Chronic exposure to glyphosate induces transcriptional changes in honey bee larva : A toxicogenomic study

The honey bee Apis mellifera is the most abundant managed pollinator in diverse crops worldwide. Consequently, it is exposed to a plethora of environmental stressors, among which are the agrochemicals. In agroecosystems, the herbicide glyphosate (GLY) is one of the most applied. In laboratory assessments, GLY affects the honey bee larval development by delaying its moulting, among other negative effects. However, it is still unknown how GLY affects larval physiology when there are no observable signs of toxicity. We carried out a longitudinal experimental design using the in vitro rearing procedure. Larvae were fed with food containing or not a sub-lethal dose of GLY in chronic exposure (120 h). Individuals without observable signs of toxicity were sampled and their gene expression profile was analyzed with a transcriptomic approach to compare between treatments. Even though 29% of larvae were asymptomatic in the exposed group, they showed transcriptional changes in several genes after the GLY chronic intake. A total of 19 transcripts were found to be differentially expressed in the RNA-Seq experiment, mainly linked with defensive response and intermediary metabolism processes. Furthermore, the enriched functional categories in the transcriptome of the exposed asymptomatic larvae were linked with enzymes with catalytic and redox activity. Our results suggest an enhanced catabolism and oxidative metabolism in honey bee larvae as a consequence of the sub-lethal exposure to GLY, even in the absence of observable symptoms.Centro Regional de Estudios Genómico

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

The molecular sensory machinery of a Chagas disease vector: expression changes through imaginal moult and sexually dimorphic features

The triatomine bug Rhodnius prolixus is a main vector of Chagas disease, which affects several million people, mostly in Latin-America. Host searching, pheromone communication, and microclimatic preferences are aspects of its behaviour that depend on multimodal sensory inputs. The molecular bases of these sensory processes are largely unknown. The expression levels of genes transcribed in antennae were compared between 5th instar larvae, and female and male adults by means of RNA-Seq. The antennae of R. prolixus showed increased expression of several chemosensory-related genes in imaginal bugs, while both sexes had similar expression patterns for most target genes. Few cases suggest involvement of target genes in sexually dimorphic functions. Most odorant and ionotropic receptor genes seemed to be expressed in all libraries. OBPs and CSPs showed very high expression levels. Other sensory-related genes such as TRPs, PPKs and mechanoreceptors had consistent levels of expression in all libraries. Our study characterises most of the sensory gene repertoire of these insects, opening an avenue for functional genetics studies. The increase in expression of chemosensory genes suggests an enhanced role in adult bugs. This knowledge allows developing new behaviour interfering strategies, increasing the options for translational research in the vector control field.Centro Regional de Estudios Genómico

Trypanosomes Modify the Behavior of Their Insect Hosts: Effects on Locomotion and on the Expression of a Related Gene

Background As a result of evolution, the biology of triatomines must have been significantly adapted to accommodate trypanosome infection in a complex network of vector-vertebrate-parasite interactions. Arthropod-borne parasites have probably developed mechanisms, largely still unknown, to exploit the vector-vertebrate host interactions to ensure their transmission to suitable hosts. Triatomines exhibit a strong negative phototaxis and nocturnal activity, believed to be important for insect survival against its predators. Methodology/Principal Findings In this study we quantified phototaxis and locomotion in starved fifth instar nymphs of Rhodnius prolixus infected with Trypanosoma cruzi or Trypanosoma rangeli. T. cruzi infection did not alter insect phototaxis, but induced an overall 20% decrease in the number of bug locomotory events. Furthermore, the significant differences induced by this parasite were concentrated at the beginning of the scotophase. Conversely, T. rangeli modified both behaviors, as it significantly decreased bug negative phototaxis, while it induced a 23% increase in the number of locomotory events in infected bugs. In this case, the significant effects were observed during the photophase. We also investigated the expression of Rpfor, the triatomine ortholog of the foraging gene known to modulate locomotion in other insects, and found a 4.8 fold increase for T. rangeli infected insects. Conclusions/Significance We demonstrated for the first time that trypanosome infection modulates the locomotory activity of the invertebrate host. T. rangeli infection seems to be more broadly effective, as besides affecting the intensity of locomotion this parasite also diminished negative phototaxis and the expression of a behavior-associated gene in the triatomine vector

