ABSTRACT Harmful algal blooms (HABs) can negatively affect coastal water quality, aquatic fauna, and human health from the consumption of toxin-contaminated seafood. Estuaries are especially prone to the incidence of these noxious events and sensitive to the associated damage. This study represents a comprehensive overview of previous investigations reporting the occurrence of harmful algae and/or phycotoxins in the Paranaguá Estuarine Complex (CEP), in Southern Brazil. Secondary data from 2002 to 2021 were combined with new results obtained from widely spaced sampling campaigns performed from 2018 to 2019 to assess the periods and estuarine sectors at greater risk for the incidence of HABs. In total, about 600 water/sediment samples containing harmful microalgae and 675 tissue samples of phycotoxin-contaminated fauna were analyzed. The most frequent and abundant species of potentially toxic microalgae were dinoflagellates belonging to the species complexes Dinophysis acuminata and Prorocentrum lima, producers of diarrhetic shellfish toxins (DSTs), and diatoms of the genus Pseudo-nitzschia, producers of amnesic shellfish toxins (ASTs). Okadaic acid, a DST, was by far the most common toxin in the fauna, reaching higher levels in primary consumers such as bivalve mollusks (mussels, oysters, and clams), zooplankton, and suspension-feeding crustaceans (ghost shrimps), and moderate to low levels in cephalopods, fishes, gastropods, echinoderms, sea turtles, seabirds, and cetaceans. Less rainy periods (winter-early spring) were associated with the highest incidence of DSTs and blooms of their producing algae P. lima and Dinophysis spp., mainly in the middle region of Paranaguá Bay and on the shallow continental platform adjacent to the estuarine mouth. However, certain HAB-forming species may be more successful in inner estuarine areas ( Prorocentrum cordatum) and during different periods, such as late summer—early fall ( Pseudo-nitzschia spp.). Continuous monitoring of harmful algae and toxins is therefore highly recommended to mitigate the effects of HABs in the entire estuary. (HABs quality toxincontaminated contaminated seafood damage andor or CEP, CEP , (CEP) Brazil 200 202 201 total 60 watersediment sediment 67 phycotoxincontaminated phycotoxin analyzed DSTs, (DSTs) Pseudonitzschia, Pseudonitzschia Pseudo nitzschia, nitzschia ASTs. ASTs . (ASTs) acid DST mussels, mussels (mussels oysters clams, clams clams) zooplankton suspensionfeeding suspension feeding ghost shrimps, shrimps shrimps) cephalopods fishes gastropods echinoderms turtles seabirds cetaceans winterearly winter early spring P spp spp. mouth However HABforming HAB forming cordatum summerearly summer spp.. spp.) estuary (CEP 20 6 (DSTs (ASTs 2

Abstract The quantitative real-time polymerase chain reaction is a widely used method for gene expression analysis requiring carefully selected reference genes to ensure data validity. Regardless of several studies on gene expression bivalves and particularly Crassostrea gigas, has not been fully investigated regarding the evaluation of reference genes suitable for normalization of expression analysis. In this study, five candidate reference genes: actin, β tubulin, α subunit of elongation factor 1, glyceraldehyde-3-phosphate dehydrogenase and 28S ribosomal RNA were analyzed, to determine the most suitable reference genes, after of Crassostrea gigas spat were fed with Gymnodinium catenatum and Prorocentrum lima in mixed and compared to non-toxic diet Isochrysis galbana. The results showed that β-tub and ef-1α were the most stable genes for oysters feed with a mixed diet of P. lima and I. galbana. The gapdh and 28S rRNA were the most stable genes for oysters feed with G. catenatum and I. galbana. In addition, the selection of optimal reference genes during dinoflagellates exposure was verified by analyzing the expression level of trypsin and cytochrome c oxidase I target genes. Our study could be beneficial for future studies on gene expression in C. gigas.

