Evaluation of the mitochondrial system in the gonad-digestive gland complex of Biomphalaria glabrata (Mollusca, Gastropoda) after infection by Echinostoma paraensei (Trematoda, Echinostomatidae).

The effect of infection by Echinostoma paraensei on the mitochondrial physiology of Biomphalaria glabrata was investigated after exposure to 50 miracidia. The snails were dissected one, two, three and four weeks after infection for collection and mechanical permeabilization of the gonad-digestive gland (DGG) complex. The results obtained indicate that prepatent infection by this echinostomatid fluke significantly suppresses the phosphorylation state (respiratory state 3) and basal oxygen consumption of B. glabrata, demonstrating that the infection reduces the ability of the intermediate host to carry out aerobic oxidative reactions. Additionally, relevant variations related to the uncoupled mitochondrial (state 3u) of B. glabrata infected by E. paraensei were observed. Four weeks after exposure, a significant reduction in mitochondrial oxygen consumption after addition of ADP (3.68±0.26pmol O2/mg proteins) was observed in the infected snails in comparison with the respective control group (5.14±0.25). In the uncoupled state, the infected snails consumed about 62% less oxygen than the infected snails (7.87±0.84pmol O2/mg proteins) in the same period. These results demonstrate a reduction in oxidative decarboxylation rate of the tricarboxylic acid cycle and faster anaerobic degradation of carbohydrates in the infected snails. The possible mechanisms that explain this new metabolic condition in the infected organisms are discussed.

Physiological alterations in Bradybaena similaris (Stylommatophora: Bradybaenidae) induced by the entomopathogenic nematode Heterorhabditis indica (Rhabditida: Heterorhabditidae) strain LPP1.

Heterorhabditis is a nematode found in the soil that is used as an important biological control agent against various organisms. However, few studies have been performed of its use against snails and the present study is the first to investigate the effect of experimental exposure of Bradybaena similaris to Heterorhabditis indica LPP1. Two groups of 16 snails were formed: the control group (not exposed) and the treatment, which was exposed for three weeks to infective juveniles (J3) of H. indica LPP1. The entire experiment was conducted in duplicate, using a total of 64 snails. After this period, the snails were dissected to collect the hemolymph to evaluate the possible physiological alterations, namely total proteins, uric acid and hemolymph urea, as well as the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as a result of the infection. The terrariums were analyzed on alternate days throughout the experiment to count the dead snails. Intense proteolysis was observed in the infected snails. An increase in the level of uric acid and reduction of the hemolymph urea content indicated that the infection by H. indica results in the inversion of the excretion pattern of the host snail. Variations in the aminotransferase activities were also observed, with the infected group presenting significantly higher values (p<0.05) than the control group for both ALT and AST. The exposure to H. indica LPP1 caused 55% mortality, with the highest rate observed in the first week after exposure (30%). These results suggest that the use of H. indica LPP1 is a feasible alternative for the biological control of B. similaris.

Cyathostomin larvae: presence on Brachiaria humidicola grass during the rainy and dry seasons of Brazil.

The presence of cyathostomin larvae is directly associated to climatic conditions of each region. This study aimed to evaluate the ecology of infective larvae on Brachiaria humidicola during the dry and rainy seasons from October 2007 to September 2008 in a tropical region, Rio de Janeiro state, southeastern Brazil. Stools were collected from the rectum of horses naturally infected with cyathostomins at the beginning of the rainy season (October to March) and dry season (April to September). They were divided into four samples of 500 g and deposited on a grass patch of B. humidicola. Seven days later and every 15 days thereafter samples of feces and grass were collected and processed by the Baermann technique. The mean number of larvae recovered from the grass varied according to the season, with greater recovery of larvae during the peak of the dry season (14,700 L3.kg-¹ DM). There was a statistically significant difference between L3 recovered from feces and grass, but not between L3 recovered from the grass base and apex. These results show that the region's climate favors the development and survival of infective cyathostomin larvae throughout the year, with a greater number of larvae during the dry season.

