Parasitism by a Protozoan in the Hemolymph of the Giant Clam,Tridacna crocea

JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.

71, 193–198 (1998)

IN974747

Parasitism by a Protozoan in the Hemolymph of the Giant Clam, Tridacna crocea Koji Nakayama,1 Miyuki Nishijima, and Tadashi Maruyama1 Marine Biotechnology Institute, Shimizu Laboratories, 1900 Sodeshi, Shimizu, Shizuoka 424, Japan Received July 14, 1997; accepted December 10, 1997

A parasitism by a protozoan was found in the giant clam, Tridacna crocea. The parasites were spindleshaped, 8.6 6 0.5 mm in length and 2.5 6 0.3 mm in width. Structural features of the apical complex of the parasite and a molecular phylogenetic analysis of its 18S rRNA gene sequence indicate that the protozoan belongs to the Apicomplexa. No flagellum was observed in the parasitic protozoan. It infected the eosinophilic granular hemocyte, one of the three types of hemocytes in the clam hemolymph, but it is not known whether it influenced the growth of the clam. r 1998 Academic Press

Key Words: infection; Tridacna crocea; hemocyte; Apicomplexa; protozoan.

INTRODUCTION

Recently, giant clams are grown by mariculture in various tropical and subtropical countries in the IndoPacific Ocean region. They are able to grow to a large size and provide a valuable source of food protein. Giant clams harbor photosynthetic zooxanthellae, symbiotic microalgae, in siphonal mantle (Goreau et al., 1973; Norton et al., 1992). While much attention has been focused on the symbiotic relationship between the host clam and its zooxanthellae (Trench et al., 1981), reproduction (Braley, 1984), and mariculture techniques (Heslinga et al., 1984), little is known about parasitism in giant clams. A trematode, Bucephalus sp., infects the gonad, kidney, digestive gland, and gill of Tridacna crocea on the Great Barrier Reef (Shelley et al., 1988). A flagellate apicomplexan, Perkinsus sp., has been observed in T. maxima at Lizard Island, North Queensland, Australia (Perkins, 1985, 1988), and in Tridacnidae clams on the Great Barrier Reef (Goggin and Lester, 1987). Translocation of giant clams for mariculture possibly mediates the transmission of infec-

tious pathogens including parasites from one location to another. A parasitism by a protozoan was found in the hemolymph of T. crocea during the investigation of its hemocytes (Nakayama et al., 1997). The parasitic protozoan has been characterized by light and transmission electron microscopy, and its small-subunit ribosomal RNA (18S rRNA) gene has been sequenced. MATERIALS AND METHODS

Animals Tridacna crocea (8–10 cm shell-length) collected at Nago Bay, Okinawa, Japan, was purchased from a local supplier and was maintained in filtered seawater in a shallow aquarium in a green house at 25°C for up to 3 months until use. Hemolymph Collection The clams were chilled on ice for 30 min before use in order to prevent rapid aggregation of hemocytes. Hemolymph was withdrawn from the pericardial chamber with a 10-ml syringe and a 22-gauge needle. Cytochemical Staining Methods Hemocytes were fixed with a mixture of 2% glutaraldehyde and 1.5% formalin for 18 h at 4°C. After the hemocytes and parasites obtained by filtration (Cytoshuttle, Cancer Diagnostics Inc., VA) were transferred to a glass slide, they were stained by the May– Gru¨nwald–Giemsa method. For visualization of acid phosphatase activity by the method of Burstone (1958) using naphthol AS-BI phosphate as the substrate, the hemocytes was fixed with 90% methanol plus 10% formalin after incubation in a 2-well type chamber slide (Nunc Co., IL) for 1 h at 25°C. Transmission Electron Microscopy

1 Current address: Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi, Iwate 026, Japan.

For transmission electron microscopy (TEM), hemocytes, and parasites were first fixed at 4°C with 2.5%

