Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho utilizou-se a técnica fluorescência de raios X usando radiação síncrotron (SR-TXRF) para estudar, quantitativamente, o transporte de cloro, potássio e cálcio na hemolinfa, urina e túbulos de Malpighi (TM) em ninfas de quinto estágio do Rhodnius prolixus (R. prolixus), considerando a excreção destes elementos em diferentes dias após o repasto sanguíneo. R. prolixus é um dos principais vetores do Trypanosoma cruzi, agente causador da doença de Chagas. R. prolixus fornece um sistema modelo particularmente útil porque seus TMs tanto secretam quanto reabsorvem íons a taxas elevadas. Os TMs filtram a hemolinfa e secretam um líquido que é muitas vezes comparado com a urina primária em vertebrados. Os resultados obtidos mostram que a concentração de potássio na urina é substancialmente maior do que na hemolinfa. A concentração de cloro na hemolinfa é menor do que na urina, mas a diferença não é tão marcada como no caso do potássio. No caso do Rhodnius é razoável interpretar a elevada concentração de potássio na urina como adaptativo para o problema de excreção imediato do inseto. A concentração de cálcio nos TMs é substancialmente maior em comparação com os valores encontrados na hemolinfa e urina. Este resultado mostra que o cálcio é retido no corpo do R. prolixus e pouco eliminado. Os resultados obtidos estão coerentes com a literatura. Avaliou-se também o efeito no transporte de Cl, K e Ca após um repasto de sangue de coelho contaminado com HgCl2 de modo a avaliar o efeito da presença deste metal tóxico no balanço iônico nos fluidos de excreção urina e hemolinfa e também pelo principal órgão de transporte, os túbulos de Malpighi. As excreções de Cl e K pela urina são afetadas pela ingestão. Este resultado é esperado levando-se em consideração a ingestão de excesso de Cl através do HgCl2. O transporte de Cl, K e Ca na hemolinfa do Rhodnius prolixus não é afetada pela ingestão de HgCl2. Nos túbulos de Malpighi, as altas concentrações de Ca obtidas foram comparáveis àquelas encontradas nos insetos controle. Pode-se concluir que SR-TXRF é um método muito promissor para análises diretas, rápidas e confiáveis para a quantificação simultânea de elementos envolvidos na regulação do transporte e em todo o sistema excretor de insetos. Além disso, o estudo do transporte e a excreção de elementos no inseto Rhodnius prolixus abrem oportunidade para a maior compreensão de efeitos da poluição em espécies de invertebrados. / In this work, we investigated changes in the concentrations of Cl, K and Ca, in 5th instar using total reflection X-ray fluorescence Rhodnius prolixus with synchrotron radiation (SR-TXRF). The elements were quantified using urine, hemolymph and Malpighian tubules samples collected on different days after a blood meal. Rhodnius prolixus is one of the most important vectors of the Trypanosoma cruzi, causative agent of Chagas? disease. R. prolixus provides a particularly useful model system because its MTs both secrete and reabsorb ions at high rates. The TMs filter hemolymph and secrete a liquid that is often compared with the primary urine in vertebrates. The experimental results showed that the concentration of potassium in the urine is substantially greater than in the hemolymph. The concentration of chlorine in the hemolymph is generally less than in the urine, but the difference is not so marked as in the case of potassium. In the case of Rhodnius, it is reasonable to interpret the high concentration of potassium in the urine as adaptive to the animals? immediate excretory problem. The concentration of calcium in the TMs is substantially greater than in both the hemolymph and the urine. This result shows that that calcium is retained in the body and not eliminated. These results are in accordance with the literature. We also investigated whether dietary mercury contamination may influence the transport of Cl, K and Ca by the hemolymph, urine and Malpighian tubules of R. prolixus fed on blood containing HgCl2. The results suggested that dietary Hg contamination may influence the Cl and K contents during excretion of the urine. It was expected considering the large amounts of chlorine ingested by Rhodnius prolixus in its meals of blood containing HgCl2. Statistical analysis showed no significant variation in all elements contents for hemolymph samples. The main conclusion which can be drawn from the results is that in all the insects studied calcium is deposited in Malpighian tubules. These observations point out that the analytical approach of the SR-TXRF method can be efficiently used to measure elements involved in the transport regulation into insect Malpighian tubules and also provides useful data concerning the biological effects of pollution on invertebrate species.