Cyathostomin larvae: presence on Brachiaria humidicola grass during the rainy and dry seasons of Brazil / Larvas de ciatostomíneos: disponibilidade em gramínea Brachiaria humidicola nas estações chuvosa e seca do Brasil

The presence of cyathostomin larvae is directly associated to climatic conditions of each region. This study aimed to evaluate the ecology of infective larvae on Brachiaria humidicola during the dry and rainy seasons from October 2007 to September 2008 in a tropical region, Rio de Janeiro state, southeastern Brazil. Stools were collected from the rectum of horses naturally infected with cyathostomins at the beginning of the rainy season (October to March) and dry season (April to September). They were divided into four samples of 500 g and deposited on a grass patch of B. humidicola. Seven days later and every 15 days thereafter samples of feces and grass were collected and processed by the Baermann technique. The mean number of larvae recovered from the grass varied according to the season, with greater recovery of larvae during the peak of the dry season (14,700 L3.kg-¹ DM). There was a statistically significant difference between L3 recovered from feces and grass, but not between L3 recovered from the grass base and apex. These results show that the region’s climate favors the development and survival of infective cyathostomin larvae throughout the year, with a greater number of larvae during the dry season.

A disponibilidade de larvas de ciatostomíneos está diretamente relacionada com as condições climáticas de cada região. Para avaliar o comportamento das larvas infectantes nos períodos seco e chuvoso em gramínea Brachiaria humidicola, realizou-se um estudo, no período de outubro/2007 a setembro/2008, na região da Baixada Fluminense, RJ, de clima tropical. Amostras de fezes foram coletadas diretamente do reto de equinos naturalmente infectados por ciatostomíneos, no início do período chuvoso (outubro a março) e seco (abril a setembro), divididas em quatro amostras de 500 g e depositadas em um canteiro formado por gramínea B. humidicola. Sete dias após o depósito e, posteriormente, a cada 15 dias, amostras de fezes e gramíneas, foram coletadas às 8 horas e processadas pela técnica de Baermann. O número médio de larvas recuperadas da gramínea variou conforme os períodos, ocorrendo maior recuperação no ápice do período seco (14.700 L3.kg-1. MS). Diferença significativa ocorreu entre a contagem de L3 recuperadas das fezes e gramínea e não significativa entre as de L3 recuperadas nas duas alturas da gramínea. Concluiu-se que as condições climáticas da região favorecem o desenvolvimento e a sobrevivência de larvas infectantes de ciatostomíneos durante todo o ano, com maior disponibilidade no período seco.

Influence of Echinostoma paraensei (Lie and Basch, 1967) infection on the calcium content in Biomphalaria glabrata (Say, 1818).

The calcium content in the hemolymph and shell of Biomphalaria glabrata (Say, 1818) was determined after exposure to different parasite burdens (5 and 50 miracidia) of Echinostoma paraensei (Lie and Basch, 1967). The snails were dissected 1, 2, 3, and 4 weeks after infection to collect the hemolymph and shell. An increase in calcemia was observed in snails infected with both miracidial doses. A significant decrease in the calcium ions in the shell was observed, coinciding with the calcemia peak in the hemolymph. This indicates greater mobilization of calcium between the shell and hemolymph to regulate the calcium content in the body when the snail is exposed to stress conditions, as has also been observed in some other infected snail species. The results obtained indicate that in this model, the calcium metabolism depends on the miracidial dose used.

Lipid levels in Biomphalaria glabrata infected with different doses of Echinostoma paraensei miracidia.

