Detoxificación de insecticidas en triatominos: un enfoque transcriptómico

La Enfermedad de Chagas afecta a millones de personas en Argentina y Latinoamérica. Se considera que el control vectorial de los insectos triatominos que la transmiten es el método más efectivo para controlarla. Sin embargo, el surgimiento de focos de resistencia a insecticidas en Triatoma infestans constituye un obstáculo en la ecorregión del Gran Chaco, donde 1 de cada 16 personas se encuentra infectada. El estudio de las causas de resistencia a insecticidas resulta importante para lograr un correcto manejo del vector. Se han hallado mutaciones en el sitio de acción de los insecticidas piretroides (el canal de sodio dependiente de voltaje presente en membranas de las células excitables) que explican en buena parte la resistencia. Además, se ha observado que la estructura cuticular y el rol de algunas enzimas detoxificativas de estos insectos podrían contribuir al fenómeno. Sin embargo, es poco lo que se conoce sobre la respuesta transcriptómica global de estos insectos frente a la intoxicación.Teniendo en cuenta las desventajas de los insecticidas neurotóxicos, se ha planteado la necesidad de desarrollar insecticidas de nueva generación que sean específicos para el organismo que se desea controlar, con un bajo impacto en la salud humana y el medio ambiente. Los neuropéptidos, moléculas clave para la regulación de diversos procesos fisiológicos en insectos, podrían ofrecer nuevos blancos para el diseño de estos insecticidas. Por todo lo expuesto, conocer la secuencia de los genes y su nivel de expresión constituye el primer paso para iniciar estudios moleculares y funcionales. Las tecnologías de secuenciación de nueva generación, en complemento con técnicas moleculares tales como el silenciamiento génico por ARN de interferencia (ARNi) ofrecen herramientas que permiten brindar información valiosa sobre la función génica.En este trabajo de Tesis se propuso realizar un análisis comparativo del repertorio de las principales superfamilias de enzimas detoxificativas de insectos (Citocromos P450, Carboxil/Colinesterasas y Glutatión Transferasas) presentes en los transcriptomas de Triatoma infestans, Triatoma dimidiata y Triatoma pallidipennis, así como en el genoma de Rhodnius prolixus. Asimismo, se exploró la expresión de estos genes en diferentes tejidos provenientes de bases de datos transcriptómicas disponibles. Se hallaron características distintivas en estas superfamilias en triatominos que podrían impactar en la respuesta a los tóxicos. Por otra parte, se identificaron candidatos interesantes a estudiar en relación a los fenómenos detoxificativos.Por otra parte, se realizaron búsquedas de las secuencias de neuropéptidos presentes en los transcriptomas de Triatoma spp., a fin de analizarlas comparativamente con las de otros insectos. Como resultado, se observó una conservación estructural de algunas familias de neuropéptidos, mientras que en otros se evidenciaron variaciones incluso dentro de la subfamilia Triatominae.Con el fin de conocer la respuesta transcriptómica global en T. infestans causada por la intoxicación con el piretroide deltametrina, se realizó un análisis de expresión diferencial mediante la técnica RNA-seq. El análisis de los resultados evidenció modulación significativa de transcriptos involucrados en procesos tales como desarrollo post-embrionario, metabolismo, organización del citoesqueleto, transporte y señalización celular, estrés oxidativo, inmunidad, cutícula y sistema sensorial y cognitivo.Por último, se evaluó la contribución del neuropéptido ITG-like a la respuesta a la intoxicación con deltametrina en R. prolixus, utilizando la técnica de ARNi. Los resultados apuntaron a un rol de este neuropéptido en la respuesta a deltametrina. En conjunto, mediante la aplicación de análisis genómicos, transcriptómicos, filogenéticos y moleculares se obtuvo información novedosa que permite ampliar las fronteras del conocimiento en el marco de la respuesta detoxificativa en triatominos. Asimismo, a partir de los resultados obtenidos en este trabajo de Tesis podrán plantearse nuevas hipótesis en pos de profundizar el conocimiento sobre las causas genéticas subyacentes al fenómeno de resistencia a insecticidas.Fil: Traverso, Lucila María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas; Argentina. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentin

Chemosensory proteins in Triatoma infestans: gene annotation and comparative analysis across hemipterans

Triatoma infestans is the main vector of Trypanosoma cruzi, the causative agent of Chagas disease in the southern cone. Resistance to pyrethroid insecticides reported in T. infestans populations could be one of the main causes of the persistence of vectorial transmission in the Argentinean Gran Chaco ecoregion. Chemosensory proteins (CSPs) are small soluble proteins from arthropods, with a studied role in olfaction. Recent evidence points to a role of these proteins in insecticide susceptibility and resistance. Accordingly, previous results from our group showed that some members of T. infestans CSP family are modulated four hours after an intoxication with deltamethrin.Para acceder a la videoconferencia completa, hacer clic en "Enlace externo".Sociedad Latinoamericana de Ecología de Vectore

Comparative analysis of detoxification-related gene superfamilies across five hemipteran species

Hemiptera is one of the most speciose orders of insects, and the most speciose considering Hemimetabola. Through their evolutive history, hemipterans with different feeding habits have adapted to deal with different chemical challenges. Three major gene families are involved in xenobiotic detoxification in insects: the cytochromes P450 (CYPs), carboxyl/cholinesterases (CCEs), and glutathione transferases (GSTs). Here we perform a comparative analysis on the complement of these gene superfamilies across five hemipteran species; four heteropterans (the pentatomid plant feeders Nezara viridula and Halyomorpha halys; the hematophagous Cimex lectularius, Cimicidae, and Rhodnius prolixus, Reduviidae), and one Auchenorrhyncha plant feeder (Nilaparvata lugens).Fil: Volonté, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnol.conicet - la Plata. Centro de Endocrinología Exp.y Aplicada (i). Grupo Vinculado Cenexa-fcex-unlp; Argentina. Centro Regional de Estudios Genomicos (creg) ; Facultad de Cs.exactas ; Universidad Nacional de la Plata;Fil: Traverso, Lucila María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Endocrinología Experimental y Aplicada. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Endocrinología Experimental y Aplicada; Argentina. Centro Regional de Estudios Genomicos (creg) ; Facultad de Cs.exactas ; Universidad Nacional de la Plata;Fil: Latorre Estivalis, Jose Manuel. Centro Regional de Estudios Genomicos (creg) ; Facultad de Cs.exactas ; Universidad Nacional de la Plata; . Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Cunha Almeida, Francisca. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; ArgentinaFil: Ons, Sheila. Centro Regional de Estudios Genomicos (creg) ; Facultad de Cs.exactas ; Universidad Nacional de la Plata

Characterization of the sensory gene repertoire of Triatoma infestans and the effect of blood ingestion on its antennal expression

Currently, Triatoma infestans is the main vector of Chagas disease in Argentina, Paraguay, and Bolivia. As many T. infestans populations present insecticide resistance, directly impacting control campaigns, alternative control methods, like those based on behavioral manipulation are needed. Bug nutritional and developmental status modulate bug responsiveness to host-related sensory cues. In order to understand the molecular bases of this modulation, we sequenced the antennal transcriptome of T. infestans and compared the gene expression profiles between unfed and fed insects.Para acceder a la videoconferencia completa, hacer clic en "Enlace externo".Sociedad Latinoamericana de Ecología de Vectore

Comparative and functional triatomine genomics reveals reductions and expansions in insecticide resistance-related gene families

Background: Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas’ disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas’ disease. Methods and findings: The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas’ disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion. Conclusions and significance: Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas’ disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms.Centro Regional de Estudios GenómicosCentro de Endocrinología Experimental y Aplicad

Comparative and functional triatomine genomics reveals reductions and expansions in insecticide resistance-related gene families

Background: Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas’ disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas’ disease. Methods and findings: The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas’ disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion. Conclusions and significance: Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas’ disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms.Centro Regional de Estudios GenómicosCentro de Endocrinología Experimental y Aplicad

Comparative and functional triatomine genomics reveals reductions and expansions in insecticide resistance-related gene families

Background: Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas’ disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas’ disease. Methods and findings: The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas’ disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion. Conclusions and significance: Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas’ disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms.Centro Regional de Estudios GenómicosCentro de Endocrinología Experimental y Aplicad

Molecular evolutionary trends and feeding ecology diversification in the Hemiptera, anchored by the milkweed bug genome.

