Effects of the toxic dinoflagellate Protoceratium reticulatum and its yessotoxins on the survival and feed ingestion of Argopecten purpuratus veliger larvae.

The effects of yessotoxins (YTXs) produced by the dinoflagellate Protoceratium reticulatum in the early stages of bivalves have not been studied in detail. The present study evaluates the effects of P. reticulatum and YTXs on the survival and feed ingestion of veliger larvae of Argopecten purpuratus. Larvae were 96 h-exposed to 500, 1000 and 2000 P. reticulatum cells mL-1, and their equivalent YTX extract was prepared in methanol. Results show a survival mean of 82 % at the highest density of dinoflagellate, and 38 % for larvae with the highest amount of YTX extract. Feed ingestion is reduced in the dinoflagellate exposure treatments as a function of cell density. Therefore, the effect of YTXs on A. purpuratus represents a new and important area of study for investigations into the deleterious effects of these toxins in the early stages of the life cycle of this and, potentially, other bivalves.

Interannual variability in mesoscale distribution of Dinophysis acuminata and D. acuta in Northwestern Patagonian fjords.

Dinophysis acuminata and D. acuta, which produce diarrheogenic toxins and pectenotoxins in southern Chile, display site-specific differences in interannual variability (2006 - 2018) in Reloncaví, Pitipalena and Puyuhuapi fjords (41 - 46 °S), Chilean Patagonia. Linear Models show decreasing trends in rainfall and river discharge. Latitudinal decreasing gradients in SST temperature and vertical salinity gradients were observed. A brackish water layer (FW salinity <11 psu), permanently present in Reloncaví, decreased in thickness with time in Pitipalena and was usually absent in Puyuhuapi, the only fjord where D. acuta reached bloom (>103 cells L‒1) densities every season. Dinophysis acuminata, associated with toxin profiles in shellfish that include only pectenotoxins, bloomed everywhere with a poleward increasing gradient. Absence of the FW layer provides a possible index of risk for D. acuta blooms. An apparent poleward shift of D. acuta populations, responsible for DSP outbreaks in Reloncaví in the 1970s, and the recent EU deregulation of pectenotoxins will have a positive impact on the mussel industry in Los Lagos Region. Changes to ongoing monitoring protocols to improve risk assessment capabilities are suggested.

Feeding response and dynamic of intoxication and detoxification in two populations of the flat oyster Ostrea chilensis exposed to paralytic shellfish toxins (PST).

Juvenile oysters (Ostrea chilensis) from two populations (Quempillén estuary and Pullinque bay) were exposed to a toxic diet containing paralytic shellfish toxins (PST), produced by Alexandrium catenella, followed by a detoxification period. Feeding behaviour, toxin profile, dynamics of intoxication/detoxification, and survival were evaluated over the entire experimental period. Both populations reduced their feeding rates during the 30-day exposure to the toxic diet. This negative effect was reversible when the diet was switched to the non-toxic one. Oysters from the estuary accumulated PST more rapidly than the population from the bay, suggesting their increased ability to cope with more adverse conditions. Both populations showed low detoxification capacity. Survival was significantly higher in oysters from the estuary, compared to those from the bay. Due to the increasing frequency and intensity of A. catenella blooms in southern Chile, it is necessary to better understand the responses of O. chilensis in different environments. This is important not only because of the ecological and commercial relevance of the bivalve, but also in consideration of expected climate change scenarios, where the new environmental conditions could favour the frequency and intensity of harmful algal bloom events.

Lipophilic Toxins in Chile: History, Producers and Impacts.

A variety of microalgal species produce lipophilic toxins (LT) that are accumulated by filter-feeding bivalves. Their negative impacts on human health and shellfish exploitation are determined by toxic potential of the local strains and toxin biotransformations by exploited bivalve species. Chile has become, in a decade, the world's major exporter of mussels (Mytilus chilensis) and scallops (Argopecten purpuratus) and has implemented toxin testing according to importing countries' demands. Species of the Dinophysis acuminata complex and Protoceratium reticulatum are the most widespread and abundant LT producers in Chile. Dominant D. acuminata strains, notwithstanding, unlike most strains in Europe rich in okadaic acid (OA), produce only pectenotoxins, with no impact on human health. Dinophysis acuta, suspected to be the main cause of diarrhetic shellfish poisoning outbreaks, is found in the two southernmost regions of Chile, and has apparently shifted poleward. Mouse bioassay (MBA) is the official method to control shellfish safety for the national market. Positive results from mouse tests to mixtures of toxins and other compounds only toxic by intraperitoneal injection, including already deregulated toxins (PTXs), force unnecessary harvesting bans, and hinder progress in the identification of emerging toxins. Here, 50 years of LST events in Chile, and current knowledge of their sources, accumulation and effects, are reviewed. Improvements of monitoring practices are suggested, and strategies to face new challenges and answer the main questions are proposed.

Multiscale physical background to an exceptional harmful algal bloom of Dinophysis acuta in a fjord system.

Dinophysis acuta produces diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins (PTX). It blooms in thermally-stratified shelf waters in late summer in temperate to cold temperate latitudes. Despite its major contribution to shellfish harvesting bans, little effort has been devoted to study its population dynamics in Chilean Patagonia. In 2017-2018, mesoscale distribution of harmful algal species (75 monitoring stations) revealed the initiation (late spring) and seasonal growth of a dense D. acuta population in the Aysén region, with maximal values at Puyuhuapi Fjord (PF). Vertical phytoplankton distribution and fine-resolution measurements of physical parameters along a 25-km transect in February 16th identified a 15-km (horizontal extension) subsurface thin layer of D. acuta from 4 to 8 m depth. This layer, disrupted at the confluence of PF with the Magdalena Sound, peaked at the top of the pycnocline (6 m, 15.9 °C, 23.4 psu) where static stability was maximal. By February 22nd, it deepened (8 m, 15.5 °C; 23.62 psu) following the excursions of the pycnocline and reached the highest density ever recorded (664 × 103 cells L-1) for this species. Dinophysis acuta was the dominant Dinophysis species in all microplankton net-tows/bottle samples; they all contained DSP toxins (OA, DTX-1) and PTX-2. Modeled flushing rates showed that Puyuhuapi, the only fjord in the area with 2 connections with the open sea, had the highest water residence time. Long term climate variability in the Southern hemisphere showed the effects of a Southern Annular Mode (SAM) in positive mode (+1.1 hPa) overwhelming a moderate La Niña. These effects included positive spring precipitation anomalies with enhanced salinity gradients and summer drought with positive anomalies in air (+1 °C) and sea surface (+2 °C) temperature. Locally, persistent thermal stratification in PF seemed to provide an optimal physical habitat for initiation and bloom development of D. acuta. Thus, in summer 2018, a favourable combination of meteorological and hydrographic processes of multiple scales created conditions that promoted the development of a widespread bloom of D. acuta with its epicentre at the head of Puyuhuapi fjord.

Protocols for Monitoring Harmful Algal Blooms for Sustainable Aquaculture and Coastal Fisheries in Chile.

Harmful algae blooms (HABs) cause acute effects on marine ecosystems due to their production of endogenous toxins or their enormous biomass, leading to significant impacts on local economies and public health. Although HAB monitoring has been intensively performed at spatiotemporal scales in coastal areas of the world over the last decades, procedures have not yet been standardized. HAB monitoring procedures are complicated and consist of many methodologies, including physical, chemical, and biological water sample measurements. Each monitoring program currently uses different combinations of methodologies depending on site specific purposes, and many prior programs refer to the procedures in quotations. HAB monitoring programs in Chile have adopted the traditional microscopic and toxin analyses but not molecular biology and bacterial assemblage approaches. Here we select and optimize the HAB monitoring methodologies suitable for Chilean geography, emphasizing on metabarcoding analyses accompanied by the classical tools with considerations including cost, materials and instrument availability, and easiness and efficiency of performance. We present results from a pilot study using the standardized stepwise protocols, demonstrating feasibility and plausibility for sampling and analysis for the HAB monitoring. Such specific instructions in the standardized protocol are critical obtaining quality data under various research environments involving multiple stations, different analysts, various time-points, and long HAB monitoring duration.

