Molecular characterisation of a haplosporidian parasite infecting rock oysters Saccostrea cuccullata in north Western Australia

A haplosporidian parasite was identified in rock oysters (Saccostrea cuccullata Born, 1778) from the Montebello Islands (latitude -20.4'S longitude 115.53'E) off the northern coast of Western Australia by histopathological examination, PCR amplification and DNA sequencing of a segment of the SSU region of the parasite's rRNA gene. An oligonucleotide probe was constructed from the parasite's SSU rRNA gene in order to confirm its presence by in situ hybridisation. The parasite was disseminated throughout the gonad follicles of the host and to a lesser extent in the gills. The only parasite life stages thus far observed in this study were a uninucleate naked cell assumed to be a precursor to multinucleate plasmodial stages and a binucleate plasmodial stage. Whilst no parasite spores were detected in affected rock oysters, a phylogenetic analysis of the SSU region of the parasite's rRNA gene indicates the parasite belongs to the genus Minchinia. A PCR and in situ hybridisation assay for the Minchinia sp. was used to identify haplosporidians described by Hine and Thorne [Hine, P.M.., Thorne, T., 2002. Haplosporidium sp. (Haplosporidia: Haplosporidiidae) associated with mortalities among rock oysters Saccostrea cuccullata in north Western Australia. Dis. Aquat. Organ. 51, 123-13], in archived rock oyster tissues from the same coastline.     

Spore ornamentation of Minchinia occulta n. sp. (Haplosporidia) in rock oysters Saccostrea cuccullata (Born, 1778)

A Minchinia sp. (Haplosporidia: Haplosporidiidae) parasite was identified infecting rock oysters and morphologically described by Hine and Thorne (2002) using light microscopy and transmission electron microscopy (TEM). The parasite was associated with up to 80% mortality in the host species and it is suspected that the parasite would be a major impediment to the development of a tropical rock oyster aquaculture industry in northern Western Australia. However, attempts to identify the parasite following the development of a specific probe for Haplosporidium nelsoni were unsuccessful. The SSU region of the parasite's rRNA gene was later characterized in our laboratory and an in situ hybridization assay for the parasite was developed. This study names the parasite as Minchinia occulta n sp. and morphologically describes the parasite using histology, scanning electron microscopy and transmission electron microscopy. The non-spore stages were unusual in that they consisted primarily of uninucleate stages reminiscent of Bonamia spp. The parasite's spores were ovoid to circular shaped and measured 4.5 microm-5.0 microm x 3.5-4.1 microm in size. The nucleus of the sporoplasm measured 1.5-2.3 microm and was centrally located. The spores were covered in a branching network of microtubule-like structures that may degrade as the spore matures.     

Detection of Minchinia sp., in rock oysters Saccostrea cuccullata (Born, 1778) using DNA probes

Haplosporidian parasites infect various invertebrate hosts including some commercially important shellfish. Haplosporidium nelsoni (along with Perkinsus marinus) has severely affected Eastern oyster production on the eastern seaboard of the United States and flat oyster production in Europe has been severely impacted by Bonamia ostreae. These parasites are also often present at a very low prevalence and there are a variety of morphologically similar species that can be difficult to differentiate during cytological or histological diagnosis hence the need to develop specific tests. Recently, a Minchinia sp. was described affecting rock oysters (Saccostrea cuccullata) in north Western Australia. In this study, two in situ hybridisation (ISH) assays and a PCR assay have been developed and optimised for use in investigating these parasites. The first ISH assay used a 166bp polynucleotide probe while the second used a 30bp oligonucleotide probe. The specificity of each ISH assay was assessed by applying each probe to a variety of haplosporidian (5), a paramyxian (1) or ciliophora (1) parasites. The polynucleotide probe produced strong hybridisation signals against all of the haplosporidian parasites tested (Minchinia sp., Minchinia teredinis, Bonamia roughleyi, H. nelsoni and Haplosporidium costale) while the oligonucleotide probe recognised only the Minchinia sp. Both probes failed to detect the paramyxian (Marteilia sp.) or the Rhynchodid-like ciliate. The PCR assay amplifies a 220bp region and detected Minchinia sp. DNA from 50ng of genomic DNA extracted from the tissues of infected oysters and 10fg of amplified Minchinia sp. DNA. The assay did not react to oysters infected with H. nelsoni or H. costale. The ability of the PCR and oligonucleotide ISH assay to diagnose Minchinia sp. infected oysters was compared to histological examination from a sample of 56 oysters. The PCR assay revealed 26 infections while histological examination detected 14 infections. The oligonucleotide ISH assay detected 29 infections. The oligonucleotide ISH and PCR assays were found to be significantly more sensitive than histology for detecting the parasite.     

Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata)

Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in other molluscs, PO was found to exist as a pro-enzyme (proPO) in hemocytes. ProPO could be activated to PO by exogenous proteases (trypsin and chymotrypsin), exposure of hemocytes to pathogen-associated molecular patterns (PAMPs) and by the detergents, Triton X-100 and sodium dodecyl sulphate (SDS). Inhibition studies confirmed the proPO activating system of Sydney rock oysters is a proteinase cascade in which Ca²⁺ dependent serine proteinases proteolytically convert proPO into active PO. Activated PO was found to be a tyrosinase-like enzyme that is responsible for both monophenolase and diphenolase activity. The bifunctional PO had higher affinity for the monophenol, hydroquinine monomethyl ether (4HA) (K_m = 4.45 ± 1.46 mM) than for the diphenol, l-DOPA (K_m = 10.27 ± 1.33 mM). Maximum enzyme activity was evident at 37 °C, pH 8 and at salinities of between 30 and 37 ppt. Melanogenesis catalysed by the active enzyme is a composite of eumelanin and the product of a sclerotin pathway combining DOPA decarboxylase with PO activity.     

Bonamia exitiosus n. sp. (Haplosporidia) infecting flat oysters Ostrea chilensis in New Zealand.

Bonamia sp. is a pathogenic parasite that occurs in the haemocytes of dredge oysters Ostrea chilensis Philippi in New Zealand. Ultrastructurally it resembles other haplosporidians in the possession of haplosporosomes, haplosporogenesis, persistence of mitotic microtubules during interphase and of the nuclear envelope during mitosis, and occurrence of a diplokaryotic or multi-nucleate plasmodial stage. Another stage containing a large vacuole derived from enlargement of 1 or more mitochondria has not previously been described from other haplosporidians. It most closely resembles B. ostreae Pichot et al., 1979, which parasitises and is pathogenic in haemocytes of European flat oysters, O. edulis. However, B. ostreae is smaller and denser, and has fewer lipoid bodies and haplosporosomes. We have nearly completely sequenced the small ribosomal gene of the organism from O. chilensis. Initial comparisons of these sequences with those of other protozoans showed similarities to B. ostreae. Polymorphism within Bonamia sp. was confirmed by restriction fragment length polymorphism analysis. On the basis of ultrastructural and molecular considerations it is proposed that this organism be named Bonamia exitiosus sp. nov.     

Haplosporidium raabei n. sp. (Haplosporidia): a parasite of zebra mussels, Dreissena polymorpha (Pallas, 1771)

Extensive connective tissue lysis is a common outcome of haplosporidian infection. Although such infections in marine invertebrates are well documented, they are relatively rarely observed in freshwater invertebrates. Herein, we report a field study using a comprehensive series of methodologies (histology, dissection, electron microscopy, gene sequence analysis, and molecular phylogenetics) to investigate the morphology, taxonomy, systematics, geographical distribution, pathogenicity, and seasonal and annual prevalence of a haplosporidian observed in zebra mussels, Dreissena polymorpha. Based on its genetic sequence, morphology, and host, we describe Haplosporidium raabei n. sp. from D. polymorpha - the first haplosporidian species from a freshwater bivalve. Haplosporidium raabei is rare as we observed it in histological sections in only 0·7% of the zebra mussels collected from 43 water bodies across 11 European countries and in none that were collected from 10 water bodies in the United States. In contrast to its low prevalences, disease intensities were quite high with 79·5% of infections advanced to sporogenesis.     

