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.

Experimental infection of Rhodnius robustus Larrousse, 1927 (Hemiptera, Reduviidae, Triatominae) with Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae) IV.

Vector competence of triatomines (kissing bugs) for Trypanosoma cruzi transmission depends on the parasite-vector interaction and the genetic constitution of both. This study evaluates the susceptibility and vector competence of Rhodnius robustus experimentally infected with T. cruzi IV (TcIV). Nymphs were fed on infected mice or an artificial feeder with blood containing culture-derived metacyclic trypomastigotes (CMT) or blood trypomastigotes (BT). The intestinal contents (IC) and excreta of the insects were examined by fresh examination and kDNA-PCR. The rate of metacyclogenesis was also determined by differential counts. Fifth instar nymphs fed with CMT ingested a greater blood volume (mean of 74.5 µL) and a greater amount of parasites (mean of 149,000 CMT/µL), and had higher positivity in the fresh examination of the IC. Third instar nymphs fed with CMT had higher positivity (33.3%) in the fresh examination of the excreta. On the 20th day after infection (dai), infective metacyclic trypomastigote (MT) forms were predominant in the excreta of 3/4 experimental groups, and on the 30th dai, the different parasitic forms were observed in the IC of all the groups. Higher percentages of MT were observed in the excreta of the 5th instar nymphs group (84.1%) and in the IC of the 3rd instar nymphs group (80.0%). Rhodnius robustus presented high susceptibility to infection since all nymphs were infected, regardless of the method used for blood meal, in addition these insects demonstrated vector competence for TcIV with high rates of metacyclogenesis being evident.

Multidisciplinary approach detects speciation within the kissing bug Panstrongylus rufotuberculatus populations (Hemiptera, Heteroptera, Reduviidae).

BACKGROUND: Panstrongylus rufotuberculatus (Hemiptera-Reduviidae) is a triatomine species with a wide geographic distribution and a broad phenotypic variability. In some countries, this species is found infesting and colonising domiciliary ecotopes representing an epidemiological risk factor as a vector of Trypanosoma cruzi, etiological agent of Chagas disease. In spite of this, little is known about P. rufotuberculatus genetic diversity. METHODS: Cytogenetic studies and DNA sequence analyses of one nuclear (ITS-2) and two mitochondrial DNA sequences (cyt b and coI) were carried out in P. rufotuberculatus individuals collected in Bolivia, Colombia, Ecuador and Mexico. Moreover, a geometric morphometrics study was applied to Bolivian, Colombian, Ecuadorian and French Guiana samples. OBJECTIVES: To explore the genetic and phenetic diversity of P. rufotuberculatus from different countries, combining chromosomal studies, DNA sequence analyses and geometric morphometric comparisons. FINDINGS: We found two chromosomal groups differentiated by the number of X chromosomes and the chromosomal position of the ribosomal DNA clusters. In concordance, two main morphometric profiles were detected, clearly separating the Bolivian sample from the other ones. Phylogenetic DNA analyses showed that both chromosomal groups were closely related to each other and clearly separated from the remaining Panstrongylus species. High nucleotide divergence of cyt b and coI fragments were observed among P. rufotuberculatus samples from Bolivia, Colombia, Ecuador and Mexico (Kimura 2-parameter distances higher than 9%). MAIN CONCLUSIONS: Chromosomal and molecular analyses supported that the two chromosomal groups could represent different closely related species. We propose that Bolivian individuals constitute a new Panstrongylus species, being necessary a detailed morphological study for its formal description. The clear morphometric discrimination based on the wing venation pattern suggests such morphological description might be conclusive.

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.

Do the new triatomine species pose new challenges or strategies for monitoring Chagas disease? An overview from 1979-2021.

