Immunological indices of Giant African Land snails (Archachatina marginata) improved with fixed dose of vitamin C during acute heat stress

The study determined immunological indices of Giant African Land snail (Archachatina marginata) improved with fixed dose of vitamin C under acute heat stress (AHS). Prior to the AHS, vitamin C was administered for four weeks to two treatment groups, while other two treatment groups were not. Each treatment was monitored, haemolymph collected at 0, 30 and 60 minutes exposure times. Immunological cytokines: interferon gamma (IFN-γ) and interleukin 2 (IL-2); and total haemocyte counts (THC) were determined. Under AHS, vitamin C elevated (p <0.05) IFN-γ production (606.33 ± 302.86) compared to other groups with or without vitamin C administration (7.20 ± 1.58 vs. 73.20 ± 32.23 vs. 7.80 ± 1.36). IL-2 was not affected (p >0.05) by vitamin C under AHS. Highest (p <0.05) THC values was obtained with vitamin C administration under AHS, but reduced under no AHS. Exposure time affected (p <0.05) IFN-γ production and THC values, but not IL-2 (p >0.05). With fixed dose of vitamin C and exposure time, highest (p <0.05) IFN-γ values were obtained under AHS with vitamin C administration at 30 minutes and at 60 minutes in THC, compared to other groups. The study concluded that fixed dose of vitamin C at 150 mg kg-1 of feed was appropriate under AHS to boost the immune system of the animals.(AU)

Genetic characterization of silkworm (Bombyx mori L. ) strains (Lepidoptera: Bombicidae) with different geographical originon the basis of isozyme markers

The aim of the present study was to assess genetics variations within and among strains with different geographical origin, belonging to Bulgarian germplasm bank of mulberry silkworm (Bombyx moriL.)and to establish their relationships using isoenzyme markers. Polyacrilamide gel electrophoresis (PAGE) was used to study the isoenzymes of nonspecific esterases (EST), malate dehydrogenase (MDH) and acid phosphatase (ACP) from haemolymph, phosphoglucomutase (PGM) and hexokinase (HK) from silk glands and alkaline phosphatase (ALP) from midgut of mulberry silkworm (Bombyx moriL.). Variability was evident in all of these enzyme systems among twelve strains from Bulgarian germplasm resources of B. mori.Total of nine lociwere detected. All of them (100%) were polymorphic. “Null” alleles in four lociwere determined. Intra-and inter-strain polymorphism was obtained. The degree of polymorphism ranged from 0% to 77.80%. Low levels of observed heterozygosity in comparison with the expected one have been calculated in almost all of strainsas well as deviations from Hardy-Weinberg equilibrium in almost all analyzed loci, resultingof excess of homozygotes. The value of FSTwas 0.4903. The dendrogram constructed with the values of genetic distance manifests that Romanian strain Cislau Tokay is formed one main clade while the rest strains studied (from Bulgaria, Japan, China, Vietnam, Spain, Syria and Egypt) are formed the other clade withsubclades. The genetic data of the tested strains from different geographical regions, would be used for identifying suitable parents for breeding programs with a view to yield improvement in this species with economic significance.

Phenoloxidase activity and haemolymph cytology in honeybees challenged with a virus suspension (deformed wings virus DWV) or phosphate buffered suspension (PBS) / Atividade fenoloxidase e citologia de hemolinfa em abelhas infectadas com uma suspensão de vírus (DWV deformed wings) ou suspensão tamponada com fosfato (PBS)

The innate immune system of honeybees mainly consists in antimicrobial peptides, cellular immunity and melanisation. In order to investigate the immune response of honeybees to immune stressors, three stress degrees were tested. Newly emerged bees naturally DWV-infected were collected from a Varroa mite-free apiary and divided into three experimental groups: naturally DWV infected bees, PBS injected bees, and artificially DWV super infected bees. Phenoloxidase activity and haemolymph cellular subtype count were investigated. Phenoloxidase activity was highest (P<0.05) in DWV-superinfected bees, and the haemocyte population differed within the three observed groups. Although, immune responses following DWV infection have still not been completely clarified, this investigation sheds light on the relation between cell immunity and the phenoloxidase activity of DWV-naturally infected honeybees exposed to additional stress such as injury and viral superinfection.(AU)

O sistema imune inato das abelhas consiste principalmente em peptídeos antimicrobianos, imunidade celular e melanização. Para investigar a resposta imune das abelhas a estressores imunológicos, foram testados três graus de estresse. Abelhas recém-emergidas naturalmente infectadas por DWV foram coletadas de um apiário livre de Varroa e divididas em três grupos experimentais: abelhas naturalmente infectadas por DWV, abelhas injetadas com PBS e abelhas superinfectadas artificialmente com DWV. A atividade de fenoloxidase e a contagem de subtipos celulares de hemolinfa foram investigadas. A atividade da fenoloxidase foi maior (P<0,05) nas abelhas super-infectadas com DWV, e a população de hemócitos diferiu entre os três grupos observados. Embora as respostas imunes após a infecção pelo DWV ainda não tenham sido completamente esclarecidas, esta investigação lança luz sobre a relação entre a imunidade celular e a atividade da fenoloxidase das abelhas infectadas naturalmente pelo DWV, expostas a estresse adicional, como lesão e superinfecção viral.(AU)

Low water temperature retards white spot syndrome virus replication in Astacus leptodactylus crayfish

