DISEASES OF AQUATIC ORGANISMS Published August 15 Dis Aquat Org

When the venerid clam Tapes decussatus is parasitized by the protozoan Perkinsus sp. it synthesizes a defensive polypeptide that is closely related to p225

Juan F. Montes, Merce Durfort, Jose Garcia-Valero*

Departament de Biologia Cel-lular i Vegetal, Facultat de Biologia. Universitat de Barcelona, Avda. Diagonal, 647, E-08028Barcelona. Spain

ABSTRACT: Molluscs, like other invertebrates, have primitive defense systems. These are based on chemotaxis, recognition and facultative phagocytosis of foreign elements. Previously, we have described one of these systems: a cellular reaction involving infiltrated granulocytes against Perkinsus sp. parasitizing the Manila clam Tapes semidecussatus, in which the parasites are encapsulated by a defensive host product, the polypeptide p225. The aim of this study is to determine the similarities between the defense mechanisms of 2 venerid clams, T semidecussatus and T. decussatus, when they are infected by Perkinsus sp. The hemocytes of both species infiltrate the connective tissue, redifferen- tiate, and ultimately, express and secrete the polypeptide which constitutes the main product of the capsule that surrounds the parasites. The main secretion product of T. decussatus shows a high degree of homology to that of T semidecussatus, since it has a similar electrophoretic mobility and the polypeptide is recognized by the polyclonal serum against p225 from T. semidecussatus, as confirmed by Western blotting and immunocytochemistry. In conclusion, we demonstrate the existence of 2 polypeptides that are closely related at the molecular and functional level, and are specific in the defense of some molluscs against infection by these protozoan parasites.

KEY WORDS: Defense mechanisms . Encapsulation . Granulocytes . Parasitism . Perkinsus sp. . Tapes decussatus . Tapes semidecussatus .

INTRODUCTION Figueras et al. 1992, Goggin 1992, Navas et al. 1992) and the introduced species T. semidecussatus (= T. Protozoa belonging to the Perkinsus (formerly philippinarum = T japonica) (Villalba & Navas 1988, Dermocystidium or Labyrinthomyxa) (Apicomplexa, Sagrista et al. 1991, 1995, Goggin 1992, Navas et al. Perkinsea) (Levine 1978) have been described as dis- 1992, Montes et al. 1995a). ease agents in 63 species of bivalves and 4 species of Recently, Bachere et al. (1995) have reviewed the gastropods (Perkins 1993). In Europe, during the last defense mechanisms that are present in bivalve mol- 10 yr, Perkinsus spp. trophozoites have been associ- luscs. There is general agreement that the molluscs ated with mass mortalities of commercially important have cellular effectors and immune mechanisms. How- venerid clams of the genus Tapes (=Ruditapes ever, it is necessary to consider that, whereas verte- = Venerupis) (, ), such as the indiye- brates have an immune system, invertebrates have nous species T. decussatus (Da Ros & Canzonier 1985, more primitive defense systems, which often rely Chagot et al. 1987, Comps & Chagot 1987, Gonzalez chiefly on phagocytic cells (Alberts et al. 1994). Those et al. 1987, Villalba & Navas 1988, Azevedo 1989, immune mechanisms are based on chenlotaxis and recognition of foreign elements by lectin-like mole- cules, and subsequent phagocytosis. However, Chin- 'Addressee for reprint requests. tala et al. (1994) concluded that serum agglutinins, the E-mail: [email protected] putative lectin-like molecules, did not play a role in

O Inter-Research 1996 Resale of full article not permitted 150 Dis Aquat Org 26:

oyster defense against Perkinsus marinus or Hap- SDS-PAGE, polypeptides were transferred to nitrocel- losporidium nelsoni. lulose membranes. Transfer was realized at 15 V for 2 h In the venerid clams, Tapes semidecussatus and T. in a Trans-blot semi-dry transfer cell (Bio Rad, Rich- decussatus, Perkinsus spp. parasitism activates the mond, CA, USA). Membranes were blocked with 10% host defense mechanisms and provokes an inflamma- non-fat dried milk in TBST [l0 mM Tris-HC1 (pH = 8), tory response based on the infiltration of hemocytes, 150 rnM NaC1,0.05 % Tween 20) for 30 min and then in- the cellular effectors. In the butterfish (also named cubated with the polyclonal serum against p225 from carpet-shell) clam T. decussatus from Portugal, Comps Tapes semidecussatus (diluted 1:800 in TBST with l0 O/o & Chagot (1987) and Chagot et al. (1987) reported an non-fat dried milk) (Montes et al. 1995b) overnight at inflammatory reaction constituted by granulocytes 4°C. After 3 washes in TBST the membranes were incu- causing the encapsulation of the trophozoites by a bated with peroxidase-conjugated swine anti-rabbit periodic Acid-Schiff (PAS) positive substance. Recent- IgG (Dako, Glostrup, Denmark) for 2 h at room temper- ly, we have described a similar cellular reaction, ature. After washing, the membranes were developed involving infiltrated granulocytes, against this patho- in a substrate solution of 3,3'-diaminobenzidine gen in the Manila (also named Japanese littleneck) tetrahydrochloride (DAB; Sigma Chemical CO, St. clam l. semidecussatus from the Spanish ivieaiier- Louis, biO, USAj db ~ii~u~iioyeii,and finally recorded ranean coast. This cellular reaction is polarized. The on Technical Pan film (Kodak, Hemel Hempstead, UK). trophozoites are encapsulated by the secretory product Immunocytochemistry. Abscesses from parasitized which is released after the death of the granulocytes gills (Tapes decussatus and T. semidecussatus) were that are closest to the parasites (Montes et al. 1995a). fixed in 4 % paraformaldehyde and 0.1 % glutaralde- The main component of this specific defense product is hyde in 0.1 M phosphate-buffered saline (PBS). Sam- a slightly glycosylated polypeptide of about 225 kDa ples were prepared for Lowicryl K4M resin embedding (p225). Moreover, the absence of this polypeptide in (Chemische Werke Lowi, Waldkraiburg, Germany) fol- non-parasitized Manila clam indicates the exclusive lowing Carlemalm et al. (1982). Immunolabeling for association of p225 with the parasitosis (Montes et al. p225 was achieved as previously described (Montes 1995b). et al. 1995b). In brief, the grids were incubated with The aim of the present study is to determine the sim- serum against p225 (1:3000 dilution), and then bound ilarities between 2 Tapes species (T. semidecussatus polyclonal antibodies were visualized following incu- and T. decussatus) in relation to their defense mecha- bation with 10 nm or 15 nm protein A gold (pAg; nisms. Our purpose is to correlate the main component Sigma). Finally, thin sections were observed with a of the cellular reaction at both cellular (infiltrated gran- Reichert-Jung Polyvar 2 optical microscope and the ulocytes) and molecular (secretory product) levels. The ultrathin sections were examined on a Hitachi H-600 results allow us to establish a high homology between AB transmission electron microscope. the cellular reactions of these closely related molluscan Quantitative evaluation. The label density (LD) was species against Perkinsus sp. parasitism. estimated as the number of gold particles per sectioned area of granules and capsule profiles. The area para- meter was estimated by stereological methods (Weibel MATERIALS AND METHODS 1919) and significance of mean differences between experimental groups was tested by ANOVA. . Specimens of parasitized and non-para- sitized clams Tapes decussatus and T. semidecussat~~s were collected in the delta of the River Ebro. Tarrag- RESULTS ona (NE Spain), at the Mediterranean Sea. Gill tissue processing. Abscesses from parasitized Histological examination gills of Tapes decussatus and T. semidecussatus, iso- lated under a stereoscopic microscope, and non-para- Light microscopy of semi-thin sections of gill ab- sitized gills of T. decussatus were prepared for SDS- scesses from the butterfish clam Tapes decussatus PAGE and Western blot as previously described revealed the presence of trophozoites of Perkinsus sp. (Montes et al. 1995b). in the connective tissue. Parasites, isolated or grouped, SDS-PAGE and Western blot. SDS-PAGE was per- were surrounded by closely packed granulocytes, formed as described, by Laemmli (1970) in 9% acryl- which constituted the cellular reaction of this organism amide gels under redwing conditions. Polypeptides against Perkinsus sp, trophozoites. Parasites were seen were resolved by sil.ver staining according to the totally or partially encapsulated by a dense homoge- method of Merril et al. (1981). Western blot analysis neous substance, giving rise to the formation of cysts was performed following Towbin et al. (1979). After (Fig. l). Montes et al.. Tapes decussatus paras~tizedby Perkinsus sp. 151

Figs. 1 to 4. Tapes decussatus parasitized by Perkinsus sp. trophozoites. Lowicryl semithin sections from gills of the clam. Closely packed granulocytes constitute the only cell type observed in the cellular reaction against the parasites. Cysts (c) consist of a variable number of trophozoites and the capsule, which surrounds the parasites totally or partially. x470. Scale bar = 50 pm. Fig. 1 inset. Mitotic figure from an infiltrated granulocyte of the cellular reaction (asterisk). x 1325. Fig Cytoplasm gela- tion (asterisk) and lysosomes (ly) in some granulocytes. X 830. Scale bar = 20 pm. Fig. Trophozoite proliferation by binary fission in a cyst (arrow). x810. Scale bar = 20 pm. Cysts containing a variable number of trophozoites. Some dead trophozoites are seen in several cysts (asterisks). x720. Scale bar = 20 pm 152 Dis Aquat Org 26: 149-157, 1996

Infiltrated granulocytes constituted the only cell type 1 2 3 4 observed in the cellular reaction, which was organized 330 - r p 225 as a single cellular mass without cells or fibers from 180 b ------: connective tissue. Moreover, processes of cell division were occasionally observed in these infiltrated cells (Fig. 1 inset). Granulocytes showed a variable profile and diameter, with the cytoplasm filled by granules. Lysosomes were recognized as dense granules (Fig. 2). Furthermore, cytoplasm condensation in a single, undifferentiated mass was observed in some granulo- cytes (Figs. 2, 3 & 4). The nuclei were circular in section and often situated eccentrically. Abundant clots of het- erochromatin were located in the nucleoplasm, where- as peripheral heterochromatin was scarce (Figs. 1, 2, 3 & 4). Cysts contained one to severai tropnozoires sur- rounded by a non-cellular, non-fibrillar capsule. Unin- Fig. 5. Lane 1: SDS-PAGE of abscesses from Perkinsus sp.- ucleated trophozoites were characterized by the pres- paiasitized gills of Tapes decussatus. Lanes 2 to 4: western ence of a vacuole that occupied up to 90% of the cell blotting with the serum against p225 from T. semidecussatus. Lane 2: abscesses from parasitized gills of T. decussatus;lane volume. Parasites were circular in section with a diam- 3: non-parasitized gills of 7 decussatus;lane 4: abscesses eter ranging between 5 and 12 pm (Fig. 1). In some from parasitized gills of 7. semidecussatus. The migration instances, trophozoite division by binary fission was positions of molecular mass standards are indicated in kDa on noticed (Fig. 3). On the other hand, dead trophozoites the left. In addition, the position of band p225 is indicated on were also seen in the cysts, which appeared as dam- the right aged cells with dense cytoplasm, shrunken aspect and spindle shape (Fig. 4). In the infiltrated granulocytes, labeling by the serum against p225 was restricted to granules. Ultrastructural characterlstics and LD of the granules were variable Western blotting according to the maturation stage of the granulocytes. Granulocytes in early and medium stages of matura- Incubation with the against levealed a tion showed the cytoplasm filled by granules with a abscesses decussatus(Fig' 5)' No apparent nal membranes. The LD for these granules was high differences were observed between these 2 clam spe- and the label was uniformly distributed & cies in molecular weight or in antibody recognition of AUtophagosomes with low LD were characteristic of the P~~YP~P~~~~'The band was not detected in the lane intermediate maturation stages (Fig, 8).Granulocytes corresponding to the non-parasitlzed gills. that lay