Rhodnius prolixus, an insect vector of Chagas disease, ingests blood meals several-fold its own body weight. This nutritional overload triggers specific adaptive responses that prevent or remediate damage from multiple stressors, including oxidative, osmotic, and microbial challenges. Transcriptomic analysis of first-instar nymph digestive apparatus revealed five members of the small Heat Shock Protein (sHSP) family that were highly expressed after blood meals but were downregulated upon...

Insects of the subfamily Triatominae, commonly known as kissing bugs, are obligate blood-feeding vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Rhodnius prolixus is among the most epidemiologically important vectors in Latin America, whereas Triatoma rubida frequently invades homes and is a potential vector in the southern United States and northern Mexico. Triatomines likely evolved from predatory reduviid assassin bugs through a transition from feeding on arthropods associated with vertebrate hosts to feeding directly on vertebrate blood. To investigate the genomic basis of this ecological and dietary shift, we generated highly contiguous, near chromosome-level genome assemblies and structural gene annotations for R. prolixus and T. rubida. The new R. prolixus assembly improves scaffold N50 more than 40-fold over the current reference genome, from 1.1 to 43.9 Mb, while reducing assembly gaps by several orders of magnitude. Both assemblies exceed 97% BUSCO completeness. Comparative analyses with representative hemipteran genomes revealed expansions of gene families associated with chemosensation and metabolism, including detoxification, protein degradation, and digestion, together with signatures of positive selection in genes involved in digestive and sensory functions. These assemblies represent the most contiguous and complete genomic resources available for Triatominae and provide a robust foundation for investigating vector biology, host adaptation, and the evolutionary origins of blood feeding within Reduviidae.

Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. However, transgenesis and gene editing...

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Insects of the subfamily Triatominae, commonly known as kissing bugs, are obligate blood-feeding vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Rhodnius prolixus is among the most epidemiologically important vectors in Latin America, whereas Triatoma rubida frequently invades homes and is a potential vector in the southern United States and northern Mexico. Triatomines likely evolved from predatory reduviid assassin bugs through a transition from feeding on arthropods associated with vertebrate hosts to feeding directly on vertebrate blood. To investigate the genomic basis of this ecological and dietary shift, we generated highly contiguous, near chromosome-level genome assemblies and structural gene annotations for R. prolixus and T. rubida. The new R. prolixus assembly improves scaffold N50 more than 40-fold over the current reference genome, from 1.1 to 43.9 Mb, while reducing assembly gaps by several orders of magnitude. Both assemblies exceed 97% BUSCO completeness. Comparative analyses with representative hemipteran genomes revealed expansions of gene families associated with chemosensation and metabolism, including detoxification, protein degradation, and digestion, together with signatures of positive selection in genes involved in digestive and sensory functions. These assemblies represent the most contiguous and complete genomic resources available for Triatominae and provide a robust foundation for investigating vector biology, host adaptation, and the evolutionary origins of blood feeding within Reduviidae.

Bartonella henselae is the primary human pathogen species of this Gram-negative bacterial genus, associated with several clinical manifestations including cat scratch disease, bacillary angiomatosis, endocarditis, and myocarditis. This pathogen was recently detected in Triatoma sordida, a vector of Chagas disease in Brazil. Here, we investigated the presence of B. henselae DNA in Rhodnius prolixus, another triatomine vector of Trypanosoma cruzi maintained in an insectary and in ducks (Cairina...

Feeding behavior in blood-sucking insects relies on gustatory evaluation to decide on sustained ingestion, yet quantifying this process from electromyogram (EMG) recordings is labor-intensive. Here we developed MyoRec, an automated computational framework employing machine learning to analyse EMG signals from the triatomine bug Rhodnius prolixus. Using recordings under appetitive and aversive conditions, a convolutional neural network detected ingestion events with 97.7% accuracy. Automated analysis revealed distinct feeding dynamics, with prolonged ingestion and higher pumping frequency under appetitive stimuli, compared to rapid feeding cessation under aversive stimuli. MyoRec substantially reduces analysis time while maintaining accuracy, providing a scalable tool to investigate how gustatory cues modulate feeding decisions in hematophagous insects.

Feeding behavior in blood-sucking insects relies on gustatory evaluation to decide on sustained ingestion, yet quantifying this process from electromyogram (EMG) recordings is labor-intensive. Here we developed MyoRec, an automated computational framework employing machine learning to analyse EMG signals from the triatomine bug Rhodnius prolixus. Using recordings under appetitive and aversive conditions, a convolutional neural network detected ingestion events with 97.7% accuracy. Automated analysis revealed distinct feeding dynamics, with prolonged ingestion and higher pumping frequency under appetitive stimuli, compared to rapid feeding cessation under aversive stimuli. MyoRec substantially reduces analysis time while maintaining accuracy, providing a scalable tool to investigate how gustatory cues modulate feeding decisions in hematophagous insects.

Rhodnius prolixus is a primary insect vector of Trypanosoma cruzi, the causative agent of Chagas disease, a neglected parasitosis endemic in Latin American countries. It has been estimated that Chagas disease affects 7-8 million people worldwide and is responsible for approximately 1000 deaths per year. Genetic and molecular studies in this species remain challenging due to its life cycle and feeding habits, thus hindering the development of new strategies to control their populations and reduce the diffusion of Chagas disease. Recently, two stable cell lines - RPE/LULS53 and RPE/LULS57 - were derived from Rhodnius embryos, which represent promising new tools to investigate the genetics of this insect vector. Here, we describe their gene expression landscapes through transcriptomic approaches. We show that 8,968 expressed genes are shared between the two cell lines, whereas 391 and 1,088 genes are uniquely expressed in RPE/LULS53 and RPE/LULS57, respectively. Although key components of primary developmental, immune and redox signaling pathways are expressed in both cell lines, some genes such as Frizzled-10-a-like and catalase show marked differences in expression. Our results strongly suggest that RPE/LULS53 and RPE/LULS57 likely represent two different cell phenotypes. Consistent with this, gene ontology analysis reveals that RPE/LULS53 is enriched for animal organ morphogenesis and stress response, while RPE/LULS57 for DNA-directed RNA polymerase activity, among others. Despite these differences, both cell lines express comparable levels of transcripts from resident transposable elements, including the highly abundant Mariner and LINE/I elements, as well as horizontally transferred transposons. Our findings shed light on the nature of the RPE/LULS53 and RPE/LULS57 embryo-derived cell lines and provide valuable transcriptomic resources for future genetic and functional studies in Rhodnius and other triatomine insect vectors.

Rhodnius prolixus is a primary insect vector of Trypanosoma cruzi, the causative agent of Chagas disease, a neglected parasitosis endemic in Latin American countries. It has been estimated that Chagas disease affects 7-8 million people worldwide and is responsible for approximately 1000 deaths per year. Genetic and molecular studies in this species remain challenging due to its life cycle and feeding habits, thus hindering the development of new strategies to control their populations and reduce the diffusion of Chagas disease. Recently, two stable cell lines - RPE/LULS53 and RPE/LULS57 - were derived from Rhodnius embryos, which represent promising new tools to investigate the genetics of this insect vector. Here, we describe their gene expression landscapes through transcriptomic approaches. We show that 8,968 expressed genes are shared between the two cell lines, whereas 391 and 1,088 genes are uniquely expressed in RPE/LULS53 and RPE/LULS57, respectively. Although key components of primary developmental, immune and redox signaling pathways are expressed in both cell lines, some genes such as Frizzled-10-a-like and catalase show marked differences in expression. Our results strongly suggest that RPE/LULS53 and RPE/LULS57 likely represent two different cell phenotypes. Consistent with this, gene ontology analysis reveals that RPE/LULS53 is enriched for animal organ morphogenesis and stress response, while RPE/LULS57 for DNA-directed RNA polymerase activity, among others. Despite these differences, both cell lines express comparable levels of transcripts from resident transposable elements, including the highly abundant Mariner and LINE/I elements, as well as horizontally transferred transposons. Our findings shed light on the nature of the RPE/LULS53 and RPE/LULS57 embryo-derived cell lines and provide valuable transcriptomic resources for future genetic and functional studies in Rhodnius and other triatomine insect vectors.

Triatomines are the vectors of Chagas disease, one of the main endemic diseases from South to North America, now expanding to other continents. These hemimetabolous insects have been considered poorly visual animals. However, recent findings challenge this idea. Here, we used Rhodnius prolixus as a model species to comprehensively characterize triatomine compound eyes. We found that in the adult stage, eye size significantly exceeds the dimensions predicted by the nymphal eye growth rate. Moreover, while the compound eye grows symmetrically in its dorsal and ventral directions throughout the nymphal instars, in the adult, the eye undergoes greater ventral growth, resulting in a dorsoventrally asymmetrical eye. By studying a bright pseudopupil induced by fluorescence in natural mutant animals, we observed no major differences in sampling resolution between the last nymphal instar and the adult stage. However, the adult eye possesses significantly larger ommatidia, particularly in its ventral region, shifting the area of highest sensitivity from the equatorial region in the nymphal instars to the ventral region in the adult. A similar eye growth pattern was observed in Triatoma infestans and Panstrongylus megistus. The analysis of photographic records from 39 species across 10 genera indicates that an asymmetrical eye is the predominant phenotype in adult triatomines. Notable exceptions in wingless adults of Mepraia spinolai, reveal a tight association between possessing a large asymmetrical eye and the presence of wings. This suggests that vision might support triatomine dispersal flights among other visual behaviors.

Triatomines are the vectors of Chagas disease, one of the main endemic diseases from South to North America, now expanding to other continents. These hemimetabolous insects have been considered poorly visual animals. However, recent findings challenge this idea. Here, we used Rhodnius prolixus as a model species to comprehensively characterize triatomine compound eyes. We found that in the adult stage, eye size significantly exceeds the dimensions predicted by the nymphal eye growth rate. Moreover, while the compound eye grows symmetrically in its dorsal and ventral directions throughout the nymphal instars, in the adult, the eye undergoes greater ventral growth, resulting in a dorsoventrally asymmetrical eye. By studying a bright pseudopupil induced by fluorescence in natural mutant animals, we observed no major differences in sampling resolution between the last nymphal instar and the adult stage. However, the adult eye possesses significantly larger ommatidia, particularly in its ventral region, shifting the area of highest sensitivity from the equatorial region in the nymphal instars to the ventral region in the adult. A similar eye growth pattern was observed in Triatoma infestans and Panstrongylus megistus. The analysis of photographic records from 39 species across 10 genera indicates that an asymmetrical eye is the predominant phenotype in adult triatomines. Notable exceptions in wingless adults of Mepraia spinolai, reveal a tight association between possessing a large asymmetrical eye and the presence of wings. This suggests that vision might support triatomine dispersal flights among other visual behaviors.

The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected tropical disease that mostly affects the population of Latin American countries, with an estimated 7 million infected people and more than 10,000 deaths per year worldwide. T. cruzi is typically transmitted by hematophagous triatomine insects, with Rhodnius prolixus being a major insect vector in South America. While the microbiome of triatomine insects has been investigated to a certain extent, the ternary...

Rhodnius prolixus is an insect vector of the protozoan Trypanosoma cruzi, the causative agent of debilitating Chagas disease, which is transmitted to humans during blood feeding. Identifying germline markers is a critical step in advancing vector control and transgenic technologies of these medically important insects. Transmission of genetic traits to the next generation requires proper differentiation of the germline that gives rise to gametes. Germline precursors are established during early stages of development as the primordial germ cell (PGC) population. Among the genes required for this process, vasa homologues exert a conserved role in germline specification. Here, we characterize and validate the genomic structure of the R. prolixus Rp-vasa locus and assess its expression during early embryogenesis. We observe widespread Rp-vasa expression in preblastoderm embryos. Later, during the cellular blastoderm and at the beginning of gastrulation, Rp-vasa and Rp-piwi2 expression is restricted to PGCs, morphologically identifiable as a cluster of cells at the posterior of the embryo. We also report, for the first time, the use of R. prolixus regulatory sequences to drive the expression of exogenous genes. We identify the Rp-vasa regulatory region and show that these cis-regulatory sequences are sufficient to drive Cas9 and dsRed expression in the early embryo. Together, these findings demonstrate that Rp-vasa has great potential for use as a PGC marker and as a driver for gene expression in transgenic and gene editing approaches for Triatomine vectors.

Rhodnius prolixus is an insect vector of the protozoan Trypanosoma cruzi, the causative agent of debilitating Chagas disease, which is transmitted to humans during blood feeding. Identifying germline markers is a critical step in advancing vector control and transgenic technologies of these medically important insects. Transmission of genetic traits to the next generation requires proper differentiation of the germline that gives rise to gametes. Germline precursors are established during early stages of development as the primordial germ cell (PGC) population. Among the genes required for this process, vasa homologues exert a conserved role in germline specification. Here, we characterize and validate the genomic structure of the R. prolixus Rp-vasa locus and assess its expression during early embryogenesis. We observe widespread Rp-vasa expression in preblastoderm embryos. Later, during the cellular blastoderm and at the beginning of gastrulation, Rp-vasa and Rp-piwi2 expression is restricted to PGCs, morphologically identifiable as a cluster of cells at the posterior of the embryo. We also report, for the first time, the use of R. prolixus regulatory sequences to drive the expression of exogenous genes. We identify the Rp-vasa regulatory region and show that these cis-regulatory sequences are sufficient to drive Cas9 and dsRed expression in the early embryo. Together, these findings demonstrate that Rp-vasa has great potential for use as a PGC marker and as a driver for gene expression in transgenic and gene editing approaches for Triatomine vectors.

