41 results on '"Silva, Carlos Peres"'
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2. Metarhizium anisopliae blastospores are highly virulent to adult Aedes aegypti, an important arbovirus vector
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de Paula, Adriano Rodrigues, Silva, Leila Eid Imad, Ribeiro, Anderson, da Silva, Gerson Adriano, Silva, Carlos Peres, Butt, Tariq M., and Samuels, Richard Ian
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- 2021
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3. Effect of brown seaweeds on Pacific white shrimp growth performance, gut morphology, digestive enzymes activity and resistance to white spot virus
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Schleder, Delano Dias, Peruch, Luiz Guilherme Buglione, Poli, Moisés Angel, Ferreira, Tamiris Henrique, Silva, Carlos Peres, Andreatta, Edemar Roberto, Hayashi, Leila, and do Nascimento Vieira, Felipe
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- 2018
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4. Potential use of mealworms as an alternative protein source for Pacific white shrimp: Digestibility and performance
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Panini, Roseane L., Freitas, Luiz Eduardo Lima, Guimarães, Ariane M., Rios, Cristina, da Silva, Maria Fernanda O., Vieira, Felipe Nascimento, Fracalossi, Débora M., Samuels, Richard Ian, Prudêncio, Elane Schwinden, Silva, Carlos Peres, and Amboni, Renata D.M.C.
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- 2017
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5. The Potential of Metarhizium anisopliae Blastospores to Control Aedes aegypti Larvae in the Field.
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Gomes, Simone Azevedo, Carolino, Aline Teixeira, Teodoro, Thais Berçot Pontes, Silva, Gerson Adriano, Bitencourt, Ricardo de Oliveira Barbosa, Silva, Carlos Peres, Alkhaibari, Abeer M., Butt, Tariq M., and Samuels, Richard Ian
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METARHIZIUM anisopliae ,AEDES aegypti ,LARVAE ,BIOLOGICAL pest control agents ,ENTOMOPATHOGENIC fungi - Abstract
Entomopathogenic fungi are promising as an environmentally benign alternative to chemical pesticides for mosquito control. The current study investigated the virulence of Metarhizium anisopliae blastospores against Aedes aegypti under both laboratory and field conditions. Virulence bioassays of conidia and blastospores were conducted in the laboratory, while field simulation bioassays were conducted under two conditions: totally shaded (TS) or partially shaded (PS). In the first bioassay (zero h), the larvae were added to the cups shortly after the preparation of the blastospores, and in the subsequent assays, larvae were added to the cups 3, 6, 9, and 12 days later. The survival of the larvae exposed to blastospores in the laboratory was zero on day two, as was the case for the larvae exposed to conidia on the sixth day. Under TS conditions, zero survival was seen on the third day of the bioassay. Under PS conditions, low survival rates were recorded on day 7. For the persistence bioassay under PS conditions, low survival rates were also observed. Metarhizium anisopliae blastospores were more virulent to Ae. aegypti larvae than conidia in the laboratory. Blastospores remained virulent under field simulation conditions. However, virulence rapidly declined from the third day of field bioassays. Formulating blastospores in vegetable oil could protect these propagules when applied under adverse conditions. This is the first time that blastospores have been tested against mosquito larvae under simulated field conditions, and the current study could be the basis for the development of a new biological control agent. [ABSTRACT FROM AUTHOR]
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- 2023
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6. Freshwater catfish jundiá (Rhamdia quelen) larvae are prepared to digest inert feed at the exogenous feeding onset: physiological and histological assessments
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Silveira, Jenniffer, Silva, Carlos Peres, Cargnin-Ferreira, Eduardo, Alexandre, Daniel, Elias, Mariele Abádia, and Fracalossi, Débora Machado
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- 2013
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7. Additional file 1 of Metarhizium anisopliae blastospores are highly virulent to adult Aedes aegypti, an important arbovirus vector
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de Paula, Adriano Rodrigues, Silva, Leila Eid Imad, Ribeiro, Anderson, da Silva, Gerson Adriano, Silva, Carlos Peres, Butt, Tariq M., and Samuels, Richard Ian
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Additional file 1: Table S1. Percentage germination of Metarhizium anisopliae (ESALQ 818 and LEF 2000) blastospores when formulated with and without sunflower oil. Fig S1. Radial growth (mm) of Metarhizium anisopliae (ESALQ 818 and LEF 2000) blastospores formulated with and without sunflower oil (20%) over a 10 day period. Photograph of ovitrap used to collect Aedes aegypti eggs in the field.
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- 2021
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8. Production of conidia using different culture media modifies the virulence of the entomopathogenic fungus Metarhizium against Aedes aegypti larvae.
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Carolino, Aline Teixeira, Pontes Teodoro, Thais Berçot, Gomes, Simone Azevedo, Silva, Carlos Peres, and Samuels, Richard Ian
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- 2021
9. Dietary Protein Requirement of Juvenile Cachara Catfish, Pseudoplatystoma reticulatum.
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Henrique Gomes Cornélio, Fernando, da Cunha, Douglas Amaral, Silveira, Jennifer, Alexandre, Daniel, Silva, Carlos Peres, and Fracalossi, Débora Machado
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CATFISHES ,FISH food ,LOW-protein diet ,FISHERIES ,FISH growth ,FISH breeding ,PHYSIOLOGY - Abstract
Cachara, Pseudoplatystoma reticulatum, is a high commercial value carnivorous catfish in Brazil, but whose dietary protein requirement is still unknown. Aiming to determine this requirement, groups of 15 juveniles (16.08 ± 1.13 g) were fed isoenergetic diets (4600 kcal/kg gross energy) with increasing levels of crude protein (30, 35, 40, 45, 50, and 55%). After 60 d, regression analysis revealed a quadratic effect ( P < 0.05) of increasing dietary crude protein concentration on growth variables. The highest weight gain and specific growth rate as well as the best feed conversion were shown by fish fed the 50% crude-protein diet. Similarly, protease activities were significantly higher ( P < 0.05) in fish fed 50% crude protein. However, the highest protein retention was observed in fish fed the 45% crude-protein diet. Protein and dry matter digestibilities did not differ ( P > 0.05) for diets containing 40, 45, or 50% crude protein. Therefore, based on weight gain and at a dietary energy concentration of 4600 kcal/kg, the estimated protein requirement for juvenile cachara between 16 and 85 g is 49.25% crude protein. This is equivalent to 44.79% digestible protein and a gross energy to digestible protein ratio of 10.27 kcal/g. [ABSTRACT FROM AUTHOR]
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- 2014
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10. Freshwater catfish jundiá (Rhamdia quelen) larvae are prepared to digest inert feed at the exogenous feeding onset: physiological and histological assessments.
