Análisis genómico y molecular de la embriogénesis de <i>Rhodnius prolixus</i> (Stähl, 1859) (Hemíptera, Reduviidae): implicancias morfológico-evolutivas en insectos

Todos los animales que poseen simetría bilateral se encuentran definidos por dos ejes de simetría ortogonales, el eje anteroposterior (A-P) que corre de la boca al ano y un eje perpendicular a este, el eje dorsoventral (D-V). A pesar de la gran variedad de modos de desarrollo embrionario y formas finales encontradas en los animales, las redes regulatorias y factores de transcripción que dan origen a estos ejes se encuentran muy conservados. De aquí surge una pregunta central, cómo estas redes regulatorias tan conservadas crean tanta diversidad morfológica, se adaptan a nuevos ambientes y de qué manera las novedades evolutivas se incorporan en un sistema de patronamiento ya establecido. El eje DV es un buen sistema de estudio ya que se conoce en detalle en Drosophila melanogaster pero no en otros insectos. Los insectos además presentan una gran variedad de especies y modos de desarrollo embrionario lo que nos permite estudiar de qué manera las redes regulatorias se adaptan a novedades evolutivas y la existencia de varias técnicas que permiten testear el funcionamiento de los genes y sus interacciones. En este contexto hemos utilizado a Rhodnius prolixus como modelo para el estudio del establecimiento del eje DV en un embrión de banda germinal intermedia donde al final del desarrollo el embrión posee la misma forma que el adulto. Hemos estudiado en detalle el desarrollo embrionario de R. prolixus para una mejor comprensión de los patrones de expresión de los genes estudiados y su función. Además, se han buscado y anotado varios genes envueltos en la formación del eje DV: toll, dorsal, decapentaplegic, zerknült, twist y zelda. Mostraremos el patrón de expresión y los fenotipos resultado de ARNi parental de toll, dpp y dorsal, los cuales representan puntos clave en la regulación de la cascada D-V.All animals with bilateral symmetry are defined by two orthogonal axes, the anterior-posterior (A-P) that runs from the mouth to the anus and the dorsoventral (D-V). Despite the variety of modes of embryonic development and animal forms, the regulatory networks and transcription factors, which give rise to these axes, are conserved. This fact gives rise to a central question, how these conserved regulatory networks adapt to a new environment and how evolutionary novelties are incorporated into a patterning system already established? The D-V axis represents a good system to study. It is known in detail in Drosophila melanogaster, but not in other insects. The insects present a variety of species and modes of embryogenesis that allows us to study how the regulatory networks adapt to evolutionary novelties and the existence of several techniques to test the function of genes and their interactions makes of them the ideal model. In this context we have used Rhodnius prolixus as a model to study the establishment of the DV axis in an intermediate germ band embryo were it reaches at the end of its development the same shape of the adult. We have study the embryonic development of R. prolixus in detail to a better understanding of the expression patterns of genes studied and their function. Aldow, we have searched and annotated several genes involved in DV axis formation: toll, dorsal, decapentaplegic, zerknült, twist and zelda. We will show the expression pattern and the phenotypes that result after parental RNAi of toll, dpp and dorsal, which represent key regulatory points of the cascade.Facultad de Ciencias Naturales y Muse

Análisis genómicos y molecular de la embriogénesis de Rhodnius prolixus (Stähl, 1859) (Hemíptera, Reduviidae) : implicancias morfológico-evolutivas en insectos

Tesis presentada para optar al Grado de Doctor en Ciencias NaturalesFil: Pagola, Lucía Elena. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo; Argentin

Genoteca de cDNA normalizada de Rhodnius prolixus

El objetivodel presente trabajo es generar una genoteca normalizada de Rhodnius prolixus, enriquecida en fragmentos de cDNA largos, utilizando un método de normalización basado en una nucleasa termoestable (nucleasa específica de ADN de doble cadena del cangrejo de Kamchatka o Kamchatka Crab Duplex-Specific Nuclease, KCDSN, (Zhulidov et al., 2004), y LD-PCR (Long Distance-PCR) de acuerdo al protocolo descripto por Anisimova et al., (2006). El método se basa en la degradación de la fracción ds cDNA mediante el uso de una nucleasa que tiene especificidad por la doble cadena (DSN). Este método involucra la desnaturalización y renaturalización del cDNA, la degradación de la fracción doble cadena de cDNA (dsDNA), por parte de la nucleasa, y la posterior amplificación de la fracción ds cDNA. El proceso de reasociación de cadenas tiene una cinética de primer orden, en la que los trasncriptos más abundantes se asocian más rápidamente que los de menor concentración. Por lo tanto al cabo de un cierto tiempo la nucleasa cortara en mayor proporcion, transcriptos abundantes. Posteriormente, la nucleasa es inactivada y el ADNc no cortado es amplificado en dos ciclos de PCR. La DSN tiene su máxima actividad a una temperatura de entre 60 – 70 °C, y además se caracteriza por tener una mayor afinidad por el ds cDNA y por los híbridos de ARN-ADN, que por el ss cDNA (Zhulidov, 2004).Eje: Biotecnología. Salud públicaCentro Regional de Estudios Genómico