Morphological and mitochondrial-DNA analysis of the Indo-West Pacific rock oysters (Ostreidae: Saccostrea species)

Species of Saccostrea are common oysters on Indo-Pacific rockyshores. The taxonomy of this genus is unclear, however, becauseof morphological plasticity among its constituent species. Oystersamples with distinct morphotypes were collected from shoresexperiencing different degrees of wave exposure in Japan, Taiwan,China, Vietnam, Singapore and Australia. Intraspecific and interspecificvariations in shell characters have been identified. Only S.kegaki could be readily differentiated from S. cucullata-B,S. cucullata-C and S. mordax by multivariate shell allometricanalyses. Phylogenetic analyses using partial mitochondrial16S DNA sequences of approximately 513 base pairs identifiedtwo clades that correspond to superspecies, i.e. putative S.cucullata and putative S. mordax, comprising nine and two lineagesfrom the Indo-West Pacific, respectively. These are S. cucullata-A,B, C, D, E, F, G, S. kegaki and S. glomerata in one clade, andS. mordax-A and B in the other. Interspecific sequence divergence(Kimura-2-parameter values) between lineages ranged from 1.02%to 11.74%. Both S. cucullata-C and S. kegaki are restrictedto the northern hemisphere, whereas S. glomerata seems to bea temperate species from the southern hemisphere. S. cucullata-Doccurs only in Taiwan. Other species, i.e. S. cucullata-A, B,D, E and F and S. mordax-A and B, have a wide range on rockyshores in the tropical and subtropical Western Pacific and inboth northern and southern hemispheres. (Received 7 January 2005; accepted 4 November 2005)     

Relations between variable rates of growth, metabolic costs and growth efficiencies in individual Sydney rock oysters (Saccostrea commercialis)

Rock oysters from a mass selection trial were compared with wild-caught (control) oysters of the same age to determine the physiological basis for faster growth rates amongst the selected individuals, and to describe the associated flexibility in phenotypic traits of feeding, metabolism and growth. In confirmation of earlier studies, fast growth was associated with faster rates of feeding, reduced metabolic rates and lower metabolic costs of growth. Selected individuals deposited more protein, at a lower metabolic cost, than the controls. Control oysters, however, deposited more lipid than the selected oysters, though the unit costs of lipid deposition did not differ between categories. The results indicated a wide plasticity of physiological rates and efficiencies and demonstrated how, by selection, interactions between physiological traits can serve to enhance growth. If differences in lipid deposition observed here were indicative of different rates of gametogenesis, then the results also suggest that selection alters the balance between growth and reproduction. Whether these differences can be termed compensatory with respect to the life history of the species remains to be determined, but the results indicate some of the ways in which physiological flexibility may be expressed to effect different patterns of energy allocation.     

Sporulation of Marteilioides branchialis n. sp. (Paramyxea) in the Sydney rock oyster, Saccostrea commercialis: an electron microscope study.

The ultrastructure of sporulation of a new parasite, Marteilioides branchialis (Paramyxea), in the Sydney Rock oyster, Saccostrea commercialis, is described. The development is typical of other Paramyxea whereby a stem cell internally cleaves a secondary cell contained within a vacuole. It differs from other species in the phylum in that each secondary cell produces a single spore composed of two concentric cells, one within a vacuole of the other. This type of sporulation represents the simplest of all known Paramyxea. Infection results in focal gill lesions and was observed concurrently with an epizootic of another paramyxean, Marteilia sydneyi.     

Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function

In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.     

