Caracterización genómico funcional de los neuropéptidos Allatotropina y Allatostatina-C y sus receptores en insectos vectores de la enfermedad de Chagas

Allatotropina (AT) y Allatostatina-C (AST-C), son neurohormonas de naturaleza proteica caracterizadas por su capacidad de estimular o inhibir la síntesis de hormonas juveniles en insectos, respectivamente. Estos péptidos han demostrado ser pleiotrópicos. AT cumple diferentes funciones a nivel del sistema nervioso central como neuromodulador además de controlar ritmos circadianos, estimula la síntesis de enzimas y controla el transporte de iones a nivel del tubo digestivo, intervienen en la actividad fagocítica de las células del sistema inmune, y se encuentra regulando la frecuencia de contracción de la musculatura asociada al tubo digestivo y del vaso dorsal. Por otro lado, la información sobre las funciones que desempeña AST-C es escasa. Se ha descripto que modula negativamente las contracciones de la musculatura visceral del intestino anterior en lepidópteros y del vaso dorsal de algunos dípteros. Rhodnius prolixus (Hemiptera:Reduvidae), es un insecto hematófago vector del Mal de Chagas, enfermedad endémica de América, la cual provoca patologías coronarias y asociadas al tubo digestivo. El causante de esta enfermedad en el parásito Trypanosoma cruzi y la trasmisión del mismo ocurre cuando los insectos se alimentan. En ese momento se desencadena el proceso de diuresis post ingesta, generando la eliminación de grandes cantidades de orina y heces sobre el huésped. El parásito ingresa al mismo a través de estos desechos por la injuria provocada por el aparato sucto-picador. El proceso de diuresis es controlado por mecanismos neuroendocrinos, para reestablecer el desbalance hídrico-mineral provocado durante la ingesta. Nuestro laboratorio ha demostrado en la especie afín, Triatoma infestans, que AT interviene en la modulación de la frecuencia cardíaca y de la tasa de ondas peristálticas en el tubo digestivo durante este proceso, lo cual propicia la recirculación de la hemolinfa. Nos es relevante entonces, caracterizar la actividad de este péptido y analizar el efecto del péptido allatostático AST-C, así como la expresión de sus receptores en R. prolixus, en el sistema digestivo, circulatorio y reproductor durante momentos fisiológicos cruciales en la vida del insecto como lo son el proceso de diuresis post-prandial, la cópula y la oviposición en la hembra adulta. Se evaluó la capacidad miomoduladora de AT y AST-C durante distintos momentos fisiológicos. AT demostró poseer la capacidad de sinergizar el efecto estimulador de la serotonina, incrementando la frecuencia de contracción de la aorta en insectos machos ayunados, mientras que AST-C antagonizó dicha actividad, disminuyendo significativamente la frecuencia de este órgano. Así mismo, este péptido disminuyo la actividad contráctil de manera dosis dependiente durante el proceso de diuresis post ingesta tanto en la aorta como en el buche. Mediante el uso de microscopía de barrido laser confocal se demostró la presencia de AST-C en los ganglios nerviosos ventrales (órganos neurohemales), en la hemolinfa del interior del corazón, en los nefrocitos asociados a los músculos alares, y en las células secretoras del epitelio del buche. El efecto miomodulador de AT y AST-C en el sistema reproductor de la hembra fue analizado en diferentes estados fisiológicos (hembras ayunadas, vírgenes y copuladas - alimentadas y grávidas). Cada uno de los péptidos respondió de manera dosis dependiente. Los eventos de la cópula y la ingesta sanguínea demostraron ser condicionantes para que cada péptido desempeñe su actividad. Más aún, cada órgano que se encuentra conformando este sistema respondió de manera diferencial ante la presencia de los péptidos. AT demostró ser mioestimulador en ambos casos, mientras que no hubo respuesta sobre los órganos en hembras vírgenes que no habían recibido alimento. Por otro lado, AST-C provocó un descenso significativo en la frecuencia de contracción de los ovarios de las hembras grávidas. Los receptores de ambos péptidos fueron clonados, secuenciados y registrados en el Genbank. Su expresión fue evidenciada en los órganos en estudios además de los tubos de Malpighi y recto. Los mismos pertenecen a la familia de los GPCRs (receptores de membrana acoplados a proteína G). Mediante el análisis genómico y bioinformática identificamos que las características propias de estos receptores se encuentran altamente conservadas entre las secuencias homólogas de insectos. Dichos análisis arrojaron como resultado que el receptor de AT de R. prolixus comparte características estructurales con los receptores de orexina de mamíferos, tales como la secuencia ubicada en el segundo dominio intracelular DRW, mientras que el receptor de AST-C es homólogo con aquellas secuencias que se encuentran relacionadas evolutivamente con los receptores de somatostatina de mamíferos, ya que poseen no solo la secuencia de DRY, característica de la familia de los GPCR, sino que además presenta el motivo secuencial YSNSAMNPILYA, altamente conservado en los receptores tipo somatostatina. En conclusión, utilizando diferentes disciplinas y metodologías, el presente trabajo de investigación cuenta con el primer reporte en cuanto a la relación funcional de péptidos allatotrópicos como moduladores del a musculatura visceral. Se ha profundizado en el entendimiento de las funciones mioestimuladoras que cumple AT, mientras que se ha descripto por primera vez el efecto antagónico de AST-C sobre el mismo. Se demuestra, además, su distribución en insectos triatominos explicando su probable fuente de síntesis y acción. Siendo el genoma de esta especie accesible llevamos a cabo la caracterización genómica de ambos receptores, cuya familia a la que pertenecen es de sumo interés ya que podrían ser utilizadas como potenciales alternativas para el desarrollo de nuevos agentes