at the periphery- - of the cellular reaction showed immature granules and were similar to the cir- Immunolocalization culating granulocytes. These granules were identified by their uniform size, floccular content, absence of By electron microscopy, granulocytes of the cellular internal membranes and low LD (Fig. 9). reaction in Tapes decussatus were characterized by Mature granulocytes, located in the inner regions of the presence of numerous secretory membrane-bound the cellular reaction, were typified by a regression of granules with a homogeneous content (Figs. 6, 7 & 8) the endomembranous compartment and the presence and large mitochondria with tubular crlstae and an of granules with a variable size, dense content, internal extended matrix (Fig. 6). The granulocyte cytoplasm membranes and high LD (70.6 * 9.4 gold particles also showed a well-developed endomembranous com- pm-2). This LD was nevertheless significantly lower partment distributed in 2 populations: round cisternae than LD obtained for the mature granules of Tapes and vesicular-tubular saccules, the former rela.ted to semidecussatus (116.9 + 11.5 gold particles pm-2) the rough endoplasmic reticulum and the latter to the (Table 1). Giant granules resulting from fusion were Golgi apparatus and the endosomal pathway (Fig. 7). occasionally observed in these mature granulocytes Moreover, autophagosomes derived from granules (Fig. 10). The LD for these giant granules was lower were occasionally seen, which showed a heteroge- than the LD for unfused mature granules. On the other neous content with internal membranes and residual hand, some mature granulocytes were observed with a bodies (Fig. 8). single granule that enclosed all granules in the cell. urrt I = 1eq apss .0000~x,aldso1nue16 X~olaisas-aide uoij urse~doldspalelab aql U! pauleluoa sa[nuei6 paIaqe1 d16uolls url L = ieq alms .OOOELX .palnq!i~s~p A[snoauaE~ola~aq sr Iaqe? .a?Aso~nuelBe JO salnuei6 ne Bu~sopuaamue~6 618 W.B~J 'wrl I = leq aless '0000~~'[aqel jo p!oAap ale sa[nue16 punouns leql sauelquraw .a~nuelBaBle1 e U! pau~eluossalnue~b news LeiaAaS .OL.61g wrl s.0 = 1eq aIesg '000~~~.lua?uos leIns2oU e 6u!moqs 'salds -01nuelB aql jo salnuer6 aJnle- paIaqe7 6 .url 1 = ieq aIe3S .ooog~X .uoge!lualaJpp JO sabels ale1 aqlulal~~opuei6e uroij IaqeI paia?~~~'yeam pue lualuo3 snoauabo~alaq'saueiquraur [euialu! ql!~(ysualse) auroso6eqdo~nv .urrl I = 1eq aIeaS ,000 11X .sa1nuei6 uo!lalsas paIaqe1 pue (Ba) sapsal\ 161oB/aurosopua '(ia) wnlna!lal s!urse~dopua qBnox .aldaolnueiB e wo~jsauelqurauropux wrl s.0 = ieq aleas .OOOEZX-paIaqel ainlsnlls d[uo aql S! lualuos a~nue~baqL ,(h)sa1nuel6 uo!laiaas punoq-aueiquraur Aq paII!j wseldold~aldao1nuei3 .snlessn2ap!uras 1 uroij szzd 1su1e6e urnlas aql qljm Peal leql snjessnaap L JO sainlanqs aldaolnuel6 jo uogez!le3oIounwurI .ds SnSU!~Bddq paz~l!seiedsnlessnmp sade~'~1 01 g ,s61.~r~ 154 DIS Aquat Org

Table 1 Label dens~tyexpressed as number of gold particles ulum and residual bodies, whlch were labeled by the pm L labelling p225 In mature granules and in capsules from serum. In these cases, the trophozoite wall was devoid both Tapes semidecussatus and T decussatus paras~tizedby Perkinsus sp Values are means t SEM from at least 30 mea- of label (Fig 15),thus indicat~ngthat the labeling fre- surements performed on 3 specimens for each clam species quently observed in the trophozoite wall (Figs. 13, 14 & The s~gnlficanceof mean differences between groups was 16) was a consequence of diffusion processes of this tested by ANOVA, F = 5 03 polypeptide.

