], T. sherlocki [27], T. lenti [27] and T. bahiensis [28, 29]), for example, was confirmed from experimental crossing and analysis of pre-zygotic and post-zygotic reproductive barriers.
Thus, taking into account that the study of experimental crosses and of the resulting hybrids can (a) help to understand the systematics of this vectors complex, (b) can be used to analyze the isolation mechanisms that limit gene flow between the different species, (c) as well as can be used to establish the role of natural hybridization in the generation of new variants (which can lead to adaptive evolution and/or the foundation of new evolutionary lineages) [58, 60], we performed interspecific experimental crosses between T. lenti and other species and subspecies of the T. brasiliensis complex and perform morphological analysis of the gonads (with emphasis in the gonadal dysgenesis) and cytogenetic analysis in the homeologous chromosomes of the hybrids of first generation (F1) (with emphasis in the interspecific genomic compatibility). Besides that, we rescue all the literature data associated with the study of reproductive barriers in this monophyletic complex.
Methods
Reciprocal experimental crosses were conducted between T. b. brasiliensis, T. b. macromelasoma, T. juazeirensis and T. melanica with T. lenti (Table 1) to assess whether there is an interspecific pre-zygotic reproductive barrier (phenomena that prevent copulation or interspecific fertilization) [55–57] installed between these species and subspecies of the T. brasiliensis complex. The insects used in the experiment came from colonies kept in the Triatominae insectary of the School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, São Paulo, Brazil. These species and subspecies were identified with the help of the dichotomous keys developed by Costa et al. [42] and Dale et al. [51]. The experimental crosses were conducted in the Triatominae insectary of the School of Pharmaceutical Sciences, according to the experiments of Costa et al. [25] and Mendonça et al. [27]: the insects were sexed as 5th instar nymphs, and males and females were kept separately until they reached the adult stage in order to guarantee the virginity of the insects used in the crosses. For the experimental crosses, three couples from each set were placed in plastic jars (diameter 5 cm × height 10 cm) and kept at room temperature. Furthermore, intraspecific crosses were also performed for group control (Table 1). The eggs were collected weekly throughout the female’s oviposition periods and the egg fertility rate was calculated (Table 1).
The gonads morphology of at least two adult male hybrids F1 from each cross (Table 1) was analyzed using a Leica MZ APO stereoscope microscope with Motic Advanced 3.2 plus image analysis system to assess the presence of the phenomenon of gonadal dysgenesis (which can be uni or bilateral) [61] and, posteriorly, cytogenetic analysis with Lacto-Acetic Orcein (De Vaio et al. [62], with modifications according to Alevi et al. [14]) were performed on slides prepared with these gonads and examined using Jenaval light microscopy (Zeiss), coupled to the digital camera and the Axio Vision LE 4.8 image analyzer system (Copyright © 2006–2009 Carl Zeiss Imaging Solutions Gmb H) to assess the degree of pairing between homeologous chromosomes (as established by Mendonça et al. [27]). Lastly, all data of the literature referring to experimental crossing studies in the T. brasiliensis complex were compiled (Table 2), with emphasis in the analysis of the presence/absence of pre-zygotic reproductive barrier.
Results and discussion
The first experimental crossings performed with species and subspecies of the T. brasiliensis complex were between T. petrocchiae and T. b. brasiliensis [59]. Posteriorly, Costa et al. [25], Almeida et al. [33], Correia et al. [26] and Mendonça et al. [27] performed crossings between different populations of T. b. brasiliensis [20] and between T. sherlocki and members of the T. brasiliensis complex [26, 27, 33], respectively, and observed reproductive compatibility between the species and subspecies of the complex. Finally, Neves et al. [18] performed a cross between T. melanocephala Neiva and Pinto (1923), T. tibiamaculata (Pinto, 1926) and T. vitticeps (Stal, 1859) with T. b. brasiliensis and confirmed that these species are unrelated to the T. brasiliensis complex (as initially suggested by Schofield and Galvão [10]).
For all crosses performed (Table 1), interspecific copulas occurred (Fig 1) [showing absence of mechanical isolation] and hybrids were obtained (Fig 2). Mechanical isolation, a pre-zygotic reproductive barrier characterized by incompatibility of the genitals [55, 56], has been reported several times in the subfamily Triatominae (acting in one or both directions of the crossings) [6, 63]. This reproductive compatibility observed between T. lenti and the species and subspecies of the T. brasiliensis complex confirms the inclusion of the species in the complex (as well as Correia et al. [26] confirmed the relationship of T. sherlocki with the T. brasiliensis complex).
