Effects of the toxic dinoflagellate, Alexandrium pacificum, on the marine diatom, Chaetoceros muelleri, and mussel (Perna canaliculus) sperm and hemocytes.

Harmful algal blooms (HABs) of Alexandrium pacificum have affected the Marlborough Sounds in New Zealand since 2010, posing a threat to green-lipped mussel (GLM, Perna canaliculus) farming. Previous studies have shown A. pacificum has negative effects GLM embryos and larvae. To further investigate these toxic mechanisms, in vitro bioassays were conducted on GLM spermatozoa, hemocytes, and the diatom, Chaetoceros muelleri. The three cell types were exposed to several treatments of A. pacificum for 2 h and responses were measured using flow cytometry and pulse amplitude-modulated fluorometry. Significant spermatozoa mortality was recorded in treatments containing A. pacificum cells or fragments, while hemocyte and C. muelleri mortality was recorded in cell-free treatments of A. pacificum which contained paralytic shellfish toxins (PSTs). Variation in sensitivity between cell types as well as the sublethal effects observed, emphasise the diverse toxic mechanisms of A. pacificum on co-occurring species in the environment.

Spatial distribution of lipophilic shellfish toxins in seawater and sediment in the Bohai Sea and the Yellow Sea, China.

Lipophilic shellfish toxins (LSTs) are widely distributed in marine environments worldwide, potentially threatening marine ecosystem health and aquaculture safety. In this study, two large-scale cruises were conducted in the Bohai Sea and the Yellow Sea, China, in spring and summer 2023 to clarify the composition, concentration, and spatial distribution of LSTs in the water columns and sediments. Results showed that okadaic acid (OA), dinophysistoxin-1 (DTX1) and/or pectenotoxin-2 (PTX2) were detected in 249 seawater samples collected in spring and summer. The concentrations of ∑LSTs in seawater were ranging of ND (not detected) -13.86, 1.60-17.03, 2.73-17.39, and 1.26-30.21 pmol L-1 in the spring surface, intermediate, bottom water columns and summer surface water layers, respectively. The detection rates of LSTs in spring and summer seawater samples were 97% and 100%, respectively. The high concentrations of ∑LSTs were mainly distributed in the north Yellow Sea and the northeast Bohai Sea in spring, and in the northeast Yellow Sea, the waters around Laizhou Bay and Rongcheng Bay in summer. Similarly, only OA, DTX1 and PTX2 were detected in the surface sediments. Overall, the concentration of ∑LSTs in the surface sediments of the northern Yellow Sea was higher than that in other regions. In sediment cores, PTX2 was mainly detected in the upper sediment samples, whereas OA and DTX1 were detected in deeper sediments, and LSTs can persist in the sediments for a long time. Overall, OA, DTX1 and PTX2 were widely distributed in the water column and surface sediments in the Bohai Sea and the Yellow Sea, China. The results of this study contribute to the understanding of spatial distribution of LSTs in seawater and sediment environmental media and provide basic information for health risk assessment of phycotoxins.

Occurrence and Exposure Assessment of Lipophilic Shellfish Toxins in the Zhejiang Province, China.

Although lipophilic shellfish toxins (LSTs) pose a significant threat to the health of seafood consumers, their systematic investigation and risk assessment remain scarce. The goals of this study were as follows: (1) analyze LST levels in commercially available shellfish in Zhejiang province, China, and determine factors influencing LST distribution; (2) assess the acute dietary risk of exposure to LSTs for local consumers during the red tide period; (3) explore potential health risks of LSTs in humans; and (4) study the acute risks of simultaneous dietary exposure to LSTs and paralytic shellfish toxins (PSTs). A total of 546 shellfish samples were collected. LSTs were detected in 89 samples (16.3%) at concentrations below the regulatory limits. Mussels were the main shellfish species contaminated with LSTs. Spatial variations were observed in the yessotoxin group. Acute exposure to LSTs based on multiple scenarios was low. The minimum tolerable exposure durations for LSTs calculated using the mean and the 95th percentile of consumption data were 19.7 and 4.9 years, respectively. Our findings showed that Zhejiang province residents are at a low risk of combined exposure to LSTs and PSTs; however, the risk may be higher for children under 6 years of age in the extreme scenario.

