Sudden peak in tetrodotoxin in French oysters during the summer of 2021: Source investigation using microscopy, metabarcoding and droplet digital PCR.

Tetrodotoxin (TTX) is a potent neurotoxin causing human intoxications from contaminated seafood worldwide and is of emerging concern in Europe. Shellfish have been shown to contain varying TTX concentrations globally, with concentrations typically higher in Pacific oysters Crassostrea gigas in Europe. Despite many decades of research, the source of TTX remains unknown, with bacterial or algal origins having been suggested. The aim of this study was to identify potential source organisms causing TTX contamination in Pacific oysters in French coastal waters, using three different techniques. Oysters were deployed in cages from April to September 2021 in an estuary where TTX was previously detected. Microscopic analyses of water samples were used to investigate potential microalgal blooms present prior or during the peak in TTX. Differences in the bacterial communities from oyster digestive glands (DG) and remaining flesh were explored using metabarcoding, and lastly, droplet digital PCR assays were developed to investigate the presence of Cephalothrix sp., one European TTX-bearing species in the DG of toxic C. gigas. Oysters analysed by liquid chromatography-tandem mass spectrometry contained quantifiable levels of TTX over a three-week period (24 June-15 July 2021), with concentrations decreasing in the DG from 424 µg/kg for the first detection to 101 µg/kg (equivalent to 74 to 17 µg/kg of total flesh), and trace levels being detected until August 13, 2021. These concentrations are the first report of the European TTX guidance levels being exceeded in French shellfish. Microscopy revealed that some microalgae bloomed during the TTX peak, (e.g., Chaetoceros spp., reaching 40,000 cells/L). Prokaryotic metabarcoding showed increases in abundance of Rubritaleaceae (genus Persicirhabdus) and Neolyngbya, before and during the TTX peak. Both phyla have previously been described as possible TTX-producers and should be investigated further. Droplet digital PCR analyses were negative for the targeted TTX-bearing genus Cephalothrix.

Differential responses of selectively bred mussels (Perna canaliculus) to heat stress-survival, immunology, gene expression and microbiome diversity.

New Zealand's green-lipped mussel (Perna canaliculus) is an ecologically and economically important species. Marine heatwaves are increasing in frequency around NZ's coastline, and these events are correlated with increased stress and mortality of some aquaculture species. This study aimed to identify general biomarkers of heat stress in P. canaliculus and to assess whether responses differed between genetically distinct selectively bred mussels. We exposed three families of selectively bred mussels (families A, B and C) to three seawater temperature regimes in the laboratory: 1) a "control" treatment (ambient 12°C), 2) a 26°C heat challenge with a subsequent recovery period, and 3) a sustained 26°C heat challenge with no recovery. We investigated whether the survival, immune response (hemocyte concentration and viability, oxidative stress and total antioxidant capacity), hemocyte gene expression and gill microbiome differed between the families during the temperature challenges. In the sustained heat-stress treatment, family A had the highest survival rate (42% compared with 25% and 5% for families C and B, respectively). Gene expression levels significantly shifted during thermal stress and differed between families, with family A more dissimilar than families B and C. Family C had substantially more genes impacted by temperature treatment and timepoint than the other families, while family B had very little genes/pathways that responded to thermal stress. Genes related to heat shock proteins and immune responses (e.g., AIF1, CTSC, TOLL8, CASP9, FNTA, AHCY, CRYAB, PPIF) were upregulated in all families during heat stress. Microbiome species-richness differed between families before and during heat-stress, with family A having a distinctly different microbiome flora than the other families. Microbial diversity changed similarly in all families exposed to prolonged heat-stress, with species of Vibrio and Campylobacter increasing in these mussels. Our study highlights the use of non-lethal sampling of hemocytes as a diagnostic tool to explore the immune response and gene expression of selectively bred mussels, to predict their response to ocean warming. This approach can identify potential thermotolerant candidates for further selective breeding, which may increase the resilience of the mussel aquaculture industry in a warming ocean.

Effects of Harmful Algal Blooms on Fish and Shellfish Species: A Case Study of New Zealand in a Changing Environment.

Harmful algal blooms (HABs) have wide-ranging environmental impacts, including on aquatic species of social and commercial importance. In New Zealand (NZ), strategic growth of the aquaculture industry could be adversely affected by the occurrence of HABs. This review examines HAB species which are known to bloom both globally and in NZ and their effects on commercially important shellfish and fish species. Blooms of Karenia spp. have frequently been associated with mortalities of both fish and shellfish in NZ and the sub-lethal effects of other genera, notably Alexandrium spp., on shellfish (which includes paralysis, a lack of byssus production, and reduced growth) are also of concern. Climate change and anthropogenic impacts may alter HAB population structure and dynamics, as well as the physiological responses of fish and shellfish, potentially further compromising aquatic species. Those HAB species which have been detected in NZ and have the potential to bloom and harm marine life in the future are also discussed. The use of environmental DNA (eDNA) and relevant bioassays are practical tools which enable early detection of novel, problem HAB species and rapid toxin/HAB screening, and new data from HAB monitoring of aquaculture production sites using eDNA are presented. As aquaculture grows to supply a sizable proportion of the world's protein, the effects of HABs in reducing productivity is of increasing significance. Research into the multiple stressor effects of climate change and HABs on cultured species and using local, recent, HAB strains is needed to accurately assess effects and inform stock management strategies.

Deciphering the molecular signal from past and alive bacterial communities in aquatic sedimentary archives.

Lake sediments accumulate information on biological communities thus acting as natural archives. Traditionally paleolimnology has focussed on fossilized remains of organisms, however, many organisms do not leave fossil evidence, meaning major ecosystem components are missing from environmental reconstructions. Many paleolimnology studies now incorporate molecular methods, including investigating microbial communities using environmental DNA (eDNA), but there is uncertainty about the contribution of living organisms to molecular inventories. In the present study, we obtained DNA and RNA inventories from sediment spanning 700 years to investigate the contribution of past and active communities to the molecular signal from sedimentary archives. Additionally, a droplet digital PCR (ddPCR) targeting the 16S ribosomal RNA (16S rRNA) gene of the photosynthetic cyanobacterial genera Microcystis was used to explore if RNA signals were from legacy RNA. We posit that the RNA signal is a mixture of legacy RNA, dormant cells, living bacteria and modern-day trace level contaminants that were introduced during sampling and preferentially amplified. The presence of legacy RNA was confirmed by the detection of Microcystis in sediments aged to ~200 years ago. Recent comparisons between 16S rRNA gene metabarcoding and traditional paleo proxies showed that past changes in bacterial communities can be reconstructed from sedimentary archives. The recovery of RNA in the present study has provided new insights into the origin of these signals. However, caution is required during analysis and interpretation of 16S rRNA gene metabarcoding data especially in recent sediments were there are potentially active bacteria.

A bacterial index to estimate lake trophic level: National scale validation.

Lakes and their catchments have been subjected to centuries to millennia of exploitation by humans. Efficient monitoring methods are required to promote proactive protection and management. Traditional monitoring is time consuming and expensive, which limits the number of lakes monitored. Lake surface sediments provide a temporally integrated representation of environmental conditions and contain high microbial biomass. Based on these attributes, we hypothesized that bacteria associated with lake trophic states could be identified and used to develop an index that would not be confounded by non-nutrient stressor gradients. Metabarcoding (16S rRNA gene) was used to assess bacterial communities present in surface sediments from 259 non-saline lakes in New Zealand encompassing a range of trophic states from alpine microtrophic lakes to lowland hypertrophic lakes. A subset of lakes (n = 96) with monitoring data was used to identify indicator amplicon sequence variants (ASVs) associated with different trophic states. A total of 10,888 indicator taxa were identified and used to develop a Sediment Bacterial Trophic Index (SBTI), which signficantly correlated (r2 = 0.842, P < 0.001) with the Trophic Lake Index. The SBTI was then derived for the remaining 163 lakes, providing new knowledge of the trophic state of these unmonitored lakes. This new, robust DNA-based tool provides a rapid and cost-effective method that will allow a greater number of lakes to be monitored and more effectively managed in New Zealand and globally. The SBTI could also be applied in a paleolimnological context to investigate changes in trophic status over centuries to millennia.

Winter diet of Japanese macaques from Chubu Sangaku National Park, Japan incorporates freshwater biota.

