Lethal effects of ichthyotoxic raphidophytes, Chattonella marina, C. antiqua, and Heterosigma akashiwo, on post-embryonic stages of the Japanese pearl oyster, Pinctada fucata martensii.

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5×102cellsml-1), C. antiqua (103cellsml-1), and H. akashiwo (5×103cellsml-1) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5×103cellsml-1 and C. marina at 8×103cellsml-1. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 105cellsml-1 more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8×103cellsml-1, 24h), C. marina (8×103cellsml-1, 24h), and H. akashiwo (104cellsml-1, 24h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (103cellsml-1, 24h),C. marina (8×103cellsml-1, 24h), and H. akashiwo (5×104cellsml-1, 24h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5×102-8×103cellsml-1, 48-86h) and C. antiqua (103-8×103cellsml-1, 72-86h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.

Detection of Minchinia occulta in samples of pearl oysters Pinctada maxima infected by Haplosporidium hinei.

OBJECTIVE: To determine if juvenile pearl oysters (Pinctada maxima) infected with Haplosporidium hinei are also infected with another haplosporidian parasite, Minchinia occulta. DESIGN: Archived samples of pearl oysters infected with H. hinei were examined using polymerase chain reaction (PCR) assays and in situ hybridisation (ISH) to analyse and identify haplosporidians. A 144-bp and 220-bp region of Minchinia DNA were targeted by PCR and amplified DNA from formalin-fixed H. hinei-infected pearl oyster samples was sequenced. A 25-bp oligonucleotide probe targeting a variable section of the parasite's small subunit rRNA gene was used in ISH. RESULTS: The results of DNA-based diagnostic assays supported each other. The sequences obtained by PCR were found to be almost identical to M. occulta from rock oysters and the ISH assay demonstrated infection with M. occulta in affected pearl oysters. ISH indicated a prevalence of infection of 26.7% in one of the previous outbreaks. CONCLUSION: Pearl oyster spat are susceptible to infection by a Minchinia parasite, most likely M. occulta, which was recently identified in rock oysters within the pearl-producing zones of Western Australia and is associated with mortalities of up to 80% in this species. The occurrence of haplosporidian co-infections in pearl oysters suggests the immunocompetence of juvenile oysters may be an important factor in preventing infection and therefore preventing mortalities such as those occurring in the recent outbreaks of pearl oyster oedema disease.

Intracellular ciliated protozoal infection in silverlip pearl oysters, Pinctada maxima (Jameson, 1901).

The pathology associated with an intracellular ciliate infection in the digestive gland of pearl oysters Pinctada maxima (Jameson, 1901) is described. Histopathological and transmission electron microscopic examination were used to characterise the organism and its location within host cells. The parasite is tear-drop shaped measuring 5.53 microm (range of 2.73-7.47 microm, n=9) in width and 11.15 microm (range of 9.02-16.2 microm) in length with a centrally located lobulated nucleus and a large nucleus:cytoplasmic ratio. The ciliate has nine evenly spaced rows of cilia running obliquely along the length of cell, converging on the pointed end. Infected digestive glands typically had a moderate to severe infiltration with mononuclear hemocyte. A strong correlation existed between the burden of ciliates and the host response; (p<0.001, C=0.315 Pearson Correlation). The use of a single tissue section upon microscopic examination was found to detect only 38-50% of the infections. However, examination of serial haematoxylin and eosin stained sections improved the reliability of detecting infection.

Spore ornamentation of Haplosporidium hinei n. sp. (Haplosporidia) in pearl oysters Pinctada maxima (Jameson, 1901).

An infection of pearl oysters, Pinctada maxima, attributed to a Haplosporidium sp. by Hine and Thorne (1998) has been detected on 3 occasions and is considered to represent a serious concern to the pearling industry in Australia. The spore ornamentation of the parasite was determined by scanning electron microscopy and transmission electron microscopy. Spores of the parasite were pleomorphic, or elongated 3.5-4 microm x 2.5-3.0 microm in size. Two filaments were wound around the spore and originated from 2 'knob-like' posterior thickenings. Both filaments passed up one side of the spore together until just below the operculum whereupon each split and passed obliquely under the lip of the opercula lid. Each filament wrapped around the spore 4 times. The posterior thickenings seem to appear late in the development of the spore and were composed of spore wall material. A second set of branching tubular filaments composed of a different material was observed on the spore body although not on mature spores possessing a 'knob-like' posterior thickening. The ornamentation on the spores of the pearl oyster parasite was unique amongst described haplosporidian species where spore ornamentation is known. The parasite is named in this manuscript as Haplosporidium hinei n. sp.

Differential community development of fouling species on the pearl oysters Pinctada fucata, Pteria penguin and Pteria chinensis (Bivalvia, Pteriidae).

A field experiment documented the development of fouling communities on two shell regions, the lip and hinge, of the pearl oyster species Pinctada fucata, Pteria penguin and Pteria chinensis. Fouling communities on the three species were not distinct throughout the experiment. However, when each species was analysed separately, fouling communities on the lip and hinge of P. penguin and P. chinensis were significantly different during the whole sampling period and after 12 weeks, respectively, whereas no significant differences could be detected for P. fucata. There was no significant difference in total fouling cover between shell regions of P. fucata and P. chinensis after 16 weeks; however, the hinge of P. penguin was significantly more fouled than the lip. The most common fouling species (the hydroid Obelia bidentata, the bryozoan Parasmittina parsevalii, the bivalve Saccostrea glomerata and the ascidian Didemnum sp.) showed species-specific fouling patterns with differential fouling between shell regions for each species. The role of the periostracum in determining the community development of fouling species was investigated by measuring the presence and structure of the periostracum at the lip and hinge of the three pearl oyster species. The periostracum was mainly present at the lip of the pearl oysters, while the periostracum at the hinge was absent and the underlying prismatic layer eroded. The periostracum of P. fucata lacked regular features, whereas the periostracum of P. penguin and P. chinensis consisted of a regular strand-like structure with mean amplitudes of 0.84 microm and 0.65 microm, respectively. Although the nature and distribution of fouling species on the pearl oysters was related to the presence of the periostracum, the periostracum does not offer a fouling-resistant surface for these pearl oyster species.