Jeffs, ANguyen, Suong2019-04-102018http://hdl.handle.net/2292/46379The flat oyster (Bluff oyster, dredge oyster) Ostrea chilensis is an iconic seafood species in New Zealand that forms the basis of one of the oldest commercial fisheries in the country and is under development for aquaculture. However, the productivity of commercially important flat oyster populations has been severely hampered by an apicomplexan parasite called apicomplexan-X (APX) which can act alone, or together with bonamiosis. Despite the commercial significance of this disease, the biology of APX is virtually unknown, with an absence of knowledge of hosts, life cycle and taxonomic identity. There are also no specific detection or control measures available to deal with disease outbreaks. Therefore, the aim of this research was to begin to fill the gaps in our knowledge of APX to assist in understanding and combating the disease. Two unique APX 18S rRNA gene sequences (~1.8kb) were isolated from APX in flat oysters by firstly applying generic apicomplexan primers followed by sequencing, and then further extension of the obtained sequences using specific primers. BLASTN search of the GenBank database and phylogenetic analysis confirmed the apicomplexan origin of the obtained sequences. Both sequences were confirmed to be specifically isolated from APX using their complementary in situ hybridisation probes, which exclusively and clearly bound to the APX zoites in histological sections of infected oysters. Based on the generated APX 18S rRNA gene portions, this current study provides an effective PCR method for specific APX detection. A specific forward primer APX-For and reverse primer APX-Rev were designed based on the two in situ hybridisation probes previously identified from regions which have the highest variability, discriminating the APX 18S rRNA sequences from partial 18S rRNA sequences of flat oyster, Pacific oyster (Crassostrea gigas), Bonamia ostreae and Bonamia exitiosa. Several approaches, including using a dilution series method, direct comparisons with histology results, and testing on DNA of other apicomplexan species were performed to prove that the PCR method has strong analytical and diagnostic sensitivity, as well as good analytical specificity for the reliable detection of APX. The new PCR method was then used to confirm the close affinity of APX-like cells seen by histology in other bivalve hosts including green-lipped mussels (Perna canaliculus), Mediterranean mussels (Mytilus galloprovincialis) and hairy mussels (Modiolus areolatus). Amplification followed by sequencing and phylogenetic analysis indicated that APX-similar structures observed in mussels are genetical identitical (99-100%) to APX in flat oysters, thereby extending the range of hosts for disease. Samples of green-lipped, Mediterranean and hairy mussels positive with APX were collected from widely geographically distributed locations in New Zealand, indicating the wide range of the parasite. However, histology showed low APX intensity and mild host response in the examined mussels compared to APX occurring in flat oyster. The results of this study have greatly extended our knowledge of APX and delivered a useful molecular tool for reliably detecting the disease which will help with detecting and managing APX in commercially important shellfish populations in New Zealand.Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher.https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htmThesisCopyright: The authorhttp://purl.org/eprint/accessRights/OpenAccessQ112937694