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glutaraldehyde in artificial Pacific seawater (APSW) (Borowitzka and Larkum, 1976). The fixed hemocytes were then postfixed with 1% OsO4 in 50% APSW, dehydrated through a graded ethanol series, and embedded in Epon 812 resin (TAAB Co., Berkshire, UK). Thin sections were cut with an ultramicrotome (Ultracut N, Reichert-Nissei, Tokyo, Japan) using a diamond knife. Silver sections were counterstained with saturated aqueous uranyl acetate and Reynold’s lead citrate and then observed in a Hitachi H-7000 electron microscope operated at 75 kV. 18S rRNA Analysis In order to lyse the hemocytes to release the parasites, infected hemocytes collected from two giant clams were mixed and kept for 24 h at 25°C. Genomic DNA was isolated from a mixture of hemocytes and parasites by a QIAamp tissue kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instruction. The 18S rRNA gene sequence of the parasite was amplified by a LA-PCR kit (Takara Shuzo, Kyoto, Japan) with universal eukaryotic primers, ss5 primer (58-GGTTGATCCTGCCAGTAGTCATATGCTTG-38) and ss3 primer (58-GATCCTTCCGCAGGTTCACCTACGGAAACC-38) (Herzog and Maroteaux, 1986; Rowan and Powers, 1992), and ligated into pCR-SCRIPT (Stratagene, CA). Three independent clones were sequenced in both directions. The nucleotide sequences were determined by using an Applied Biosystem 373A DNA sequencer with a Taq DyeDeoxy terminator cycle sequencing kit (Perkin–Elmer, CA). Nucleotide sequences were examined by the GENETYX system (Software Development Co., Tokyo, Japan) and a nucleotide sequence homology search was performed using the FASTA program (Pearson and Lipman, 1988) through the WWW homepage of DDBJ, the DNA Database Bank of Japan (Shizuoka, Japan). A phylogenetic tree was constructed by distance matrix methods using the programs DNADIST, NEIGHBOR, CONSENSE in PHYLIP Version 3.5c (Felsenstein, 1989) from sequence data aligned by Clustal W Version 1.6 (Thompson et al., 1994) and gap-excluded. The phylogenetic tree was drawn by TreeView version 1.4 (Page, 1996). Bootstrap resampling was used to quantify relative support for branches. RESULTS

Light Microscopy A parasite was found in the hemolymph of the giant clam, Tridacna crocea and found in 77 clams of 99

tested clams. The parasites were spindle-shaped (Fig. 1). The fresh parasites were 8.6 6 0.5 µm in length and 2.5 6 0.3 µm in width (mean 6 SD, n 5 19). Three or four parasites were usually found in each infected hemocyte. It moved by a wavy motion, although no flagellum was seen. A complex structure, dark under phase-contrast optics, was observed near the anterior end, and there were also small granules which were stained purplish red by the May–Gru¨nwald–Giemsa method (Fig. 2). Acid phosphatase activity was detected in the granules of hemocytes with parasites but not on the parasite (Fig. 3). When the withdrawn hemolymph was incubated in a slide chamber for 1 day, the parasites burst out of the infected hemocytes. Transmission Electron Microscopy Observation In longitudinal sections of parasites in hemocytes, micronemes could be seen in the apical complex as well as electron-lucent granules (Fig. 4). The nucleus was situated near the posterior end. The surface of the parasitic protozoan was uneven and in some areas undulant. The apical complex was constructed from conoid, micronemes, and rhoptries (Fig. 5). Small electron-dense granules were often observed in the cytoplasm of the infected hemocytes. In the hemocytes, the parasite cells were not enclosed by a hemocyte cell membrane. 18S rRNA Gene Sequence Analysis Two 18S rRNA gene sequences were amplified with ss5 and ss3 primers and sequenced. One was the 18S rRNA gene sequence of T. crocea and the other was thought to be that of the parasitic protozoan. The length of amplified 18S rRNA gene sequence of the parasite including the primers was 1786 bp. The G/C content of this sequence was 44% (A 5 490, C 5 336, G 5 453, and U 5 507). The nucleotide sequence data was deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under Accession No. AB000912. A homology search by the FASTA program in DDBJ showed that this is most closely related to 16S-like ribosomal RNA gene of Sarcocystis muris (Apicomplexa: Coccidiasina) (Gajaghar et al., 1991), with an 83.3% identity. No sequence having more than a 90% identity was found in the database. Further, the 18S rRNA gene sequence of the parasitic protozoan was compared with other 18S rRNA gene sequences by a homology search program (Lipman and Pearson, 1985) in GENETYX and found to be more than 80% identical with those of dinoflagellates including a zooxanthella

