Immunorecognition in the gastropod molluscs with particular reference to the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata)

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1 Bolletino di zoologia ISSN: (Print) (Online) Journal homepage: Immunorecognition in the gastropod molluscs with particular reference to the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata) Enzo Ottaviani To cite this article: Enzo Ottaviani (1992) Immunorecognition in the gastropod molluscs with particular reference to the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata), Bolletino di zoologia, 59:2, , DOI: / To link to this article: Copyright Taylor and Francis Group, LLC Published online: 2 Jan Submit your article to this journal Article views: 33 View related articles Citing articles: 12 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 21 December 2017, At: 07:16

2 Boll. Zool. 59: (1992) Immunorecognition in the gastropod molluscs with particular reference to the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata) ENZO OTTAVIANI Dipanimento di Biologia animale, Università di Modena, via Berengario 14, Modena (Italy) ABSTRACT The internal defence system of gastropod molluscs is able to discriminate between self and non-self. The recognition is carried out principally by both the cellular and humoral components of the haemolymph. Together with freely circulating haemocytes, other defence cells are found scattered throughout the tissue or localized in organs. The present review refers primarily to Planorbarius corneus, since the defence mechanisms presented by this animal are typical of those of the other gastropods studied. P. corneus presents two circulating haemocytes: the spreading (SH) and the round haemocytes (RH); in other gastropods only one cell type is described, and this can be considered as a spreading haemocyte. The haemocytes derive both from a haematopoietic organ and from mature circulating haemocytes. The SH show phagocytic properties, adhere to glass, produce agglutinins, bind Con A and contain muramic acid. The RH have non phagocytic properties, do not adhere to glass, form rosettes with sheep red blood cells, are stimulated to proliferate by PHA and present numerous typical markers of vertebrate T lymphocytes. RH are also able to lyse 51 Cr pre-labeled K562 target cells in a classical, short-term, natural cytotoxicity test, and this function is modulated by human recombinant interleukin-2. Furthermore, SH and RH play a role in the recognition of foreign tissue, the SH are able to encapsulate and phagocytize foreign material, and the RH, with their NK (natural killer)-like activity, may act like the vertebrate cytotoxic T lymphocytes or NK cells. Thus, it is possible to conclude that RH have characteristics reminiscent of vertebrate T lymphocytes, while SH belong to the category of macrophages. With regards to the humoral component, different factors, such as lysosomal enzymes, lysins and agglutinins or lectins, have been described. In P. corneus, a natural glycoprotein agglutinin has been isolated, whose carbohydrate component contains muramic rather than sialic acid. Moreover, an induced bacterial agglutinin has been purified, although this induction is relatively rare in gastropods. Lysozyme-like molecules have also been detected and they act like alarm molecules in inflammatory reactions. Taken together, the humoral and cellular investigations, the bacterial clearance studies and the specific responses observed in transplantation experiments are all in favour of the presence of a memorytype response of short duration. Finally, interrelations appear to exist between the immune and neuroendocrine systems. ACTH and ß-endorphin-immunoreactive molecules have been detected in serum and SH, and these molecules appear to play a physiological role in the process of phagocytosis and in stress response. KEY WORDS: Gastropod molluscs - Planorbarius corneus - Internal defence mechanisms - Cellular and humoral components. ACKNOWLEDGEMENTS This work was supported by M.U.R.S.T. (60%) and C.N.R. grants to E.O. INTRODUCTION Gastropod molluscs have an internal defence system with a well-developed capacity to discriminate between self and non-self. Recognition in these creatures, as in all animals characterized by coelomic cavities, is principally carried out by both the cellular and humoral components of the haemolymph. Together with freely circulating haemocytes, there are different types of defence cells that may be scattered throughout