Background: White spot syndrome virus is a pathogen of major economic importance to cultured penaeid shrimp industries globally. White spot disease can cause mortalities reaching 100% within 3-10 days of gross signs appearing. WSSV replicates in tissues from mesoderm and ectoderm embryonic origin and characteristically induces cell nuclei hypertrophy and intra nuclear inclusion bodies. WSSV also has an extremely broad host range including marine and freshwater crabs and crayfishes, copepods and other arthropods in addition to shrimp. Water temperature can affect the progress of WSD in crustaceans but there have been conflicting reports of higher temperatures protecting Litopenaeus vannamei shrimp but lower temperatures protecting Marsupenaeus japonicas. Here we have examined how 2 water temperatures affect the progression of WSD in the freshwater crayfish Astacus leptodactylus. Materials, Methods & Results: Freshwater Astacus leptodactylus crayfi sh (20 ± 0.5 g) were obtained from Aras dam, Iran. Crayfish were acclimated for 10 days in an aerated indoor cement tank with flow-through of dechlorinated freshwater with the flow rate set at 0.5 L/s, water temperature 15 ± 1ºC and dissolved oxygen 5.2 ppm. Two groups of 25 crayfish were allocated to tanks being supplied 15 ± 1°C water and 2 groups of 25 crayfish were allocated to tanks being supplied 25 ± 1°C water. Each crayfish was injected intramuscularly into soft tissue at the base of swimming legs with either 50 µL inoculum containing 103.2 lethal units/mL. A negative control group of crayfish was injected with PBS. Mortality amongst groups was monitored for 30 days and WSSV DNA present in haemolymph collected on Days 3, 5 and 10 post-injection was detected by nested-PCR. Morbidity and mortalities amongst crayfish held in lower temperature water were delayed and WSSV DNA was detected by nested-PCR at Day 10 pi compared to being clearly detected at Days 3, 5 and 10 pi amongst crayfish held in higher temperature water. Evidence of eosinophilic intranuclear inclusions detected by histology correlated with when WSSV was first detected by nested-PCR. Discussion: The data indicate that low water temperature retards WSSV replication in A. leptodactylus crayfi sh. Similar temperature-related effects on WSD progression have been reported in other freshwater crayfish species. For example, no mortality occurred amongst WSSV-infected Pacifastacus leniusculus held in either 4 ± 2°C or 12 ± 2°C water, but all crayfish died with WSD symptoms after they were transferred to 22 ± 2°C water. In another study, Procambarus clarkia held in 24 ± 1°C water all died by Day 9 post-challenge with WSSV but amongst crayfish held in 18 ± 1°C water, mortality only started on Day 10 post-challenge and in took until Day 22 before all had died, and amongst crayfish held in 10 ± 1°C water, no deaths occurred up to Day 24 post-challenge when the bioassay was terminated. A. leptodactylus injected with WSSV and maintained in 15 ± 1°C water only became moribund and nested-PCR-positive for WSSV by Day 10 pi in contrast to crayfish held in 25 ± 1°C water that started to die and were PCR-positive on Day 3 pi. Unlike WSD in shrimp, no histological evidence of basophilic intra nuclear inclusions was observed in WSSV-infected crayfish tissues even in the late stages of disease. This might be attributed to the over expression of anti-lipopolysaccharide factor in crayfish and/or the low numbers of apoptotic haemocytes that develop in WSSV-infected crayfish compared to the shrimp. Whether the over-expression of anti-lipopolysaccharide factor in combination with lowered WSSV replication induced by lowered water temperature both contributed to slowing the progress of WSD in Astacus leptodactylus warrants further investigation.

EFFECT OF TEMPERATURE AND FOOD PROTEIN AND LIPID IN STAGES OF OVARIAN MATURATION AND STAGES OF MOLTING Macrobrachium acanthurus (WIEGMANN, 1836) / EFEITO DA TEMPERATURA E DOS ALIMENTOS PROTÉICO E LIPÍDICO NOS ESTÁDIOS DE MATURAÇÃO OVARIANA E ESTÁGIOS DE MUDA DO Macrobrachium acanthurus (WIEGMANN, 1836)

Knowing the reproductive physiology of the prawn Macrobrachium acanthurus in relation to controlled factors, such as processed food and temperature variations, would help to develop culture techniques, assuring its survival. Aiming at the finding some data on this subject, females of M. acanthurus were submitted to the effect of three temperatures (20, 25 and 30oC) and three types of food (lipidic, proteic and commercial). Following this procedure, the effects of haemolymph glucose, total protein and total lipid levels on ovarian maturation as well as in the moulting stages were analyzed. Results showed that nutrient levels usually increased as the animals reached D stage and the temperature increased. The nutrient levels also increased as animals reached stage III of ovarian maturation. Glucose and total protein levels increased with increasing temperature, while total lipid levels diminished at 30oC. Lipid food produced an increase in the haemolymph glucose and total proteins, while lipid and commercial food produced an increase in total lipids. In spite of that, results showed no significant differences among moulting stages and ovarian maturation, when measured at the same temperature.KEYWORDS: glucose; haemolymph; physiology; total lipid; total protein.