FIG. 1. A live parasite viewed with phase-contrast optics. p, parasite; eg, eosinophilic granular hemocyte. Scale bar, 10 µm. FIG. 2. Light microscopy of the parasite. The parasites were stained by the May-Gru¨nwald-Giemsa method. p, parasite; eg, eosinophilic granular hemocyte; ac, agranular cell; mc, morula-like cell. Scale bar, 10 µm. FIG. 3. Light microscopy of the infected hemocyte, stained by a method for acid phosphatase activity and counterstained by haematoxylin. p, parasite; eg, eosinophilic granular hemocyte. Scale bar, 10 µm.

PARASITISM IN THE GIANT CLAM

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FIG. 4. Transmission electron micrograph of the infected hemocyte. ne, nucleus of the eosinophilic granular hemocyte; np, nucleus of the parasite; mi, microneme; lp, small electron-lucent granule of the parasite. Scale bar, 1 µm.

FIG. 5. Transmission electron micrograph of the apical complex in the parasite. c, conoid; mi, microneme; rh, rhoptry. Scale bar, 0.5 µm.

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TABLE 1 Percentage of Identities among 18S rRNA Gene Sequences Apicomplexa Coccidiasina

The parasite in Tridacna crocea Sarcocystis muris Toxoplasma gondhii Eimeria tennera Cytauxzoon felis Babesia rodhaini Theileria annulata Perkinsus marinus Zooxanthella Prorocentrum micans Tetrahymena corlissi Anophyroides haemophila

Piroplasmasina

S. mu

T. go

E. te

C. fe

B. ro

T. an

85.1

84.8 95.6

81.9 87.1 87.5

84.1 85.2 85.2 82.3

80.8 80.5 80.3 78.3 88.8

80.0 74.7 80.4 77.6 91.6 89.3

Perkinsasida P. ma 82.2 83.7 84.9 81.8 82.7 77.9 69.4

Dinoflagellida

Ciliophora

Zx

P. mi

T. co

A. ha

84.2 86.2 86.5 83.4 83.9 79.8 80.8 86.7

84.9 87.1 87.2 84.5 84.9 80.5 80.7 87.7 93.6

75.7 76.0 76.7 73.7 75.7 74.6 76.1 76.6 76.1 76.6

79.4 81.7 81.5 78.8 80.6 79.7 81.2 81.6 81.8 82.3 83.1

Mollusca T. cr 71.7 72.0 69.8 72.1 69.4 63.8 62.8 72.0 71.7 71.0 66.4 70.6

Note. Homology searches were done by the Lipman–Pearson method with GENETYX-MAC program. 18S rRNA gene sequence of zooxanthella was determined with zooxanthellae isolated from the giant clam T. gigas (Accession No. AB004828). Abbreviations: S. mu, Sarcocystis muris (M64244, M34846); T. go, Toxoplasma gondhii (M97703); E. te, Eimeria tennera (U67121); C. fe, Cytauxzoon felis (L19080); B. ro, Babesia rodhaini (M87565); T. an, Theileria annulata (M64243, M34845); P. ma, Parkinsus marinus (X75762); Zx, zooxanthella; P. mi, Prorocentrum micans (M14649); T. co, Tetrahymena corlissi (U17356); A. ha, Anophyroides haemophila (U51554); T. cr, Tridacna crocea (D88908).

isolated from T. gigas as well as those of apicomplexans (Table 1). For phylogenetic analysis of the 18S rRNA gene sequences, multiple alignments with the 11 taxa provided 1593 phylogenetically informative sites. While the result of our analysis indicated that the parasitic protozoan was closely related to the apicomplexans of the suborder Eimeriorina (genera Eimeria, Sarcocystis, and Toxoplasma) and Piroplasmorida (genera Babesia, Cytauxzoon, and Theilelia), the parasitic protozoan was not monophyletic with them (Fig. 6). The suborder Eimeriorina and Piroplasmorida formed a clade, respectively. The dinoflagellates, Prorocentrum micans and

FIG. 6. Phylogenetic tree constructed from distance matrix analysis of 18S rRNA sequences. The numbers are percentages of bootstrap possibilities from 1000 cycles replications.