Bar: 5 cm.
Bar: 5 cm.
Although hybrids were obtained for all directions of the crosses (Table 1), the hatch rates varied greatly, being the lowest rate observed for the crossing between T. b. brasiliensis and T. lenti (both directions) and the highest rates related to crosses between T. juazeirensis female and T. lenti male and between T. melanica and T. lenti (both directions). The only species of the T. brasiliensis complex that present genetic distance very close to the minimum stipulated to consider a species as valid are T. melanica and T. lenti [28]. However, it is not possible to correlate the low interspecific distance with the hatching rate because the genetic distance between T. juazeirensis and T. lenti is higher than 10% [28] (close to the observed distance between T. lenti and T. infestans [28] which are phylogenetically distant species [64] and do not produce hybrids [65]).
Justi et al. [64] suggest that climate change resulting from the Andean elevation [which occurred approximately 14 million years ago (Mya)] are associated with allopatric events responsible for the origin of the T. brasiliensis complex in the Caatinga biome, as well as the other complexes present in several biomes in the Latin America: T. rubrovaria in the Pampa, T. infestans in the Chaco and T. sordida, T. matogrossensis and part of T. maculata in the Cerrado. Triatoma b. brasiliensis when crossed with species of the T. sordida [65], T. infestans [65] and T. vitticeps complexes [18] do not result in fertile eggs. On the other hand, considering the biological concept of species [55–57], the absence of pre-zygotic reproductive barriers among practically all species and subspecies of the T. brasiliensis complex (Table 2) indicates that the taxa of this group may have diverged recently (being necessary the action of post-zygotic barriers—unfeasibility, sterility or collapse of the hybrid [56, 57]—to break the hybrids in nature).
Patterson and Gaunt [66] demonstrate that triatomines have diversified 95 Mya, putatively linking the origin of haematophagous behavior to the origin of South America. Monteiro et al. [39] performed an estimate of the divergence time associated with the speciation of the members of the T. brasiliensis complex (with emphasis on the relationship between T. b. brasiliensis and T. melanica) and suggested 5.2 Myr of independent evolution between these taxa. Considering the origin of Triatominae [66], the 5.2 Myr dating confirms the recent diversification of the species and subspecies of the T. brasiliensis complex.
Taking into account that to occur pairing between the genome of different species there must be at least 80% homeology between chromosomes (which allows to evaluate the evolutionary relationship of the species) [67], we cytogenetically analyzed the genomic compatibility of the T. brasiliensis species and subspecies complex, through the degree of pairing of the homeologous chromosomes of the hybrids in metaphase I. The results obtained demonstrated 100% pairing between the chromosomes of the hybrids (Fig 3).
Note that 100% of the chromosomes were paired. X: X sex chromosome; Y: Y sex chromosome; Bar: 10 μm.
Mendonça et al. [27] analyzed cytogenetically hybrids from interspecific crosses between T. lenti and T. sherlocki and, as reported in the present study, also observed genomic compatibility between the species (100% pairing between homeologous chromosomes of the hybrids in metaphase I). Riley [68] points out that two species have different genomes when their chromosomes are different so that there is no pairing between one or more pairs of homeologous during hybrid meiosis I, which results in sterility (reproductive isolation) and, consequently, genetic isolation between species (absence of gene flow). However, we suggest that the interspecific genomic compatibility observed, together with absence of pre-zygotic reproductive barrier (Table 2) come from the recent diversification of the taxa of this group, which does not unviable the specific status of the species of this complex (since all species of the T. brasiliensis complex present genetic distance to the Cyt B mitochondrial gene greater than 2% [28, 39, 40]).
All species and subspecies of the T. brasiliensis complex share the same chromosomal characteristics [15, 28, 34–38, 69]. Costa et al. [70] suggested that T. b. macromelasoma resulted from the natural crossing between T. b. brasiliensis and T. juazeirensis by the phenomenon of homoploidal hybridization. Recently, Costa et al. [71], through phenotypic and genotypic analyzes suggested the existence of a natural hybridization zone where interspecific crossings may occur between T. b. brasiliensis and T. juazeirensis, which supports the origin of T. b. macromelasoma by homoploidal evolution. Natural hybridization events can be more common than previously thought: recently Antunes et al. [72] demonstrated that the choice for the copula is not always towards conspecific females (which may result in increased genotypic and phenotypic variability).
Although T. b. macromelasoma is considered as an incipient species, the low interspecific genetic distance observed between T. b. macromelasoma, T. b. brasiliensis and T. juazeirensis [69] together with the production capacity of experimental hybrids (Table 2), point to the possibility of natural hybridization events followed by introgression during the evolution of these taxa [69] (which highlights the need for attention of the vector programs with T. juazeirensis–mainly in view of the recent catches of this species infected with T. cruzi in human dwellings [73]–(since genes associated with the high vector capacity of T. b. brasiliensis can be fixed in this species by introgression) and with its possible hybrids (that thus the hybrids resulting from the cross between T. sherlocki and T. juazeirensis [33] can present higher fitness than their parents).
None of the male hybrids analyzed presented the phenomenon of gonadal dysgenesis (Fig 4), that is, the testicles were normal (without atrophy) when compared to the morphology of the testis of Triatominae [74]. This result can be confirmed by the birth of hybrids in all directions of the crossings (Table 1), which demonstrates that both gonads and gametogenesis are normally occurring in first generation hybrids (F1). Gonadal dysgenesis, a phenomenon quite common in drosophilids [75], has never been reported in the subfamily Triatominae. On the other hand, the sterility of the hybrid from the abnormal gametogenesis (chromosomal pairing errors) has been observed several times in these vectors [58, 76, 77].
Note that the gonads do not have gonadal dysgenesis. Bar: 10 mm.
Although the crosses between T. b. brasiliensis female and T. petrocchiae male performed by Espínola [59] did not result in hybrids, it is worth mentioning that this author reported hatching of a nymph resulting from the cross between T. petrocchiae female and T. b. brasiliensis male (Table 2). This result demonstrates that there is still genomic compatibility between these two taxa (even if it’s extremely low), which corroborates the sharing of common ancestry between T. petrocchiae and species and subspecies of the T. brasiliensis complex [22], even though T. petrocchiae is the most distant species of the group, possibly being the taxon with more derived genetic and genomic characteristics.
Recently, was observed that reptile blood is the main food source for T. petrocchiae [78]. On the other hand, T. b. brasiliensis is mainly associated with rodents [49]. Taking into account that these two taxa live in sympathy [50], it is likely that the ecological (or habitat) isolation is the main pre-zygotic barrier that makes unfeasible the formation of natural hybrids between T. petrocchiae and T. b. brasiliensis.
Alevi et al. [29] observed pairing error between a pair of homeologous chromosomes of hybrids from the cross between T. bahiensis and T. lenti, which highlighted the specific status of T. bahiensis (for resulting in unviable gametes). The incompatibility of a pair of chromosomes was extremely important from a taxonomic point of view [79]. However, these results emphasize the evolutionary relationship between these species belonging to the T. brasiliensis complex by the pairing of 90% of the autosomes of these vectors.
Conclusions
Thus, we demonstrate that there are no pre-zygotic reproductive barriers installed between T. lenti and the species and subspecies of the T. brasiliensis complex. In addition, we demonstrate that the hybrids obtained between these crosses have high genomic compatibility (100% pairing between homeologous chromosomes) and the absence of gonadal dysgenesis. These results point to reproductive compatibility between T. lenti and species and subspecies of the T. brasiliensis complex (confirming their inclusion in the complex) and lead us to suggest a possible recent diversification of the taxa of this monophyletic group.
References
- 1.
WHO (World Health Organizarion). Chagas disease (American trypanosomiasis); 2020 [Cited 2020 August 20] https://www.who.int/chagas/disease/en/.