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Silveira, Jenniffer, Silva, Carlos Peres, Cargnin-Ferreira, Eduardo, Alexandre, Daniel, Elias, Mariele Abádia, and Fracalossi, Débora Machado
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CATFISHES ,FISH larvae ,FISH feeds ,DIGESTIVE organs ,DIGESTIVE enzymes ,SERINE proteinases ,EGG incubation - Abstract
This study assessed the morphological development of jundiá larvae’s digestive system and digestive proteolytic activity. Specific serine proteinases activities varied over time, with the highest peak at 12 h after hatching (AH), which corresponded to 296.38 ± 84.20 mU mg
−1 for trypsin and 315.45 ± 42.16 mU mg−1 for chymotrypsin. Specific aspartic proteinases activities increased up to the start of weaning, oscillated during that phase, but showed a consistent increase after that, resulting in the highest specific activity at 252 h AH (7.88 ± 0.68 mU mg−1 ). Gel assays showed different molecular forms, especially of serine proteinases. Histology showed the gastrointestinal tract development onset at 0 h AH and open mouth at 4 h AH. At 16 h AH, the following differentiation of the digestive tract was evident: oropharyngeal cavity, esophagus, liver, pancreas, stomach, and intestine. At 40 h AH, zymogen granules in the pancreas were observed, and at 48 h AH, mucus in the digestive tract and gastric glands in the stomach. Findings indicate that jundiá has a functional stomach before the end of vitelline reserves. Therefore, jundiá larvae are probably capable to digest inert feed at the exogenous feeding onset. [ABSTRACT FROM AUTHOR]- Published
- 2013
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11. Alpha-Glucosidase Promotes Hemozoin Formation in a Blood-Sucking Bug: An Evolutionary History.
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Mury, Flávia Borges, da Silva, José Roberto, Ferreira, Ligia Souza, dos Santos Ferreira, Beatriz, de Souza-Filho, Gonçalo Apolinário, de Souza-Neto, Jayme Augusto, Ribolla, Paulo Eduardo Martins, Silva, Carlos Peres, do Nascimento, Viviane Veiga, Machado, Olga Lima Tavares, Berbert-Molina, Marília Amorim, and Dansa-Petretski, Marilvia
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RHODNIUS prolixus ,GLUCOSIDASES ,AMINO acid sequence ,BIOMINERALIZATION ,BINDING sites ,GENE expression ,GENETIC engineering ,GLUCOSIDASE inhibitors ,BLOOD proteins - Abstract
Background: Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated. Methodology/Principal Findings: Hz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes. Conclusions/Significance: Herein the Hz formation is shown to be associated to an α-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance. [ABSTRACT FROM AUTHOR]
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- 2009
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12. Hydrolytic enzymes of leaf-cutting ant fungi
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Erthal, Milton, Silva, Carlos Peres, Cooper, Richard M., and Samuels, Richard Ian
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HYDROLASES , *LEAF-cutting ants , *SYMBIOSIS , *CHITINASE , *PECTINS , *FUNGUS ants , *SCANNING electron microscopy - Abstract
Abstract: The production of enzymes and the colonization of leaves by Leucoagaricus gongylophorus were investigated to further understand the digestive interactions of leaf-cutting ant colonies. The enzymes detected were indicative of a saprophytic origin of this fungus, producing all the enzymes necessary for plant tissue breakdown. Enhanced activities of certain enzymes in the fungus garden extracts may be due to the particular behaviour of the adult worker ants that concentrate fungal acquired enzymes in the rectal fluid and subsequently defaecate these enzymes onto the leaves. The production of chitinases by the fungus may be an ancestral vestige of lower attines, and may have a role as agonists of invading microbes. Growth of the fungus on plant cell wall medium resulted in highest enzyme activity against pectin, reflecting the fact that polygalacturonans comprise the main matrix of the primary plant cell wall. SEM shows that L. gongylophorus does not form specialized structures for cell wall penetration, but gains access to the inner plant tissue at the cut edges of the leaf fragments. Enzymes secreted by the fungus were compared to those seen in larval and adult leaf-cutting ants, demonstrating the inter-dependence of the symbiotic relationship between the ants and their fungi. [Copyright &y& Elsevier]
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- 2009
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13. Effects of a Reserve Protein on Spodoptera frugiperda Development: A Biochemical and Molecular Approach to the Entomotoxic Mechanism.
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Oliveira, Carolina Turatti, Machado, Suzy Wider, Bezerra, Cézar da Silva, Cardoso, Marlon Henrique, Franco, Octávio Luiz, Silva, Carlos Peres, Alves, Demetrio Gomes, Rios, Cristina, and Macedo, Maria Lígia R.
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FALL armyworm ,PROTEINS ,INSECT pests ,DIGESTIVE enzymes ,PLANT defenses ,INSECT development ,PEPTIDASE - Abstract
Talisin is a storage protein from Talisia esculenta seeds that presents lectin-like and peptidase inhibitor properties. These characteristics suggest that talisin plays a role in the plant defense process, making it a multifunctional protein. This work aimed to investigate the effects of chronic intake of talisin on fifth instar larvae of Spodoptera frugiperda, considered the main insect pest of maize and the cause of substantial economic losses in several other crops. The chronic intake of talisin presented antinutritional effects on the larvae, reducing their weight and prolonging the total development time of the insects. In addition, talisin-fed larvae also showed a significant reduction in the activity of trypsin-like enzymes. Midgut histology analysis of talisin-fed larvae showed alterations in the intestinal epithelium and rupture of the peritrophic membrane, possibly causing an increase of aminopeptidase activity in the midgut lumen. Talisin also proved to be resistant to degradation by the digestive enzymes of S. frugiperda. The transcription profile of trypsin, chymotrypsin and aminopeptidase genes was also analyzed through qPCR technique. Talisin intake resulted in differential expression of at least two genes from each of these classes of enzymes. Molecular docking studies indicated a higher affinity of talisin for the less expressed enzymes. [ABSTRACT FROM AUTHOR]
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- 2020
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14. Entomotoxic properties of Dioclea violacea lectin and its effects on digestive enzymes of Anagasta kuehniella (Lepidoptera).