Alexandrium catenella cyst accumulation by passive and active dispersal agents: Implications for the potential spreading risk in Chilean Patagonian fjords.

The dinoflagellate Alexandrium catenella is responsible for paralytic shellfish poisoning and negative socioeconomic impacts on the fishing industry and aquaculture. In Chilean Patagonia, the reasons underlying the significant increase in the geographical extension (from south to north) of A. catenella blooms during the last five decades are not well understood. To assess the potential spreading risk of A. catenella during an intense austral summer bloom, we conducted an in situ experiment in a "hotspot" of this dinoflagellate in southern Chile. The objective was to assess the accumulation of A. catenella resting cysts in passive (fishing nets) and active (mussels) dispersal agents during the phase of bloom decline. Large numbers of resting cysts were detected in fishing nets (maximum of 5334 cysts net-1 per month) at 5 m depth and in mussels (maximum of 16 cysts g-1 of digestive gland) near Vergara Island. The potential of these vectors to serve as inoculum sources and the implications of our findings for A. catenella population dynamics are discussed.

Accumulation and biotransformation dynamics of the neurotoxic complex, saxitoxin, in different life stages of Ostrea chilensis.

The neurotoxic complex saxitoxin, is a group of marine toxins that historically has significantly impacted human health and the ability to utilize marine resources. A steady increase in the distribution and intensity of Alexandrium catenella blooms in Chile, and around the world, has caused major ecological and socioeconomic impacts, putting this type of dinoflagellate, and its toxicity, in the spotlight. Ostrea chilensis is a commercially and ecologically important resource harvested from wild populations and farmed in centers of southern Chile, where it is exposed to large harmful algal blooms of the type that can cause paralysis in humans. This study contributes to our understanding about the transfer of toxins from A. catenella cells to juvenile and adult Ostrea chilensis by tracking transformations of the neurotoxic complex until it reaches its most stable molecular form in the intracellular environment of O. chilensis tissues. These biotransformations are different in O. chilensis juveniles and adults, indicating a differentiated response for these two life stages of this bivalve species. These studies can be used for similar analyses in other ecologically and commercially important species of filter feeding organisms, providing greater understanding of the specific interactions of bivalves in scenarios of toxic dinoflagellate proliferations (e.g. A. catenella blooms).

Next Generation Sequencing and mass spectrometry reveal high taxonomic diversity and complex phytoplankton-phycotoxins patterns in Southeastern Pacific fjords.

In fjord systems, Harmful Algal Blooms (HABs) not only constitute a serious problem when affecting the wildlife and ecosystems, but also human health and economic activities related to the marine environment. This is mostly due to a broad spectrum of toxic compounds produced by several members of the phytoplankton. Nevertheless, a deep coverage of the taxonomic diversity and composition of phytoplankton species and phycotoxin profiles in HAB prone areas are still lacking and little is known about the relationship between these fundamental elements for fjord ecosystems. In this study, a detailed molecular and microscopic characterization of plankton communities was performed, together with an analysis of the occurrence and spatial patterns of lipophilic toxins in a HAB prone area, located in the Southeastern Pacific fjord region. Microscopy and molecular analyses based on the 18S rRNA gene fragment indicated high diversity and taxonomic homogeneity among stations. Four toxigenic genera were identified: Pseudo-nitzschia, Dinophysis, Prorocentrum, and Alexandrium. In agreement with the detected species, liquid chromatography coupled with mass spectrometry revealed the presence of domoic acid (DA), pectenotoxin-2 (PTX-2), dinophysistoxin-2 (DTX-2), and 13-desmethyl spirolide C (SPX-1). Furthermore, a patchy distribution among DA in different net haul size fractions was found. Our results displayed a complex phytoplankton-phycotoxin pattern and for the first time contribute to the characterization of high-resolution phytoplankton community composition and phycotoxin distribution in fjords of the Southeastern Pacific region.

Trophic ecology of two co-existing Sub-Antarctic limpets of the genus Nacella: spatio-temporal variation in food availability and diet composition of Nacella magellanica and N. deaurata.

Interactions between algae and herbivores can be affected by various factors, such as seasonality and habitat structure. Among herbivores inhabiting marine systems, species of the order Patellogastropoda are considered key organisms in many rocky coasts of the world. Nacella species are one of the most dominant macro-herbivores on the rocky shores of the sub-Antarctic ecoregion of Magellan. However, the importance of its key role must be associated with its trophic ecology. The objective of this work was to evaluate spatial and temporal variabilities in the dietary composition of two intertidal Nacella species, considering grazing on macro- (macroalgae) and microscopic (periphyton) food. The composition of periphyton and the availability of macroalgae in the winter and summer seasons were examined at two localities of the Magellanic province, alongside the gut contents of N. magellanica and N. deaurata. The dietary composition differed between the two Nacella species, as well as between seasons and locations. The differences observed in the diet of the two species of Nacella may be mainly due to their respective distributions in the intertidal zone. Both species presented a generalist strategy of grazing, which is relationed to the seasonality of micro- and macroalgae availability and to the variability of the assemblages between the localities. This research was the first to perform a detailed study of the diet of intertidal Nacella species.

Dinophysis toxins: causative organisms, distribution and fate in shellfish.

Several Dinophysis species produce diarrhoetic toxins (okadaic acid and dinophysistoxins) and pectenotoxins, and cause gastointestinal illness, Diarrhetic Shellfish Poisoning (DSP), even at low cell densities (<103 cells·L⁻¹). They are the main threat, in terms of days of harvesting bans, to aquaculture in Northern Japan, Chile, and Europe. Toxicity and toxin profiles are very variable, more between strains than species. The distribution of DSP events mirrors that of shellfish production areas that have implemented toxin regulations, otherwise misinterpreted as bacterial or viral contamination. Field observations and laboratory experiments have shown that most of the toxins produced by Dinophysis are released into the medium, raising questions about the ecological role of extracelular toxins and their potential uptake by shellfish. Shellfish contamination results from a complex balance between food selection, adsorption, species-specific enzymatic transformations, and allometric processes. Highest risk areas are those combining Dinophysis strains with high cell content of okadaates, aquaculture with predominance of mytilids (good accumulators of toxins), and consumers who frequently include mussels in their diet. Regions including pectenotoxins in their regulated phycotoxins will suffer from much longer harvesting bans and from disloyal competition with production areas where these toxins have been deregulated.

Evaluation of passive samplers as a monitoring tool for early warning of Dinophysis toxins in shellfish.

From June 2006 to January 2007 passive samplers (solid phase adsorbing toxin tracking, SPATT) were tested as a monitoring tool with weekly monitoring of phytoplankton and toxin content (liquid chromatography-mass spectrometry, LC-MS) in picked cells of Dinophysis and plankton concentrates. Successive blooms of Dinophysis acuminata, D. acuta and D. caudata in 2006 caused a long mussel harvesting closure (4.5 months) in the Galician Rías (NW Spain) and a record (up to 9246 ng·g resin-week-1) accumulation of toxins in SPATT discs. Best fit of a toxin accumulation model was between toxin accumulation in SPATT and the product of cell densities by a constant value, for each species of Dinophysis, of toxin content (average) in picked cells. Detection of Dinophysis populations provided earlier warning of oncoming diarrhetic shellfish poisoning (DSP) outbreaks than the SPATT, which at times overestimated the expected toxin levels in shellfish because: (i) SPATT accumulated toxins did not include biotransformation and depuration loss terms and (ii) accumulation of toxins not available to mussels continued for weeks after Dinophysis cells were undetectable and mussels were toxin-free. SPATT may be a valuable environmental monitoring and research tool for toxin dynamics, in particular in areas with no aquaculture, but does not provide a practical gain for early warning of DSP outbreaks.

