Trypanosoma cruzi infection follow-up in a sylvatic vector of Chagas disease: Comparing early and late stage nymphs.

Chagas disease is caused by Trypanosoma cruzi and transmitted by the triatomine Mepraia spinolai in the southwest of South America. Here, we examined the T. cruzi-infection dynamics of field-caught M. spinolai after laboratory feeding, with a follow-up procedure on bug populations collected in winter and spring of 2017 and 2018. Bugs were analyzed twice to evaluate T. cruzi-infection by PCR assays of urine/fecal samples, the first evaluation right after collection and the second 40 days after the first feeding. We detected bugs with: the first sample positive and second negative (+/-), the first sample negative and second positive (-/+), and with both samples positive or negative (+/+; -/-). Bugs that resulted positive on both occasions were the most frequent, with the exception of those collected in winter 2018. Infection rate in spring was higher than winter only in 2018. Early and late stage nymphs presented similar T. cruzi-infection rates except for winter 2017; therefore, all nymphs may contribute to T. cruzi-transmission to humans. Assessment of infection using two samples represents a realistic way to determine the infection a triatomine can harbor. The underlying mechanism may be that some bugs do not excrete parasites unless they are fed and maintained for some time under environmentally controlled conditions before releasing T. cruzi, which persists in the vector hindgut. We suggest that T. cruzi-infection dynamics regarding the three types of positive-PCR results detected by follow-up represent: residual T. cruzi in the rectal lumen (+/-), colonization of parasites attached to the rectal wall (-/+), and presence of both kinds of flagellates in the hindgut of triatomines (+/+). We suggest residual T. cruzi-infections are released after feeding, and result 60-90 days after infection persisting in the rectal lumen after a fasting event, a phenomenon that might vary between contrasting seasons and years.

TriatoScore: an entomological-risk score for Chagas disease vector control-surveillance.

BACKGROUND: Triatomine bugs transmit Chagas disease across Latin America, where vector control-surveillance is increasingly decentralized. Locally run systems often deal with highly diverse native-vector faunas-plus, in some areas, domestic populations of non-native species. Flexible entomological-risk indicators that cover native and non-native vectors and can support local decision-making are therefore needed. METHODS: We present a local-scale entomological-risk score ("TriatoScore") that leverages and builds upon information on the ecology-behavior and distribution-biogeography of individual triatomine bug species. We illustrate our approach by calculating TriatoScores for the 417 municipalities of Bahia state, Brazil. For this, we (i) listed all triatomine bug species recorded statewide; (ii) derived a "species relevance score" reflecting whether each species is native/non-native and, if native, whether/how often it invades/colonizes dwellings; (iii) mapped each species' presence by municipality; (iv) for native vectors, weighted presence by the proportion of municipal territory within ecoregions occupied by each species; (v) multiplied "species relevance score" × "weighted presence" to get species-specific "weighted scores"; and (vi) summed "weighted scores" across species to get municipal TriatoScores. Using standardized TriatoScores, we then grouped municipalities into high/moderate/low entomological-risk strata. RESULTS: TriatoScores were higher in municipalities dominated by dry-to-semiarid ecoregions than in those dominated by savanna-grassland or, especially, moist-forest ecoregions. Bahia's native triatomines can maintain high to moderate risk of vector-borne Chagas disease in 318 (76.3%) municipalities. Historical elimination of Triatoma infestans from 125 municipalities reduced TriatoScores by ~ 27% (range, 20-44%); eight municipalities reported T. infestans since Bahia was certified free of Trypanosoma cruzi transmission by this non-native species. Entomological-risk strata based on TriatoScores agreed well with Bahia's official disease-risk strata, but TriatoScores suggest that the official classification likely underestimates risk in 42 municipalities. Of 152 municipalities failing to report triatomines in 2006-2019, two and 71 had TriatoScores corresponding to, respectively, high and moderate entomological risk. CONCLUSIONS: TriatoScore can help control-surveillance managers to flexibly assess and stratify the entomological risk of Chagas disease at operationally relevant scales. Integrating eco-epidemiological, demographic, socioeconomic, or operational data (on, e.g., local-scale dwelling-infestation or vector-infection frequencies, land-use change and urbanization, housing conditions, poverty, or the functioning of control-surveillance systems) is also straightforward. TriatoScore may thus become a useful addition to the triatomine bug control-surveillance toolbox.

Effect of habitat fragmentation on rural house invasion by sylvatic triatomines: A multiple landscape-scale approach.

After the decrease of the relative importance of Triatoma infestans, a number of studies reported the occurrence of sylvatic triatomines dispersing actively to domestic environments in the dry western Chaco Region of Argentina. Anthropic modification of the landscape is mentioned as one of the main causes of the increase in domicile invasion. The aim of this study was to describe the occurrence and frequency of sylvatic triatomines invading rural houses, and to evaluate the effect of habitat fragmentation and other ecological factors on the invasion of rural houses in central Argentina. We hypothesized that the decrease in food sources and the loss of wild ecotopes, as a consequence of habitat fragmentation, increase the chances of invasion by triatomines. The entomological data was collected by community-based vector surveillance during fieldwork carried out between 2017-2020, over 131 houses located in fourteen rural communities in the northwest of Córdoba Province (central Argentina). We used generalized linear models to evaluate the effect of (i) the environmental anthropic disturbance in the study area, (ii) the composition and configuration of the landscape surrounding the house, (iii) the spatial arrangement of houses, (iv) and the availability of artificial refuges and domestic animals in the peridomicile, on house invasion by triatomines. We report the occurrence of seven species of triatomines invading rural houses in the study area -T. infestans, T. guasayana, T. garciabesi, T. platensis, T. delpontei, T. breyeri and P. guentheri-. Study data suggest that invasion by triatomines occurs with higher frequency in disturbed landscapes, with houses spatially isolated and in proximity to subdivided fragments of forest. The availability of domestic refuges in the peridomestic structures as well as the presence of a higher number of domestic animals increase the chances of invasion by triatomines.

Trends in evolution of the Rhodniini tribe (Hemiptera, Triatominae): experimental crosses between Psammolestes tertius Lent & Jurberg, 1965 and P. coreodes Bergroth, 1911 and analysis of the reproductive isolating mechanisms.

BACKGROUND: The tribe Rhodniini is a monophyletic group composed of 24 species grouped into two genera: Rhodnius and Psammolestes. The genus Psammolestes includes only three species, namely P. coreodes, P. tertius and P. arthuri. Natural hybridization events have been reported for the Rhodniini tribe (for genus Rhodnius specifically). Information obtained from hybridization studies can improve our understanding of the taxonomy and systematics of species. Here we report the results from experimental crosses performed between P. tertius and P. coreodes and from subsequent analyses of the reproductive and morphological aspects of the hybrids. METHODS: Crossing experiments were conducted between P. tertius and P. coreodes to evaluate the pre- and post-zygotic barriers between species of the Rhodniini tribe. We also performed cytogenetic analyses of the F1 hybrids, with a focus on the degree of pairing between the homeologous chromosomes, and morphology studies of the male gonads to evaluate the presence of gonadal dysgenesis. Lastly, we analyzed the segregation of phenotypic characteristics. RESULTS: Interspecific experimental crosses demonstrated intrageneric genomic compatibility since hybrids were produced in both directions. However, these hybrids showed a high mortality rate, suggesting a post-zygotic barrier resulting in hybrid unviability. The F1 hybrids that reached adulthood presented the dominant phenotypic segregation pattern for P. tertius in both directions. These insects were then intercrossed; the hybrids were used in the cross between P. tertius ♀ × P. coreodes ♂ died before oviposition, and the F1 hybrids of P. coreodes ♀ x P. tertius ♂ oviposited and their F2 hybrids hatched (however, all specimens died after hatching, still in first-generation nymph stage, pointing to a hybrid collapse event). Morphological analyses of male gonads from F1 hybrids showed that they did not have gonadal dysgenesis. Cytogenetic analyses of these triatomines showed that there were metaphases with 100% pairing between homeologous chromosomes and metaphases with pairing errors. CONCLUSION: The results of this study demonstrate that Psammolestes spp. have intrageneric genomic compatibility and that post-zygotic barriers, namely unviability of hybrid and hybrid collapse, resulted in the breakdown of the hybrids of P. tertius and P. coreodes, confirming the specific status of species based on the biological concept of species.

