Karyotype Evolution in Triatominae (Hemiptera, Reduviidae): The Role of Chromosomal Rearrangements in the Diversification of Chagas Disease Vectors.

Several cytogenetic studies have already been performed in Triatominae, such that different karyotypes could be characterized (ranging from 2n = 21 to 25 chromosomes), being the changes in the number of chromosomes related mainly to fusion and fission events. These changes have been associated with reproductive isolation and speciation events in other insect groups. Thus, we evaluated whether different karyotypes could act in the reproductive isolation of triatomines and we analyzed how the events of karyotypic evolution occurred along the diversification of these vectors. For this, experimental crosses were carried out between triatomine species with different karyotypes. Furthermore, based on a phylogeny with 88 triatomine taxa (developed with different molecular markers), a reconstruction of ancestral karyotypes and of anagenetic and cladogenetic events related to karyotypic alterations was performed through the ChromoSSE chromosomal evolution model. All crosses performed did not result in hybrids (prezygotic isolation in both directions). Our modeling results suggest that during Triatominae diversification, at least nine cladogenetic events may be associated with karyotype change. Thus, we emphasize that these alterations in the number of chromosomes can act as a prezygotic barrier in Triatominae (karyotypic isolation), being important evolutionary events during the diversification of the species of Chagas disease vectors.

A review of the taxonomy and biology of Triatominae subspecies (Hemiptera: Reduviidae).

Due to its public health importance, as vectors of Chagas disease, the subfamily Triatominae, has received the attention of taxonomists. Knowing how to correctly identify these insects is of paramount importance for the surveillance and control of these vectors. Over many years, more than 40 triatomine subspecies were proposed. Infraspecific taxa are accepted as real entities in nature and recognized as biologically significant. Infraspecific diversity and population subdivision are frequently confused with the underlying mechanisms that lead to the formation of new species and species-level diversity. The distinction between infraspecific and interspecific divergence processes has received far less attention than species delimitation. In this work, new literature data on subspecies included in the subfamily Triatominae are presented. Data were compiled about Triatoma brasiliensis, Triatoma breyeri, Triatoma circummaculata, Triatoma dimidiata, Triatoma incrassata, Triatoma infestans, Triatoma phyllosoma, Triatoma protracta, Triatoma recurva, Triatoma rubida, Triatoma sanguisuga, Triatoma vitticeps, Panstrongylus megistus, Paratriatoma hirsuta, and Paratriatoma lecticularia subspecies. Although several of these subspecies have been synonymized, herein, we emphasize that some subspecies may be valid. Therefore, further studies are needed on the taxonomy, evolution, phylogeny, biogeography, ecology, physiology, and behavior of species to reinforce, or to invalidate the infraspecific status in the subfamily.

Biology of Chagas disease vectors: biological cycle and emergence rates of Rhodnius marabaensis Souza et al., 2016 (Hemiptera, Reduviidae, Triatominae) under laboratory conditions.

In Latin America, Chagas disease has been mostly transmitted to humans by contact with the feces or urine of triatomine species infected with the protozoan Trypanosoma cruzi. There are currently 156 species in the subfamily Triatominae, distributed in 18 genera and five tribes. The prolixus group of the genus Rhodnius is composed of 11 species. Rhodnius marabaensis was the last species described and considered in this grouping of vectors. Knowledge about the biology, ecology, and behavior of these vectors is of great epidemiological importance, and in order to expand the knowledge of the biology of R. marabaensis, this paper describes the biological cycle and emergence rates of the species under laboratory conditions. The experiment was carried out at temperatures ranging from 15.5 to 29 °C (average of 24 °C) and humidity ranging from 51.4 to 72.2 (average of 63). For each of the fifteen couples, the egg emergence rate was calculated throughout the oviposition period. The oviposition period lasted from February to September, and the emergence rate varied between 13.9 and 53.3%. R. marabaensis presented an emergence rate of 46.7% and a total biological cycle of 193 days (the mean time required for emergence (25.1 days), 1st nymphal instar (19.4 days), 2nd nymphal instar (22.1 days), 3rd nymphal instar (26.2 days), 4th nymphal instar (29.3 days), and 5th nymphal instar (70.9 days)). Based on the biological cycle of R. marabaensis and 14 other Rhodnius species already described in the literature, it was also possible to calculate the averages for the groups prolixus, pictipes, and pallescens and, mainly, for the genus Rhodnius, contributing to the knowledge of this important group of Chagas disease vectors.

