Testing a health baseline during a bivalve mollusc mortality event: An investigation into die-offs of pipi Paphies australis from Aotearoa New Zealand.

Disease is a major threat to the economic, ecological and cultural services provided by wild bivalve populations. Over the past decade anecdotal reports on declining health of native bivalve populations around Aotearoa New Zealand have been supported by increasing observations of mass die-offs. Causes of declining health and mass die-offs of wild bivalves are not clear and could be due to a number of interactive and cumulative factors, including declining water quality, climate change, or disease. Pipi/kokota (Paphies australis) within the Whangarei area (northern New Zealand) have suffered repeated die-offs and declining health since at least 2009. Baseline health data for wild native bivalve populations are scarce making it difficult to identify changes in pathogen infection prevalence and intensity and infer their importance to host health. This research aimed to examine and document the health of pipi in Whangarei with the objective of identifying factors that may contribute to their ill health and lack of population recovery. We sampled pipi from four sites within Whangarei, eight times across two years (total n = 640) to establish a health baseline using histopathology, general bacteriology, and qPCR for the intracellular bacteria Endozoicomonas spp. Three pipi mass die-offs occurred during the sampling window that were opportunistically sampled to compare against the health baseline established using healthy pipi. An increase in bacterial growth and a decrease in the abundance of Endozoicomonas spp. in mortality pipi was observed compared with the health baseline. Establishing a health baseline for pipi from Whangarei provided a benchmark to assess changes in a pipi population experiencing high mortality. Such data can help identify factors contributing to die-offs and to help inform what mitigation, if any, is possible in wild shellfish populations.

Perkinsus olseni in green-lipped mussels Perna canaliculus: diagnostic evaluation, prevalence, and distribution.

Perkinsus olseni (Perkinsidae) is a molluscan parasite notifiable to the World Organisation for Animal Health that is reported in several shellfish hosts in New Zealand, including the native green-lipped mussel Perna canaliculus. Green-lipped mussels comprise over half of New Zealand's aquaculture export value and have historically been considered free of serious diseases based on extensive histology-based surveillance. The discovery of P. olseni in green-lipped mussels has raised questions about future disease threats to green-lipped mussels, particularly under changing ocean climatic conditions. Using mussels collected from farmed (n = 358) and wild (n = 236) populations, we aimed to determine the distribution and prevalence of P. olseni in green-lipped mussels around New Zealand, and assess the performance of diagnostic tests, including real-time PCR, conventional PCR, and culture using Ray's fluid thioglycolate medium (RFTM). Prevalence and diagnostic test performance was evaluated using Bayesian latent class analysis with informative priors. The prevalence of P. olseni was 0-3%, except for 1 wild population from a harbour where prevalence was 22%. Real-time PCR had the highest diagnostic sensitivity (87%) compared to 62 and 21% for conventional PCR and RFTM, respectively. Diagnostic specificity was similar among all methods (96-98%). No mortality was observed during the study. Our results suggest that real-time PCR is the diagnostic test best suited for surveillance of P. olseni in subclinically infected green-lipped mussels under New Zealand conditions.

Limited impact of a bioeroding sponge, Cliona sp., on Ostrea chilensis from Foveaux Strait, New Zealand.

Bioeroding sponges can cause extensive damage to aquaculture and wild shellfish fisheries. It has been suggested that heavy sponge infestations that reach the inner cavity of oysters may trigger shell repair and lead to adductor detachment. Consequently, energy provision into shell repair could reduce the energy available for other physiological processes and reduce the meat quality of commercially fished oysters. Nevertheless, the impacts of boring sponges on oysters and other shellfish hosts are inconclusive. We studied the interaction between boring sponges and their hosts and examined potential detrimental effects on an economically important oyster species Ostrea chilensis from Foveaux Strait (FS), New Zealand. We investigated the effect of different infestation levels with the bioeroding sponge Cliona sp. on commercial meat quality, condition, reproduction, and disease susceptibility. Meat quality was assessed with an index based on visual assessments used in the FS O. chilensis fishery. Meat condition was assessed with a common oyster condition index, while histological methods were used to assess sex, gonad stage, reproductive capacity, and pathogen presence. Commercial meat quality and condition of O. chilensis were unaffected by sponge infestation. There was no relationship between sex ratio, gonad developmental stage, or gonad index and sponge infestation. Lastly, we found no evidence that sponge infestation affects disease susceptibility in O. chilensis. Our results suggest that O. chilensis in FS is largely unaffected by infestation with Cliona sp. and therefore reinforces the growing body of evidence that the effects of sponge infestation can be highly variable among different host species, environments, and habitats.

Intracellular bacteria in New Zealand shellfish are identified as Endozoicomonas species.