Trends in taxonomy of Triatomini (Hemiptera, Reduviidae, Triatominae): reproductive compatibility reinforces the synonymization of Meccus Stål, 1859 with Triatoma Laporte, 1832.

BACKGROUND: Meccus' taxonomy has been quite complex since the first species of this genus was described by Burmeister in 1835 as Conorhinus phyllosoma. In 1859 the species was transferred to the genus Meccus and in 1930 to Triatoma. However, in the twentieth century, the Meccus genus was revalidated (alteration corroborated by molecular studies) and, in the twenty-first century, through a comprehensive study including more sophisticated phylogenetic reconstruction methods, Meccus was again synonymous with Triatoma. Events of natural hybridization with production of fertile offspring have already been reported among sympatric species of the T. phyllosoma subcomplex, and experimental crosses demonstrated reproductive viability among practically all species of the T. phyllosoma subcomplex that were considered as belonging to the genus Meccus, as well as between these species and species of Triatoma. Based on the above, we carried out experimental crosses between T. longipennis (considered M. longipennis in some literature) and T. mopan (always considered as belonging to Triatoma) to evaluate the reproductive compatibility between species of the T. phyllosoma complex. In addition, we have grouped our results with information from the literature regarding crosses between species that were grouped in the genus Meccus with Triatoma, in order to discuss the importance of experimental crosses to confirm the generic reorganization of species. RESULTS: The crosses between T. mopan female and T. longipennis male resulted in viable offspring. The hatching of hybrids, even if only in one direction and/or at low frequency, demonstrates reproductive compatibility and homeology between the genomes of the parents. CONCLUSION: Considering that intergeneric crosses usually do not result in viable offspring in Triatominae, the reproductive compatibility observed between the T. phyllosoma subcomplex species considered in the Meccus genus with species of the Triatoma genus shows that there is "intergeneric" genomic compatibility, which corroborates the generic reorganization of Meccus in Triatoma.

Under pressure: phenotypic divergence and convergence associated with microhabitat adaptations in Triatominae.

BACKGROUND: Triatomine bugs, the vectors of Chagas disease, associate with vertebrate hosts in highly diverse ecotopes. It has been proposed that occupation of new microhabitats may trigger selection for distinct phenotypic variants in these blood-sucking bugs. Although understanding phenotypic variation is key to the study of adaptive evolution and central to phenotype-based taxonomy, the drivers of phenotypic change and diversity in triatomines remain poorly understood. METHODS/RESULTS: We combined a detailed phenotypic appraisal (including morphology and morphometrics) with mitochondrial cytb and nuclear ITS2 DNA sequence analyses to study Rhodnius ecuadoriensis populations from across the species' range. We found three major, naked-eye phenotypic variants. Southern-Andean bugs primarily from vertebrate-nest microhabitats (Ecuador/Peru) are typical, light-colored, small bugs with short heads/wings. Northern-Andean bugs from wet-forest palms (Ecuador) are dark, large bugs with long heads/wings. Finally, northern-lowland bugs primarily from dry-forest palms (Ecuador) are light-colored and medium-sized. Wing and (size-free) head shapes are similar across Ecuadorian populations, regardless of habitat or phenotype, but distinct in Peruvian bugs. Bayesian phylogenetic and multispecies-coalescent DNA sequence analyses strongly suggest that Ecuadorian and Peruvian populations are two independently evolving lineages, with little within-lineage phylogeographic structuring or differentiation. CONCLUSIONS: We report sharp naked-eye phenotypic divergence of genetically similar Ecuadorian R. ecuadoriensis (nest-dwelling southern-Andean vs palm-dwelling northern bugs; and palm-dwelling Andean vs lowland), and sharp naked-eye phenotypic similarity of typical, yet genetically distinct, southern-Andean bugs primarily from vertebrate-nest (but not palm) microhabitats. This remarkable phenotypic diversity within a single nominal species likely stems from microhabitat adaptations possibly involving predator-driven selection (yielding substrate-matching camouflage coloration) and a shift from palm-crown to vertebrate-nest microhabitats (yielding smaller bodies and shorter and stouter heads). These findings shed new light on the origins of phenotypic diversity in triatomines, warn against excess reliance on phenotype-based triatomine-bug taxonomy, and confirm the Triatominae as an informative model system for the study of phenotypic change under ecological pressure .

