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Immunorecognition in the gastropod molluscswith particular reference to the freshwater snailPlanorbarius corneus (L.) (Gastropoda, Pulmonata)Enzo Ottaviani aa Dipanimento di Biologia animale, Università di Modena, via Berengario 14, Modena, I‐41100, ItalyVersion of record first published: 28 Jan 2009.

To cite this article: Enzo Ottaviani (1992): Immunorecognition in the gastropod molluscs with particular reference to thefreshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata), Bolletino di zoologia, 59:2, 129-139

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Boll. Zool. 59: 129-139(1992)

Immunorecognition inthe gastropod molluscs with particularreference to the freshwater snailPlanorbarius corneus (L.)(Gastropoda, Pulmonata)

ENZO OTTAVIANIDipanimento di Biologia animale, Università di Modena,via Berengario 14, 1-4 1100 Modena (Italy)

ABSTRACT

The internal defence system of gastropod molluscs is able todiscriminate between self and non-self. The recognition is carriedout principally by both the cellular and humoral components of thehaemolymph. Together with freely circulating haemocytes, otherdefence cells are found scattered throughout the tissue or localizedin organs. The present review refers primarily to Planorbarius cor-neus, since the defence mechanisms presented by this animal aretypical of those of the other gastropods studied. P. corneus presentstwo circulating haemocytes: the spreading (SH) and the roundhaemocytes (RH); in other gastropods only one cell type isdescribed, and this can be considered as a spreading haemocyte. Thehaemocytes derive both from a haematopoietic organ and frommature circulating haemocytes. The SH show phagocytic properties,adhere to glass, produce agglutinins, bind Con A and containmuramic acid. The RH have non phagocytic properties, do notadhere to glass, form rosettes with sheep red blood cells, arestimulated to proliferate by PHA and present numerous typicalmarkers of vertebrate T lymphocytes. RH are also able to lyse 51Crpre-labeled K562 target cells in a classical, short-term, naturalcytotoxicity test, and this function is modulated by human recom-binant interleukin-2. Furthermore, SH and RH play a role in therecognition of foreign tissue, the SH are able to encapsulate andphagocytize foreign material, and the RH, with their NK (naturalkiller)-like activity, may act like the vertebrate cytotoxic T lym-phocytes or NK cells. Thus, it is possible to conclude that RH havecharacteristics reminiscent of vertebrate T lymphocytes, while SHbelong to the category of macrophages. With regards to the humoralcomponent, different factors, such as lysosomal enzymes, lysins andagglutinins or lectins, have been described. In P. corneus, a naturalglycoprotein agglutinin has been isolated, whose carbohydrate com-ponent contains muramic rather than sialic acid. Moreover, an in-duced bacterial agglutinin has been purified, although this inductionis relatively rare in gastropods. Lysozyme-like molecules have alsobeen detected and they act like alarm molecules in inflammatory reac-tions. Taken together, the humoral and cellular investigations, the bac-terial clearance studies and the specific responses observed in tran-splantation experiments are all in favour of the presence of a memory-type response of short duration. Finally, interrelations appear to existbetween the immune and neuroendocrine systems. ACTH andß-endorphin-immunoreactive molecules have been detected in serumand SH, and these molecules appear to play a physiological role in theprocess of phagocytosis and in stress response.

KEY WORDS: Gastropod molluscs - Planorbarius corneus - Inter-nal defence mechanisms - Cellular and humoral components.

ACKNOWLEDGEMENTS

This work was supported by M.U.R.S.T. (60%) and C.N.R. grantsto E.O.