First detection of Pectenotoxin-11 and confirmation of OA-D8 diol-ester in Dinophysis acuta from European waters by LC-MS/MS.

During November 2005, a dense bloom of Dinophysis spp. dominated (>97%) by Dinophysis acuta in the Galician Rías Baixas (NW Spain), provided a unique opportunity to describe the full toxin profile - including toxins that represent a low percentage and escape detection in analyses of single-cell isolates - in plankton concentrates rich in this species. Detection and identification of toxins were carried out by liquid chromatography coupled to mass spectrometry (LC-MS/MS), a method that is based on their retention times (RT) and the fragmentation patterns of their mass spectra. OA-D8 diol-ester and PTX11 were detected in co-occurrence with okadaic acid analogues (OA, DTX2) and PTX2 in plankton concentrates dominated by D. acuta. The presence of a PTX11-isomer, which was suggested to be PTX13 or a novel PTX11-isomer, released in the sea water, was also confirmed in samples obtained after deployment of passive samplers (SPATT) in situ at the time of the D. acuta bloom maximum. The amount of PTX11 per cell of D. acuta, estimated as PTX2 equivalents, ranged between not detected and 2.2pg cell(-1), and represented a maximum of 2.9% of the total toxin content. The variation in PTX11 content per cell of D. acuta, during a daily cycle, followed the same pattern than that of PTX2, with maxima at 21:00 and 03:00h (dark hours), but the amounts per cell were one order of magnitude lower. This is the first report of PTX11, together with the confirmation of OA-D8 diol-ester in D. acuta populations from Europe.

Extraction of microalgal toxins by large-scale pumping of seawater in Spain and Norway, and isolation of okadaic acid and dinophysistoxin-2.

Marine biotoxins from microalgae can accumulate in shellfish and lead to poisoning of human consumers as well as fish, marine mammals and sea birds. Toxicological assessment of the toxins and development of analytical methods require large amounts of high-purity toxins and their metabolites. Although these toxins can be obtained in limited amounts from contaminated shellfish or from microalgal cultures, difficulties arise when the toxin-producing microalga is difficult to culture or its identity is not known. To circumvent this problem, we have developed a large-scale method for solid-phase extraction (SPE) of lipophilic biotoxins from natural microalgal blooms in seawater. To enhance subsequent purification of toxins adsorbed on the column, we included a filtration step to release the toxins from the cells while removing insoluble compounds and cellular debris. The efficacy of the method was illustrated by extraction and purification of okadaic acid and dinophysistoxin-2 from a high-density Dinophysis acuta bloom in Spain and from a mixed bloom containing low densities of D. acuta in Norway. Isolation of the toxins adsorbed on the SPE column was simple and efficient, and results obtained so far indicate that the method is potentially applicable to a wide range of microalgal toxins such as azaspiracids, pectenotoxins, spirolides and microcystins. The method should also be useful for harvesting toxins from large-scale microalgal cultures, and for bioprospecting for and isolation of bioactive natural products from marine and freshwater environments.

Characterisation of okadaic acid related toxins by liquid chromatography coupled with mass spectrometry.

In the Diarrhetic Shellfish Poisoning (DSP) phenomena, the parent toxins, namely okadaic acid (OA) and/or dinophysistoxin-2 (DTX2), are predominantly found esterified. Therefore, a toxicity assessment of a sample can only be performed after an alkaline hydrolysis step in order to recover the parent molecules in their free form. The presence of several OA diol esters has already been confirmed in Prorocentrum lima and Prorocentrum belizeanum cultures. This paper reports on the analysis of OA diol esters using liquid chromatography coupled with mass spectrometry (LC-MS/MS), and establishes a method for their detection and identification based upon their retention times (RT) and the fragmentation patterns of their mass spectra.