Chagas disease persists as one of the most important, and yet most neglected, diseases in the world, and several changes in its epidemiological aspects have been recorded since its discovery. Currently, some of the most relevant changes are related to: (i) the reduction in the incidence of the endemic due to the control of the most important vectors, Triatoma infestans and Rhodnius prolixus, in many countries; (ii) the migration of human populations spreading cases of the disease throughout the world, from endemic to non-endemic areas, transforming Chagas disease into a global threat; and (iii) new acute cases and deaths caused by oral transmission, especially in the north of Brazil. Despite the reduction in the number of cases, new challenges need to be responded to, including monitoring and control activities aiming to prevent house infestation by the secondary vectors from occurring. In 1979, Lent & Wygodzinsky(1) published the most complete review of the subfamily Triatominae, encompassing 111 recognised species in the taxon. Forty-two years later, 46 new species and one subspecies have been described or revalidated. Here we summarise the new species and contextualise them regarding their ecology, epidemiologic importance, and the obstacles they pose to the control of Chagas disease around the world.

Identification of new Corynebacterium pseudotuberculosis antigens by immunoscreening of gene expression library.

BACKGROUND: Caseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans. The etiologic agent is the Corynebacterium pseudotuberculosis bacillus. The objective of this study was to build a gene expression library from C. pseudotuberculosis and use immunoscreening to identify genes that encode potential antigenic proteins for the development of DNA and subunit vaccines against CLA. RESULTS: A wild strain of C. pseudotuberculosis was used for extraction and partial digestion of genomic DNA. Sequences between 1000 and 5000 base pairs (bp) were excised from the gel, purified, and the digested DNA fragments were joined to bacteriophage vector ZAP Express, packaged into phage and transfected into Escherichia coli. For immunoscreening a positive sheep sera pool and a negative sera pool for CLA were used. Four clones were identified that strongly reacted to sera. The clones were confirmed by polymerase chain reaction (PCR) followed by sequencing for genomic comparison of C. pseudotuberculosis in GenBank. The genes identified were dak2, fagA, fagB, NlpC/P60 protein family and LPxTG putative protein family. CONCLUSION: Proteins of this type can be antigenic which could aid in the development of subunit or DNA vaccines against CLA as well as in the development of serological tests for diagnosis. Immunoscreening of the gene expression library was shown to be a sensitive and efficient technique to identify probable immunodominant genes.

Heterochromatin base pair composition and diversification in holocentric chromosomes of kissing bugs (Hemiptera, Reduviidae).

The subfamily Triatominae (Hemiptera, Reduviidae) includes 150 species of blood-sucking insects, vectors of Chagas disease or American trypanosomiasis. Karyotypic information reveals a striking stability in the number of autosomes. However, this group shows substantial variability in genome size, the amount and distribution of C-heterochromatin, and the chromosome positions of 45S rDNA clusters. Here, we analysed the karyotypes of 41 species from six different genera with C-fluorescence banding in order to evaluate the base-pair richness of heterochromatic regions. Our results show a high heterogeneity in the fluorescent staining of the heterochromatin in both autosomes and sex chromosomes, never reported before within an insect subfamily with holocentric chromosomes. This technique allows a clear discrimination of the heterochromatic regions classified as similar by C-banding, constituting a new chromosome marker with taxonomic and evolutionary significance. The diverse fluorescent patterns are likely due to the amplification of different repeated sequences, reflecting an unusual dynamic rearrangement in the genomes of this subfamily. Further, we discuss the evolution of these repeated sequences in both autosomes and sex chromosomes in species of Triatominae.

Genes encoding defensins of important Chagas disease vectors used for phylogenetic studies.

Insects possess both cellular and humoral immune responses. The latter makes them capable to recognize and control invading pathogens after synthesis of a variety of small proteins, also known as antimicrobial peptides. Defensins, cysteine-rich cationic peptides with major activity against Gram-positive bacteria, are one ubiquitous class of antimicrobial peptides, widely distributed in different animal and plant taxa. Regarding triatomines in each of the so far analyzed species, various defensin gene isoforms have been identified. In the present study, these genes were sequenced and used as a molecular marker for phylogenetic analysis. Considering the vectors of Chagas disease the authors are reporting for the first time the presence of these genes in Triatoma sordida (Stål, 1859), Rhodnius nasutus (Stål, 1859), and Panstrongylus megistus (Burmeister, 1835). Members of the Triatoma brasiliensis species complex were included into the study to verify the genetic variability within these taxa. Mainly in their mature peptide, the deduced defensin amino acid sequences were highly conserved. In the dendrogram based on defensin encoding nucleotide, sequences the Triatoma Def3/4 genes were separated from the rest. In the dendrogram based on deduced amino acid sequences the Triatoma Def2/3/4 together with Rhodnius DefA/B pre-propeptides were separated from the rest. In the sub-branches of both the DNA and amino acid dendrograms, the genus Triatoma was separated from the genus Rhodnius as well as from P. megistus.

Geometric morphometry of the Rhodniusprolixus complex (Hemiptera, Triatominae): patterns of intraspecific and interspecific allometry and their taxonomic implications.

In the subfamily Triatominae, the genus Rhodnius is one of the most studied, not only because of its epidemiological importance, but also because of the difficulty in differentiating its species. Currently, one of the strategies to control Chagas disease, besides other initiatives such as the analysis of donated blood, is focused on fighting the vector. Correctly identifying triatomines is essential for the entomoepidemiological surveillance of Chagas disease. The objective of the present work was to compare the species of the R.prolixus complex using geometric morphometry of hemelytra and heads to evaluate the patterns of intraspecific and interspecific allometry and their taxonomic implications. This method can help in the diagnosis of close species, whose morphological characteristics are insufficient for correct identification. Specimens from five different collections were used, covering the species included in the R.prolixus complex (R.barretti, R.dalessandroi, R.domesticus, R.marabaensis, R.milesi, R.montenegrensis, R.nasutus, R.neglectus, R.neivai, R.prolixus and R.robustus). Morphometric analyses indicated that the hemelytra are not structures with good resolution for separating species and, for this reason, the use of the heads proved to be more adequate for this group (thus allowing differentiation of all species of the R.prolixus complex). The results suggest that R.milesi is a variant of R.neglectus and confirms that R.prolixus and R.robustus are distinct species. Furthermore, we propose the creation of the R.neivai complex comprising R.domesticus and R.neivai.

Comparative morphology of eggs of Rhodnius domesticus Neiva & Pinto, Rhodnius neglectus Lent and Rhodnius prolixus Stål (Hemiptera: Reduviidae: Triatominae).

Rhodnius species are potential vectors of the etiological agent of Chagas disease (CD), the protozoan Trypanosoma cruzi. CD impacts around seven million people in Latin America, resulting in approximately fourteen thousand deaths per year. Several species of Rhodnius are notable not only for their epidemiological relevance, but also for the challenging distinction between their species. Rhodnius has twenty species, each with its specific epidemiological importance. Rhodnius neglectus and Rhodnius prolixus are found with colonies in domiciliary environments. The observation of eggs in human dwellings signals the colonization process of these insects, increasing the risk of contamination of the population, since correct identification of eggs is necessary to help more effective vector control programs. Here we highlight diagnostic characters of eggs for these three species.

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.

Entomological indicators and food sources of triatomines in the Brazilian semi-arid region.

BACKGROUND: Triatomines are biological vectors of Trypanosoma cruzi, the etiological agent of Chagas Disease (CD) and have various mammalian hosts. This study evaluated the entomological indicators and food sources of triatomines in Petrolina in the semi-arid region of Brazil, where CD is endemic. METHODS: Triatomines were captured indoors and outdoors through an active search and entomological indices (household and natural infections) were calculated. Parasitological analyses were performed through microscopic visualization using Giemsa-stained insect feces, and DNA sequencing was employed to identify food sources from the gut contents of 82 insects (9.05%) that were better preserved. RESULTS: We captured triatomines (906) in peridomicile (807) and intradomicile (99): Triatoma brasiliensis (84.7%, 767 specimens), Triatoma spp. (8.2%, 74 specimens), T. pseudomaculata (6.5%, 59 specimens), Rhodnius spp. (0.4%, four specimens), R. nasutus (0.1%, one specimen), and T. sordida (0.1%, one specimen). The household infestation index is 11.8%. Thirty-five triatomines were infected (33 T. brasiliensis and two T. pseudomaculata), corresponding to a natural infection index of 3.8%. The identified food sources were human T. pseudomaculata and T. brasiliensis, dogs for T. brasiliensis and rodents (Mus musculus) for T. brasiliensis. CONCLUSIONS: The results reinforce the need to intensify CD diagnosis, surveillance, and control actions, as an increase in entomological indices was recorded. Blood from humans and domestic and synanthropic animals was detected in the infected triatomines, suggesting a risk of CD vector transmission in Petrolina. As CD is a zoonosis, multidisciplinary and intersectoral CD surveillance must be conducted in the context of the One Health.

Chagas Disease Vectors of Alagoas, Brazil: First Report of Panstrongylus geniculatus (Latreille, 1811) (Hemiptera, Triatominae) in the Brazilian State and Update of the Dichotomous Key Based on Cytogenetic Data.

Panstrongylus geniculatus is the most widely distributed species of Panstrongylus in Brazil and merits attention from vector control programs due to its potential for domiciliation. Specimens infected with Trypanosoma cruzi have already been reported in both peridomiciliary and domiciliary environments. Building on these findings, we report, for the first time, the presence of P. geniculatus in the state of Alagoas and provide an updated dichotomous key (based on cytogenetic data) for species in Alagoas. Panstrongylus geniculatus has been identified in Boca da Mata, Joaquim Gomes, and Novo Lino. In light of the recent notification of Rhodnius domesticus and this record of P. geniculatus, we present an updated identification key enabling the differentiation of all species in Alagoas. Emphasis is placed on the importance of correctly identifying triatomine species because it is crucial for the development of effective control measures, thereby aiding in the mitigation of Chagas disease.

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).

Unveiling the overlooked: Current and future distribution dynamics of kissing bugs and palm species linked to oral Chagas disease transmission.

Chagas disease, a neglected global health concern primarily transmitted through the bite and feces of kissing bugs, has garnered increasing attention due to recent outbreaks in northern Brazil, highlighting the role of oral transmission facilitated by the kissing bugs species Rhodnius robustus and Rhodnius pictipes. These vectors are associated with palm trees with large crowns, such as the maripa palm (Attalea maripa) and moriche palm (Mauritia flexuosa). In this study, we employ maximum entropy (MaxEnt) ecological niche models to analyze the spatial distribution of these vectors and palm species, predicting current and future climate suitability. Our models indicate broader potential habitats than documented occurrences, with high suitability in northern South America, southern Central America, central Africa, and southeast Asia. Projections suggest increased climate suitability by 2040, followed by a reduction by 2080. This study identifies present and future areas suitable for kissing bugs and palm tree species due to climate change, aiding in the design of prevention and management strategies.

Chromosomal divergence and evolutionary inferences in Rhodniini based on the chromosomal location of ribosomal genes.

In this study, we used fluorescence in situ hybridisation to determine the chromosomal location of 45S rDNA clusters in 10 species of the tribe Rhodniini (Hemiptera: Reduviidae: Triatominae). The results showed striking inter and intraspecific variability, with the location of the rDNA clusters restricted to sex chromosomes with two patterns: either on one (X chromosome) or both sex chromosomes (X and Y chromosomes). This variation occurs within a genus that has an unchanging diploid chromosome number (2n = 22, including 20 autosomes and 2 sex chromosomes) and a similar chromosome size and genomic DNA content, reflecting a genome dynamic not revealed by these chromosome traits. The rDNA variation in closely related species and the intraspecific polymorphism in Rhodnius ecuadoriensis suggested that the chromosomal position of rDNA clusters might be a useful marker to identify recently diverged species or populations. We discuss the ancestral position of ribosomal genes in the tribe Rhodniini and the possible mechanisms involved in the variation of the rDNA clusters, including the loss of rDNA loci on the Y chromosome, transposition and ectopic pairing. The last two processes involve chromosomal exchanges between both sex chromosomes, in contrast to the widely accepted idea that the achiasmatic sex chromosomes of Heteroptera do not interchange sequences.