INTRODUCTION

Gastropod molluscs have an internal defence systemwith a well-developed capacity to discriminate betweenself and non-self. Recognition in these creatures, as in allanimals characterized by coelomic cavities, is principallycarried out by both the cellular and humoral componentsof the haemolymph. Together with freely circulatinghaemocytes, there are different types of defence cellsthat may be scattered throughout the connective tissueor localized in particular organs, such as the digestivegland, where cells with phagocytic activity are present.Such cells represent a fixed phagocyte system in Helixpomatia (Reade, 1968), while in Planorbarius corneusthey make up almost the entire gland (Ottaviani, 1990).Furthermore, we recall the antigen-trapping cells whichhave been described in the blood sinuses of the kidney,digestive gland, and foot of H. pomatia. The surface car-bohydrate receptors of these cells bind foreign substan-ces which are then phagocytized by the circulatinghaemocytes (Renwrantz et al., 1981). Finally, we alsodraw attention to the fixed phagocytes of the connectivetissues described in Lymnaea stagnalis (Sminia et al.,1979b).

In the present review, it seemed appropriate to offertwo separate descriptions of the components of thehaemolymph in P. corneus, even if such a division doesnot really exist, as every immune response is the result ofthe co-operation between the cellular and the humoralcomponent.

CELLULAR COMPONENT

The great majority of gastropods, including P. corneus,present two circulating haemocytes: spreadinghaemocytes (SH) and round haemocytes (RH) (seeSminia, 1981, for a review; Ottaviani, 1983), each havinga characteristic morphology. The presence of two celltypes in Helix aspersa (Prowse & Tait, 1969), Bulinusguernei (Krupa et al., 1977), Bulinus trunca tus roblfsi(Cheng & Guida, 1980) and Biompbalaria glabrata(Harris, 1975; Cheng & Auld, 1977; Cheng & Garrabrant,1977; Yoshino, 1986) has been inferred by functionalaspects and surface marker analysis, in addition to mor-phological studies. In those cases in which only one typeis described, the cell type has the characteristics of SH(Ottaviani, 1989a; Franchini & Ottaviani, 1990). In L.stagnalis, a single cell type with both morphologies hasbeen described, where the round form is the young stageand the spreading form the mature stage. These two for-ms have functional capacities which differ only quan-titatively (Sminia et al., 1983; see Sminia & van derKnaap, 1986, for a review). In this case, too, it has beenpossible to find similarities between the mature stage andthe SH type (Ottaviani & Franchini, 1988).

In the haemolymph of P. corneus between 800 and1400 haemocytes/mm3 are present; SH constituting about

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130 E. OTTAVIANI

i-'-.

Fig. I - Light micrograph of spreading haemocyte of P. corneusstained with May-Griinwald Giemsa, Bar, 5 urn.

Fig. 2 - Light micrograph of round haemocyte of P. corneus stainedwith May-Grünwald Giemsa. Bar, 5 urn.

80%, and RH the remaining 20%. These haemocytesderive from mature circulating haemocytes and fromprecursor cells situated in a haemocyte-producing organ(HPO), where the haemoblasts transform intohaemocytes which then migrate into the sinuses of HPO;there after they are transported to all parts of the body(Ottaviani, 1983; 1988a). The HPO has been found inseveral other species of Planorbids and Lymnaeapalustris (Kinoti, 1971; Lie et al., 1975; Rachford, 1976;Jeong et al., 1983) and, as in P. corneus, it lies betweenthe mantle cavity and the pericardium.

Examination of the haemocytes in freshly drawnhaemolymph stained with May-Grünwald Giemsa showsthat SH have an irregular form with abundant cytoplasm,rich in pseudopodia of varying length and shape. Thenucleus is crinkled or kidney-shaped (Fig. 1). Thesehaemocytes tend to form clumps. The RH present around nucleus with scant cytoplasm, evident as a slenderring around the nucleus. Sometimes the cytoplasm is notclearly visible (Fig. 2) (Ottaviani, 1983). Ultrastructuralexamination shows the presence in SH of both smoothand rough endoplasmic reticulum, free ribosomes andpolyribosomes, several Golgi complexes in an activephase, lysosome-like structures, and clumps of a-glucogen granules. Moreover, vacuoles are present in theouter cytoplasm, indicating active endocytosis (Fig. 3).The RH, in contrast to SH, have predominantly rough en-doplasmic reticulum and mitochondria whose matrixcontain large electron-dense granules (Fig. 4). This celltype, therefore, sometimes shows nuclear andcytoplasmic damage that is a feature of cellular apoptosis,considered to be physiological tissue turnover (Ottaviani& Franchini, 1988). As reported above, SH presentultrastructural similarities with the spreadingamoebocytes of L. stagnalis (Stang-Voss, 1970; Sminia,

Fig. 3 - Electron micrograph of spreading haemocyte of P. corneus.x 32500. (Reported by Ottaviani & Franchini, 1988)

1972), the granulocytes of B. guernei (Krupa et al.,1977), and the granulocytes of B. glabrata (Harris, 1975,Joky et al., 1983). Together with similar morphologicalcharacteristics, these blood cells, which bear differentnames, such as granulocyte, amoebocyte, leukocyte orhaemocyte, also present the same functional patterns, e.g.spreading properties, substrate adhesion, phagocytosis

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IMMUNORECOGNITION IN THE GASTROPOD MOLLUSCS 131

.©•

* .

Fig. 4 - Electron micrograph of round haemocyte of P. corneus.x 19000. (Reported by Ottaviani & Franchini, 1988)

and receptors for concanavalin A (Con A) (Prowse & Tait,1969; Anderson & Good, 1976; Jeong & Heuneman,1976; Cheng & Garrabrant, 1977; Renwrants & Cheng,1977, Schoenberg & Cheng, 1980; 1981; Yoshino, 1981;Ottaviani, 1983; Ottaviani & Franchini, 1986). The RH ofP. corneus is comparable only to the ultrastructure of theround amoebocytes described for L. stagnalis (Sminia,1972; see Sminia, 1981 for a review). The other cell typefound by other researchers, the hyalinocyte, differs mor-phologically from RH in its electron transparent feature,

but these two cell types do share some behaviouralcharacteristics; they remain spherical, they do not emitpseudopodia and they have no phagocytic capacity(Cheng & Garrabrant, 1977; see Sminia, 1981 for areview; Ottaviani, 1983).

The functional aspects are reported in the Table I. Thetwo cell types in the haemolymph of P. corneus presentdifferent functions which, on the one hand, are charac-teristic of phagocytic cells (SH) and, on the other, ofcytotoxic cells (RH).

Both in vitro and in vivo, SH can phagocytize latex par-ticles, bacteria and apoptotiç RH (Ottaviani, 1983; Ot-taviani & Franchini, 1988; Ottaviani, 1989b). The role ofthe lysosomal enzymes during phagocytosis is supportedby the increase of acid phosphatase in SH of P. corneuschallenged with bacteria (Ottaviani et al., 1986), a fact,moreover, which confirms results obtained by otherauthors (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, 1983).

The RH are able to lyse 51Cr pre-labeled K562 targetcells in a classical, short-term, natural cytotoxicity test,and this function is modulated by human recombinantinterleukin-2 (hrlL-2) (Franceschi et al., 1991).

Moreover, both SH and RH are involved in therecognition of foreign tissue, although in different ways.Alio- and xenograft implants (ganglia from animals of thesame species or from Helix lucorum) are initially encap-sulated and then phagocytized. Only haemolymph cellsare involved in this phenomenon. The RH reach the graftfirst, while the SH are responsible for the formation ofthe capsule and the destruction and removal byphagocytosis of the foreign cells. Encapsulation does notoccur in cases of auto-transplantation. The sequence ofencapsulation is similar in alio- and xeno-transplants, butin 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 adhesionPhagocytosisChemotactic activityRosette with SRBCResponsiveness to PHARecognition of auto-, alio- and xenoimplantsReactivity to pAb anti-muramic acidReactivity to pAb anti-human-lysozymeReactivity to pAb anti-human-ß3-microglobulinReactivity to Con ACytotoxicityResponsiveness to hrlL-2