Assay for okadaic acid O-acyl transferase using HPLC-FLD.

Dinophysistoxin 1 (DTX1, 1) and okadaic acid (OA, 2), produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp., are primary diarrhetic shellfish toxins (DSTs), which may cause gastric illness in people consuming such as bivalves. Both compounds convert to dinophysistoxin 3 (DTX3, 3; generic name for 1 and 2 with fatty acids conjugated at 7-OH) in bivalves. The enzyme okadaic acid O-acyl transferase (OOAT) is a membrane protein found in the microsomes of the digestive glands of bivalves. In this study, we established an in vitro enzymatic conversion reaction using 4-nitro-2,1,3-benzoxadiazole (NBD)-OA (4), an OA derivative conjugated with (R)-(-)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (NBD-APy) on 1-CO2H, as a substrate. We detected the enzymatically produced 3, NBD-7-O-palmitoyl-OA (NBD-Pal-OA), using high-performance liquid chromatography-fluorescence detection. We believe that an OOAT assay using 4 will facilitate the fractionation and isolation of OOAT in the future.

Seasonal variability of trace elements bioaccumulation in Pacific Oysters (Crassostrea gigas) from an experimental pilot farm in the Calich Lagoon (Sardinia, Italy).

BACKGROUND: Metals pollution is a worldwide environmental issue due to their persistence in the ecosystems, non-degradability, and bioaccumulation in marine biota. Pacific Oysters (Crassostrea gigas) are highly nutritious bivalve representing an important dietary constituent but may accumulate metals through feeding on suspended sediments from surrounding water, then represent a suitable tool for biomonitoring. MATERIALS AND METHODS: The occurrence of trace elements (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Rb, Se, Sn, V, Zn) was investigated in Pacific Oysters (Cassostrea gigas) collected from Calich Lagoon in each season of 2019. Samples were homogenized and subjected to microwave acid digestion before being analyzed by inductively coupled plasma-mass spectrometer (ICP-MS). RESULTS: The results showed a significant seasonal variation for temperature, dissolved oxygen, chlorophyll, and pH. Moreover, high significant seasonal variation in concentrations of Cd, Mn, Ni, and V was recorded. The highest values were found for Fe (128 mg kg⁻1 w.w.), and Al (112 mg kg⁻1 w.w.) in October, for Zn (113 mg kg⁻1 w.w.) in March and May. CONCLUSIONS: Pacific Oysters were confirmed as suitable bioindicators of the health status of coastal lagoons; trace elements concentrations were highly affected by season of collection, and according to literature the highest values were recorded in autumn and summer. The EU legal limits for Cd and Pb were not exceeded, then the farmed oysters were safe to consumers.

Okadaic acid as a major problem for the seafood safety (Mytilus galloprovincialis) and the dynamics of toxic phytoplankton in the Slovenian coastal sea (Gulf of Trieste, Adriatic Sea).

This article presents the first results on shellfish toxicity in the Slovenian sea (Gulf of Trieste, Adriatic Sea) since the analytical methods for the detection of biotoxins (PSP, ASP, DSP and other lipophilic toxins) in bivalve molluscs were included in the national monitoring program in 2013. In addition to toxins, the composition and abundance of toxic phytoplankton and general environmental characteristics of the seawater (surface temperature and salinity) were also monitored. During the 2014-2019 study period, only lipophilic toxins were detected (78 positive tests out of 446 runs), of which okadaic acid (OA) predominated in 97 % of cases, while dinophysistoxin-2 and yessotoxins only gave a positive result in one sampling event each. The number of samples that did not comply with the EC Regulation for the OA group was 17 or 3.8 % of all tests performed, all of which took place from September to November, while a few positive OA tests were also recorded in December, April, and May. This toxicity pattern was consistent with the occurrence pattern of the five most common DSP-producing dinoflagellates, which was supported by the development of warm and thermohaline stratified waters: Dinophysis caudata, D. fortii, D. sacculus, D. tripos and Phalacroma rotundatum. The strong correlation (r = 0.611, p < 0.001) between D. fortii, reaching abundances of up to 950 cells L-1, and OA suggests that D. fortii is the main cause of OA production in Slovenian waters. Strong interannual variations in OA and phytoplankton dynamics, exacerbated by the effects of anthropogenic impacts in this coastal ecosystem, reduce the predictability of toxicity events and require continuous and efficient monitoring. Our results also show that the introduction of the LC-MS/MS method for lipophilic toxins has improved the management of aquaculture activities, which was not as accurate based on mouse bioassays.

