Population genetics and genomics of Triatoma brasiliensis (Hemiptera, Reduviidae) in an area of high pressure of domiciliary infestation in Northeastern Brazil.

Understanding the population dynamics of vectors is crucial for effective control of vector-borne diseases. In the Northeastern Brazilian semi-arid region, Triatoma brasiliensis persists as the most significant Chagas disease vector, frequently displaying recurrent domiciliary infestations. This situation raises relevant public health concerns in the municipality of Currais Novos in the state of Rio Grande do Norte. This area has experienced a high prevalence of peridomiciliary re-infestations by T. brasiliensis, coupled with elevated rates of Trypanosoma cruzi infection. Therefore, we assessed the distribution of genetic variation via mitochondrial Cytochrome b gene (MT-CYB) sequencing (n = 109) and single nucleotide polymorphisms (SNPs, n = 86) to assess the gene flow among distinct populations distributed in varied geographic spots and environments, mainly sylvatic and peridomiciliary. Insects were collected from rural communities at Currais Novos, enclosed within a 16 km radius. Sampling included 13 populations: one intradomiciliary, eight peridomiciliary, and four sylvatic. Furthermore, an external population located 220 km from Currais Novos was also included in the study. The method employed to obtain SNP information relied on ddRAD-seq genotyping-by-sequencing (GBS), enabling a genome-wide analysis to infer genetic variation. Through AMOVA analysis of MT-CYB gene variation, we identified four distinct population groups with statistical significance (FCT= 0.42; p<0.05). We identified a total of 3,013 SNPs through GBS, with 11 loci showing putative signs of being under selection. The variation based on 3,002 neutral loci evidenced low genetic structuration based on low FST values (p>0.05), indicating local panmixia. However, resampling algorithms pointed out that three samples from the external population were assigned (>98 %) in a cluster contrasting from the ones putatively under local panmixia - validating the newly applied genome-wide marker for studies on the population genetics at finer-scale resolution for T. brasiliensis. The presence of population structuring in some of the sampled points, as suggested by the mitochondrial marker, leads us to assume that infestations were probably initiated by small populations of females - demographic event poses a risk for rapid re-infestations. The local panmictic pattern revealed by the GBS marker poses a challenge for vector control measures, as re-infestation foci may be distributed over a wide geographical and ecological range. In such instances, vectors exhibit reduced susceptibility to conventional insecticide spraying operations since sylvatic populations are beyond the reach of these interventions. The pattern of infestation exhibited by T. brasiliensis necessitates integrating innovative strategies into the existing control framework, holding the potential to create a more resilient and adaptive vector control program. In our dataset, the results demonstrated that the genetic signals from both markers were complementary. Therefore, it is essential to consider the nature and inheritance pattern of each marker when inferring the pattern of re-infestations.

From molecules to ecosystems: Insights into a network of interactions for a Chagas disease outbreak using Triatoma brasiliensis as natural samplers.

Exploring the dynamics of disease transmission involves an understanding of complex interactions within the eco-epidemiologic framework. In the context of Chagas disease (CD), elements are mainly represented by the interactions among the pathogen, insect vector, host, humans and the environment. We performed quantitative and qualitative analyses on a dataset derived from 98 Triatoma brasiliensis infected by trypanosomatids, which were linked to a CD outbreak in the semi-arid region of northeastern Brazil. We extracted invertebrate-derived DNA (iDNA) from these insects, comprising 18 populations around the outbreak area, each indicative of various strata of anthropogenic influence. Food source (FS) diversity, representing potential parasite reservoirs, was determined through mitochondrial gene (cyt b) sequencing of vertebrates, and parasite genotyping was accessed using fluorescent amplified fragment barcodes (FFLB) of trypanosomatids. We also assessed the residents' awareness of breeding sites for CD vectors in the inspected houses. The quantification of Trypanosoma cruzi was estimated via real-time PCR and is denominated here as the average parasite load (PL) per insect (T. cruzi/intestinal unit). We aimed to address vector-parasite-host-environment interactions that were discussed based on their significance among the components. Notably, among the significant interactions, we observed that the PL in the insects was significantly influenced by FS. Infected insects that fed on the classic reservoir, Didelphis albiventris, and Galea spixii exhibited higher PLs, compared to those that fed on Kerodon rupestris (p < 0.04)-a primary host. While D. albiventris is already recognized as a synanthropic species, we propose that G. spixii may also be undergoing a synanthropic process. Conversely, domestic cats are frequently identified as FS in infected insects from the sylvatic environment, suggesting a possible change in their behavior towards a wild state. Therefore, we propose that neglected anthropogenic actions have facilitated the reciprocal (sylvatic-peridomestic) circulation of T. cruzi-especially noted for TcI because it was predominant in insects found in peridomestic environments. Residents are often unaware of the existence of insect breeding grounds near their homes, particularly when it involves the storage of materials without planning for use, such as piles of tiles, bricks and wood. Although indirect inferences about the interaction among vector-parasite-host-environment are still incipient, we highlight the potential use of vectors as natural samplers of biological and ecological components in transmitting the disease.

