Análisis genómico-funcional de la embriología de <i>Rhodnius prolixus</i> (Ståhl, 1859) (Hemíptera, Reduviidae)

La genómica y biología del desarrollo de Rhodnius prolixus (vector de la enfermedad del Chagas) no fue estudiada sino hasta recientemente. El genoma de R. prolixus fue secuenciado y actualmente se encuentra en proceso de anotación. En este contexto, y dentro del consorcio de secuenciación del genoma de R. prolixus, se llevó a cabo la construcción de una genoteca normalizada de ADNc incluyendo todos los estadíos de desarrollo, desde huevos hasta insectos adultos, tanto hembras como machos. Tanto esta genoteca como la última versión de anotación del genoma de R. prolixus fueron utilizados para la búsqueda de genes de segmentación. Los genes identificados y caracterizados fueron los siguientes: giant, krüppel, hunchback, knirps, tailless, orthodenticle, empty-spiracles, forkhead, hairy, even skipped, runt y engrailed. A partir de la genoteca normalizada de ADNc se clonaron los genes Rp-gt y Rp-Kr, los cuales fueron caracterizados, mostrando su patrón de expresión y función durante el desarrollo embrionario. Mediante el análisis bioinformático de la región genómica upstream de las unidades transcripcionales de Rp-gt y Rp-Kr, se pudo identificar (para cada uno de estos genes) un potencial elemento regulatorio. Estos resultaron ser similares en posición y composición a los sitios de unión a factores de transcripción analizados en Drosophila melanogaster. Como referencia también se analizo la región equivalente en Tribolium castaneum. Rp-gt muestra expresión materna, de forma tal que el transcripto se encuentra en ovarios y oocitos sin fertilizar. En embriones en estadío de pre-blastodermo la distribución del transcripto es en parches formando un gradiente posterior. La expresión cigótica de Rp-gt se da en dos dominios, uno cefálico y otro abdominal. Mediante ARNi parental, se pudo ver que Rp-gt es requerido para la correcta formación de la cabeza y el abdomen. La cabeza pierde los apéndices mandibulares y maxilares, se reduce la longitud del clípeo-labro y el abdomen pierde segmentos anteriores. La expresión de Rp-Kr es cigótica. Durante el proceso de invaginación del embrión, Rp-Kr se expresa en la mitad posterior del huevo, mientras que durante el estadío de banda germinal se expresa en la parte central del embrión, desde el segmento torácico T2 hasta los primeros segmentos abdominales. Los embriones interferidos para Rp-Kr presentan una alteración del patrón de segmentación. Estas alteraciones corresponden a la zona media del embrión, donde se pierden el 2do y 3er segmento torácico y el abdomen se reduce de diez a seis segmentos. Además, los embriones con fenotipo interferido, muestran dificultades en la formación del segmento labial y T1, y cambios homeóticos, en los cuales aparece un peine tibial ectópico en la tibia de la pata T2. En la presente tesis doctoral, se muestra un análisis bioinformático de una genoteca normalizada de ESTs y la caracterización de la mayoría de los ortólogos de genes de VI segmentación para R. prolixus. Mostramos las potenciales regiones regulatorias para los genes Rp-gt y Rp-Kr, cuya posición se encuentra evolutivamente conservada tanto en D. melanogaster como en T. castaneum. Por último, se demuestra tanto estructural como funcionalmente que Rp-gt y Rp-Kr son verdaderos genes gap, el primero para la región cefálica y abdominal, y el segundo en la región central del embrión de R. prolixus.Facultad de Ciencias Naturales y Muse

Análisis genómico-funcional de la embriología de Rhodnius prolixus (Sthl, 1859) (Hemiptera-Reduviidae)

Tesis presentada para optar al Grado de Doctor en Ciencias NaturalesFil: Lavore, Andrés E.. 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

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

Comparative genomics analysis of triatomines reveals common first line and inducible immunity-related genes and the absence of Imd canonical components among hemimetabolous arthropods

Background: Insects operate complex humoral and cellular immune strategies to fend against invading microorganisms. The majority of these have been characterized in Drosophila and other dipterans. Information on hemipterans, including Triatominae vectors of Chagas disease remains incomplete and fractionated. Results: We identified putative immune-related homologs of three Triatominae vectors of Chagas disease, Triatoma pallidipennis, T. dimidiata and T. infestans (TTTs), using comparative transcriptomics based on established immune response gene references, in conjunction with the predicted proteomes of Rhodnius prolixus, Cimex lecticularis and Acyrthosiphon pisum hemimetabolous. We present a compressive description of the humoral and cellular innate immune components of these TTTs and extend the immune information of other related hemipterans. Key homologs of the constitutive and induced immunity genes were identified in all the studied hemipterans. Conclusions: Our results in the TTTs extend previous observations in other hemipterans lacking several components of the Imd signaling pathway. Comparison with other hexapods, using published data, revealed that the absence of various Imd canonical components is common in several hemimetabolous species.Centro Regional de Estudios Genómico

Adaptations in energy metabolism and gene family expansions revealed by comparative transcriptomics of three Chagas disease triatomine vectors

Background: Chagas disease is a parasitic infection caused by Trypanosoma cruzi. It is an important public health problem affecting around seven to eight million people in the Americas. A large number of hematophagous triatomine insect species, occupying diverse natural and human-modified ecological niches transmit this disease. Triatomines are long-living hemipterans that have evolved to explode different habitats to associate with their vertebrate hosts. Understanding the molecular basis of the extreme physiological conditions including starvation tolerance and longevity could provide insights for developing novel control strategies. We describe the normalized cDNA, full body transcriptome analysis of three main vectors in North, Central and South America, Triatoma pallidipennis, T. dimidiata and T. infestans. Results: Two-thirds of the de novo assembled transcriptomes map to the Rhodnius prolixus genome and proteome. A Triatoma expansion of the calycin family and two types of protease inhibitors, pacifastins and cystatins were identified. A high number of transcriptionally active class I transposable elements was documented in T. infestans, compared with T. dimidiata and T. pallidipennis. Sequence identity in Triatoma-R. prolixus 1:1 orthologs revealed high sequence divergence in four enzymes participating in gluconeogenesis, glycogen synthesis and the pentose phosphate pathway, indicating high evolutionary rates of these genes. Also, molecular evidence suggesting positive selection was found for several genes of the oxidative phosphorylation I, III and V complexes. Conclusions: Protease inhibitors and calycin-coding gene expansions provide insights into rapidly evolving processes of protease regulation and haematophagy. Higher evolutionary rates in enzymes that exert metabolic flux control towards anabolism and evidence for positive selection in oxidative phosphorylation complexes might represent genetic adaptations, possibly related to prolonged starvation, oxidative stress tolerance, longevity, and hematophagy and flight reduction. Overall, this work generated novel hypothesis related to biological adaptations to extreme physiological conditions and diverse ecological niches that sustain Chagas disease transmission.Centro Regional de Estudios Genómico