A comparison of the MPN and pour plate methods for estimating shellfish contamination by Escherichia coli.

AIMS: Shellfish production areas are classified for suitability for human consumption using counts of E.coli in shellfish samples. Two alternative laboratory methods are approved in the EU and UK for measuring E. coli in shellfish samples; the MPN and pour plate methods. These methods have inherently different statistical uncertainty and may give different counts for the same sample. Using two approaches: simulated data and spiking experiments, we investigate the theoretical properties of the two methods to determine their reliability for shellfish waters classification. METHODS AND RESULTS: Assuming a Poisson distribution of E. coli in shellfish samples, we simulate concentrations in 10,000 samples using the MPN and pour plate methods. We show that for higher concentrations of E. coli the pour plate method becomes increasingly more reliable than the MPN method. The MPN method has higher probabilities than pour plate of generating results exceeding shellfish classification thresholds, while conversely having higher probabilities of failing to detect counts that exceed regulatory thresholds. The theoretical analysis also demonstrates that the MPN method can produce genuine extreme outliers, even when E. coli are randomly distributed within the sampled material. A laboratory spiking experiment showed results consistent with the theoretical analysis, suggesting the Poisson assumption used in the theoretical analysis is reasonable. CONCLUSION: The large differences in statistical properties between the pour plate and MPN methods should be taken into consideration in classifying shellfish beds, with the pour plate method being more reliable over the crucial range of E. coli concentrations used to determine class boundaries.

Insights into the combined toxicity and mechanisms of BDE-47 and PFOA in marine blue mussel: An integrated study at the physiochemical and molecular levels.

The coexistence of multiple emerging contaminants imposes a substantial burden on the ecophysiological functions in organisms. The combined toxicity and underlying mechanism requires in-depth understanding. Here, marine blue mussel (Mytilus galloprovincialis L.) was selected and exposed to 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and perfluorooctanoic acid (PFOA) individually and in combination at environmental related concentrations to elucidate differences in stress responses and potential toxicological mechanisms. Characterization and comparison of accumulation, biomarkers, histopathology, transcriptomics and metabolomics were performed. Co-exposure resulted in differential accumulation patterns, exacerbated histopathological alterations, and different responses in oxidative stress and biomarkers for xenobiotic transportation. Moreover, the identified differentially expressed genes (DEGs) and differential metabolites (DEMs) in mussels were found to be annotated to different metabolic pathways. Correlation analyses further indicated that DEGs and DEMs were significantly correlated with the above biomarkers. BDE-47 and PFOA altered the genes and metabolites related to amino acid metabolism, energy and purine metabolism, ABC transporters, and glutathione metabolism to varying degrees, subsequently inducing accumulation differences and combined toxicity. Furthermore, the present work highlighted the pivotal role of Nrf2-keap1 detoxification pathway in the acclimation of M. galloprovincialis to reactive oxygen species (ROS) stress induced by BDE-47 and PFOA. This study enabled more comprehensive understanding of combined toxic mechanism of multi emerging contaminants pollution.

Estimating the risks of exposure to harmful algal toxins among Scottish harbour seals.

Harmful algal bloom (HAB) toxins consumed by marine predators through fish prey can be lethal but studies on the resulting population consequences are lacking. Over the past approximately 20 years there have been large regional declines in some harbour seal populations around Scotland. Analyses of excreta (faeces and urine from live and dead seals and faecal samples from seal haulout sites) suggest widespread exposure to toxins through the ingestion of contaminated prey. A risk assessment model, incorporating concentrations of the two major HAB toxins found in seal prey around Scotland (domoic acid (DA), and saxitoxins (STX)), the seasonal persistence of the toxins in the fish and the foraging patterns of harbour seals were used to estimate the proportion of adults and juveniles likely to have ingested doses above various estimated toxicity thresholds. The results were highly dependent on toxin type, persistence, and foraging regime as well as age class, all of which affected the proportion of exposed animals exceeding toxicity thresholds. In this preliminary model STX exposure was unlikely to result in mortalities. Modelled DA exposure resulted in doses above an estimated lethal threshold of 1900 µg/kg body mass affecting up to 3.8 % of exposed juveniles and 5.3 % of exposed adults. Given the uncertainty in the model parameters and the limitations of the data these conclusions should be treated with caution, but they indicate that DA remains a potential factor involved in the regional declines of harbour seals. Similar risks may be experienced by other top predators, including small cetaceans and seabirds that feed on similar prey in Scottish waters.

Transcriptome response of a marine copepod in response to environmentally-relevant concentrations of saxitoxin.

Paralytic shellfish toxins (PSTs) can pose a serious threat to human health. Among them, saxitoxin (STX) is one of the most potent natural neurotoxins. Here, the copepod Tigriopus japonicus, was exposed to environmentally relevant concentrations (2.5 and 25 µg/L) STX for 48 h. Although no lethal effects were observed at both concentrations, the transcriptome was significantly altered, and displayed a concentration-dependent response. STX exposure decreased the copepod's metabolism and compromised immune defense and detoxification. Additionally, STX disturbed signal transduction, which might affect other cellular processes. STX exposure could inhibit the copepod's chitin metabolism, disrupting its molting process. Also, the processes related to damage repair and protection were up-regulated to fight against high concentration exposure. Collectively, this study has provided an early warning of PSTs for coastal ecosystem not only because of their potent toxicity effect but also their bioaccumulation that can transfer up the food chain after ingestion by copepods.

Effect of temperature, salinity and nutrients on the growth and toxin content of the dinoflagellate Gymnodinium catenatum from the southwestern Mediterranean.

The dinoflagellate Gymnodinium catenatum is considered the primary cause of recurrent paralytic shellfish toxins (PSTs) in shellfish on the Moroccan Mediterranean coasts. The impacts of key environmental factors on the growth, cell yield, cell size and PST content of G. catenatum were determined. Results indicated that increasing salinity from 32 to 39 and nitrate concentrations from 441 µM to 1764 µM did not significantly (ANOVA, P-value >0.63) modify the growth rate of the studied species. Gymnodinium catenatum exhibited the highest growth rate at 24 °C. Cells arrested their division at 15 °C and at ammonium concentration above 441 µM, suggesting that this nitrogen form is toxic for G. catenatum. Furthermore, G. catenatum was unable to assimilate urea as a nitrogen source. In G. catenatum cells, eight analogues of saxitoxin were detected, belonging to the N-sulfocarbamoyl (C1-4, B1 and B2) and decarbamoyl (dc-GTX2/3) toxins. C-toxins contributed 92 % to 98 % of the molar composition of the PSTs. During the exponential growth, C2 tended to dominate, while C3 prevailed during the stationary phase. Toxin content per cell (ranging from 5.5 pg STXeq.cell-1 to 22.4 pg STXeq.cell-1) increased during the stationary growth phase. Cell toxin content increased with the concentrations of nitrate, ranging from 12.1 pg STXeq.cell-1 at 441 µM to 22.4 pg STXeq.cell-1 at 1764 µM during the stationary growth phase. The toxin content of G. catenatum showed the highest values measured at the highest tested temperatures, especially during the stationary phase, where toxicity reached 17.8 pg STXeq.cell-1 and 16.4 pg STXeq.cell-1 at 24 °C and 29 °C, respectively. The results can help understand the fluctuations in the growth and PST content of G. catenatum in its habitat in response to changing environmental variables in the Mediterranean Sea when exposed to increases in warming pressure and eutrophication.

Characterization of MLST-99 Salmonella Typhimurium and the monophasic variant I:4,[5],12:i:- isolated from Canadian Atlantic coast shellfish.