The gap gene Krüppel of Rhodnius prolixus is required for segmentation and for repression of the homeotic gene sex comb-reduced

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.Facultad de Ciencias Exacta

The gap gene giant of Rhodnius prolixus is maternally expressed and required for proper head and abdomen formation

The segmentation process in insects depends on a hierarchical cascade of gene activity. The first effectors downstream of the maternal activation are the gap genes, which divide the embryo in broad fields. We discovered a sequence corresponding to the leucine-zipper domain of the orthologue of the gene giant (Rp- gt) in traces from the genome of Rhodnius prolixus, a hemipteran with intermediate germ-band development. We cloned the Rp- gt gene from a normalized cDNA library and characterized its expression and function. Bioinformatic analysis of 12.5. kbp of genomic sequence containing the Rp- gt transcriptional unit shows a cluster of bona fide regulatory binding sites, which is similar in location and structure to the predicted posterior expression domain of the Drosophila orthologue. Rp-gt is expressed in ovaries and maternally supplied in the early embryo. The maternal contribution forms a gradient of scattered patches of mRNA in the preblastoderm embryo. Zygotic Rp-gt is expressed in two domains that after germ band extension are restricted to the head and the posterior growth zone. Parental RNAi shows that Rp-gt is required for proper head and abdomen formation. The head lacks mandibulary and maxillary appendages and shows reduced clypeus-labrum, while the abdomen lacks anterior segments. We conclude that Rp-gt is a gap gene on the head and abdomen and, in addition, has a function in patterning the anterior head capsule suggesting that the function of gt in hemipterans is more similar to dipterans than expected.Facultad de Ciencias Exacta

The gap gene giant of Rhodnius prolixus is maternally expressed and required for proper head and abdomen formation

The segmentation process in insects depends on a hierarchical cascade of gene activity. The first effectors downstream of the maternal activation are the gap genes, which divide the embryo in broad fields. We discovered a sequence corresponding to the leucine-zipper domain of the orthologue of the gene giant (Rp- gt) in traces from the genome of Rhodnius prolixus, a hemipteran with intermediate germ-band development. We cloned the Rp- gt gene from a normalized cDNA library and characterized its expression and function. Bioinformatic analysis of 12.5. kbp of genomic sequence containing the Rp- gt transcriptional unit shows a cluster of bona fide regulatory binding sites, which is similar in location and structure to the predicted posterior expression domain of the Drosophila orthologue. Rp-gt is expressed in ovaries and maternally supplied in the early embryo. The maternal contribution forms a gradient of scattered patches of mRNA in the preblastoderm embryo. Zygotic Rp-gt is expressed in two domains that after germ band extension are restricted to the head and the posterior growth zone. Parental RNAi shows that Rp-gt is required for proper head and abdomen formation. The head lacks mandibulary and maxillary appendages and shows reduced clypeus-labrum, while the abdomen lacks anterior segments. We conclude that Rp-gt is a gap gene on the head and abdomen and, in addition, has a function in patterning the anterior head capsule suggesting that the function of gt in hemipterans is more similar to dipterans than expected.Facultad de Ciencias Exacta

The gap gene Krüppel of Rhodnius prolixus is required for segmentation and for repression of the homeotic gene sex comb-reduced

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.Facultad de Ciencias Exacta

Smoking flies: testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster

Studies about the relationship between substances consumed by humans and their impact on health, in animal models, have been a challenge due to differences between species in the animal kingdom. However, the homology of certain genes has allowed extrapolation of certain knowledge obtained in animals. Drosophila melanogaster, studied for decades, has been widely used as model for human diseases as well as to study responses associated with the consumption of several substances. In the present work we explore the impact of tobacco consumption on a model of ‘smoking flies’. Throughout these experiments, we aim to provide information about the effects of tobacco consumption on cardiac physiology. We assessed intracellular calcium handling, a phenomenon underlying cardiac contraction and relaxation. Flies chronically exposed to tobacco smoke exhibited an increased heart rate and alterations in the dynamics of the transient increase of intracellular calcium in myocardial cells. These effects were also evident under acute exposure to nicotine of the heart, in a semi-intact preparation. Moreover, the alpha 1 and 7 subunits of the nicotinic receptors are involved in the heart response to tobacco and nicotine under chronic (in the intact fly) as well as acute exposure (in the semi-intact preparation). The present data elucidate the implication of the intracellular cardiac pathways affected by nicotine on the heart tissue. Based on the probed genetic and physiological similarity between the fly and human heart, cardiac effects exerted by tobacco smoke in Drosophila advances our understanding of the impact of it in the human heart. Additionally, it may also provide information on how nicotine-like substances, e.g. neonicotinoids used as insecticides, affect cardiac function.Centro de Investigaciones CardiovascularesCentro de Estudios Parasitológicos y de Vectore

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.La lista completa de autores que integran el documento puede consultarse en el archivoEste documento tiene una corrección (ver documento relacionado).Centro Regional de Estudios GenómicosInstituto de Investigaciones Bioquímicas de La Plat