DOPA decarboxylase is essential for cuticle tanning in Rhodnius prolixus (Hemiptera: Reduviidae), affecting ecdysis, survival and reproduction

Cuticle tanning occurs in insects immediately after hatching or molting. During this process, the cuticle becomes dark and rigid due to melanin deposition and protein crosslinking. In insects, different from mammals, melanin is synthesized mainly from dopamine, which is produced from DOPA by the enzyme DOPA decarboxylase. In this work, we report that the silencing of the RpAadc-2 gene, which encodes the putative Rhodnius prolixus DOPA decarboxylase enzyme, resulted in a reduction in nymph survival, with a high percentage of treated insects dying during the ecdysis process or in the expected ecdysis period. Those treated insects that could complete ecdysis presented a decrease in cuticle pigmentation and hardness after molting. In adult females, the knockdown of AADC-2 resulted in a reduction in the hatching of eggs; the nymphs that managed to hatch failed to tan the cuticle and were unable to feed. Despite the failure in cuticle tanning, knockdown of the AADC-2 did not increase the susceptibility to topically applied deltamethrin, a pyrethroid insecticide. Additionally, our results showed that the melanin synthesis pathway did not play a major role in the detoxification of the excess (potentially toxic) tyrosine from the diet, an essential trait for hematophagous arthropod survival after a blood meal.Fil: Sterkel, Marcos. 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: 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; ArgentinaFil: Oliveira, Pedro L.. Universidade Federal do Rio de Janeiro; Brasi

DOPA decarboxylase is essential for cuticle tanning in Rhodnius prolixus (Hemiptera: Reduviidae), affecting ecdysis, survival and reproduction

Cuticle tanning occurs in insects immediately after hatching or molting. During this process, the cuticle becomes dark and rigid due to melanin deposition and protein crosslinking. In insects, different from mammals, melanin is synthesized mainly from dopamine, which is produced from DOPA by the enzyme DOPA decarboxylase. In this work, we report that the silencing of the RpAadc-2 gene, which encodes the putative Rhodnius prolixus DOPA decarboxylase enzyme, resulted in a reduction in nymph survival, with a high percentage of treated insects dying during the ecdysis process or in the expected ecdysis period. Those treated insects that could complete ecdysis presented a decrease in cuticle pigmentation and hardness after molting. In adult females, the knockdown of AADC-2 resulted in a reduction in the hatching of eggs; the nymphs that managed to hatch failed to tan the cuticle and were unable to feed. Despite the failure in cuticle tanning, knockdown of the AADC-2 did not increase the susceptibility to topically applied deltamethrin, a pyrethroid insecticide. Additionally, our results showed that the melanin synthesis pathway did not play a major role in the detoxification of the excess (potentially toxic) tyrosine from the diet, an essential trait for hematophagous arthropod survival after a blood meal.Centro Regional de Estudios Genómico

The role of neuropeptides in regulating ecdysis and reproduction in the hemimetabolous insect Rhodnius prolixus

In ecdysozoan animals, moulting entails the production of a new exoskeleton and shedding of the old one during ecdysis. It is induced by a pulse of ecdysone that regulates the expression of different hormonal receptors and activates a peptide-mediated signalling cascade. In Holometabola, the peptidergic cascade regulating ecdysis has been well described. However, very little functional information regarding the neuroendocrine regulation of ecdysis is available for Hemimetabola, which display an incomplete metamorphosis. We use Rhodnius prolixus as a convenient experimental model to test two hypotheses: (1) the role of neuropeptides that regulate ecdysis in Holometabola is conserved in hemimetabolous insects; and (2) the neuropeptides regulating ecdysis play a role in the regulation of female reproduction during the adult stage. The RNA interference-mediated reduction of ecdysis triggering hormone (ETH) mRNA levels in fourth-instar nymphs resulted in lethality at the expected time of ecdysis. Unlike in holometabolous insects, knockdown of eth and orcokinin isoform A (oka) did not affect oviposition in adult females, pointing to a different endocrine regulation of ovary maturation. However, eth knockdown prevented egg hatching. The blockage of egg hatching appears to be a consequence of embryonic ecdysis failure. Most of the first-instar nymphs hatched from the eggs laid by females injected with dsRNA for eclosion hormone (dsEH), crustacean cardioactive peptide (dsCCAP) and dsOKA died at the expected time of ecdysis, indicating the crucial involvement of these genes in post-embryonic development. No phenotypes were observed upon corazonin (cz) knockdown in nymphs or adult females. The results are relevant for evolutionary entomology and could reveal targets for neuropeptide-based pest control tools.Fil: Sterkel, Marcos. 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: Volonté, Mariano. 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: Albornoz, Maximiliano Gastón. 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: Wulff, Juan Pedro. 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: Sanchez Matias, Mariana del Huerto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Terán, Paula Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Ajmat, María Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Ons, Sheila. 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; Argentin

