Translational control is a key step in gene expression regulation during apoptosis. To understand the mechanisms of mRNA translation of a pro‐apoptotic gene, reaper (rpr), we adapted the ...tobramycin‐aptamer technique described by Hartmuth et al. (Proc. Natl. Acad. Sci. USA 2002, 99, 16719–16724) for the analysis of proteins interacting with rpr 5' untranslated region (UTR). We assembled ribonucleoprotein complexes in vitro using translation extracts derived from Drosophila embryos and purified the RNA‐protein complexes for mas spectrometry analysis. We identified the proteins bound to the 5' UTR of rpr. One of them, the La antigen, was validated by RNA‐crosslinking experiments using recombinant protein and by the translation efficiency of reporter mRNAs in Drosophila cells after RNAinterference experiments. Our data provide evidence of the involvement of La antigen in the translation of rpr and set a protocol for purification of tagged‐RNA‐protein complexes from cytoplasmic extracts.
Stinkbugs (Hemiptera: Pentatomidae) are of major economic importance as pest of crops. Among the species composing the stinkbug complex, Nezara viridula is one of the most abundant in Brazil, ...Argentina and the Southern USA. However, this species has been poorly characterized at the genetic and physiological level. Here we sequenced and analyzed the complete transcriptome of N. viridula male and female adults. We identified neuropeptide precursor genes and G-protein coupled receptors for neuropeptides in this transcriptome. Mature neuropeptides were identified in N. viridula brain extracts by liquid chromatography-tandem mass spectrometry. We also analyzed the neuropeptide precursor complement in the genome sequence of Halyomorpha halys, another pentatomid of economic relevance. We compared the results in both pentatomids with the well-characterized neuropeptide repertoire from the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae). We identified both group-specific features (which could be related to the different feeding habits) and similarities that could be characteristic of Heteroptera. This work contributes to a deeper knowledge of the genetic information of these pests, with a focus on neuroendocrine system characterization.
The importance of Chagas disease motivated the scientific effort to obtain the complete genomic sequence of the vector species Rhodnius prolixus, this information is also relevant to the ...understanding of triatomine biology in general. The central nervous system is the key regulator of insect physiology and behavior. Neurohormones (neuropeptides and biogenic amines) are the chemical messengers involved in the regulation and integration of neuroendocrine signals. In insects, this signaling is mainly mediated by the interaction of neurohormone ligands with G protein coupled receptors (GPCRs). The recently sequenced R. prolixus genome provides us with the opportunity to analyze this important family of genes in triatomines, supplying relevant information for further functional studies. Next-generation sequencing methods offer an excellent opportunity for transcriptomic exploration in key organs and tissues in the presence of a reference genome as well as when a reference genome is not available. We undertook a genomic analysis to obtain a genome-wide inventory of opsines and the GPCRs for neurohormones in R. prolixus. Furthermore, we performed a transcriptomic analysis of R. prolixus central nervous system, focusing on neuropeptide precursor genes and neurohormone and opsines GPCRs. In addition, we mined the whole transcriptomes of Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis – three sanitary relevant triatomine species – to identify neuropeptide precursors and GPCRs genes. Our study reveals a high degree of sequence conservation in the molecular components of the neuroendocrine system of triatomines.
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•We identified GPCRs for neurohormones and opsins in Rhodnius prolixus genome sequence.•Thirty-seven of the identified proteins had matching EST evidence in R. prolixus CNS.•ESTs from GPCRs were identified in Triatoma spp.•We describe the transcriptome of R. prolixus central nervous system.•The study reveals a high degree of conservation in the neuroendocrine system in triatomines.
In hematophagous insects, blood intake triggers a prompt response mediated by neuropeptides, which regulates a variety of physiological processes. Here we report a quantitative proteomic analysis of ...the postfeeding response in the central nervous system of Rhodnius prolixus, a vector of Chagas disease. The concentration of neuropeptides NVP-like, ITG-like, kinin-precursor peptide, and neuropeptide-like precursor 1 (NPLP1) significantly changes in response to blood intake. We also performed a neuropeptidomic analysis of other feeding-related organs, namely salivary glands and gut. We identified NPLP1 in salivary glands and myosuppressin in midgut. This is the first report suggesting a role for NPLP1, involving the peptides processed from this precursor in the hormonal control of the production and/or release of saliva. Our results contribute to the understanding of the postprandial neuroendocrine response in hematophagous and provide important information for physiological and pharmacological studies aimed to the design of next-generation insecticides such as peptidomimetics.
The Drosophila body organizer Bicoid (Bcd) is a maternal homeodomain protein. It forms a concentration gradient along the longitudinal axis of the preblastoderm embryo and activates early zygotic ...segmentation genes in a threshold‐dependent fashion. In addition, Bcd acts as a translational repressor of maternal caudal (cad) mRNA in the anterior region of the embry o. This process involves a distinct Bcd‐binding region (BBR) in the 3′ untranslated region (UTR) of cad mRNA. Using cotransfection assays, we found that Bcd represses translation in a cap‐dependent manner. Bcd‐dependent translational repression involves a portion of the PEST motif of Bcd, a conserved protein motif best known for its function in protein degradation. Rescue experiments with Bcd‐deficient embryos expressing transgene‐derived Bcd mutants indicate that amino acid replacements within the C‐terminal portion of the PEST motif prevent translational repression of cad mRNA but allow for Bcd‐dependent transcriptional activation. Thus, Bcd contains separable protein domains for transcriptional and translational regulation of target genes. Maternally‐derived cad protein in the anterior region of embryos interferes with head morphogenesis, showing that cad mRNA suppression by Bcd is an important control event during early Drosophila embryogenesis.
