A New Bioassay for the Detection of Paralytic and Amnesic Biotoxins Based on Motor Behavior Impairments of Zebrafish Larvae.

The global concern about the increase of harmful algal bloom events and the possible impacts on food safety and aquatic ecosystems presents the necessity for the development of more accessible techniques for biotoxin detection for screening purposes. Considering the numerous advantages that zebrafish present as a biological model and particularly as a toxicants sentinel, we designed a sensitive and accessible test to determine the activity of paralytic and amnesic biotoxins using zebrafish larvae immersion. The ZebraBioTox bioassay is based on the automated recording of larval locomotor activity using an IR microbeam locomotion detector, and manual assessment of four complementary responses under a simple stereoscope: survival, periocular edema, body balance, and touch response. This 24 h acute static bioassay was set up in 96-well microplates using 5 dpf zebrafish larvae. For paralytic toxins, a significant decrease in locomotor activity and touch response of the larvae was detected, allowing a detection threshold of 0.1-0.2 µg/mL STXeq. In the case of the amnesic toxin the effect was reversed, detecting hyperactivity with a detection threshold of 10 µg/mL domoic acid. We propose that this assay might be used as a complementary tool for environmental safety monitoring.

Metabarcoding reveals high genetic diversity of harmful algae in the coastal waters of Texas, Gulf of Mexico.

Environmental-DNA (eDNA) for metabarcoding is a rapid and effective means to investigate microplankton community composition and species diversity. The objective of this study was to examine the genetic diversity of the phytoplankton community in the Gulf of Mexico, with particular emphasis on harmful algal bloom species. Samples were collected at stations along the coast of Texas in September-October 2017 that were inundated by low salinity waters in the aftermath of Hurricane Harvey. Metabarcodes were generated from the eDNA targeting both the V4 and V8-V9 regions of the 18S rDNA gene. Evaluation of the metabarcodes revealed an unexpectedly high number of harmful algal species during this short period, including five that had not been documented in this region previously. A total of 36 harmful algal species could be differentiated based on V4 and V8-V9 metabarcode markers. Using a phylogenetic approach, the taxonomic resolution of each marker differed and not all species could be differentiated using solely one marker. The V4 region resolved species within some genera (e.g., Heterocapsa), while the V8-V9 marker was necessary to resolve species within other genera (e.g., Chattonella). In other cases, species differentiation within a genus required a combination of both markers (e.g., Prorocentrum, Karenia), or another marker will be needed to resolve all species (e.g., Alexandrium, Dinophysis). We conclude that no single marker can delineate all species, so it is recommended HAB monitoring programs use more than one marker. Overall, the observed diversity of HAB species along the Texas coast using metabarcoding exceeded reports from other parts of the world.

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.

Floración algal nociva del dinoflagelado Blixaea quinquecornis en el Perú / Harmful algal bloom of dinoflagellate Blixaea quinquecornis in Peru

Resumen En el presente trabajo, se registra por primera vez floraciones algales nocivas del dinoflagelado Blixaea quinquecornis (Abé) Gottschling, 2017 para aguas costeras del mar peruano. La determinación taxonómica de esta especie se realizó mediante microscopia de luz, electrónica de barrido y contraste interferencial diferencial (DIC). La fórmula tecal encontrada fue: pp, 3', 2a, 7'',5c, 5"' y 2''' 4s. Blixaea quinquecornis fue registrada en las bahías Sechura, Miraflores y Paracas entre el 2014 y 2017, usualmente con altas densidades de 3.2 x105 cel.L-1 (Miraflores) y 2.11x105 cel.L-1 (Paracas) en verano y 4.11 x104 (Paracas) en otoño 2016. Blixaea quinquecornis estuvo alternando con floraciones algales del dinoflagelado atecado Akashiwo sanguinea. El florecimiento de B. quinquecornis se observó con TSM entre 18.5 a 26.6 °C, salinidades entre 34.70 y 35.32 ups, pH entre 7.32 y 8.82 y oxígeno disuelto entre 3.8 y 12.5 mL/L. En Bahía Miraflores, la floración de B. quinquecornis fue asociado positivamente con el incremento de la TSM, salinidad y negativamente con la concentración de nitratos, mientras que las floraciones algales de A. sanguinea se correlacionó positivamente con la concentración de fosfatos y oxígeno disuelto. El registro de la floración de B. quinquecornis en las bahías de Miraflores y Paracas, coincidió con eventos de inusual calentamiento del mar asociado con el evento cálido El Niño 2015-2016.