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Oliveira, Carolina Turatti, Kunz, Daniele, Silva, Carlos Peres, and Macedo, Maria Lígia Rodrigues
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MEDITERRANEAN flour moth , *LECTINS , *DIGESTIVE enzymes , *LEGUMES , *BIOLOGICAL insecticides - Abstract
Entomotoxic plant lectins have been extensively studied in the past two decades, yet the exact mechanisms underlying their toxic effects remain unknown. This study investigated the effects of Dioclea violacea lectin (DVL) on larval development in Anagasta kuehniella . Chronic exposure of larvae (from neonates to the fourth instar) demonstrated that DVL interfered with larval growth, retarding development and decreasing larval mass without affecting survival. DVL decreased trypsin-like, chymotrypsin-like, and α-amylase activities and proved resistant to proteolysis by midgut proteases up to 24 h. Shorter exposures to dietary DVL had no effect on midgut enzyme activity. Feeding fourth-instar larvae with fluorescently-labeled DVL revealed lectin binding to the peritrophic membrane. [ABSTRACT FROM AUTHOR]
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- 2015
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15. Efeito da dieta à base de inseto (Nauphoeta cinerea) no crescimento, sistema imune, atividade enzimática, capacidade antioxidante e microbiota em Litopenaeus vannamei
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Rios, Cristina, Universidade Federal de Santa Catarina, and Silva, Carlos Peres
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Bioquímica ,Microbiota ,Entomofagia ,Litopenaeus vannamei ,Sistema imunológico - Abstract
Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2022. O cultivo de camarões marinhos é uma atividade muito lucrativa e uma das espécies mais produzidas devido a fatores zootécnicos favoráveis é o camarão-branco-do-pacífico, Litopenaeus vannamei (Decapoda: Penaeidae). Um dos grandes entraves da sua produção, além da fácil disseminação de agentes patogênicos, é o alto custo dos insumos com a alimentação. Em geral, na nutrição de espécies aquícolas, é utilizada a farinha de peixe como ingrediente proteico principal. No entanto, buscam-se fontes alternativas de proteína sustentáveis e econômicas. Com isso, tem sido elaborados estudos com o uso de farinha de insetos na alimentação de camarões. Este trabalho tem por objetivo analisar o perfil de enzimas digestivas, análise da microbiota intestinal, parâmetros imunológicos e estresse oxidativo de camarões juvenis da espécie L. vannamei (n=9), com peso inicial de 6,10 ± 0,52g (média, ± desvio padrão), peso final de 9,05 ± 0,49g e com um ganho de peso de 2,60± 0,74 g, em experimento realizado durante vinte dias. Os camarões foram alimentados com farinhas de Nauphoeta cinerea (NC), em diferentes níveis de substituição, com 25%, 50%, 75% e 100% e o controle (farinha de peixe). O experimento foi realizado no Laboratório de Camarões Marinhos da UFSC (LCM) iniciando com o cultivo dos camarões juvenis em aquários, com fornecimento diário das rações. Após esse período, os animais foram eutanasiados, sendo dissecados o hepatopâncreas e esses mantidos em nitrogênio líquido e freezer -80 ºC. Os hepatopâncreas foram homogeneizados e centrifugados para a realização dos ensaios enzimáticos para determinação de atividades proteolíticas do tipo tripsina, do tipo quimotripsina, lipases e a-amilases. Zimogramas utilizando-se géis SDS-PAGE foram obtidos para fracionamento das atividades proteolíticas e amilásicas. Também foram analisadas as enzimas catalase, contagem de hemócitos e atividade aglutinante na hemolinfa. Os dados obtidos mostram que o ganho de peso, taxa de crescimento específico e taxa de sobrevivência não foram afetados com a substituição de farinha de peixe por farinha de inseto na dieta dos camarões. Os resultados de atividade absoluta e específica de tripsina, quimotripsina, lipase e a-amilase demonstram não haver diferenças significativas entre o controle e os tratamentos, assim como nos padrões das atividades enzimáticas encontradas nos zimogramas. Com relação aos parâmetros imunológicos da hemolinfa a concentração proteica, a contagem de hemócitos, a atividade de fenoloxidase e hemaglutinante, demonstram não haver diferenças siginificativas entre o controle e os tratamentos. O mesmo foi observado com relação às dosagens de catalase. Com relação à microbiota intestinal, nossos resultados mostraram que os filos dominantes em ordem crescente foram: Proteobacteria, Bacteriodetes, Actinobacteria, Firmicutes, Verrucomicrobia e Planctomycetes, nesta ordem. No entanto, embora não tenham sido encontradas diferenças na diversidade bacteriana, o nível de substituição NC-100 quando comparado com o controle, apresenta uma maior diversidade de gêneros. Contudo, foi possível observar um padrão semelhante de microbiota entre os outros tratamentos. Abstract: The cultivation of marine shrimp is a very lucrative activity and one of the most produced species due to favorable zootechnical factors is the Pacific white shrimp, Litopenaeus vannamei (Decapoda: Penaeidae). One of the major barriers to its production, in addition to the easy dissemination of pathogenic agents, is the high cost of inputs with feed. Overall, in the nutrition of aquaculture species, fishmeal is used as the main protein ingredient. However, sustainable and economical alternative sources of protein are sought. Therefore, studies have been carried out with the use of insect flour in shrimp feed. This work aims to analyze the profile of digestive enzymes, analysis of the intestinal microbiota, immunological parameters and oxidative stress of Litopennaeus vannamei juveniles (n=9), with an initial weight of 6.10 ± 0.52g (mean, ± standard deviation), final weight of 9.05 ± 0.49 g and with a weight gain of 2.60 ± 0.74 g, in an experiment held out for twenty days. Shrimps were fed with Nauphoeta cinerea (NC) flour, at different levels of substitution, with 25%, 50%, 75% and 100% and the control (fishmeal). The experiment was carried out at the Laboratório de Camarões Marinhos da UFSC (LCM) starting with the cultivation of shrimp juveniles in aquariums, with a daily supply of feed. After this period, the animals were euthanized, the hepatopancreas being dissected and kept in liquid nitrogen and freezer -80 ºC. The hepatopancreas were homogenized and centrifuged for enzymatic assays to determine trypsin-like, chymotrypsin-like, lipase and a-amylase activities. Zymograms using SDS-PAGE gels were obtained to fractionate ?-amylases. The following parameters were also analysed in hemolymph: catalase activity, total hemocyte count and hemagglutinating activity. The data showed that the weight gain, specific growth rate and survival rate were not affected with the replacement of fishmeal by insect meal in the shrimp diet. The results of absolute and specific activity of trypsin, chymotrypsin, lipase and a-amylase demonstrated that there were no differences between the control and treatments, as well as the patterns of enzymatic activities found in the zymograms. In relation to immunological parameters of hemolymph, such as protein concentration, hemocyte count, phenoloxidase and hemagglutinating activity, there were no significant differences between control and treatments. The same was observed in relation to catalase activity. Regarding the intestinal microbiota, our results showed that the dominant phyla in ascending order were: Proteobacteria, Bacteriodetes, Actinobacteria, Firmicutes, Verrucomicrobia and Planctomycetes. However, although no differences were found in bacterial diversity, the NC-100 substitution level presents a greater diversity of genus. However, it was possible to observe a similar microbiota pattern among the other treatments.
