J. Eukarvot. Microbiol.. 51(2), 2004 pp. 214219 0 2004 by the Scciety of Protozoologists

Nematopsis gigas n. sp. (Apicomplexa), a Parasite of Nerita ascencionis (Gastropoda, Neritidae) from Brazil

CARLOS AZEVEDOa,b, and ISkRAS PADOVAN‘ =Department of Cell Biology, Institute of Biomedical Sciences, University of Oporto, Lg. A. Salazar no. 2, 4099-003 Porto, Portugal

bCIIMAR-Center for Marine and Environmental Research, University of Oporto, Porto, Portugal, and ‘Department of Histology and Embryology, Center of Biological Sciences, Federal University of Pernambuco, Recife, Brazil

ABSTRACT. A new species of Nematopsis (Apicomplexa, Porosporidae) is described from the mantle tissues of the seawater gastro- pod, Nerita ascencionis (Neritidae), collected in the Atlantic North off the coast of “Fernando de Noronha” Island (3” 47’ 57” S, 32” 25’ 12” W) situated about 350 km from the northeast coast of Brazil. Numerous oocysts, each contained in a parasitophorous vacuole, were found in the cytoplasm of phagocytes in the mantle tissue of the host. The phagocytes were surrounded by a thin wall composed of lucent material. The phagocyte cytoplasm contained a nucleus surrounded by numerous vesicles and some dense masses. The oocysts were 21.9 Z 0.5 pm long, and 11.5 2 0.6 pm wide. The oocyst wall was 0.18425 pm thick, and the apical zone contained a micropyle, 1.0-1.2 pm in diameter, covered by a canopy-like operculum about 0.25 p,m thick. Externally, the oocyst wall was surrounded by numerous anastomosing microfibrils attached to the wall and extending towards the periphery of the parasitophorous vacuole. Some microfibrils formed a dense complex network that surrounded the~oocyst in the middle of the parasitophorous vacuole, which opened only at the apical zone near the external region of the opercular system.

On the basis of the data obtained by light and transmission electron microscopy and host specificity, the gregarine Nematopsis gigas is distinguished from the nearest species as a new species. The taxonomic affinities and morphological comparisons with other similar species of the same genus are discussed.

Key Words. Nematopsis gigas, new species, oocyst, phagocyte, sea water, ultrastructure.

N the order Eugregarinida LCger, 1900, the family Porospor- I idae LabbB, 1899, with two genera, Porospora Schneider, 1875 and Nematopsis Schneider, 1892, includes several species that occur in commercially important marine molluscs (Brad- bury 1994; Cheng 1967; Lauckner 1983; Perkins 1991; Sat0 et al. 1996; Schneider 1892; Sprague 1970). Taxonomic differ- entation of these two genera has been in a state of confusion (Desportes, Vivarts and ThBodoridbs 1977; Sprague 1970). Originally considered synonymous, some authors have sup- ported these two genera; the genus Nematopsis has been re- ported to have monozoic oocysts (spores or sporocysts) with a thick wall enclosing a single sporozoite, while the genus Po- rospora has oocysts without a wall (Azevedo and Matos 1999; Desportes, VivarBs, and ThCodoridts 1977; Sprague 1970; Sprague and Orr 1955; ThBodoridbs 1962).

Nematopsis is a heteroxenous gregarine with its vegetative and reproductive phase in crabs, and sporogony in lamelli- branch molluscs (Prytherch 1938, 1940; Sprague 1970; Th60- dorides 1962). Sporogonic stages have been reported rarely in gastropods (Hatt 1927, 1931). In the present paper we describe the ultrastructure of the oocyst of Nematopsis gigas n. sp., a gregarine parasite of a gastropod. This is the first detailed de- scription of the genus Nematopsis from a gastropod, and the first report of a gregarine from “Fernando de Noronha” island near the Brazilian coast.

MATERIALS AND METHODS Twenty-five specimens of Nerita ascencionis (Gastropoda,

Neritidae) were collected in the intertidal Atlantic off the north coast of “Fernando de Noronha” Island (3” 47‘ 57” S, 32“ 25’ 12” W) (Brazil). Small fragments of fresh infected mantle were examined and photographed using Nomarski differential inter- ference-contrast (DIC) optics. Measurements were made di- rectly on living oocysts or from photomicrographs of living oocysts. For transmission electron microscopy (TEM), small fragments of the infected mantle tissues were fixed in 3.0% (v/ v) glutaraldehyde in 0.2 M sodium cacodylate buffer, pH 7.2 for 10-12 h at 4 “C, washed overnight in the same buffer at 4

Corresponding Author: C. Azevedo-Telephone number: + 35 1.22. 206.22.00; Fax number: + 351.22.206.2232/33; E-mail: azevedoc@ icbas.up.pt

“C, and post-fixed in 2.0% osmium tetroxide for 3 h in the same buffer and at the same temperature. After dehydration in a grad- ed ethanol series and propylene oxide (8-10 h in each change), the tissue was embedded in Epon. Semithin sections (about 1 ,um thick) for light microscopy were stained with methylene blue-azur II. Ultrathin sections (50-60 p,m thick) were sec- tioned using a diamond knife, double-stained with uranyl ace- tate and lead citrate, and observed in a JEOL 100 CXII TEM operated at 60 kV.

RESULTS Light microscopy. The parasite, represented by several oo-

cysts, was observed in semithin sections and in squash prepa- rations of the mantle tissues of the gastropod N. ascencionis (Fig. 1-3). Isolated or grouped oocysts were irregularly dis- persed within parasitophorous vacuoles (PV) in the phagocyte (Fig. 1-3). Five of 25 (20%) molluscs contained several para- sitized phagocytes, each containing from 1-7 oocysts (Fig. 1). Each oocyst contained only one sporozoite (Fig. 2, 3), but some

+ Fig. 1-6. Light and transmission electron microscopy of the api-

complexan gregarine, Nematopsis gigas n. sp., a parasite found in the mantle tissue of the gastropod Nerita ascencionis (all scale bars in pm). 1. Semithin section showing some host phagocytes (*) each containing some oocysts (OC) located in a lighter area (parasitophorous vacuole). Between each oocyst and the periphery of the parasitophorous vacuole a dense structure (arrowheads) was observed. The “phagocyte” walls are indicated by double arrows. 2. Six living oocysts (OC) located with- in the phagocytes (*) observed with Nomarski differential interference contrast (DIC) optics. The “phagocyte” wall is indicated by double arrows. 3. Three living oocysts (OC) in the same phagocyte (*) ob- served in DIC. Note the oocyst wall (W) and the enclosed sporozoite (S). 4. Ultrathin longitudinal section of an oocyst. Note the oocyst wall (W), the sporozoite (S) with its nucleus (N), the parasitophorous vacuole (PV), and the dense network of microfibrils (arrowheads) of the PV. 5. Ultrathin section of a transverse section of the oocyst (OC) showing the wall (W) and the sporozoite (S) with its nucleus (N). The parasitopho- rous vacuole (PV) contains numerous radial anastomosing microfibrils. The “phagocyte” wall is thin (double arrows). 6. Ultrathin section showing details of the oocyst wall (W), the complex anastomosing mi- crofibrils located in the parasitophorous vacuole (PV) and the surround- ing dense network of microfibrils (arrowheads).

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fused PVs were observed (Fig. 1) . The oocysts were surrounded by a wall-like structure (Fig. 1). Oocysts were 21.9 2 0.5 pm long and 11.5 i 0.6 pm wide (n = 50) when observed in squash preparations (Fig. 2, 3).

Transmission electron microscopy. Each PV containing a single oocyst (Fig. 4, 5) was formed by a PV matrix in close contact with the cytoplasm of the phagocyte (Fig. 5, 6). The cytoplasm of the phagocyte contained a complex group of ve- sicular structures with different diameters, interspersed with some dense masses (Fig. 6). The matrix of the PV consisted of a complicated network of irregular and anastomosed microfi- brils that projected from the oocyst wall towards the periphery of the PV. The wall of the PV was composed of a dense net- work of aggregated microfibrils, forming a continuous structure (Fig. 6-9). This structure was open at the apical region of the oocyst (Fig. 7-9), and sometime was observed near or in con- tact with the oocyst wall (Fig. 9).

Each oocyst wall contained a single vermiform uninucleated sporozoite accommodated in the oocyst lumen (Fig. 4, 5 ) The oocyst wall was 0.40 pm thick and limited by two homogenous layers (Fig. 5, 6). The apical end of the wall contained a circular micropyle covered by an operculum attached to the PV mem- brane by a cord (Fig. 7-9).

Based on the morphological and ultrastructural aspects of the phagocyte, PV, oocysts and host specificity, we propose the establishment of a new species according to the differences found in relation to the previously described species:

Phylum Apicomplexa Levine, 1970 Class Sporozoa Leuckart, 1879 Order Eugregarinida LCger, 1900 Family Porosporidae Schneider, 1899

Genus Nematopsis Schneider, 1892 Nematopsis gigas n. sp. (Fig. 1-12) Life history stages. Host mollusc phagocytes in the mantle

tissues contain a variable number of PVs (up to 7), each con- taining an oocyst (Fig. 1-3).

Description. Oocyst is - 22 (21.4-22.4) pm by width - 11.5 (10.9-12.1) pm (n = 50), each containing an uninucleate sporozoite (Fig. 2-5). Oocyst wall is 0.18-0.25 pm thick with a circular micropyle, 1.0-1.2 pm in diameter, covered by an operculum (Fig. 6-9). The matrix of the PV is occupied by a complicated network of numerous anastomosing microfibrils that project from the oocyst wall towards the phagocyte cyto- plasm (Fig. 10-11). These microfibrils were aggregated in the central region of the PV matrix forming a wall-like structure surrounding the oocysts (Fig. 6, 10, 11).

Type host. The oocysts were observed in phagocytes of the mantle tissues of N. ascencionis (Gastropoda, Neritidae).

Type locality. North coast of “Fernando de Noronha” Is- land (3” 47’ 57” S , 32” 25’ 12” W), Brazil.

Prevalence. (20%). (5/25) Type specimens. Glass slides with holotype and with sem-

ithin sections were deposited in the International Protozoan

Type Slide Collection at the Smithsonian Institution, Washing- ton, D. C., USA (USMN No. 1018788).

Etymology. The specific epithet “gigas” derives from Greek “gigas”, meaning “giant”, and relates to the dimen- sions of the oocyst.

DISCUSSION

The ultrastructural morphology of the oocysts described in the present work corresponds to the same life cycle stage of eugregarines of the genus Nematopsis Schneider, 1892, of which the ultrastructure was previously described (Azevedo and Cachola 1992; Azevedo and Matos 1999; Padovan et al. 2003). The genus Nematopsis was defined as having oocysts with a thick wall enclosing a single sporozoite (Sprague 1970). The majority of oocysts, referred to as spores by Sprague (1970), of almost all Nematopsis sp. have been described by light mi- croscopy (Sato et al. 1996; Sprague 1949, 1970) or illustrated by schematic drawings based on light microscopy (Belofastova 1996; Hatt 1927; Prytherch 1938, 1940; Sprague 1970). Only a few have been studied by TEM (Azevedo and Cachola 1992; Azevedo and Matos 1999; Padovan et al. 2003).

The most obvious differences with previously described spe- cies were the dimensions of the oocysts and the ultrastructural organization of the PV, particularly the complicated anasto- mosing microfibrils that form a wall-like structure around the oocyst wall in the central region of the PV. Until now, the two largest Nematopsis oocysts described in bivalve species were Nematopsis prytherch, about 19 X 16 pm (Sprague 1949) and Nematopsis duorari about 18.9 X 9.8 pm (Kruse 1966). 00- cysts of N. gigas are considerably larger, 22 X 11.5 pm.

Finally, the ultrastructural organization of the cytoplasm of the phagocytes and the different host species contrast with pre- viously described Nematopsis species. The vesicular organiza- tion of phagocyte cytoplasm infected by N. gigas showed great morphological differences compared to host cell cytoplasm in- fected by Nematopsis species, which contained numerous gly- cogen granules (Azevedo and Matos 1999; Padovan et al. 2003).

Based on these differences, and host specificity, we justify the establishment of a new species, N. gigas n. sp., the first recorded protozoan parasite from this Atlantic island.

ACKNOWLEDGMENTS

This study was partially supported by Eng” Ant6nio de Al- meida Foundation (Porto-Portugal) and Propesq-UFPe (Recife- Brazil). We would like to thank Prof. J. C. Nascimento and Mr. Sidney Vieira da Silva (UFRPe-Recife) for their help in col- lecting material. We thank Mrs. Laura Corral and Mr. J. Car- valheiro for excellent technical assistance, and the anonymous reviewers and Associate Editor for their most helpful comments and suggestions.

t

Fig. 7-11. Light (LM) and transmission electron microscopy (TEM) of the apicomplexan gregarine Nernatopsis gigas n. sp., a parasite in the mantle tissue of the gastropod Nerita ascencionis (All scale bars in km). 7, 8. Two similar aspects of the oocysts observed by LM (7) and TEM (8) showing the surrounding parasitophorous vacuole (PV), the oocyst wall (W), sporozoite ( S ) , a dense network of microfibrils (arrowheads) and the opercular system (OP). Note the “phagocyte” wall (double arrows) in Fig. 7. 9. A longitudinal section of the opercular region of an oocyst, located within the parasitophorous vacuole (PV), showing the operculum (OP), the oocyst wall (W), the sporozoite ( S ) , and the surrounding dense network of microfibrils (arrowheads), here in contact with the oocyst wall (W). 10. Ultrastructural detail of parts of the phagocyte. Note the ‘‘phagocyte” wall (double arrows), the phagocyte nucleus (N) located eccentrically and surrounded by vesicular structures of the cytoplasm, the parasitophorous vacuole (PV) occupied by numerous anastomosing microfibrils and the dense network of microfibrils (arrowheads), and the oocyst wall (W). 11. Ultrastructural details of the parasitophorous vacuole (PV) showing the numerous anastomosing microfibrils and dense network of microfibrils (arrowheads) surrounding the oocyst wall (W).

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Fig. 12. Schematic drawing of a longitudinal section of an oocyst of Nematopsis gigas n. sp. and the surrounding structures of the parasito- phorous vacuole (PV). Note the numerous anastomosing microfibrils of the PV, some of which form a dense and complex network (arrowheads). The phagocyte has an eccentric nucleus (N), a vesicular cytoplasm, and is surrounded by the “phagocyte” wall (double arrows).

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Received OW1 2/03; accepted 11/13/03