Abstract In the present work, harmful algal blooms of the dinoflagellate Blixaea quinquecornis (Abé) Gottschling, 2017 are recorded for the first time for coastal waters of the Peruvian sea. The taxonomic determination of this species was done with light microscopy, Scanning Electron Microscopy (SEM), and Differential Interference Contrast (DIC) microscopy. The thecal formula is: pp, 3', 2a, 7'', 5c, 5"' and 2''' 4s. Blixaea quinquecornis was recorded in Sechura, Miraflores and Paracas bays between 2014 - 2016, mainly with algal blooms of 3.2 x105 cel.L-1 (Miraflores) and 2.11x105 cel.L-1 (Paracas) and 4.11 x104 cel.L-1 (Paracas) in autumn of 2016. Blixaea quinquecornis was found alternating with algal bloom produced by the athecate dinoflagellate Akashiwo sanguinea. The physicochemical variables associated with the bloom of B. quinquecornis were SST (18.5 to 26.6° C), salinities (34.7 to 35.325), pH (7.32 to 12.5), and dissolved oxygen (3.8 to 12.5 mL/L). The bloom of B. quinquecornis in Miraflores Bay was positively associated with the increase in SST, salinity and negatively with the concentration of nitrates, whereas the algal blooms of A. sanguinea were positively correlated with the concentration of phosphates and oxygen. The record of the bloom of B. quinquecornis in the 3 bays coincided with unusual sea warming event associated with the El Niño phenomenon 2015-2016.

Interactive effects of temperature and salinity on the growth and cytotoxicity of the fish-killing microalgal species Heterosigma akashiwo and Pseudochattonella verruculosa.

Fish-killing blooms of Heterosigma akashiwo and Pseudochattonella verruculosa have been devastating for the farmed salmon industry, but in Southern Chile the conditions that promote the growth and toxicity of these microalgae are poorly understood. This study examined the effects of different combinations of temperature (12, 15, 18 °C) and salinity (10, 20, 30 psu) on the growth of Chilean strains of these two species. The results showed that the optimal growth conditions for H. akashiwo and P. verruculosa differed, with a maximum rate of 0.99 day-1 obtained at 15 °C and a salinity of 20 psu for H. akashiwo, and a maximum rate of 1.06 day-1 obtained at 18 °C and a salinity of 30 psu for P. verruculosa. Cytotoxic assays (2 × 101 - 2 × 105 cell mL-1; cells, filtrates, and cell lysates) performed at salinities of 20 and 30 psu showed a 100% reduction in the viability of embryonic fish cells exposed to intact cells of H. akashiwo and a 39% reduction following exposure to culture filtrates of P. verruculosa. Differences in the fish-killing mechanisms (direct cell contact vs. extracellular substances) and physiological traits of H. akashiwo and P. verruculosa explain the recent occurrence of very large blooms under contrasting (cold-brackish vs. hot-salty) extreme climate conditions in Chile.

The effect of iron on Chilean Alexandrium catenella growth and paralytic shellfish toxin production as related to algal blooms.