- 2. Alevi KCC, Oliveira J, Garcia ACC, Cristal DC, Delgado LMG, Bittinelli IF, et al. Triatoma rosai sp. nov. (Hemiptera, Triatominae): A new species of Argentinian Chagas disease vector described based on integrative taxonomy. Insects. 2020; 11: 830. pmid:33255910
- 3. Zhao Y, Galvão C, Cai W. Rhodnius micki, a new species of Triatominae (Hemiptera, Reduviidae) from Bolivia. ZooKeys. 2021; 1012: 71–93. pmid:33584109
- 4. Costa J, Dale C, Galvão C, Almeida CE, Dujardin JP. Do the new triatomine species pose new challenges or strategies for monitoring Chagas disease? An overview from 1979–2021. Mem Inst Oswaldo Cruz. 2021; 116: e210015. pmid:34076075
- 5. Dale C, Justi SA, Galvão C. Belminus santosmalletae (Hemiptera: Heteroptera: Reduviidae): New Species from Panama, with an Updated Key for Belminus Stål, 1859 Species. Insects. 2021; 12: 686. pmid:34442252
- 6. Usinger RL, Wygodzinsky P, Ryckman RE. The biosystematics of Triatominae. Annu Rev Entomol. 1966; 11: 309–330. pmid:5321579
- 7. Lucena D. Estudos sobre a doença de Chagas no Brasil. Rev Bras Malar Doen Trop. 1970; 22: 3–173.
- 8. Lent H, Wygodzinsky P. Revision of the Triatominae (Hemiptera, Reduviidae), and their significance as vectors of Chagas’ disease. Bull Am Mus Nat Hist. 1979; 163: 123–520.
- 9. Carcavallo RU, Jurberg J, Lent H, Noireau F, Galvão C. Phylogeny of the Triatominae (Hemiptera: Reduviidae). Proposals for taxonomic arrangements. Entomol Vect. 2000; 7: 1–99.
- 10. Schofield CJ, Galvão C. Classification, evolution, and species groups within the Triatominae. Acta Trop. 2009; 110: 88–100. pmid:19385053
- 11.
ICNZ (The International Code of Zoological Nomenclature). The International Code of Zoological Nomenclature. [Cited 2020 August 20]. https://www.iczn.org/.
- 12. Justi SA, Russo CAM, Reis J, Obara MT, Galvão C. Molecular phylogeny of Triatomini (Hemiptera: Reduviidae: Triatominae). Parasit Vectors. 2014; 7: 149. pmid:24685273
- 13. Noireau F, Menezes S, Gumiel M, Dujardin J, Soares S, Ubaldo R, et al. Phylogenetic relationships within the oliveirai complex (Hemiptera: Reduviidae: Triatominae). Acta Trop. 2002; 2: 11–17.
- 14. Alevi KCC, Mendonça PP, Pereira NP, Rosa JA, Azeredo-Oliveira MTV. Karyotype of Triatoma melanocephala Neiva and Pinto (1923). Does this species fit in the Brasiliensis subcomplex? Infect Genet Evol. 2012; 12: 1652–1653. pmid:22760157
- 15. Alevi KCC, Rosa JA, Azeredo-Oliveira MTV. Cytotaxonomy of the Brasiliensis subcomplex and the Triatoma brasiliensis complex (Hemiptera: Reduviidae: Triatominae). Zootaxa. 2014; 3838: 583–589. pmid:25081800
- 16. Alevi KCC, Oliveira J, Azeredo-Oliveira MTV, Rosa JA. Triatoma vitticeps subcomplex (Hemiptera, Reduviidae, Triatominae): a new grouping of Chagas disease vectors from South America. Parasit Vectors. 2017; 10: 180. pmid:28407782
- 17. Pita S, Lorite P, Nattero J, Galvão C, Alevi KCC, Teves SC, et al. New arrangements on several species subcomplexes of Triatoma genus based on the chromosomal position of ribosomal genes (Hemiptera—Triatominae). Infect Genet Evol. 2016; 43: 225–231. pmid:27245153
- 18. Neves SJM, Sousa PS, Oliveira J, Ravazi A, Madeira FF, et al. Prezygotic isolation confirms the exclusion of Triatoma melanocephala, T. vitticeps and T. tibiamaculata of the T. brasiliensis subcomplex (Hemiptera, Triatominae). Infect Genet Evol. 2020; 79: 104149. pmid:31864008
- 19. Belintani T, Oliveira J, Pinotti H, Silva LA, Alevi KAC, Galvão C, et al. Phylogenetic and phenotypic relationships of the Triatoma sordida subcomplex (Hemiptera: Reduviidae: Triatominae). Act Trop. 2020; 2012: 105679.
- 20. Forattini OP. Biogeografia Origem e Distribuição da Domiciliação de triatomíneos no Brasil. Rev Saúde Públ. 1980; 14: 265–299.
