A biosensor monitoring approach for toxic algae: Construction of calibration curves to infer cell numbers in field material.

A variety of shellfish toxin-producing Harmful Algal Blooms (HABs) occur every year in coastal temperate waters worldwide. These toxic HABs may cause lengthy (months) harvesting bans of mussels and other suspension feeding bivalves exposed to their blooms. To safeguard public health and the shellfish industry, European Union regulations request periodic monitoring of potentially toxic microalgae in seawater and phycotoxins in live bivalve molluscs from shellfish production areas. Monitoring of other toxic microalgae, e.g., fish killers, is based solely on cell counts. Morphological identification and quantification of microalgal cells with light microscopy is time-consuming, requires a good expertise, and accurate identification to species level (e.g., Pseudo-nitzschia species) may require electron microscopy. Toxicity varies among morphologically similar species; there are toxic and non-toxic strains of the same species. Molecular techniques using ribosomal DNA sequences offer a possibility to identify and detect precisely the potentially toxic genus/species. In an earlier project (MIDTAL), specific probes against rRNA sequences of all HAB taxa, known at the time of the project, affecting shellfish areas worldwide were designed, and those affecting Europe were tested and calibrated against rRNA extracts of clonal cultures and field samples. Microarray technology was adopted to relate to cell numbers the fluorescence signal from the reaction of all target species probes spotted in the microarray slides with those present in a single sample extract. The EMERTOX project aimed to develop a more automatic "Lab on a chip" (LOC) technology, including a non- (cell) disruptive water concentration system and biosensors for HAB cells detection. Here, calibration curves are presented against toxic microalgae (cultures and field samples) causing endemic and emerging toxicity events in Galicia (NW Spain) and Portugal. Results here relating cell numbers to electrochemical signals will be used in an early warning biosensor for toxic algae.

Targeted and non-targeted mass spectrometry to explore the chemical diversity of the genus Gambierdiscus in the Atlantic Ocean.

Dinoflagellates of the genus Gambierdiscus have been associated with ciguatera, the most common non-bacterial fish-related intoxication in the world. Many studies report the presence of potentially toxic Gambierdiscus species along the Atlantic coasts including G. australes, G. silvae and G. excentricus. Estimates of their toxicity, as determined by bio-assays, vary substantially, both between species and strains of the same species. Therefore, there is a need for additional knowledge on the metabolite production of Gambierdiscus species and their variation to better understand species differences. Using liquid chromatography coupled to mass spectrometry, toxin and metabolomic profiles of five species of Gambierdiscus found in the Atlantic Ocean were reported. In addition, a molecular network was constructed aiming at annotating the metabolomes. Results demonstrated that G. excentricus could be discriminated from the other species based solely on the presence of MTX4 and sulfo-gambierones and that the variation in toxin content for a single strain could be up to a factor of two due to different culture conditions between laboratories. While untargeted analyses highlighted a higher variability at the metabolome level, signal correction was applied and supervised multivariate statistics performed on the untargeted data set permitted the selection of 567 features potentially useful as biomarkers for the distinction of G. excentricus, G. caribaeus, G. carolinianus, G. silvae and G. belizeanus. Further studies will be required to validate the use of these biomarkers in discriminating Gambierdiscus species. The study also provided an overview about 17 compound classes present in Gambierdiscus, however, significant improvements in annotation are still required to reach a more comprehensive knowledge of Gambierdiscus' metabolome.

Lipophilic Shellfish Poisoning Toxins in Marine Invertebrates from the Galician Coast.

For the purpose of assessing human health exposure, it is necessary to characterize the toxins present in a given area and their potential impact on commercial species. The goal of this research study was: (1) to screen the prevalence and concentrations of lipophilic toxins in nine groups of marine invertebrates in the northwest Iberian Peninsula; (2) to evaluate the validity of wild mussels (Mytilus galloprovincialis) as sentinel organisms for the toxicity in non-bivalve invertebrates from the same area. The screening of multiple lipophilic toxins in 1150 samples has allowed reporting for the first time the presence of 13-desmethyl spirolide C, pinnatoxin G, okadaic acid, and dinophysistoxins 2 in a variety of non-traditional vectors. In general, these two emerging toxins showed the highest prevalence (12.5-75%) in most of the groups studied. Maximum levels for 13-desmethyl spirolide C and pinnatoxin G were found in the bivalves Magallana gigas (21 µg kg-1) and Tellina donacina (63 µg kg-1), respectively. However, mean concentrations for the bivalve group were shallow (2-6 µg kg-1). Okadaic acid and dinophysistoxin 2 with lower prevalence (1.6-44.4%) showed, on the contrary, very high concentration values in specific species of crustaceans and polychaetes (334 and 235 µg kg--1, respectively), to which special attention should be paid. Statistical data analyses showed that mussels could be considered good biological indicators for the toxicities of certain groups in a particular area, with correlations between 0.710 (for echinoderms) and 0.838 (for crustaceans). Polychaetes could be an exception, but further extensive surveys would be needed to draw definitive conclusions.