The dinoflagellate Alexandrium catenella is a well-known paralytic shellfish toxin producer that forms harmful algal blooms (HABs) worldwide. Blooms of this species have repeatedly brought severe ecological and economic impacts to Chile, especially in the southern region, where the shellfish and salmon industries are world-famous. The mechanisms of such HABs have been intensively studied but are still unclear. Nutrient overloading is one of the often-discussed drivers for HABs. The present study used the A. catenella strain isolated from southern Chile to investigate how iron conditions could affect their growth and toxin production as related to HAB. Our results showed that an optimum concentration of iron was pivotal for proper A. catenella growth. Thus, while excess iron exerted a toxic effect, low iron media led to iron insufficiency and growth inhibition. In addition, the study shows that the degree of paralytic shellfish toxin production by A. catenella varied depending on the iron concentration in the culture media. The A. catenella strain from southern Chile produced GTX1-4 exclusively in the fmol cell-1 scale. Based on these findings, we suggest that including iron and paralytic shellfish toxin measurements in the fields can improve the current HAB monitoring and contribute to an understanding of A. catenella bloom dynamics in Chile.

Evaluation of 24-h screen deployments as a standardized platform to monitor Gambierdiscus populations in the Florida Keys and U.S. Virgin Islands.

Anchored mesh screens have been suggested as a standardized approach to monitor the cell abundances of epiphytic dinoflagellates in benthic habitats, including toxigenic members of the Gambierdiscus genus responsible for ciguatera poisoning (CP). Here we deployed screens for 24h at eight sites in the Florida Keys and St. Thomas (US Virgin Islands) to evaluate their performance relative to the traditional method of assessing Gambierdiscus abundance in which cell counts are normalized to wet weight of host algae. The 30-month study (April 2013 - August 2015) involved monthly sampling at sites where screens were suspended at near-bottom locations for a 24h period and retrieved, with concurrent collections of macrophytes; including Halimeda, Laurencia, and Thalassia in the Florida Keys, and Dictyota in both regions. Gambierdiscus cells were identified and enumerated in the screen and macrophyte samples, and several regression techniques were evaluated (linear regression using untransformed and log-transformed data; negative binomial distribution (NBD) regression) to determine how well the screen-derived data could estimate algal cell concentrations on the host algae. In all cases, the NBD models performed the best based on Akaike Information Criteria values, although 38% of the regressions were not statistically-significant, including all of the St. Thomas sites. The r2 values were all < 0.75 and averaged 0.36, indicating relatively poor fit of the screen data. False negative results (regression models underestimating actual cell abundances) were common occurrences, ranging from 5 to 74% of the scenarios tested. In summary, these results indicate that 24h screen deployments do not appear to be consistent in all situations. Caution is therefore needed when considering 24h screens as a standardized monitoring approach for quantifying Gambierdiscus population dynamics across geography and ecosystems. Furthermore, neutral (artificial) substrates may not adequately capture either the host preference or palatability that likely influence the initial vector of toxin incorporation in the food web via herbivory on these macrophytes.

Combined characterization of a new member of Marivita cryptomonadis strain LZ-15-2 isolated from cultivable phycosphere microbiota of highly toxic HAB dinoflagellate Alexandrium catenella LZT09.

During our conveying the microbial structures of phycosphere microbiota (PM) derived from diverse marine harmful algal bloom (HAB) dinoflagellates, a new rod-sharped, white-colored cultivable bacterial strain, designated as LZ-15-2, was isolated from the PM of highly toxic Alexandrium catenella LZT09. Phylogenetic analysis of 16S rRNA gene sequence indicated that strain LZ-15-2 belonged to the genus Marivita within the family Rhodobacteraceae, and demonstrated the highest gene similarity of 99.2% to M. cryptomonadis CL-SK44T, and less than 98.65% with other type strains of Marivita. Phylogenomic calculations on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the new isolate and M. cryptomonadis CL-SK44T were 99.86% and 99.88%, respectively. Genomic comparison of strain LZ-15-2 with available genomes of Marivita species further verified its taxonomic position within the genus of Marivita. Moreover, comparative genomics analysis showed a proximal similarity of strain LZ-15-2 with M. cryptomonadis CL-SK44T, and it also revealed an open pan-genome status based on constructed gene accumulation curves among Marivita members with 9,361 and 1,712 genes for the pan- and core-genome analysis, respectively. Based on combined polyphasic taxonomic characteristics, strain LZ-15-2 represents a new member of M. cryptomonadis, and proposed as a potential candidate for further exploration of the detailed mechanisms governing the dynamic cross-kingdom algae-bacteria interactions (ABI) between PM and their algal host LZT09.