T semidec~~ssatus T decussat~ls DISCUSSION Mature granules 116.9 t 11.5 706+94"' Capsule 107.1 t 9.1 51 5 + 11 8" Several species belonging to the genus Perkinsus "p < 0.01 and "'p < 0.001 comparing values in homolo- have been described as disease agents in infectious gous compartments from both species processes affecting several molluscan species (Lauck- ner 1983, Perkins 1988, 1993). These infections are -. characterized by high mortalities of the host species. In I nese singie granuies, wnicn snowed a heterogeneous the preseni siuciy, we si~uwii~e si~~lilu~iiies beivv-eeii content and labeling (Fig. 1 l), constituted intermediate the defensive responses, at both the cellular and mole- stages between giant granules and autophagosomes, cular level, that 2 venerid clams present against the and were never secreted around the trophozoites or parasitism by Perkinsus sp. the capsule. Finally, late stages of granulocyte matura- The butterfish clam Tapes decussatus is a native spe- tion were sometimes charactenzed by gelation of the cies from European coasts and closely related with the cytoplasm, leading to the formation of a matrix around Manila clam T semidecussatus. The Manila clam was the granules, which showed high LD (Fig. 12). introduced into Europe from natural Indo-Pacific pop- By electron microscopy, the trophozoites were char- ulation~for commercial purposes, since its size and acterized by the presence of a euchromatic nucleus, growth rate are greater than those of T decussatus. positioned eccentrically, which contained a large Although these venerid species are phenotypically nucleolus; in addition, the vacuolar compartment was similar, they constitute 2 distinct entities with a repro- constituted by a voluminous vacuole and some smaller ductive mating barrier (Gerard 1978), striking kary- elements placed In the peripheral cytoplasm (Figs. 13, ological differences and high genetic distance (Borsa & 14 & 15).Furthermore, mitochondria with an expanded Thiriot-Quievreux 1990). matrix, cisternae of endoplasmic reticulum, polysomes, Azevedo (1989) described the trophozoltes para- an endomembranous network and lipid droplets were sitizing Tapes decussatns from Portugal as a new spe- recognized. Trophozoites were surrounded by a homo- cies of the genus Perkinsus, P. atlanticus. This differs geneous thln wall (Fig. 13). from the other known species, P. marinr~s(Mackin et Trophozoites were frequently enclosed by a capsule al. 1950), P. olseni (Lester & Davis 1981) and P, (Figs. 13, 14 & 16) which originated from the holocrlne karlssonl (McGladdery et al. 1991), in zoospore ultra- secretion of the infiltrated granulocytes. As a conse- structure, host identity and host response. The struc- quence of this, the outermost regions of the capsule tural characteristics of the trophozoites and defensive were characterized by the presence of unfused gran- responses noted in T decussatus and T. semidecussa- ules and remnants of granulocyte membranes (Fig. 14). tus (Montes et al. 1995a) from the Spanish Mediter- On the other hand, the innermost regions of the cap- ranean coast indicate that this paraslte is homologous sule showed an amorphous content with remnants of to P. atlantic~~s. granule membranes and, frequently, paracrystalline In Perkinsus sp.-parasitized Tapes decussatus from inclusions (Fig. 16). Finally, dead trophozoites were Portugal, Comps & Chagot (1987) and Chagot et al. observed in advanced stages of encapsulation (1987) reported an uncommon host reaction In compar- (Fig 16). The capsule and the trophozoite wall were ison with those described in other molluscs against labeled by the serum (Figs. 13, 14 & 16).The LD for the infectious agents. The host reaction consisted of an capsule was similar to that observed in the granules of inflammatory response by infiltrated granulocytes wlth both species (Table l). Moreover, the LD obtained for PAS-positive granules, in which the trophozoites were Tapes semidecussatus was higher than that obtained frequently encysted by a PAS-positive substance. for T. decussatus (Table 1). Recently, we have described a similar host reactlon in Finally, trophozoites were observed, in some in- T semidecussatus from the Spanish Mediterranean stances, surrounded by a dense matrix lined by the coast. The Manila clam reacts against paras~tismby plasma membrane of the granulocytes (Fig. 15). This Perkinsus sp. trophozoites by recruiting blood granulo- matrix contained round cisternae of endoplasmic retic- cytes, which redifferentiate and constitute a cellular Montes et al.: Tapes decussatus parasitized by Perkinsus sp. 155