BACKGROUND: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. RESULTS: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding. CONCLUSIONS: With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus's strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes

Molecular evolutionary trends and feeding ecology diversification in the Hemiptera, anchored by the milkweed bug genome

Background: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order whose members offer a close outgroup to the Holometabola, with high diversity within the order for feeding ecology and excellent experimental tractability for molecular genetics. Sequenced genomes have recently become available for hemipteran pest species such as phloem-feeding aphids and blood-feeding bed bugs. To complement and build upon these resources, we present the genome sequence and comparative analyses centered on the large milkweed bug, Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. Results: The 926-Mb genome of Oncopeltus is relatively well represented by the current assembly and official gene set, which supports Oncopeltus as a fairly conservative hemipteran species for anchoring molecular comparisons. We use our genomic and RNA-seq data not only to characterize features of the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins and of intron gain and turnover in the Hemiptera. These analyses also weigh the relative importance of lineage and genome size as predictors of gene structure evolution in insects. Furthermore, we identify enzymatic gains and losses that correlate with hemipteran feeding biology, particularly for reductions in chemoreceptor family size and loss of metabolic reactions within species with derived, fluid-nutrition feeding modes. Conclusions: With the milkweed bug genome, for the first time we have a critical mass of sequenced species representing a hemimetabolous insect order, substantially improving the diversity of insect genomics beyond holometabolans such as flies and ants. We use this addition to define commonalities among the Hemiptera and then delve into how hemipteran species' genomes reflect their feeding ecology types. Our novel and detailed analyses integrate global and rigorous manual approaches, generating hypotheses and identifying specific sets of genes for future investigation. Given Oncopeltus's strength as an experimental research model, we take particular care to evaluate the sequence resources presented here, augmenting its foundation for molecular research and highlighting potentially general considerations exemplified in the assembly and annotation of this medium-sized genome.La lista completa de autores puede consultarse en el documento.Centro Regional de Estudios Genómico

Neuropeptidomics in Triatoma infestans. Comparative transcriptomic analysis among triatomines

Chagas’ disease, affecting up to 6–7 million people worldwide, is transmitted to humans through the feces of triatomine kissing bugs. From these, Rhodnius prolixus, Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis are important vectors distributed throughout the Latin American subcontinent. Resistance to pyrethroids has been developed by some triatomine populations, especially T. infestans, obstructing their control. Given their role in the regulation of physiological processes, neuroendocrine-derived factors have been proposed as a source of molecular targets for new-generation insecticides. However, the involvement of neuropeptides in insecticide metabolism and resistance in insects has been poorly studied. In the present work, the sequences of 20 neuropeptide precursor genes in T. infestans, 16 in T. dimidiata, and 13 in T. pallidipennis detected in transcriptomic databases are reported, and a comparative analysis in triatomines is presented. A total of 59 neuropeptides were validated by liquid chromatography-tandem mass spectrometry in brain and nervous ganglia from T. infestans, revealing the existence of differential post-translational modifications, extended and truncated forms. The results suggest a high sequence conservation in some neuropeptide systems in triatomines, whereas remarkable differences occur in several others within the core domains. Comparisons of the basal expression levels for several neuropeptide precursor genes between pyrethroid sensitive and resistant population of T. infestans are also presented here, in order to introduce a proof of concept to test the involvement of neuropeptides in insecticide resistance. From the precursors tested, NVP and ITG peptides are significantly higher expressed in the resistant population. To our knowledge, this is the first report to associate differential neuropeptide expression with insecticide resistance. The information provided here contributes to creating conditions to widely extend functional and genetic studies involving neuropeptides in triatomines.Fil: Traverso, Lucila María. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Sierra, Ivana Samanta. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Sterkel, Marcos. Universidade Federal do Rio de Janeiro; BrasilFil: Francini, Flavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Endocrinología Experimental y Aplicada. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Endocrinología Experimental y Aplicada; ArgentinaFil: Ons, Sheila. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentin