Mapping the development of a Dinophysis bloom in a shellfish aquaculture area using a novel molecular qPCR assay.

Diarrhetic shellfish toxins produced by certain species of the marine dinoflagellate Dinophysis can accumulate in shellfish in high concentrations, representing a significant food safety issue worldwide. This risk is routinely managed by monitoring programs in shellfish producing areas, however the methods used to detect these harmful marine microbes are not usually automated nor conducted onsite, and are often expensive and require specialized expertise. Here we designed a quantitative real-time polymerase chain reaction (qPCR) assay based on the ITS-5.8S ribosomal region of Dinophysis spp. and evaluated its specificity, efficiency, and sensitivity to detect species belonging to this genus. We designed and tested twenty sets of primers pairs using three species of Dinophysis - D. caudata, D. fortii and D. acuminata. We optimized a qPCR assay using the primer pair that sufficiently amplified each of the target species (Dacu_11F/Dacu_11R), and tested this assay for cross-reactivity with other dinoflagellates and diatoms in the laboratory (11 species) and in silico 8 species (15 strains) of Dinophysis, 3 species of Ornithocercus and 2 species of Phalacroma. The qPCR assay returned efficiencies of 92.4% for D. caudata, 91.3% for D fortii, and 91.5% for D. acuminata, while showing no cross-reactivity with other phytoplankton taxa. Finally, we applied this assay to a D. acuminata bloom which occurred in an oyster producing estuary in south eastern Australia, and compared cell numbers inferred by qPCR to those determined by microscopy counts (max abund. ∼6.3 × 103 and 5.3 × 103 cells L-1 respectively). Novel molecular tools such as qPCR have the potential to be used on-farm, be automated, and provide an early warning for the management of harmful algal blooms.

Using qPCR and high-resolution sensor data to model a multi-species Pseudo-nitzschia (Bacillariophyceae) bloom in southeastern Australia.

Harmful algal blooms, including those caused by the toxic diatom Pseudo-nitzschia, can have significant impacts on human health, ecosystem functioning and ultimately food security. In the current study we characterized a bloom of species of Pseudo-nitzschia that occurred in a south-eastern Australian oyster-growing estuary in 2019. Using light microscopy, combined with molecular (ITS/5.8S and LSU D1-D3 rDNA regions) and toxicological evidence, we observed the bloom to consist of multiple species of Pseudo-nitzschia including P. cf. cuspidata, P. hasleana, P. fraudulenta and P. multiseries, with P. cf. cuspidata being the only species that produced domoic acid (3.1 pg DA per cell). As several species of Pseudo-nitzschia co-occurred, only one of which produced DA, we developed a rapid, sensitive and efficient quantitative real-time polymerase chain reaction (qPCR) assay to detect only species belonging to the P. pseudodelicatissima complex Clade I, to which P. cf. cuspidata belongs, and this indicated that P. cuspidata or closely related strains may have dominated the Pseudo-nitzschia community at this time. Finally, using high resolution water temperature and salinity sensor data, we modeled the relationship between light microscopy determined abundance of P. delicatissima group and environmental variables (temperature, salinity, rainfall) at two sites within the estuary. A total of eight General Linear Models (GLMs) explaining between 9 and 54% of the deviance suggested that the temperature (increasing) and/or salinity (decreasing) data were generally more predictive of high cell concentrations than the rainfall data at both sites, and that overall, cell concentrations were more predictive at the more oceanic site than the more upstream site, using this method. We conclude that the combination of rapid molecular methods such as qPCR and real-time sensor data modeling, can provide a more rapid and effective early warning of harmful algal blooms of species of Pseudo-nitzschia, resulting in more beneficial regulatory and management outcomes.

A Comparative Analysis of Methods (LC-MS/MS, LC-MS and Rapid Test Kits) for the Determination of Diarrhetic Shellfish Toxins in Oysters, Mussels and Pipis.

