Vinícius Menezes Tunholi-Alves

Activation of anaerobic metabolism in Biomphalaria glabrata (Mollusca: Gastropoda) experimentally infected by Angiostrongylus cantonensis (Nematoda, Metastrongylidae) by high-performance liquid chromatography.

The activity of lactate dehydrogenase and the concentrations of glucose in the hemolymph and of glycogen in the digestive gland and cephalopedal mass of Biomphalaria glabrata experimentally infected with Angiostrongylus cantonensis were evaluated. Additionally, high performance liquid chromatography (HPLC) was used to determine the hemolymph concentrations of some carboxylic acids (oxalic, piruvic, lactic and succinic). After one, two and three weeks of infection, the snails were dissected to collect the hemolymph and separate the tissues. A significant reduction of the levels of glucose in the hemolymph was observed as of the first week of infection in relation to the control group. The lactate dehydrogenase activity of the infected group was significantly higher than the average of the control group. This increase was accompanied by a reduction of the levels of piruvic acid and an increase in the levels of lactic acid in the hemolymph of the parasited snails, confirming the acceleration of the anaerobic metabolism, necessary for the host to obtain energy and maintain its redox balance. In parallel, there was a decrease in the glycogen content of the storage tissues, with that reduction being significantly greater in the cephalopedal mass than the digestive gland, demonstrating that in this interaction system, the mobilization of glycogen was not sufficient to maintain and reestablish the normal glycemia of the infected snails.

Effects of infection by larvae of Angiostrongylus cantonensis (Nematoda, Metastrongylidae) on the metabolism of the experimental intermediate host Biomphalaria glabrata

The effect of infection by Angiostrongylus cantonensis on the activity of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the concentration of total proteins, uric acid and urea in the hemolymph of Biomphalaria glabrata were investigated. The snails were dissected after 1, 2 and 3 weeks of infection to collect the hemolymph. The infection by A. cantonensis induced severe changes in the host snails metabolism, triggering physiological mechanisms to minimize the deleterious effects caused by the larvae. There was a significant decrease in the concentration of total proteins in the infected snails, which occurred gradually as the infection advanced. This change was accompanied by an increase in the concentrations of urea and a decrease in the levels of uric acid in the hemolymph, suggesting that in this model the infection induces proteolysis and inversion of the excretion pattern of the infected snails. Besides this, variations in the activities of the aminotransferases were observed, with significantly higher levels in the infected groups than in the control group. These results indicate an increase in the protein metabolism of the infected snails, since there was an increase in nitrogen catabolites such as urea.

Changes in the reproductive biology of Biomphalaria glabrata experimentally infected with the nematode Angiostrongylus cantonensis.

This study showed for the first time changes in the reproductive biology of Biomphalaria glabrata experimentally infected with Angiostrongylus cantonensis. The values of all the parameters analyzed (total number of eggs, number of egg masses, number of eggs/mass, number of eggs/snail, percentage of viable eggs and galactogen content in albumen gland) changed with progressive infection. The results indicate the occurrence of partial parasitic castration of B. glabrata by A. cantonensis larvae, probably in response to the depletion of energy reserves, with no injuries to the gonadal tissues.

Biochemical profile of Biomphalaria glabrata (Mollusca: Gastropoda) after infection by Echinostoma paraensei (Trematoda: Echinostomatidae)

The effect of infection by Echinostoma paraensei on the activity of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the concentration of total proteins, uric acid and urea in the hemolymph of Biomphalaria glabrata were investigated after exposure to five or 50 miracidia. The biochemical concentrations were measured weekly until the end of the fourth week after exposure. There was a significant decrease in the concentrations of total proteins in the snails exposed both to five and 50 miracidia, as well as an increase in the nitrogenous products of excretion, ALT and AST activities. The higher ALT activity in the hemolymph of the snails after infection with 50 miracidia suggests highest energetic requirement in these snails in relation to snails exposed to five miracidia. The results also suggest an increase in the use of total proteins, since there was increased formation of nitrogenous catabolites, in conformity with an increase in the aminotransferase activities, frequently associated with tissue damages. This can be explained by damage due to penetration by the miracidia and subsequent development of intramolluscan sporocysts and rediae.