O conhecimento da fisiologia reprodutiva do camarão Macrobrachium acanthurus em relação a fatores controlados, como alimentos processados e variações de temperaturas, ajudaria a desenvolver técnicas de cultivo, garantindo sua sobrevivência. Com o objetivo de contribuir para esse conhecimento, fêmeas de M. acanthurus foram submetidas ao efeito das temperaturas de 20, 25 e 30oC e de três alimentos (lipídico, protéico e comercial). Em seguida, os efeitos nos níveis de glicose, proteínas totais e lipídios totais na hemolinfa, tanto na maturação ovariana como nos estágios de muda, foram analisados. Os resultados mostraram que, nos estágios de muda, os níveis dos nutrientes na hemolinfa, em geral, aumentaram conforme os animais chegam ao estágio de muda D, com o aumento da temperatura. Nos estádios de maturação ovariana, os nutrientes na hemolinfa aumentaram conforme se aproximavam ao estádio III. Os níveis de glicose e proteínas totais aumentaram com o incremento da temperatura, enquanto que os níveis de lipídios totais diminuíram a 30oC. Dentre os alimentos, o lipídico induziu ao aumento de glicose e proteínas totais na hemolinfa e os alimentos comercial e protéico induziram ao aumento de lipídios totais.PALAVRAS-CHAVE: fisiologia; glicose; hemolinfa; lipídios total; proteína total.

Seasonal variations in the intermediate metabolism of the crayfish Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) in the natural environment and experimental culture

The goal of this study was to evaluate the effects of seasonal variations on energy metabolism in different tissues of the freshwater crayfish Parastacus brasiliensis (von Martens, 1869). Crayfish were collected monthly from January 2001 to January 2003 in São Francisco de Paula, Rio Grande do Sul, Brazil, in a stream and in a culture tank. Haemolymph samples were collected from each crayfish in the field with a syringe, by puncturing the membrane at the base of the chelipeds. Hepatopancreas, gills, and abdominal muscle were removed for determination of free glucose, glycogen, total lipids, and triglycerides. The haemolymph samples were used for determination of glucose, total proteins, total lipids, and triglycerides. Statistical analysis revealed significant differences in biochemical composition in crayfish collected in the stream compared to the experimental tank during the year, principally in glucose and triglycerides in haemolymph, glycogen and total lipids in all tissues study, and triglycerides only in abdominal muscle. The regular food intake partially modified these seasonal variations of the metabolic pattern. Environmental conditions (e.g., food availability and water temperature) and reproductive period appeared to be the main factors influencing the seasonal patterns of variation in energy metabolism.

O objetivo deste estudo foi avaliar o efeito das variações sazonais no metabolismo energético em diferentes tecidos do lagostim de água-doce Parastacus brasiliensis (von Martens, 1869). Os lagostins foram coletados mensalmente de Janeiro de 2001 a Janeiro de 2003 em São Francisco de Paula, Rio Grande do Sul, Brasil, no riacho e no tanque de cultivo. Amostras de hemolinfa foram coletadas de cada lagostim em campo com seringa, por punção na membrana da base dos quelípodos. Hepatopâncreas, brânquias e músculo abdominal foram removidos para a determinação dos níveis de glicose livre, de glicogênio, de lipídios totais e de triglicerídeos. As amostras de hemolinfa foram utilizadas para a determinação dos níveis de glicose, de proteínas totais, de lipídios totais e de triglicerídeos. As análises estatísticas revelaram uma variação significativa na composição bioquímica dos lagostins coletados no riacho quando comparados com os do cultivo experimental durante o ano, principalmente da glicose e dos triglicerídeos na hemolinfa, glicogênio e lipídios totais em todos os tecidos estudados, e dos triglicerídeos somente no músculo abdominal. O aporte regular de alimento modificou parcialmente estas variações sazonais do padrão metabólico. As condições ambientais (ex.; disponibilidade de alimento e a temperatura da água) e o período reprodutivo parecem ser os principais fatores a influenciar os padrões sazonais da variação do metabolismo energético.

Seasonal variations in the intermediate metabolism of the crayfish Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) in the natural environment and experimental culture

The goal of this study was to evaluate the effects of seasonal variations on energy metabolism in different tissues of the freshwater crayfish Parastacus brasiliensis (von Martens, 1869). Crayfish were collected monthly from January 2001 to January 2003 in São Francisco de Paula, Rio Grande do Sul, Brazil, in a stream and in a culture tank. Haemolymph samples were collected from each crayfish in the field with a syringe, by puncturing the membrane at the base of the chelipeds. Hepatopancreas, gills, and abdominal muscle were removed for determination of free glucose, glycogen, total lipids, and triglycerides. The haemolymph samples were used for determination of glucose, total proteins, total lipids, and triglycerides. Statistical analysis revealed significant differences in biochemical composition in crayfish collected in the stream compared to the experimental tank during the year, principally in glucose and triglycerides in haemolymph, glycogen and total lipids in all tissues study, and triglycerides only in abdominal muscle. The regular food intake partially modified these seasonal variations of the metabolic pattern. Environmental conditions (e.g., food availability and water temperature) and reproductive period appeared to be the main factors influencing the seasonal patterns of variation in energy metabolism.

O objetivo deste estudo foi avaliar o efeito das variações sazonais no metabolismo energético em diferentes tecidos do lagostim de água-doce Parastacus brasiliensis (von Martens, 1869). Os lagostins foram coletados mensalmente de Janeiro de 2001 a Janeiro de 2003 em São Francisco de Paula, Rio Grande do Sul, Brasil, no riacho e no tanque de cultivo. Amostras de hemolinfa foram coletadas de cada lagostim em campo com seringa, por punção na membrana da base dos quelípodos. Hepatopâncreas, brânquias e músculo abdominal foram removidos para a determinação dos níveis de glicose livre, de glicogênio, de lipídios totais e de triglicerídeos. As amostras de hemolinfa foram utilizadas para a determinação dos níveis de glicose, de proteínas totais, de lipídios totais e de triglicerídeos. As análises estatísticas revelaram uma variação significativa na composição bioquímica dos lagostins coletados no riacho quando comparados com os do cultivo experimental durante o ano, principalmente da glicose e dos triglicerídeos na hemolinfa, glicogênio e lipídios totais em todos os tecidos estudados, e dos triglicerídeos somente no músculo abdominal. O aporte regular de alimento modificou parcialmente estas variações sazonais do padrão metabólico. As condições ambientais (ex.; disponibilidade de alimento e a temperatura da água) e o período reprodutivo parecem ser os principais fatores a influenciar os padrões sazonais da variação do metabolismo energético.