selectivos para el control de plagas.Allatotropin (AT) and Allatostatin-C (AST-C) are neuropeptides characterized by their ability to stimulate or inhibit the juvenile hormones synthesis in insects, respectively. They are pleiotropic too: AT performs different functions at the level of the central nervous system as a neuromodulator and as a controller of circadian rhythms. Also, AT stimulates the synthesis of enzymes and regulate the transport of ions at the level of the digestive tract ; it is implicated in the phagocytic activity in the cells of the immune system, and stimulates the frequency of contraction of the wall muscles of the gut and dorsal vessel. On the other hand, little is known about functions that AST-C could perfom. It acts as a myoinhibitory peptide on the spontaneous contractions of the anterior gut in lepidopterous and in the heart in dipterous. The Chagas disease vector, Rhodnius prolixus (Hemiptera: Reduvidae), is a haematophagous insect useful to design physiological experiments to increase the knowledge about the biology of this insect with health importance. This illness is endemic of America and is caused by the parasite Trypanosoma cruzi, which provoke coronary and gut pathologies. The transmission occurs when the insect feeds on blood. At that moment the process of diuresis post prandial starts, provoking the elimination of large amounts of urine and feces on the host. The parasite enters through these wastes by the injury caused by the insect when its feeds. During the diuresis process, the mineral hydric balance is altered and controlled by neuroendocrine mechanisms. Our laboratory has shown in the related specie, Triatoma infestans, that AT is involved as miomodulator in the aorta´s frequency and peristaltic wave´s rate of the gut during the diuresis process. The movements of the wall of these organs cause the re-circulation of the haemolymph during this process. The aim of this research is to increase de knowledge about the functional characterization of the neuropeptides AT and AST-C as well as the expression of their receptors in R. prolixus, in the digestive, circulatory and reproductive system during the crucial physiological events in the life cycle of the insects like diuresis process, the mate and oviposition in the adult female. The ability of AT and AST-C to modulate the visceral muscles was evaluated during different physiological states. AT synergizes the stimulatory effect of serotonin, increasing the frequency of contraction of the aorta in unfed male insects, whereas AST-C antagonized this effect, reducing significantly the frequency of this organ. AST-C inhibitory effect was also evaluated over the frequency of peristaltic waves of the crop as well as the aorta during the diuresis process. Images of confocal laser scanning microscopy showed the presence of AST-C in the ventral nerves ganglions (neurohaemals organs), in the haemolymph inside the heart, in the nephrocytes associated with the allary muscles, and in the secretory cells of the crop epithelium. The myomodulatory effect of AT and AST-C on the female reproductive system was evaluated at different physiological states, both of them acted as dose dependent way. Mating and feeding events are factors that modulate the activity of both peptides. Moreover, each organ of this system showed to be affected on different way when the peptides had been applied. Myostimulatory effect of AT was carry out in mated unfed females as well as on gravid ones, whereas there was no response on the organs in unfed virgin females. On the other hand, AST-C caused a significative decrease in the frequency of contraction of gravid ovaries. AT and AST-C receptors were sequenced and annotated in Genbank. They belong to the GPCRs family (G protein coupled receptors). The genomic and bioinformatic analysis let us identify that the structure characteristics of these receptors are highly conserved among the homologous insects sequences. R. prolixus AT receptor shares structural features with mammalian orexin receptors, such as the aminoacid sequence DRW located in the second intracellular domain, whereas the AST-C receptor seems to be homologous with those sequences which are found to be related to mammalian somatostatin receptors, because they possess not only the DRY sequence, characteristic of the GPCR family, but also presents the sequential motif YSNSAMNPILYA, highly conserved in somatostatin like receptors. In summary, by the use of different methods and disciplines, this research has the first report on the functional relationship of allatotropic peptides as myoregulators over the aorta, crop and the reproductive female system. We deepen the understanding of the myostimulatory functions of AT, while the antagonistic effect of AST-C over the AT activity has been described for the first time. Couse the genome of this insect is available, we carry out the genomic characterization of both receptors. Nowadays, GPCR family to which they belong has a great interest because they just could be use as new selective agents for pest control. In summary, by the use of different methods and disciplines, this research work has the first report on the functional relationship of allatotropic peptides as myoregulators over de aorta, crop and the reproductive female system. We deepen the understanding of the myostimulatory functions of AT, while the antagonistic effect of AST-C over the AT activity it has been described for the first time. It is also demonstrated that the distribution of this peptide in triatomine insects that explain its probable source of synthesis and activity on the studied organs. Couse the genome of this insect is available, we carry out the genomic characterization of both receptors. Nowadays, GPCR family to which they belong has a great interest because they just could be use as new selective agents for pest control.Facultad de Ciencias Naturales y Muse