zooxanthella were supported as a clade, with Perkinsus marinus as its sister taxon. DISCUSSION

A parasitism by a protozoan was found in the hemolymph and hemocytes of Tridacna crocea. The apical complex at the anterior position of the protozoan in a hemocyte indicates that it belongs to Apicomplexa (Levine, 1988). The presence of acid phosphatase activity in the granule of the infected hemocyte indicates that the parasitic protozoan invaded the eosinophilic granular hemocytes, since it was detected only in this type of hemocyte (Nakayama et al., 1997). The presence of small electron-dense granules in the infected hemocyte also indicated that the parasites infected this type of hemocytes. Although these eosinophilic granular hemocytes are phagocytic, the parasites were not in the phagosomes because of the absence of a cell membrane surrounding them and the absence of an acid phosphatase activity around them. A parasite belonging to the genus Perkinsus, with an apical complex consisting of an open-sided conoid (Perkins, 1976, 1988), was found in Tridacnidae clams on the Great Barrier Reef (Goggin and Lester, 1987), but the closed conoid of the parasitic protozoan in T. crocea indicates that it is distinct from Perkinsus. The life cycle of this parasitic protozoan remains obscure. It appears to have little influence on the growth of the giant clams, because the infected clams seemed to be healthy for at least several months in our aquarium. The homology search with the 18S rRNA gene se-

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quence of the parasitic protozoan also indicates that it belongs to Apicomplexa. It is reasonable that 18S rRNA gene sequences of apicomplexans should be similar to those of zooxanthellae, because they are reported to be evolutionarily related to dinoflagellates (Gajadhar et al., 1991; Goggin and Barker, 1993; Flores et al., 1996). The homology comparison also shows that the parasitic protozoan in this study is distinct from Perkinsus marinus (82.2% identical) and Perkinsus sp. (83.1% identical). The molecular phylogenetic analysis shows that the parasitic protozoan formed a monophyletic group with the other euapicomplexans but it deeply branched from the others. While the parasitic protozoan may be closer to a gregarine apicomplexan, no 18S rRNA gene sequences of gregarine apicomplexan is now available to compare. ACKNOWLEDGMENTS We are grateful to Dr. A. Miyata of the Oita Medical University (Oita, Japan) and to Dr. K. Nagasawa of the National Research Institute of Far Seas Fisheries (Shizuoka, Japan) for useful discussions. Ms. S. Suzuki is acknowledged for DNA sequence determinations. This work was performed as a part of the Industrial Science and Technology Frontier Program supported by the New Energy and Industrial Technology Development Organization (Tokyo, Japan). REFERENCES Borowitzka, M. A., and Larkum, A. W. D. 1976. Calcification in the green alga Halimeda. II. The exchange of Ca21 and the occurrence of age gradients in calcification and photosynthesis. J. Exp. Bot. 27, 864–878. Braley, R. D. 1984. Reproduction in the giant clams Tridacna gigas and T. derasa in situ on the North-Central Great Barrier Reef and Papua New Guinea. Coral Reefs 3, 221–227. Burstone, M. S. 1958. Histochemical comparison of naphthol ASphosphates for the demonstration of phosphatases. J. Nat. Cancer Inst. 20, 601–615. Felsenstein, J. 1989. PHYLIP Phylogeny inference package (Version 3.2). Cladistics 5, 164–166. Flores, B. S., Siddall, M. E., and Burreson, E. M. 1996. Phylogeny of the Haplosporidia (Eukaryota: Alveolata) based on small subunit ribosomal RNA gene sequence. J. Parasitol. 82, 616–623. Gajadhar, A. A., Marquardt, W. C., Hall, R., Gunderson, J., AriztiaCarmona, E. V., and Sogin, M. L. 1991. Ribosomal RNA sequences of Sarcocystis muris, Theileria annulata and Crypthecodinium cohnii reveal evolutionary relationships among apicomplexans, dinoflagellates, and ciliates. Mol. Biochem. Parasitol. 45, 147–154. Goggin, C. L., and Lester, R. J. G. 1987. Occurrence of Perkinsus species (Protozoa, Apicomplexa) in bivalves from the Great Barrier Reef. Diseases in Aqua. Org. 3, 113–117.

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