OKB, a novel family of brain-gut neuropeptides from insects

In insects, neuropeptides play a central role in the control of most physiological processes. The knowledge and characterization of new neuropeptide families, is of interest on the fields of Genetics, Genomics, Neurobiology, Endocrinology and Evolution. This knowledge also provides the tools for the design of peptidomimetics, pseudopeptides or small molecules, capable of disrupting the physiological processes regulated by the signaling molecules and their receptors. This is a promising target for a novel generation of insecticides. Using database searches, mass spectrometry and RACE-PCR, we identified a neuropeptide precursor transcript encoding a new family of insect neuropeptides in the hemipteran Rhodnius prolixus. We named this precursor Orcokinin B, because is originated by the alternative splicing of the Orcokinin gen. EST and genomic data suggests that Orcokinin B is expressed in the nervous system and gut from several insect species, with the exception of Drosophila sp. (Diptera) and Acyirthosiphon pisum (Hemiptera). Mass spectrometry and RT-PCR confirmed the expression of Orcokinin B in brain and anterior midgut of R. prolixus. Furthermore, we identified orthologues of this new family of peptides in genomic and EST databases from Arachnids and Crustaceans.Centro Regional de Estudios Genómico

Orcokinin neuropeptides regulate ecdysis in the hemimetabolous insect Rhodnius prolixus

To grow and develop insects must undergo ecdysis. During this process, the individual sheds the old cuticle to emerge as the following developmental stage. During ecdysis, different programed behaviors are regulated by neuropeptidergic pathways. In general, components of these pathways are better characterized in crustacean and holometabolous insects than in hemimetabola. In insects, the orkoninin gene produces two different neuropeptide precursors by alternative splicing: orcokinin A and orcokinin B. Although orcokinins are well conserved in insect species, their physiological role remains elusive. Here we describe a new splicing variant of the orcokinin gene in the hemimetabolous triatomine Rhodnius prolixus. We further analyze the expression pattern and the function of the alternatively spliced RhoprOK transcripts by means of immunohistochemistry and RNAi-mediated gene silencing. Our results indicate that orkoninis play an essential role in the peptidergic signaling pathway regulating ecdysis in the hemimetabolous insect Rhodnius prolixus.Centro Regional de Estudios GenómicosCentro de Endocrinología Experimental y Aplicad

Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (?702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immunedeficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.Fil: Calderón Fernández, Gustavo Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Esponda Behrens, Natalia Irene. 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: Juarez, Marta Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Latorre Estivalis, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Oswaldo Cruz; Brasil. Instituto Nacional de Ciencia e Tecnologia en Entomología Molecular; BrasilFil: Lavore, Andres Esteban. 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: Lazzari, Claudio Ricardo. Université François Rabelais; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lorenzo, Marcelo Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Oswaldo Cruz; Brasil. Instituto Nacional de Ciencia e Tecnologia en Entomología Molecular; BrasilFil: 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; ArgentinaFil: Pagola, Lucia Elena. 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: Pascual, Agustina. 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: Pedrini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Sterkel, Marcos. Universidade Federal do Rio de Janeiro; Brasil. 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

Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes

Genómica y proteómica de neuropéptidos en Rhodnius prolixus

Sterkel, M. (2011). Genómica y proteómica de neuropéptidos en Rhodnius prolixus (Tesis de Doctorado). Universidad Nacional de Quilmes, Bernal, Argentina.La enfermedad de Chagas afecta a 10 millones de personas en el continente americano, mientras que 80 millones se encuentran en riesgo de contraerla. El agente causante de la enfermedad es el protozoo Tripanosoma cruzi, transmitido por insectos hematófagos del orden Hemiptera, siendo las especies más importantes en la transmisión Rhodinus prolixus y Triatoma infestans. Debido a la falta de tratamiento en la etapa crónica de la enfermedad, el control de las poblaciones del vector mediante la fumigación con insecticidas piretroides ha sido, y sigue siendo, la principal estrategia de prevención. Sin embargo, en los últimos años se han detectado poblaciones con una altísima resistencia a este tipo de insecticidas (Picollo et al, 2005). Dado que los neuropéptidos se encuentran involucrados en la regulación de todos los procesos fisiológicos de vida de un insecto (ver Nässel, 2002), se ha propuesto la manipulación de los sistemas de señalización peptídicos como una posible estrategia para el diseño de nuevos insecticidas. En el presente trabajo hemos realizado una caracterización exhaustiva de los neuropéptidos presentes en R. prolixus a nivel de secuencia y estructura, utilizando técnicas bioinformáticas, proteómicas y de biología molecular. Por otra parte, hemos aplicado técnicas de peptidómica cuantitativa para caracterizar la respuesta post-ingesta sanguínea, a fin de comenzar la caracterización fisiológica de los neuropéptidos identificados. Nuestros resultados indican que el neuropeptidoma de R. prolixus es apenas diferente de los demás insectos con el genoma secuenciado y ensamblado, todos ellos holometábolos, presentando particularidades únicas a nivel de secuencia. Este trabajo es el punto de partida para posteriores estudios a nivel fisiológico, y sienta las bases necesarias para posteriores estudios funcionales, así como también para estudios de interacción con los receptores y para estudios comparativos y/o evolutivos con otros artrópodos. Se trata del primer trabajo de este tipo realizado en un insecto vector de enfermedadesBeca del Consejo Nacional de Investigaciones Científicas y Tecnológicas. Beca Arancel, Universidad Nacional de Quilme

Functional proteomics of neuropeptidome dynamics during the feeding process of Rhodnius prolixus

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.Fil: Sterkel, Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; ArgentinaFil: Urlaub, Henning. Institut Max Planck fuer Bioanorganische Chemie; AlemaniaFil: Rivera Pomar, Rolando. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; ArgentinaFil: Ons, Sheila. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin