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Abstract The chemical control of Triatoma infestans , the major Chagas disease vector in southern South America, has been threatened in the last years by the emergence of pyrethroid-resistant bug populations. As an alternative approach, the efficacy of the entomopathogenic fungus Beauveria bassiana to control T. infestans populations (regardless their pyrethroid susceptibility) has been demonstrated. Growing research efforts on the interaction between T. infestans and B. bassiana by molecular, ecological, biochemical and behavioral traits has allowed framing such interaction as an evolutionary arms race. This review will focus on the relationships established in this particular host-pathogen system, compiling available data on the relevance of fungal pathogenesis, insect behavior, population dynamics and human intervention to favor fungal dissemination in bug populations. The current snapshot shows the fungus ahead in the evolutionary arms race and predicts a promissory landscape for the biological control of Chagas disease vectors. Highlights • The interaction between triatomines and their fungal pathogens is reviewed. • Some mechanisms involved in fungal attack and insect defense are identified. • Such interaction can be framed within an evolutionary arms race. • The fungus seems to be ahead since it overcome whatever barrier its host interposes. • B. bassiana is an effective tool to control pyrethroid-resistant T. infestans. [ABSTRACT FROM AUTHOR]

• We evaluated the toxicity of binary mixtures of permethrin and a natural monoterpene in Triatoma infestans. • The monoterpenes eugenol and menthol synergized the speed of action of permethrin. • Menthyl acetate did not modify the speed of action of permethrin. • Identification of new synergists could help to reduce the use of conventional insecticides. The blood-sucking bug , Triatoma infestans, is one of the main vectors of Chagas disease in America. It is usually controlled with pyrethroids, but the emergence of resistance to these insecticides creates the need to look for alternative products. Eugenol, menthol and menthyl acetate are botanical monoterpenes, which produce lethal and sublethal effects on insects. The purpose of this work was to determine what type of toxicological interactions occur when binary mixtures, formed by the pyrethroid permethrin and sublehtal doses of eugenol, menthol or menthyl acetate, are applied to T. infestans. First instar nymphs were exposed to filter papers impregnated with the insecticides. The number of knocked down insects was registered at different times and Knock Down Time 50% (KT 50) values were calculated. The following KT 50 values with their corresponding 95% Confidence Intervals were obtained: permethrin, 47.29 (39.92 - 56.32) min; permethrin + eugenol, 34.08 (29.60 - 39.01) min; permethrin + menthol, 27.54 (23.28 - 32.55) min; permethrin + menthyl acetate, 43.62 (39.99 - 47.59) min. Eugenol and menthol increased the speed of action of permethrin (synergism), but menthyl acetate had no effect on it (additivity). These results provide the basis to further explore interactions between conventional insecticides and plant monoterpenes as potential tools for controlling T. infestans. [Display omitted] [ABSTRACT FROM AUTHOR]

Rhodnius prolixus (Hemiptera: Reduviidae) is a hematophagous insect native from South America. By the end of the 20th century, it was one of the main vectors of Chagas disease in Venezuela, Colombia, several Central American countries and southern Mexico. The aim of the present article is to review the literature regarding R. prolixus toxicology. British entomologist Vincent B. Wigglesworth carried out the first studies on this subject over seventy years ago. A wide bibliographical search allowed to locate one hundred and thirty scientific articles describing the effects of different insecticides on R. prolixus . About one-third of these articles report the acute toxicity and/or sublethal effects produced by the main synthetic neurotoxic families of insecticides (organochlorines, organophosphates, carbamates and pyrethroids). Only a couple of these studies have regarded the toxicokinetics or toxicodynamics of these insecticides. Insect growth or development disruptors, such as juvenoids, chitin synthesis inhibitors, precocenes, azadirachtin and lignoids, have been thoroughly studied in R. prolixus . Important aspects on the mode of action of ureases were also described in this species. By the end of the 1960’s, resistance to insecticides was detected in R. prolixus from Venezuela. Some years later, the existence of pyrethroid-resistant individuals was also reported. Control programmes for R. prolixus in countries where Chagas is endemic have only used synthetic neurotoxic insecticides. In 2011, Central America and southern Mexico were declared free of this insect. The recent sequencing of the R. prolixus genome will provide valuable information to understand the molecular basis of insecticide resistance in this species. [ABSTRACT FROM AUTHOR]