Parasite × vector relationship in Chagas disease: does Trypanosoma cruzi (Chagas, 1909) infection affect the spermatogenesis of Triatoma infestans (Klug, 1834)?

The parasite-vector interaction of Chagas disease is still poorly understood and the understanding of this relationship can help in the development of new strategies to control Trypanosoma cruzi transmission, which is the etiological agent of this disease. Considering the need to know if T. cruzi can cause some pathology in the reproductive system of the Chagas disease vectors, we investigated the spermatogenesis of Triatoma infestans infected by T. cruzi through histological and cytogenetic analysis. Trypanosoma cruzi Bolivia strain infection was not pathogenic for the reproductive system of T. infestans, because all the analyzed males had normal spermatogenesis, with all phases (spermatocytogenesis, meiosis and spermiogenesis) happening without any change. Thus, we demonstrated that the presence of T. cruzi Bolivia strain does not have influence in the spermatogenesis of T. infestans and we suggest that the influences on reproductive system observed for other species were a result of the action of the parasite on gametogenesis of females.

Nucleolar persistence during spermatogenesis of the genus Rhodnius (Hemiptera, Triatominae).

The Triatominae subfamily is comprised of 18 genera and six tribes. The tribe Rhodniini is comprised of two genera (Rhodnius and Psammolestes). Nucleolar persistence is defined by the presence of the nucleolus or nucleolar corpuscles during the meiotic metaphase. To date, this phenomenon has been described for 13 species of triatomine that are included in the genera Triatoma, Rhodnius, and Panstrongylus. Thus, because the phenomenon of nucleolar persistence has been described in only two species of the genus Rhodnius, we have analyzed the nucleolar behavior during spermatogenesis of eight species of the genus Rhodnius (R. colombiensis, R. montenegrensis, R. nasutus, R. neglectus, R. neivai, R. pictipes, R. prolixus, and R. robustus), with a focus on nucleolar persistence. By means of cytogenetic analysis with silver ions, nucleolar behavior during spermatogenesis is described in the eight species of Rhodnius analyzed. In all of them nucleolar behavior was similar and the phenomenon of nucleolar persistence was often observed. Therefore, we confirm nucleolar persistence as a peculiarity of the genus Rhodnius. However, it is emphasized that new cytogenetic analysis should be performed in the Triatominae subfamily, more specifically among the 15 genera that do not exhibit the nucleolar behavior described, in order to assess whether this phenomenon is truly a synapomorphy of these hematophagous insects.

Taxonomic reassessment of Rhodnius zeledoni Jurberg, Rocha & Galvão: a morphological and morphometric analysis comparing its taxonomic relationship with Rhodnius domesticus Neiva & Pinto.

BACKGROUND: Rhodnius zeledoni was described from a single specimen. Since its description, doubts have arisen regarding the taxonomic status of this species in relation to Rhodnius domesticus. METHODS: The present study reviewed and compared R. zeledoni with R. domesticus based on morphological analysis and head geometric morphometrics. RESULTS: Our analysis revealed the absence of distinctive diagnostic characters between the two species at specific levels. Rhodnius zeledoni and R. domesticus show morphological and morphometric similarity, with only minor differences in coloration observed between them. Contrary to previous statements, our analysis showed that R. zeledoni and R. paraensis are not closely related species, not corroborating previous studies with such an assumption. CONCLUSIONS: Therefore, we formally propose R. zeledoni as a junior synonym of R. domesticus.

Analysis of the maternal inheritance hypothesis of the exochorium in eggs from hybrids of Chagas disease vectors.