Submitted by PPG Medicina e Ci?ncias da Sa?de (medicina-pg@pucrs.br) on 2018-09-17T18:15:32Z No. of bitstreams: 1 KARINE_KAPPAUN.pdf: 4206975 bytes, checksum: b450fa28646271e7dfac9389de975825 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-09-18T12:20:56Z (GMT) No. of bitstreams: 1 KARINE_KAPPAUN.pdf: 4206975 bytes, checksum: b450fa28646271e7dfac9389de975825 (MD5) / Made available in DSpace on 2018-09-18T12:54:15Z (GMT). No. of bitstreams: 1 KARINE_KAPPAUN.pdf: 4206975 bytes, checksum: b450fa28646271e7dfac9389de975825 (MD5) Previous issue date: 2018-07-06 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Ureases (urea amidohydrolases, EC 3.5.1.5) are nickel-dependent enzymes, widely spread in bacteria, fungi and plants, which catalyze the hydrolysis of urea to ammonia and carbon dioxide. Jaburetox is a peptide derived from a urease isoform of the Canavalia ensiformis plant. Structurally, jaburetox is an intrinsically disordered peptide, demonstrated by bioinformatics tools, and experimentally, by circular dichroism (CD) and nuclear magnetic resonance (NMR). Several biological properties of jaburetox have already been described, such as insecticidal activity, ability to interact with lipid bilayers, fungitoxic effect, among others. Soyuretox is a peptide colinear to jaburetox derived from the soybean ubiquitous urease. One of the objectives of this thesis was to carry out structural studies of the soyuretox peptide by CD and NMR, which revealed its intrinsically disordered nature and that its secondary structure content is modified in the presence of SDS micelles. Biological properties of soyuretox were evaluated. The peptide has entomotoxic activity, inducing aggregation of hemocytes from Rhodnius prolixus, the vector of Chagas disease, both in vivo and in vitro. The fungitoxic effect of soyuretox on yeast Candida albicans involves the production of superoxide anions, and the peptide was immunolocalized on the surface of the yeast. Finally, the behavioral and morphological effects of soyuretox on zebrafish larvae (Danio rerio) were evaluated, aiming to deepen the understanding of the mechanism of toxic action and the toxicological profile of this molecule, which presents biotechnological potential as a biopesticide. / Ureases (ureia amido-hidrolases; EC 3.5.1.5) s?o enzimas n?quel dependentes, amplamente distribu?das em bact?rias, fungos e plantas, que catalisam a hidr?lise da ureia ? am?nia e di?xido de carbono. O jaburetox ? um pept?deo derivado de uma isoforma de urease da planta Canavalia ensiformis. Estruturalmente, o jaburetox ? um pept?deo intrinsecamente desordenado, demonstrado por ferramentas in silico de bioinform?tica, e experimentalmente, por dicro?smo circular (CD) e resson?ncia magn?tica nuclear (RMN). Diversas propriedades biol?gicas do jaburetox j? foram descritas, tais como atividade inseticida, capacidade de interagir com bicamadas lip?dicas, efeito fungit?xico, dentre outras. O soyuretox ? um pept?deo colinear ao jaburetox, por?m derivado da urease ub?qua de soja. Um dos objetivos dessa tese foi realizar estudos estruturais do pept?deo soyuretox por CD e RMN, que revelaram sua natureza intrinsecamente desordenada e aumento no conte?do de estrutura secund?ria na presen?a de micelas de SDS. Propriedades biol?gicas do soyuretox foram avaliadas. O pept?deo tem atividade entomot?xica, induzindo agrega??o de hem?citos de Rhodnius prolixus, o vetor da doen?a de Chagas, tanto in vivo como in vitro. O efeito fungit?xico do soyuretox na levedura Candida albicans envolve a produ??o de ?nions super?xido, sendo o pept?deo imunolocalizado ligado na superf?cie da levedura. Por fim, foram avaliados os efeitos comportamentais e morfol?gicos do soyuretox em larvas de zebrafish (Danio rerio), visando aprofundar o entendimento do mecanismo de a??o t?xica e o perfil toxicol?gico dessa mol?cula, que apresenta potencial biotecnol?gico como biopesticida.

In insects, ecdysis is an important feature of growth and development and is tightly controlled by a variety of neuropeptides. In holometabolous insects, crustacean cardioactive peptide (CCAP) is one of many factors that regulate ecdysis behaviours; however, not much is known about the control of ecdysis in hemimetabolous insects. In this thesis, the CCAP-signaling pathway is shown to be essential for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus. The cDNA sequence of the CCAP gene has been cloned from the R. prolixus central nervous system (CNS) and the functional role of CCAP as a neuromodulator/neurotransmitter demonstrated. Specifically, the expression of RhoprCCAP in CNS neurons producing extensive CCAP-like immunoreactive processes within the neuropile indicates that CCAP plays central roles in coordination of other neurons. RhoprCCAP also acts as a neurohomone/neuromodulator released peripherally to coordinate many tissues. Thus, CCAP-like immunoreactive processes are found in neurohemal sites and also on peripheral tissues. The RhoprCCAP receptor (RhoprCCAPR) has been cloned and shown to be a G-protein coupled receptor (GPCR). RhoprCCAPR expression is observed in the CNS and certain peripheral tissues of R. prolixus. Also, CCAP stimulates hindgut contractions and increases the heartbeat rate in a dose-dependent manner. The involvement of CCAP in R. prolixus ecdysis has been investigated. Up-regulation of the RhoprCCAP transcript in the CNS and the RhoprCCAP receptor (RhoprCCAPR) transcript in the CNS and specific peripheral tissues was observed immediately prior to ecdysis. Also, decreasing staining intensity of CCAP-like immunoreactivity in neurons immediately following ecdysis indicates the release of CCAP during ecdysis. The critical importance of the CCAP-signalling pathway was further demonstrated by knockdown of the RhoprCCAP and RhoprCCAPR transcripts utilizing double stranded RNA interference. Insects with these transcripts knocked down have high mortality (up to 84%), typically at the expected time of ecdysis, or have ecdysis extremely delayed. Taken together, this thesis demonstrates that RhoprCCAP plays a crucial role in regulating ecdysis behaviours in R. prolixus, and clearly shows the conserved nature of the CCAP-signaling pathway in ecdysis for both holometabolous and hemimetabolous insects.

In insects, ecdysis is an important feature of growth and development and is tightly controlled by a variety of neuropeptides. In holometabolous insects, crustacean cardioactive peptide (CCAP) is one of many factors that regulate ecdysis behaviours; however, not much is known about the control of ecdysis in hemimetabolous insects. In this thesis, the CCAP-signaling pathway is shown to be essential for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus. The cDNA sequence of the CCAP gene has been cloned from the R. prolixus central nervous system (CNS) and the functional role of CCAP as a neuromodulator/neurotransmitter demonstrated. Specifically, the expression of RhoprCCAP in CNS neurons producing extensive CCAP-like immunoreactive processes within the neuropile indicates that CCAP plays central roles in coordination of other neurons. RhoprCCAP also acts as a neurohomone/neuromodulator released peripherally to coordinate many tissues. Thus, CCAP-like immunoreactive processes are found in neurohemal sites and also on peripheral tissues. The RhoprCCAP receptor (RhoprCCAPR) has been cloned and shown to be a G-protein coupled receptor (GPCR). RhoprCCAPR expression is observed in the CNS and certain peripheral tissues of R. prolixus. Also, CCAP stimulates hindgut contractions and increases the heartbeat rate in a dose-dependent manner. The involvement of CCAP in R. prolixus ecdysis has been investigated. Up-regulation of the RhoprCCAP transcript in the CNS and the RhoprCCAP receptor (RhoprCCAPR) transcript in the CNS and specific peripheral tissues was observed immediately prior to ecdysis. Also, decreasing staining intensity of CCAP-like immunoreactivity in neurons immediately following ecdysis indicates the release of CCAP during ecdysis. The critical importance of the CCAP-signalling pathway was further demonstrated by knockdown of the RhoprCCAP and RhoprCCAPR transcripts utilizing double stranded RNA interference. Insects with these transcripts knocked down have high mortality (up to 84%), typically at the expected time of ecdysis, or have ecdysis extremely delayed. Taken together, this thesis demonstrates that RhoprCCAP plays a crucial role in regulating ecdysis behaviours in R. prolixus, and clearly shows the conserved nature of the CCAP-signaling pathway in ecdysis for both holometabolous and hemimetabolous insects.

Rhodnius prolixus, the principal Chagas disease vector, requires a blood meal to complete its moult cycle into the next stage. Allatotropins (ATs), a family of peptides first isolated from Manduca sexta, have been shown to regulate the biosynthesis of juvenile hormone, an insect growth and development hormone; however, ATs, being multimodal peptides, also exhibit myotropic effects on some insect visceral muscles. Here, this AT family of peptides has been examined in R. prolixus. Genomic analysis revealed a cDNA fragment of 973bp encoding one mature amidated AT tridecapeptide (Rhopr-AT) with high transcript levels observed, via RT-PCR, in the central nervous system (CNS) and pool of fat body and trachea. AT-like immunoreactive neurons were found throughout the CNS and AT-like immunoreactive processes were present on some peripheral tissues. Bioassays using hindgut and dorsal vessel contraction, however, failed to demonstrate any effect of Rhopr-AT on these tissues. Future work will examine the effects of Rhopr-AT on JH production.

Rhodnius prolixus, the principal Chagas disease vector, requires a blood meal to complete its moult cycle into the next stage. Allatotropins (ATs), a family of peptides first isolated from Manduca sexta, have been shown to regulate the biosynthesis of juvenile hormone, an insect growth and development hormone; however, ATs, being multimodal peptides, also exhibit myotropic effects on some insect visceral muscles. Here, this AT family of peptides has been examined in R. prolixus. Genomic analysis revealed a cDNA fragment of 973bp encoding one mature amidated AT tridecapeptide (Rhopr-AT) with high transcript levels observed, via RT-PCR, in the central nervous system (CNS) and pool of fat body and trachea. AT-like immunoreactive neurons were found throughout the CNS and AT-like immunoreactive processes were present on some peripheral tissues. Bioassays using hindgut and dorsal vessel contraction, however, failed to demonstrate any effect of Rhopr-AT on these tissues. Future work will examine the effects of Rhopr-AT on JH production.

Thesis (Ph.D.)--McMaster University, 2004. / Supervisor: Michael J. O'Donnell. Includes bibliographical references (leaves 193-208).

Orientador: João Aristeu da Rosa / Banca: Marcos Takashi Obara / Banca: Mara Cristina Pinto / Resumo: Descreve-se R. marabaensis n. sp. coletada em maio/2014 na reserva Murumurú, município de Marabá, Estado do Pará, Brasil. Observaram-se caracteres morfológicos da cabeça, tórax, abdômen e de ovos. Foram consultadas as descrições morfológicas de 19 espécies de Rhodnius, porém exame mais detalhado foi realizado comparativamente com R. prolixus e R. robustus que ocorrem na região Norte do Brasil. Observou-se também exemplares de 18 espécies de Rhodnius depositadas no Laboratório Nacional e Internacional de Referência de Taxionomia de Triatomíneos do Instituto Oswaldo Cruz, Rio de Janeiro, Brasil. Os caracteres morfológicos mais notáveis que distinguem R. marabaensis n. sp. são o vértice da cabeça em forma de quilha, o comprimento do segundo segmento da antena, forma do prosterno, mesosterno, metasterno, conjunto de manchas no abdômen, genitália masculina, faces posterior e ventral da genitália externa feminina e os caracteres morfológicos de ovos. A análise filogenética do fragmento do gene Cytb também mostrou diferenças com outras sete espécies de Rhodnius avaliadas. Como foram examinados exemplares de Rhodnius jacundaensis Serra, Serra & Von Atzingen, 1980 [nomen nudum] depositados na Casa da Cultura de Marabá essa espécie entra em sinonímia com R. marabaensis n. sp., pois foi verificado que se tratavam da mesma espécie. / Abstract: The species Rhodnius marabaensis sp. is described. It was collected in May 2014 in the Murumurú Environmental Reserve in the city of Marabá, Pará State, Brazil. The morphological characteristics of the head, thorax, abdomen, and eggs have been determined. The morphological descriptions of 19 previously described Rhodnius species were consulted; however, R. prolixus and R. robustus, which also occur in the northern region of Brazil, were examined in more detail. The exam included specimens from 18 Rhodnius species held in the Brazilian National and International Triatomine Taxonomy Reference Laboratory at the Oswaldo Cruz Institute in Rio de Janeiro, Brazil. The most notable morphological features that distinguish R. marabaensis sp. nov. are the keel-shaped apex of the head, the length of the second segment of the antennae, the shapes of the prosternum, the mesosternum, and the metasternum, the set of spots on the abdomen, the male genitalia, the posterior and ventral surfaces of the external female genitalia, and the morphological characteristics of the eggs. A phylogenetic analysis revealed that this species' cyt-b gene fragment differed from the other seven Rhodnius species that were evaluated. Rhodnius jacundaensis Serra, Serra & Von Atzingen, 1980 [nomen nudum] specimens deposited in the Marabá Cultural Center were examined and were found to be synonymous with R. marabaensis sp. nov. / Mestre