The Japanese macaque (Macaca fuscata) is native to the main islands of Japan, except Hokkaido, and is the most northerly living non-human primate. In the Chubu Sangaku National Park of the Japanese Alps, macaques live in one of the coldest areas of the world, with snow cover limiting the availability of preferred food sources. Winter is typically a bottleneck for food availability potentially resulting in marked energy deficits, and mortality may result from famine. However, streams with groundwater upwelling flow during the winter with a constant water temperature of about 5 °C are easily accessible for Japanese macaques to search for riverine biota. We used metabarcoding (Cytochrome c oxidase I) of fecal samples from Japanese macaques to determine their wintertime diet. Here we provide the first robust evidence that Japanese macaques feed on freshwater biota, including brown trout, riverine insects and molluscs, in Chubu Sangaku National Park. These additional food sources likely aid their winter survival.

A Microencapsulation Method for Delivering Tetrodotoxin to Bivalves to Investigate Uptake and Accumulation.

Most marine biotoxins are produced by microalgae. The neurotoxin tetrodotoxin (TTX) has been reported in many seafood species worldwide but its source is unknown, making accumulation and depuration studies in shellfish difficult. Tetrodotoxin is a water-soluble toxin and cannot be directly ingested by shellfish. In the present study, a method was developed which involved binding TTX to solid particles of humic acid and encapsulating them in agar-gelatin capsules. A controlled quantity of TTX-containing microcapsules (size range 20-280 µm) was fed to Paphies australis, a bivalve known to accumulate TTX in the wild. The TTX-containing microcapsules were fed to P. australis every second day for 13 days. Ten P. australis (including five controls fed non-toxic microalgae) were harvested after 7 days and ten after 13 days. Paphies australis accumulated TTX, reaching concentrations of up to 103 µg kg-1 by day 13, exceeding the European Food Safety Authority recommended concentration of 44 µg kg-1 in shellfish. This novel method will allow future studies to explore the effects, accumulation and depuration rates of TTX in different animals and document how it is transferred through food webs.

Comparing sediment DNA extraction methods for assessing organic enrichment associated with marine aquaculture.

Marine sediments contain a high diversity of micro- and macro-organisms which are important in the functioning of biogeochemical cycles. Traditionally, anthropogenic perturbation has been investigated by identifying macro-organism responses along gradients. Environmental DNA (eDNA) analyses have recently been advocated as a rapid and cost-effective approach to measuring ecological impacts and efforts are underway to incorporate eDNA tools into monitoring. Before these methods can replace or complement existing methods, robustness and repeatability of each analytical step has to be demonstrated. One area that requires further investigation is the selection of sediment DNA extraction method. Environmental DNA sediment samples were obtained along a disturbance gradient adjacent to a Chinook (Oncorhynchus tshawytscha) salmon farm in Otanerau Bay, New Zealand. DNA was extracted using four extraction kits (Qiagen DNeasy PowerSoil, Qiagen DNeasy PowerSoil Pro, Qiagen RNeasy PowerSoil Total RNA/DNA extraction/elution and Favorgen FavorPrep Soil DNA Isolation Midi Kit) and three sediment volumes (0.25, 2, and 5 g). Prokaryotic and eukaryotic communities were amplified using primers targeting the 16S and 18S ribosomal RNA genes, respectively, and were sequenced on an Illumina MiSeq. Diversity and community composition estimates were obtained from each extraction kit, as well as their relative performance in established metabarcoding biotic indices. Differences were observed in the quality and quantity of the extracted DNA amongst kits with the two Qiagen DNeasy PowerSoil kits performing best. Significant differences were observed in both prokaryotes and eukaryotes (p < 0.001) richness among kits. A small proportion of amplicon sequence variants (ASVs) were shared amongst the kits (~3%) although these shared ASVs accounted for the majority of sequence reads (prokaryotes: 59.9%, eukaryotes: 67.2%). Differences were observed in the richness and relative abundance of taxonomic classes revealed with each kit. Multivariate analysis showed that there was a significant interaction between "distance" from the farm and "kit" in explaining the composition of the communities, with the distance from the farm being a stronger determinant of community composition. Comparison of the kits against the bacterial and eukaryotic metabarcoding biotic index suggested that all kits showed similar patterns along the environmental gradient. Overall, we advocate for the use of Qiagen DNeasy PowerSoil kits for use when characterizing prokaryotic and eukaryotic eDNA from marine farm sediments. We base this conclusion on the higher DNA quality values and richness achieved with these kits compared to the other kits/amounts investigated in this study. The additional advantage of the PowerSoil Kits is that DNA extractions can be performed using an extractor robot, offering additional standardization and reproducibility of results.

Survey of Tetrodotoxin in New Zealand Bivalve Molluscan Shellfish over a 16-Month Period.

Tetrodotoxin (TTX) is a heat-stable neurotoxin typically associated with pufferfish intoxications. It has also been detected in shellfish from Japan, the United Kingdom, Greece, China, Italy, the Netherlands and New Zealand. A recent European Food Safety Authority (EFSA) scientific opinion concluded that a level of <0.044 mg TTX/kg in marine bivalves and gastropods, based on a 400 g portion size, does not result in adverse effects in humans. There have been no reports of human illness attributed to the consumption of New Zealand shellfish containing TTX. To obtain a greater understanding of its presence, a survey of non-commercial New Zealand shellfish was performed between December 2016 and March 2018. During this period, 766 samples were analysed from 8 different species. TTX levels were found to be low and similar to those observed in shellfish from other countries, except for pipi (Paphies australis), a clam species endemic to New Zealand. All pipi analysed as part of the survey were found to contain detectable levels of TTX, and pipi from a sampling site in Hokianga Harbour contained consistently elevated levels. In contrast, no TTX was observed in cockles from this same sampling site. No recreationally harvested shellfish species, including mussels, oysters, clams and tuatua, contained TTX levels above the recommended EFSA safe guidance level. The levels observed in shellfish were considerably lower than those reported in other marine organisms known to contain TTX and cause human intoxication (e.g., pufferfish). Despite significant effort, the source of TTX in shellfish, and indeed all animals, remains unresolved making it a difficult issue to understand and manage.

Seasonal and Spatial Variations in Bacterial Communities From Tetrodotoxin-Bearing and Non-tetrodotoxin-Bearing Clams.

Tetrodotoxin (TTX) is one of the most potent naturally occurring compounds and is responsible for many human intoxications worldwide. Paphies australis are endemic clams to New Zealand which contain varying concentrations of TTX. Research suggests that P. australis accumulate the toxin exogenously, but the source remains uncertain. The aim of this study was to identify potential bacterial TTX-producers by exploring differences in bacterial communities in two organs of P. australis: the siphon and digestive gland. Samples from the digestive glands of a non-toxic bivalve Austrovenus stutchburyi that lives amongst toxic P. australis populations were also analyzed. Bacterial communities were characterized using 16S ribosomal RNA gene metabarcoding in P. australis sourced monthly from the Hokianga Harbor, a site known to have TTX-bearing clams, for 1 year, from ten sites with varying TTX concentrations around New Zealand, and in A. stutchburyi from the Hokianga Harbor. Tetrodotoxin was detected in P. australis from sites all around New Zealand and in all P. australis collected monthly from the Hokianga Harbor. The toxin averaged 150 µg kg-1 over the year of sampling in the Hokianga Harbor but no TTX was detected in the A. stutchburyi samples from the same site. Bacterial species diversity differed amongst sites (p < 0.001, F = 5.9) and the diversity in siphon samples was significantly higher than in digestive glands (p < 0.001, F = 65.8). Spirochaetaceae (4-60%) and Mycoplasmataceae (16-78%) were the most abundant families in the siphons and the digestive glands, respectively. The bacterial communities were compared between sites with the lowest TTX concentrations and the Hokianga Harbor (site with the highest TTX concentrations), and the core bacterial communities from TTX-bearing individuals were analyzed. The results from both spatial and temporal studies corroborate with previous hypotheses that Vibrio and Bacillus could be responsible for the source of TTX in bivalves. The results from this study also indicate that marine cyanobacteria, in particular picocyanobacteria (e.g., Cyanobium, Synechococcus, Pleurocapsa, and Prochlorococcus), should be investigated further as potential TTX producers.

Elucidating Biodiversity Shifts in Ballast Water Tanks during a Cross-Latitudinal Transfer: Complementary Insights from Molecular Analyses.