Morphological studies applied to the taxonomy of the Triatominae cover various structures (head, wing, thorax, genitalia, and eggs). Exochorial structures of hybrid eggs were characterized and compared with the parents, demonstrating that hybrids presented characteristics identical to the exochorial pattern observed in the females of the crosses, which resulted in the hypothesis that the pattern of triatomine eggs is possibly a characteristic inherited from females. Thus, we characterized the exochorium of the eggs of several triatomine hybrids and compared them with the parents, to assess the pattern of segregation and test the hypothesis of maternal inheritance. Hybrids were obtained in at least one direction from all crosses. The analysis of the exochorium of the eggs of the hybrids showed different patterns of segregation: "exclusively paternal", "predominantly maternal", "predominantly paternal", "mutual", and "differential". Curiously, none of the hybrids evaluated presented characteristics that segregated exclusively from the female parental species. Thus, we demonstrate that the hypothesis of maternal inheritance of the exochorium pattern of eggs is not valid and we emphasize the importance of alternative/combined tools (such as integrative taxonomy) for the correct identification of these insect vectors (mainly in view of possible natural hybridization events due to climate and environmental changes).

Mapping the Silent Threat: A Comprehensive Analysis of Chagas Disease Occurrence in Riverside Communities in the Western Amazon.

Chagas disease (CD) is a typical tropical illness caused by Trypanosoma cruzi. The objective of this study was to assess the prevalence of Chagas disease in communities in two states of the Brazilian Amazon. Data collection occurred in July in the Alto Juruá region of Acre and in December in the communities of Humaitá, Amazonas, in 2019. A total of 477 participants were included in the study. In the communities of Alto Juruá, triatomine collections and analyses of T. cruzi infection were also carried out. All confirmed cases were found in the state of Acre, resulting in a total prevalence of 1.67. Of these eight cases, seven underwent ECG, all of which were concluded as normal by the physician team's cardiologists. Seventeen triatomine bugs, all belonging to the Rhodnius genus, were captured. The natural infection rate by T. cruzi was 25% in the Nova Cintra community and 66.67% in the Boca do Moa community (Alto Juruá). This research found that more than 1% of the studied population exhibited positive serological results for Chagas disease in the riverine communities during the study period, representing a small portion of cases among those who have not yet been diagnosed.

Triatominae (Hemiptera: Reduviidae) fauna in bird nests: the case of Morrinhos, Ceará, Brazil.

Triatominae are associated with various Brazilian habitats, including bird nests, animal burrows, and peridomestic structures. Despite extensive studies on triatomines in domiciliary environments in Ceará, Brazil, there has been limited research on their presence in the wild. This study focuses on the municipality of Morrinhos in Ceará, which is characterized by a Caatinga biome and riparian forests along the Acaraú River. During the study, a total of 185 nests of Pseudoseisura cristata were analyzed in rural locations and triatomines were collected in 26 nests from 12 different locations. A total of 117 triatomines was collected, belonging to three species: Psammolestes tertius, Rhodnius nasutus, and Triatoma pseudomaculata. Rhodnius nasutus was the only species found in a nest inhabited by Didelphis albiventris. Nests of P. cristata serve as shelters for various animals, providing an ideal environment for triatomines to establish colonies due to their proximity to food sources and their generalist feeding habits. The incorporation of anthropogenic materials by birds in nest construction can indirectly affect the presence of other animals that may serve as food sources for triatomines. Understanding the interactions between triatomines, birds, and their habitats is essential for assessing the risks associated with triatomine infestation in wild ecosystems.

Integrative taxonomy and phylogenetic systematics of the Triatoma rubrovaria subcomplex (Hemiptera, Triatominae).

Triatoma rubrovaria subcomplex consists of T. carcavalloi, T. circummaculata, T. klugi, T. limai, T. oliveirai, T. pintodiasi, T. rubrovaria, T. patagonica and T. guasayana, which can be vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease. In this study, morphological, morphometric, and genetic characters of T. circummaculata, T. pintodiasi, T. carcavalloi, T. klugi, and T. rubrovaria were analyzed in view of the integrative taxonomy and phylogeny of the T. rubrovaria subcomplex. Molecular studies were carried out through the sequencing and analysis of the mitochondrial genes COI and CytB, nuclear genes ITS I, ITS 2, 16S, and 28S from rDNA and rescued a monophyletic group. Furthermore, differential morphological characters were found among the five species in the pronotum, scutellum, stridulatory sulcus, male genitalia, and external female genitalia. Finally, morphometric analyses made it possible to differentiate the five species. Phylogenetic analyzes rescued the relationship of T. pintodiasi with members of the T. rubrovaria subcomplex and demonstrated that this subcomplex is a monophyletic group composed of the species T. carcavalloi, T. circummaculata, T. klugi, T. guasayana, T. limai, T. oliveirai, T. patagonica, T. pintodiasi, and T. rubrovaria. Furthermore, through integrative taxonomy, it was possible to confirm the specific status of the species T. carcavalloi, T. circummaculata, T. pintodiasi, T. klugi, and T. rubrovaria, offering new useful morphological characters for the differentiation and characterization of these potential vectors and distributed in Southern Brazil.