The effect of experimental exposure of Biomphalaria glabrata to different doses (5 and 50) of Echinostoma paraensei miracidia on the total levels of cholesterol and triglycerides circulating in the hemolymph and the neutral lipids in the digestive gland-gonad (DGG) complex were studied. The snails were dissected one, two, three and four weeks after infection to collect the hemolymph and DGG tissue, to measure the levels of cholesterol and triglycerides in the hemolymph and neutral lipids in the tissue. The results for the hemolymph showed a similar order of variation for both substrates tested in the first week after infection. The reduced levels of these lipids in the infected snails indicate intense use of these substrates both by the intermediate host and the parasite, suggesting its probable participation in the energy metabolism and structural construction of the developing larval stages. Alterations in the profile of neutral lipids in the DGG were also found. The results obtained indicate that in this model, the lipid metabolism depends on the miracidial dose used.

Changes in the reproductive biology of Biomphalara glabrata infected with different doses of Echinostoma paraensei miracidia.

The egg-laying rate, number of egg masses, number of eggs/mass, number of eggs hatched/snail and egg viability of Biomphalaria glabrata exposed to different doses (5 and 50) of Echinostoma paraensei miracidia were analyzed as indicators of reproductive activity. Polystyrene plates were placed in aquariums containing the snails and every other day for four weeks after infection the plates were removed to count the number of egg masses and eggs laid. After this, the plates were numbered individually and placed in new aquariums free of snails and the egg masses were observed daily to determine the hatching rate. On average there was an increase in the parameters evaluated in the infected snails in relation to the controls (uninfected snails), except for egg viability, which was significantly lower in the groups infected with 50 miracidia. These findings indicate that when infected, this host snail is able to increase its reproductive activity, suggesting an ecological strategy to maintain the species.

Pathological findings in the caeca of naturally infected ostriches, Struthio camelus Linnaeus, 1758 (Aves, Struthionidae) parasitized by Codiostomum struthionis (Horst, 1885) Railliet and Henry, 1911 (Nematoda, Strongylidae).

In order to characterize lesions associated with Codiostomum struthionis in ostriches, 10 caeca were examined on both macro- and microscopic levels. Parasites were found in the distal third of the caecum and characterized as C. struthionis. Thickened mucosa was identified macroscopically where parasites were observed in high concentrations. Nodular areas were also observed in the distal third of the infected caeca, as well as hemorrhagic areas abutting small ulcers surrounded by edema. These findings were not observed in healthy controls. The concentration of C. struthionis found in infected animals was directly correlated with the severity of lesions observed in each caecum. These results allowed us to infer that C. struthionis is responsible for lesions in ostrich caeca.

Further study of Codiostomum struthionis (Horst, 1885) Railliet and Henry, 1911 (Nematoda, Strongylidae) parasite of ostriches (Struthio camelus Linnaeus, 1758) (Aves, Struthioniformes).

Codiostomum struthionis is a nematode parasite of the ostrich caecum. Little is known about its pathology, being considered by many authors as a non-pathogenic parasite. Infections by C. struthionis are sometimes overlooked because its eggs are indistinguishable from another ostrich nematode, Libyostrongylus spp. Fecal cultures and infective larvae identification are necessary for proper identification. The aim of this study is to provide improved morphological characterization of adults and infective larvae of C. struthionis. Ten caeca of adult ostriches were collected and washed in 0.09% saline solution. Male and female nematodes were collected and quantified separately. Nematodes were fixed in A.F.A. for optical microscopy or fixed in Karnovsky solution for scanning electron microscopy. To obtain infective larvae, fecal samples were collected at sites of high concentration of parasites in the caeca and fecal cultured. The resultant larvae were identified and measured with light microscope at 400x. Nine of the 10 slaughtered ostriches were parasitized by C. struthionis. All nematodes were found in the distal third of the caeca. A total of 566 parasites were recovered (234 males and 332 females). All the cultured larvae had characteristics of C. struthionis (rounded cephalic region with a flat extremity, an acute larvae tail termination and a long and filamentous sheath tail). All the adult parasites were characterized as C. struthionis. Through the analysis of the infective larvae it was determined that the morphology of the larvae tail was the best trait to use in the distinction of this species (live bird diagnosis).