In this study, the evolution of ballast water (BW) assemblages across different trophic levels was characterized over a 21 day cross-latitudinal vessel transit using a combination of molecular methods. Triplicate BW samples were collected every second day and size-fractionated (<2.7, 10, >50 µm). Measurements of adenosine triphosphate (ATP) and metabarcoding of environmental nucleic acid (DNA and RNA) analyses, complemented by microscopy and flow cytometry, were performed on each sample. Measured ATP concentrations exhibited high variance between replicates and a strong negative trend in the large (≥50 µm) fraction over the voyage. In concert with microscopy, the metabarcoding data indicated a die-off of larger metazoans during the first week of study and gradual reductions in dinoflagellates and ochrophytes. The ATP and metabarcoding data signaled persistent or increased cellular activity of heterotrophic bacteria and protists in the BW, which was supported by flow cytometry. The metabarcoding showed the presence of active bacteria in all size fractions, suggesting that the sequential filtration approach does not ensure taxonomical differentiation, which has implications for BW quality assessment. Although our data show that ATP and metabarcoding have potential for indicative BW screening for BW compliance monitoring, further research and technological development is needed to improve representativeness of sampling and deliver the unequivocal response criteria required by the international Ballast Water Management Convention.

Assessment of Ciguatera and Other Phycotoxin-Related Risks in Anaho Bay (Nuku Hiva Island, French Polynesia): Molecular, Toxicological, and Chemical Analyses of Passive Samplers.

Ciguatera poisoning is a foodborne illness caused by the consumption of seafood contaminated with ciguatoxins (CTXs) produced by dinoflagellates from the genera Gambierdiscus and Fukuyoa. The suitability of Solid Phase Adsorption Toxin Tracking (SPATT) technology for the monitoring of dissolved CTXs in the marine environment has recently been demonstrated. To refine the use of this passive monitoring tool in ciguateric areas, the effects of deployment time and sampler format on the adsorption of CTXs by HP20 resin were assessed in Anaho Bay (Nuku Hiva Island, French Polynesia), a well-known ciguatera hotspot. Toxicity data assessed by means of the mouse neuroblastoma cell-based assay (CBA-N2a) showed that a 24 h deployment of 2.5 g of resin allowed concentrating quantifiable amounts of CTXs on SPATT samplers. The CTX levels varied with increasing deployment time, resin load, and surface area. In addition to CTXs, okadaic acid (OA) and dinophysistoxin-1 (DTX1) were also detected in SPATT extracts using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), consistent with the presence of Gambierdiscus and Prorocentrum species in the environment, as assessed by quantitative polymerase chain reaction (qPCR) and high-throughput sequencing (HTS) metabarcoding analyses conducted on passive window screen (WS) artificial substrate samples. Although these preliminary findings await further confirmation in follow-up studies, they highlight the usefulness of SPATT samplers in the routine surveillance of CP risk on a temporal scale, and the monitoring of other phycotoxin-related risks in ciguatera-prone areas.

Local factors drive bacterial and microeukaryotic community composition in lake surface sediment collected across an altitudinal gradient.

Lake surface sediments are dominated by microorganisms that play significant roles in biogeochemical cycling within lakes. There is limited knowledge on the relative importance of local environmental factors and altitude on bacterial and microeukaryotic community richness and composition in lake sediments. In the present study, surface sediment samples were collected from 40 lakes along an altitude gradient (2-1215 m). Microbial communities were characterized using 16S (bacteria) and 18S (microeukaryotes) rRNA gene metabarcoding. Bacterial and microeukaryotic richness were not correlated with altitude but instead to environmental variables (e.g. area of water in the catchment (bacteria: R = -0.43). For both bacteria and microeukaryotes, dissimilarity in the community structure had a higher correlation to combined environmental variables (without altitude) (bacteria: R = 0.53; microeukaryotes: R = 0.55) than altitude alone (bacteria: R = 0.34; microeukaryotes: R = 0.47). Sediment sulfur and productive grassland were important variables in determining the relative abundance of sulfate reducing bacteria. Nitrospira, was positively related to altitude but negatively to water column total organic carbon and the proportion of productive grassland in the catchment. Little overlap in amplicon sequence variants was shown amongst lakes. This has important considerations for management decisions, suggesting that to protect biodiversity, conservation of numerous lakes and lake types is required.

Release and degradation of environmental DNA and RNA in a marine system.