Machine-learning model led design to experimentally test species thermal limits: The case of kissing bugs (Triatominae).

Species Distribution Modelling (SDM) determines habitat suitability of a species across geographic areas using macro-climatic variables; however, micro-habitats can buffer or exacerbate the influence of macro-climatic variables, requiring links between physiology and species persistence. Experimental approaches linking species physiology to micro-climate are complex, time consuming and expensive. E.g., what combination of exposure time and temperature is important for a species thermal tolerance is difficult to judge a priori. We tackled this problem using an active learning approach that utilized machine learning methods to guide thermal tolerance experimental design for three kissing-bug species: Triatoma infestans, Rhodnius prolixus, and Panstrongylus megistus (Hemiptera: Reduviidae: Triatominae), vectors of the parasite causing Chagas disease. As with other pathogen vectors, triatomines are well known to utilize micro-habitats and the associated shift in microclimate to enhance survival. Using a limited literature-collected dataset, our approach showed that temperature followed by exposure time were the strongest predictors of mortality; species played a minor role, and life stage was the least important. Further, we identified complex but biologically plausible nonlinear interactions between temperature and exposure time in shaping mortality, together setting the potential thermal limits of triatomines. The results from this data led to the design of new experiments with laboratory results that produced novel insights of the effects of temperature and exposure for the triatomines. These results, in turn, can be used to better model micro-climatic envelope for the species. Here we demonstrate the power of an active learning approach to explore experimental space to design laboratory studies testing species thermal limits. Our analytical pipeline can be easily adapted to other systems and we provide code to allow practitioners to perform similar analyses. Not only does our approach have the potential to save time and money: it can also increase our understanding of the links between species physiology and climate, a topic of increasing ecological importance.

Juvenile hormone mediates lipid storage in the oocytes of Dipetalogaster maxima.

Triatomines are vectors of Chagas disease and important model organisms in insect physiology. "Kissing bugs" are obligatory hematophagous insects. A blood meal is required to successfully complete oogenesis, a process primarily controlled by juvenile hormone (JH). We used Dipetalogaster maxima as an experimental model to further understand the roles of JH in the regulation of vitellogenesis and oogenesis. A particular focus was set on the role of JH controlling lipid and protein recruitment by the oocytes. The hemolymph titer of JH III skipped bisepoxide increased after a blood meal. Following a blood meal there were increased levels of mRNAs in the fat body for the yolk protein precursors, vitellogenin (Vg) and lipophorin (Lp), as well as of their protein products in the hemolymph; mRNAs of the Vg and Lp receptors (VgR and LpR) were concomitantly up-regulated in the ovaries. Topical administration of JH induced the expression of Lp/LpR and Vg/VgR genes, and prompted the uptake of Lp and Vg in pre-vitellogenic females. Knockdown of the expression of LpR by RNA interference in fed females did not impair the Lp-mediated lipid transfer to oocytes, suggesting that the bulk of lipid acquisition by oocytes occurred by other pathways rather than by the endocytic Lp/LpR pathway. In conclusion, our results strongly suggest that JH signaling is critical for lipid storage in oocytes, by regulating Vg and Lp gene expression in the fat body as well as by modulating the expression of LpR and VgR genes in ovaries.