Rapid methods for the detection of biotoxins in shellfish can assist the seafood industry and safeguard public health. Diarrhetic Shellfish Toxins (DSTs) are produced by species of the dinoflagellate genus Dinophysis, yet the comparative efficacy of their detection methods has not been systematically determined. Here, we examined DSTs in spiked and naturally contaminated shellfish-Sydney Rock Oysters (Saccostrea glomerata), Pacific Oysters (Magallana gigas/Crassostrea gigas), Blue Mussels (Mytilus galloprovincialis) and Pipis (Plebidonax deltoides/Donax deltoides), using LC-MS/MS and LC-MS in 4 laboratories, and 5 rapid test kits (quantitative Enzyme-Linked Immunosorbent Assay (ELISA) and Protein Phosphatase Inhibition Assay (PP2A), and qualitative Lateral Flow Assay (LFA)). We found all toxins in all species could be recovered by all laboratories using LC-MS/MS (Liquid Chromatography-tandem Mass Spectrometry) and LC-MS (Liquid Chromatography-Mass Spectrometry); however, DST recovery at low and mid-level concentrations (<0.1 mg/kg) was variable (0-150%), while recovery at high-level concentrations (>0.86 mg/kg) was higher (60-262%). While no clear differences were observed between shellfish, all kits delivered an unacceptably high level (25-100%) of falsely compliant results for spiked samples. The LFA and the PP2A kits performed satisfactorily for naturally contaminated pipis (0%, 5% falsely compliant, respectively). There were correlations between spiked DSTs and quantitative methods was highest for LC-MS (r2 = 0.86) and the PP2A kit (r2 = 0.72). Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time.

Overview of Australian and New Zealand harmful algal species occurrences and their societal impacts in the period 1985 to 2018, including a compilation of historic records.

Similarities and differences between Australia and New Zealand in Harmful Algal species occurrences and Harmful Algal Events impacting on human society (HAEDAT) are reported and factors that explain their differences explored. Weekly monitoring of harmful phytoplankton and biotoxins commenced in Australia in 1986 and in New Zealand in 1993. Anecdotal historic HAB records in both countries are also catalogued. In Australia, unprecedented highly toxic Paralytic Shellfish Toxin (PST)-producing blooms of Alexandrium catenella have impacted the seafood industry along the 200 km east coast of Tasmania from 2012 to present. Toxic blooms in 1986-1993 by Gymnodinium catenatum in Tasmania were effectively mitigated by closing the affected area for shellfish farming, while a bloom by this same species in 2000 in New Zealand caused significant economic damage from restrictions on the movement of greenshell mussel spat. The biggest biotoxin event in New Zealand was an unexpected outbreak of Neurotoxic Shellfish Poisoning (NSP) in 1993 in Hauraki Gulf (putatively due to Karenia cf. mikimotoi) with 180 reported cases of human poisonings as well as reports of respiratory irritation north of Auckland. Strikingly, NSP never recurred in New Zealand since and no NSP events have ever been reported in Australia. In New Zealand, Paralytic Shellfish Poisoning (PSP) was the predominant seafood toxin syndrome, while in Australia Ciguatera Fish Poisoning (CFP) was the major reported seafood toxin syndrome, while no CFP has been recorded from consumption of New Zealand fish. In Australia, Diarrhetic Shellfish Poisoning (DSP) illnesses were recorded from two related outbreaks in 1997/98 following consumption of beach harvested clams (pipis) from a previously non-monitored area, whereas in New Zealand limited DSP illnesses are known. No human illnesses from Amnesic Shellfish Poisoning (ASP) have been reported in either Australia or New Zealand. Selected examples of HABs appearing and disappearing (NSP in New Zealand, Alexandrium catenella in Tasmania), species expanding their ranges (Noctiluca, Gambierdiscus), and reputed ballast water introductions (Gymnodinium catenatum) are discussed. Eutrophication has rarely been invoked as a cause except for confined estuaries and fish ponds and estuarine cyanobacterial blooms. No trend in the number of HAEDAT events from 1985 to 2018 was discernible.

First Detection of Paralytic Shellfish Toxins from Alexandrium pacificum above the Regulatory Limit in Blue Mussels (Mytilus galloprovincialis) in New South Wales, Australia.

In 2016, 2017 and 2018, elevated levels of the species Alexandrium pacificum were detected within a blue mussel (Mytilus galloprovincialis) aquaculture area at Twofold Bay on the south coast of New South Wales, Australia. In 2016, the bloom persisted for at least eight weeks and maximum cell concentrations of 89,000 cells L-1 of A. pacificum were reported. The identity of A. pacificum was confirmed using molecular genetic tools (qPCR and amplicon sequencing) and complemented by light and scanning electron microscopy of cultured strains. Maximum reported concentrations of paralytic shellfish toxins (PSTs) in mussel tissue was 7.2 mg/kg PST STX equivalent. Elevated cell concentrations of A. pacificum were reported along the adjacent coastal shelf areas, and positive PST results were reported from nearby oyster producing estuaries during 2016. This is the first record of PSTs above the regulatory limit (0.8 mg/kg) in commercial aquaculture in New South Wales since the establishment of routine biotoxin monitoring in 2005. The intensity and duration of the 2016 A. pacificum bloom were unusual given the relatively low abundances of A. pacificum in estuarine and coastal waters of the region found in the prior 10 years.

Four recent ciguatera fish poisoning incidents in New South Wales, Australia linked to imported fish

Between 2015 and 2017, the NSW Food Authority investigated 4 ciguatera fish poisoning incidents linked to fish caught in tropical Australian and international waters and imported to New South Wales. Three of the implicated fish were caught near the Capel Bank Seamount, part of the Lord Howe Island Seamount Chain off the coast of Queensland. The fourth incident, in 2017, involved a fish caught between Cooktown and Lizard island, also off the Queensland coast. Thirteen individuals were affected. Early identification of potential Ciguatera fish poisoning (CFP) cases can facilitate a faster incident response, patient diagnosis and treatment. While different fish species were involved, and the severity and duration of symptoms varied between individuals, 3 of the 4 incidents were distinguished by those affected having consumed a fish meal prepared with the head of the fish. Affected individuals also described a sensation of hot/cold temperature reversal or a painful 'sharpness' when in contact with or drinking cold water. This paper outlines a series of incidents where a case's symptoms, which can vary substantially in conjunction with their previous exposure history, the type of fish consumed and how the fish was cooked, can be used to identify potential CFP cases and hence CFP risks that may need to be addressed. Keywords: Ciguatera fish poisoning, ciguatoxins, seafood borne illness, New South Wales, Australia, Pacific ciguatoxin 1-B (P-CTX-1B).

Diarrhetic Shellfish Toxin Monitoring in Commercial Wild Harvest Bivalve Shellfish in New South Wales, Australia.

An end-product market survey on biotoxins in commercial wild harvest shellfish (Plebidonax deltoides, Katelysia spp., Anadara granosa, Notocallista kingii) during three harvest seasons (2015⁻2017) from the coast of New South Wales, Australia found 99.38% of samples were within regulatory limits. Diarrhetic shellfish toxins (DSTs) were present in 34.27% of 321 samples but only in pipis (P. deltoides), with two samples above the regulatory limit. Comparison of these market survey data to samples (phytoplankton in water and biotoxins in shellfish tissue) collected during the same period at wild harvest beaches demonstrated that, while elevated concentrations of Dinophysis were detected, a lag in detecting bloom events on two occasions meant that wild harvest shellfish with DSTs above the regulatory limit entered the marketplace. Concurrently, data (phytoplankton and biotoxin) from Sydney rock oyster (Saccostrea glomerata) harvest areas in estuaries adjacent to wild harvest beaches impacted by DSTs frequently showed elevated Dinophysis concentrations, but DSTs were not detected in oyster samples. These results highlighted a need for distinct management strategies for different shellfish species, particularly during Dinophysis bloom events. DSTs above the regulatory limit in pipis sampled from the marketplace suggested there is merit in looking at options to strengthen the current wild harvest biotoxin management strategies.

Management of Ciguatoxin Risk in Eastern Australia.

Between 2014 and 2016, five cases of ciguatera fish poisoning (CFP), involving twenty four individuals, were linked to Spanish Mackerel (Scomberomorus commerson) caught in the coastal waters of the state of New South Wales (NSW) on the east coast of Australia. Previously, documented cases of CFP in NSW were few, and primarily linked to fish imported from other regions. Since 2015, thirteen individuals were affected across four additional CFP cases in NSW, linked to fish imported from tropical locations. The apparent increase in CFP in NSW from locally sourced catch, combined with the risk of CFP from imported fish, has highlighted several considerations that should be incorporated into risk management strategies to minimize CFP exposure for seafood consumers.

Clinical diagnosis and chemical confirmation of ciguatera fish poisoning in New South Wales, Australia.