- Published
- 2022
16. Mecanismos da endocitose de vicilinas em células epiteliais do intestino médio das larvas do caruncho Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae)
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Kunz, Daniele, Universidade Federal de Santa Catarina, and Silva, Carlos Peres
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Bioquímica ,Endocitose ,Proteínas ,Celulas epiteliais ,Gorgulho do feijao-de-corda - Abstract
Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2017. O transporte de proteínas através do epitélio intestinal dos insetos é ainda pouco conhecido. Há evidências de que vicilina, uma importante proteína de armazenamento de sementes do feijão-de-corda (Vigna unguiculata), é internalizada em larvas do bruquíneo Callosobruchus maculatus. Tem sido relatado que esta globulina de armazenamento interage com proteínas presentes nas membranas microvilares ao longo do trato digestivo das larvas. Na presente Tese, as vias celulares envolvidas na endocitose de vicilina em larvas do caruncho C. maculatus foram estudadas. Vicilina marcada com FITC (purificada a partir de sementes do feijão-de-corda) foi incorporada na dieta das larvas de C. maculatus na concentração fisiológica (0,5% m/m). O destino das globulinas marcadas ou não-marcadas foi monitorado por microscopia confocal, imunohistoquímica e Western blotting. A proteína microvilar de ligação à vicilina foi purificada usando cromatografia de afinidade em uma coluna de vicilina-Sepharose seguida por espectrometria de massa MALDI-TOF. A absorção de vicilinas é um caso de endocitose mediada por receptor. O receptor de vicilina foi purificado e mostrou uma elevada homologia com as proteínas da superfamília SEC14, principalmente a proteína transportadora de ?-tocoferol. Estas proteínas estão presentes no intestino das larvas nas microvilosidades das células epiteliais, associadas a vesículas de endocitose. Vesículas endocíticas e cisternas foram encontradas em toda a extensão das células epiteliais do intestino médio. O tipo de transcitose destas macromoléculas foi confirmado através da utilização de inibidores específicos da via endocítica mediada por clatrina ou via mediada por caveolina. Os inibidores filipina III, nistatina e wortmanina inibiram significativamente a endocitose de vicilina, sugerindo que a via endocítica é principalmente mediada por caveolina. Este trabalho mostrou que a transcitose de vicilina através das células do intestino médio de larvas do inseto C. maculatus é mediada por um membro da família SEC14 homóloga à proteína transportadora de ?-tocoferol. É possível sugerir que vicilina é internalizada por endocitose dependente majoritariamente por caveolina. Em relação à faseolina foi observada dificuldade de internalização pelas larvas de C. maculatus. Estas globulinas favoreceram a geração de espécies reativas de oxigênio, onde o aumento da peroxidação lipídica foi determinado pelo método TBARS e o uso de anticorpos anti malondialdeído e 4- hidroxinonenal. Abstract : The transport of proteins across the intestinal epithelium of insects is still little known. There is evidence that vicilin, a major storage protein of cowpea seeds (Vigna unguiculata), is internalized in larvae of the bruchid C. maculatus. It has been reported that this storage globulin interacts with proteins present in the microvillar membranes along the digestive tract of the larvae. In the present Thesis, the cellular routes involved in the endocytosis of vicilin in larval C. maculatus was investigated. FITC-labeled vicilin (purified from C. maculatus susceptible seeds of cowpea) were incorporated into the diet of the larvae at physiological concentration (0.5% m/m). The fate of labeled or non-labeled globulins was monitored by confocal microscopy, immunohistochemistry and western blotting. The microvillar vicilin-binding protein was purified by using affinity chromatography on a vicilin-Sepharose column followed by MALDI-TOF mass spectrometry. The absorption of vicilins is a case of receptor-mediated endocytosis. The putative vicilin receptor was purified and showed high homology with proteins from the SEC14 superfamily. These proteins are present in the luminal surface of the midgut cell microvilli and inside these epithelial cells, associated to endocytic vesicles. Endocytic vesicles and cisternae were found throughout the extent of midgut epithelial cells. The type of transcytosis of these macromolecules was confirmed through the use of specific inhibitors of clathrin or caveolin-mediated pathways. The inhibitors filipin III, nystatin and wortmannin significantly inhibited the endocytosis of vicilin, suggesting that the endocytic pathway is mediate mainly by caveolin. In this Thesis it was shown that the transcytosis of vicilin through the midgut cells of larval C. maculatus is mediated by a member of the SEC14 family homologous to the a-tocopherol transfer protein. It is possible to suggest that vicilin is internalized by endocytosis dependent on caveolin. In relation to the phaseolin, it was observed difficulty of internalization by the larvae of C. maculatus. These globulins favor the generation of reactive oxygen species, where the increase of lipid peroxidation was determined by the TBARS method and the use of anti-malondialdehyde and 4-hydroxynenal antibodies.