Structure and function of the stylets of hematophagous Triatominae (Hemiptera: Reduviidae), with special reference to Dipetalogaster maxima.

Kissing bugs (Hemiptera: Reduviidae: Triatominae) are able to bend their rod-like maxillae while searching for blood vessels in the tissue of their vertebrate hosts. Little is known about the working mechanisms of these bending movements and the distal opening of the food channel. We compared the morphological structure of the stylets (mandibles and maxillae) of four triatomine species and analyzed the feeding process of Dipetalogaster maxima (Uhler, 1894). The maxillae of triatomine bugs are interlocked by a tongue-and-groove system, allowing longitudinal sliding. While penetrating the host tissue, the animals perform rapid alternate back and forth movements of the maxillae. The resistance of the surrounding tissue pushes the asymmetric apex of the maxillae away from its straight path, i.e., if one individual maxilla is protracted alone, its tip curves inwards, and the other maxilla follows. Once a blood vessel is tapped, the spine-like tip of the left maxilla splays outwards. Apically, each of the maxillae features an abutment, the left one exhibiting a notch that presumably facilitates splaying. The mechanical interaction of the two maxillary abutments enables the distal opening of the food channel but might also support the movements of the maxillary bundle attributable to different bending moment distributions.

Behavioral response mediated by feces in Triatoma infestans (Hemiptera: Reduviidae: triatominae) susceptible and resistant to deltamethrin.

Chemical cues from feces promote aggregation behavior in Triatoma infestans nymphs and adults. Given the importance of T. infestans resistant to pyrethroids in several areas of Argentina and Bolivia, it would be important to know if there is an association with specific attraction and aggregation behaviors. These behaviors, to and surrounding refuges, play an important role in triatomine population dynamics, an important factor to consider and model for vector control strategies. The aim of the present study was to analyze the behavior of orientation to chemical signals emitted by feces from deltamethrin resistant (R) and susceptible (S) T. infestans. The behavioral assays were performed in a circular glass arena divided in two equal sectors. Fecal signals emitted by both S and R feces are attractants to fifth-instar nymphs of both S and R populations. Both toxicological phenotypes remained significantly longer on R feces, as compared to S feces. This is the first evidence in a triatomine, for the association of an aggregation behavior and insecticide resistance and may be the result of pleiotropic effects surrounding resistance genes.

Alien Invasion: The Case of the Western Conifer-Seed Bug (Heteroptera: Coreidae) in Chile, Overreaction, and Misidentifications.

Leptoglossus occidentalis (Heidemann) is an invasive species introduced in Europe, Asia, and most recently South America. In the present study, we report the overreaction situation caused by this bug in Chile, as it has been confused with kissing bugs (Reduviidae: Triatominae), Chagas' disease vectors. During 2018 and first months of 2019, we received 74 alleged cases of kissing bugs to confirm identification. From these, a total of 63 were identified as L. occidentalis, representing a 85% of the total denounces. Additionally, the first bite case in a human is described. The situation caused by L. occidentalis in Chile is discussed, and an illustrated table is provided to correctly identify this species and tell it apart from Triatomines. It is concluded that L. occidentalis is well established in Chile, and it is necessary to educate the population on recognition of the bug and to avoid overreaction as the species can inflict painful bites, but not transmit any disease.

Wide distribution of Trypanosoma cruzi-infected triatomines in the State of Bahia, Brazil.

