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Biomphalaria glabrata snails were infected with Schistosoma mansoni and maintained at different dilutions of artificial ocean water for up to 4 weeks. Glucose and maltose concentration of the digestive gland-gonad complex were analyzed by high-performance thin-layer chromatography at different stages of the infection. B. glabrata snails were divided into three experimental groups: Group A, snails with early prepatent infection (10 days post-infection); Group B, snails with late prepatent infection (22 days post-infection); and Group C, snails with patent infection (45 days post-infection). Infected snails in A were maintained at different salinities for 2 weeks and then necropsied, and their two main simple sugars, i.e., glucose and maltose, were analyzed. Groups B and C contained two subgroups: the first subgoups were analyzed after 2 weeks, and the second after 4 weeks. Controls for these experiments were maintained identically in either deionized water or artificial spring water. Maltose and glucose were extracted from the digestive gland-gonad complex in ethanol-water (70:30). 1-Butanol-glacial acetic acid-diethyl ether-deionized water (27:18:5:3) mobile phase was used to separate sugars on EMD Millipore silica gel preadsorbent plates. Sugars were detected using α-naphthol-sulfuric acid reagent and quantified with a CAMAG TLC Scanner 3 at 515 nm. The obtained data were compared using analysis of variance (ANOVA) single factor statistical analysis. Statistical differences were not found in any sugars in Group A snails. For glucose, a significant difference was found after 4 weeks in both B and C snails. For maltose, a significant difference was found after 4 weeks in B snails and after 2 weeks in C snails. Different salinity levels affect the maltose and glucose concentrations of adult B. glabrata snails infected with S. mansoni.
Silica gel high-performance thin-layer chromatography (HPTLC) was used to study the effects of both Schistosoma mansoni infection and high temperatures on the neutral and polar lipid content of whole bodies of Biomphalaria glabrata snails. Neutral lipids were determined using petroleum ether-diethyl ether-glacial acetic acid (80:20:1) mobile phase, phosphomolybdic acid detection reagent, densitometry at 610 nm, and polar lipids with chloroform-methanol-water (65:25:4) mobile phase, cupric sulfate-phosphoric acid reagent, and scanning at 370 nm. The high-temperature experiments were done at ambient (22–24°C), 28°C, and 34°C. Snails were maintained at these temperatures for 7 days prior to necropsy. Extracts of their bodies were then analyzed by HPTLC to determine changes that occurred in the lipid content as a function of temperature and to compare unexposed to exposed cultures at each temperature. At 4 weeks postinfection (PI), the 34°C exposed snails had significantly lower amounts of free sterols than the unexposed culture. At 4 weeks PI, the 34°C exposed snails also had significantly lower amounts of free sterols than the ambient and 28°C exposed snails. At 6 weeks PI, ambient exposed snails had significantly lower free fatty acids and significantly higher phosphatidylcholine than unexposed snails. The 28°C exposed snails had significantly lower amounts of free sterols and phosphatidylethanolamine than the unexposed snails. The 28°C exposed snails also had significantly higher amounts of free sterols, triacylglycerols, and phosphatidylcholine than the ambient snails and significantly lower amounts of free fatty acids than the ambient temperature snails. The ambient exposed snails had significantly lower amounts of free sterols than the 28°C and 34°C snails. The 34°C exposed snails had significantly lower amounts of triacylglycerols than the ambient temperature and 28°C exposed snails. At 8 weeks PI, the 28°C exposed snails had significantly higher amounts of phosphatidylcholine than the unexposed snails. These findings suggest that high temperature and S. mansoni infection had individual and combined deleterious effects on the lipid metabolism of the snails.
Summary
Different thin-layer chromatography (TLC) systems were evaluated for analysis of 21 biologically important essential and nonessential amino acids in complex mixtures such as biological tissues and fluids. Amino acids were visualized on the layers by derivatization with ninhydrin reagent, and R F values were determined by slit-scanning densitometry. The five systems found to be most useful for analysis of amino acids were cellulose and silica gel high-performance TLC (HPTLC) plates developed with either 2-butanol-pyridine-glacial acetic acid-deionized water, 39:34:10:26, or 2-butanol-pyridine-25% ammonia-deionized water, 39:34:10:26, and ion exchange TLC plates developed with citrate buffer, pH 3.3. Using these five systems with ninhydrin detection, identification of all amino acids except for leucine and isoleucine in complex mixtures is possible, and quantification can be achieved if the amino acid to be quantified is well separated from adjacent components of the mixture. Example chromatograms are illustrated for separation and identification of amino acids in a snail tissue sample on a cellulose HPTLC plate.