Impact of the COVID-19 Pandemic in Spain in the Successive Pandemic Waves on Hemodialysis Patients and Healthcare Personnel.

(1) Background: The impact of SARS-CoV-2 has been variable over the time course of the pandemic and in different populations. The aim was to analyze the impact of COVID-19 infection in a known population of hemodialysis (HD) patients and professionals in Spain at different times of the pandemic. (2) Methods: We conducted an observational, descriptive study with a follow-up from 3 March 2020 to 23 April 2022 (776 days), using in average of 414 professionals and 1381 patients from 18 HD units in Spain. The data from the positive PCR or the rapid antigen detection test (RADT) subject were analyzed and segmented into six periods (waves). (3) Results: Of 703 positive COVID-19 tests, 524 were HD patients (74.5%), and 179 were HD professionals (25.5%). Overall, 38% of staff and 43% of patients were affected. Differences were observed in regard to incidence (21% vs. 13%), mortality (3.5% vs. 0%), and symptomatology between the patients and professionals and throughout the pandemic. (4) Conclusions: COVID-19 severity varied during different pandemic waves, with a greater impact seen in the first wave. HD professionals and patients had similar infection rates, but patients had higher mortality rates. Community transmission was the primary route of infection.

First Report of Two Gymnodimines and Two Tetrodotoxin Analogues in Invertebrates from the North Atlantic Coast of Spain.

Gymnodimine D (GYM D), 16-desmethyl gymnodimine D (16-desmethyl GYM D), and two tetrodotoxin analogues have been found in invertebrates obtained from the north Atlantic coast of Spain from May 2021 to October 2022. It is the first report of GYMD and 16-desmethyl GYM D in invertebrates worldwide and of the tetrodotoxin analogues, 5,6,11 trideoxy tetrodotoxin (5,6,11 trideoxy TTX) and its isomer (referred to as 5,6,11 trideoxy-epi-TTX), in the north Atlantic Coast of Spain. In this study, we also report for the first time the detection of tetrodotoxin (TTX) in three species (the cnidaria Calliactis parasitica, an unidentified species, and the bivalve Tellina donacina). The prevalence was medium for GYM D and 16-desmethyl GYM D and low for TTXs overall. The concentrations recorded were variable, with maximum values of GYM D in the bivalve Cerastoderma edule (8.8 µg GYM A equivalents kg-1), of 16-desmethyl GYM D in the bivalve Magellana gigas (10 µg GYM A equivalents kg-1) and of TTX and 5,6,11 trideoxy TTX in the cnidaria C. parasitica (49.7 and 233 µg TTX equivalents kg-1, respectively). There is very scarce information about these compounds. Therefore, the reporting of these new detections will increase the knowledge on the current incidence of marine toxins in Europe that the European Food Safety Authority (EFSA), in particular, and the scientific community, in general, have. This study also highlights the importance of analyzing toxin analogues and metabolites for effective monitoring programs and adequate health protection.

Twenty-Five Years of PSP Toxicity in Galician (NW Spain) Bivalves: Spatial, Temporal, and Interspecific Variations.

Twenty-five years of paralytic shellfish poisoning (PSP) toxicity in Galician bivalves have been studied. PSP was detected in 4785 out of 73,740 samples of the commercially important bivalve species analyzed from 1995 to 2020. Its general prevalence in the area was 6.5%. Only 1.6% of all samples tested were over the regulatory limit (incidence). The maximum level of PSP in the area, 40,800 µg STX 2HCl-eq kg-1, was recorded in raft mussels from Bueu (PON-II, Pontevedra) in December 2005. The highest maximum PSP values were found in mussels, which were mostly affected by Gymnodinium catenatum, but not those of prevalence and incidence which were recorded in clams, mostly affected by Alexandrium. Average levels in mussels were higher than in any other studied species. Spatially, in general, the prevalence, incidence, maximum, and average PSP toxicity during episodes tend to decrease from south to northeast, but some hot points with high levels can be identified. PCA analysis separates the southern rías, associated to G. catenatum blooms, from the middle and northern ones, associated to Alexandrium blooms. Along the year, two main peaks of the four variables are observed, the first one in late autumn-winter and the other in summer, the summer peak being much more important for the infaunal species than for raft mussels. In the seasonal pattern obtained by time series analysis of the average PSP toxicity, the autumn-winter peak was only maintained (and very reduced) in the southern rías, indicating that this peak is seasonally much less important than the summer peak. The observed seasonality is expected based on the timing of the blooms of the two PSP-producing phytoplankton groups present in the area. Over the 25 years of monitoring, large differences in PSP toxicity have been observed. Apart from some special years, an ascending trend in prevalence and incidence seems to be present from 2011 to 2020. No trend seems to exist during the same period for average or maximum toxicity.

Enzymatic Biotransformation of 13-desmethyl Spirolide C by Two Infaunal Mollusk Species: The Limpet Patella vulgata and the Cockle Cerastoderma edule.

The presence of a 13-desmethyl Spirolide C isomer (Iso-13-desm SPX C) is very common in some infaunal mollusks in Galicia contaminated with this toxin. Its possible origin by biological transformation was investigated by incubating homogenates of the soft tissues of limpets and cockles spiked with 13-desmethyl Spirolide C (13-desm SPX C). The involvement of an enzymatic process was also tested using a raw and boiled cockle matrix. The enzymatic biotransformation of the parent compound into its isomer was observed in the two species studied, but with different velocities. The structural similarity between 13-desm SPX C and its isomer suggests that epimerization is the most likely chemical process involved. Detoxification of marine toxins in mollusks usually implies the enzymatic biotransformation of original compounds, such as hydroxylation, demethylation, or esterification; however, this is the first time that this kind of transformation between spirolides in mollusks has been demonstrated.

Okadaic Acid Depuration from the Cockle Cerastoderma edule.

The cockle Cerastoderma edule is a commercially important species in many European Countries. It can accumulate okadaic acid (OA) and other toxins in its group, which makes it unsuitable for human consumption, producing harvesting bans to avoid intoxications. The duration of those bans depends in part on the depuration kinetics of the toxin in this species. In this work, this kinetics was studied by means of fitting different models to depuration data experimentally obtained, using naturally contaminated cockles. Cockles depurated OA faster than most other bivalve species studied. Models that include Michaelis-Menten kinetics describe the depuration better than those using a first order exponential decrease to describe the first (or the only) compartment. One-compartment models were not able to describe the final part of the depuration curve, in which OA was depurated very slowly. Therefore, two-compartment models were needed. Esters were depurated at a much faster rate than the free form of the toxin; however, no significant esterification was detected during the process. The slow depuration rate suggests that other bivalve species could be used as sentinels to monitor cockle populations, but caution should be taken when toxin concentrations are very high.

Spirolides in Bivalve Mollusk of the Galician (NW Spain) Coast: Interspecific, Spatial, Temporal Variation and Presence of an Isomer of 13-Desmethyl Spirolide C.

Spirolides are cyclic imines whose risks to human health have not been sufficiently evaluated. To determine the possible impact of these compounds in Galicia (NW Spain), their presence and concentration in bivalve mollusk were studied from 2014 to 2021. Only 13-desmethyl spirolide C (13desmSPXC) and an isomer have been detected, and always at low concentrations. Mussel, Mytilus galloprovincialis, was the species which accumulated more spirolides, but the presence of its isomer was nearly restricted to cockle, Cerastoderma edule, and two clam species, Venerupis corrugata and Polititapes rhomboides. On average, the highest 13desmSPXC levels were found in autumn-winter, while those of its isomer were recorded in spring-summer. Both compounds showed decreasing trends during the study period. Geographically, the concentration tends to decrease from the southern to the north-eastern locations, but temporal variability predominates over spatial variability.

Latitudinal Variation in the Toxicity and Sexual Compatibility of Alexandrium catenella Strains from Southern Chile.

The bloom-forming toxic dinoflagellate Alexandrium catenella was first detected in southern Chile (39.5-55° S) 50 years ago and is responsible for most of the area's cases of paralytic shellfish poisoning (PSP). Given the complex life history of A. catenella, which includes benthic sexual cysts, in this study, we examined the potential link between latitude, toxicity, and sexual compatibility. Nine clones isolated from Chilean Patagonia were used in self- and out-crosses in all possible combinations (n = 45). The effect of latitude on toxicity, reproductive success indexes, and cyst production was also determined. Using the toxin profiles for all strains, consisting of C1, C2, GTX4, GTX1, GTX3, and NeoSTX, a latitudinal gradient was determined for their proportions (%) and content per cell (pg cell-1), with the more toxic strains occurring in the north (-40.6° S). Reproductive success also showed a latitudinal tendency and was lower in the north. None of the self-crosses yielded resting cysts. Rather, the production of resting cysts was highest in pairings of clones separated by distances of 1000-1650 km. Our results contribute to a better understanding of PSP outbreaks in the region and demonstrate the importance of resting cysts in fueling new toxic events. They also provide additional evidence that the introduction of strains from neighboring regions is a cause for concern.