Salmonella enterica subsp. enterica Typhimurium and its monophasic variant I 1;4,[5],12:i:- (MVST) are responsible for thousands of reported cases of salmonellosis each year in Canada, and countries worldwide. We investigated S. Typhimurium and MVST isolates recovered from raw shellfish harvested in Atlantic Canada by the Canadian Food Inspection Agency (CFIA) over the past decade, to assess the potential impact of these isolates on human illness and to explore possible routes of shellfish contamination. Whole-genome sequence analysis was performed on 210 isolates of S. Typhimurium and MVST recovered from various food sources, including shellfish. The objective was to identify genetic markers linked to ST-99, a sequence type specifically associated with shellfish, which could explain their high prevalence in shellfish. We also investigated the genetic similarity amongst CFIA ST-99 isolates recovered in different years and geographical locations. Finally, the study aimed to enhance the molecular serotyping of ST-99 isolates, as they are serologically classified as MVST but are frequently misidentified as S. Typhimurium through sequence analysis. To ensure recovery of ST-99 from shellfish was not due to favourable growth kinetics, we measured the growth rates of these isolates relative to other Salmonella and determined that ST-99 did not have a faster growth rate and/or shorter lag phase than other Salmonella evaluated. The CFIA ST-99 isolates from shellfish were highly clonal, with up to 81 high-quality single nucleotide variants amongst isolates. ST-99 isolates both within the CFIA collection and those isolated globally carried numerous unique deletions, insertions and mutations in genes, including some considered important for virulence, such as gene deletions in the type VI secretion system. Interestingly, several of these genetic characteristics appear to be unique to North America. Most notably was a large genomic region showing a high prevalence in genomes from Canadian isolates compared to those from the USA. Although the functions of the majority of the proteins encoded within this region remain unknown, the genes umuC and umuD, known to be protective against UV light damage, were present. While this study did not specifically examine the effects of mutations and insertions, results indicate that these isolates may be adapted to survive in specific environments, such as ocean water, where wild birds and/or animals serve as the natural hosts. Our hypothesis is reinforced by a global phylogenetic analysis, which indicates that isolates obtained from North American shellfish and wild birds are infrequently connected to isolates from human sources. These findings suggest a distinct ecological niche for ST-99, potentially indicating their specialization and adaptation to non-human hosts and environments, such as oceanic habitats.

Insights into pollution characteristics and human health risks of plasticizer phthalate esters in shellfish species.

The ubiquitous application of phthalate esters (PAEs) as plasticizers contributes to high levels of marine pollution, yet the contamination patterns of PAEs in various shellfish species remain unknown. The objective of this research is to provide the first information on the pollution characteristics of 16 PAEs in different shellfish species from the Pearl River Delta (PRD), South China, and associated health risks. Among the 16 analyzed PAEs, 13 were identified in the shellfish, with total PAE concentrations ranging from 23.07 to 3794.08 ng/g dw (mean = 514.35 ng/g dw). The PAE pollution levels in the five shellfish species were as follows: Ostreidae (mean = 1064.12 ng/g dw) > Mytilus edulis (mean = 509.88 ng/g dw) > Babylonia areolate (mean = 458.14 ng/g dw) > Mactra chinensis (mean = 378.90 ng/g dw) > Haliotis diversicolor (mean = 335.28 ng/g dw). Dimethyl phthalate (DMP, mean = 69.85 ng/g dw), diisobutyl phthalate (DIBP, mean = 41.39 ng/g dw), dibutyl phthalate (DBP, mean = 130.91 ng/g dw), and di(2-ethylhexyl) phthalate (DEHP, mean = 226.23 ng/g dw) were the most abundant congeners. Notably, DEHP constituted the most predominant fraction (43.98 %) of the 13 PAEs detected in all shellfish from the PRD. Principal component analysis indicated that industrial and domestic emissions served as main sources for the PAE pollution in shellfish from the PRD. It was estimated that the daily intake of PAEs via shellfish consumption among adults and children ranged from 0.004 to 1.27 µg/kgbw/day, without obvious non-cancer risks (< 0.034), but the cancer risks raised some alarm (2.0 × 10-9-1.4 × 10-5). These findings highlight the necessity of focusing on marine environmental pollutants and emphasize the importance of ongoing monitoring of PAE contamination in seafood.

Occurrence of microplastics in the Haima cold seep area of the South China Sea.