Evidence that hematophagous triatomine bugs may eat plants in the wild.

Blood feeding is a secondary adaptation in hematophagous bugs. Many proteins are secreted in the saliva that are devoted to coping with the host's defense and to process the blood meal. Digestive enzymes that are no longer required for a blood meal would be expected to be eventually lost. Yet, in many strictly hematophagous arthropods, α-amylase genes, which encode the enzymes that digest starch from plants, are still present and transcribed, including in the kissing bug Rhodnius prolixus (Hemiptera, Reduviidae) and its related species, which transmit the Chagas disease. We hypothesized that retaining α-amylase could be advantageous if the bugs occasionally consume plant tissues. We first checked that the α-amylase protein of Rhodnius robustus retains normal amylolytic activity. Then we surveyed hundreds of gut DNA extracts from the sylvatic R. robustus to detect traces of plants. We found plant DNA in 8% of the samples, mainly identified as Attalea palm trees, where R. robustus are usually found. We suggest that although of secondary importance in the blood-sucking bugs, α-amylase may be needed during occasional plant feeding and thus has been retained.

Revisiting the Hybridization Processes in the Triatoma brasiliensis Complex (Hemiptera, Triatominae): Reproductive Isolation between Triatoma petrocchiae and T. b. brasiliensis and T. lenti.

Triatoma petrocchiae is a species morphologically similar to T. b. brasiliensis (which resulted in a synonymization event); despite this similarity, genetic, morphological, and experimental crossbreeding studies confirmed the specific status of T. petrocchiae. Considering that both species have been reported living in sympatry and that, for a long time, most species of the T. brasiliensis complex were considered only chromatic variants of T. b. brasiliensis, we carried out experimental crosses between T. b. brasiliensis and T. petrocchiae (to confirm whether these species are reproductively isolated) and between T. lenti and T. petrocchiae (to assess whether T. petrocchiae also presents prezygotic isolation with the other species of the T. brasiliensis complex). Reciprocal experimental crosses were conducted, and weekly, the eggs were collected, counted, and separated in new containers to assess the hatch rate. Neither cross resulted in hybrids, demonstrating that there are pre-zygotic reproductive barriers installed between T. petrocchiae and the other species of the T. brasiliensis complex. On the basis of the results above, we demonstrated that T. petrocchiae is reproductively isolated from T. b. brasiliensis and T. lenti. Furthermore, we suggest that T. petrocchiae is the species most derived from the T. brasiliensis complex.

Dynamics of food sources, ecotypic distribution and Trypanosoma cruzi infection in Triatoma brasiliensis from the northeast of Brazil.