Caracterización genómico funcional del neuropéptido Allatotropina y Allatostatina-C y su receptor en insectos vectores de la enfermedad de Chagas

Tesis presentada para optar al Grado de Doctor en Ciencias NaturalesFil: Villalobos Sambucaro, María José. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo; Argentin

Allatostatin-C antagonizes the synergistic myostimulatory effect of allatotropin and serotonin in <i>Rhodnius prolixus</i> (Stal)

Haematophagous insects can ingest large quantities of blood in a single meal producing a large quantity of urine in the following hours to eliminate the excess of water and mineral ions incorporated. The excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of the intensification of aorta contractions, combined with an increase of anterior midgut peristaltic waves. We have recently shown that haemolymph circulation during post-prandial diuresis is modulated by the synergistic activity of allatotropin (AT) and serotonin, resulting in an increase in aorta and crop contraction rates. In the present study we describe the antagonistic effect of allatostatin-C (AST-C) on the increase of aorta frequency of contractions induced by serotonin/AT in Rhodnius prolixus. The administration of AST-C counteracted the increase in the frequency induced by the treatment with serotonin/ AT, but did not affect the increase in frequency induced by the administration of serotonin alone, suggesting that AST-C is altering the synergism between serotonin and AT. Furthermore, the administration of AST-C during post-prandial diuresis decreases the number of peristaltic waves of the anterior midgut. The AST-C putative receptor is expressed in the hindgut, midgut and dorsal vessel, three critical organs involved in post-prandial diuresis. All together these findings provide evidence that AST-C plays a key role as a myoregulatory and cardioregulatory peptide in R. prolixus.Facultad de Ciencias Naturales y MuseoCentro Regional de Estudios Genómico

The Evolutionary History of The Orexin/Allatotropin GPCR Family: From Placozoa and Cnidaria to Vertebrata