- Published
- 2017
17. Qualidade pós-despesca do camarão marinho Litopenaeus vannamei alimentado com farinha de larva de Tenebrio molitor
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Panini, Roseane Lucia, Universidade Federal de Santa Catarina, Amboni, Renata Dias de Mello Castanho, and Silva, Carlos Peres
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Criação ,Ciência dos alimentos ,Alimentação e rações ,Litopenaeus vannamei - Abstract
Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Ciência dos Alimentos, Florianópolis, 2017. Estudos recentes têm destacado o valor da farinha de inseto como substituto parcial ou total de farinha de peixe para a alimentação aquícola. Do ponto de vista nutricional, dependendo da espécie e/ou fase de vida, os insetos são ricos em proteínas e lipídeos. O objetivo deste trabalho foi investigar o efeito da utilização da farinha de larva de Tenebrio molitor (TM) na dieta do camarão branco do Pacífico (Litopeneaus vannamei), em substituição à farinha de peixe, no que diz respeito ao desempenho zootécnico e qualidade pós-despesca. Inicialmente, foi determinada a composição centesimal e os perfis de aminoácidos e ácidos graxos da TM. Os coeficientes da digestibilidade aparente (CDAs) da TM foram determinados para o camarão L. vannamei e a partir dos resultados dos CDAs da TM foram preparadas cinco dietas com diferentes níveis de substituição de farinha de peixe por TM (0, 25, 50, 75 e 100 %), as quais foram empregadas na avaliação do desempenho zootécnico e qualidade do camarão L. vannamei após seis semanas de cultivo. A TM apresentou teores elevados de proteína(558,2 g kg-1), lipídeos (346,4 g kg-1) e baixo teor de cinzas (30,3 g kg- 1). Possui todos os aminoácidos essenciais, sendo a metionina encontrada em menor quantidade. Os ácidos graxos mais abundantes daTM são o ácido oleico (C18:1n9), seguido do ácido palmítico (C16:0) edo ácido linoleico (C18:2n6), que constituíram 47,17, 17,78 e 15,80 % do total de ácidos graxos, respectivamente. O ácido linolênico foi detectado em uma baixa quantidade, enquanto os ácidos eicosapentaenóico (EPA) e docosahexaenóico (DHA) não foram detectados. Os valores dos CDAs da TM para o camarão L. vannamei foram 45,9 % para matéria seca, 66,5 % para energia, 76,1 % para proteína e para os aminoácidos essenciais os valores variaram de 72 a 86%. O aminoácido metionina mostrou ser o aminoácido limitante da TM para o camarão L. vannamei. Os parâmetros de crescimento avaliados como ganho de peso, taxa de crescimento específico, ingestão alimentar, conversão alimentar, retenção proteica e sobrevivência não foram afetados pela substituição da farinha de peixe por TM (p > 0,05). Em relação à qualidade do camarão L. vannamei, o teor de proteína do músculo não foi afetado (p > 0,05) com a substituição da farinha de peixe pela TM. Contudo, a substituição afetou o teor de lipídeos e o perfil de ácidos graxos (p < 0,05) do camarão. Apesar dos lipídeos serem o menor constituinte do músculo, o seu teor aumentou significativamente com o aumento dos níveis de substituição, enquanto, que os ácidos graxos poli-insaturados EPA e DHA diminuíram linearmente. A cor e a firmeza mantiveram-se inalteradas. Desta forma, com este estudo, sugere-se que a farinha de larva de T. molitor tem um grande potencial para ser utilizada como um ingrediente alternativo à farinha de peixe na dieta do camarão L. vannamei, embora o aminoácido metionina seja limitante. Além disso, é importante melhorar o perfil dos ácidos graxos da larva de T. molitor, uma vez que estes refletem no perfil dos ácidos graxos do camarão, possivelmente por meio da inclusão de fontes de ácidos graxos poli-insaturados na dieta do inseto. Abstract : Studies on replacing the use of fishmeal in fish diets with insects are showing promising results and have encouraged further research. Insects are a highly nutritious food source, rich in proteins and lipids, depending on the species and stages of development. This study investigated the use of mealworm (MW) as a fishmeal substitute for the farmed shrimp Litopenaeus vannamei, especially the possible influence on performance and postharvest quality. Firstly, the proximate composition, essential amino acid and fatty acid profiles of MW were determined. MW was evaluated for apparent crude protein, aminoacids, and energy digestibility when fed to L. vannamei juvenile. Subsequently, the digestible values of MW were evaluated after six weeks of shrimp culture using five diets containing different proportions of fishmeal replaced by MW (0, 25, 50, 75 and 100 %), the performance and the postharvest quality were evaluated. MW protein values observed were558.2 g kg-1 and lipid values were 346.4 g kg-1. The MW essential amino acids profile showed a low amount of methionine. The fatty acidcompositions of MW showed elevated levels of oleic acid (18:1n9),palmitic acid (16:0) and linoleic acid (18:2n6), corresponding to 47.17,17.78 and 15.8 % of total fatty acids, respectively. MW had a low level of linolenic acid (18:3n3) and no long-chain PUFAs (i.e., EPA and DHA). The values for ADC were: 45.9 % for dry matter, 66.5 % for energy and 76.1 % for crude protein while the ADC value for essential amino acids ranged from 72 to 86 %. Methionine was the only limiting amino acid in MW when used to culture L. vannamei. Weight gain, specific growth rate, feed intake, feed conversion, survival and protein retention were not affected when fishmeal was replaced by MW (p > 0.05). The protein content of shrimps showed no significant differences (p > 0.05) between the treatments. However, the replacement affected the shrimp lipid content and fatty acid profile (p < 0.05). EPA and DHA fatty acids decreased linearly with increasing levels of fishmeal substitution by MW. Colour and firmness were unchanged between the treatments. The results suggested that mealworm is a suitable feed ingredient to replace fishmeal in diets for L. vannamei, although methionine is a limiting amino acid. It is also important to optimize the fatty acid profile of MW, since it reflects in the shrimp?s fatty acid profile, possibly by addiction of polyunsaturated fatty acid sources in the insect diet.
- Published
- 2017
18. Perfil de enzimas digestivas em juvenis do camarão-branco-do-pacífico Litopenaeus vannamei alimentados com dietas contendo diferentes níveis de substituição de farinha de peixe por farinha das larvas do inseto Tenebrio molitor
- Author
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Rios, Cristina, Universidade Federal de Santa Catarina, and Silva, Carlos Peres
- Subjects
Bioquímica ,Criação ,Farinha de peixe como racao ,Litopenaeus vannamei - Abstract
Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2017. A carcinicultura é um dos setores da aquicultura mais rentáveis e o camarão branco do pacífico, Litopenaeus vannamei (Decapoda: Penaeidae) é uma das espécies mais cultivadas mundialmente devido à sua rusticidade, rápido crescimento, fácil manejo e aceitação comercial. O Brasil encontra-se como um grande produtor, favorecido por sua ampla faixa costeira, porém alguns fatores como enfermidade virais e insumos acarretam custos na produção, pois demandam gastos altos de manutenção. A nutrição de espécies é um deles, sendo considerada um grande entrave da carcinicultura, por seu alto custo, em particular devido à utilização da farinha de peixe como um dos componentes nas rações. Contudo, tem-se buscando formas econômicas e sustentáveis de fontes proteicas alternativas para substituição da farinha de peixe. Para isso, tem-se elaborado estudos no fortalecimento do uso de farinha de inseto na alimentação de camarões sem perdas financeiras. O presente trabalho tem por objetivo analisar o perfil de enzimas digestivas de juvenis da espécie de camarões marinhos L. vannamei (n=9), com peso inicial de 4,42 ± 0,02g (média, ± desvio padrão) peso final de 9,23 ± 0,31g (média, ± desvio padrão) e com um ganho de peso de 4,81± 0,30 g (média, ± desvio padrão). Os camarões alimentados com farinhas das larvas do inseto Tenebrio molitor, em diferentes níveis de substituição, com 25%, 50%, 75% e 100% e o controle (farinha de resíduo de salmão). O experimento foi realizado no Laboratório de Camarões Marinhos da UFSC (LCM) iniciando com o cultivo dos camarões em tanques de engorda fornecendo diariamente as rações durante 42 dias. Após esse período de engorda, os animais foram eutanasiados, sendo dissecados o hepatopâncreas e esses mantidos em freezer a -25 ºC em tubos cônicos contendo 1 mL de solução salina 3%. Os hepatopâncreas foram homogeneizados e centrifugados para a realização dos ensaios enzimáticos para determinação de atividades proteolíticas do tipo tripsina (substrato usado bz-R-pNA), quimotripsina (substrato usado suc-AAPF-pNA) e sobre o substrato proteico azoalbumina, além de amilases e lipases. Zimogramas utilizando-se géis SDS-PAGE foram obtidos para fracionamento das atividades proteolíticas e amilásicas. Os dados obtidos mostram que houve um ganho de peso e que a taxa de sobrevivência não foi afetada com a substituição de farinha de inseto na dieta dos camarões. Os resultados deatividade absoluta de tripsina, quimotripsina, lipases e amilases demonstram não haver diferenças significativas entre o controle e os tratamentos, assim como nos padrões das atividades enzimáticas encontradas nos zimogramas. Já as determinações das atividades especificas de quimotripsina e amilases demonstraram diferenças significativas entre a dieta contendo 25% de substituição e a dieta controle. Já na atividade sobre azoalbumina, foi encontrada diferença nos tratamentos 75% para atividade absoluta e 25% para atividade especifica, e no tratamento com 100% para as duas atividades. Abstract : Shrimp farming is one of the most profitable sectors of aquaculture, and the white Pacific shrimp, Litopenaeus vannamei (Decapoda: Penaeidae) is one of the most cultivated species worldwide due to its rusticity, rapid growth, easy handling and commercial acceptance. Brazil is a major producer, favored by its wide coastal area, but factors such as viral illnesses and inputs cause costs in production, as they demand high maintenance costs. The nutrition of species is one of them, being considered a great hindrance of shrimp farming, due to its high cost, in particular due to the use of fish meal as one of the components in the feeds. However, we have been looking for economic and sustainable forms of alternative protein sources for fish meal substitution. For this purpose, studies have been carried out to strengthen the use of insect meal in shrimp feed without financial losses. The objective of the present work was to analyze the digestive enzymes profile of juveniles of the marine shrimp L. vannamei (n = 9), with initial weight of 4.42 ± 0.02 g (mean, standard deviation), 23 ± 0.31 g (mean ± standard deviation) and a weight gain of 4.81 ± 0.30 g (mean ± standard deviation). The shrimp fed the larvae of the mealworm Tenebrio molitor, at different replacement levels, with 25%, 50%, 75% and 100% and control (commercial fish meal). The experiment was carried out in the Laboratório de Camarões Marinhos da UFSC (LCM), starting with the cultivation of shrimp in fattening tanks, providing daily feed for 42 days. After this fattening period, the animals were euthanized and the hepatopancreas were dissected and kept in a freezer at -25 ºC in conical tubes containing 1 mL of 3% saline solution. The hepatopancreas were homogenized and centrifuged for the enzymatic assays to determine protease activities trypsin-like (bz-R-pNA as the substrate), chymotrypsin-like (suc-AAPF-pNA as the substrate) and against the proteinaceous substrate azoalbumin, as well as amylases and lipases. Zymograms using SDS-PAGE gels were obtained for fractionation of proteolytic and amylase activities. The data obtained showed that there the a weight gain and the survival rate were not altered with the incorporation of insect meal in the shrimp diet in addition. The results of the absolute activity of trypsin, chymotrypsin, lipases and amylase demonstrated no significant differences between control and treatments, as well as in the patterns of enzymatic activities found in thezymograms. The determinations of the specific activities of chymotrypsin and amylases showed significant differences between the diet containing 25% of incorporation and the control. In the azoalbumin activity, a difference was found in treatments 75% for absolute activity and 25% for specific activity, and in the treatment with 100% for both activities.
- Published
- 2017
19. Estudo da antibiose e destino da faseolina (Globulina - Phaseolus vulgaris) em larvas do caruncho Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae)
- Author
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Kunz, Daniele, Universidade Federal de Santa Catarina, and Silva, Carlos Peres
- Subjects
Bioquimica ,Coleoptero ,fungi ,food and beverages ,Gorgulho do feijao-de-corda ,Feijao comum - Abstract
Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2014. Abstract : This work confirmed the high toxicity of the storage protein from the seeds of the common bean Phaseolus vulgaris (phaseolin) on the larvae of the seed beetle Callosobruchus maculatus. When incorporated into the diet of the larvae at physiological concentrations (0.5 %) larval mortality of C. maculatus increased. The main factor involved seems to be the accumulation of vesicles containing phaseolin in the enterocytes leading to the impairment of vesicular traffic. The fate of the seed storage globulins from cowpea Vigna unguiculata (vicilin) and from P. vulgaris (phaseolin) was monitored by fluorescence microscopy after cryostat microtomy. By feeding bean beetle larvae with vicilin/ FITC and phaseolin/ FITC noticed a difference in the absorptive process of globulins among the tested diets. The intestinal epithelium of larvae fed phaseolin/ FITC showed higher fluorescence than larvae fed vicilin. A membrane bound protein has been isolated from the intestinal epithelium of larval C. maculatus by using an affinity column where the vicilin was covalently mobilized to sepharose. This protein has a high homology with the a-tocopherol (vitamin E precursor) transfer protein. These data suggest that the vicilin molecule binds to receptors in the microvillar membrane and subsequently it may be absorbed inside vesicles within the epithelial cells of the larval midgut. This process may be a case of receptor-mediated endocytosis. These findings suggest a new mechanism of antibiosis, involving accumulation of vicilin variants and phaseolin in the intestinal epithelium of the C. maculatus larvae and commitment of endocytic processes. This mechanism probably involves the binding of vicilin/phaseolin protein homologous to a a-tocopherol transfer protein, thereby killing the insect by the lack of absorption of important nutrients for development,such as the vitamin E.
- Published
- 2014
20. Estudo da digestão de proteínas com ênfase na distribuição espacial das proteinases digestivas do camarão branco do pacífico Litopenaeus vannamei
- Author
-
Alexandre, Daniel, Universidade Federal de Santa Catarina, and Silva, Carlos Peres
- Subjects
Bioquímica ,Digestao ,Litopenaeus vannamei - Abstract
Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2014. Os camarões possuem importância econômica, distribuídos por todo tropical e subtropical em todas as regiões do mundo. Por causa da pressão contínua sobre os estoques pesqueiros de camarões para consumo humano, a carcinicultura em muitos países se desenvolveu como uma importante ferramenta para suprir fonte de proteína de crustáceos. A espécie Litopenaeus vannamei ou camarão branco do Pacífico é uma espécie oriunda do pacífico que foi introduzida com maior sucesso nas fazendas de cultivo devido suportarem grande variação de salinidade. Possui intestino anterior e posterior de origem ectodérmica e intestino médio de origem endodérmica o que faz esta região uma região de secreção e absorção de nutrientes. No intestino médio dos peneídeos, assim como em insetos, ocorre a presença da membrana peritrófica, sendo que nos insetos lhe foi aferida uma função fisiológica importante: a circulação endo ectoperitrófica. Na presente tese, através do conhecimento da bioquímica da digestão de proteínas e pelo conhecimento do comportamento das enzimas ao longo do intestino médio do camarão, foi possível identificar o mesmo padrão de decréscimo de atividade ao logo deste, que foi desfeito, ao ser suplementado a dieta com proteínas (ovoalbumina nas concentrações de 60% e 80% m/m). Estas atividades enzimáticas foram observadas através de reação de digestão com substratos sintéticos (suc-AAPF-pNA, bz-R-pNA e L-pNA), substrato proteico (Azoalbumina) e zimograma em SDS-PAGE 12% contendo 0,1% de gelatina. Na tentativa de purificar e identificar algumas enzimas foi utilizado cromatografia de troca iônica em coluna do tipo Mono - Q. Em outros experimentos que corroboram a hipótese de circulação endo ectoperitrófica em camarões foi utilizado o processo de gavagem que através de filmagens foi possível visualizar o processo acontecendo, fundamentando assim a circulação endo ectoperitrófica em camarões. Abstract: Prawns and shrimps have economic importance, distributed throughout tropical and subtropical regions in the world. Because of continued pressure on fish stocks of shrimps for human consumption, shrimp farming in many countries has developed as an important tool to meet protein source crustaceans. The Litopenaeus vannamei species or Pacific white shrimp is a species from Pacific which was introduced more successfully in cultivation farms due withstand large variations in salinity. Shrimps have anterior and posterior intestine from ectodermal and endodermal origin, which it makes this region a region of secretion and absorption of nutrients. The midgut of penaeids, as in insects, the presence of the peritrophic membrane occurs, and it was measured in insects an important physiological function: the endo ectoperitrophic circulation. In this thesis, through the knowledge of the biochemistry of protein digestion and knowledge of the behavior of the enzymes along the midgut of juvenile shrimp, it was possible to identify the same pattern of decrease of the enzyme activities, which were shifted, when the diet was supplemented with a protein (ovalbumin at the concentrations of 60% and 80% w / w). These enzyme activities were assessed by digestion reaction with synthetic substrates (suc-AAPF-pNA, bz-R-L-pNA and pNA) protein substrate (azoalbumina) and zymogram on SDS-12% PAGE containing 0.1% gelatin. In an attempt to purify and identify some enzymes it was used ion-exchange chromatography on Mono-type column - Q. Other experiments support the hypothesis that the endo ectoperitrophic circulation takes place in shrimps by using the gavage process, through which it was possible to document the process.
- Published
- 2014
21. Identificação de poligalacturonases expressas no sistema digestório de Callosobruchus maculatus (Coleoptera chrysomelidae:bruchinae)
- Author
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Santana, Mychelle Carneiro, Universidade Federal de Santa Catarina, and Silva, Carlos Peres
- Subjects
Bioquimica ,Feijao-de-corda ,Gorgulho do feijao-de-corda ,Pectinase - Abstract
Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2013. As paredes celulares das plantas contém uma complexa mistura de polissacarídeos e proteínas, demandando uma série de enzimas para degradá-las. Organismos capazes de secretar enzimas de degradação podem aproveitar as paredes celulares como fonte de nutrientes, agindo como verdadeiros patógenos vegetais, gerando perdas nas produções agrícolas e consequentemente à economia. A busca por novas técnicas de controle de pragas tem impulsionado estudos baseados na sua biologia, visando diminuir o uso de substâncias químicas, sabidamente prejudiciais ao meio ambiente e aos consumidores. Trabalhos anteriores detectaram sequências gênicas supostamente codificadoras de poligalacturonases em larvas de C. maculatus, besouro praga do feijão caupi. Estas enzimas degradam o ácido poligalacturônico (PGA) presente na matriz de pectina na parede celular vegetal. A partir destes dados, investigamos se os genes estavam sendo expressos e se as enzimas tinham atividade funcional no animal. Amostras de homogeneizados intestinais do inseto foram ensaiadas contra PGA em teste de difusão radial, onde a atividade detectada foi proporcional à quantidade de amostra aplicada, demonstrando que a enzima além de ser expressa, é também funcional. A distribuição espacial da enzima foi ensaiada através da dosagem dos açúcares redutores, cujo tampão mais adequado foi o citrato-fosfato, com atividade ótima no pH 6,5. Os resultados mostraram maior atividade nas amostras do conteúdo luminal, confirmando que a enzima é secretada no intestino do inseto, embora os valores das atividades absoluta e específica tenham se mostrado baixos. SDS-PAGE foi preparado para confirmação do número de isoformas encontradas em estudos anteriores. Apenas duas isoformas foram detectadas, com formação de bandas em diferentes faixas de pHs. Atividade da enzima pectinase também foi ensaiada através do teste de difusão radial contra pectina, apresentando atividade e proporcionalidade em relação à quantidade de amostra aplicada, constituindo um novo ponto a ser abordado na espécie quanto às enzimas celulolíticas. Abstract : Plant cell walls contain a complex mixture of proteins and polysaccharides, requiring a number of enzymes to degrade them. Organisms capable of secreting enzymes active on the cell wall constituents can use it as a source of nutrients, acting as true plant pathogens, generating losses in agricultural production and consequently in the economy. The search for new methods of pest control has driven studies