Histopathology of Gymnophallus sp. sporocysts in the edible mytilid, Modiolus barbatus.

ABSTRACT Infection of Gymnophallus sp. in the bearded horse mussel (Modiolus barbatus) was studied in samples collected during 1 year in the Mall Ston Bay (eastern Adriatic Sea). Mean prevalence of sporocyst was low (3.5%), which peaked in February (7.1%), whereas the mean metacercariae prevalence was 8.1%, peaking in September (16.7%). Histologically the Gymnophallus sp. sporocyst infection in bearded horse mussel resulted in clear reaction of bivalve tissue, where the induced changes depend strongly on the site of trematode infection. Retardation of gametogenesis, necrosis of connective tissue and hemocytic infiltration are the main histological features of infection in this new host.

KEY WORDS: gymnophallus, histopathology, Modiolus barbatus, sporocyst, metacercaria

INTRODUCTION

Parasites may affect host populations by using a part of its resources, causing costly immune responses and decrease in host reproduction or by affecting the behavior of its paratenic host (Blanchet et al. 2003), thus accelerating the voyage to its final destination and at the end inducing mortality (Holopainen et al. 1997).

Family Gymnophallidae (Odhner 1905) comprises a small group of digeneans that live in the intestine, gall-bladder and bursa Fabricii of Charadriiformes (shore birds) and Anseriformes (diving ducks) that feed primarily on intertidal lamellibranchs, gastropods and benthic polychaetes (Nolso 2002). The exceptions are 2 taxa, Gymnophalloides seoi and G. heardi, found parasitizing the digestive system of mammals, including humans (Chai et al. 2001). A remarkable feature of family Gymnophallidae consists in the flexibility of its life style. Natural selection has favored abbreviation or truncation of the parasite life cycle, so that for certain genera of Gymnophallidae, the mollusc represents the first and the second intermediate host (Paulin & Cribb 2002). However their life cycle diversity is reflected in the capability of assuming one of the following strategies: gymnophallids that involve bivalves as first and second intermediate hosts; copepods as the first and bivalves and polychaetes as second intermediate hosts or bivalves as the only hosts. In rare cases gastropods or branchipods are also suitable hosts (Scholz 2002).

Because M. barbatus (Linneus, 1758) is a commercially valuable bivalve in the Adriatic, and as such is a possible candidate for aquaculture, it is important to evaluate potential pathologic effects of Gymnophallus sp. on this species. This is the first record of Gymnophallus sp. in the bearded horse mussel and is also a new geographical distribution for this parasitic genus.

MATERIALS AND METHODS

Bivalve samples were collected by SCUBA diver in the Mali Ston Bay, eastern Adriatic Sea, from January 2004 to January 2005. Specimens were transported live to the laboratory and left for seven days in a flow-through system. The samples comprised of 30-40 individuals monthly (total n = 396).

Before the parasitologic examinations, mussels were left for an hour in 7% Mg[Cl.sub.2] to relax adductor muscles. Then they were dissected under a dissecting lens and the tissue was pressed between two compression glasses and observed under the microscope at magnifications of x20 and x100. Sporocysts and metacercariae were counted in both mantles.

Tissues containing mantle, ctenidia, labial palps and digestive system infected with sporocysts was immediately fixed in Davidson fixative prepared with seawater. Standard histological procedure was performed to embed tissue in paraffin blocks, after the alcohol dehydration series. The blocks were cut at 5 [micro]m. Sections were stained with Mayer hematoxylin and counterstained with eosin. Slides were examined using a compound microscope at x200, x400 and x1,000 magnification.

Prevalence and abundance were calculated following Bush et al. (1997). Abundance was calculated as the number of Gymnophallus metacercaria divided by the number of bearded horse mussels sampled.