BACKGROUND: The identification of Trypanosoma cruzi and blood-meal sources in synanthropic triatomines is important to assess the potential risk of Chagas disease transmission. We identified T. cruzi infection and blood-meal sources of triatomines caught in and around houses in the state of Bahia, northeastern Brazil, and mapped the occurrence of infected triatomines that fed on humans and domestic animals. METHODS: Triatominae bugs were manually captured by trained agents from the Epidemiologic Surveillance team of Bahia State Health Service between 2013 and 2014. We applied conventional PCR to detect T. cruzi and blood-meal sources (dog, cat, human and bird) in a randomized sample of triatomines. We mapped triatomine distribution and analyzed vector hotspots with kernel density spatial analysis. RESULTS: In total, 5906 triatomines comprising 15 species were collected from 127 out of 417 municipalities in Bahia. The molecular analyses of 695 triatomines revealed a ~10% T. cruzi infection rate, which was highest in the T. brasiliensis species complex. Most bugs were found to have fed on birds (74.2%), and other blood-meal sources included dogs (6%), cats (0.6%) and humans (1%). Trypanosoma cruzi-infected triatomines that fed on humans were detected inside houses. Spatial analysis showed a wide distribution of T. cruzi-infected triatomines throughout Bahia; triatomines that fed on dogs, humans, and cats were observed mainly in the northeast region. CONCLUSIONS: Synanthropic triatomines have a wide distribution and maintain the potential risk of T. cruzi transmission to humans and domestic animals in Bahia. Ten species were recorded inside houses, mainly Triatoma sordida, T. pseudomaculata, and the T. brasiliensis species complex. Molecular and spatial analysis are useful to reveal T. cruzi infection and blood-meal sources in synanthropic triatomines, identifying areas with ongoing threat for parasite transmission and improving entomological surveillance strategies.

Biological characteristics of Meccus phyllosomus pallidipennis (Hemiptera: Reduviidae: Triatominae) fed on two different hosts.

INTRODUCTION: Different blood meal sources can affect biology of triatomines. METHODS: We studied the influence of two different blood meal sources (rabbits and hens) on five biological parameters in Meccus phyllosomus pallidipennis and collected biological parameters. RESULTS: The cohort fed on rabbits had a shorter median life-cycle and lower mortality rate than that fed on hens. Both cohorts required a similar number of blood meals to reach adulthood. Median longevity and number of blood meals for adults were similar between both cohorts. CONCLUSIONS: The studied parameters reflect the high grade of adaptation of M. p. pallidipennis feeding on different hosts.

Potential impact of climate change on the geographical distribution of two wild vectors of Chagas disease in Chile: Mepraia spinolai and Mepraia gajardoi.

BACKGROUND: Mepraia gajardoi and Mepraia spinolai are endemic triatomine vector species of Trypanosoma cruzi, a parasite that causes Chagas disease. These vectors inhabit arid, semiarid and Mediterranean areas of Chile. Mepraia gajardoi occurs from 18° to 25°S, and M. spinolai from 26° to 34°S. Even though both species are involved in T. cruzi transmission in the Pacific side of the Southern Cone of South America, no study has modelled their distributions at a regional scale. Therefore, the aim of this study is to estimate the potential geographical distribution of M. spinolai and M. gajardoi under current and future climate scenarios. METHODS: We used the Maxent algorithm to model the ecological niche of M. spinolai and M. gajardoi, estimating their potential distributions from current climate information and projecting their distributions to future climatic conditions under representative concentration pathways (RCP) 2.6, 4.5, 6.0 and 8.5 scenarios. Future predictions of suitability were constructed considering both higher and lower public health risk situations. RESULTS: The current potential distributions of both species were broader than their known ranges. For both species, climate change projections for 2070 in RCP 2.6, 4.5, 6.0 and 8.5 scenarios showed different results depending on the methodology used. The higher risk situation showed new suitable areas, but the lower risk situation modelled a net reduction in the future potential distribution areas of M. spinolai and M. gajardoi. CONCLUSIONS: The suitable areas for both species may be greater than currently known, generating new challenges in terms of vector control and prevention. Under future climate conditions, these species could modify their potential geographical range. Preventive measures to avoid accidental human vectorial transmission by wild vectors of T. cruzi become critical considering the uncertainty of future suitable areas projected in this study.

