Studies on Bonamia parasites (Haplosporidia) in the New Zealand flat oyster Ostrea chilensis

Abstract

The haplosporidian parasites Bonamia exitiosa and B. ostreae have caused significant and consequential marine disease epizootics of oysters around the world. Due to the severity of disease that these parasites can cause, B. exitiosa and B. ostreae are listed as notifiable pathogens to the World Organisation for Animal Health (OIE). Bonamia exitiosa is endemic to the New Zealand flat oyster, Ostrea chilensis, where two epizootics in the Foveaux Strait, New Zealand, reduced the oyster population to 9% of its pre-disease level. Until this thesis research, no other Bonamia parasite had been detected in New Zealand. This thesis reports on the detection of the exotic B. ostreae in New Zealand for the first time, and presents a series of studies on Bonamia parasite dispersal, pathology, and laboratory diagnostics.

Until 2015, B. ostreae had only been detected in the European flat oyster O. edulis from the Northern Hemisphere, including Europe and North America. Following a report of high-level mortality within two O. chilensis farms from the Marlborough Sounds, New Zealand, histological examination, PCR, and DNA sequencing confirmed the detection of B. ostreae in New Zealand infecting O. chilensis. In addition to detecting B. ostreae, concurrent infections of B. exitiosa and B. ostreae were also reported. Histologically, there was little observed difference in infection intensity and presentation of pathology between the epidemiological subpopulations.

Diagnostic pooling is a common laboratory practice to increase efficiency and reduce expenses. Following the report of B. ostreae, a New Zealand-wide targeted surveillance was initiated that resulted in a high number of samples for testing. I investigated the efficacy of two published SYBR Green real-time PCR assays when used to detect B. ostreae in pooled-samples of infected oyster tissue. Each PCR targets a different gene within the B. ostreae genome: the actin 1 gene and the 18S rRNA gene. Experimental pools of 3, 5, and 10 individuals with ten replicates of each were created. The PCR targeting the actin 1 gene could not reproducibly detect B. ostreae in any pool size, whereas the 18S rRNA gene PCR assay reproducibly detected B. ostreae in pools of 5. This study highlights that detection efficacy is not comparable between individual and pooled-sample testing and that validation data are imperative before pooled-sample testing is implemented.

The large range extension of B. ostreae to New Zealand raised questions of dispersal, specifically whether B. ostreae is a recent arrival into New Zealand and from which Northern Hemisphere population it originated. I used internal transcribed spacer (ITS) rDNA sequences from New Zealand B. ostreae (n = 29) and compared them to published Northern Hemisphere B. ostreae sequences from Maine, USA (n = 7), California, USA (n = 18), and the Netherlands (n = 6) to investigate intraspecific variation. Low ITS rDNA variation within New Zealand isolates combined with no detection of B. ostreae in archived O. chilensis samples suggests that B. ostreae is a recent arrival to New Zealand. Because of the high level of B. ostreae sequence variation from Northern Hemisphere sites, inferences of dispersal origin could not be made and increased sampling of different B. ostreae isolates is required to elucidate the genetic relationship among them.

How Bonamia parasites disperse within the environment is uncertain. Bonamia exitiosa and B. ostreae do not produce spores and are obligate parasites of oysters. Bonamia ostreae can infect oyster larvae, so the hypothesis that Bonamia spp. can disperse between oyster beds during the pelagic larval phase of the host was tested. Out of 12 sites from around New Zealand, I detected B. exitiosa and B. ostreae in O. chilensis from three sites and one site, respectively. Using B. exitiosa ITS rDNA and O. chilensis mitochondrial cytochrome oxidase subunit 1 (CO1) gene sequences, I compared the genetic structure of host and parasite. Bonamia exitiosa displayed genetic structure across three sites, although some haplotypes were shared between them. Genetic structure was detected in O. chilensis except for gene flow between Tasman Bay-Marlborough Sounds-Chatham Islands. Considering the pattern of genetic structure, it seems that O. chilensis may experience long distance dispersal, which is likely influenced by oceanographic factors. A failure to detect B. exitiosa in genetically connected O. chilensis populations as well as haplotype sharing between B. exitiosa populations, despite strong genetic structuring among respective O. chilensis populations, suggest that long-distance co-dispersal of Bonamia parasites with O. chilensis is unlikely. Instead, the dispersal of Bonamia parasites among oyster beds is more likely influenced by anthropogenic factors.