- 21. Costa J, Barth OM, Marchon-Silva V, Almeida CE, Freitas-Sibajev MGR, Panzera F. Morphological Studies on the Triatoma brasiliensis Neiva, 1911 (Hemiptera, Reduviidae, Triatominae) Genital Structures and Eggs of Different Chromatic Forms. Mem Inst Oswaldo Cruz. 1997; 92: 493–498.
- 22. Oliveira J, Marcer PL, Takiya DM, Mendonça VJ, Belintani T, Bargues MD, et al. Combined phylogenetic and morphometric information to delimit and unify the Triatoma brasiliensis species complex and the Brasiliensis subcomplex. Acta Trop. 2017; 170: 140–148. pmid:28219669
- 23. Oliveira J, Almeida CE, Mendonça VJ, Alevi KCC, Costa J, Rosa JA. Triatoma brasiliensis Species Complex: Characterization of the external female genitalia. J Vec Ecol. 2020; 45: 57–68. pmid:32492272
- 24. Costa J, Marchon-Silva V. Período de intermuda e resistência ao jejum de diferentes populações de Triatoma brasiliensis (Hemiptera: Reduviidae: Triatominae). Entomol Vectores, 1998; 5: 23–34.
- 25. Costa J, Almeida CE, Dujardin JP, Beard CB. Crossing Experiments Detect Genetic Incompatibility among Populations of Triatoma brasiliensis Neiva, 1911 (Heteroptera, Reduviidae, Triatominae). Mem Inst Oswaldo Cruz. 2003; 98: 637–639. pmid:12973530
- 26. Correia N, Almeida CE, Lima-Neiva V, Gumiel M, Lima MM, Medeiros LMO, et al. Crossing experiments confirm Triatoma sherlocki as a member of the Triatoma brasiliensis species complex. Acta Trop. 2013; 128: 162–167. pmid:23850508
- 27. Mendonça VJ, Alevi KCC, Medeiros LMO, Nascimento JD, Azeredo-Oliveira MTV, Rosa JA. Cytogenetic and morphologic approaches of hybrids from experimental crosses between Triatoma lenti Sherlock & Serafim, 1967 and T. sherlocki Papa et al. 2002 (Hemiptera: Reduviidae). Infect Genet Evol. 2014; 26: 123–131. pmid:24861813
- 28. Mendonça VJ, Alevi KCC, Pinotti H, Gurgel-Gonçalves R, Pita S, Guerra AL, et al. Revalidation of Triatoma bahiensis Sherlock & Serafim, 1967 (Hemiptera: Reduviidae) and phylogeny of the T. brasiliensis species complex. Zootaxa; 2016: 239–254. pmid:27394816
- 29. Alevi KCC, Pinotti H, Araújo RF, Azeredo-Oliveira MTV, Rosa JA, Mendonça VJ. Hybrid colapse confirm the specific status of Triatoma bahiensis Sherlock and Serafim, 1967 (Hemiptera, Triatominae). Am J Trop Med Hyg. 2018; 98: 475–477. pmid:29260653
- 30. Costa J, Ribeiro De Almeida J, Britto C, Duarte R, Marchon-Silva V, Pacheco RDS. Ecotopes, Natural Infection and Trophic Resources of Triatoma brasiliensis (Hemiptera, Reduviidae, Triatominae). Mem Inst Oswaldo Cruz. 1998; 93: 7–13.
- 31. Costa J, Dornak LL, Almeida CE, Peterson AT. Distributional potential of the Triatoma brasiliensis species complex at present and under scenarios of future climate conditions. Parasit Vect. 2014; 7: 238. pmid:24886587
- 32. Costa J, Freitas-Sibajev MGR, Marchon-Silva V, Pires MQ, Pacheco RS. Isoenzymes Detect Variation in Populations of Triatoma brasiliensis (Hemiptera: Reduviidae: Triatominae). Mem Inst Oswaldo Cruz. 1997; 92: 459–464. pmid:9361737
- 33. Almeida CE, Oliveira HL, Correia N, Dornak LL, Gumiel M, Neiva VL, et al. Dispersion capacity of Triatoma sherlocki, Triatoma juazeirensis and laboratory-bred hybrids. Acta Trop. 2012; 122: 71–79. pmid:22210440
- 34. Panzera F, Pérez R, Nicolini P, Hornos S, Costa J, Borges E, et al. Chromosome homogeneity in populations of Triatoma brasiliensis Neiva 1911 (Hemiptera–Reduviidae–Triatominae). Cad Saúde Pública. 2000; 16: 83–88.
- 35. Alevi KCC, Mendonça PP, Succi M, Pereira NP, Rosa JA, Azeredo-Oliveira MTV. Karyotype and spermatogenesis in Triatoma lenti (Hemiptera: Triatominae), a potential Chagas vector. Genet Mol Res. 2012; 11: 4278–4284. pmid:23315807
- 36. Alevi KCC, Mendonça PP, Pereira NP, Guerra AL, Facina CH, Rosa JA, et al. Distribution of constitutive heterochromatin in two species of triatomines: Triatoma lenti Sherlock and Serafim (1967) and Triatoma sherlocki Papa, Jurberg, Carcavallo, Cerqueira & Barata (2002). Infect Genet Evol. 2013; 13: 301–303. pmid:23183310
- 37. Alevi KCC, Rosa JA, Azeredo-Oliveira MTV. Spermatogenesis in Triatoma melanica Neiva and Lent, 1941 (Hemiptera, Triatominae). J Vector Ecol. 2014; 39: 231–233. pmid:24820580
- 38. Alevi KCC, Bittinelli IF, Delgado LMG, Madeira FF, Oliveira J, Lilioso M, et al. Molecular cytotaxonomy of the Triatoma brasiliensis species subcomplex (Hemiptera, Triatominae). Acta. Trop. 2020; 201: 105225. pmid:31654646
- 39. Monteiro FA, Donnelly MJ, Beard CB, Costa J. Nested clade and phylogeographic analyses of the Chagas disease vector Triatoma brasiliensis in Northeast Brazil. Mol Phylogenet Evol. 2004; 32: 46–56. pmid:15186796
- 40. Mendonça VJ, Silva MTA, Araújo RF, Júnior JM, Júnior MB, Almeida CE, et al. Phylogeny of Triatoma sherlocki (Hemiptera: Reduviidae: Triatominae) inferred from two mitochondrial genes suggests its location within the Triatoma brasiliensis complex. Am J Trop Med Hyg. 2009; 81: 858–864. pmid:19861622
- 41. Gardim S, Almeida C.E, Takiya DM, Oliveira J, Araújo RF, Cicarelli RMB, et al. Multiple mitochondrial genes of some sylvatic Brazilian Triatoma: Non-monophyly of the T. brasiliensis subcomplex and the need for a generic revision in the Triatomini. Infect Genet Evol. 2014; 23: 74–79. pmid:24508245
- 42. Costa J, Correia NC, Neiva VL, Cristina T, Gonçalves M, Felix M. Revalidation and redescription of Triatoma brasiliensis macromelasoma Galvão, 1956 and an identification key for the Triatoma brasiliensis complex (Hemiptera: Reduviidae: Triatominae). Mem Inst Oswaldo Cruz. 2013; 108: 785–789. pmid:24037202
- 43. Costa J, Argolo AM, Felix M. Redescription of Triatoma melanica Neiva & Lent, 1941, New Status (Hemiptera: Reduviidae: Triatominae). Zootaxa. 2006; 385: 47–52.
- 44. Costa J, Felix M. Triatoma juazeirensis sp. nov. from the state of Bahia, Northeastern Brazil (Hemiptera: Reduviidae: Triatominae). Mem Inst Oswaldo Cruz. 2007; 102: 87–90.
- 45.
Costa J, Neiva L, Almeida CE. O complexo Triatoma brasiliensis (Hemiptrera, Reduviidae, Triatominae) como modelo de estudo: uma abordagem multidisciplinar e ecoepidemiológica. In: Oliveira J, Alevi KCC, Camargo LMA, Meneguetti DUL, editors. Atualidades em Medicina Tropical no Brasil: Vetores; 2020, pp. 99–101.
- 46. Silveira AC, Vinhaes MC. Elimination of vector-borne transmission of Chagas disease. Mem Inst Oswaldo Cruz. 1999; 94: 405–411. pmid:10677766
- 47. Costa J, Almeida CE, Dotson EM, Lins A, Vinhaes M, Silveira AC, et al. The Epidemiologic Importance of Triatoma brasiliensis as a Chagas Disease Vector in Brazil: A Revision of Domiciliary Captures during 1993–1999. Mem Inst Oswaldo Cruz. 2003; 98: 443–449. pmid:12937751
- 48. Monsalve-Lara K, Liloso M, Valença-Barbosa C, Thyssen P, Miguel DC, Limeira C, et al. The risk of oral transmission in an area of a Chagas disease outbreak in the Brazilian northeast evaluated through entomological, socioeconomic and schooling indicators. Acta Trop. 2020; 2015: 105803.
- 49. Lilioso M, Reigada C, Pires-Silva D, Fontes FVHM, Limeira C, Monsalve-Lara J, et al. Dynamics of food sources, ecotypic distribution and Trypanosoma cruzi infection in Triatoma brasiliensis from the northeast of Brazil. PLoS Negl Trop Dis. 2020; 14: e0008735. pmid:32986738
- 50. Lima-Oliveira TM, Fontes FVHM, Lilioso M, Pires-Silva D, Teixeira MMG, Meza JGV, et al. Molecular eco-epidemiology on the sympatric Chagas disease vectors Triatoma brasiliensis and Triatoma petrocchiae: ecotopes, genetic variation, natural infection prevalence by trypanosomatids and parasite genotyping. Acta Trop.; 2020: 201: 105188. pmid:31545949
- 51. Dale C, Almeida CE, Mendonça VJ, Oliveira J, Rosa JA, Galvão C, et al. An updated and illustrated dichotomous key for the Chagas disease vectors of Triatoma brasiliensis species complex and their epidemiologic importance. Zookeys. 2018; 805: 33–43.
- 52. Sherlock IA, Serafim M. Triatoma lenti sp. n., Triatoma pessoai sp. n. e Triatoma bahiensis sp. n. do estado da Bahia, Brasil (Hemiptera, Reduviidae). Gaz Méd Bahia. 1967; 67: 75–92.
- 53.
Galvão C. Vetores da Doença de Chagas no Brasil. Curitiba: Sociedade Brasileira de Zoologia Press; 2014.
- 54. Almeida CE, Folly-Ramos E, Peterson AT, Lima-Neiva V, Gumiel M, Duarte R, et al. Could the bug Triatoma sherlocki be vectoring Chagas disease in small mining communities in Bahia, Brazil. Med Vet Entomol. 2009; 23; 410–417. pmid:19941607
- 55.
Mayr E. Animal Species and Evolution. Cambridge: Harvard University Press; 1963.
- 56.
Mayr E. Populations, Species, and Evolution. Cambridge: Harvard University Press; 1970.
- 57.
Dobzhansky T. Genetics of the Evolutionary Process. New York: Columbia University Press; 1970.
- 58. Pérez R, Hernández M, Quintero O, Scvortzoff E, Canale D, Méndez L, et al. Cytogenetic analysis of experimental hybrids in species of Triatominae (Hemiptera-Reduviidae). Genetica. 2005; 125: 261–270. pmid:16247698
- 59. Espínola H. Reproductive isolation between Triatoma brasiliensis Neiva, 1911 and Triatoma petrochii Pinto & Barretto, 1925 (Hemiptera Reduviidae). Rev Bras Biol. 1971; 31: 277–281. pmid:4945961
- 60.
Arnold ML. Natural Hybridization and Evolution. New York: University Press; 1997.
- 61. Ravazi A, Oliveira J, Campos FF, Madeira FF, Reis YV, Oliveira ABB, et al. Trends in evolution of the Rhodniini tribe (Hemiptera, Triatominae): experimental crosses between Psammolestes tertius Lent & Jurberg, 1965 and P. coreodes Bergroth, 1911 and analysis of the reproductive isolating mechanisms. Parasit Vectors. 2021; 14: 350. pmid:34215287
- 62. De Vaio ES, Grucci B, Castagnino AM, Franca ME, Martinez ME. Meiotic differences between three triatomine species (Hemiptera: Reduviidae). Genetica. 1985; 67: 185–191.
- 63. Abalos JW. Sobre híbridos naturales y experimentales de Triatoma. An Inst Med Reg. 1948; 2: 209–223.