the connective tissue or localized in particular organs, such as the digestive gland, where cells with phagocytic activity are present. Such cells represent a fixed phagocyte system in Helix pomatia (Reade, 196), while in Planorbarius corneus they make up almost the entire gland (Ottaviani, 1990). Furthermore, we recall the antigen-trapping cells which have been described in the blood sinuses of the kidney, digestive gland, and foot of H. pomatia. The surface carbohydrate receptors of these cells bind foreign substances which are then phagocytized by the circulating haemocytes (Renwrantz et al., 191). Finally, we also draw attention to the fixed phagocytes of the connective tissues described in Lymnaea stagnalis (Sminia et al., 1979b). In the present review, it seemed appropriate to offer two separate descriptions of the components of the haemolymph in P. corneus, even if such a division does not really exist, as every immune response is the result of the co-operation between the cellular and the humoral component. CELLULAR COMPONENT The great majority of gastropods, including P. corneus, present two circulating haemocytes: spreading haemocytes (SH) and round haemocytes (RH) (see Sminia, 191, for a review; Ottaviani, 193), each having a characteristic morphology. The presence of two cell types in Helix aspersa (Prowse & Tait, 1969), Bulinus guernei (Krupa et al., 1977), Bulinus trunca tus roblfsi (Cheng & Guida, 190) and Biompbalaria glabrata (Harris, 1975; Cheng & Auld, 1977; Cheng & Garrabrant, 1977; Yoshino, 196) has been inferred by functional aspects and surface marker analysis, in addition to morphological studies. In those cases in which only one type is described, the cell type has the characteristics of SH (Ottaviani, 199a; Franchini & Ottaviani, 1990). In L. stagnalis, a single cell type with both morphologies has been described, where the round form is the young stage and the spreading form the mature stage. These two forms have functional capacities which differ only quantitatively (Sminia et al., 193; see Sminia & van der Knaap, 196, for a review). In this case, too, it has been possible to find similarities between the mature stage and the SH type (Ottaviani & Franchini, 19). In the haemolymph of P. corneus between 00 and 1400 haemocytes/mm 3 are present; SH constituting about

3 130 E. OTTAVIANI i-'-. Fig. I - Light micrograph of spreading haemocyte of P. corneus stained with May-Griinwald Giemsa, Bar, 5 urn. 0%, and RH the remaining 20%. These haemocytes derive from mature circulating haemocytes and from precursor cells situated in a haemocyte-producing organ (HPO), where the haemoblasts transform into haemocytes which then migrate into the sinuses of HPO; there after they are transported to all parts of the body (Ottaviani, 193; 19a). The HPO has been found in several other species of Planorbids and Lymnaea palustris (Kinoti, 1971; Lie et al., 1975; Rachford, 1976; Jeong et al., 193) and, as in P. corneus, it lies between the mantle cavity and the pericardium. Examination of the haemocytes in freshly drawn haemolymph stained with May-Grünwald Giemsa shows that SH have an irregular form with abundant cytoplasm, rich in pseudopodia of varying length and shape. The nucleus is crinkled or kidney-shaped (Fig. 1). These haemocytes tend to form clumps. The RH present a round nucleus with scant cytoplasm, evident as a slender ring around the nucleus. Sometimes the cytoplasm is not clearly visible (Fig. 2) (Ottaviani, 193). Ultrastructural examination shows the presence in SH of both smooth and rough endoplasmic reticulum, free ribosomes and polyribosomes, several Golgi complexes in an active phase, lysosome-like structures, and clumps of a- glucogen granules. Moreover, vacuoles are present in the outer cytoplasm, indicating active endocytosis (Fig. 3). The RH, in contrast to SH, have predominantly rough endoplasmic reticulum and mitochondria whose matrix contain large electron-dense granules (Fig. 4). This cell type, therefore, sometimes shows nuclear and cytoplasmic damage that is a feature of cellular apoptosis, considered to be physiological tissue turnover (Ottaviani & Franchini, 19). As reported above, SH present ultrastructural similarities with the spreading amoebocytes of L. stagnalis (Stang-Voss, 1970; Sminia, Fig. 2 - Light micrograph of round haemocyte of P. corneus stained with May-Grünwald Giemsa. Bar, 5 urn. Fig. 3 - Electron micrograph of spreading haemocyte of P. corneus. x (Reported by Ottaviani & Franchini, 19) 1972), the granulocytes of B. guernei (Krupa et al., 1977), and the granulocytes of B. glabrata (Harris, 1975, Joky et al., 193). Together with similar morphological characteristics, these blood cells, which bear different names, such as granulocyte, amoebocyte, leukocyte or haemocyte, also present the same functional patterns, e.g. spreading properties, substrate adhesion, phagocytosis

