Larvae of hatchery-reared Pacific oysters Crassostrea gigas Thunberg ceased feedlng 3 to 4 d after spawning, and 60 to l00 O/ O mortalities occurred after 7 to l l d. Up to 25 '!?o of moribund larvae had putative phagocyte precursors and fibroblasts with enlarged nuclei of abnormal shape and chromatin pattern, which contained ovoid to hexagonal, non-enveloped, 97 + 4 nm diameter capsids and nucleocapsids of a herpes-like virus. Nucleocapsids became enveloped and de-enveloped as they passed through the nuclear membranes. Some became enveloped in intranuclear sacs containing tubular elements Naked cytoplasmic nucleocapsids became enveloped by budding into vesicles or Golgi cisternae, and probably acquired a tegument from large dense granular masses near Golgi complexes. Extracellular enveloped virions usually had a tegument, and were 131 + 9 nm In diameter Extracellular naked nucleocapsids were thought to denve from lysed cells. Non-enveloped ovoid to hexagonal 86 t 12 nm virions, thought to be degraded herpesvirions, were abundant in inter-digitating processes of putative phagocytes in > 5 0 % of moribund larvae. Comparison w~t h groups within the Herpesviridae was made, and similarities to cytomegaloviruses noted. Elevated temperature and crowding may increase susceptibility of oyster larvae to herpes-like virus infection.

Veligers removed from brooding T~ostrea chilens~s (Philippi, 1845) experienced -95% mortalities over 3 to 4 d at 16 to 18°C that appeared to be associated with a herpes-like virus. Ultrastructural observations of post-removal veligers showed the presence of early viral replication or putative latent stages at 4 h, all stages of replication at low levels at 24 h, which increased to high levels at 48 h, followed by mortalities at 72 h onwards. Initially, infected interstitial or epithelia1 cells had an enlarged nucleus with a wavy outline in which heterochromatin was marginated. With continued increase in size, nuclei became smooth in outline with reduction or loss of heterochromatin. Capsids with lucent cores (LCC) and empty capsids appeared in the nucleus, often in association with tubular structures -65 nm in diameter that were composed of subunits in a helical configuration that contained a tubular core -35 nm in diameter. Empty capsids and LCC sometimes occurred in paracrystalline arrays. Partial nucleolar disaggregation and encapsidation of dense fibrillar material preceded envelopment entering and de-envelopment leaving the perinuclear cisterna, tegumentation in cytoplasmic vesicles, and egress. Groups of dense cytoplasmic filaments 30 to 35 nm in diameter occurred in some infected cells. Apparently normal cells with a few intranuclear empty capsids and/or LCC at 4 h postremoval may represent latent infections. Replication was not observed in larvae held at 24 to 27'C. but a few cells had enlarged hypochromatic karyolytic nuclei, and 1 to 2 capsids were observed in them, at 48 h. Viral replication was similar to that of ranid herpesvirus 1 (Lucke tumour) infections. This is the fifth ostreid species from which herpesviruses have been reported.

Reticular structures, confronting cisternae (CC), including cylindrical confronting cisternae (CCC), an unusual surface projection, formation of haplosporosome-like bodies (H-LB) and haplosporogenesis are described from Bonamia sp. infecting oysters Tiostrea chilensis from New Zealand. Formation of H-LB began when balls of putative nuclear material derived from indentations in the nuclear surface were encircled by cisternae of Golgi bearing a layer of dense material on their inner, concave surface. Also. H-LB were formed when CC split longitudinally and putative nuclear material filled the lumen of the resultant tubular structure, which subsequently pinched off H-LB. Haplosporogenesis began with the formation of cytoplasmic multi-vesicular bodies (MVB), which filled with dark osmiophilic material derived from nearby Golgi. The characteristic membranes of haplosporosomes appeared to form within these MVBs. It is suggested that H-LB are unusual structures in haplosporidans and that their core is formed from nuclear material. The CC from which they may form are also unusual, and in mammals CC occur in rapidly divid~ng or diseased cells. As CCC can only be induced in virally infected, transformed or neoplastic mammalian cells, it is considered that in Bonamia sp. CCC may be overt signs of an underlying disease. The significance of this is discussed in relation to the sudden appearance and pathogenicity of Bonamia sp. and, possibly, Haplosporidium nelsonj (MSX).

Bonamia sp., a serious pathogen of oysters Tiostrea chilensis, was studied using the zinc iodide-osmium tetroxide technique (ZIO), the imidazole-osmium technique for lipids (IM), and reactivity for acid phosphatases [P-glycerophosphatase (P-GPase), cytidine monophosphatase at pH 5.0 (CMPase 5.0) and pH 2.7 (CMPase 2.7), thiamine pyrophosphatase (TPPase)], and P-galactosidase to try and determine the function of cytoplasmic organelles. Z10 reacted with the nuclear membrane, Golgi, endoplasmic reticulum (ER), cytoplasmic vesicles, and variably with putative phagosomes, the mitochondrial matrix, haplosporosomes and multivesicular bodies, but not lipoid bodies. IM strongly labelled lipoid bodies, and lipids in the nuclear membrane, ER except anastomosing ER (aER) and mitochondrial membranes and cristae, but was weak in aER and on haplosporosomes. P-GPase occurred in the nuclear membrane, some elements of ER, Golgi and mitochondrla, and in lipoid bodies. CMPase 5.0 was variable in Golgi and ER, but was stronger at pH 2.7 and occurred in the nuclear membrane. TPPase only occurred in a single Golgi cisterna, or very weakly in a few lipoid bodies. P-galactosidase was rarely seen, and only within Golgi. As there was considerable metabolism of unsaturated lipids and little hydrolytic enzyme content, it was concluded that Bonarnla sp. may synthesise lipids from triglycerides hydrolysed by haemocyte enzymes, reducing the need for parasite hydrolytic enzymes.

ABSTRACT. Green-lip mussel Perna canaliculus spat (15 to 30 m m length) in the outer Marlborough Sounds, New Zealand, suffered 50 to 100% mortal~t)! during January to April 1994 (summer/autumn) following thinning and reseeding of the mussel lines by farmers Adult mussel ~nortalities of 2 to 5 % continued from February to early h4ay 1994 Histolog~cal examination showed extensive haemocytosis and multifocal liquefact~on necrosls of i n t e r s t~t~a l cells, basal cells and digest~ve tubule ep~thelial cells. Sloughed pyknot~c 01-karyolyt~c digestive tubule epithelia1 cells formed charactenst~c rounded granular b o d~e s 10 to 15 pm diameter both In digestive tubules and free in lesions. No v~r a l inclusion bodies were observed Ultrastructural examination showed highly mod~fied rough endoplasn~ic reticulum assoc~ated with small, 25 to 45 nm diameter, electron-dense uncoated virus-like particles Identical cell damage and virus-like particles were subsequently found In moribund adult (75 to 110 mm length) P canallculus and stunted (25 to 47 mm length) subtidal 12-li'tilus galloprov~ncial~s from the same area.Following purification of extracts of the moribund spat b}! isopycnlc centl-ifugation in CsCI, large numbers of 25 nm diameter, unenveloped, virus pal-t~cles were scvn by electron mlcroscopy These part~cles had a density of 1.364 g cm-3 A broad band at a d e n s~t y c~xpected for enveloped particles (1 21 to 1 24 g cm ') was also observed but contained few v~rus-like particles Cell damage a n d mussel mortalit~es are thus likely due to a small unenveloped virus.

The interaction between the haplosporidian pathogen Bonamia sp. and the haemocytes of oysters Tiostrea chilensis was studied ultrastructurally and using ultracytochemistry for the lysosomal hydrolases, P-glycerophosphatase (P-GPase), cytidine monophosphatase (CMPase), thiamine pyrophosphatase (TPPase), and P-galactosidase. Although fine and coarse granulocytes, serous (brown) cells and hyalinocytes were present, agranular precursors of fine granulocytes (progranulocytes), degranulated coarse granulocytes, hyalinocytes and serous cells commonly phagocytosed Bonamia sp. A sequence of infection from initial occupation of tight phagosomes to formation of an enlarged phagosome or parasitophorous vacuole (PV), which sometimes broke down, occurred in all infected haemocytes. Enlargement of the phagosome membrane to eventually form a PV coincided with formation of bilaminar vesicles in the lipoid bodies of parasites and their release from an indented area of the parasite surface. Too few infected granulocytes were observed to determine whether phagosome modification blocked lysosome-phagosome fusion. Vesicular CMPase and TPPase were produced by agranular haemocytes containing phagocytosed Bonamia sp. and passed into tight phagosomes. Despite some endocytosis of these enzymes by Bonamia sp.. very few parasites appeared dense or moribund. From the orientation of lipoid bodies toward the phagosome lumen, and release of P-GPase into the lumen from the lipold bodies, parasite hydrolytic enzymes in lipoid bod~es may function In paraslte nutrition, by extracellular digestion of host haemocyte cytoplasm. KEY WORDS: Bonam~a sp.. Oyster haemocytes. Phagosome:lysosome interactions. Enzymes. Parasitophorous vacuole

Apparent replication of small DNA-negative vlrus-like particles (VLPs) is described from digestive and secretory (= basiphil) cells of scallops Pecten novaezelandiae, Reeve, 1853 and tohcroa Paphies ventricosunl (Gray, 1843) sampled during mass rnortalities, and compared w~t h apparently healthy individuals. In scallop digestive cells with putative VLPs, endocytotic and smooth membrane vesicles increased, endoplasmic reticulum (ER) proliferated, and VLPs 22 to 30 nrn across were seen in an orderly array on the surfaces of the outer nuclear membrane and along ER. Proliferating ER membranes, lined with VLPs and enclosing a dense matnx, were arranged in a reticulated configuratlon. The ER cisternae dilated to form vacuolar inclusions (VI) containing elongated bodles, spherical in section, in a flocculent matrix which were ornated with VLPs arrays on the external membrane. Enclosed bodies also formed by budding of cytoplasm into the VI. In scallop secretory cells VLPs replaced ribosomes on ER, and ER cisternae dilated, but V1 seldom formed Toheroa diverticular epithelium showed similar changes, but secretory cells differed in that the outel-membrane of the nucleus and Golgi cisternae, rather than ER, proliferated. In addit~on, complete V1 were apparently not formed. The cytological changes observed in both bivalves are similar to those associated with enteroviruses (Picornaviridae) and caliciviruses. The possible role of VLPs In bivalve pathology is discussed.

No abstract