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 (0.86 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 tim

Hybridisation capture allows DNA damage analysis of ancient marine eukaryotes

Marine sedimentary ancient DNA (sedaDNA) is increasingly used to study past ocean ecosystems, however, studies have been severely limited by the very low amounts of DNA preserved in the subseafloor, and the lack of bioinformatic tools to authenticate sedaDNA in metagenomic data. We applied a hybridisation capture ‘baits’ technique to target marine eukaryote sedaDNA (specifically, phyto- and zooplankton, ‘Planktonbaits1’; and harmful algal bloom taxa, ‘HABbaits1’), which resulted in up to 4- and 9-fold increases, respectively, in the relative abundance of eukaryotes compared to shotgun sequencing. We further used the bioinformatic tool ‘HOPS’ to authenticate the sedaDNA component, establishing a new proxy to assess sedaDNA authenticity, “% eukaryote sedaDNA damage”, that is positively correlated with subseafloor depth. We used this proxy to report the first-ever DNA damage profiles from a marine phytoplankton species, the ubiquitous coccolithophore Emiliania huxleyi. Our approach opens new avenues for the detailed investigation of long-term change and evolution of marine eukaryotes over geological timescales

Unprecedented Alexandrium blooms in a previously low biotoxin risk area of Tasmania, Australia.

During October 2012, a shipment of blue mussels (Mytilus galloprovincialis) from the poorly monitored east coast of Tasmania, Australia, was tested by Japanese import authorities and found to be contaminated with unacceptable levels of Paralytic Shellfish Toxins (PSTs; 10 mg/kg). Subsequently local oysters, scallops, clams, the viscera of abalone and rock lobsters were also found to be contaminated. This led to a global product recall and loss to the local economy of AUD 23M. Following low toxicity during 2013 and 2014 and implementation of minimal shellfish farm closures, a more severe bloom event occurred during July-November 2015 and again June-September 2016 (up to 300,000 Alexandrium cells/L; 24 mg/kg PST in mussels, 6 mg/kg in Crassostrea gigas oysters), also causing 4 human illnesses resulting in hospitalization after consumption of wild shellfish. While Alexandrium tamarense had been detected in low concentrations in southeastern Australia since 1987, all cultured strains belonged to the mostly non-toxic group 5 (now designated A. australiense; detected since 1987) and weakly toxic group 4 (A. pacificum; detected in 1997). In contrast, the 2012 to 2016 outbreaks were dominated by highly toxic group 1 (A. fundyense) never detected previously in the Australian region. Molecular analyses suggest that A. fundyense may have been a cryptic ribotype previously present in Tasmania, but newly stimulated by altered water column stratification conditions driven by changing rainfall and temperature patterns. Increased seafood and plankton monitoring of the area now include the implementation of Alexandrium qPCR, routine Neogen™ immunological and HPLC PST tests, but ultimately may also drive change in harvesting strategies and aquaculture species selection by the local seafood industry

Phytoplankton calcification as an effective mechanism to prevent cellular calcium poisoning

Marine phytoplankton have developed the remarkable ability to tightly regulate the concentration of free calcium ions in the intracellular cytosol at a level of ~ 0.1 μmol L−1 in the presence of seawater Ca2+ concentrations of 10 mmol L−1. The low cytosolic calcium ion concentration is of utmost importance for proper cell signalling function. While the regulatory mechanisms responsible for the tight control of intracellular Ca2+ concentration are not completely understood, phytoplankton taxonomic groups appear to have evolved different strategies, which may affect their ability to cope with changes in seawater Ca2+ concentrations in their environment on geological timescales. For example, the Cretaceous (145 to 66 Ma), an era known for the high abundance of coccolithophores and the production of enormous calcium carbonate deposits, exhibited seawater calcium concentrations up to 4 times present-day levels. We show that calcifying coccolithophore species (Emiliania huxleyi, Gephyrocapsa oceanica and Coccolithus braarudii) are able to maintain their relative fitness (in terms of growth rate and photosynthesis) at simulated Cretaceous seawater calcium concentrations, whereas these rates are severely reduced under these conditions in some non-calcareous phytoplankton species (Chaetoceros sp., Ceratoneis closterium and Heterosigma akashiwo). Most notably, this also applies to a non-calcifying strain of E. huxleyi which displays a calcium sensitivity similar to the non-calcareous species. We hypothesize that the process of calcification in coccolithophores provides an efficient mechanism to alleviate cellular calcium poisoning and thereby offered a potential key evolutionary advantage, responsible for the proliferation of coccolithophores during times of high seawater calcium concentrations. The exact function of calcification and the reason behind the highly ornate physical structures of coccoliths remain elusive

Technical note: A comparison of methods for estimating coccolith mass

The fossil record of coccolithophores dates back approximately 225 million years, and the production of their calcite platelets (coccoliths) contributes to the global carbon cycle over short and geological timescales. Variations in coccolithophore parameters (e.g. community composition, morphology, size and coccolith mass) are a key factor for ocean biogeochemical dynamics (e.g. biological carbon pump) and have been used as a palaeoproxy to understand past oceanographic conditions. Coccolith mass has been frequently estimated with different methods with electron microscopy being the most applied. Here, we compared the electron microscopy (EM) method with the Coulter multisizer (CM) (i.e. electric field disturbance) and bidirectional circular polarization (BCP) methods to estimate coccolith masses (pg CaCO3) in controlled laboratory experiments with two ecotypes of Emiliania huxleyi. Average coccolith mass estimates were in good agreement with literature data. However, mass estimates from the CM were slightly overestimated compared to EM and BCP estimates, and a correction factor (cf=0.8) is suggested to compensate for this discrepancy. The relative change in coccolith mass triggered by morphotype-specific structures and environmental parameters (i.e. seawater carbonate chemistry) was suitably captured by each of the three techniques.</p

Mixotrophic uptake of organic compounds by coccolithophores

Osmotrophy is one of the main modes of mixotrophic acquisition of carbon by phytoplankton, but historically it has been under‐investigated and its physiological and ecological relevance remains poorly understood. Here, we investigate osmotrophy in coccolithophores. Coccolithophores are one of the major contributors to the ocean biomass inhabiting both euphotic and subeuphotic depths in the marine environment. Coccolithophores demonstrate the potential to utilize a wide array of organic compounds in darkness. In experiments with BioLog Ecoplates, we screened a wide array of organic compounds as potential carbon sources, and observed that the major types of organic compounds taken up by coccolithophores were primarily carbohydrates along with a few amino acids and polymers. Furthermore, in subsequent radiotracer experiments, the uptake rates of 14C‐labeled dissolved organic carbon compounds in the dark were low relative to the maximal rates of photosynthetic carbon fixation in the light. The time course of uptake for some compounds suggests constitutive capacity for their transport, while for others the transport appears to be activated. Nonetheless, the collective slow uptake rate of a large array of organic compounds found in seawater, might be the only way that osmotrophy could fuel significant coccolithophore growth in the deep euphotic and subeuphotic zones in the sea

Field validation of the southern rock lobster paralytic shellfish toxin monitoring program in Tasmania, Australia

Paralytic shellfish toxins (PST) are found in the hepatopancreas of Southern Rock Lobster Jasus edwardsii from the east coast of Tasmania in association with blooms of the toxic dinoflagellate Alexandrium catenella. Tasmania’s rock lobster fishery is one of the state’s most important wild capture fisheries, supporting a significant commercial industry (AUD 97M) and recreational fishing sector. A comprehensive 8 years of field data collected across multiple sites has allowed continued improvements to the risk management program protecting public health and market access for the Tasmanian lobster fishery. High variability was seen in toxin levels between individuals, sites, months, and years. The highest risk sites were those on the central east coast, with July to January identified as the most at-risk months. Relatively high uptake rates were observed (exponential rate of 2% per day), similar to filter-feeding mussels, and meant that lobster accumulated toxins quickly. Similarly, lobsters were relatively fast detoxifiers, losing up to 3% PST per day, following bloom demise. Mussel sentinel lines were effective in indicating a risk of elevated PST in lobster hepatopancreas, with annual baseline monitoring costing approximately 0.06% of the industry value. In addition, it was determined that if the mean hepatopancreas PST levels in five individual lobsters from a site were −1, there is a 97.5% probability that any lobster from that site would be below the bivalve maximum level of 0.8 mg STX equiv. kg−1. The combination of using a sentinel species to identify risk areas and sampling five individual lobsters at a particular site, provides a cost-effective strategy for managing PST risk in the Tasmanian commercial lobster fishery

The roles of endolithic fungi in bioerosion and disease in marine ecosystems. II. Potential facultatively parasitic anamorphic ascomycetes can cause disease in corals and molluscs

Anamorphic ascomycetes have been implicated as causative agents of diseases in tissues and skeletons of hard corals, in tissues of soft corals (sea fans) and in tissues and shells of molluscs. Opportunist marine fungal pathogens, such as Aspergillus sydowii, are important components of marine mycoplankton and are ubiquitous in the open oceans, intertidal zones and marine sediments. These fungi can cause infection in or at least can be associated with animals which live in these ecosystems. A. sydowii can produce toxins which inhibit photosynthesis in and the growth of coral zooxanthellae. The prevalence of many documented infections has increased in frequency and severity in recent decades with the changing impacts of physical and chemical factors, such as temperature, acidity and eutrophication. Changes in these factors are thought to cause significant loss of biodiversity in marine ecosystems on a global scale in general, and especially in coral reefs and shallow bays