Influence of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) Infection on Mortality of the Sylvatic Triatomine Vector, Mepraia spinolai (Heteroptera: Reuviidae), Under Fasting.

The etiologic agent of Chagas disease, Trypanosoma cruzi, is transmitted by hematophagous insect vectors that subsist on repeated blood meals over their lives separated by periods of fasting. Using naturally infected Mepraia spinolai, we measured the influence of parasite infection on this host vector's mortality during regular feeding and after fasting. After their capture, the insects were fed twice with uninfected mice to evaluate parasitic infection in their fecal samples by microscopic observation and PCR. Then the insects were subjected to a fasting period, followed by a third (final) feeding. After each feeding, a fecal sample was obtained to evaluate T. cruzi infection. To determine its progress through ontogeny, mortality and ecdysis of the infected and uninfected nymphs and adults were recorded on three occasions, over 140 d, and analyzed. Detections of infection by T. cruzi between the two first feedings increased, but this detection level was generally reduced after final feeding unless reinfected. For nymphs (stages III-V), their mortality was highest when infected after the fasting period, whereas adults were equally resistant to death after fasting when infected with T. cruzi. Metacyclic trypomastigotes were principally excreted in the fecal samples. Our results confirm that T. cruzi is pathogenic to its invertebrate hosts under nutritional stress conditions, when nymphs' mortality is higher while infected than uninfected when they were hungry. These results are epidemiologically important because T. cruzi harms the fasting vector M. spinolai, reducing its lifespan and competence as a disease vector, and thereby its rates of parasite transmission.

Reproductive Aspects of Chagas Disease Vectors: Evidence of Transcriptional Activity during the Nucleolar Persistence Phenomenon in the Spermatogenesis of Triatomines.

The subfamily Triatominae currently consists of 154 species, most of them being of great importance for public health because they are considered potential vectors of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas disease. In addition to their epidemiological importance, these insects are considered important biological models for cell studies because they have peculiar characteristics in their cells, for example, persistence of the nucleolus during spermatogenesis. This phenomenon is characterized by the presence of the nucleolus or nucleolar corpuscles during all phases of meiosis. To date, all knowledge is restricted to the study of the presence/absence of the nucleolus during the triatomine meiosis, so the present work aimed to analyze if this persistent nucleolar material has transcriptional activity. Analysis of the meiotic metaphases of Rhodnius montenegrensis and Panstrongylus megistus by using fluorochrome acridine orange made it possible to characterize the presence of RNA in the nucleolar material. Thus, it was demonstrated, for the first time, that the persistent nucleolar material during triatomine meiosis is transcriptionally active, supporting the hypothesis of the relationship between nucleolar persistence during meiosis of these insects and the formation of the chromoid body, an organelle responsible for the support of all transcriptional activities during spermiogenesis.

Spermathecae: Morphofunctional features and correlation with fat bodies and trachea in six species of vectors of Chagas disease.

Since spermatheca is able to transport spermatozoa and maintain a specific microenvironment for the storage of viable sperm cells for long periods of time, specific morphofunctional features must be involved in this capacity, and an efficient nutritional and oxygen supply must be required. In this study, we investigated the histological features of spermathecae and fat bodies in six species of three genera of epidemiological importance for Chagas' disease. The association of the reproductive system with the fat bodies and tracheal system was also focused in these species. The reproductive system, tracheae and fat bodies were fixed in 4% formaldehyde, and embedded in glycol methacrylate. The sections were stained with H.E., picrosirius red and Periodic-Acid Schiff methods for morphological analyses. Paraffin-embedded spermatheca sections were submitted to immunofluorescence for detection of V-ATPase. In P. lignarius, R. montenegrensis and R. prolixus, the spermatheca contains a slightly dilated tubular distal portion. In P. megistus and T. tibiamaculata, the spermatheca shows a large bulbous distal portion, and in T. infestans, a large oval-shaped distal portion. In all species, this portion was surrounded by a thin muscular layer, and the epithelial height varied according to the shape of this terminal portion. All spermathecal proximal portions showed simple columnar epithelium surrounded by a thick muscular layer. The epithelial cells of spermathecae showed PAS-positive cytoplasm and V-ATPase immunofluorescence in the apical surface. Tracheoles and polysaccharide-rich fat body cells were found next or in close contact to the oviduct or spermathecal tissues. The results indicate that the spermatheca proximal portion is related to contraction and sperm transport, whose oxygen and energy supply is guaranteed by the associated tracheal branches and fat bodies. In the storage portion, fat bodies and tracheae seem to be crucial for the maintenance of an optimal spermathecal microenvironment and storage of viable sperm cells. The participation of V-ATPase in the spermathecae epithelial cells may contribute for the maintenance of an optimal luminal milieu to spermatozoa, by alkalinization and/or acidification of lumen, similarly to the other epithelial cell types in insects. Further studies are necessary to clarify the role of this proton pump in the spermathecal epithelial cells.

Survivorship of wild caught Mepraia spinolai nymphs: The effect of seasonality and Trypanosoma cruzi infection after feeding and fasting in the laboratory.

Chagas disease is caused by Trypanosoma cruzi. Vector survival is an important variable affecting vectorial capacity to determine parasite transmission risk. The aims of this study are to evaluate vector survival under fasting/starvation conditions of wild-caught Mepraia spinolai after feeding and fasting, the pathogenicity of T. cruzi infection, the parasite burden and seasonal variation in parasite discrete typing units (DTU). The survivorship of M. spinolai nymphs after two continuous artificial feedings was evaluated, assessing their infection with microscopic observation of fecal samples and PCR. Later, insects were fasted/starved until death. We performed qPCR analyses of parasite load in the fecal samples and dead specimens. T. cruzi genotyping was performed using conventional PCR amplicons and hybridization tests. Infection rate was higher in M. spinolai nymphs in summer and spring than in fall. Parasite burden varied from 3 to 250,000 parasites/drop. Survival rate for starved nymph stage II was lower in insects collected in the spring compared to summer and fall. TcII was the most frequent DTU. Mainly metacyclic trypomastigotes were excreted. We conclude that M. spinolai infection rate in nymphs varies among seasons, suggesting higher transmission risk in warmer seasons. However, nymphs stage II collected in spring are more sensitive to starvation compared to other seasons. TcII in single or mixed infection does not seem relevant to determine vector pathogenicity. These results of vector survivorship after fasting/starvation are important to determine the competence of M. spinolai as a vector of T. cruzi, since they excrete metacyclic trypomastigotes and the parasitism with T. cruzi seems to be poorly pathogenic to the vector under a severe fasting/starvation condition.

Genital morphology and copulatory behavior in triatomine bugs (Reduviidae: Triatominae).

Triatomines (Heteroptera: Reduviidae) include around 139 species, widely known as vectors of Chagas disease. Our aim is to review the existing knowledge of the genital morphology and sexual behavior and provide some functional analysis of these traits in triatomines. A complex set of traits comprise genitalia and these are highly variable among species. The components of the phallus and seminal products (secreted by action of testes and two accessory glands) interact to allow successful sperm transfer to the female spermathecae (usually a pair of blind tubes that emerge from the common oviduct). Seminal products may inhibit female physiology and extend mating duration. Mating behavior in triatomines is best characterized as scramble competition. We suggest that males may evaluate female condition prior to copulation, given that female fitness is largely affected by food (blood) source. Although rearing several triatomine species may be difficult and discourage from undertaking studies on this group, any further investigation on sexual behavior and mating interactions may provide data for applicative studies including Chagas disease vectors control.