Innovative approaches used to combat Chagas disease transmission tend to combine a set of comprehensive efforts to understand the ecology of local vectors. In this work we identified molecularly the blood meal of 181 Triatoma brasiliensis, distributed in 18 populations (8 sylvatic and 10 peridomestic), which were collected across a range of 240 km (East-West) and 95 km (North-South) in the semi-arid region of northeastern, Brazil. We used the vertebrate mitochondrial gene (cytochrome B) sequencing applied to DNA isolated from bug midgut to identify the insect blood meal sources via the BLAST procedure. The peridomestic populations were classified according to two main hypotheses of site-occupancy for T. brasiliensis: the first says that the infestation is mainly driven by structures that resemble its natural habitat (stony-like ecotopes) and the second assumes that it is associated with key-hosts (rodents and goats). Rodents of the Caviidae family (Galea spixii and Kerodon rupestris) were identified as the key-host of T. brasiliensis, but also the potential Trypanosoma cruzi reservoir-able to connect the sylvatic and domestic T. cruzi cycle. Cats also deserve to be studied better, as potential T. cruzi reservoirs. By modeling the food sources + site-occupancy + T. cruzi natural infection, we identified man-made ecotopes suitable for forming dense triatomine infestations with high rates of T. cruzi natural infection, which may be taken into account for vector control measures.

Drivers of molecular and morphometric variation in Triatoma brasiliensis (Hemiptera: Triatominae): the resolution of geometric morphometrics for populational structuring on a microgeographical scale.

BACKGROUND: The protozoan Trypanosoma cruzi circulates in semiarid areas of northeastern Brazil in distinct ecotopes (sylvatic, peridomestic and domestic) where Triatoma brasiliensis Neiva, 1911 is the most important Chagas disease vector. In this study, we analyzed microevolutionary and demographic aspects of T. brasiliensis populations at the ecotypic, micro and macro-geographic scales by combining morphometrics and molecular results. Additionally, we aimed to address the resolution of both markers for delimiting populations in distinct scales. METHODS: We sampled populations of T. brasiliensis from distinct ecotypic and geographic sites in the states Rio Grande do Norte (RN) and Paraíba (PB). The geometric morphometry was carried out with 13 landmarks on the right wings (n = 698) and the genetic structure was assessed by sequencing a region of cytochrome b mitochondrial gene (n = 221). Mahalanobis distance (MD) and coefficient of molecular differentiation (ΦST) were calculated among all pairs of populations. The results of comparisons generated MD and ΦST dendrograms, and graphics of canonical variate analysis (CVA). RESULTS: Little structure was observed for both markers for macro-geographic scales. Mantel tests comparing geographic, morphometric and genetic matrices showed low correlation (all R2 < 0.35). The factorial graphics built with the CVA evidenced population delimitation for the morphometric data at micro-geographic scales. CONCLUSIONS: We believe that T. brasiliensis carries in its genotype a source of information to allow the phenotypical plasticity across its whole distribution for shaping populations, which may have caused a lack of population delimitation for CVAs in morphometric analysis for macro-geographic scale analysis. On the other hand, the pattern of morphometric results in micro-geographic scales showed well-defined groups, highlighting the potential of this tool to inferences on the source for infestation.

Triatoma petrocchiae (Hemiptera, Reduviidae, Triatominae): A Chagas disease vector of T. brasiliensis species complex associated to reptiles.

In semi-arid areas of northeastern Brazil, Chagas disease vectors of Triatoma brasiliensis species complex comprise a monophyletic group of kissing bugs that inhabit rock outcrops. Most of them exhibit allopatric or parapatric distribution; the exception is T. petrocchiae, which is found in cohabitation with T. brasiliensis in rock outcrops. We used vertebrate mitochondrial gene sequencing applied to DNA isolated from bug midgut to identify the insect blood meal sources via BLAST procedure. Fourteen sylvatic insects from four geographic districts in the states of Rio Grande do Norte and Paraíba had their blood meal sources detected. While T. brasiliensis is recorded to be associated mainly (52-71%) with rodents, T. petrocchiae samples were strongly associated (86%) with reptiles of Tropidurus and Hemidactylus genera. We suggest that T. petrocchiae is the single member within this complex to be associated with reptiles, indicating a distinct niche occupation related to the trophic resources.

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.