The hemipteran subfamily Triatominae includes 150 blood-sucking species, vectors of Chagas disease. By far the most specious genus is Triatoma , assembled in groups, complexes and subcomplexes based on morphological similarities, geographic distribution and genetic data. However, many molecular studies questioned the species integration of several subcomplexes as monophyletic units. In triatomines, chromosomal position of major ribosomal DNA (rDNA) loci is extremely variable but seems to be species-specific and an evolutionary conserved genetic trait, so that closely related species tend to have ribosomal clusters in the same chromosomal location. Considering that the autosomal position as the ancestral character for all heteropteran species, including triatomines, we suggest that the movement of rDNA loci from autosomes to sex chromosomes rapidly established reproductive barriers between divergent lineages. We proposed that the rDNA translocation from the autosomes to the sex chromosomes restrict reproductive compatibility and eventually promote speciation processes. We analyzed the chromosomal position of 45S rDNA clusters in almost all species of the matogrossensis, rubrovaria, maculata and sordida subcomplexes. The fluorescent in situ hybridization results are discussed considering the available genetic data and we proposed new arrangements in the species that constitute each one of these subcomplexes. [ABSTRACT FROM AUTHOR]

High Throughput Sequencing capabilities have made the process of assembling a transcriptome easier, whether or not there is a reference genome. But the quality of a transcriptome assembly must be good enough to capture the most comprehensive catalog of transcripts and their variations, and to carry out further experiments on transcriptomics. There is currently no consensus on which of the many sequencing technologies and assembly tools are the most effective. Many non-model organisms lack a reference genome to guide the transcriptome assembly. One question, therefore, is whether or not a reference-based genome assembly gives better results than de novo assembly. The blood-sucking insect Rhodnius prolixus —a vector for Chagas disease—has a reference genome. It is therefore a good model on which to compare reference-based and de novo transcriptome assemblies. In this study, we compared de novo and reference-based genome assembly strategies using three datasets (454, Illumina, 454 combined with Illumina) and various assembly software. We developed criteria to compare the resulting assemblies: the size distribution and number of transcripts, the proportion of potentially chimeric transcripts, how complete the assembly was (completeness evaluated both through CEGMA software and R. prolixus proteome fraction retrieved). Moreover, we looked for the presence of two chemosensory gene families (Odorant-Binding Proteins and Chemosensory Proteins) to validate the assembly quality. The reference-based assemblies after genome annotation were clearly better than those generated using de novo strategies alone. Reference-based strategies revealed new transcripts, including new isoforms unpredicted by automatic genome annotation. However, a combination of both de novo and reference-based strategies gave the best result, and allowed us to assemble fragmented transcripts. [ABSTRACT FROM AUTHOR]

The thermal sense of triatomine bugs, vectors of Chagas disease, is unique among insects. Not only do these bugs exhibit the highest sensitivity to heat known in any animal up to date, but they can also perceive the infrared radiation emitted by the body of their warm-blooded hosts. The sensory basis of this capacity has just started to be unravelled. To shed additional light on our understanding of thermosensation, we initiated an analysis of the genetic basis of the thermal sense in Rhodnius prolixus . We tested the hypothesis that a TRPV (transient receptor potential vanilloid) channel receptor is involved in the evaluation of heat in this species. Two different approaches were adopted. Initially, we analysed the expression of a TRPV candidate for this function, i.e., RproIav , in different tissues. Subsequently, we tested the effects of capsaicin and capsazepine, two molecules known to interact with mammal TRPV1, using three different behavioural protocols for evaluating thermal responses: (1) proboscis extension response (PER), (2) thermopreference in a temperature gradient and (3) spatial learning in an operant conditioning context. Bioinformatic analyses confirmed that the characteristic features typical of the TRPV channel subfamily are found in the RproIav protein sequence. Molecular analysis showed that RproIav is expressed in R. prolixus , not only in the antennae, but also in other body structures bearing sensory organs. Behavioural experiments consistently revealed that capsaicin treated insects are less responsive to heat stimuli and prefer lower temperatures than non-treated insects, and that they fail to orient in space. Conversely, capsazepine induces the opposite behaviours. The latter data suggest that triatomine thermoreception is based on the activation of a TRP channel, with a similar mechanism to that described for mammal TRPV1. The expression of RproIav in diverse sensory structures suggests that this receptor channel is potentially involved in bug thermoreception. This constitutes solid evidence that thermosensation could be based on the activation of TRP receptors that are expressed in different tissues in R. prolixus . Whether RproIav channel is a potential target for the compounds tested and whether it mediates the observed effects on behaviour still deserves to be confirmed by further research. [ABSTRACT FROM AUTHOR]