Over the last decade, there has been growing interest in the analysis of environmental DNA (eDNA) to infer the presence of organisms in aquatic environments. The efficacy of eDNA/eRNA based tools are highly depend on the turnover rate of the molecule (their release and degradation). Environmental DNA has been shown to persist for days, weeks or years in environmental samples. Environmental RNA (eRNA) is thought to degrade faster than eDNA, however to our knowledge, no experimental studies have explored this. Here we present an aquarium study to investigate eDNA and eRNA shedding rates and degradation for two sessile marine invertebrates. The copy numbers for eDNA and eRNA were assessed using droplet digital PCR targeting the mitochondrial Cytochrome c Oxidase subunit 1 (COI) gene. Environmental RNA persisted after organism removal for much longer than expected with detections for up to 13 h. In contrast, eDNA was detected is samples collected up to 94 h after organism removal. There was no evidence that the decay rates constants for eDNA and eRNA were different (p = 0.6, Kruskal-Wallis tests). Both eDNA and eRNA was detected in biofilms collected at the end of the experiment (day 21). This suggests binding with organic or inorganic compounds or stabilization of these molecules in the biofilm matrix. The finding of the prolonged persistence of eRNA may provide new opportunities for improved biodiversity surveys through reducing false positives caused by legacy DNA and could also facilitate new research on environmental transcriptomics.

Tetrodotoxin in marine bivalves and edible gastropods: A mini-review.

Tetrodotoxin (TTX) is a potent neurotoxin responsible for countless human intoxications and deaths around the world. The distribution of TTX and its analogues is diverse and the toxin has been detected in organisms from both marine and terrestrial environments. Increasing detections seafood species, such as bivalves and gastropods, has drawn attention to the toxin, reinvigorating scientific interest and regulatory concerns. There have been reports of TTX in 21 species of bivalves and edible gastropods from ten countries since the 1980's. While TTX is structurally dissimilar to saxitoxin (STX), another neurotoxin detected in seafood, it has similar sodium channel blocking action and potency and both neurotoxins have been shown to have additive toxicities. The global regulatory level for the STX group toxins applied to shellfish is 800 µg/kg. The presence of TTX in shellfish is only regulated in one country; The Netherlands, with a regulatory level of 44 µg/kg. Due to the recent interest surrounding TTX in bivalves, the European Food Safety Authority established a panel to assess the risk and regulation of TTX in bivalves, and their final opinion was that a concentration below 44 µg of TTX per kg of shellfish would not result in adverse human effects. In this article, we review current knowledge on worldwide TTX levels in edible gastropods and bivalves over the last four decades, the different methods of detection used, and the current regulatory status. We suggest research needs that will assist with knowledge gaps and ultimately allow development of robust monitoring and management protocols.

Spatial variability and depuration of tetrodotoxin in the bivalve Paphies australis from New Zealand.

Tetrodotoxin (TTX) is a potent neurotoxin responsible for many human intoxications globally. Despite its potency and widespread occurrence in taxonomically diverse species, the primary source of TTX remains uncertain. Paphies australis, an endemic clam found in New Zealand, has been found to contain TTX in several locations. However, it is unknown if this represents endogenous production or accumulation from an external source. To address this question, the concentrations of TTX in whole P. australis and dissected organs (siphons, foot, digestive gland and the 'rest') from thirteen sites around New Zealand were determined using liquid chromatography-tandem quadrupole mass spectrometry analysis (LC-MS/MS). Depuration rate of TTX was also investigated by harvesting and measuring concentrations in P. australis maintained in captivity on a toxin-free diet every three to 15 days for 150 days. The LC-MS/MS analyses of the spatial samples showed that TTX was present in P. australis from all regions tested, with significantly (p < 0.001) higher concentrations (15-50 µg kg-1) observed at lower latitudes of the North Island compared with trace levels (0.5-3 µg kg-1) in the South Island of New Zealand. Tetrodotoxin was detected in all the dissected organs but the siphons contained the highest concentrations of TTX at all sites analysed. A linear model of the depuration data identified a significant (p < 0.001) decline in total TTX concentrations in P. australis over the study period. The siphons maintained the highest amount of TTX across the entire depuration study. The digestive glands contained low concentrations at the start of the experiment, but this depurated rapidly and only traces remained after 21 days. These results provide evidence to suggest that P. australis does not produce TTX endogenously but obtains the neurotoxin from an exogenous source (e.g., diet) with the source more prevalent in warmer northern waters. The association of higher TTX concentrations in shellfish with warmer environments raises concerns that this toxin's distribution and abundance could become an increasing human health issue with global warming.

Anatoxins are consistently released into the water of streams with Microcoleus autumnalis-dominated (cyanobacteria) proliferations.