Physiological changes in Biomphalaria glabrata Say, 1818 (Pulmonata: Planorbidae) caused by sub-lethal concentrations of the latex of Euphorbia splendens var. hislopii N.E.B (Euphorbiaceae).

Molluscides have been used as one of the strategies to control schistosomiasis. Many plant extracts with molluscidal effects have been tested, but the action of the latex of Euphorbia splendens var. hislopii is considered the most promising because it meets the recommendations of the World Health Organization (WHO). The objective of this study was to determine the lethal dose and identify the effects of the different doses of latex of E. splendens var. hislopii on the physiology of Biomphalaria glabrata submitted to treatment for 24 h. The concentrations of glucose, uric acid and total proteins in the hemolymph and of glycogen in the digestive gland and cephalopodal mass were determined. The LD50 value was 1 mg/l. The highest escape index was found to be at a concentration of 0.6 mg/l. The results showed that the latex of E. splendens var. hislopii caused a sharp reduction in the reserves of glycogen in the digestive gland and elevation of the protein content in the hemolymph of B. glabrata.

Physiological changes in Biomphalaria glabrata say, 1818 (Pulmonata: Planorbidae) caused by sub-lethal concentrations of the latex of Euphorbia splendens var. hislopii N.E.B (Euphorbiaceae)

Molluscides have been used as one of the strategies to control schistosomiasis. Many plant extracts with molluscidal effects have been tested, but the action of the latex of Euphorbia splendens var. hislopii is considered the most promising because it meets the recommendations of the World Health Organization (WHO). The objective of this study was to determine the lethal dose and identify the effects of the different doses of latex of E. splendens var. hislopii on the physiology of Biomphalaria glabrata submitted to treatment for 24 h. The concentrations of glucose, uric acid and total proteins in the hemolymph and of glycogen in the digestive gland and cephalopodal mass were determined. The LD50 value was 1 mg/l. The highest escape index was found to be at a concentration of 0.6 mg/l. The results showed that the latex of E. splendens var. hislopii caused a sharp reduction in the reserves of glycogen in the digestive gland and elevation of the protein content in the hemolymph of B. glabrata.

Ausência da atividade anti-helmíntica de plantas em frangos de corte naturalmente infectados com Heterakis gallinarum (Schranck, 1788) Madsen,1949 / Absence of the anthelminthic activity of plants in cut chickens naturally infected with Heterakis gallinarum (Schranck, 1788) Madsen,1949

Estudou-se a atividade anti-helmíntica das plantas; Allium sativum, Punica granatum, Tynnanthus fasciculatus e Cocos nucifera. Foram utilizados 70 frangos infectados naturalmente com Heterakis gallinarum, divididos em grupos de dez animais, com um controle positivo (20) e um negativo (10). Administraram-se as plantas na forma de extrato aquoso e suco por gavage e trituradas incorporadas à ração nas doses de 2, 3 e 10g kg-1 dia-1, durante três dias consecutivos. Os resultados foram avaliados através de teste não. O A. sativum, P. granatum, T. fasciculatus e C. nucifera eliminaram: 6,70; 4,12; 1,25 e 0,22 por cento , respectivamente. Os percentuais de eliminação do A. sativum e do T. fasciculatus, corresponderam respectivamente a 1/4 e 1/6 do controle positivo, sendo o C. nucifera e o P. granatum semelhantes ao controle negativo. Desta forma, as plantas nas doses empregadas não apresentaram atividade significativa (P<0,05) sobre o helminto H. gallinarum .