Seasonal variation of the effect of high-carbohydrate and high-protein diets on the intermediate metabolism of Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) maintained in the laboratory

The goal of this study was to evaluate the effect of a high-carbohydrate diet (HC) and a high-protein diet (HP) on the metabolism of the crayfish Parastacus brasiliensis (Von Martens, 1869), collected in different seasons and maintained in the laboratory for 15 days. Crayfish were collected monthly from January 2002 to January 2004 at São Francisco de Paula, Southern Brazil, in Guarapirá stream. In the laboratory, the animals were kept submerged in aquariums under controlled conditions. They were fed ad libitum, for 15 days with either a HC or HP diet. At the end of this period, haemolymph samples were collected, as were hepatopancreas, gills, and abdominal muscle that were removed for determination of glycogen, free glucose, lipids, and triglycerides. The haemolymph samples were used for determination of glucose, proteins, lipids, and triglycerides. Statistical analysis (ANOVA) revealed significant seasonal differences in biochemical composition in crayfish maintained on HC or HP diets. Independent of the diets offered to the animals and the controlled conditions for 15 days, the indications of seasonality were unchanged. The observed changes seemed to be related to the reproductive period. Moreover, the HC diet increased all energy reserves in adult parastacids, which may aid in reproduction.

O objetivo deste estudo foi avaliar o efeito da dieta rica em carboidratos (HC) e da dieta rica em proteínas (HP) sobre o metabolismo do Parastacus brasiliensis (Von Martens, 1869) coletados em diferentes estações e mantidos durante 15 dias em laboratório. Os lagostins foram coletados mensalmente de Janeiro de 2002 a Janeiro de 2004 em São Francisco de Paula, Sul do Brasil, no riacho Guarapirá. No laboratório, os animais foram mantidos submersos em aquários sob condições controladas. Eles foram alimentados ad libitum por 15 dias com a dieta HC ou HP. Após o final do período, as amostras de hemolinfa foram coletadas, assim como o hepatopâncreas, as brânquias e o músculo abdominal foram removidos para a determinação do glicogênio, glicose livre, lipídios e triglicerídeos. As amostras de hemolinfa foram usadas para a determinação da glicose, proteínas, lipídios e triglicerídeos. As análises estatísticas (ANOVA) revelaram uma diferença sazonal significativa na composição bioquímica nos lagostins mantidos com dieta HC ou HP. Independente da dieta oferecida para os animais e das condições controladas por 15 dias, não foi possível alterar os perfis metabólicos encontrados sazonalmente nos animais em campo. As mudanças observadas parecem estar relacionadas ao período reprodutivo. Além disso, a dieta HC aumentou todas as reservas energéticas nos parastacídeos adultos, o que pode auxiliar na reprodução.

Seasonal variation of the effect of high-carbohydrate and high-protein diets on the intermediate metabolism of Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) maintained in the laboratory

The goal of this study was to evaluate the effect of a high-carbohydrate diet (HC) and a high-protein diet (HP) on the metabolism of the crayfish Parastacus brasiliensis (Von Martens, 1869), collected in different seasons and maintained in the laboratory for 15 days. Crayfish were collected monthly from January 2002 to January 2004 at São Francisco de Paula, Southern Brazil, in Guarapirá stream. In the laboratory, the animals were kept submerged in aquariums under controlled conditions. They were fed ad libitum, for 15 days with either a HC or HP diet. At the end of this period, haemolymph samples were collected, as were hepatopancreas, gills, and abdominal muscle that were removed for determination of glycogen, free glucose, lipids, and triglycerides. The haemolymph samples were used for determination of glucose, proteins, lipids, and triglycerides. Statistical analysis (ANOVA) revealed significant seasonal differences in biochemical composition in crayfish maintained on HC or HP diets. Independent of the diets offered to the animals and the controlled conditions for 15 days, the indications of seasonality were unchanged. The observed changes seemed to be related to the reproductive period. Moreover, the HC diet increased all energy reserves in adult parastacids, which may aid in reproduction.

O objetivo deste estudo foi avaliar o efeito da dieta rica em carboidratos (HC) e da dieta rica em proteínas (HP) sobre o metabolismo do Parastacus brasiliensis (Von Martens, 1869) coletados em diferentes estações e mantidos durante 15 dias em laboratório. Os lagostins foram coletados mensalmente de Janeiro de 2002 a Janeiro de 2004 em São Francisco de Paula, Sul do Brasil, no riacho Guarapirá. No laboratório, os animais foram mantidos submersos em aquários sob condições controladas. Eles foram alimentados ad libitum por 15 dias com a dieta HC ou HP. Após o final do período, as amostras de hemolinfa foram coletadas, assim como o hepatopâncreas, as brânquias e o músculo abdominal foram removidos para a determinação do glicogênio, glicose livre, lipídios e triglicerídeos. As amostras de hemolinfa foram usadas para a determinação da glicose, proteínas, lipídios e triglicerídeos. As análises estatísticas (ANOVA) revelaram uma diferença sazonal significativa na composição bioquímica nos lagostins mantidos com dieta HC ou HP. Independente da dieta oferecida para os animais e das condições controladas por 15 dias, não foi possível alterar os perfis metabólicos encontrados sazonalmente nos animais em campo. As mudanças observadas parecem estar relacionadas ao período reprodutivo. Além disso, a dieta HC aumentou todas as reservas energéticas nos parastacídeos adultos, o que pode auxiliar na reprodução.

Protein eletropherogram of haemolymph from Apis mellifera L. bees submitted to royal jelly production / Eletroferograma de proténas de hemolinfa de abelhas Apis mellifera L. submetidos à produção de geléia real

The objective of this research was to study the eletropherogram of the proteins of the haemolymph in Apis mellifera L. worker bees by utilizing eletroforese in polyacrylamide gel. Haemolymph samples were obtained from 18 beehives of africanized honey bees and their hybrids, italian and carniolas, which were submitted to royal jelly production, either with or without access to 20% pollen protein. Haemolymph sample from recently emerged adult bees aged, 12, 18 and 24 days was obtained. These samples were extracted by introducing a 50 L micropipette tip into the dorsal heart of each bee, through the intrasegmental membrane, between the third and the fourth abdomen segments. In order to inhibit the tirosinase activity, haemolymph samples were treated with three drops of fenyltio-ureia. The dosage absorbance rate of protein for each sample were determined by the use of the spectrophotometer and the biochemical biureto method. The absorbance rate was measured at 540 nm. The PhastSystem and 8-25% gradient mini polyacrylamide gels were used in the sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) technique. Five hundred ng of protein were applied to each well. After one run of the electrophoresis, the gels were stained with silver according to the manufacturus specifications. In the haemolymph of newly emerged adult bees, the electrophoresis pattern is evenly distribute

O objetivo do presente trabalho foi estudar o eletroferograma de proteínas da hemolinfa de operárias de Apis mellifera L., através de eletroforese em gel de poliacrilamida. As amostras de hemolinfa foram obtidas de 18 colônias de abelhas africanizadas e suas híbridas, italianas e cárnicas, submetidas à produção de geléia real, com e sem acesso a 20% de proteína proveniente de pólen. As amostras de hemolinfa de abelhas adultas recém-emergidas, de 12, 18 e 24 dias de idade foram obtidas pela introdução de uma ponteira de aplicação de polietileno de 50 L no coração dorsal, através da membrana intrasegmental, entre o terceiro e o quarto segmentos abdominais de cada abelha. A hemolinfa extraída foi misturada com três gotas de feniltio-uréia, para inibir a atividade da tirosinase. A dosagem de proteínas foi determinada em espectrofotômetro pelo método bioquímico de biureto. A absorbância foi medida em 540 nm, e após calculada a quantidade de proteína de cada amostra. No fracionamento eletroforético das proteínas foram empregados o PhastSystem e mini géis de poliacrilamida em gradiente 8-25% na técnica de SDSPAGE. Foram aplicados 500 ng de proteína em cada ponto de aplicação. Após a corrida, os géis foram corados com prata, de acordo com as especificações do fabricante. Na hemolinfa das abelhas adultas recém emergidas, o padrão eletroforético está distribuído uniformemente entre band

Protein eletropherogram of haemolymph from Apis mellifera L. bees submitted to royal jelly production / Eletroferograma de proténas de hemolinfa de abelhas Apis mellifera L. submetidos à produção de geléia real

The objective of this research was to study the eletropherogram of the proteins of the haemolymph in Apis mellifera L. worker bees by utilizing eletroforese in polyacrylamide gel. Haemolymph samples were obtained from 18 beehives of africanized honey bees and their hybrids, italian and carniolas, which were submitted to royal jelly production, either with or without access to 20% pollen protein. Haemolymph sample from recently emerged adult bees aged, 12, 18 and 24 days was obtained. These samples were extracted by introducing a 50 L micropipette tip into the dorsal heart of each bee, through the intrasegmental membrane, between the third and the fourth abdomen segments. In order to inhibit the tirosinase activity, haemolymph samples were treated with three drops of fenyltio-ureia. The dosage absorbance rate of protein for each sample were determined by the use of the spectrophotometer and the biochemical biureto method. The absorbance rate was measured at 540 nm. The PhastSystem and 8-25% gradient mini polyacrylamide gels were used in the sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) technique. Five hundred ng of protein were applied to each well. After one run of the electrophoresis, the gels were stained with silver according to the manufacturus specifications. In the haemolymph of newly emerged adult bees, the electrophoresis pattern is evenly distribute

O objetivo do presente trabalho foi estudar o eletroferograma de proteínas da hemolinfa de operárias de Apis mellifera L., através de eletroforese em gel de poliacrilamida. As amostras de hemolinfa foram obtidas de 18 colônias de abelhas africanizadas e suas híbridas, italianas e cárnicas, submetidas à produção de geléia real, com e sem acesso a 20% de proteína proveniente de pólen. As amostras de hemolinfa de abelhas adultas recém-emergidas, de 12, 18 e 24 dias de idade foram obtidas pela introdução de uma ponteira de aplicação de polietileno de 50 L no coração dorsal, através da membrana intrasegmental, entre o terceiro e o quarto segmentos abdominais de cada abelha. A hemolinfa extraída foi misturada com três gotas de feniltio-uréia, para inibir a atividade da tirosinase. A dosagem de proteínas foi determinada em espectrofotômetro pelo método bioquímico de biureto. A absorbância foi medida em 540 nm, e após calculada a quantidade de proteína de cada amostra. No fracionamento eletroforético das proteínas foram empregados o PhastSystem e mini géis de poliacrilamida em gradiente 8-25% na técnica de SDSPAGE. Foram aplicados 500 ng de proteína em cada ponto de aplicação. Após a corrida, os géis foram corados com prata, de acordo com as especificações do fabricante. Na hemolinfa das abelhas adultas recém emergidas, o padrão eletroforético está distribuído uniformemente entre band

Haemolymph electrophoretic pattern of Ascia monuste orseis larvae (Lepidoptera: pieridae) parasitized by Cotesia glomerata (Hymenoptera: braconidae)

Cotesia glomerata is a natural enemy of the vegetable plague Ascia monuste orseis and preferably parasites 2nd, 3rd and 4th instar larvae. Parasitism effects on the haemolymph protein profile of Ascia monuste orseis larvae from the 2nd to 7th days were analyzed both qualitatively and quantitatively by SDS-PAGE and Coomassie-Blue binding methods. Quantitative analysis showed a progressive increase in the protein content of about 6.5 and 12.5 times in parasitized and non-parasitized larvae from the 2nd to 5th days, respectively. On the 6th day, a decrease in protein content was observed in both groups, although this decrease was significantly less than the control group that continued to metamorphosis. Meanwhile, parasitized larvae had one more day (7th day) in their larval period to complete parasitoid development, justified by the fact that parasitoid is koinobiont and allows host feeding. On this day, a drastic increase in protein content was detected when the parasitoids left the host. The SDS-PAGE showed proteins of high molecular weight (>120 kDa) on the 5th day of the non-parasitized larvae when they entered pre-pupa stage and on the 7th day of parasitized larvae. Proteins with MW lower than 62 kDa and higher than 27 kDa were absent on the 5th day in control larvae (pre-pupa phase), but present in parasitized larvae. This could indicate a possible relation between these proteins and the host juvenile hormone. Therefore, the presence of C. glomerata influences Ascia monuste orseis development, but its own physiological development is apparently independent of the host, which tends to die when parasitism succeeds.

Ultra-estrutura de glândulas abdominais tegumentares em Oxaea flavescens (Hymenoptera, Andrenidae, Oxaeinae)

The sternal glands of the abdomen of Oxaea flavescens (Klug, 1807) consist of class III glandular cells around a reservoir constituted by branched folds of the intersegmental membrane of segments III, IV and V. The gland cells are rich in rough endoplasmic reticulum and produce a secretion with mucous aspect. The treatment with oxidated osmium and ruthenium red showed numerous Golgi regions in the cell and carbohydrates absorption from the haemolymph, respectively. The high degree of development of the glands suggests an important function to the species, although still unknown.

Ultra-estrutura de glândulas abdominais tegumentares em Oxaea flavescens (Hymenoptera, Andrenidae, Oxaeinae)

The sternal glands of the abdomen of Oxaea flavescens (Klug, 1807) consist of class III glandular cells around a reservoir constituted by branched folds of the intersegmental membrane of segments III, IV and V. The gland cells are rich in rough endoplasmic reticulum and produce a secretion with mucous aspect. The treatment with oxidated osmium and ruthenium red showed numerous Golgi regions in the cell and carbohydrates absorption from the haemolymph, respectively. The high degree of development of the glands suggests an important function to the species, although still unknown.

EFFECT OF TEMPERATURE AND FOOD PROTEIN AND LIPID IN STAGES OF OVARIAN MATURATION AND STAGES OF MOLTING Macrobrachium acanthurus (WIEGMANN, 1836) / EFEITO DA TEMPERATURA E DOS ALIMENTOS PROTÉICO E LIPÍDICO NOS ESTÁDIOS DE MATURAÇÃO OVARIANA E ESTÁGIOS DE MUDA DO Macrobrachium acanthurus (WIEGMANN, 1836)

Knowing the reproductive physiology of the prawn Macrobrachium acanthurus in relation to controlled factors, such as processed food and temperature variations, would help to develop culture techniques, assuring its survival. Aiming at the finding some data on this subject, females of M. acanthurus were submitted to the effect of three temperatures (20, 25 and 30oC) and three types of food (lipidic, proteic and commercial). Following this procedure, the effects of haemolymph glucose, total protein and total lipid levels on ovarian maturation as well as in the moulting stages were analyzed. Results showed that nutrient levels usually increased as the animals reached D stage and the temperature increased. The nutrient levels also increased as animals reached stage III of ovarian maturation. Glucose and total protein levels increased with increasing temperature, while total lipid levels diminished at 30oC. Lipid food produced an increase in the haemolymph glucose and total proteins, while lipid and commercial food produced an increase in total lipids. In spite of that, results showed no significant differences among moulting stages and ovarian maturation, when measured at the same temperature.KEYWORDS: glucose; haemolymph; physiology; total lipid; total protein.

O conhecimento da fisiologia reprodutiva do camarão Macrobrachium acanthurus em relação a fatores controlados, como alimentos processados e variações de temperaturas, ajudaria a desenvolver técnicas de cultivo, garantindo sua sobrevivência. Com o objetivo de contribuir para esse conhecimento, fêmeas de M. acanthurus foram submetidas ao efeito das temperaturas de 20, 25 e 30oC e de três alimentos (lipídico, protéico e comercial). Em seguida, os efeitos nos níveis de glicose, proteínas totais e lipídios totais na hemolinfa, tanto na maturação ovariana como nos estágios de muda, foram analisados. Os resultados mostraram que, nos estágios de muda, os níveis dos nutrientes na hemolinfa, em geral, aumentaram conforme os animais chegam ao estágio de muda D, com o aumento da temperatura. Nos estádios de maturação ovariana, os nutrientes na hemolinfa aumentaram conforme se aproximavam ao estádio III. Os níveis de glicose e proteínas totais aumentaram com o incremento da temperatura, enquanto que os níveis de lipídios totais diminuíram a 30oC. Dentre os alimentos, o lipídico induziu ao aumento de glicose e proteínas totais na hemolinfa e os alimentos comercial e protéico induziram ao aumento de lipídios totais.PALAVRAS-CHAVE: fisiologia; glicose; hemolinfa; lipídios total; proteína total.

Transmissão transovariana de Babesia bovis em Boophilus microplus: obtenção de cepa de carrapato livre de Babesia spp.

In this experiment part of the life cycle of Babesia bovis in its invertebrate host, the tick Boophilus microplus was studied. In order to evaluate the capacity of infection and transmission of B. bovis were collected semi-engorged females of B. microplus fed on carrier and ill bovines. In the 18th day after infestation, semi-engorged females were collected directly from bovine bodies and after 21st day engorged females dropped on the ground. All the collected groups were incubated at 27°C and relative humidity greater than 70%. At the 5th day of oviposition the diagnosis was made by direct examination of haemolymph smears. The biological test reveled that B. bovis transovarial transmission doesn't happer in semi-engorged females. The results offer a simple techique to obtain strains of ticks free of B. bovis.

O presente trabalho objetivou o estudo de parte do ciclo da Babesia bovis no seu hospedeiro invertebrado, o carrapato Boophilus microplus. Analisou-se a capacidade de infeccção e transmissão transovariana de B. bovis em partenóginas de B. microplus, alimentadas em bovinos portadores e enfermos por esse protozoário. No 18º dia após a infestação, coletaram-se partenóginas diretamente do corpo dos bovinos e teleóginas após o desprendimento natural, a partir do 21º dia. Todos os grupos foram incubados a 27ºC e umidade relativa superior a 70%. No 5º dia após o início da postura, realizou-se o exame de hemolinfa a fim de diagnosticar a infecção dos ínstares por B. bovis. A ausência de infecção detectada no exame de hemolinfa foi confirmada posteriormente com o teste biológico, revelando que partenóginas não transmitem B. bovis transovarianamente. Esses resultados oferecem uma técnica simplificada para a obtenção de cepas de carrapatos livres de B. bovis.

Transmissão transovariana de Babesia bovis em Boophilus microplus: obtenção de cepa de carrapato livre de Babesia spp

In this experiment part of the life cycle of Babesia bovis in its invertebrate host, the tick Boophilus microplus was studied. In order to evaluate the capacity of infection and transmission of B. bovis were collected semi-engorged females of B. microplus fed on carrier and ill bovines. In the 18th day after infestation, semi-engorged females were collected directly from bovine bodies and after 21st day engorged females dropped on the ground. All the collected groups were incubated at 27°C and relative humidity greater than 70%. At the 5th day of oviposition the diagnosis was made by direct examination of haemolymph smears. The biological test reveled that B. bovis transovarial transmission doesn't happer in semi-engorged females. The results offer a simple techique to obtain strains of ticks free of B. bovis.

O presente trabalho objetivou o estudo de parte do ciclo da Babesia bovis no seu hospedeiro invertebrado, o carrapato Boophilus microplus. Analisou-se a capacidade de infeccção e transmissão transovariana de B. bovis em partenóginas de B. microplus, alimentadas em bovinos portadores e enfermos por esse protozoário. No 18º dia após a infestação, coletaram-se partenóginas diretamente do corpo dos bovinos e teleóginas após o desprendimento natural, a partir do 21º dia. Todos os grupos foram incubados a 27ºC e umidade relativa superior a 70%. No 5º dia após o início da postura, realizou-se o exame de hemolinfa a fim de diagnosticar a infecção dos ínstares por B. bovis. A ausência de infecção detectada no exame de hemolinfa foi confirmada posteriormente com o teste biológico, revelando que partenóginas não transmitem B. bovis transovarianamente. Esses resultados oferecem uma técnica simplificada para a obtenção de cepas de carrapatos livres de B. bovis.

Influência da temperatura sobre os tipos celulares presentes na hemolinfa de larvas e ninfas de Rhipicephalus sanguineus / Influence of temperature on cellular types present in the Rhipicephalus sanguineus larvae and nymphs haemolymph

Este trabalho foi realizado com o intuito de se verificar a influência de três temperaturas (18, 27 e 32ºC) sobre os tipos celulares presentes na hemolinfa de larvas e ninfas de Rhipicephalus sanguineus (Latreille, 1806). Seis tipos celulares básicos foram encontrados na hemolinfa: prohemócitos (PR), plasmatócitos (PL), granulócitos (GR), esferulócitos (ES), adipohemócitos (AD) e oenocitóides (OE). Os ES foram divididos em dois subtipos (ES I e ES II), devido às variações morfológicas encontradas. O efeito da temperatura foi mais acentuado sobre o estádio de larva. Ocorreu queda acentuada de GR e aumento de ES II na temperatura de 18ºC, enquanto que à 27 e 32ºC, os valores foram semelhantes. GR foi o tipo celular mais abundante, indicando o seu envolvimento no metabolismo. Para ninfas este também foi o tipo celular mais abundante, porém não ocorreram variações acentuadas segundo a temperatura, indicando que cada estádio de desenvolvimento apresenta determinada necessidade metabólica que se expressa nos tipos celulares presentes na hemolinfa.(AU)