The establishment of the anterior–posterior segmentation in insects requires the concerted action of a hierarchical gene network. Here, we study the orthologue of Krüppel gap gene in the hemipteran ...Rhodnius prolixus (Rp-Kr). We characterized its structure, expression pattern and function. The genomic sequence upstream of the Rp-Kr transcriptional unit shows a putative regulatory region conserved in the orthologue genes from Drosophila melanogaster and Tribolium castaneum. Rp-Kr expression is zygotic and it is expressed in the anterior half of the embryo (the posterior half of the egg) during the blastoderm stage and germ band formation; later, during germ band extension, it is expressed in a central domain, from T2 to A3. The Rp-Kr loss of function phenotypes shows disrupted thoracic and abdominal segmentation. Embryos with weak segmentation phenotypes show homeotic transformations, in which an ectopic tibial comb, typical of T1 leg, appears in T2, which correlates with the ectopic expression of Rp-sex-comb reduced in this leg.
•We identify the Krüppel ortholog gene in R. prolixus.•A Rp-Kr conserved regulatory cluster corresponds to Drosophila CD1.•RNAi experiments evidence a classical gap phenotype.•Rp-Kr-embryos show a homeotic phenotype likely due to derepression of Scr.
The establishment of the anterior-posterior segmentation in insects requires the concerted action of a hierarchical gene network. Here, we study the orthologue of Krueppel gap gene in the hemipteran ...Rhodnius prolixus (Rp-Kr). We characterized its structure, expression pattern and function. The genomic sequence upstream of the Rp-Kr transcriptional unit shows a putative regulatory region conserved in the orthologue genes from Drosophila melanogaster and Tribolium castaneum. Rp-Kr expression is zygotic and it is expressed in the anterior half of the embryo (the posterior half of the egg) during the blastoderm stage and germ band formation; later, during germ band extension, it is expressed in a central domain, from T2 to A3. The Rp-Kr loss of function phenotypes shows disrupted thoracic and abdominal segmentation. Embryos with weak segmentation phenotypes show homeotic transformations, in which an ectopic tibial comb, typical of T1 leg, appears in T2, which correlates with the ectopic expression of Rp-sex-comb reduced in this leg.
The process of body prepatterning during Drosophila blastoderm formation relies on the localized activities of zygotic segmentation genes, which are controlled by asymmetrically distributed maternal ...determinants. The anterior determinant bicoid, a homeodomain transcription factor, forms an anterior-to-posterior concentration gradient. It interacts with the maternal transcription factor hunchback to activate the anterior zygotic patterning genes, including the central gap gene Krüppel (Kr). In contrast, the posterior maternal system does not provide such a decisive transcription factor, but rather prevents the repressor hunchback from acting in the posterior half so that the gap genes giant (gt) and knirps (kni) are activated by an as yet unknown transcription factor. Here we show that caudal, a conserved homeodomain protein that forms a posterior-to-anterior concentration gradient, and the anterior determinant bicoid cooperate to form a partly redundant activator system in the posterior region of the embryo.
► We predicted the sequence of the region IIS4–IIS6 from the para-type sodium channel gene in the triatomine Rhodnius prolixus. ► We sequenced the IIS4–IIS6 region in the triatomine Triatoma ...infestans. ► We identified a point mutation in the IIS4–IIS6 region that is associated with pyrethroid resistance in T. infestans. ► We designed and developed a method based in PCR for the detection of the kdr mutation in T. infestans from the field.
The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in the domain II region of the channel have been implicated in pyrethroid resistance of several insect species. We identified the sequence of domain II from the para sodium channel in Rhodnius prolixus, a vector of Chagas’ disease. With this information, we cloned and sequenced the domain II of the sodium channel from the other main Chagas’ disease vector: Triatoma infestans. We also identified the presence of a resistance-conferring mutation (L1014F) in a pyrethroid-resistant population of T. infestans from Argentina, and present a PCR-based method to detect this mutation in individuals from field populations. These findings have important implications for the implementation of strategies for resistance management, and for the rational design of campaigns for the control of Chagas’ disease transmission.
Background Chagas' disease is an important public health concern in Latin America. Despite intensive vector control efforts using pyrethroid insecticides, the elimination of Triatoma infestans has ...failed in the Gran Chaco, an ecoregion that extends over Argentina, Paraguay, Bolivia and Brazil. The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in domain II region of the channel have been implicated in pyrethroid resistance of several insect species. Methods and Findings In the present paper, we identify L925I, a new pyrethroid resistance-conferring mutation in T. infestans. This mutation has been found only in hemipterans. In T. infestans, L925I mutation occurs in a resistant population from the Gran Chaco region and is associated with inefficiency in the control campaigns. We also describe a method to detect L925I mutation in individuals from the field. Conclusions and Significance The findings have important implications in the implementation of strategies for resistance management and in the rational design of campaigns for the control of Chagas' disease transmission.
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