Abstract: This paper documents the morphology of previously undescribed adult reproductive accessory glands in eight species of blood-feeding Hemiptera, vectors of Chagas disease. These species are three Triatoma (T. dimidiata, T. klugi, T. sordida), three Rhodnius (R. brethesi, R. nasutus, R. pictipes), and one species each from Nesotriatoma (N. bruneri) and Panstrongylus (P. megistus). This survey shows that the male reproductive systems between species of four genera of Reduviidae adhere to the same general plan seen in previously described vectors of Chagas disease. This morphological similarity suggests that reproductive success of the male is contingent on the delivery of a vital set of male accessory gland secretions to the female in conjunction with material from the testes and seminal vesicle. However, variations were observed in the accessory glands of females, especially at the level of the genus. The spermathecae are morphologically distinct, and the posterior accessory glands are absent in some. The differences in spermathecae morphology likely reflect physiological adaptations associated with speciation driven by cryptic female choice in which the female determines which sperm are used for fertilization. Differences in the posterior reproductive accessory gland can be correlated with variations in ovipositioning behaviour. Since reproductive physiology is important for species success, this information also augments epidemiological studies by providing a comparison to R. prolixus, a Chagas disease vector for which the physiology is well known. [Copyright &y& Elsevier]

Abstract: The adaptation to light of compound eyes in insects has been extensively documented and their adaptive role is well understood. Much less attention has been paid, however, to the control of ocelli sensitivity, a study which could help us to understand the functional role of these simple eyes. We analyzed the dynamic changes in the distribution of screening pigments which occur in the ocelli of the haematophagous bug, Triatoma infestans, when the insects are subjected either to light/dark cycles (LD), to constant darkness (DD) or constant light (LL). We then compared these changes with those occurring in the compound eyes of the same individuals and found that, while compound eyes are subject to the control of an endogenous circadian clock, the adaptation of the ocelli is entirely dependent on environmental illumination. In addition, we have observed that environmental temperature is not involved in the control of screening pigments in either ocelli or compound eyes as a direct stimulus, nor as a Zeitgeber. The existence of a differential control in the components of the dual visual system represents an adaptive advantage in the adjustment of visual sensitivity in insects exposed to quick changes in lighting conditions in their natural habitat. We discuss the implications of our findings with regards to the biology of triatomines and with respect to the general understanding the functional role of insect ocelli. [Copyright &y& Elsevier]

Abstract: Heat is the principal host-associated cue for the blood-sucking bug Rhodnius prolixus. It is both necessary and sufficient to trigger the “proboscis extension response” (PER), an essential element of the feeding behaviour of this insect. The aim of this study was to determine whether the temperature of an object itself or the thermal contrast between the object and the environmental background is responsible for triggering the PER. Thermal stimuli at 25–50°C were presented in thermal environments of 20–40°C. The results showed that stimuli at 30 and 35°C trigger the highest rates of response, provided that they were presented in thermal backgrounds at temperatures of below 35°C. Thus, bugs display a preference for objects at temperatures corresponding to those at the surface of their vertebrate hosts (birds and mammals). However, this preference disappears if no heat exchange between the bug and its potential host is possible (i.e. if they are at the same temperature) and may even become negative if the insect and the surrounding environment are at a temperature above 35°C). In these situations, and when the object was too warm to be a potential host, PER rates were much lower. These findings have potential implications for the feeding strategies adopted by triatomine bugs in the natural tropical areas they inhabit. [Copyright &y& Elsevier]

Abstract: All known significant insect vectors of Trypanosoma cruzi are members of the Reduviidae, subfamily Triatominae. Infections with this parasite are the cause of Chagas disease, the single most costly parasitic disease in the Western Hemisphere. The Triatominae are almost completely restricted to the Americas, with >130 species distributed in several foci of species richness and endemism; nevertheless, the processes involved in the diversification of this group remain poorly understood. The Triatoma brasiliensis species complex was recently proposed based on geography, morphology, ecology, and molecular data, and is believed to comprise two species and two subspecies. Here, we report results from a broad series of studies, in which first-generation offspring of experimental crosses were studied in terms of wing morphometry and phylogenetic position. Morphometrics, morphological, ecological and geographic analyses were consistent with the hypothesis of T. brasiliensis macromelasoma as a product of hybridization between two others (T. brasiliensis brasiliensis and T. juazeirensis). Although evidence is supportive of the hypothesis of speciation via hybridization as a mode of triatomine diversification, the case is not as-yet conclusive, and confirmation via molecular markers is necessary. [Copyright &y& Elsevier]

Panstrongylus species are widely distributed throughout the Americas, where they act as vectors of Trypanosoma cruzi, agent of Chagas disease. Their intraspecific relationships, taxonomic position and phylogeny in relation to other Triatomini were explored using ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS-2) sequence polymorphisms and maximum parsimony, distance and maximum likelihood analyses of 10 populations representing six species of the genus (P. megistus, P. geniculatus, P. rufotuberculatus, P. lignarius, P. herreri and P. chinai). At the subspecific level, P. megistus appeared more homogeneous than P. rufotuberculatus and P. geniculatus (both with broader distribution). Several dinucleotide microsatellites were detected in the sequences of given species. Many of these microsatellites (GC, TA, GT and AT) showed different number of repeats in different populations and thus, may be very useful for population differentiation and dynamics analyses in future studies. The sequences of P. lignarius (considered sylvatic) and P. herreri (a major disease vector in Peru) were identical, suggesting that these species should be synonymised. Intrageneric analysis showed a clear separation of P. rufotuberculatus, with closest relationships between P. geniculatus and P. chinai, and P. megistus occupying a separate branch. Genetic distances between Panstrongylus species (0.11585–0.22131) were higher than those between Panstrongylus and other Triatomini (16 species from central and North America and South America) (0.08617–0.11039). The distance between P. megistus and P. lignarius/herreri (0.22131) was the largest so far recorded in the tribe. The pronounced differences in length and nucleotide composition suggest a relatively old divergence of Panstrongylus species. P. rufotuberculatus was closer to Mesoamerican Triatoma, Meccus and Dipetalogaster species than to other Panstrongylus. All Panstrongylus clustered with the Mesoamerican clade; P. rufotuberculatus clustered with the phyllosoma complex and T. dimidiata, with D. maxima and T. barberi in a basal position. The rest of Panstrongylus appeared paraphyletically in the tree. This is evidence suggesting polyphyly within the genus Panstrongylus, whose species may be related to the ancestors giving rise to central and North American Triatomini. [Copyright &y& Elsevier]