Ottaviani, 1983Ottaviani, 1983; Ottaviani & Franchini, 1988; Ottaviani, 1989bOttaviani et al., 1990bOttaviani, 1983Ottaviani, 1988bOttaviani & Verginc, 1990, Ottaviani et al., 1991bOttaviani & Montagnani, 1989Ottaviani, 1991aOttaviani et al, 1990dOttaviani & Franchini, 1986Franceschi et al., 1991Franceschi et al, 1991

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132 E. OTTAVIANI

Vergine, 1990; Ottaviani et al., 1991b). These studiessuggest, first, that in P. corneus, recognition and rejec-tion of transplants involves two cell types, i.e. RH, whichhave NK (natural killer)-like activity and could act like thecytotoxic T lymphocytes in vertebrates, and SH, whichperform the double function of encapsulation andphagocytosis. Secondly, the response to different graftsis characterized by specificity.

Observation by flow cytometry and im-munofluorescence procedures showed that SH, but notRH, were positive for anti-N-acetyl-mu'ramic acid (Ot-taviani & Montagnani, 1989). The presence of this acidhas also been demonstrated in glycoconjugates of severaltissues of other gastropod molluscs. N-acetyl-neuraminicacid (sialic acid) is not found in these tissues. Ingastropods N-acetyl-muramic acid possibly substitutessialic acid and has a similar role and function to that invertebrate glycoproteins. Moreover, both molecules areC9 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 theonly cell type present in the haemolymph of Viviparusater. These cells have the same functions as the spreadinghaemocytes found in other gastropods (Ottaviani,1989a).

As reported in Table II, several investigations by im-munocytochemistry, RIA- and flow cytometry have

TABLE II - Bioactivepeptide (BAP)-Uke molecules in spreading (SH)and round haemocytes (RH) in Planorbarius corneus.

BAP SH RH

ACTHß-endorphina,-antichymotrypsinBombesinCalcitoninCCK-8 (INC)CCK-8 (Peninsula)CCK-39GastrinGlucagonInsulin (CRL)Insulin (Peninsula)Met-enkephalinNeurotensinOxytocinProlactinSomatostatinSubstance PThyroglobulinThyroxin (T4)VasopressinVIP

ar . ' " * > .

*.. «•«!.•. --

Reported and modified by Ottaviani & Cossarizza (1990)

Fig. 5 - Spreading haemocytes positive (a) and a round haemocytenegative (b) to anti-ACTH antibody. Each group of spreadinghaemocytes presents five cells. Bar, 10 um. (Reported by Ottavianietal, 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 thehaemocytes probably plays a role in the modulation ofthe 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 (Ot-taviani et al., 1990c). It is interesting to note that similarmolecules have been described in unicellular organismsand invertebrates (Le Roith et al., 1982), despite the ab-sence of anatomic targets usually associated with theneuroendocrine system. Opioid-like molecules in inver-tebrates appear to be a general phenomenon, asuggestion supported by the fact that the only cell typepresent in the haemolymph of another snail, i.e.amoebocytes in L. stagnalis (Sminia, 1972), is alsopositive to the anti-ACTH antibody (Ottaviani et al.,1991a). These results are in agreement with previous ob-servations indicating presence of certain chemically iden-tified opioids in the bivalve Mytilus edulis (Leung &Stefano, 1984; Stefano & Leung, 1984).

The presence of irACTH and irß-endorphin moleculesonly in cells capable of phagocytic activity suggests thatthese molecules may play a physiological role in theprocess of phagocytosis. Indeed, ACTH and ß-endorphinexert chemotactic activity towards SH (Ottaviani et al.,1990b). The target cell SH probably responds to the ex-tracellular ACTH signal by varying the intracellular cyclicAMP level. In order to migrate towards a chemoattrac-tant, a cell has to be able to recognize the substance,presumably by specific receptors, and to modify the

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cytoskeletal components involved in cell migration. In-deed, after incubation with ACTH, modification of theactin microfilament network is evident in SH. Changes inthe sites of microfilament-membrane interactions and theformation of new adhesive structures are probably alsoinvolved (Franchini et al, 1990). Furthermore, ACTH in-duces the release of biogenic amines in the serum and thephagocytic activity of SH towards Staphylococcus aureusincreases with an increase in the ACTH dose (Ottaviani etal, 1991a).

On the whole, these data suggest that these opioidneuropeptides are involved in a complex set of ancestralimmune responses including the stress response. An in-tegral part of this phenomenon is the release of biogenicamines.

The analysis of the RH and SH phenotype by flowcytometry has allowed further characterization of thesecell types. Mouse anti- human mAbs recognizing CD(cluster of differentiation) antigens that define lym-phocyte subsets were used (Franceschi et al, 1991).

I am well aware that a positive staining with a mouseanti-human mAb only means that the epitope recognizedby the antibody is probably present, and does notdefinitely prove the existence of a given molecule.

TABLE III - Cytofluorimetric analysis of spreading (SH) arid roundbaemocytes (RH) of Planorbarius corneus using mouse anti-humanmAbs.

mAb anti- SH RH

CDla, CD16, CD26, CD29, CD56

CD5, CD34, CD45RA, CD54, CD61, CD71CD2, CD3, CD4, CD7, CD8, CDlla, CDllb,CDllc, CD13, CD18, CD19, CD20, CD21, CD22,CD23, CD25, CD33, CD38, 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 showsthat the haemocytes react with antibodies recognizingepitopes present on molecules typical of human NK cells,such as CD16 and CD56, and epitopes present onmolecules found on human T lymphocytes such asCDla, CD26 and CD29. Moreover, positivity for epitopespresent on molecules involved in cell-cell interactionssuch as CDla (MHC precursor), CD29 (intregrin ß, chain),CD56 (an N-CAM isoform) and CD61 (integrin ßä chain),is also evident.

Although there is some uncertainly on the degree ofhomology of immunoreactive molecules in man and the

snail, our results nevertheless strongly suggest thatprimitive invertebrate haemocytes present the sameepitopes, and possibly also the same molecules, as verysophisticated immune cells, such as mammalian lym-phocytes.

HUMORAL COMPONENT

In the serum of gastropods, several humoral factors areinvolved in immune responses. Agglutinins or Jectins,opsonins, lysins, lysosomal enzymes, and others havebeen described (see Ratcliffe et al., 1985; Olafsen, 1986;Sminia & van der Knaap, 1986, for reviews). These fac-tors are naturally present or may be induced afterstimulation (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, 1982;Ottaviani, 1984, and others).

The molluscan agglutinins that seem to act similarly toantibodies are protein or glycoprotein (Hammarström &Kabat, 1969;Jenkin& Rowley, 1970; Pauleyeffl/., 1971a;Hardy et al., 1977; Baldo et al., 1978; Renwrantz & Stah-mer, 1983). They may also have, opsonic properties(Pauley et al, 1971b; Harm & Renwrantz, 1980; van derKnaap et al, 1982; Renwrantz & Stahmer, 1983), andneed divalent cations (in particular, Ca++) to manifestbiological activity (Hammarström & Kabat, 1969; Baldoet al, 1978; Renwrantz & Stahmer, 1983). They can alsomediate chemotaxis (Schmid, 1975) and bind specificcarbohydrates (Renwrantz, 1983).

In P. corneus, agglutination was observed with theerythrocytes of rabbit and goldfish, and with blood cellsof V. ater and the storage cells of Macrobiotus ricbtersi(Ottaviani, 1986).

An agglutinin against goldfish erythrocytes has beenisolated from the haemolymph of P. corneus by affinitychromatoghaphy on Sephadex gel G 150 (Fig. 6) (Ot-taviani & Tarugi, 1986). This molecule is an acidglycoprotein with a mol wt of 130 KD. The acid part isnot composed of the usual sialic acid but of N-acetyl-muramic acid. This agglutinin is synthesized in the SH.Indeed, using immunoperoxidase techniques with an an-tibody against P. corneus haemolymph serum and ananti-N-acetyl-muramic acid antibody, only the SH reactpositively (Ottaviani, 1988c; Ottaviani & Montagnani,1989). Similar results were obtained in L. stagnalis,where the amoebocytes synthesize agglutinin/opsoninand bear it on their surface as a receptor for foreignmaterials (van der Knaap et al, 1981).

Natural bacterial agglutinins have not been found in P.corneus, and bacterial agglutinins are induced only afterinjection of the bacteria (Ottaviani, 1986; Ottaviani &Tarugi, 1989). In particular, an induced bacterialagglutinin with a mol wt of 330-350 KD has been purifiedfollowing repeated injections of S. aureus. The induc-tion of agglutinin? and antibacterial substances in

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134 E. OTTAVIANI

21.5K

14K

A BFig. 6 - SDS-polyacrylamide gel electrophoresis of isolated agglutininfrom P. corneus: A) mol wt standards; B) isolated agglutinin indicatedby arrow. (Reported by Ottaviani & Tarugi, 1986).

specifically to foreign challenge. It is, therefore, possibleto hypothesize that the increase in the irlysozymemolecule 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, irlysozymematerial is probably involved in the initial steps whichcontrol the promotion of inflammatory reactions (Ot-taviani, 1991a). Indeed, as a result of aspecific stimuli,these molecules increase within a few hours in the serumand are induced by bacterial antigens. The increase inirlysozyme molecule levels a few hours after injection ofthe various antigens is probably the result of their releasefrom storage sites, whereas the increase after 24 hrs inresponse to bacterial stimulation is the result of new syn-thesis. The persistence of the irmolecules only after bac-terial injection cannot be a specific response, and onemay suppose that the inflammatory reaction followingbacterial injection lasts longer than that provoked byother foreign substances, and, as a result, inducesirlysozyme molecule production. Furthermore, there is acorrelation between irlysozyme molecules andphagocytosis, since these molecules are only present inSH, and, as ACTH and ß-endorphin-like molecules, theyshow chemotactic activity towards SH.

In P. corneus, a decrease in haemolymph phagocyticcells (SH) was observed after bacterial injection, and thisdrop seems to be mediated by humoral factor(s),probably including irlysozyme, irACTH and irß-endorphin molecules, released into the haemolymph.

TABLE IV - Concentration (nmol/ml) of free amino acids (FAA) inthe serum of Planorbarius corneus before and after (2 h) challengewith living Staphylococcus aureus.

molluscs appears to be relatively rare. To date, there hasbeen only one report of induced bactericidal response inabalones challenged with a formalin-killed suspension ofbacterial culture EMB-1 (Cushing et al., 1971). This maybe a result of limited knowledge, or these animals maypresent different immunological strategies. Natural andinduced haemolytic activity is evident in the haemolym-ph of Mercenaria mercenaria, and this haemolysinprobably has a protective role related to antimicrobial ac-tivity (Anderson, 1981). Moreover, Cheng et al. (1975;1977) have suggested that the presence of lysozyme mayindicate a non-specific inducible humoral defencemechanism. Indeed, B. glabrata challenged with bac-teria presents increased haemolymph lysozyme levels.However, the significance of haemolymph lysozymelevels is still not clear; in Crassostrea virginica lysozymelevels were higher following infection with certainparasites but lower following infection with others (Feng& Canzonier, 1970). In P. Corneus irlysozyme moleculesseem also to be inducible molecules responding non-

FAA

Aspartic acidThreonineSerineGlutamic acidProlineGlycineAlanine1/2 CystineValineMethionineIsoleucineLeucineTyrosinePhenylalanineLysineHistidineArginine

Controls

4.17 * 1.2351.78 ±2.3967.84 ± 4.028.44 s 0.68

22.99 * 3.2734.44 £ 2.0431.13*1.4514.73 * 1.3542.33 * 5.876.91 ± 0.30

21.40 ±4.1024.75 ±3.19

8.47 ± 2.1618.95 ± 3.4644.28 ± 15.9814.81 ±0.6110.28 ± 3.27

Challenged

0.89 ± 0.1530.08* 1.8619.95 ± 0.3713-45 * 1.4815.12 ±3.1110.47 ± 0.9515.26 ± 7.6711.61 ±3:8240.54 * 6.766.94 ±2.17

10.92 ± 2.5522.18 ±4.046.52*1.65

17.19 ± 2.64 .39.13 ± 8.4712.72 ± 0.427.04 ± 0.56

The tests were carried out on three samples.(Reported by Ottaviani et al., 1986).

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These substances, which show chemotactic activitytowards SH, play a physiological role in phagacytosis.Furthermore, SH may be removed from the circulatinghaemolymph as a result of the release of «depletion fac-tors» (Ottaviani, 1989b), suggesting that in P. corneus, asin H. pomatia (Renwrantz et al., 1981), the circulatinghaemocytes are not involved in the 1st phase of theclearence event (Ottaviani, 1990). Indeed, Renwrantz etal. (1981) report that bacterial elimination in H. pomatiais characterized by a two-phase clearance event, in whichthe 1st phase does not involve the circulatinghaemocytes and is characterized by the accumulation ofinvading cells in various organs, whereas the 2nd phaseinvolves the attraction of circulating haemocytes byorgan-trapped foreign cells.

As far as the response of enzymes and free amino acids(FAA) in the haemolymph to bacteria is concerned, it hasbeen observed that both acid and alkaline phosphataseactivity decrease 30 min after bacterial injection, andreturn to initial levels after bacterial elimination (192 hrs).Lactate-dehydrogenase (LDH), on the other hand, reactsin the opposite way, even if this increase is not verypronounced. Finally, the a-amylase shows no variation,indicating that this emzyme is not involved in bacterialclearance (Ottaviani et al., 1986).

The reduction in acid phosphatase activity can be at-tributed to a low level of release of the enzyme into theserum, to inhibition induced by NADH + H + , as obser-ved in in vitro reactions with the purifed enzyme, or toa combination of these factors. With respect to alkalinephosphatase, the causes are still unknown. The increasein LDH activity is probably related to the fact that theenergy for phagocytosis derives from glycolysis, as ob-served in insects (Anderson et al., 1973) and molluscs(Cheng, 1976).

The comparison, 2 hrs after bacterial injection, ofnumerous FAA with controls (Table IV) shows an in-crease in glutamic acid and a more or less pronouncedfall in all other FAA. The increase in glutamate and thedecrease in aspartate are probably connected to theanaerobic metabolism. Indeed, the former has beenshown to be a substrate (Ebberink et al., 1979), and thelatter a final product of anaerobic metabolism (De Zwaan,1977; Livingstone, 1978). Finally, the reduction in theother FAA may be due to their utilization by P. corneus,as suggested by Gilberston et al. (1967) for Australorbisglabratus infected with parasites, but does not excludeuse of the FAA by injected bacteria themselves.

MEMORY-TYPE RESPONSE

The problem of the presence of some kind of memorycomponent in invertebrates and, in particular, ingastropods is still debated. Humoral and cellular in-vestigations and bacterial clearance studies, togetherwith the specific responses observed in transplantationsin P. corneus are in favour of the presence of a memory-

20 -

'C41

10 -

.3'S

60 90

Time (min)

120 150

Fig. 7 - In vitro phagocytosis of Stapbylococcus aureus by P. corneushaemocytes. a: control; b: treated. One experiment representative ofthree is shown. (Reported by Ottaviani, 1991b).

TABLE V - Agglutinin titers in Planorbarius corneus.

Bacteria

Stapbylococcusaureus

Escberichiacoli

Injection

first(day 0)

second(day 14)

first(day 0)

second(day 14)

Agglutination

direct

cross

direct

cross

direct

cross

direct

cross

after 38

4 - 8

16

8

8

8

16

8

Titer

h after 24 h16-32

8

64

8

16

8

32 -64

8

Reported by Ottaviani, 1991b.

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136 E. OTTAVIANI

800 •

| 6 0 0 -

"3

J-400e

I2OO -

0.5 1 2 4 8 24 48 72 96 168192

Time (h)

1000 -i

0.5 1 2 4 8 24 48 72 96

3000 -i

0.5 1 2 4 8 24 48 72 96

Time (h)

Fig. 8 - 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., 1986).

type response of short duration. These studies, therefore,suggest that there could be various types of memorywhich differ according to their degree of inducibility and •specificity. In view of the fact that this mechanism in in-vertebrates is not based on clonal selection (i.e., absenceof lymphocytes), the term «invertebrate memory», ratherthan «immunological memory», is suggested, to contrastwith «vertebrate memory» (Ottaviani et al., 1986; Ot-taviani, 1991b). As far as the humoral response is concer-ned (Table V), repeated injection with the two bacteria(S. aureus and Escherichia colt) induces agglutininswhich were not present in the two groups of animalsprior to injection. It should be noted that there are bothspecific agglutinins, which are found in directagglutination (agglutination tests performed with thesame bacteria used to immunize the animals) and whosetiter increases in the second injection, and aspecificagglutinins, found in cross-agglutination (tests performedwith bacteria used to immunize the other group ofanimals), whose titer remains constant. In vitro bacterialphagocytosis experiments were carried out to determinethe cellular response, and, as shown in Fig. 7, bacterialelimination was higher when the haemocytes from theanimals which had been used as controls had alreadycome into contact with the bacteria to be phagocytized.Indeed, after 30 min 18% of bacteria are alive in the con-trols in comparison to 5.3% in the treated animals, thisdifference remaining relatively constant with time.

The bacterial clearance experiments show (Fig. 8a, b, c)that, in contrast to results after the first injection, theclearance rate is faster and the clearance patternsmarkedly different following the second (14 days) andthird (73 days) bacterial injections. On the whole, allthese phenomena are similar to an anamnestic response.

CONCLUSIONS

On the whole, it can be concluded that:(i) the gastropod immune system presents the three func-tional 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 roundhaemocytes (RH), have characteristics reminiscent of ver-tebrate Tlymphocytes; and the spreading haemocytes(SH), which belong to the category of macrophagelineage;(iii) the RH present numerous typical markers of T lym-phocytes in vertebrates such as man or rodents. Currentdata do not yet allow in-depth analysis, but suggest thatcells such as the RH of P. corneus might be the ancestralprecursors of T lymphocytes. These cells could be amodel for studying the evolutionary origin of T lym-phocytes;(iv) the functional characteristics and behaviour of thespreading haemocytes point to the possibility that thiscell type could represent the ancestral cellular com-

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IMMUNORECOGNITION IN THE GASTROPOD MOLLUSCS 137

ponent of internal defence mechanisms in gastropods.Phagocytosis, the oldest immune response, is evident inthese cells, and, although cellular and molecular struc-tures involved in defence have undergone markedmodifications during evolution, phagocytosis maintainsuniform characteristics from protozoa to man (seeAzzolina et al., 1985 for a review). Furthermore, the onlyblood cell found in some species of gastropods is thespreading haemocyte (Cuénot, 1892; Pan, 1958; Ot-taviani, 1989a). Lastly, spreading haemocytes presentN-acetyl-muramic acid on the surface membrane, andthis is a constituent part of the exposed glycoproteingroups in lower forms of life such as bacteria;(v) interrelations appear to exist between the immuneand neuroendocrine systems, suggesting the hypothesisthat Nature, as in other cases, followed a similar strategyin the construction of the neuroendocrine and immunesystems, i.e. the major systems responsible for theacquisition, elaboration, and memorization of infor-mation essential for maintaining the homeostasis of theorganism with respect to the internal and external en-vironment.

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