This work was carried out in order to verify the effect of three temperatures (18, 27 and 32°C) on cellular types present in Rhipicephalus sanguineus (Latreille, 1806) larva and nymph hemolymph. Six cellular types were found in the hemolymph: prohemocytes (PR), plasmatocytes (PL), granulocytes (GR), spherulocytes (ES), adipohemocytes (AD) and oenocytoids (OE), being the ES divided into two subtypes (ES I and ES II). Effect of temperature was more intense in the larva stage in which a GR leukopenia and ES II leukocytosis occurred at 18°C. At 27 and 32°C the amount of these cells was similar, being the GR the most abundant cell type. This indicates the participation of these cells in the metabolism of this stage. It was also the most abundant cell type found in the nymphs, however, without the occurrence of marked variations among the distinct temperatures. This indicates that each development stage presents a specific metabolic need, which is expressed by the cell types present in the hemolymph. In some cases, the presence os cells is independent on the temperature.(AU)

Identificação e quantificação de hemócitos de fêmeas ingurgitadas de Boophilus microplus inoculados com fungos Metarhizium anisopliae, Beauveria bassiana, Penicillium corylophilum e Fusarium oxysporum / Identification and quantification of hemocytes obtained from engorged females of Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) inoculated with Metarhizium anisopliae, Beauveria bassiana, Penicillium corylophilum and Fusarium oxysporum

O controle biológico do carrapato Boophilus microplus com a utilização de Metarhizium anisopliae e Beauveria bassiana tem sido avaliado por diversos pesquisadores. A seleção de cepas resistentes é um mecanismo adotado pelos artrópodes para sobrevivência da espécie e sua manutenção no ambiente. Apesar de estar esclarecida a forma como o fungo penetra no hospedeiro e comprovada sua patogenicidade sobre diferentes fases de seu ciclo biológico, a sua resposta imune frente a estes agentes agressores necessita de maiores estudos. O presente trabalho teve como objetivo: identificar e quantificar os tipos celulares envolvidos na resposta imune celular de B. microplus inoculados com fungos entomopatogênicos não entomopatogênicos. Na experimentação foram utilizados 60 fêmeas ingurgitadas de Boophilus microplus, representando seis tratamentos cada um contendo 10 espécimes. Para os grupos inoculados com fungos entomopatogênicos foi utilizada a suspensão aquosa dos fungos Metarhizium anisopliae (isolado Ma 959) e Beauveria bassiana (isolado Bb 986). Nos tratamentos com fungos não entomopatogênicos, Penicillium corylophilum e Fusarium oxysporum, foram formados ainda um grupo testemunha (não recebeu inoculação) e um grupo inoculado com solução de tween 80 a 0,1% em água destilada estéril, considerado controle negativo. Nos grupos dos tratamentos com suspensão fúngica ou solução a inoculação foi feita na região posterior do idiossoma do carrapato. As coletas de hemolinfa foram realizadas durante todo o período de vida dos espécimes, tendo inicio 24 horas após inoculação. As amostras de hemolinfa foram fixadas com metanol e coradas com Giemsa. Em todos os períodos estudados, tanto nos espécimes inoculados com fungos como nos controles, foram observados pró-hemócitos, plasmatócitos, granulócitos, esferulócitos e oenocitóides. Pró-hemócitos, plasmatócitos e esferulócitos foram às células mais numerosas na hemolinfa. Foi observada a ausência de hemócitos no grupo inoculado com B. bassiana 72 horas após a inoculação, como também a morte dos espécimes inoculados com fungos entomopatogênicos a partir deste período. A ausência de células evidência o efeito imunossupressor do fungo sobre os carrapatos estudados. Característica não observada para Metarhizium anisopliae, mesmo tendo provocado acentuada mortalidade dos carrapatos. Os fungos não entomopatogênicos não afetaram de forma significativa o ciclo de vida destes carrapatos, sendo rapidamente eliminados do organismo

Biological control of the tick Boophilus microplus with Metarhizium anisopliae and Beauveria bassiana has been evaluated by several researchers. The selection of specimens resistant to chemical products is a mechanism used by arthropods for survival and maintenance in the environment. It is already known how fungi penetrate the host and their pathogenic potential at different phases of their biological cycle, however, ticks immune response against these agents need more detailed studies. The present work had as objectives: to identify and quantify the cellular types involved in the cellular response of B. microplus inoculated with entomopathogenic (M. anisopliae and B. bassiana) and non-entomopathogenic fungi (Penicillium corylophilum and Fusarium oxysporum). In this study 60 engorged females of B. microplus were used, representing six treatment groups with 10 specimens each. Ticks were inoculated with aqueous suspension of conidia. There were two control groups: in the first one ticks were inoculated with Tween 80 0,1% aqueous solution (negative control), in the second one ticks were not inoculated (testimony group). Fungi suspension or Tween 80 solution were applied in the back part of the idiosoma of the tick. The hemolinfa samples were collected during the whole period of life of the specimens, beginning 24 hours after inoculation. The haemolymph samples were fixed in methanol and stained with Giemsa. In all studied periods, cells like pro-hemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids were observed in the specimens of all groups. Prohemocytes, plasmatocytes and spherulocytes were the most numerous cells observed in the hemolinfa. Hemocytes were absent in the group inoculated with B. bassiana 72 hours after inoculation. After this period, specimens inoculated with entomopathogenic fungi were dead. The absence of cells suggests the immune-suppressor effect of the fungi on this tick species. It was not observed when ticks were treated with M. anisopliae due to accentuated mortality caused by this fungus species. The nonentomopathogenic fungi did not affect the life cycle of this tick, being quickly eliminated of the organism. It was suggested based on the absence of conidia during sampling