Diplobulbus brayi n. sp. (Digenea: Opecoelidae) from Odax acroptilus (Perciformes: Odacidae) off Western Australia

Diplobulbus brayi n. sp. is described from the small intestine of Odax acroptilus (Perciformes: Odacidae) from Point Peron in southern Western Australia. One of the five previously described species of Diplobulbus, D. vitellosus Bilqees, 1972 from Tetraodon lunaris (now Lagocephalus lunaris), is synonymised with Alloheterolebes indicus (Gupta, 1968) Hafeezullah, 1971, which is known from the same host in the Indian Ocean. D. brayi n. sp. can be distinguished from the remaining four species of Diplobulbus by the distinct interruption of the vitelline follicles at the ventral sucker, its more elongate form and its long post-testicular space. Diplobulbus is so far restricted to the Indo-Pacific region and appears to be stenoxenic to labroid fishes. This is the first record of a trematode from an odacid fish.     

Widespread survey finds no evidence of Haplosporidium nelsoni (MSX) in Gulf of Mexico oysters

The advent of molecular detection assays has provided a set of very sensitive tools for the detection of pathogens in marine organisms, but it has also raised problems of how to interpret positive signals that are not accompanied by visual confirmation. PCR-positive results have recently been reported for Haplosporidium nelsoni (MSX), a pathogen of the oyster Crassostrea virginica in 31 of 40 oysters from 6 sites in the Gulf of Mexico and the Caribbean Sea. Histological confirmation of the PCR results was not undertaken, and no haplosporidian has been reported from the numerous histological studies and surveys of oysters in the region. To further investigate the possibility that H. nelsoni is present in this region, we sampled 210 oysters from 40 sites around the Gulf of Mexico and Puerto Rico using PCR and 180 of these using tissue-section histology also. None of the oysters showed evidence of H. nelsoni by PCR or of any haplosporidian by histology. We cannot, therefore, confirm that H. nelsoni is present and widespread in the Gulf of Mexico and the Caribbean Sea. Our results do not prove that H. nelsoni is absent from the region, but taken together with results from previous histological surveys, they suggest that for the purposes of controlling oyster importation, the region should continue to be considered free of the parasite.     

Physiological components of growth differences between individual oysters (Crassostrea gigas) and a comparison with Saccostrea commercialis

Pacific oysters (Crassostrea gigas) of identical age from two genetically distinct lines, one fast growing and the other slow growing, were held at three levels of ration and analysed for physiological traits to explain differences in their rates of growth. The data supported three hypotheses; faster growth was associated with faster rates of consumption of food, reduced metabolic rate at maintenance (i.e., at zero growth), and reduced metabolic costs of growth. A comparison with the Sydney rock oyster, Saccostrea commercialis, based on similar experiments on the two species, indicated that faster growth of Pacific oysters depended on similar physiological differences; the mean metabolic costs of growth, however, were similar in the two species. It is suggested that a general model for genetically linked differences in the growth rate of bivalve molluscs will need to include the processes of metabolic control rather than relying solely on an analysis of the individual components of the energetics of growth.     

Notes onDiphucephala affinis (Coleoptera:Scarabaeidae) associated with flowers ofHibbertia andAcacia in Western Australia

Notes are provided on the association of the beetleDiphucephala affinis (Scarabaeidae) with yellow flowers ofHibbertia hypericoides, H. huegelii (Dilleniaceae),Acacia pulchella, andA. stenoptera (Mimosaceae). Observations were undertaken during September 9–19, 1979 at S. Perth, Western Australia. They indicated thatD. affinis is not a pollinator ofHibbertia as suggested in the literature, but may play a small role in the pollination of someAcacia species.     

Ostrincola humesi sp. nov., a myicolid copepod (Poecilostomatoida) parasitic in rock oysters cultured in the Gulf of Thailand

A new species of poecilostomatoid copepod, Ostrincola humesi (Myicolidae), is described, based on specimens collected from the mantle cavity of the rock oyster, Saccostrea cucullata (Born), cultured in the Gulf of Thailand. A close comparison was made between the new species and Ostrincola koe Tanaka with which the new species was previously confused. A key to the nine species of Ostrincola is provided.     

Micropleura australiensis n. sp. (Nematoda: Micropleuridae) from the body cavity of Crocodylus johnsoni in Western Australia

A new nematode species, Micropleura australiensis n. sp., is described on the basis of specimens found in the peritoneal cavity of the Australian freshwater crocodile, Crocodylus johnsoni Krefft, from the Ord River area, Western Australia. The new species is mainly characterized by the length of spicules (0.360-0.366 mm) and gubernaculum (0.096-0.105 mm), the number and arrangement of male caudal papillae (4 preanal and 6 postanal pairs), and the postequatorial vulva. To date, it is the first species of Micropleura reported from Australia. Micropleura trionyxi Agrawal, 1966, and M. lissemysia Chattervati, 1985, are considered junior synonyms of M. indica Khera, 1951.     

Studies of the Liagoraceae (rhodophyta) of Western Australia: Gloiotrichus fractalis gen. et sp. nov. and Ganonema helminthaxis sp. nov.

Two new taxa of Liagoraceae (Nemaliales) are described from Western Australia. Gloiotrichus fractalis gen. et sp. nov. has been collected from 3–20 m depths at the Houtman Abrolhos, Western Australia. Plants are calcified, extremely lubricous, and grow to 17 cm in length. Carpogonial branches are straight, 6 or 7 cells in length, arise from the basal or lower cells of cortical fascicles, and are occasionally compound. Branched sterile filaments of narrow elongate cells arise on the lower cells of the carpogonial branch prior to gonimoblast initiation, at first on the basal cells, then on progressively more distal cells. Following presumed fertilisation the carpogonium divides transversely, with both cells giving rise to gonimoblast filaments. The distal cells of the carpogonial branch then begin to fuse, with fusion progressing proximally until most of the cells of the carpogonial branch are included. As fusion extends, the filaments on the carpogonial branch are reduced to the basal 2 or 3 cells. The gonimoblast is compact and bears terminal carposporangia. Spermatangial clusters arise on subterminal cells of the cortex, eventually displacing the terminal cells. The sequence of pre- and post-fertilisation events occurring in the new genus separates it from all others included in the Liagoraceae, although it appears to have close affinities with the uncalcified genus Nemalion. Ganonema helminthaxis sp. nov. was collected from 12 m depths at Rottnest Island, Western Australia. Plants are uncalcified and mucilaginous, the axes consisting of a few (< 10) primary medullary filaments, each cell of which gives rise to a cortical fascicle at alternate forks of the pseudodichotomies borne on successive medullary cells. Subsidiary (adventitious) filaments and rhizoids comprise the bulk of the thallus. Carpogonial branches are straight, (3-)4(-6) cells in length, arise on the basal 1–4 cells of the cortical fascicles, and are frequently compound. Carposporophytes develop from the upper of two daughter cells formed by a transverse division of the fertilised carpogonium. Ascending and descending sterile filaments girdle the carpogonial branch cells and arise mostly on the supporting cell prior to fertilisation. Ganonema helminthaxis is the first completely non-calcified member of the genus, and its reproductive and vegetative morphology supports the recognition of Ganonema as a genus independent from Liagora. Liagora codii Womersley is a southern Australian species displaying features of Ganonema, to which it is transferred.     

Dactylomyza gibsoni n. g., n. sp. (Digenea: Opecoelidae) from Schuettea woodwardi (Waite) (Monodactylidae) from off Western Australia

An opecoelid digenean, Dactylomyza gibsoni n. g., n. sp. is described and figured from Schuettea woodwardi (Waite), a monodactylid from off the coast of Western Australia. The new genus conforms to the concept of the opecoelid subfamily Opecoelinae. The resemblance of the new genus to three other opecoelid genera, Pseudopecoeloides Yamaguti, 1940, Opecoeloides Odhner, 1928 and Poracanthium Dollfus, 1948, is discussed. Dactylomyza n. g. is distinguished from these morphologically similar worms on the basis of its median genital pore, ventral sucker appendages, uroproct and the absence of an accessory sucker. Pseudopecoeloides equesi Manter, 1947 is transferred to the new genus as Dactylomyza equesi (Manter, 1947) n. comb.