Triatoma petrocchiae is the newly member of the Triatoma brasiliensis species complex. This species overlaps with T. brasiliensis in geographic and ecotypic occupation in the sylvatic habitat because both inhabit rocky outcrops in the semi-arid portion of Brazilian northeast. In this region T. brasiliensis is the most important Chagas disease vector because it constantly colonizes domiciles. In contrast, T. petrocchiae is rarely found in peri or intradomiciliary habitats - reason why little is known about this species. Therefore, Here, we present information for the first time on. the T. petrocchiae ecotopes, genetic diversity, Trypanosoma cruzi prevalence/genotyping in comparison to T. brasiliensis. We found T. brasilensis (N = 223) and T. petrocchiae (N = 69) in co-habitation in rocky outcrops in three Districts of Paraíba and Rio Grande do Norte states. Forty-tree T. petrocchiae insects of eleven sampling spots (composing three geographic populations) were genotyped for the mitochondrial Cyt B gene and little geographic structure was observed. Tajima's D test suggested that species is evolving toward a mutation-drift equilibrium in our collection range. Sylvatic T. petrocchiae had 4% (3/68) of infected insects by T. cruzi, whereas T. brasiliensis had 26% (59/223). Fluorescent Fragment Length Barcoding demonstrated that all three T. petrocchiae harbored TcI whereas T. brasiliensis had TcI, but also TcIII, TcII/TcVI and T. rangeli genotype A, sometimes under mixed infections. None of infected T. petrocchiae were carrying mixed infections. However, this result should be confirmed using a larger pool of infected bugs. We here presented the first documentation of T. rangeli infecting T. brasiliensis. The finding of infected T. petrocchiae calls for constant vector monitoring because the epidemiologic scenario is dynamic and sylvatic vectors are progressively found in adaptation to anthropic environments.

Molecular cytotaxonomy of the Triatoma brasiliensis species subcomplex (Hemiptera, Triatominae).

The Triatoma genus is paraphyletic, and its species are grouped into complexes and subcomplexes. Given the fact that species that make up a given subcomplex generally share chromosomal traits, we analyzed the distribution of AT- and CG-rich DNA of the T. brasiliensis species subcomplex, in order to establish affinities among members of the T. brasiliensis subcomplex based on chromatin and chromosome traits and develop an identification key for the four monophyletic Triatoma subcomplexes from South America. All species exhibited a CG-rich X sex chromosome and autosomes, as well as an AT-rich Y sex chromosome. This feature can be used as a diagnostic characteristic to determine whether a given species is a member of the T. brasiliensis subcomplex, because it enables the differentiation of these species from all Triatoma of South America. Thus, we confirmed the chromosomal relationship of the T. brasiliensis species subcomplex and developed a dichotomous key based on the chromocenter to differentiate the species from this subcomplex from the other monophyletic Triatoma subcomplexes from South America.

High Triatoma brasiliensis Densities and Trypanosoma cruzi Prevalence in Domestic and Peridomestic Habitats in the State of Rio Grande do Norte, Brazil: The Source for Chagas Disease Outbreaks?

AbstractA total of 2,431 Triatoma brasiliensis were collected from 39 populations of Paraíba (PB) and Rio Grande do Norte (RN) states, Brazil. In PB, Trypanosoma cruzi infection was not detected in either peridomestic or domestic vector populations. In contrast, in RN, T. brasiliensis was detected with high parasite prevalence in these ecotopes (30.7-40.0%). Moreover, peridomicile insect population densities were more than double the average densities of all other settings evaluated (19.17 versus < 8.94 triatomine/man-hour). Genotyped parasites evidenced a mix of T. cruzi lineages circulating in both peridomestic and sylvatic populations. Although vector control efforts have dramatically decreased Chagas disease transmission to humans, recent outbreaks have been detected in four municipalities of RN state. Our results clearly evidence a worrisome proximity between infected vectors and humans in RN. Indeed, finding of infected T. brasiliensis inside homes is routinely recorded by local vector control surveillance staff around the outbreak area, challenging the current and conventional view that vector transmissions are controlled in northeastern Brazil. This scenario calls for strengthening vector control surveillance and interventions to prevent further Chagas transmission, especially in RN State.

Combined phylogenetic and morphometric information to delimit and unify the Triatoma brasiliensis species complex and the Brasiliensis subcomplex.

"Triatoma brasiliensis species complex" was defined as a monophyletic group of the species: T. brasiliensis, T. juazeirensis, T. melanica, and T. sherlocki. An alternative grouping scheme proposed the concept of "Brasiliensis subcomplex" which included the former species together with T. melanocephala, T. petrocchiae, T. lenti, T. tibiamaculata, and T. vitticeps. To evaluate the relationship among these taxa we combined the results obtained with four mitochondrial genes (12S, 16S, COI and Cytb, adding to 1811bp) and geometric morphometric analysis of wings and heads. Panstrongylus megistus was included in the analysis as it was previously found related to T. tibiamaculata, T. melanocephala and T. vitticeps. The results of both molecular and morphometric approaches clearly grouped the species analyzed into two monophyletic units, supported by both genetic and wing variability. The first one (G1) comprises the four species originally included in the T. brasiliensis species complex plus T. lenti and T. petrocchiae. The second group (G2) was composed by T. melanocephala, T. tibiamaculata and T. vitticeps, and remarkably, P. megistus if considering wing variability and phylogenetic results. Nevertheless, geometric morphometrics of heads provided a quantitative measurement that discriminates Panstrongylus from the Triatoma species based on the position of the antennal insertion relative to eyes, as it is used as the generic distinctive character. The discrepancy among approaches questions the validity of this character to define Panstrongylus genus. Independently of the chosen group definition -"T. brasiliensis species complex" or "Brasiliensis subcomplex"-we propose to delimit it to species of G1 that are all associated with the Caatinga biome in the Brazilian Northeast. G2 are the ones associated with the Atlantic Forest biome.

Vector capacity of members of Triatoma brasiliensis species complex: The need to extend Chagas disease surveillance to Triatoma melanica.

We conducted a lab-based comparative study on vector capacity features of two species of triatomines: Triatoma brasiliensis and T. melanica. Both are members of the T. brasiliensis species complex. The former is the most important Chagas disease vector in the northeastern region of Brazil. To date, no transmission via T. melanica has been recorded. Immature insects exhibited distinct intermoult periods without a direct relationship to a given species. Females of T. brasiliensis consumed an average of 1.9 times more meals (mean = 12.92 vs 6.63) and survived for a shorter period (mean =330.8 days) than T. melanica (mean = 365.2 days), probably due to the cost of reproduction (all significant at P<0.05). These data support the idea that T. brasiliensis is more adapted to lab conditions and is more able to infest domiciles than T. melanica. We also found significant distinctions in other features between these species, such as the elapsed time without eating before molting, which was higher for the second, third, and fifth nymph stages of T. melanica. Regarding features analyzed related to vector capacity, insects of all life stages of both species were considered competent to transmit Trypanosoma cruzi because they needed many feedings (mean =1.5-3.82) to moult and because a high proportion (>39%) of insects defecated rapidly (<30 s) after feeding. Overall, results highlight the need to extend vector surveillance to T. melanica.

Triatoma rubrovaria (Blanchard, 1843) (Hemiptera-Reduviidae-Triatominae) IV: bionomic aspects on the vector capacity of nymphs.

Triatoma rubrovaria has become the most frequently captured triatomine species since the control of T. infestans in the state of Rio Grande do Sul (RS), Brazil. The aim of this study was to evaluate aspects of the vectorial competence of T. rubrovaria using nymphs raised in laboratory under environmental conditions of temperature and humidity and fed on mice. The average developmental period of T. rubrovaria was 180.1 days. The percentage of defecation shortly after feeding was still higher than previous studies in which samples of T. rubrovaria subjected to a slight starvation period before the blood meal were used. The obtained results support former indication that T. rubrovaria presents bionomic characteristics propitious to be a good vector of Trypanosoma cruzi to man. Therefore its domiciliary invasion process must be continuously monitored.

A Triatoma maculata (Hemiptera, Reduviidae, Triatominae) population from Roraima, Amazon region, Brazil, has some bionomic characteristics of a potential Chagas disease vector.