Exploring the Hidden World of Vectors of Chagas Disease: A Fascinating Look at the Taxonomic Aspects of the Psammolestes Genus (Hemiptera, Triatominae).

Chagas disease (CD) is a neglected illness affecting approximately seven million individuals, with vector transmission occurring via triatomine bugs. The Rhodniini tribe comprises 24 species, grouped into the Rhodnius and Psammolestes genera. Given the importance of accurately identifying CD vectors, the taxonomy of Psammolestes spp. was revisited using morphological and morphometric data. Specimens of P. tertius, P. coreodes, and P. arthuri were collected, and the morphological characteristics of the head, thorax, abdomen, and eggs were analyzed. Morphometric studies of eggs were also conducted. Dichotomous keys allowing for the differentiation of Psammolestes spp. were elaborated based on adult insect and egg morphological characteristics. Through these studies, it was possible to differentiate the three Psammolestes species and confirm that this genus should not be classified under the Rhodnius genus, contributing to Rhodniini taxonomy.

First evidence of gonadal hybrid dysgenesis in Chagas disease vectors (Hemiptera, Triatominae): gonad atrophy prevents events of interspecific gene flow and introgression.

BACKGROUND: Hybridization events between Triatoma spp. have been observed under both natural and laboratory conditions. The ability to produce hybrids can influence different aspects of the parent species, and may even result in events of introgression, speciation and extinction. Hybrid sterility is caused by unviable gametes (due to errors in chromosomal pairing [meiosis]) or by gonadal dysgenesis (GD). All of the triatomine hybrids analyzed so far have not presented GD. We describe here for the first time GD events in triatomine hybrids and highlight these taxonomic and evolutionary implications of these events. METHODS: Reciprocal experimental crosses were performed between Triatoma longipennis and Triatoma mopan. Intercrosses were also performed between the hybrids, and backcrosses were performed between the hybrids and the parent species. In addition, morphological and cytological analyzes were performed on the atrophied gonads of the hybrids. RESULTS: Hybrids were obtained only for the crosses T. mopan♀ × T. longipennis♂. Intercrosses and backcrosses did not result in offspring. Morphological analyses of the male gonads of the hybrids confirmed that the phenomenon that resulted in sterility of the hybrid was bilateral GD (the gonads of the hybrids were completely atrophied). Cytological analyses of the testes of the hybrids also confirmed GD, with no germ cells observed (only somatic cells, which make up the peritoneal sheath). CONCLUSIONS: The observations made during this study allowed us to characterize, for the first time, GD in triatomines and demonstrated that gametogenesis does not occur in atrophied gonads. The characterization of GD in male hybrids resulting from the crossing of T. mopan♀ × T. longipennis♂ highlights the importance of evaluating both the morphology and the cytology of the gonads to confirm which event resulted in the sterility of the hybrid: GD (which results in no gamete production) or meiotic errors (which results in non-viable gametes).

New in morphometry: Geometric morphometry of the external female genitalia of Triatominae (Hemiptera: Reduviidae).

The study of geometric morphometry has an impact on Triatominae studies. Currently, several taxonomic and systematic studies use this approach. The Triatominae subfamily comprises three fossil species and 154 extant species potentially capable of transmitting Trypanosoma cruzi, the causative agent of Chagas disease. This study aims to evaluate the external female genitalia of adult triatomines using multivariate geometric morphometric approaches, not only for validation but also for systematic inferences. Specimens belonging to the genera Panstrongylus, Psammolestes, Rhodnius, and Triatoma were evaluated, in addition to two species previously included in Triatoma: T. longipennis and T. phyllosoma. The results show that the external female genitalia have operational morphology and allow characterization of the species and the genera of the Triatominae. In addition, the multivariate technique enabled delimitation of the phylogenetic relationships of the subfamily, presenting results consistent with systematic studies. It can be concluded that the external female genitalia evaluated by geometric morphometry is a useful character for the taxonomy and systematics of Triatominae.