The pollution of deep-sea microplastics has received increasing attention. As a special ecosystem in the deep sea, the cold seep area is of great significance for studying the distribution of microplastics in the deep sea. In this work, the distribution and characteristics of microplastics in seawater, sediments, and shellfish in the Haima cold seep area and the correlation between the characteristics of microplastics in different media and the type of media were studied. Microplastics were found in all three media. The abundance of microplastics in different samples from the Haima cold seep area ranged 1.8-3.8 items/L for the seawater, 11.47-96.8 items/kg (d.w.) for the surface sediments, and 0-5 items/individual (0-0.714 items/g) for the shellfish. The amount of microplastics ingested by shellfish varied among different species. The microplastics in these three media were mainly fibrous, dark-colored, small-sized rayon, polyethylene terephthalate (PET), and polyethylene (PE). In the correlation analysis of microplastic characteristics among the three media, it was found that the characteristics of microplastics in different media in the same area were closely related, and each pair of variables showed a significant positive correlation (P ≤ 0.05). The distinctive geographical conditions would accelerate the interchange of microplastics among various media. Principal component analysis showed that habitat contribute to microplastic feature differences in shellfish. Differences in correlation were observed between the characteristics of shellfish microplastics in different regions and the characteristics of microplastics in surrounding seawater and sediments.

Monitoring of bivalve mollusk harvesting areas: the relevance of Salmonella spp.

The microbiological monitoring of bivalve mollusk harvesting areas in the Marche region is based on the parameters of Escherichia coli and Salmonella spp. However, Regulation EU/2019/627 stated criteria based on E. coli only to determine the health status of these areas. Therefore, the reason for Salmonella spp. monitoring, as provided in the Marche region, could be aimed at reducing the risk of placing on the market contaminated bivalve mollusks. This study, using the results of microbiological monitoring carried out in the Marche region from 2015 to 2022 and the methods based on Bayes' theorem and Poisson's distribution, evaluated the effectiveness and efficiency of Salmonella spp. monitoring in reducing the risk to the consumer. The results show that i) the use of a single sample unit significantly reduced the possibility of detecting non-compliance with the microbiological safety criterion; ii) the time taken to report positive results (average of approximately 10 days) did not allow the timely implementation of control measures; iii) the prevalence of positive outcomes was quite sporadic: a random trend of positivity is recognizable on a geographical and monthly basis for mussels and a geographical basis for striped clams; iv) considering the predictive value of E. coli against Salmonella spp., the specificity is very high and the negative predictive value versus Salmonella spp. would be >80%. In conclusion, the study shows that the monitoring of Salmonella spp. has a limited effect on reducing the risk to the consumer; however, in the cost/benefit assessment, other aspects not covered by this study should be considered.

Discovery of a series of portimine-A fatty acid esters in mussels.

Vulcanodinium rugosum is a benthic dinoflagellate known for producing pinnatoxins, pteriatoxins, portimines and kabirimine. In this study, we aimed to identify unknown analogs of these emerging toxins in mussels collected in the Ingril lagoon, France. First, untargeted data acquisitions were conducted by means of liquid chromatography coupled to hybrid quadrupole-orbitrap mass spectrometry. Data processing involved a molecular networking approach, and a workflow dedicated to the identification of biotransformed metabolites. Additionally, targeted analyses by liquid chromatography coupled to triple quadrupole mass spectrometry were also implemented to further investigate and confirm the identification of new compounds. For the first time, a series of 13-O-acyl esters of portimine-A (n = 13) were identified, with fatty acid chains ranging between C12:0 and C22:6. The profile was dominated by the palmitic acid conjugation. This discovery was supported by fractionation experiments combined with the implementation of a hydrolysis reaction, providing further evidence of the metabolite identities. Furthermore, several analogs were semi-synthesized, definitively confirming the discovery of these metabolization products. A new analog of pinnatoxin, with a molecular formula of C42H65NO9, was also identified across the year 2018, with the highest concentration observed in August (4.5 µg/kg). The MS/MS data collected for this compound exhibited strong structural similarities with PnTX-A and PnTX-G, likely indicating a substituent C2H5O2 in the side chain at C33. The discovery of these new analogs will contribute to deeper knowledge of the chemodiversity of toxins produced by V. rugosum or resulting from shellfish metabolism, thereby improving our ability to characterize the risks associated with these emerging toxins.

