Free Amino Acids of Australorbis glabratus Hemolymph: Comparison of Four Geographic Strains and Effect of Infection by Schistosoma mansoni

Abstract

Concentrations of free amino acids in the hemolymph of uninfected Australorbis glabratus from Surinam, Venezuela, Puerto Rico, and Brazil were determined by automatic recording column chromatography. Additionally, the hemolymphs of normal and of Schistosoma mansoni-infected Puerto Rican A. glabratus were compared with respect to concentration of free amino acids and protein. The concentrations of certain free amino acids varied considerably in the different strains of uninfected snails, but similar variations also occurred in duplicate samples prepared from one of the strains. Total free amino acids of the hemolymph of infected snails varied only slightly. Levels of all free amino acids decreased during the course of infection with S. mansoni. The total free amino acid concentration in the hemolymph of snails with mature infections was approximately one-half that of normal snails. Total hemolymph protein varied among individual uninfected snails and was significantly decreased among infected snails. Several recent studies have dealt with the biochemical nature of snail hemolymph. Qualitative studies of amino acids of Australorbis glabratus hemolymph have been made (Dusanic and Lewert, 1963; Targett, 1962), and the hemolymph proteins have been characterized (Dusanic and Lewert, 1963; Wright and Ross, 1963; Michelson, 1966). This study was designed to determine quantitatively the hemolymph amino acids in four geographic strains of A. glabratus, one of which is highly refractory to infection by Schistosoma mansoni. Additionally, the free amino acid concentrations of the hemolymph of one of the strains were determined during the course of infection by S. mansoni, and the total protein concentrations of the hemolymph of infected and normal A. glabratus were compared. MATERIALS AND METHODS Maintenance and infection of snails A. glabratus from Venezuela (Ag-Ven) and Surinam (Ag-Sur) were obtained from the Tropical Research Medical Laboratory in San Juan, Puerto Rico, in 1962; the Puerto Rican strain of A. glabratus (Ag-PR) was obtained in 1958 from the National Institutes of Health; the Brazilian strain Received for publication 23 December 1966. * This study was supported by a NASA Predoctoral Traineeship to the senior author. t Present address: Department of Tropical Public Health, Harvard School of Public Health, 25 Shattuck Street, Boston, Massachusetts 02115. (Ag-Bah) originated in Bahia and was supplied to us in 1964 by Dr. W. L. Paraense. The Puerto Rican NIH strain of S. mansoni was used to infect Ag-PR. Exposures en masse to 20 to 40 miracidia per snail resulted in infection rates of over 90%. Infected and normal snails were maintained at 27 C in groups of 25 or less in 1-gal glass jars. Water was aerated continuously and changed every 10 to 14 days, leaf lettuce was provided as food, and the snails were fed a supplement once a week (Etges and Gresso, 1965). Collection and preparation of hemolymph Hemolymph for amino acid analysis was obtained from snails 14 to 17 mm in diameter. Snails were starved for 24 hr, rinsed with tap water, and allowed to air-dry. Hemolymph, obtained by puncturing the heart, was collected in 1-ml centrifuge tubes in ice water. Debris was removed by centrifugation (1,000 g/10 min), and the hemolymph was stored in sealed glass vials at -10 C for use within 6 weeks. Samples of 6 ml each, representing the hemolymph pooled from 175 to 225 snails, were used for each analysis. Single pools from uninfected Ag-Bah, Ag-Sur, and Ag-Ven, and duplicate pools from Ag-PR, were analyzed. Hemolymph was drawn from separate groups of snails (Ag-PR) 6, 16, and 32 days postexposure to determine the effect of infection. In addition, fresh hemolymph samples from 50 normal and 20 infected (shedding) Ag-PR, each 15 mm in diameter, were withdrawn for protein determinations. Hemolymph for amino acid analysis was deproteinized by dialysis. Each sample was filtered through a Millipore filter (0.45 J porosity) into a cellophane dialysis bag and allowed to dialyze at 3 to 5 C for 3 days against 50 ml of distilled water, which was changed daily. The total dialysate was