RESULTS

Gymnophallidae sporocysts were noticeable as bright red filiform masses throughout the gonads and mantle. They were sausage-like, with a prominent anterior opening and characteristic reddish granules. Sporocyst prevalence was low, with mean value of 3.5% and a peak in February of 7.1% (Fig. 1). Metacercarial prevalence did not follow the pattern of sporocyst prevalence, peaking in September (16.7%), with an annual mean value of 8.1%. The mean abundance of metacercariae was only 0.09. It was impossible to count all mother sporocysts, thus their abundance was not calculated. Complete depletion of both sporocysts and metacercariae, when their prevalence reached zero, occur in July during the peak of M. barbatus spawning.

[FIGURE 1 OMITTED]

The tissue reaction of bivalves infected with sporocysts differed, primarily depending on the infection site. The most common site was the connective tissue around the digestive gland and gonads, but sporocysts could also be isolated from the intrapallial part of the mantle and isthmus. In the vicinity of the digestive gland, flattened layers of the connective tissue cells were present around the sporocysts. These layers were rich in cellular elements and infiltrated by hemocytes, but with scarce collagen fibers; thus no real encapsulation by the host tissue occurred (Fig. 2). When sporocysts were localized near gonadal tissue, a considerable retardation in oocytes development was noticed (Fig. 3). In these cases, oocytes were small and embedded in thick connective tissue. They were in the first stage of oogenesis, whereas non-infected gonads already had developed fully mature oocytes (Fig. 4). However, no degenerative or necrotic changes were noticeable in gametes, even when the retardation of physiologic gametogenesis was evident. Regardless, of the infection site, the hemocyte infiltration was always present and observed in all infected tissues. Hematocytic infiltration was accompanied by hypertrophic and hyperplastic changes of the connective tissue (Fig. 5).

[FIGURES 2-5 OMITTED]

In cases of severe infections, fragmentation of the host tissue occurred presumably by mechanical destruction caused by sporocysts movements that left clumps of hypertrophied cells, with small and decentralized nuclei. Debris from necrotic cells along with few hemocytes was present on the edges of fragmented tissue. The presence of sporocysts in the intrapallial or isthmatic part of the mantle induced the mildest changes in the bivalve tissue--hematocytic infiltration with hypertrophy and hyperplasia of connective tissue cells.

Because of low abundance of unencysted metacercaria in the host (0.09), no significant pathologic effect was observed.

DISCUSSION

Gymnophallid infections are described in bivalves from Baltic, North Sea, East Atlantic coast as well as from Mediterranean (Lauckner 1984, Russell-Pinto 1990, Russell-Pinto & Bartoli 1992, Russell-Pinto & Bowers 1998, Montandouin et al. 2000). This is the first record of Gymnophallus sp. in the Adriatic Sea. Further, M. barbatus represents a new host for this trematode. The parasite was previously considered as oioxenic, using specifically cockles, mainly Cerastoderma edule, C. glaucum (Lauckner 1971) and the clam Tapes philippinarum as hosts (Montaudouin et al. 2000). However, in our study the trematode did not reach the prevalences in M. barbatus population, as are found in cockles. The plotted values of prevalences of sporocysts and metacercariae in Figure 1 show that the temporal distribution of the trematode over 1 year had opposing patterns. Metacercariae clearly displayed two peaks and two declines in prevalence over 12 months, with lowest values in spring and autumn, and with highest values in April and September. At the same time, monthly prevalences of sporocyst were on average 2-fold lower. The increase of sporocysts prevalence was mirrored in metacercariae decrease and vice versa, meaning that sporocysts regress after generating a new metacercarial population. This is best illustrated during the winter months when sporocyst had maximum values from December to February, and when metacercariae number collapsed. The decreases in prevalence of sporocysts and metacercariae occurred when host gametes were released (Mladineo et al. in prep), suggesting that the digenean assisted its own spread to the spread of a new bivalve generation.

Trematodes have different strategies for maintenance and spread in the host population. Gymnophallus mother sporocysts harbor large numbers of different developmental stages, from germ balls to cercariae, assuring continuous and asynchronous cercarial production and enabling constant release of infective stages and reinfection. Cercariae are also equipped with a long bifurcated tail that enables transmission of neighboring bivalves. In spite of these adaptations for dispersion in the environment, the prevalence and abundance of the trematode in M. barbatus still remained low.

