Legislative and regulatory aspects of molluscan health management.

The world population is growing quickly and there is a need to make sustainable protein available through an integrated approach that includes marine aquaculture. Seafood is already a highly traded commodity but the production from capture fisheries is rarely sustainable, which makes mollusc culture more important. However, an important constraint to its continued expansion is the potential for trade movements to disseminate pathogens that can cause disease problems and loss of production. Therefore, this review considers legislative and regulatory aspects of molluscan health management that have historically attempted to control the spread of mollusc pathogens. It is argued that the legislation has been slow to react to emerging diseases and the appearance of exotic pathogens in new areas. In addition, illegal trade movements are taken into account and possible future developments related to improvements in areas such as data collection and diagnostic techniques, as well as epidemiology, traceability and risk analysis, are outlined.

Ultrastructural comparison of Bonamia spp. (Haplosporidia) infecting ostreid oysters.

The ultrastructure of Bonamia from Ostrea angasi from Australia, Crassostrea ariakensis from the USA, O. puelchana from Argentina and O. edulis from Spain was compared with described Bonamia spp. All appear conspecific with B. exitiosa. The Bonamia sp. from Chile had similarities to the type B. exitiosa from New Zealand (NZ), but less so than the other forms recognized as B. exitiosa. Two groups of ultrastructural features were identified; those associated with metabolism (mitochondrial profiles, lipid droplets and endoplasmic reticulum), and those associated with haplosporogenesis (Golgi, indentations in the nuclear surface, the putative trans-Golgi network, perinuclear granular material and haplosporosome-like bodies). Metabolic features were regarded as having little taxonomic value, and as the process of haplosporogenesis is not understood, only haplosporosome shape and size may be of taxonomic value. However, the uni-nucleate stages of spore-forming haplosporidians are poorly known and may be confused with Bonamia spp. uni-nucleate stages. The many forms of NZ B. exitiosa have not been observed in other hosts, which may indicate that it has a plastic life cycle. Although there are similarities between NZ B. exitiosa and Chilean Bonamia in the development of a larger uni-nucleate stage and the occurrence of cylindrical confronting cisternae, the clarification of the identity of Chilean Bonamia must await molecular studies.

Bonamia exitiosa transmission among, and incidence in, Asian oyster Crassostrea ariakensis under warm euhaline conditions.

Previously reported in Australia, New Zealand, and more recently in Europe, the protistan parasite Bonamia exitiosa was also reported in the mid-Atlantic region of the USA after causing serious mortalities there in the Asian oyster Crassostrea ariakensis. At the time, this oyster was being considered for introduction, and the potential consequences of introducing this species were being assessed using field and laboratory studies. B. exitiosa emerged as the most serious disease threat for this oyster species, especially under warm euhaline conditions and for oysters <50 mm in size. To better evaluate how quickly this parasite may be able to spread among C. ariakensis, we investigated B. exitiosa transmission and incidence in C. ariakensis. During a first trial, potential direct transmission of B. exitiosa was evaluated by cohabitating infected C. ariakensis with uninfected C. ariakensis under in vivo quarantine conditions. In a second experiment, B. exitiosa incidence was estimated in situ by determining its prevalence in C. ariakensis deployed in an enzootic area after 4, 7, 14, 21 and 28 d of exposure. Results suggest that under warm euhaline conditions B. exitiosa can be transmitted among C. ariakensis without requiring any other parasite source and that parasite incidence may be at least as high as 40% after only 4 d exposure to an enzootic area. These results underscored the severity of the bonamiasis disease threat to C. ariakensis and provided further evidence that efforts to build an aquaculture industry based on C. ariakensis in the eastern USA might have been thwarted by parasitic disease.

Haplosporidium littoralis sp. nov.: a crustacean pathogen within the Haplosporida (Cercozoa, Ascetosporea).

Previously, we described the pathology and ultrastructure of an apparently asporous haplosporidian-like parasite infecting the common shore crab Carcinus maenas from the European shoreline. In the current study, extraction of genomic DNA from the haemolymph, gill or hepatopancreas of infected C. maenas was carried out and the small subunit ribosomal DNA (SSU rDNA) of the pathogen was amplified by PCR before cloning and sequencing. All 4 crabs yielded an identical 1736 bp parasite sequence. BLAST analysis against the NCBI GenBank database identified the sequence as most similar to the protistan pathogen group comprising the order Haplosporida within the class Ascetosporea of the phylum Cercozoa Cavalier-Smith, 1998. Parsimony analysis placed the crab pathogen within the genus Haplosporidium, sister to the molluscan parasites H. montforti, H. pickfordi and H. lusitanicum. The parasite infecting C. maenas is hereby named as Haplosporidium littoralis sp. nov. The presence of a haplosporidian parasite infecting decapod crustaceans from the European shoreline with close phylogenetic affinity to previously described haplosporidians infecting molluscs is intriguing. The study provides important phylogenetic data for this relatively understudied, but commercially significant, pathogen group.

Inter-relationships of haplosporidians deduced from ultrastructural studies.

We reviewed papers reporting haplosporidian ultrastructure to compare inter-relationships based on ultrastructure with those based on molecular data, to identify features that may be important in haplosporidian taxonomy, and to consider parasite taxonomy in relation to host taxonomy. There were links between the following: (1) the plasmodia of an abalone parasite, Haplosporidium nelsoni and Urosporidium crescens in the release of haplosporosomes; (2) H. costale and H. armoricanum in haplosporosome shape and presence and shape of Golgi in spores; (3) basal asporous crustacean haplosporidians which form haplosporosomes from formative bodies (FBs) in vegetative stages--H. nelsoni, which forms haplosporosomes from FBs in plasmodial cytoplasm, and H. louisiana, Minchinia spp. and Bonamia perspora, which form haplosporosomes from FBs in spores; (4) crustacean haplosporidians, Bonamia spp. and M. occulta in the predominance of uni- and binucleate stages; and (5) lipid-like vesicles in sporoplasms of H. costale, H. armoricanum, H. lusitanicum, H. pickfordi, H. montforti, and B. perspora. In general, these relationships reflect phylogenies based on molecular studies. As well as spore form and ornamentation, haplosporogenesis in spores appears to be taxonomically important. Parasite and host taxonomy were linked in the infection of lower invertebrates by Urosporidium spp., the infection of oysters by Bonamia spp., and of molluscs by Minchinia spp. Haplosporidium spp. are patently an artificial, paraphyletic group probably comprising many taxa. Consequently, the taxonomy of haplosporidians needs a thorough revision.

Shellfish tissues evaluated for Perkinsus spp. using the Ray's fluid thioglycollate medium culture assay can be used for downstream molecular assays.

Ray's fluid thioglycollate medium (RFTM) culture assay is the standard, recommended method for surveillance of Perkinsus spp. infections in marine molluscs. In this assay, shellfish tissues are incubated in RFTM, stained with Lugol's iodine solution to render Perkinsus spp. cells blue-black, and evaluated microscopically to rate infection intensities. A limitation of this assay, however, is the lack of pathogen species specificity. Generally, identification of Perkinsus spp. requires DNA sequence analysis of parallel or additional samples since the exposure to iodine is believed to hamper DNA amplification from samples processed by the RFTM assay. However, we show that P. marinus DNA can be successfully amplified by PCR from Crassostrea virginica tissues cultured in RFTM and stained with Lugol's iodine. The beneficial consequence is that, where necessary, DNA sequence data may be obtained from RFTM-cultured tissues, allowing the identification of the Perkinsus sp. responsible for an observed infection. This would obviate further sampling, representing gain of time and reduction in cost, where a Perkinsus sp. is unexpectedly observed in new host(s) or location(s) but where parallel samples are not available for molecular diagnostics. Laboratories without molecular diagnostic tools for Perkinsus spp. may fix presumptive Perkinsus sp.-positive culture material in 95% ethanol for transport to, and subsequent analysis by, a laboratory that does have this capacity.

Microsatellite marker development and analysis in the eastern oyster (Crassostrea virginica): confirmation of null alleles and non-Mendelian segregation ratios.

Eighteen microsatellite markers were developed for the Crassostrea virginica nuclear genome, including di-, tri-, and tetranucleotide microsatellite repeat regions that included perfect, imperfect, and compound repeat sequences. A reference panel with DNA from the parents and four progeny of 10 full-sib families was used for a preliminary confirmation of polymorphism at these loci and indications of null alleles. Null alleles were discovered at three loci; in two instances, primer redesign enabled their amplification. Two to five representative alleles from each locus were sequenced to ensure that the targeted loci were amplifying. The sequence analysis revealed not only variation in the number of simple sequence repeat units, but also polymorphisms in the microsatellite flanking regions. A total of 3626 bp of combined microsatellite flanking region from the 18 loci was examined, revealing indels as well as nucleotide site substitutions. Overall, 16 indels and 146 substitutions were found with an average of 4.5% polymorphism across all loci. Eight markers were tested on the parents and 39-61 progeny from each of four families for examination of allelic inheritance patterns and genotypic ratios. Twenty-six tests of segregation ratios revealed eight significant departures from expected Mendelian ratios, three of which remained significant after correction for multiple tests. Deviations were observed in both the directions of heterozygote excess and deficiency.

Development of a PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the Haplosporidia.

The development of diagnostic assays more sensitive and specific than traditional histological techniques is important for the management of bonamiasis in flat oysters Ostrea edulis. A specific polymerase chain reaction (PCR) protocol was developed for the detection of very small amounts of Bonamia ostreae (Pichot et al. 1980) ribosomal DNA (rDNA) in bulk DNA from oyster gill and hemolymph. The presence of a 760 bp PCR amplification product corresponded with B. ostreae infections determined cytologically in 185 oysters from Ireland, Spain, and the USA. All (100%) 'heavily' and 'moderately' infected oysters, 86.7 % of the 'lightly' infected oysters, and 66.7 % of the 'scarcely' infected oysters were confirmed to be infected using the PCR. In addition, 37.9% of the oysters in which B. ostreae was not detected using cytology were positive using the PCR. Sampling error and the subjectivity of cytological diagnoses are the likely sources of disagreement between diagnostic methods in oysters with very light infections. The PCR assay developed here is more sensitive and less ambiguous than standard histological and cytological techniques. Phylogenetic analysis of DNA sequence data confirmed B. ostreae to be a member of the Haplosporidia.