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.

First detection of pectenotoxin-2 in shellfish associated with an intense spring bloom of Dinophysis acuminata on the central Chilean coast.

Diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins (PTX) produced by endemic species of the genus Dinophysis, mainly D. acuta and D. acuminata, pose a big threat to public health, artisanal fisheries and the aquaculture industry in Southern Chile. This work reports the first detection of lipophilic toxins, including pectenotoxin-2 (PTX-2) and gymnodimine-A (GYM-A), in hard razor clam (Tagelus dombeii) associated with an unprecedented spring bloom -38.4 × 103 cells L-1 in integrated hose sampler (0-10 m) - of Dinophysis acuminata in coastal waters of central Chile. The socio-economic challenges to small-scale fisheries are discussed. The study points to the pressing need for sound policies to face unexpected HAB event, probably due to biogeographical expansions, with a focus on fisheries management, participation of stakeholders, and development of adaptive capacities.

Distribution of dissolved iron and bacteria producing the photoactive siderophore, vibrioferrin, in waters off Southern California and Northern Baja.

Phytoplankton blooms can cause acute effects on marine ecosystems due either to their production of endogenous toxins or to their enormous biomass leading to major impacts on local economies and public health. Despite years of effort, the causes of these Harmful Algal Blooms are still not fully understood. Our hypothesis is that bacteria that produce photoactive siderophores may provide a bioavailable source of iron for phytoplankton which could in turn stimulate algal growth and support bloom dynamics. Here we correlate iron concentrations, phytoplankton cell counts, bacterial cell abundance, and copy numbers for a photoactive siderophore vibrioferrin biosynthesis gene in water samples taken from 2017 cruises in the Gulf of California, and the Pacific Ocean off the coast of northern Baja California as well as during a multiyear sampling at Scripps Pier in San Diego, CA. We find that bacteria producing the photoactive siderophore vibrioferrin, make up a surprisingly high percentage of total bacteria in Pacific/Gulf of California coastal waters (up to 9%). Vibroferrin's unique properties and the widespread prevalence of its bacterial producers suggest that it may contribute significantly to generating bioavailability of iron via photoredox reactions.

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.

Interannual variability in Dinophysis spp. abundance and toxin accumulation in farmed mussels (Perna perna) in a subtropical estuary.

This study evaluated an 8-year dataset (2007 to 2015, except 2008) in the attempt to identify the most susceptible periods for the occurrence of diarrheic shellfish poisoning (DSP) episodes associated with the presence of toxigenic dinoflagellates, Dinophysis spp., in the mussel farming area of Babitonga Bay (southern Brazil). Dinophysis acuminata complex was the most frequent (present in 66% of the samples) and abundant (max. 4100 cells L-1) taxon, followed by D. caudata (14%; max. 640 cells L-1) and D. tripos (0.9%; max. 50 cells L-1). There was a marked onset of the annual rise in Dinophysis spp. abundance during weeks 21-25 (early winter) of each year, followed by a second peak on week 35 (spring). Mussel (Perna perna) samples usually started testing positive in DSP mouse bioassays (MBA) in late winter. Positive results were more frequent in 2007 and 2011 when the mean D. acuminata complex abundance was ~ 500 cells L-1. Although positive DSP-MBA results were observed in only 11% of the samples during the studied period, the toxin okadaic acid (OA) was present in 90% of the analyzed mussels (max. 264 µg kg-1). MBA results were positive when D. acuminata complex cell densities exceed 1200 ± 300 cells L-1, while trace toxin amounts could be detected at cell densities as low as 150 ± 50 cells L-1 (free OA) to 200 ± 100 cells L-1 (conjugated OA). Low salinity and the meteorological conditions triggered by La Niña events were the main factors associated with both Dinophysis abundance and OA accumulation in mussels.