Kissing bugs are the primary vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Kissing bugs are exposed to thermal variability, including short periods of heat stress, which can induce mortality or exert sublethal effects. This study investigated Rhodnius prolixus following brief periods of high thermal stress with respect to survival, blood feeding, developmental processes, and T. cruzi infection, with a focus on sublethal effects. Our results demonstrated a significant decrease in survival for R. prolixus at 42{degrees}C for 8 hours. When exposed to sub-lethal thermal stress (40{degrees}C for 8 hours), blood ingestion (amount and proportion) was reduced after 24 hours of recovery from thermal stress. Among the bugs that fed after 24 hours, molting was not impacted by temperature exposure. The infection rate decreased after heat exposure, likely due to reduced blood volume ingested when feeding 24 hours after heat stress. A week of recovery after exposure to higher temperatures improved feeding and increased infection rates to levels comparable to those of kissing bugs not exposed to thermal stress. Our findings offer insights into how extreme temperature events may influence Chagas disease. Specifically, these studies highlight the need to clarify how temperature, particularly at sublethal levels, interacts with vector biology to alter parasite transmission.

Kissing bugs are the primary vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Kissing bugs are exposed to thermal variability, including short periods of heat stress, which can induce mortality or exert sublethal effects. This study investigated Rhodnius prolixus following brief periods of high thermal stress with respect to survival, blood feeding, developmental processes, and T. cruzi infection, with a focus on sublethal effects. Our results demonstrated a significant decrease in survival for R. prolixus at 42{degrees}C for 8 hours. When exposed to sub-lethal thermal stress (40{degrees}C for 8 hours), blood ingestion (amount and proportion) was reduced after 24 hours of recovery from thermal stress. Among the bugs that fed after 24 hours, molting was not impacted by temperature exposure. The infection rate decreased after heat exposure, likely due to reduced blood volume ingested when feeding 24 hours after heat stress. A week of recovery after exposure to higher temperatures improved feeding and increased infection rates to levels comparable to those of kissing bugs not exposed to thermal stress. Our findings offer insights into how extreme temperature events may influence Chagas disease. Specifically, these studies highlight the need to clarify how temperature, particularly at sublethal levels, interacts with vector biology to alter parasite transmission.

Rhodnius prolixus, an insect vector of Chagas disease, ingests blood meals several-fold its own body weight. This nutritional overload triggers specific adaptive responses that prevent or remediate damage from multiple stressors, including oxidative, osmotic, and microbial challenges. Transcriptomic analysis of first-instar nymph guts revealed five members of the Small Heat-Shock Protein (sHSP) family that were highly expressed after blood meals but were downregulated upon Trypanosoma cruzi infection. sHSPs are proteins involved in cellular homeostasis and stress responses. Simultaneous knockdown of all five sHSPs profoundly disrupted insect physiology, causing decreased actin filament network formation, interrupted peristalsis, blocked ER expansion, and reduced ER-mitochondria association, while increasing reactive oxygen species generation and inducing premature epithelial cell mitosis. This apparent loss of gut homeostasis was recapitulated by trypanosome infection and, conversely, sHSP silencing in infected insects increased parasite numbers. Taken together, our data reveal a paramount role for sHSPs in gut cell homeostasis following blood meals and support the hypothesis that sHSP expression down regulation represents an adaptive manipulation of the insect host by the parasite.

Rhodnius prolixus, an insect vector of Chagas disease, ingests blood meals several-fold its own body weight. This nutritional overload triggers specific adaptive responses that prevent or remediate damage from multiple stressors, including oxidative, osmotic, and microbial challenges. Transcriptomic analysis of first-instar nymph guts revealed five members of the Small Heat-Shock Protein (sHSP) family that were highly expressed after blood meals but were downregulated upon Trypanosoma cruzi infection. sHSPs are proteins involved in cellular homeostasis and stress responses. Simultaneous knockdown of all five sHSPs profoundly disrupted insect physiology, causing decreased actin filament network formation, interrupted peristalsis, blocked ER expansion, and reduced ER-mitochondria association, while increasing reactive oxygen species generation and inducing premature epithelial cell mitosis. This apparent loss of gut homeostasis was recapitulated by trypanosome infection and, conversely, sHSP silencing in infected insects increased parasite numbers. Taken together, our data reveal a paramount role for sHSPs in gut cell homeostasis following blood meals and support the hypothesis that sHSP expression down regulation represents an adaptive manipulation of the insect host by the parasite.