Aerobic to anaerobic transition in Biomphalaria glabrata (Say, 1818) infected with different miracidial doses of Echinostoma paraensei (Lie and Basch, 1967) by high-performance liquid chromatography

The glucose content in the hemolymph and glycogen content in the digestive gland-gonad complex (DGG) and cephalopedal mass of Biomphalaria glabrata exposed to different parasite doses (5 and 50 miracidia) of Echinostoma paraensei as well as the activity of lactate dehydrogenase were evaluated. HPLC (high-performance liquid chromatography) analyses were also performed to determine the concentrations of four organic acids (oxalic, succinic, pyruvic and lactic) present in the hemolymph of infected and uninfected snails, to better understand the effect of infection on the hosts energetic/oxidative metabolism. The snails were dissected 1-4 weeks after infection to collect the hemolymph and separate the tissues. There was alteration in the glycemia of the snails at both parasite doses, with a significant increase of glycemia from of the third week after infection in comparison to the control group. Changes were also observed in the lactate dehydrogenase activity, with increased activity as the infection progressed. In parallel, there was a decrease in the glycogen content in the storage tissues, with a markedly greater reduction in the digestive gland-gonad complex (larval development site) in comparison with the cephalopedal mass. Additionally, the infection by both miracidial doses resulted in an increase of oxalic and lactic acid levels, as well as in a decline of piruvic and succinic acid levels in B. glabrata, thus explaining the reduction of the oxidative decarboxylation rate in the tricarboxylic acid cycle and acceleration of the anaerobic degradation of carbohydrates in the snails, through lactic fermentation, which is essential to ensure energy supply and success of the infection.

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.

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 calcium metabolism of Biomphalaria glabrata experimentally infected with Angiostrongylus cantonensis

Levels of calcium in the haemolymph and reserves in the shell of Biomphalaria glabrata experimentally infected by Angiostrongylus cantonensis were determined for the first time. At the same time, histochemical analyses of the digestive gland of infected and uninfected snails were performed to better understand the possible changes in metabolism of calcium in these organisms. After 1, 2 and 3 weeks of infection, the snails were dissected for collection of haemolymph and separation of tissues. The highest calcium concentrations in the haemolymph were found 2 weeks after infection, with a 39.61% increase in relation to the respective control group. However, there was a significant reduction in the concentration of this ion in the haemolymph of infected snails after 1 week of infection in relation to the uninfected specimens. In parallel, intense hypocalcification was shown in the shell of infected snails 1 and 2 weeks after infection, differing significantly in relation to the respective control groups. Morphological changes in the digestive gland of infected snails were also observed, confirming the role of this ion as an important element in the parasite encapsulation process.

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.

Lipids analysis in hemolymph of African giant Achatina fulica (Bowdich, 1822) exposed to different photoperiods

The influence of different photophases (0, 6, 12, 18 and 24 hours) on the triglycerides and total cholesterol contents in the hemolymph of A. fulica was evaluated, since there is no information in the literature about the influence of this factor on lipids metabolism in mollusks. After 2 and 4 weeks of exposure the snails were dissected. The cholesterol content at the 2nd and 4th weeks post exposure only varied significantly in the groups exposed at 24 hours and 0 hour of photophase, respectively. Probably, such increase may be a result of a rise in cholesterol biosynthesis and/or remodelling of cell membranes. There were no significant differences among the content of triglycerides in the snails exposed to 6, 12, 18 and 24 hours of photophase during two weeks. The snails exposed to intermediate photophase (6 and 12 hours) had the triglycerides content increased, ranging over values near to those observed in the group exposed to 0 hour. Results showed that triglycerides metabolism in A. fulica are more influenced by photoperiod than cholesterol metabolism. A negative relation is maintained between the triglycerides content in the hemolymph and the different photophases, with lower mobilisation of triglycerides under shorter photophases.