Le métabolisme est la somme des réactions chimiques dans un organisme. L’énergie leur permet d’effectuer la biosynthèse, le maintien des fonctions vitales et l’activité physique. Si l’énergie est transformée en chaleur nous pouvons déterminer le taux de transformation d’énergie chimique en taux métabolique (TM) et nous pouvons donc calculer les besoins énergétiques d’un animal. Le principal objectif de ce travail est d’évaluer le métabolisme énergétique chez la punaise hématophage Rhodnius prolixus. Nos résultats montrent, que le TM de l’alimentation chez R. prolixus atteint jusqu’à 17 fois le TM au repos. Le quotient respiratoire est de 0,83 pendant le repos et 0,52 pendant l’alimentation. De plus, la désactivation des protéines permettant gérer le choc thermique, diminuent le TM pendant la digestion. Le coût énergétique (CE) de la marche peut atteindre jusqu’à 1,7 fois le CE pendant le repos. Finalement, le CE de la production d’un oeuf chez R. prolixus est approximativement de 11,7 J. L’ensemble de nos résultats apportent des outils qui fournissent des éléments nécessaires pour mieux contrôler la transmission de maladies vectorielles. / Metabolism is the sum of all the chemical reactions in an organism. Energy uptake allows animals to perform biosynthesis, maintenance, and external work. If the energy produced is converted into heat, we can estimate the energy metabolism as the rate of conversion of chemical energy into metabolic rate (MR), which allows the calculation of energy requirements. The main objective of this work is to assess the metabolic cost of physiological work in the blood-sucking bug Rhodnius prolixus. Our results show that feeding is costly. The MR during feeding in R. prolixus reaches up to 17 times the MR during rest. The mean respiratory quotient is 0.83 during rest and 0.52 during feeding. We showed that the deactivation of Heat Shock Proteins on R. prolixus, causes a diminution of MR during digestion. Then, we showed that the energy cost (EC) during walking can reach up to 1.7 times the EC during rest. Finally, we found that the EC of production of one egg of R. prolixus was 11.7 J. All our results provide tools to a better understanding of biology and ecology of an hematophagous insect to provide the necessary elements to better control of transmission of vector-borne diseases.

CAPA-related peptides belong to a family of neuropeptides localized to the central nervous system that can function in diverse roles in the regulation of water and salt homeostasis in insects. These peptides are known to stimulate fluid secretion by Malpighian tubules (MTs) in Dipteran species, thus serving a diuretic function. In contrast, this thesis demonstrates that members of this family of peptides in Rhodnius prolixus serve an anti-diuretic role and have multiple tissue targets, whereby they oppose the activity of diuretic hormones such as serotonin (5-Hydroxytryptamine hydrochloride; 5-HT). I have identified two genes each encoding three peptides in R. prolixus, suggesting this insect is capable of producing a greater number of CAPA-peptides compared to other insects that contain only a single CAPA gene. Interestingly, while the second peptide encoded in each R. prolixus gene (RhoprCAPA-α2/-β2) inhibits the stimulatory effects of serotonin on tissues such as the anterior midgut and Malpighian tubules, it appears the other CAPA-related and pyrokinin-related peptides do not play a major role in inhibiting the effects of serotonin on these tissues. More specifically, serotonin-stimulated fluid secretion by MTs and fluid absorption by the anterior midgut are reduced by the anti-diuretic peptide, RhoprCAPA-α2. In addition, I have also identified a G protein-coupled receptor which likely mediates the anti-diuretic effect associated with RhoprCAPA-α2 and have functionally characterized this receptor in Chinese hamster ovary cells. Spatial transcript expression analysis in fifth-instars reveals a wide distribution of the receptor in tissues associated with the rapid post-gorging diuresis. Thus, my findings suggest that numerous tissues are regulated by the CAPA peptides in R. prolixus. Gene structure and phylogenetic analyses demonstrate that this receptor is the orthologue of the D. melanogaster capa receptor (CG14575) with homologs in other insects. Taken together, my thesis demonstrates that the RhoprCAPA peptides play an integral role in the coordination and maintenance of anti-diuresis in R. prolixus. This mechanism is necessary following the rapid diuresis associated with blood-feeding by this medically-important insect.