Bloom of Trichodesmium (Oscillatoriales, Phormidiaceae) and seasonality of potentially harmful phytoplankton in San Pedro Bay, Leyte, Philippines / Proliferación de Trichodesmium (Oscillatoriales: Euphorbiaceae) y estacionalidad de fitoplancton potencialmente dañino en Bahía San Pedro, Leyte, Filipinas

AbstractSince 1983, San Pedro Bay in the Philippines had been reported to be the site of episodic Pyrodinium bahamense var. compressum blooms that caused paralytic shellfish poisoning in its nearby coastal communities. This bay is also subjected to numerous storms; the strongest was super typhoon Haiyan in November 8, 2013. For the first time, the seasonal dynamics of potentially toxic and harmful phytoplankton in this bay is elucidated. This is also the first record of a bloom of the cyanobacteria, Trichodesmium erythraeum that reached 70 000 colonies/L in April 2013 in this area. There were other 19 potentially toxic and harmful phytoplankton encountered during the sampling period. These consisted of a haptophyte, Phaeocystis globosa, the diatom Pseudonitzschia and 17 dinoflagellates. Seven of these harmful algae had densities high enough to be traced through time. Normally, diatoms abound during the dry season. But Pseudo-nitzschia increased in abundance during the wet season of 2012 and 2013. The dinoflagellates and Phaeocystis globosa behaved as expected and exhibited a relative increase in cell density during the rainy season of both years too. High nutrient availability during this season must have influenced the behavior of the phytoplankton despite differences in temperature and light intensity among seasons. Other notable but rare harmful species found only in plankton net tows during the study were Pyrodinium bahamense var. compressum, Alexandrium tamiyavanichii, Cochlodiniumpolykrikoides, and Noctiluca scintillans. Rev. Biol. Trop. 64 (2): 897-911. Epub 2016 June 01.

ResumenDesde 1983, la Bahía de San Pedro en Filipinas ha sido reportada como un sitio de proliferación de Pyrodinium bahamense var. compressum que causó intoxicación paralítica en sus comunidades costeras cercanas. Esta bahía también está sometida a numerosas tormentas; entre las más fuertes se presentó un súper tifón en Haiyan, el 8 de noviembre 2013. Por primera vez, se explica la dinámica estacional del fitoplancton potencialmente tóxico y dañino en esta bahía. Este es también el primer registro en esta área de una proliferación de cianobacterias (Trichodesmium erythraeum) que alcanzó 70 000 colonias/L en abril 2013. Durante el periodo de muestreo se presentaron otras 19 proliferaciones de fitoplancton potencialmente tóxicas y dañinas. Estos consistían en una haptófita, Phaeocystis globosa, la diatomea Pseudo-nitzschia y 17 dinoflagelados. Siete de estas algas nocivas tenían densidades suficientemente altas como para ser rastreadas a través del tiempo. Normalmente, las diatomeas abundan durante la estación seca. Pero Pseudo-nitzschia aumentó en abundancia durante la temporada de lluvias de 2012 y 2013. Los dinoflagelados y Phaeocystis globosa se comportaron como se esperaba y exhibieron un aumento relativo de la densidad celular durante la temporada de lluvias en los dos años. La alta disponibilidad de nutrientes durante esta temporada debe haber influido en el comportamiento del fitoplancton a pesar de las diferencias en la temperatura y la intensidad de la luz entre estaciones. Otras especies nocivas notables, pero raras que se encontraron sólo en las redes de arrastre de plancton durante el estudio fueron: Pyrodinium bahamense var. compressum, Alexandriumtamiyavanichii, Cochlodinium polykrikoides y Noctiluca scintillans.