Even though Chagas disease is rare in the Brazilian Amazon, the conditions for the establishment of domiciliated cycles prevail in many areas where triatomines are of frequent occurrence. In Roraima, a previous serological and entomological survey in three agricultural settlements showed the existence of all transmission cycle elements, i.e., individuals infected by Trypanosoma cruzi, triatomine species previously found harboring T. cruzi in the broader Amazon region of neighboring countries and, domicile/ peridomicile conditions favorable to triatomine colonization. Triatoma maculata was the most frequent species, found in chicken houses in the peridomicile and sporadically within residences. Aiming to investigate the possibility of T. maculata to possess the potentiality to transmit T. cruzi in the area, bionomic characteristics were studied under laboratory conditions. These were feeding frequency, time for defecation after a blood meal, time elapsed in voluntary fasting pre- and pos-ecdysis, moulting time periods, pre-oviposition and oviposition periods and index of oviposition, incubation period, egg viability, longevity and mortality rate. Results show that the Passarão population of T. maculata should be considered a potential vector of T. cruzi since it shows a capacity to infest artificial ecotopes in the peridomicile, to carry out large number of meals during the nymphal cycle, to have a relatively short developmental cycle capable of producing 2.9 generations/year, to blood source eclecticism, to defecate immediately after the blood meal while still on the host and to the fact that has been previously found naturally infected by T. cruzi.

A Triatoma maculata ( Hemiptera, Reduviidae, Triatominae ) population from Roraima, Amazon region, Brazil, has some bionomic characteristics of a potential Chagas disease vector

A doença de Chagas é de rara ocorrência na Região Amazônica Brasileira, onde contudo as condições para o estabelecimento de ciclos domésticos existem. Um estudo previamente realizado em áreas de colonização agrícola no Estado de Roraima, mostrou a possibilidade de ciclos autóctones de transmissão virem a ocorrer uma vez que todos os elementos estavam lá presentes, indivíduos infectados por Trypanosoma cruzi, espécies de triatomíneos anteriormente descritas como infectadas por T. cruzi na Região Amazônica de países fronteiriços e, ambientes domiciliares e peri-domiciliares favoráveis à colonização de triatomíneos. Triatoma maculata foi a espécie mais frequentemente encontrada, tendo sido coletada em galinheiros no peridomicílio e esporadicamente nos domicílios. Visando investigar a potencialidade de T. maculata como espécie vetora na área, algumas características bionômicas foram estudadas em condições de laboratório incluindo freqüência de alimentação, tempo de defecação pós-prandial, tempo de jejum voluntário na pré- e na pós-ecdise, período inter-mudas, períodos de pré-oviposição e de oviposição, índice de oviposição, período de incubação, viabilidade dos ovos, índices de longevidade e de mortalidade. Os resultados mostraram que a população de T. maculata da Colônia Agrícola do Passarão deve ser considerada vetora em potencial do T. cruzi uma vez que mostrou capacidade de infestar ecótopos artificiais no peridomicílio, de se alimentar com freqüência durante o período ninfal, de possuir um ciclo de desenvolvimento relativamente curto com 2,9 gerações/ano, de possuir hábitos ecléticos de alimentação, de defecar imediatamente após a hematofagia quando ainda no hospedeiro e devido ao fato de ter sido previamente encontrada infectada por T. cruzi.

Triatoma rubrovaria (Blanchard, 1843) (Hemiptera - Reduviidae - Triatominae) IV: bionomic aspects on the vector capacity of nymphs

Triatoma rubrovaria has become the most frequently captured triatomine species since the control of T. infestans in the state of Rio Grande do Sul (RS), Brazil. The aim of this study was to evaluate aspects of the vectorial competence of T. rubrovaria using nymphs raised in laboratory under environmental conditions of temperature and humidity and fed on mice. The average developmental period of T. rubrovaria was 180.1 days. The percentage of defecation shortly after feeding was still higher than previous studies in which samples of T. rubrovaria subjected to a slight starvation period before the blood meal were used. The obtained results support former indication that T. rubrovaria presents bionomic characteristics propitious to be a good vector of Trypanosoma cruzi to man. Therefore its domiciliary invasion process must be continuously monitored.