Triatoma longipennis (Usinger) is an important vector of Trypanosoma cruzi in western, central and northern Mexico, due to its wide distribution, high infection rates, and epidemiological indices. However, its population genetics has not been completely characterized. In this study, the intra-specific relationships between different T. longipennis populations were analyzed from seven states in Mexico using mitochondrial cyt B as a marker. Our results show that the population of Jalisco was the most diverse, with the highest genetic and haplotypic variation (Hd = 0.978, π = 0.099 and θ = 0.079), even 25 times higher than some other populations analyzed. Heterogeneous migration and gene flow were observed without relation to their geographical distribution, that is, nearby populations may present high values of gene flow with low migration. In contrast, remote populations have low gene flow values with high migration. Genetic isolation was apparently present in the Guanajuato population, however, Mantel's analyzes to determine when an isolation by distance is present did not show correlation between genetic (F ST) and geographic (Km) distances (P = 0.064). The STRUCTURE analyzes showed that populations such as Chihuahua, Jalisco, Nayarit and Michoacán appear to show a similar population structure, suggesting a common ancestor. Our results suggest two routes of diversification of T. longipennis highly influenced by anthropogenic effects. Elucidation of the population genetic structure of T. longipennis will help to better understand the role of gene flow and migration in the dispersal of this important Chagas disease vector. [Display omitted] • The genetic variation between different M. longipennis populations was analyzed. • It was suggested as center of dispersion and diversification the Jalisco population. • Some populations, such as Guanajuato showed genetic isolation. • Role of gene flow and migration on dispersal of this species is provided. [ABSTRACT FROM AUTHOR]

• Phylogeny of the T. sordida subcomplex is presented. • Morphometric and molecular data confirm the systematic status T. sordida subcomplex. • Triatoma guasayana, T. patagonica displaced to T. rubrovaria subcomplex. • Triatoma sordida from Argentina, Bolivia, Brazil. Chagas disease, whose etiological agent is the protozoan Trypanosoma cruzi , affects millions of people worldwide and it is mainly transmitted by infected triatomine feces. Triatoma is the most diverse genus and one of the most important from an epidemiological point of view. Species of this genus are grouped into eight complexes and nine subcomplexes. Triatoma sordida subcomplex consists of T. garciabesi, T. guasayana, T. patagonica , and, T. sordida. Given the recent discussions on their phylogenetic status, this study aims to evaluate morphometric and genetics characters that group and distinguish T. garciabesi, T. guasayana, T. patagonica , and T. sordida , as well as to infer the phylogenetic relationships within the group and evaluate the proximity with T. rubrovaria subcomplex. The results corroborate the phylogenetic relationship of T. guasayana and T. patagonica with the T. rubrovaria subcomplex. Molecular data confirm the proximity of T. jurbergi, T. matogrossensis, T. vandae as T. garciabesi, and T. sordida. Together, genetic variability was shown between T. sordida populations. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Chagas disease is caused by the Trypanosoma cruzi and transmitted mainly by triatomines. Triatoma is a paraphyletic group and the species of this genus are grouped into complexes and subcomplexes. Morphological data and geographical distribution grouped initially T. melanocephala , T. vitticeps and T. tibiamaculata in the T. brasiliensis subcomplex. However, karyotypic and phylogenetic analysis suggested the exclusion of T. melanocephala , T. vitticeps and T. tibiamaculata from this subcomplex. Considering that studies of experimental crosses can help to understand the systematics of species, we performed experimental crosses between T. melanocephala , T. vitticeps and T. tibiamaculata with T. b. brasiliensis. No crosses resulted in hybrids. Taking into account that the species of the T. brasiliensis subcomplex do not present interspecific prezygotic barriers, the characterization of reproductive barriers shows that T. melanocephala , T. vitticeps and T. tibiamaculata do not present an evolutionary proximity relationship with the species of this subcomplex. Thus, we confirmed the exclusion of these species from the T. brasiliensis subcomplex and we emphasize the importance of experimental crosses for evolutionary studies. • We detected reproductive isolation between T. melanocephala and T. b. brasiliensis. • We detected reproductive isolation between T. vitticeps and T. b. brasiliensis. • We detected reproductive isolation between T. tibiamaculata and T. b. brasiliensis. • We confirm the exclusion of these three species from the T. brasiliensis subcomplex. [ABSTRACT FROM AUTHOR]

• We analyzed the distribution of AT- and CG-rich DNA of the Triatoma brasiliensis species subcomplex. • All species exhibited a CG-rich X sex chromosome and autosomes, as well as an AT-rich Y sex chromosome. • We developed a dichotomous key based on the chromocenter to differentiate the species from this subcomplex from the other monophyletic Triatoma subcomplexes from South America. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]