Harris, Robert Adam; (1991) Haemolymph proteins and the snail immune response. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In this dissertation, the presence of a haemagglutinin of vertebrate erythrocytes in the cell-free haemolymph of the freshwater snail Bihinds nasutus 1214 has been confirmed. This agglutinating activity could be adsorbed out of the haemolymph by incubation with human erythrocytes or by miracidia, but not sporocysts or cercariae of Schistosoma margrebowiei. The agglutinating property is abrogated by heating the haemolymph to 50°C, and by prior digestion with pepsin or trypsin, indicating the proteinaceous nature of the agglutinin(s). Agglutinating activity could be inhibited by porcine mucin, but not by single monosaccharides or disaccharides, or by combinations of these carbohydrates. In attempts to isolate the haemagglutinin factor(s), several conventional protein isolation procedures were employed but because of the intrinsic problems presented by the small volumes of haemolymph available these were unsatisfactory. However, success came with the adoption of a novel combination of affinity chromatography using concanavalin A-sepharose 4B followed by FPLC gel filtration using Superose 6 (Pharmacia). By this procedure the haemagglutinating property of the haemolymph was shown to reside in a single polypeptide. This haemagglutinin has an of 135k (reduced) and is a glycoprotein. Glycoproteins of similar Mr were isolated from other species of Bulinus using an identical purification protocol. Similarities in the polypeptide backbones of these proteins were demonstrated by Cleveland mapping. An antiserum raised in Balb-C mice to B. truncatus 1521 135k protein showed cross-reactivity within the genus Bulinus, being restricted to the tnincatus/tropicus species complex. Following infection with larval schistosomes, and upon mechanical wounding, alterations in the levels of plasma proteins were observed. The 135k proteins were not detectable in haemolymph during the early stages of infection. Staining of frozen sections of schistosome-infected Bulinus natalensis 272 with the 135k antiserum indicated that the 135k molecules had bound to the invading parasites. This is evidence to support the hypothesis that the 135k haemagglutinin acts as a recognition factor in the defence mechanisms of Bulinus spp. Studies of snail haemocytes have revealed physiological and functional similarities between haemocyte populations of different snail species. The nature of the buffer utilised in in vitro haemocyte studies has been shown to be of importance, although SSS (developed specifically for haemocytes of L. stagnalis) has proved efficient for haemocytic populations of both aquatic and terrestrial species. Lectin and antibody markers have been utilised in attempts to define haemocytic subpopulations, and haemocytes from different species have been shown to possess identical surface determinants for these markers. The production of reactive oxygen intermediates (ROFs) by snail haemocytes was investigated, and shown to vary among the populations tested.

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