Peptidic messengers constitute a highly diversified group of intercellular messengers widely distributedin nature that regulate a great number of physiological processes in Metazoa. Being crucial for life, itseem that they have appeared in the ancestral group from which Metazoa evolved, and were highlyconserved along the evolutionary process. Peptides act mainly through G-protein coupled receptors(GPCRs), a family of transmembrane molecules. GPCRs are also widely distributed in nature beingpresent in metazoan, but also in Choanoflagellata and Fungi. Among GPCRs, the Allatotropin/Orexin(AT/Ox) family is particularly characterized by the presence of the DRW motif in the second intracellularloop (IC Loop 2), and seems to be present in Cnidaria, Placozoa and in Bilateria, suggesting that it waspresent in the common ancestor of Metazoa. Looking for the evolutionary history of this GPCRs wesearched for corresponding sequences in public databases. Our results suggest that AT/Ox receptorswere highly conserved along evolutionary process, and that they are characterized by the presenceof the E/DRWYAI motif at the IC Loop 2. Phylogenetic analyses show that AT/Ox family of receptorsreflects evolutionary relationships that agree with current phylogenetic understanding in Actinopterygiiand Sauropsida, including also the largely discussed position of TestudinesFil: Alzugaray, Maria Eugenia. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Bruno, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; ArgentinaFil: Villalobos Sambucaro, María José. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Ronderos, Jorge Rafael. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentin

Allatotropin modulates myostimulatory and cardioacceleratory activities in <i>Rhodnius prolixus</i> (Stal)

Haematophagous insects can ingest large quantities of blood in a single meal and eliminate high volumes of urine in the next few hours. This rise in diuresis is possible because the excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of intensification of aorta contractions combined with an increase of the anterior midgut peristaltic waves. It has been previously described that haemolymph circulation during post-prandial diuresis is stimulated by the synergistic activity of allatotropin (AT) and serotonin in the kissing bug Triatoma infestans; resulting in an increase in aorta contractions. In the same species, AT stimulates anterior midgut and rectum muscle contractions to mix urine and feces and facilitate the voiding of the rectum. Furthermore, levels of AT in midgut and Malpighian tubules increased in the afternoon when insects are getting ready for nocturnal feeding. In the present study we describe the synergistic effect of AT and serotonin increasing the frequency of contractions of the aorta in Rhodnius prolixus. The basal frequency of contractions of the aorta in the afternoon is higher that the observed during the morning, suggesting the existence of a daily rhythmic activity. The AT receptor is expressed in the rectum, midgut and dorsal vessel, three critical organs involved in post-randial diuresis. All together these findings provide evidence that AT plays a role as a myoregulatory and cardioacceleratory peptide in R. prolixus.Facultad de Ciencias Naturales y Muse

Allatotropin modulates myostimulatory and cardioacceleratory activities in <i>Rhodnius prolixus</i> (Stal)

Haematophagous insects can ingest large quantities of blood in a single meal and eliminate high volumes of urine in the next few hours. This rise in diuresis is possible because the excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of intensification of aorta contractions combined with an increase of the anterior midgut peristaltic waves. It has been previously described that haemolymph circulation during post-prandial diuresis is stimulated by the synergistic activity of allatotropin (AT) and serotonin in the kissing bug Triatoma infestans; resulting in an increase in aorta contractions. In the same species, AT stimulates anterior midgut and rectum muscle contractions to mix urine and feces and facilitate the voiding of the rectum. Furthermore, levels of AT in midgut and Malpighian tubules increased in the afternoon when insects are getting ready for nocturnal feeding. In the present study we describe the synergistic effect of AT and serotonin increasing the frequency of contractions of the aorta in Rhodnius prolixus. The basal frequency of contractions of the aorta in the afternoon is higher that the observed during the morning, suggesting the existence of a daily rhythmic activity. The AT receptor is expressed in the rectum, midgut and dorsal vessel, three critical organs involved in post-randial diuresis. All together these findings provide evidence that AT plays a role as a myoregulatory and cardioacceleratory peptide in R. prolixus.Facultad de Ciencias Naturales y Muse

The Evolutionary History of The Orexin/Allatotropin GPCR Family : From Placozoa and Cnidaria to Vertebrata

Peptidic messengers constitute a highly diversifed group of intercellular messengers widely distributed in nature that regulate a great number of physiological processes in Metazoa. Being crucial for life, it seem that they have appeared in the ancestral group from which Metazoa evolved, and were highly conserved along the evolutionary process. Peptides act mainly through G-protein coupled receptors (GPCRs), a family of transmembrane molecules. GPCRs are also widely distributed in nature being present in metazoan, but also in Choanofagellata and Fungi. Among GPCRs, the Allatotropin/Orexin (AT/Ox) family is particularly characterized by the presence of the DRW motif in the second intracellular loop (IC Loop 2), and seems to be present in Cnidaria, Placozoa and in Bilateria, suggesting that it was present in the common ancestor of Metazoa. Looking for the evolutionary history of this GPCRs we searched for corresponding sequences in public databases. Our results suggest that AT/Ox receptors were highly conserved along evolutionary process, and that they are characterized by the presence of the E/DRWYAI motif at the IC Loop 2. Phylogenetic analyses show that AT/Ox family of receptors refects evolutionary relationships that agree with current phylogenetic understanding in Actinopterygii and Sauropsida, including also the largely discussed position of Testudines.Facultad de Ciencias Naturales y Muse

Allatotropin Modulates Myostimulatory and Cardioacceleratory Activities in Rhodnius prolixus (Stal)

Fil: Villalobos-Sambucaro, María José. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra Histología y Embriología Animal; ArgentinaFil: Lorenzo-Figueiras, Alicia Nieves. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: Riccillo, Fernando Luis. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra Histología y Embriología Animal; ArgentinaFil: Diambra, Luis Anibal. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; ArgentinaFil: Noriega, Fernando Gabriel. Florida International University. Department of Biological Sciences; USAFil: Ronderos, Jorge Rafael. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra Histología y Embriología Animal; Argentin

Allatostatin-C antagonizes the synergistic myostimulatory effect of allatotropin and serotonin in <i>Rhodnius prolixus</i> (Stal)

Haematophagous insects can ingest large quantities of blood in a single meal producing a large quantity of urine in the following hours to eliminate the excess of water and mineral ions incorporated. The excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of the intensification of aorta contractions, combined with an increase of anterior midgut peristaltic waves. We have recently shown that haemolymph circulation during post-prandial diuresis is modulated by the synergistic activity of allatotropin (AT) and serotonin, resulting in an increase in aorta and crop contraction rates. In the present study we describe the antagonistic effect of allatostatin-C (AST-C) on the increase of aorta frequency of contractions induced by serotonin/AT in Rhodnius prolixus. The administration of AST-C counteracted the increase in the frequency induced by the treatment with serotonin/ AT, but did not affect the increase in frequency induced by the administration of serotonin alone, suggesting that AST-C is altering the synergism between serotonin and AT. Furthermore, the administration of AST-C during post-prandial diuresis decreases the number of peristaltic waves of the anterior midgut. The AST-C putative receptor is expressed in the hindgut, midgut and dorsal vessel, three critical organs involved in post-prandial diuresis. All together these findings provide evidence that AST-C plays a key role as a myoregulatory and cardioregulatory peptide in R. prolixus.Facultad de Ciencias Naturales y MuseoCentro Regional de Estudios Genómico

The juvenile hormone described in Rhodnius prolixus by Wigglesworth is juvenile hormone III skipped bisepoxide

Juvenile hormones (JHs) are sesquiterpenoids synthesized by the corpora allata (CA). They play critical roles during insect development and reproduction. The frst JH was described in 1934 as a “metamorphosis inhibitory hormone” in Rhodnius prolixus by Sir Vincent B. Wigglesworth. Remarkably, in spite of the importance of R. prolixus as vectors of Chagas disease and model organisms in insect physiology, the original JH that Wigglesworth described for the kissing-bug R. prolixus remained unidentifed. We employed liquid chromatography mass spectrometry to search for the JH homologs present in the hemolymph of fourth instar nymphs of R. prolixus. Wigglesworth’s original JH is the JH III skipped bisepoxide (JHSB3), a homolog identifed in other heteropteran species. Changes in the titer of JHSB3 were studied during the 10-day long molting cycle of 4th instar nymph, between a blood meal and the ecdysis to 5th instar. In addition we measured the changes of mRNA levels in the CA for the 13 enzymes of the JH biosynthetic pathway during the molting cycle of 4th instar. Almost 90 years after the frst descriptions of the role of JH in insects, this study fnally reveals that the specifc JH homolog responsible for Wigglesworth’s original observations is JHSB3.Fil: Villalobos Sambucaro, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; ArgentinaFil: Nouzova, Marcela. Florida International University; Estados UnidosFil: Ramirez, Cesar E.. Florida International University; Estados UnidosFil: Alzugaray, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; ArgentinaFil: Fernandez-Lima, Francisco. Florida International University; Estados UnidosFil: Ronderos, Jorge Rafael. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; ArgentinaFil: Noriega, Fernando. Florida International University; Estados Unido