based on their biology, aiming to reduce the use of chemicals, known to harm the environment and consumers. In larvae of Callosobruchus maculatus, known as cowpea beetle, gene sequences encoding putative polygalacturonases were detected. These enzymes degrade polygalacturonic acid (PGA) present in the matrix of pectin in the plant cell wall. To assess if the enzymes were functional in C. maculatus, samples containing intestinal homogenates were tested against PGA in radial diffusion assays, where the activity detected was proportional to the amount of sample applied. The confirmation of the origin of the activity was performed using the same test, by using a cowpea flour extract, as enzyme source, which was inactive. The spatial distribution of the enzyme among the different compartments of the gut of the insect was assayed by the DNS method, whose the most suitable buffer among the tested, was citrate-phosphate, with maximum activity at pH 6.5. Results showed greater activity in the samples of luminal contents, confirming that the enzymes are secreted in the gut of the insect, although the values of absolute and specific activities have been shown very low. SDS-PAGE was prepared to confirm the number of isoforms found in the transcriptome and in the proteome of C. maculatus, however, only two isoforms were detected with bands at different pH values. Due to the commonly presence of genes encoding plants cell wall degrading enzymes in other members of the Chrysomelidae family, pectinase activity was also assayed in intestinal homogenates of larval C. maculatus. Radial diffusion assays against pectin confirmed activity and proportionality in relation to the amount of sample applied, constituting a new point to be addressed about the cellulolytic enzymes in the species
- Published
- 2013
22. Investigação sobre a adaptação do inseto praga de cereais Tenebrio molitor a inibidores de proteinases digestivas
- Author
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Alexandre, Daniel, Universidade Federal de Santa Catarina, and Silva, Carlos Peres
- Subjects
Bioquimica ,Inseto ,Interacao animal-planta ,Inibidores de proteases ,Digestao - Abstract
Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2010 Nas larvas de Tenebrio molitor (verme do trigo) são encontrados variados tipos de proteinases digestivas serínicas e cisteínicas, quando alimentados com uma dieta controle de farinha de trigo. Quando alimentados com farinha do feijão comum (Phaseolus vulgaris), pelo menos cinco proteinases apresentam atividade mais elevadas, enquanto a expressão de três enzimas pré-existentes foi reprimida. A indução dessas proteinases ocorreu entre 30 min e 1 h após a alimentação. Aqui nós relatamos o fracionamento das proteinases envolvidas na adaptação de T. molitor a inibidores de proteinases em sementes de leguminosas incorporadas em uma dieta controle usando cromatografia de troca iônica e filtração em gel, seguida pela caracterização das enzimas com substratos e inibidores naturais e sintéticos. Os inibidores utilizados neste estudo (todos incorporados na concentração de 0,4% no farelo de trigo) foram: inibidor de tripsina de soja (STI), inibidor de tripsina e quimotripsina de soja (SBI), inibidor de tripsina de Adenanttera pavonina (ApTI) e o inibidor de tripsina de P. vulgaris (BTI). As atividades proteolíticas foram ensaiadas para azoalbumina, para os substratos fluorogênicos z-PR-MCA, z-RR-MCA e succinil-GGR-MCA e os substratos cromogênicos suc-AAPF-pNA e benzoil-R-pNA. As atividades também foram monitoradas através de zimogramas e de fracionamento em géis bidimensionais. Dos quatro inibidores testados, o ApTI foi capaz de inibir as enzimas proteolíticas majoritárias sem induzir enzimas insensíveis a este inibidor, enquanto os outros inibidores além de inibir as proteinases constitutivas causaram indução de um conjunto diferente de proteinases serínicas. Entre as proteinases induzidas, uma era de interesse particular, pois foi induzida após sessenta minutos após a ingestão de SBI, STI e BTI. Esta proteinase teve sua atividade caracterizada como uma quimotripsina através de análise eletroforética bidimensional seguida de sequenciamento por espectrometria de massas.
- Published
- 2012
23. Ontogenia do sistema digestório das larvas de jundiá, Rhamdia quelen, determinada através de estudos bioquímicos e histológicos
- Author
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Silveira, Jenniffer, Universidade Federal de Santa Catarina, Fracalossi, Débora Machado, and Silva, Carlos Peres
- Subjects
Aquicultura ,Jundiá (Peixe) ,Fisiologia ,Proteinase ,Digestao ,Ontogenia - Abstract
Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias. Programa de Pós-Graduação em Aqüicultura. Larvas de jundiá possuem saco vitelínico relativamente grande, quando comparado com outras espécies de peixes nativos, o que sugere que possam digerir alimento inerte desde o início da alimentação. Portanto, avaliou-se o desenvolvimento morfológico e a atividade proteolítica do sistema digestório de larvas de jundiá desde a eclosão até 252 horas após a eclosão (HAE). As atividades das proteinases serínicas variaram ao longo do tempo, sendo observado o maior pico às 12 HAE, quando a tripsina mostrou atividade específica de 296,38 ± 84,20 mU/mg e a quimotripsina de 315,45 ± 42,16 mU/mg. As atividades das proteinases aspárticas aumentaram até o início da transição alimentar ou desmame, oscilaram ao longo do desmame, mas voltaram a crescer constante e acentuadamente durante a alimentação com o alimento inerte, obtendo-se a maior atividade específica às 252 HAE (2,56 ± 0,10 mU/mg). Os ensaios in gel mostraram uma diversidade de formas moleculares, principalmente de proteinases serínicas. As análises histológicas mostraram que às 0 HAE as larvas já apresentam desenvolvimento do trato gastrointestinal, às 4 HAE há o início da abertura bucal e às 16 HAE as larvas apresentam diferenciação no trato digestório, quando é possível diferenciar a cavidade da bucofaringe, esôfago, fígado, pâncreas, estômago e intestino. Às 40 HAE são observados grânulos de zimógenos no pâncreas e às 48 HAE, a presença de glândulas gástricas no estômago e muco no tubo digestório. Os resultados indicam que larvas de jundiá podem receber ração desde o início da alimentação exógena.
- Published
- 2012
24. Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus.
- Author
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Bittencourt-Cunha PR, Silva-Cardoso L, Oliveira GA, Silva JR, Silveira AB, Kluck GE, Souza-Lima M, Gondim KC, Dansa-Petretsky M, Silva CP, Masuda H, Silva Neto MA, and Atella GC
- Subjects
- Animals, Female, Membrane Lipids metabolism, Rhodnius physiology, Digestive System metabolism, Lipid Metabolism physiology, Phospholipids metabolism, Rhodnius metabolism
- Abstract
In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.
- Published
- 2013
- Full Text
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