- 64. Justi SA, Galvão C, Schrago CG. Geological Changes of the Americas and their Influence on the Diversification of the Neotropical Kissing Bugs (Hemiptera: Reduviidae: Triatominae). PLoS Negl Trop Dis. 2016; 10: e0004527. pmid:27058599
- 65. Perlowagora-Szumlewics A, Correia MV. Induction of male sterility manipulation of genetic mechanisms present in vector species of Chagas disease (remarks on integrating sterile-male release with insecticidal control measures against vectors of Chagas disease). Rev Inst Med Trop São Paulo. 1972; 14: 360–371. pmid:4631330
- 66. Patterson JS, Gaunt MW. Phylogenetic multi-locus codon models and molecular clocks reveal the monophyly of haematophagous reduviid bugs and their evolution at the formation of South America. Mol Phylogenet Evol. 2010; 56: 608–621. pmid:20435148
- 67.
Dewey DR. Genomic and phylogenetic relationships among North American perennial Triticeae. In: Estes JR, Tyrl RJ, Brunken JN, editors. Grasses and Grasslands: Systematics and Ecology. Norman: University of Oklahoma Press; 1982. pp. 51–88.
- 68. Riley R. The secondary pairing of bivalents with genetically similar chromosomes. Nature. 1966; 185: 751–752.
- 69. Guerra AL, Borsatto KCC, Pagliusi ND, Madeira FF, Oliveira J, Rosa JA, et al. Revisiting the Homoploid Hybrid Speciation Process of the Triatoma brasiliensis macromelasoma Galvão, 1956 (Hemiptera, Triatominae) Using Cytogenetic and Molecular Markers. Am J Trop Med Hyg. 2019; 100: 911–913. pmid:30793690
- 70. Costa J, Peterson AT, Pierre J. Morphological evidence suggests homoploid hybridization as a possible mode of speciation in the Triatominae (Hemiptera, Heteroptera, Reduviidae). Infect Genet Evol. 2009; 9: 263–270. pmid:19135177
- 71. Costa J, Bargues MD, Neiva VL, Lawrence GG, Gumiel M, Oliveira G, et al. Phenotypic variability confirmed by nuclear ribosomal DNA suggests a possible natural hybrid zone of Triatoma brasiliensis species complex. Infect Genet Evol. 2016; 37: 77–87. pmid:26520796
- 72. Antunes A, Dias LP, Guimarães GA, Oliveira J, Rosa JA, Almeida CE, et al. Sexual Choice in Males of the Triatoma brasiliensis Complex: A Matter of Maintenance of the Species or Genetic Variability? Open Parasitol J. 2020; 8: 1–9.
- 73. Ribeiro G Jr., Santos CGS, Lanza F, Reis J, Vaccarezza F, Diniz C, et al. Wide distribution of Trypanosoma cruzi-infected triatomines in the State of Bahia, Brazil. Parasit Vectors. 2019; 12: 604. pmid:31878960
- 74. Alevi KCC, Rosa JA, Azeredo-Oliveira MTV. Coloration of the testicular peritoneal sheath as a synapomorphy of triatomines (Hemiptera, Reduviidae). Biota Neotrop. 2014; 14: e20140099.
- 75. Almeida LM, Carareto CMA. Gonadal hybrid dysgenesis in Drosophila sturtevanti (Diptera, Drosophilidae). Ilheringia. 2002; 92: 71–79.
- 76. Díaz S, Panzera F, Jaramillo-O N, Pérez R, Fernández R, Vallejo G, et al. Genetic, Cytogenetic and Morphological Trends in the Evolution of the Rhodnius (Triatominae: Rhodniini) Trans-Andean Group. PLoS ONE. 2014; 9: e87493. pmid:24498330
- 77. Villacís AG, Dujardin JP, Panzera F, Yumiseva CA, Pita S, Santillán-Guayasamín S, et al. Chagas vectors Panstrongylus chinai (Del Ponte, 1929) and Panstrongylus howardi (Neiva, 1911): chromatic forms or true species? Parasit Vectors. 2020; 13: 226. pmid:32375868
- 78. Liloso M, Pires-Silva D, Fontes FVHM, Oliveira J, Rosa JA, Vilela RV, et al. Triatoma petrocchiae (Hemiptera, Reduviidae, Triatominae): A Chagas disease vector of T. brasiliensis species complex associated to reptiles. Infect Genet Evol. 2020; 82: 103407.
- 79. Heitzmann-Fontenelle T. Bionomia comparativa de triatomíneos. VI—Híbridos de Triatoma brasiliensis Neiva, 1911 x Triatoma lenti Sherlock & Serafim, 1967 (Hemiptera, Reduviidade). Mem Inst Butantan. 1984; 47: 175–181.