Predatory activity of Arthrobotrys oligospora and Duddingtonia flagrans on pre-parasitic larval stages of cyathostominae under different constant temperatures

A atividade predatória de fungos nematófagos e o desenvolvimento de estádios pré-parasitários de nematódeos estrongilídeos säo influenciados pela temperatura. O efeito de diferentes temperaturas constantes na atividade predatória dos fungos Arthrobotrys oligospora e Dddingtonia flagrans sobre as fases larvares de vida livre dos nematódeos ciatostomíneos foi avaliado em um experimento onde fezes de cavalos contendo ovos de parasitos foram tratados com fungos e incubadas em diferentes temperaturas constantes (10 graus C, 15 graus C, 20 graus C, 25 graus C e 30 graus C). Os resultados indicaram que a temperatura ótima de desenvolvimento de ovo até L3 foi de 25 graus C. A 10 graus C o número de L3 recuperado foi praticamente zero e a 15 graus C e 20 graus C os percentuais obtidos foram inferiores a 3 por cento do total do número de ovos por grama de fezes. Quando estes cultivos foram incubados por um período adicional de 14 dias à 27 graus C permitiram o desenvolvimento até L3. Em todos os cultivos inoculados com fungos, quando as larvas estavam presentes, houve reduçäo significativa no número de larvas atribuído à açäo predatória dos fungos. A 25 graus C e 30 graus C os fungos ocasionaram reduçöes acima de 90 por cento no número de L3. Os cultivos mantidos à 10 graus C, 15 graus C, 20 graus C, 25 graus C e 30 graus C quando incubados por um período adicional de 14 dias na temperatura controle, mantiveram percentuais de reduçäo larvar superior a 90 por cento para o fungo A. oligospora, o mesmo näo ocorrendo para D. flagrans, que apresentou percentuais de 47,5 por cento e 41,8 por cento quando estimulado a 10 graus C e 20 graus C, respectivamente. Esses fungos demonstraram ser eficientes na reduçäo do número de L3 quando adicionados a fezes de eqüinos nas mesmas temperaturas ideais para o desenvolvimento dos estádios larvares pré-parasitários.

Efeito do fungo Metarhizium anisopliae sobre estádios pré-parasíticos de Cyathostominae (Nematoda: Strongylidae) / The effect of the fungus metarhizium anisopliae at free living stages of Cyathostominae (Nematoda: Strongylidae)

O efeito do fungo Metarhizium anisopliae sobre estádios de vida livre de nematóides ciatostomineos (Strongylidae: Cyathostominae) foi avaliado, visto que sua patogenicidade para insetos e carrapatos já é comprovada. Para isto, foi utilizada uma cultura deste fungo em meio de arroz, contendo 1,3X10(8) conidios de M. anisopliae por grama, que foi adicionada a coproculturas com ovos de nematóides intestinais de eqüinos, compondo dois grupos tratados com diferentes concentrações (1,3X10(8) e 1,3X10(6) conídios/grama de fezes). As coproculturas foram mantidas em câmara climatizada a ± 30°C e 90% de umidade relativa. Após quinze dias, todas as culturas foram retiradas da câmara, as larvas foram recuperadas, contadas e identificadas. Nos dois grupos tratados observou-se redução no número de larvas encontradas nas coproculturas quando comparadas com as do grupo controle. A redução do número de larvas variou de 55,4 a 85,1 %, demonstrando o provável potencial deste fungo, para utilização no controle alternativo destes nematóides.

The effects of fungus Metarhizium anisopliae in free-living stages of nematodes (Strongylidae: Cyathostominae) were evaluate as a potencial aid to reduce the number of larvae in culture of horse feces. Metarhizium anisopliae cultured in rice, contained 1,3 X 10(8) conidia per gram, were added to culture of feces containing nematode eggs, in two treatment groups with different concentrations (l,3X10(8) e 1,3X10(6) conidia per gram of feces). The cultures were maintained at temperature of ± 30°C and 90% of relative umidity. After fiftheen days the cultured larvae were recovered, counted and identified. In the treated groups it was observed a reduction of the larval number, when compared with the control group. The reduction of 55.4 to 85.1% demonstrated a possibility of the use of this fungus as an alternativo control method for nematodes.