Genome Size Variation of Chagas Disease Vectors of the Rhodniini Tribe.

The genome size of five Rhodnius species (R. milesi, R. nasutus, R. neivai, R. prolixus, and R. robustus) and two Psammolestes species (P. coroedes and P. tertius) were estimated using flow cytometry and/or k-mer distributions in genome sequences. Phylogenetic generalized linear mixed models highlighted significant genome size variations among species and between sexes, with R. prolixus showing the largest genome. In this study we provide the first data on female genome size in Triatominae. For five species, female genome size did not differ from males, except for R. robustus, where females had smaller genomes. Genome size estimations based on the k-mer distribution method were less than those estimated from flow cytometry, but both methods exhibited the same pattern of sexual differences. Further genomic studies are needed to infer whether genome size variation could be an adaptive trait in Rhodnius.

Morphological Stasis in Time? A Triatoma brasiliensis brasiliensis Study Using Geometric Morphometrics in the Long Run.

Triatoma brasiliensis brasiliensis Neiva, 1911 is one of the most important vectors of Chagas disease in the Brazilian semiarid regions in the north-east. The risk imposed by T. b. brasiliensis to the human populations, due to frequent invasions and/or colonization of the domiciles, demands constant monitoring and control actions as well as an understanding of its evolutionary process. In this context, the following research studies the pattern of shape adaptation over time using a large dataset from 102 years of specimen collections in order to identify the morphological plasticity of this vector in Brazil. This dataset was analyzed using geometric morphometrics tools and the timescale was divided into eight different groups, containing specimens from 1912 to 2014. Geometric morphometrics analysis showed an interesting morphological stasis in the wing shape of T. b. brasiliensis, which allowed us to understand the high capacity of adaptation to changes in climate condition through time, and the invasive status which Triatoma species have around the world. Moreover, these results showed novel findings as an interesting phenotypic pattern, with no modifications in more than 100 years, leading us to understand the shape evolution in Triatominae as a vector species of diseases.

Immature instars of three species of Rhodnius Stål, 1859 (Hemiptera, Reduviidae, Triatominae): morphology, morphometry, and taxonomic implications.

BACKGROUND: Among the 18 genera of the Triatominae subfamily, three stand out for their diversity and epidemiological importance: Triatoma, Panstrongylus, and Rhodnius. Rhodnius includes 21 species that can transmit Trypanosoma cruzi (the etiological agent of Chagas disease, also known as American trypanosomiasis) and Trypanosoma rangeli. The Rhodnius prolixus complex comprises seven species, including Rhodnius marabaensis, Rhodnius prolixus, and Rhodnius robustus, which occur in the northern region of Brazil. Since both adults and immatures can carry T. cruzi, in this study the five nymphal instars of the three species mentioned were dorsally characterized. METHODS: Using microscopy, morphometrics, and geometric morphometrics, the present work measures and describes the morphological characters of the five nymphal instars of R. marabaensis, R. prolixus, and R. robustus. RESULTS: The study enabled the characterization of all five nymphal instars, as well as the distinction between the three species in each of their instars. CONCLUSIONS: The morphological, morphometrics of the head, thorax, and abdomen and geometric morphometrics studies of the head enabled the specific distinction of these three species in all five instars.

Trends in evolution of the Triatomini tribe (Hemiptera, Triatominae): reproductive incompatibility between four species of geniculatus clade.

BACKGROUND: The geniculatus clade, composed by the rufotuberculatus, lignarius, geniculatus and megistus groups, relates evolutionarily the species of the genus Panstrongylus and Nesotriatoma. Several studies have shown that triatomine hybrids can play an important role in the transmission of Chagas disease. Natural hybrids between species of the geniculatus clade have never been reported to our knowledge. Thus, carrying out experimental crosses between species of the geniculatus clade can help to elucidate the taxonomic issues as well as contribute to the epidemiological knowledge of this group. METHODS: Experimental crosses were carried out between species of the megistus and lignarius groups to evaluate the reproductive compatibility between them. A phylogenetic reconstruction was also performed with data available in GenBank for the species of the geniculatus clade to show the relationships among the crossed species. RESULTS: Phylogenetic analysis grouped the species of the geniculatus clade into four groups, as previously reported. In the interspecific crosses performed there was no hatching of eggs, demonstrating the presence of prezygotic barriers between the crossed species and confirming their specific status. CONCLUSIONS: In contrast to the other groups of the Triatomini tribe, as well as the Rhodniini, there are prezygotic barriers that prevent the formation of hybrids between species of the megistus and lignarius groups. Thus, the geniculatus clade may represent an important evolutionary model for Triatominae, highlighting the need for further studies with greater sample efforts for this clade (grouping the 17 species of Panstrongylus and the three of Nesotriatoma).

Do not judge a book by its cover: would Triatoma tibiamaculata (Pinto, 1926) belong to Triatoma Laporte, 1832, or to Panstrongylus Berg, 1879, with misleading homoplasies?

BACKGROUND: Triatoma tibiamaculata is a species distributed in ten Brazilian states which has epidemiological importance as it has already been found infecting household areas. The taxonomy of this triatomine has been quite unstable: it was initially described as Eutriatoma tibiamaculata. Later, the species was transferred from the genus Eutriatoma to Triatoma. Although included in the genus Triatoma, the phylogenetic position of T. tibiamaculata in relation to other species of this genus has always been uncertain once this triatomine was grouped in all phylogenies with the genus Panstrongylus, rescuing T. tibiamaculata and P. megistus as sister species. Thus, we evaluated the generic status of T. tibiamaculata using phylogenetic and chromosomal analysis. METHODS: Chromosomal (karyotype) and phylogenetic (with mitochondrial and nuclear markers) analyses were performed to assess the relationship between T. tibiamaculata and Panstrongylus spp. RESULTS: The chromosomal and phylogenetic relationship of T. tibiamaculata and Panstrongylus spp. confirms the transfer of the species to Panstrongylus with the new combination: Panstrongylus tibiamaculatus. CONCLUSIONS: Based on chromosomal and phylogenetic characteristics, we state that P. tibiamaculatus comb. nov. belongs to the genus Panstrongylus and that the morphological features shared with Triatoma spp. represent homoplasies.

Morphological, Cytological and Molecular Studies and Feeding and Defecation Pattern of Hybrids from Experimental Crosses between Triatoma sordida and T. rosai (Hemiptera, Triatominae).

Under laboratory conditions, Triatoma rosai and T. sordida are able to cross and produce hybrids. In the face of climate and environmental changes, the study of hybrids of triatomines has evolutionary and epidemiological implications. Therefore, we performed morphological, cytological and molecular studies and characterized the feeding and defecation pattern of hybrids from crosses between T. sordida and T. rosai. The morphological characterization of the female genitalia of the hybrids showed that characteristics of both parental species segregated in the hybrids. Cytogenetic analyzes of hybrids showed regular metaphases. According to molecular studies, the mitochondrial marker Cytochrome B (CytB) related the hybrids with T. sordida and the nuclear marker Internal Transcribed Spacer 1 (ITS-1) related the hybrids with T. rosai. Both parents and hybrids defecated during the blood meal. Thus, the hybrids resulting from the cross between T. sordida and T. rosai presented segregation of phenotypic characters of both parental species, 100% homeology between homeologous chromosomes, phylogenetic relationship with T sordida and with T. rosai (with CytB and ITS-1, respectively), and, finally, feeding and defecation patterns similar to the parents.

Chagas Disease Vectors of Espírito Santo, Brazil: First Report of Triatoma infestans (Klug, 1834) (Hemiptera, Triatominae) in the Brazilian State and Development of an Identification Key Based on Cytogenetic Data.

Chagas disease is a potentially life-threatening illness caused by the protozoan Trypanosoma cruzi and transmitted, mainly, by hematophagous insects of the Triatominae subfamily. In Brazil, there are currently about 66 triatomine species distributed throughout the country's 27 states. Triatoma infestans is considered as a species of great vectorial importance, mainly because of its biological characteristics, such as the high degree of anthropophilia, adaptation to the home environment, ability to withstand long periods of fasting, and present a wide geographical distribution. Taking into account the epidemiological importance of these species, we carried out the first report of T. infestans in the Espírito Santo, Brazil, and development of an identification key for all species notified in that state, based on cytogenetic data. This information is important because they contribute to the direction of epidemiological surveillance activities carried out by vector control programs of the Espírito Santo, Brazil.