Kaimoana (shellfish, seafood) is an important food source and a significant social and cultural component of many New Zealand communities, especially the indigenous Maori. Over the past decade a decline has been detected in shellfish health and an increase in mortality events around New Zealand. Intracellular bacteria termed Rickettsia-like organisms (RLOs) have been observed in New Zealand bivalve molluscs during shellfish mortality events. Affected bivalves include cockles Austrovenus stutchburyi, ringed dosinia Dosinia anus, green-lipped mussels Perna canaliculus, pipi Paphies australis, toheroa Paphies ventricosa, tuatua Paphies subtriangulata, deepwater tuatua Paphies donacina and scallops Pecten novaezelandiae. RLOs are an informal morphology-based classification of intracellular bacteria, with the exact identification often unknown. Using shellfish collected during mortality events from 2014 to 2019 and apparently healthy samples collected in 2018 and 2019, we aimed to identify RLOs in New Zealand shellfish. Bacterial 16S rRNA gene sequences from RLO-infected shellfish showed >95% identity to published Endozoicomonas species. In situ hybridization confirmed the presence of the sequenced gene in the gill epithelium and digestive epithelium of all study species. A genus-specific quantitative PCR, targeting the 16S rRNA gene was developed to detect Endozoicomonas spp. in shellfish tissue. Prevalence of Endozoicomonas spp. in samples from mortality events and healthy shellfish analysed by quantitative PCR was high. Samples collected from mortality events, however, had a significantly higher load of Endozoicomonas spp. than the healthy samples. These results give us a greater understanding of these intracellular bacteria and their presence in populations of New Zealand shellfish.

Low internal transcribed spacer rDNA variation in New Zealand Bonamia ostreae: evidence for a recent arrival.

Bonamia ostreae is a haplosporidian parasite of oysters that was first reported to occur in the Southern Hemisphere in 2015 in the New Zealand flat oyster Ostrea chilensis. Until that report, B. ostreae had been restricted to populations of O. edulis within the Northern Hemisphere. This large range extension raised questions regarding B. ostreae dispersal, including whether B. ostreae is a recent introduction and from where it originated. The whole 18S rRNA gene of New Zealand B. ostreae revealed 99.9-100% sequence homology to other published B. ostreae 18S rDNA sequences. Internal transcribed spacer (ITS) rDNA sequences (n = 29) were generated from New Zealand B. ostreae and compared to published B. ostreae sequences from 3 Northern Hemisphere sites: California, USA (n = 18), Maine, USA (n = 7), and the Netherlands (n = 6) to investigate intraspecific variation. Low ITS rDNA variation was observed from New Zealand B. ostreae isolates, and high levels of variation were observed from Northern Hemisphere B. ostreae sequences. We hypothesise that the low ITS rDNA diversity found in New Zealand B. ostreae is the result of a founder effect resulting from a single introduction from a limited number of propagules. The high level of ITS rDNA variation from the Northern Hemisphere prevented inferences of dispersal origins. New Zealand B. ostreae were genetically differentiated from all sites, and additional genetic data are required to better determine the origin of B. ostreae in New Zealand.

Bonamia ostreae in the New Zealand oyster Ostrea chilensis: a new host and geographic record for this haplosporidian parasite.

Previous reports of the haplosporidian parasite Bonamia ostreae have been restricted to the Northern Hemisphere, including Europe, and both eastern and western North America. This species is reported for the first time in New Zealand infecting the flat oyster Ostrea chilensis. Histological examination of 149 adult oysters identified 119 (79.9%) infected with Bonamia microcells. Bonamia generic PCR of several oysters followed by DNA sequencing of a 300 bp portion of the 18S rDNA gene produced a 100% match with that of B. ostreae. All DNA-sequenced products also produced a B. ostreae PCR-restriction fragment length polymorphism (PCR-RFLP) profile. Bonamia species-specific PCRs further detected single infections of B. exitiosa (2.7%), B. ostreae (40.3%), and concurrent infections (53.7%) with these 2 Bonamia species identifying overall a Bonamia prevalence of 96.6%. Detailed histological inspection revealed 2 microcell types. An infection identified by PCR as B. ostreae histologically presented small microcells (mean ± SE diameter = 1.28 ± 0.16 µm, range = 0.9-2 µm, n = 60) commonly with eccentric nuclei. A B. exitiosa infection exhibited larger microcells (mean ± SE diameter = 2.12 ± 0.27 µm, range = 1.5-4 µm, n = 60) with more concentric nuclei. Concurrent infections of both Bonamia species, as identified by PCR, exhibited both types of microcells. DNA barcoding of the B. ostreae-infected oyster host confirmed the identification as O. chilensis. A suite of other parasites that accompany O. chilensis are reported here for the first time in mixed infection with B. ostreae including apicomplexan X (76.5%), Microsporidium rapuae (0.7%) and Bucephalus longicornutus (30.2%).

Pooled sample testing for Bonamia ostreae: A tale of two SYBR Green real-time PCR assays.

Pooled testing of samples is a common laboratory practice to increase efficiency and reduce expenses. We investigated the efficacy of 2 published SYBR Green real-time PCR assays when used to detect the haplosporidian parasite Bonamia ostreae in pooled samples of infected oyster tissue. Each PCR targets a different gene within the B. ostreae genome: the actin 1 gene or the 18S rRNA gene. Tissue homogenates (150 mg) of the New Zealand flat oyster Ostrea chilensis were spiked with ~1.5 × 103 purified B. ostreae cells to create experimental pools of 3, 5, and 10. Ten positive replicates of each pool size were assayed twice with each PCR and at 2 different amounts of DNA template. The PCR targeting the actin 1 gene was unable to reproducibly detect B. ostreae in any pool size. Conversely, the 18S rRNA gene PCR could reproducibly detect B. ostreae in pools of up to 5. Using a general linear model, there was a significant difference in the number of pools that correctly detected B. ostreae between each PCR ( p < 0.01) and each pool size ( p < 0.01). It is likely that the single copy actin 1 gene is more likely to be diluted and not detected by pooling than the multi-copy 18S rRNA gene. Our study highlights that validation data are necessary for pooled sample testing because detection efficacy may not be comparable to individual sample testing.