Proliferations of potentially toxic, mat-forming Microcoleus are increasing in streams globally. A range of cyanotoxins are produced by Microcoleus, with the neurotoxic anatoxins (anatoxin-a, dihydro-anatoxin-a, homoanatoxin-a and dihydro-homoanatoxin-a) the most commonly reported. The anatoxins produced by Microcoleus are thought to be largely contained within the cells. More knowledge on whether anatoxins are been released into the overlying stream water is required to better assess health risks to human, animals, and aquatic organisms. Field studies were conducted in three streams experiencing toxic Microcoleus autumnalis (basionym Phormidium autumnale)-dominated proliferations. Samples were collected every 1.5-3 h over a 24- or 26-h sampling period. Water samples were analyzed for total (intracellular and dissolved) and dissolved anatoxins, and time-integrated anatoxin samples were collected using solid phase adsorption tracking technology (SPATT). Anatoxins were detected in all stream water and SPATT samples (max. 0.91 ng mL-1 and 95 ng g-1 of strata-x hr-1). At two sites, anatoxins were largely dissolved, whereas at the third site only total anatoxins could be detected. Temporal variability in anatoxin concentrations was observed, but there were no evident patterns between sampling sites. Linear regression showed a very weakstatistically significant relationship (R2 = 0.24, p = 0.002) between total anatoxin concentrations in water and SPATT, however, when tested per site, only one of the three showed a significant relationship. These results highlight the potential for chronic exposure to anatoxins for humans (i.e., through drinking water) and aquatic organisms in streams with M. autumnalis proliferations. The health implications of this are unknown.

Distribution of Tetrodotoxin in the New Zealand Clam, Paphies australis, Established Using Immunohistochemistry and Liquid Chromatography-Tandem Quadrupole Mass Spectrometry.

Tetrodotoxin (TTX) is one of the most potent neurotoxins known. It was originally thought to only occur in puffer fish but has now been identified in twelve different classes of freshwater and marine organisms, including bivalves. Despite being one of the world’s most studied biotoxins, its origin remains uncertain. There is contradictory evidence regarding the source of TTX and its pathway through food webs. To date, the distribution of TTX has not been examined in bivalves. In the present study, 48 Paphies australis, a TTX-containing clam species endemic to New Zealand, were collected. Thirty clams were dissected, and organs and tissues pooled into five categories (siphons, digestive gland, adductor muscles, and the ‘rest’) and analyzed for TTX using liquid chromatography-mass spectrometry (LC-MS). The micro-distribution of TTX was visualized in the remaining 18 individuals using an immunohistological technique incorporating a TTX-specific monoclonal antibody. The LC-MS analysis revealed that siphons contained the highest concentrations of TTX (mean 403.8 µg/kg). Immunohistochemistry analysis showed TTX in the outer cells of the siphons, but also in the digestive system, foot, and gill tissue. Observing TTX in organs involved in feeding provides initial evidence to support the hypothesis of an exogenous source in P. australis.

Molecular Identification of Gambierdiscus and Fukuyoa (Dinophyceae) from Environmental Samples.

Ciguatera Fish Poisoning (CFP) is increasing across the Pacific and the distribution of the causative dinoflagellates appears to be expanding. Subtle differences in thecal plate morphology are used to distinguish dinoflagellate species, which are difficult to determine using light microscopy. For these reasons we sought to develop a Quantitative PCR assay that would detect all species from both Gambierdiscus and Fukuyoa genera in order to rapidly screen environmental samples for potentially toxic species. Additionally, a specific assay for F. paulensis was developed as this species is of concern in New Zealand coastal waters. Using the assays we analyzed 31 samples from three locations around New Zealand and the Kingdom of Tonga. Fourteen samples in total were positive for Gambierdiscus/Fukuyoa and two samples were also positive using the F. paulensis assay. Samples from the Kermadec Islands were further characterized using high-throughput sequencing metabarcoding. The majority of reads corresponded to Gambierdiscus species with three species identified at all sites (G. australes, G. honu and G. polynesiensis). This is the first confirmed identification of G. polynesiensis, a known ciguatoxin producer, in New Zealand waters. Other known toxin-producing genera were also detected, included Alexandrium, Amphidinium, Azadinium, Dinophysis, Ostreopsis, and Prorocentrum.