Histological changes induced by the parasite are evident but not devastating, perhaps reflecting the low abundance of the parasite in individual mussels. The negative effect of trematodes on host reproduction has been observed in many cases (Pekkarinen 1987, Taskinen et al. 1994, Taskinen & Valtonen 1995, Taskinen et al. 1997, Khamdan 1998, Taskinen 1998, Da Silva et al. 2002). Gonadal infections can even induce changes in the reproductive strategy of the bivalve, as in Pisidium amnicum where higher mortality and castration are compensated for higher reproductive effort and semelparity (Holopainen et al. 1997, Rantanen et al. 1998). However, in the bearded horse mussel no excessive degenerative or necrotic changes were noticeable in gametes, even when the suppression of physiologic gametogenesis was evident. Oocytes were viable, showing only a certain degree of retardation compared with oocytes of noninfected bivalves. Because of the low incidence of sporocysts in the host, this reproductive inhibition had no drastic impact on the overall bivalve population. The presence of sporocysts in the intrapallial or isthmatic part of the mantle induces the mildest changes, resulting in hematocytic infiltration with hypertrophic and hyperplastic connective tissue cells. Hemocytes are implicated in the processes of regeneration of soft tissue (Ruddell 1971) and can adopt diverse defense strategies from phagocytosis of foreign agents to production of lectins and secretion of enzymes (Chagot et al. 1992). Therefore hemocyte presence clearly indicated the invasiveness and pathogenicity of Gymnophallus sp. Similar changes were found by Robledo et al. (1994) in the mantle of mussel parasitized by sporocysts of Proctoeces maculatus. Only in severe infections, tissue fragmentation was present. On the surface of sporocyst tegumentum of 2 other members of Gymnophallidae--Lacunovermis macomae and Meiogymnophallus minutes, hydrolytic enzymes that are involved in sporocysts' metabolism, such as phosphatases and [beta]-glucuronidase, were suggested to play an important role in tissue deterioration (Pekkarinen 1987, Russell-Pinto et al. 1996), so this activity along with mechanical destructions could be the cause of tissue necrosis in severe infections.

Mantle pathologies in bivalves are mainly described in cases of free metacercariae infection in extrapallila cavity, where metaplasia and hyperplasia are common tissue reactions, with resulting lysis of mantle epithelium. These changes were observed only in relation to Gymnophallus sporocysts, and not metacercariae, which didn't induce any histological changes in the invaded tissue. However, metacercariae of other genera are able to induce other more invasive changes. For example, in the burrowing sand clam Darina solenoids, the mantle envelops metacercariae of Bartolius pierrei and initiates the secretion of a noncellular hyaline capsule (Cremonte & Ituarte 2003). If the process takes place at the inner surface of the shell, it can end up in formation of igloo-like calcareous pits (Ituarte et al. 2001) or nacrezation (Cheng 1967, Cheng & Rifkin 1970). Mechanical impairments can also result from metacercarial infection. In Cerastoderma edule and C. glaucum, Meiogymnophallus fossarum metacercariae compromise normal production of shell and ligament proteins as the large number of parasites damage the periostracum, inducing its excessive growth that results in impaired closure of shell valves (Bartoli 1973). M. minutus parasitizes the hinge area, where it induces excessive proliferation of ligament protein resulting in tissue folding and again impaired closure of shell valves (Bowers et al. 1996). In comparison with these changes, sporocysts in bearded horse mussel mantle tissue evoked only a mild tissue reaction, which did not appear to limit the host reproductive capability, a consequence that would also limit digenean spread. Instead, only mild hemocytes infiltration and cell hypertrophy permitted regular and normal development of the parasite inside the bivalve. Further, the low prevalence and abundance of the parasite in the mussel population have also shown not to be significant limiting factors for the future use in aquaculture. Nevertheless, the trematode imposes a certain degree of tissue alteration in the host, which deserves the research of gymnophallidae-host relationship during a longer time scale.

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IVONA MLADINEO * AND MELITA PEHARDA

Institute of Oceanography and Fisheries, PO Box 500, 21000 Split, Croatia

* Corresponding author. E-mail: [e-mail]