4 IMMUNORECOGNITION IN THE GASTROPOD MOLLUSCS 131. Fig. 4 - Electron micrograph of round haemocyte of P. corneus. x (Reported by Ottaviani & Franchini, 19) and receptors for concanavalin A (Con A) (Prowse & Tait, 1969; Anderson & Good, 1976; Jeong & Heuneman, 1976; Cheng & Garrabrant, 1977; Renwrants & Cheng, 1977, Schoenberg & Cheng, 190; 191; Yoshino, 191; Ottaviani, 193; Ottaviani & Franchini, 196). The RH of P. corneus is comparable only to the ultrastructure of the round amoebocytes described for L. stagnalis (Sminia, 1972; see Sminia, 191 for a review). The other cell type found by other researchers, the hyalinocyte, differs morphologically from RH in its electron transparent feature, *. but these two cell types do share some behavioural characteristics; they remain spherical, they do not emit pseudopodia and they have no phagocytic capacity (Cheng & Garrabrant, 1977; see Sminia, 191 for a review; Ottaviani, 193). The functional aspects are reported in the Table I. The two cell types in the haemolymph of P. corneus present different functions which, on the one hand, are characteristic of phagocytic cells (SH) and, on the other, of cytotoxic cells (RH). Both in vitro and in vivo, SH can phagocytize latex particles, bacteria and apoptotiç RH (Ottaviani, 193; Ottaviani & Franchini, 19; Ottaviani, 199b). The role of the lysosomal enzymes during phagocytosis is supported by the increase of acid phosphatase in SH of P. corneus challenged with bacteria (Ottaviani et al., 196), a fact, moreover, which confirms results obtained by other authors (Meuleman, 1972; Sminia, 1972; Rodrick & Cheng, 1974; Sminia et al., 1974; Harris, 1975; Harris & Cheng, 1975; Foley & Cheng, 1977; Cheng & Butler, 1979; Cooper-Willis, 1979; Cheng, 193). The RH are able to lyse 51 Cr pre-labeled K562 target cells in a classical, short-term, natural cytotoxicity test, and this function is modulated by human recombinant interleukin-2 (hrll-2) (Franceschi et al., 1991). Moreover, both SH and RH are involved in the recognition of foreign tissue, although in different ways. Alio- and xenograft implants (ganglia from animals of the same species or from Helix lucorum) are initially encapsulated and then phagocytized. Only haemolymph cells are involved in this phenomenon. The RH reach the graft first, while the SH are responsible for the formation of the capsule and the destruction and removal by phagocytosis of the foreign cells. Encapsulation does not occur in cases of auto-transplantation. The sequence of encapsulation is similar in alio- and xeno-transplants, but in the latter the reaction is more rapid (Ottaviani & TABLE I - Characteristics of spreading (SH) and round baemocytes (RH) of Planorbarius corneus. Markers or functions SH RH References Glass adhesion Phagocytosis Chemotactic activity Rosette with SRBC Responsiveness to PHA Recognition of auto-, alio- and xenoimplants Reactivity to pab anti-muramic acid Reactivity to pab anti-human-lysozyme Reactivity to pab anti-human-ß 3 -microglobulin Reactivity to Con A Cytotoxicity Responsiveness to hrll-2 Ottaviani, 193 Ottaviani, 193; Ottaviani & Franchini, 19; Ottaviani, 199b Ottaviani et al., 1990b Ottaviani, 193 Ottaviani, 19b Ottaviani & Verginc, 1990, Ottaviani et al., 1991b Ottaviani & Montagnani, 199 Ottaviani, 1991a Ottaviani et al, 1990d Ottaviani & Franchini, 196 Franceschi et al., 1991 Franceschi et al, 1991

5 132 E. OTTAVIANI Vergine, 1990; Ottaviani et al., 1991b). These studies suggest, first, that in P. corneus, recognition and rejection of transplants involves two cell types, i.e. RH, which have NK (natural killer)-like activity and could act like the cytotoxic T lymphocytes in vertebrates, and SH, which perform the double function of encapsulation and phagocytosis. Secondly, the response to different grafts is characterized by specificity. Observation by flow cytometry and immunofluorescence procedures showed that SH, but not RH, were positive for anti-n-acetyl-mu'ramic acid (Ottaviani & Montagnani, 199). The presence of this acid has also been demonstrated in glycoconjugates of several tissues of other gastropod molluscs. N-acetyl-neuraminic acid (sialic acid) is not found in these tissues. In gastropods N-acetyl-muramic acid possibly substitutes sialic acid and has a similar role and function to that in vertebrate glycoproteins. Moreover, both molecules are C9 acidic sugars with carboxylic and glycosidic functions (Bolognani Fantin & Ottaviani, 1990; see Ottaviani et al., 1990a, for a review). N-acetyl-muramic acid has also been detected in the only cell type present in the haemolymph of Viviparus ater. These cells have the same functions as the spreading haemocytes found in other gastropods (Ottaviani, 199a). As reported in Table II, several investigations by immunocytochemistry, RIA- and flow cytometry have TABLE II - Bioactivepeptide (BAP)-Uke molecules in spreading (SH) and round haemocytes (RH) in Planorbarius corneus. BAP SH RH ACTH ß-endorphin a,-antichymotrypsin Bombesin Calcitonin CCK- (INC) CCK- (Peninsula) CCK-39 Gastrin Glucagon Insulin (CRL) Insulin (Peninsula) Met-enkephalin Neurotensin Oxytocin Prolactin Somatostatin Substance P Thyroglobulin Thyroxin (T4) Vasopressin VIP Reported and modified by Ottaviani & Cossarizza (1990) *.. ««! ar.'"*>. Fig. 5 - Spreading haemocytes positive (a) and a round haemocyte negative (b) to anti-acth antibody. Each group of spreading haemocytes presents five cells. Bar, 10 um. (Reported by Ottaviani etal, 1990c). revealed the presence of several immunoreactive (ir) bioactive peptide (BAP) molecules on RH and SH of P. corneus. The presence of irbap materials in the haemocytes probably plays a role in the modulation of the immune response (Ottaviani & Cossarizza, 1990). With regard to the presence of opioid neuropeptides, such as iracth (Fig. 5) and irß-endorphin molecules, these have only been found in SH and in the serum (Ottaviani et al., 1990c). It is interesting to note that similar molecules have been described in unicellular organisms and invertebrates (Le Roith et al., 192), despite the absence of anatomic targets usually associated with the neuroendocrine system. Opioid-like molecules in invertebrates appear to be a general phenomenon, a suggestion supported by the fact that the only cell type present in the haemolymph of another snail, i.e. amoebocytes in L. stagnalis (Sminia, 1972), is also positive to the anti-acth antibody (Ottaviani et al., 1991a). These results are in agreement with previous observations indicating presence of certain chemically identified opioids in the bivalve Mytilus edulis (Leung & Stefano, 194; Stefano & Leung, 194). The presence of iracth and irß-endorphin molecules only in cells capable of phagocytic activity suggests that these molecules may play a physiological role in the process of phagocytosis. Indeed, ACTH and ß-endorphin exert chemotactic activity towards SH (Ottaviani et al., 1990b). The target cell SH probably responds to the extracellular ACTH signal by varying the intracellular cyclic AMP level. In order to migrate towards a chemoattractant, a cell has to be able to recognize the substance, presumably by specific receptors, and to modify the

6 IMMUNORECOGNITION IN THE GASTROPOD MOLLUSCS 133 cytoskeletal components involved in cell migration. Indeed, after incubation with ACTH, modification of the actin microfilament network is evident in SH. Changes in the sites of microfilament-membrane interactions and the formation of new adhesive structures are probably also involved (Franchini et al, 1990). Furthermore, ACTH induces the release of biogenic amines in the serum and the phagocytic activity of SH towards Staphylococcus aureus increases with an increase in the ACTH dose (Ottaviani et al, 1991a). On the whole, these data suggest that these opioid neuropeptides are involved in a complex set of ancestral immune responses including the stress response. An integral part of this phenomenon is the release of biogenic amines. The analysis of the RH and SH phenotype by flow cytometry has allowed further characterization of these cell types. Mouse anti- human mabs recognizing CD (cluster of differentiation) antigens that define lymphocyte subsets were used (Franceschi et al, 1991). I am well aware that a positive staining with a mouse anti-human mab only means that the epitope recognized by the antibody is probably present, and does not definitely prove the existence of a given molecule. TABLE III - Cytofluorimetric analysis of spreading (SH) arid round baemocytes (RH) of Planorbarius corneus using mouse anti-human mabs. mab anti- SH RH CDla, CD16, CD26, CD29, CD56 CD5, CD34, CD45RA, CD54, CD61, CD71 