Figs. 13 to 16. Tapes decussatus parasitized by Perkinsus sp. Immunolocalization of trophozoite-associated structures that react with the serum against p225. Fig. 13. Labeling in the capsule that surrounds a Perkinsus sp. trophozoite (tr).The internal mem- branes of the capsule (c) are devoid of label. Amount of label in the trophozoite wall (W) is low but significant. ~20000.Scale bar = 1 pm. Fig. 14. Labeled nnfused granules in the outermost region of the capsule (c),adjacent to the trophozoite wall (W). ~23000. Scale bar = 0.5 pmFig. 15. Trophozoite in close contact with a granulocyte that shows gelated cytoplasm and that contains labeled residual bodies [asterisks) and remnants of rough endoplasmic reticulum (er). Trophozoite wall (W) was unlabeled. ~23000.Scale bar = 0.5 pm Fig. 16. Trophozoites at several stages of disorganization, surrounded by a labeled capsule (c) that shows internal membranes and paracrystalline structures. X 13000. Scale bar = 1 pm reaction (Montes et al. 1995a). These infiltrated cells secretion IS exclusively located around the tropho- synthesize and secrete their main product, a slightly zoites. glycosylated polypeptide of about 225 kDa (p225), The cellular reaction of Tapes decussatus against which encapsulates the parasites (Montes et al. 1995b). Perklnsus sp. trophozoites showed the same character- The cellular reaction is functionally polarized and its istics described for T.semidecussatus (Montes et al. 156 Dis Aquat Org 26: 149-157. 1996