Description of the male genitalia of Belminus rugulosus Stål and Belminus corredori Galvão & Angulo, and comments on the holotype of Parabelminus yurupucu Lent & Wygodzinsky (Hemiptera: Heteroptera: Reduviidae: Triatominae: Bolboderini).

The male genitalia of Belminus rugulosus Stål and Belminus corredori Galvão & Angulo are described and illustrated for the first time. Comments on the holotype of Parabelminus yurupucu Lent & Wygodzinsky are presented (Hemiptera: Heteroptera: Reduviidae: Triatominae: Bolboderini). A discussion on previous data in the literature on male genitalia of species of Belminus Stål is provided.

Characterization of Triatoma infestans (Klug, 1834) (Hemiptera: Heteroptera, Reduviidae) from Residual Foci in the States of Bahia and Rio Grande do Sul, Brazil, Using Geometric Morphology.

Triatoma infestans is one of the main vectors of Chagas disease in Latin America. Although the species is under control in most Latin countries, it is still necessary to maintain epidemiological surveillance. The present study aims to characterize T. infestans populations from residual foci in Bahia and Rio Grande do Sul, Brazil, comparing them with natural populations in Argentina and Bolivia. For this, we adopt the geometric morphometry of the heads. It is possible to report the morphometric variety of the studied populations. In addition, we show that the size of the heads contributes to the differentiation between populations, while the shape has less power to discriminate groups. Furthermore, we show that some natural populations have morphometric proximity to residual populations, suggesting a relationship between these triatomines. Our data do not support the origin of residual populations but demonstrate the importance of new studies with other techniques to understand the dynamics of distribution and reintroduction of these vectors in Brazilian territory.

External quality assessment of the entomological identification of triatomines in the network of public laboratories in Rondônia, Brazil.

BACKGROUND: An external quality assessment on the identification of triatomines within the laboratory network in the state of Rondônia. METHODS: Seven laboratories participated in this evaluation. Each was provided with support materials and nine insects from the Hemiptera order for identification. RESULTS: All samples were accurately identified at the species level. However, correct sex identification was achieved for only 79% of the samples. The most significant challenges were encountered in determining the sex of predators, phytophagous species, Rhodnius robustus, and Rhodnius pictipes. CONCLUSIONS: The identified shortcomings can inform enhancements in vector control programs for Chagas disease.

Characterization of External Female Genitalia in Five Triatoma Laporte Species of South America (Hemiptera: Reduviidae: Triatominae).

Currently, there are 158 valid species of triatomines, all of which are potential vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. The correct taxonomic identification of triatomines is essential since each species hos a different epidemiological importance. The aim of the study is to compare five species of South American Triatoma. Here we present a comparative study of terminal abdominal segments in females by scanning electron microscopy (SEM) of the species Triatoma delpontei, T. jurbergi, T. infestans var. melanosoma, T. platensis, and T. vandae. The results showed diagnostic characters for the studied species. The dorsal view featured more valuable characters, with seven informative characters. Similarities were observed among T. delpontei, T. infestans var. melanosoma, and T. platensis, and between T. jurbergi and T. vandae, correlating with previous studies. Thus, female genital characters proved to be reliable and useful in the diagnosis of the Triatoma species studied here; additional studies, along with other sets of behavioral, morphological, and molecular data, helped to reinforce the hypotheses found here.