CD2, CD3, CD4, CD7, CD, CDlla, CDllb, CDllc, CD13, CD1, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD3, CD43, CD45RO, CD57, HLA-DR, TcR ct/ß, TcR y/ô Reported by Franceschi et al. (1991) Table III summarizes the results obtained by staining P. corneus haemocytes with the panel of mabs and shows that the haemocytes react with antibodies recognizing epitopes present on molecules typical of human NK cells, such as CD16 and CD56, and epitopes present on molecules found on human T lymphocytes such as CDla, CD26 and CD29. Moreover, positivity for epitopes present on molecules involved in cell-cell interactions such as CDla (MHC precursor), CD29 (intregrin ß, chain), CD56 (an N-CAM isoform) and CD61 (integrin ß ä chain), is also evident. Although there is some uncertainly on the degree of homology of immunoreactive molecules in man and the snail, our results nevertheless strongly suggest that primitive invertebrate haemocytes present the same epitopes, and possibly also the same molecules, as very sophisticated immune cells, such as mammalian lymphocytes. HUMORAL COMPONENT In the serum of gastropods, several humoral factors are involved in immune responses. Agglutinins or Jectins, opsonins, lysins, lysosomal enzymes, and others have been described (see Ratcliffe et al., 195; Olafsen, 196; Sminia & van der Knaap, 196, for reviews). These factors are naturally present or may be induced after stimulation (Boyd et al., 1966; Gilbertson & Etges, 1967; Rudolph, 1973; Cheng & Rodrick, 1974; Cheng et al., 1975; 1977; Michelson & Dubois, 1977; Sminia et al, 1979a; Stein & Basch, 1979; van der Knaap et al, 192; Ottaviani, 194, and others). The molluscan agglutinins that seem to act similarly to antibodies are protein or glycoprotein (Hammarström & Kabat, 1969;Jenkin& Rowley, 1970; Pauleyeffl/., 1971a; Hardy et al., 1977; Baldo et al., 197; Renwrantz & Stahmer, 193). They may also have, opsonic properties (Pauley et al, 1971b; Harm & Renwrantz, 190; van der Knaap et al, 192; Renwrantz & Stahmer, 193), and need divalent cations (in particular, Ca ++ ) to manifest biological activity (Hammarström & Kabat, 1969; Baldo et al, 197; Renwrantz & Stahmer, 193). They can also mediate chemotaxis (Schmid, 1975) and bind specific carbohydrates (Renwrantz, 193). In P. corneus, agglutination was observed with the erythrocytes of rabbit and goldfish, and with blood cells of V. ater and the storage cells of Macrobiotus ricbtersi (Ottaviani, 196). An agglutinin against goldfish erythrocytes has been isolated from the haemolymph of P. corneus by affinity chromatoghaphy on Sephadex gel G 150 (Fig. 6) (Ottaviani & Tarugi, 196). This molecule is an acid glycoprotein with a mol wt of 130 KD. The acid part is not composed of the usual sialic acid but of N-acetylmuramic acid. This agglutinin is synthesized in the SH. Indeed, using immunoperoxidase techniques with an antibody against P. corneus haemolymph serum and an anti-n-acetyl-muramic acid antibody, only the SH react positively (Ottaviani, 19c; Ottaviani & Montagnani, 199). Similar results were obtained in L. stagnalis, where the amoebocytes synthesize agglutinin/opsonin and bear it on their surface as a receptor for foreign materials (van der Knaap et al, 191). Natural bacterial agglutinins have not been found in P. corneus, and bacterial agglutinins are induced only after injection of the bacteria (Ottaviani, 196; Ottaviani & Tarugi, 199). In particular, an induced bacterial agglutinin with a mol wt of KD has been purified following repeated injections of S. aureus. The induction of agglutinin? and antibacterial substances in

7 134 E. OTTAVIANI 21.5K 14K A Fig. 6 - SDS-polyacrylamide gel electrophoresis of isolated agglutinin from P. corneus: A) mol wt standards; B) isolated agglutinin indicated by arrow. (Reported by Ottaviani & Tarugi, 196). molluscs appears to be relatively rare. To date, there has been only one report of induced bactericidal response in abalones challenged with a formalin-killed suspension of bacterial culture EMB-1 (Cushing et al., 1971). This may be a result of limited knowledge, or these animals may present different immunological strategies. Natural and induced haemolytic activity is evident in the haemolymph of Mercenaria mercenaria, and this haemolysin probably has a protective role related to antimicrobial activity (Anderson, 191). Moreover, Cheng et al. (1975; 1977) have suggested that the presence of lysozyme may indicate a non-specific inducible humoral defence mechanism. Indeed, B. glabrata