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

Evaluation of reference genes for insect olfaction studies

Submitted by Nuzia Santos ([email protected]) on 2016-02-01T17:29:22Z No. of bitstreams: 1 Evaluation of reference genes for insect olfaction studies.pdf: 13551079 bytes, checksum: e23c2a48fc3b69cadff7f0386fc1d505 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-02-01T17:35:45Z (GMT) No. of bitstreams: 1 Evaluation of reference genes for insect olfaction studies.pdf: 13551079 bytes, checksum: e23c2a48fc3b69cadff7f0386fc1d505 (MD5)Made available in DSpace on 2016-02-01T17:35:45Z (GMT). No. of bitstreams: 1 Evaluation of reference genes for insect olfaction studies.pdf: 13551079 bytes, checksum: e23c2a48fc3b69cadff7f0386fc1d505 (MD5) Previous issue date: 2015Department of Plant Protection Biology. Chemical Ecology Unit. SLU, Alnarp, Sweden / Bioversity International. Consultative Group for International Agricultural Research. Bujumbura, Burundi.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Comportamento do Vetor e Grupo de Interações com Patogenos. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Comportamento do Vetor e Grupo de Interações com Patógenos. Belo Horizonte, MG, Brasil.Department of Plant Protection Biology. Chemical Ecology Unit. SLU, Alnarp, Sweden.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Comportamento de Vetores e Interação com Patógenos. Belo Horizonte, MG, Brasil.Background: Quantitative reverse transcription PCR (qRT-PCR) is a robust and accessible method to assay gene expression and to infer gene regulation. Being a chain of procedures, this technique is subject to systematic error due to biological and technical limitations mainly set by the starting material and downstream procedures. Thus, rigorous data normalization is critical to grant reliability and repeatability of gene expression quantification by qRT-PCR. A number of ‘housekeeping genes’, involved in basic cellular functions, have been commonly used as internal controls for this normalization process. However, these genes could themselves be regulated and must therefore be tested a priori. Methods: We evaluated eight potential reference genes for their stability as internal controls for RT-qPCR studies of olfactory gene expression in the antennae of Rhodnius prolixus, a Chagas disease vector. The set of genes included were: α-tubulin; β-actin; Glyceraldehyde-3-phosphate dehydrogenase; Eukaryotic initiation factor 1A; Glutathione-S-transferase; Serine protease; Succinate dehydrogenase; and Glucose-6-phosphate dehydrogenase. Five experimental conditions, including changes in age,developmental stage and feeding status were tested in both sexes. Results: We show that the evaluation of candidate reference genes is necessary for each combination of sex, tissue and physiological condition analyzed in order to avoid inconsistent results and conclusions. Although, Normfinder and geNorm software yielded different results between males and females, five genes (SDH, Tub, GAPDH, Act and G6PDH) appeared in the first positions in all rankings obtained. By using gene expression data of a single olfactory coreceptor gene as an example, we demonstrated the extent of changes expected using different internal standards. Conclusions; This work underlines the need for a rigorous selection of internal standards to grant the reliability of normalization processes in qRT-PCR studies. Furthermore, we show that particular physiological or developmental conditions require independent evaluation of a diverse set of potential reference genes

Thermosensation and the TRPV channel in Rhodnius prolixus

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.Fil: Zermoglio, Paula Florencia. Université François Rabelais de Tours; Francia. 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: Latorre Estivalis, Jose Manuel. Fundación Oswaldo Cruz; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Crespo, José Emilio. Université François Rabelais de Tours; Francia. 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: Lorenzo, Marcelo Gustavo. Fundación Oswaldo Cruz; BrasilFil: Lazzari, Claudio Ricardo. Université François Rabelais de Tours; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

<i>Foraging</i> gene expression is decreased in <i>R</i>. <i>prolixus</i> infected with <i>T</i>. <i>rangeli</i>.

<p>Expression of <i>Rpfor</i> was evaluated by qPCR in uninfected and (A) <i>T</i>. <i>cruzi-</i> or (B) <i>T</i>. <i>rangeli-</i>infected <i>R</i>. <i>prolixus</i> nymphs. Bars represent the mean fold change of <i>Rpfor</i> mRNA levels normalized relative to those of uninfected control insects ± SE (converted to same arbitrary scale as the means); n = 3 pools (5 brains each) per treatment, except for <i>T</i>. <i>rangeli</i> controls, n = 2 pools (5 brains each).</p