Ciguatera fish poisoning is common in tropical and sub-tropical areas and larger fish (> 10 kg) are more susceptible to toxin accumulation with age. Although the coastal climate of northern New South Wales is considered sub-tropical, prior to 2014 there has only been 1 documented outbreak of ciguatera fish poisoning from fish caught in the region. During February and March 2014, 2 outbreaks of ciguatera fish poisoning involved 4 and 9 individuals, respectively, both following consumption of Spanish mackerel from northern New South Wales coastal waters (Evans Head and Scotts Head). Affected individuals suffered a combination of gastrointestinal and neurological symptoms requiring hospital treatment. At least 1 individual was symptomatic up to 7 months later. Liquid chromatography-tandem mass spectrometry detected the compound Pacific ciguatoxin-1B at levels up to 1.0 µg kg(-1) in fish tissue from both outbreaks. During April 2015, another outbreak of ciguatera fish poisoning was reported in 4 individuals. The fish implicated in the outbreak was caught further south than the 2014 outbreaks (South West Rocks). Fish tissue was unavailable for analysis; however, symptoms were consistent with ciguatera fish poisoning. To our knowledge, these cases are the southernmost confirmed sources of ciguatera fish poisoning in Australia. Educational outreach to the fishing community, in particular recreational fishers was undertaken after the Evans Head outbreak. This highlighted the outbreak, species of fish involved and the range of symptoms associated with ciguatera fish poisoning. Further assessment of the potential for ciguatoxins to occur in previously unaffected locations need to be considered in terms of food safety.

Warm temperature acclimation impacts metabolism of paralytic shellfish toxins from Alexandrium minutum in commercial oysters.

Species of Alexandrium produce potent neurotoxins termed paralytic shellfish toxins and are expanding their ranges worldwide, concurrent with increases in sea surface temperature. The metabolism of molluscs is temperature dependent, and increases in ocean temperature may influence both the abundance and distribution of Alexandrium and the dynamics of toxin uptake and depuration in shellfish. Here, we conducted a large-scale study of the effect of temperature on the uptake and depuration of paralytic shellfish toxins in three commercial oysters (Saccostrea glomerata and diploid and triploid Crassostrea gigas, n = 252 per species/ploidy level). Oysters were acclimated to two constant temperatures, reflecting current and predicted climate scenarios (22 and 27 °C), and fed a diet including the paralytic shellfish toxin-producing species Alexandrium minutum. While the oysters fed on A. minutum in similar quantities, concentrations of the toxin analogue GTX1,4 were significantly lower in warm-acclimated S. glomerata and diploid C. gigas after 12 days. Following exposure to A. minutum, toxicity of triploid C. gigas was not affected by temperature. Generally, detoxification rates were reduced in warm-acclimated oysters. The routine metabolism of the oysters was not affected by the toxins, but a significant effect was found at a cellular level in diploid C. gigas. The increasing incidences of Alexandrium blooms worldwide are a challenge for shellfish food safety regulation. Our findings indicate that rising ocean temperatures may reduce paralytic shellfish toxin accumulation in two of the three oyster types; however, they may persist for longer periods in oyster tissue.

A fish kill associated with a bloom of Amphidinium carterae in a coastal lagoon in Sydney, Australia.

We report on a dense bloom (~1.80 × 105 cells mL-1) of the marine dinoflagellate species Amphidinium carterae (Genotype 2) in a shallow, small intermittently open coastal lagoon in south eastern Australia. This bloom co-occurred with the deaths of >300 individuals of three different species of fish. The opening of the lagoon to the ocean, as well as localized high nutrient levels, preceded the observations of very high cell numbers. A. carterae is usually benthic and sediment-dwelling, but temporarily became abundant throughout the water column in this shallow (<2 m) sandy habitat. Histopathological results showed that the Anguilla reinhardtii individuals examined had damage to epithelial and gill epithelial cells. An analysis of the bloom water indicated the presence of a compound with a retention time and UV spectra similar to Luteophanol A, a compound known from a strain of Amphidinium. Assays with a fish gill cell line were conducted using a purified compound from cells concentrated from the bloom, and was found to cause a loss of 87% in cell viability in 6 h. The fish deaths were likely due to the low dissolved oxygen levels in the water and/or the presence of Luteophanol A-like compounds released during the bloom.

Distribution of the genus Alexandrium (Halim) and paralytic shellfish toxins along the coastline of New South Wales, Australia.

Blooms of Alexandrium species, in particular the species Alexandrium catenella, accounted for more than 50% of algal related, shellfish aquaculture harvest zone closures in New South Wales (NSW) Australia since 2005. While there are indications that species of Alexandrium are more abundant than they were formerly, there is little data available on the spatial and temporal distribution and abundance of the genus in NSW. A six and a half year dataset comprising a total of 8649 fortnightly samples from 31 estuaries spread over 2000 km of NSW coastline was analysed. The greatest abundances of Alexandrium spp. were observed during the austral Spring and Summer, in estuaries in the mid and southern latitudes of the state. In identifying these high risk zones, we propose variables such as season, temperature, rainfall and estuarine flushing to be targeted in intensive site specific studies, to support the development of predictive tools for resource managers.