1995a). The reactive cells are infiltrated granulocytes trophozoites, which are encapsulated by the holocrine that redifferentiate and synthesize a secretory product, secretion of the granulocytes. This encapsulation could stored in membrane-bound granules, which encapsu- block trophozoite dissem~nation (Rodriguez & Navas lates the parasites. However, the cellular reaction of T. 1992, 1995, Montes et al. 1995a), even though this cel- semidecussatus was organized into individualized lular reaction could obliterate the blood sinuses of the areas separated by connective tissue, consisting of spe- clam and, thus, be the eventual cause of the host death cific cells (spindle-shaped connective granulocytes) (Montes et al. 1995a). and intermingled fibres, whereas in T, decussatus, the In conclusion, we demonstrate for the first time 2 cellular reaction was formed by a single cell mass, polypeptides specifically related to clam defense without elements from the connective tissue. against infection by Perkinsus sp. These 2 polypep- Infiltrated granulocytes of Tapes decussatus showed tides are not only closely related at the molecular and a considerable set of endomembranes, such as large the functional level, but are also a specific product of mitochondria and round cisternae of the endoplasmic the reactive connective tissue. reticulum, that were not seen in the Manila clam. Granules from the butterfish clam did not show the parallel arrays of the internal membranes that are Ac~nowiea'gemenls.We are grateiui ro MS Merce santmart~ (Direccio General de Pesca Maritima, Generalitat of Catalo- characteristic in T. semidecussatus. In some cases, nia, Spain) for providing specimens for this study, to MS granulocyte granules from T. decussatus showed early Almudena Garcia and the staff of Serveis Cientifico-Tecnics fusion, giving rise to a single, giant, secretory granule. (Universitat de Barcelona) for technical assistance and to Mr This fusion was concurrent with the loss of water, Robin Rycroft for linguistic advice. This work was supported which led to the gelation of the cytoplasm. in part by a grant from the lnstitut dfEstudis Catalans. The organization of the capsule around the tropho- zoites showed the same characteristics in both clams. LITERATURE CITED However, those from Tapes decussatus were some- times incomplete, and the parasites were only partially Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1994) The immune system. In: Molecular biology of the encapsulated. It is interesting to point out that the per- cell. Garland Publishing Inc, New York, p 1196-1254 centage of dead trophozoites relative to total encapsu- Azevedo C (1989) Fine structure of Perkinsus atlanticus n. sp. lated parasites was lower for T, semidecussatus than (Apicomplexa, Perkinsea] parasite of the clam Ruditapes for T. decussatus (11 and 36% respectively; data not decussatus from Portugal. J Parasitol 75(4):627-635 shown). These data are consistent with the results Bachere E, Mialhe E,Noel D, Boulo V, Morvan A, Rodriguez J (1995) Knowledge and research prospects in marine obtained by Rodriguez et al. (1994), who stated that mollusc and crustacean immunology. Aquaculture 132: Perkinsus atlanticus spreads more easily into 7 semi- 17-32 decussatus than T. decussatus after infection by Borsa P, Thiriot-Quievreux C (1990) Karyological and allo- zoospores. zymic characterization of Ruditapes phllrppinarum, R. aureus and R. decussatus (Bivalvia, Veneridae). Aquacul- The p225 from Tapes semidecussatus and the poly- ture 90:209-227 peptide from 7. decussatus exhibit strong homology. Carlemalm E, Garavito RM, Villiger W (1982) Resin develop- First, both polypeptides have a similar apparent mole- ment for electron microscopy and an analysis of embed- cular mass, and that from T. decussatus cross-reacts ding at low temperature. J I\4icrosc 126:123-143 with the serum against p225 from T. semidecussatus. Chagot D, Comps M, Boulo V, Ruano F, Grizel H (1987) Hlsto- logical study of a cellular reaction in Ruditapes decussatus Second, at the cellular level, both are synthesized by infected by a protozoan. Aquaculture 67~260-261 infiltrated granulocytes and show the same pattern of Chintala MM, Ford SE, Fisher WS, Ashton-Alcox KA (1994) synthesis, storage and secretion. Third, at the physio- Oyster serum agglutinins and resistance to protozoan par- logical level, both synthesis products are organized as asites. J Shellfish Res 13(1):115-121 Comps M, Chagot D (1987) Une parasitose nouvclle chez la a capsule around the trophozoites. Finally, the non- palourde Ruditapes decussatus L. CR Acad Sci Paris (111) parasitized specimens were devoid of the respective 304(1):41-44 polypeptides (Montes et al. 1995b). Da Ros L, Canzonier WJ (1985)Perkinsus, a protistan threat to Moreover, regardless of the nature and mechanism bivalve culture in the Mediterranean basin. Bull Eur Ass of the clam infection by Perkinsus sp., these results Fish Pathol5(2):23-25 Figueras A, Robledo JAF, Novoa B (1992) Occurrence of allow us to speculate about the organization of the cel- haplosporidian and Perkinsus-like infections in carpet- lular reaction and the encapsulation of the trophozoites shell clams, Ruditapes decussatus (Linnaeus, 1758), of the (Montes et al. 1995a). Once the parasites have reached Ria de Vigo (Galicia, NW Spain). J Shellfish Res ll(2): the connective tissue of these clam species, hemocytes 377-382 Gerard A (1978) Recherches sur la variabilite de diverses pop- are recruited. Under parasite induction these hemo- ulation~ de Ruditapes decussatus et Ruditapes philip- cytes redifferentiate, giving rise to secretory granulo- pinarum (Veneridae, Bivalvia). PhD thesis, University of cytes. These constitute the cellular reaction around the Bretagne Occidentale, Brest Montes et al.: Tapes decussatus parasltized by Perk~nsussp. 157

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Responsible Subject Ed~tor.A. K. Sparks, Seattle, Manuscript first received: June 6, 1995 Washington, USA Rev~sedvelsion accepted: January 30, 1996