Virulence phenotypes differ between toxigenic Vibrio parahaemolyticus isolated from western coasts of Europe.

Vibrio parahaemolyticus is the leading bacterial cause of gastroenteritis associated with seafood consumption worldwide. Not all members of the species are thought to be pathogenic, thus identification of virulent organisms is essential to protect public health and the seafood industry. Correlations of human disease and known genetic markers (e.g. thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH)) appear complex. Some isolates recovered from patients lack these factors, while their presence has become increasingly noted in isolates recovered from the environment. Here, we used whole-genome sequencing in combination with mammalian and insect models of infection to assess the pathogenic potential of V. parahaemolyticus isolated from European Atlantic shellfish production areas. We found environmental V. parahaemolyticus isolates harboured multiple virulence-associated genes, including TDH and/or TRH. However, carriage of these factors did not necessarily reflect virulence in the mammalian intestine, as an isolate containing TDH and the genes coding for a type 3 secretion system (T3SS) 2α virulence determinant, appeared avirulent. Moreover, environmental V. parahaemolyticus lacking TDH or TRH could be assigned to groups causing low and high levels of mortality in insect larvae, with experiments using defined bacterial mutants showing that a functional T3SS1 contributed to larval death. When taken together, our findings highlight the genetic diversity of V. parahaemolyticus isolates found in the environment, their potential to cause disease and the need for a more systematic evaluation of virulence in diverse V. parahaemolyticus to allow better genetic markers.

Advances in the Early Warning of Shellfish Toxification by Dinophysis acuminata.

In Western Europe, the incidence of DST is likely the highest globally, posing a significant threat with prolonged bans on shellfish harvesting, mainly caused by species of the dinoflagellate genus Dinophysis. Using a time series from 2014 to 2020, our study aimed (i) to determine the concentration of D. acuminata in water at which shellfish toxin levels could surpass the regulatory limit (160 µg OA equiv kg-1) and (ii) to assess the predictability of toxic events for timely mitigation actions, especially concerning potential harvesting bans. The analysis considered factors such as (i) overdispersion in the data, (ii) distinct periods of presence and absence, (iii) the persistence of cells, and (iv) the temporal lag between cells in the water and toxins in shellfish. Four generalized additive models were tested, with the Tweedie (TW-GAM) model showing superior performance (>85%) and lower complexity. The results suggest existing thresholds currently employed (200 and 500 cells L-1) are well-suited for the Portuguese coast, supported by empirical evidence (54-79% accuracy). The developed algorithm allows for thresholds to be tailored on a case-by-case basis, offering flexibility for regional variations.

Assessing the utility of shellfish sanitation monitoring data for long-term estuarine water quality analysis.

Regular testing of coastal waters for fecal coliform bacteria by shellfish sanitation programs could provide data to fill large gaps in existing coastal water quality monitoring, but research is needed to understand the opportunities and limitations of using these data for inference of long-term trends. In this study, we analyzed spatiotemporal trends from multidecadal fecal coliform concentration observations collected by a shellfish sanitation program, and assessed the feasibility of using these monitoring data to infer long-term water quality dynamics. We evaluated trends in fecal coliform concentrations for a 20-year period (1999-2021) using data collected from spatially fixed sampling sites (n = 466) in North Carolina (USA). Findings indicated that shellfish sanitation data can be used for long-term water quality inference under relatively stationary management conditions, and that salinity trends can be used to investigate management-driven bias in fecal coliform observations collected in a particular area.

Temporal variation in the concentrations and profiles of paralytic shellfish toxins and tetrodotoxin in scallop (Mizuhopecten yessoensis) and bloody clam (Anadara broughtonii) collected from the coast of Miyagi Prefecture, Japan.

For food safety, the concentrations and profiles of paralytic shellfish toxins (PSTs) and tetrodotoxin were examined in economically important scallops and bloody clams collected from the coast of the Miyagi Prefecture, Japan. PSTs were the major toxins in both species. The tetrodotoxin concentration in scallops increased in summer, although the highest value (18.7 µg/kg) was lower than the European Food Safety Authority guideline threshold (44 µg/kg). This confirmed the safety for tetrodotoxin in this area.