challenged with bacteria presents increased haemolymph lysozyme levels. However, the significance of haemolymph lysozyme levels is still not clear; in Crassostrea virginica lysozyme levels were higher following infection with certain parasites but lower following infection with others (Feng & Canzonier, 1970). In P. Corneus irlysozyme molecules seem also to be inducible molecules responding non- B specifically to foreign challenge. It is, therefore, possible to hypothesize that the increase in the irlysozyme molecule levels in the serum is responsible, at least in part, for the lytic attack and death of bacteria (Cheng & Rodrick, 1975; Cheng et al., 1977). Moreover, irlysozyme material is probably involved in the initial steps which control the promotion of inflammatory reactions (Ottaviani, 1991a). Indeed, as a result of aspecific stimuli, these molecules increase within a few hours in the serum and are induced by bacterial antigens. The increase in irlysozyme molecule levels a few hours after injection of the various antigens is probably the result of their release from storage sites, whereas the increase after 24 hrs in response to bacterial stimulation is the result of new synthesis. The persistence of the irmolecules only after bacterial injection cannot be a specific response, and one may suppose that the inflammatory reaction following bacterial injection lasts longer than that provoked by other foreign substances, and, as a result, induces irlysozyme molecule production. Furthermore, there is a correlation between irlysozyme molecules and phagocytosis, since these molecules are only present in SH, and, as ACTH and ß-endorphin-like molecules, they show chemotactic activity towards SH. In P. corneus, a decrease in haemolymph phagocytic cells (SH) was observed after bacterial injection, and this drop seems to be mediated by humoral factor(s), probably including irlysozyme, iracth and irßendorphin molecules, released into the haemolymph. TABLE IV - Concentration (nmol/ml) of free amino acids (FAA) in the serum of Planorbarius corneus before and after (2 h) challenge with living Staphylococcus aureus. FAA Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine 1/2 Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Controls 4.17 * ± ± s * * * * ± ± ± ± ± ± ± ± 3.27 The tests were carried out on three samples. (Reported by Ottaviani et al., 196). Challenged 0.9 ± * ± * ± ± ± ±3: * ± ± ± * ± ± ± ± 0.56

8 IMMUNORECOGNITION IN THE GASTROPOD MOLLUSCS 135 These substances, which show chemotactic activity towards SH, play a physiological role in phagacytosis. Furthermore, SH may be removed from the circulating haemolymph as a result of the release of «depletion factors» (Ottaviani, 199b), suggesting that in P. corneus, as in H. pomatia (Renwrantz et al., 191), the circulating haemocytes are not involved in the 1st phase of the clearence event (Ottaviani, 1990). Indeed, Renwrantz et al. (191) report that bacterial elimination in H. pomatia is characterized by a two-phase clearance event, in which the 1st phase does not involve the circulating haemocytes and is characterized by the accumulation of invading cells in various organs, whereas the 2nd phase involves the attraction of circulating haemocytes by organ-trapped foreign cells. As far as the response of enzymes and free amino acids (FAA) in the haemolymph to bacteria is concerned, it has been observed that both acid and alkaline phosphatase activity decrease 30 min after bacterial injection, and return to initial levels after bacterial elimination (192 hrs). Lactate-dehydrogenase (LDH), on the other hand, reacts in the opposite way, even if this increase is not very pronounced. Finally, the a-amylase shows no variation, indicating that this emzyme is not involved in bacterial clearance (Ottaviani et al., 196). The reduction in acid phosphatase activity can be attributed to a low level of release of the enzyme into the serum, to inhibition induced by NADH + H +, as observed in in vitro reactions with the purifed enzyme, or to a combination of these factors. With respect to alkaline phosphatase, the causes are still unknown. The increase in LDH activity is probably related to the fact that the energy for phagocytosis derives from glycolysis, as observed in insects (Anderson et al., 1973) and molluscs (Cheng, 1976). The comparison, 2 hrs after bacterial injection, of numerous FAA with controls (Table IV) shows an increase in glutamic acid and a more or less pronounced fall in all other FAA. The increase in glutamate and the decrease in aspartate are probably connected to the anaerobic metabolism. Indeed, the former has been shown to be a substrate (Ebberink et al., 1979), and the latter a final product of anaerobic metabolism (De Zwaan, 1977; Livingstone, 197). Finally, the reduction in the other FAA may be due to their utilization by P. corneus, as suggested by Gilberston et al. (1967) for Australorbis glabratus infected with parasites, but does not exclude use of the FAA by injected bacteria themselves. MEMORY-TYPE RESPONSE The problem of the presence of some kind of memory component in invertebrates and, in particular, in gastropods is still debated. Humoral and cellular investigations and bacterial clearance studies, together with the specific responses observed in transplantations in P. corneus are in favour of the presence of a memory- 'C 41.3 'S Time (min) Fig. 7 - In vitro phagocytosis of Stapbylococcus aureus by P. corneus haemocytes. a: control; b: treated. One experiment representative of three is shown. (Reported by Ottaviani, 1991b). TABLE V - Agglutinin titers in Planorbarius corneus. Bacteria Stapbylococcus aureus Escberichia coli Injection first (day 0) second (day 14) first (day 0) second (day 14) Reported by Ottaviani, 1991b. Agglutination direct cross direct cross direct cross direct cross after Titer h after 24 h

9 136 E. OTTAVIANI "3 J-400 e I2OO i i Time (h) Time (h) Fig. - Clearance of Staphylococcus aureus from circulation in P. corneus. a: primary response; b: secondary response after 14 days; c: tertiary response after 73 days. (Reported by Ottaviani et al., 196). type response of short duration. These studies, therefore, suggest that there could be various types of memory which differ according to their degree of inducibility and specificity. In view of the fact that this mechanism in invertebrates is not based on clonal selection (i.e., absence of lymphocytes), the term «invertebrate memory», rather than «immunological memory», is suggested, to contrast with «vertebrate memory» (Ottaviani et al., 196; Ottaviani, 1991b). As far as the humoral response is concerned (Table V), repeated injection with the two bacteria (S. aureus and Escherichia colt) induces agglutinins which were not present in the two groups of animals prior to injection. It should be noted that there are both specific agglutinins, which are found in direct agglutination (agglutination tests performed with the same bacteria used to immunize the animals) and whose titer increases in the second injection, and aspecific agglutinins, found in cross-agglutination (tests performed with bacteria used to immunize the other group of animals), whose titer remains constant. In vitro bacterial phagocytosis experiments were carried out to determine the cellular response, and, as shown in Fig. 7, bacterial elimination was higher when the haemocytes from the animals which had been used as controls had already come into contact with the bacteria to be phagocytized. Indeed, after 30 min 1% of bacteria are alive in the controls in comparison to 5.3% in the treated animals, this difference remaining relatively constant with time. The bacterial clearance experiments show (Fig. a, b, c) that, in contrast to results after the first injection, the clearance rate is faster and the clearance patterns markedly different following the second (14 days) and third (73 days) bacterial injections. On the whole, all these phenomena are similar to an anamnestic response. CONCLUSIONS On the whole, it can be concluded that: (i) the gastropod immune system presents the three functional components identified by Hildemann et al. (1979) as minimal criteria for immunological competence: 1) cytotoxicity, 2) specificity, 3) memory; (ii) two cell types are present in P. corneus: the round haemocytes (RH), have characteristics reminiscent of vertebrate Tlymphocytes; and the spreading haemocytes (SH), which belong to the category of macrophage lineage; (iii) the RH present numerous typical markers of T lymphocytes in vertebrates such as man or rodents. Current data do not yet allow in-depth analysis, but suggest that cells such as the RH of P. corneus might be the ancestral precursors of T lymphocytes. These cells could be a model for studying the evolutionary origin of T lymphocytes; (iv) the functional characteristics and behaviour of the spreading haemocytes point to the possibility that this cell type could represent the ancestral cellular com-

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