Resumen La reacción en cadena de la polimerasa cuantitativa en tiempo real es un método ampliamente utilizado para el análisis de expresión génica, requiere genes de referencia cuidadosamente seleccionados para garantizar la validez de los datos. A pesar de los estudios sobre expresión génica en bivalvos y particularmente en Crassostrea gigas, existe escasa información sobre la evaluación de los genes de referencia adecuados para la normalización del análisis de expresión. En este estudio se analizaron cinco genes candidatos de referencia: actina, β tubulina, subunidad α del factor de elongación 1, gliceraldehído-3-fosfato deshidrogenasa y ARN ribosomal 28S, para determinar los genes más adecuados, en juveniles de Crassostrea gigas alimentado con una dieta mixta de Gymnodinium catenatum y Prorocentrum lima en comparación con una dieta no tóxica de Isochrysis galbana. Los resultados mostraron que β tub y ef1 α fueron los genes más estables para C. gigas alimentado con una dieta mixta de I. galbana y P. lima; los genes más estables para los ostiones alimentados con I. galbana y G. catenatum fueron gapdh y 28S rRNA. Además, la selección de los genes de referencia durante la exposición a dinoflagelados tóxicos se verificó analizando el nivel de expresión de los genes blanco tripsina y citocromo c oxidasa I. El presente estudio será de gran utilidad para futuros estudios sobre análisis de expresión génica en juveniles de C. gigas.

Abstract This study addressed the occurrence and distribution of benthic microalgae, with emphasis on toxic species, on different substrates (macroalgae, seagrass and sediments) along the outer, euhaline zone of the Estuarine Complex of Paranaguá Bay, southern Brazil, from July 2015 to May 2016. Canonical analysis of principal coordinates (CAP) was used to identify the potential environmental drivers of microphytobenthic assemblage structure. A total of 18 potentially toxic benthic microalgae species were reported, including 7 dinoflagellates, 9 cyanobacteria, one diatom and one euglenophyte. The abundance of all potentially toxic microalgae varied consistently along the investigated period. Dinoflagellates dominated the microalgae assemblage in July and September 2015. Mean densities on macrophytes ranged from 33.6 cells g-1 in May 2016 to 775.6 cells g-1 in September 2015, being positively correlated with dissolved nitrogen-nutrients, water transparency, salinity, and chlorophyll-a. Prorocentrum cf. lima was the most frequent and abundant dinoflagellate. Overall, the highest abundance of toxic species occurred in February 2016, when cyanobacterial mats dominated by Lyngbya cf. aestuarii were observed throughout the estuary associated with periods of heavier rainfall and higher turbidity (shallower Secchi depth). At that time, a selected microphytobenthic extract exhibited acute toxicity on nauplii of the brine shrimp Artemia salina in the laboratory.

ABSTRACT The effects of Gymnodynium catenatum and Prorocentrum lima, dinoflagellate species present in the Gulf of California, on marine bivalves, have demonstrated physiological alterations and changes in gene expression patterns; however, modification effects of the genes involved in immune defense are still poorly understood. This study analyzed the mRNA levels of five genes encoding for the immune defense of oyster Crassostrea gigas spat fed with a control diet (Isochrysis galbana alone) and a combination of the two toxic dinoflagellates. Expression levels of lipopolysaccharide (LPS)-binding protein 1,3-glucan and cavorting genes were higher in oysters exposed to the combined diets in all treatments compared to the non-toxic diet at day three, which was probably related with an activation of the oysters’ immediate immune response during the first 24 h. Protein 44 interferon-induced gene expression level was repressed in treatments with the highest dinoflagellate concentration and overexpressed in the diet with equal dinoflagellate concentration. Interaction protein-Toll and immunoglobulin gene transcript levels reached the highest values at day seven in oysters exposed to all combined diets. Then, the immune defense appeared to be activated in oyster spat as a response of toxins and/or extra-cellular compounds produced by the dinoflagellates.

This study examined the spatial and temporal variability of the phytoplankton communities and their physicochemical variables in three stations at Pescadores Artesanales beach, located in Chorrillos (Lima, Peru). In situ measurements of temperature and pH were made every month for a year, while salinity and dissolved oxygen for half a year. Surface water samples were collected to determine the phytoplankton composition. A total of 120 phytoplanktonic taxa were identified, belonging to 46 families and 66 genera. Phytoplankton species were dominated by diatoms (59.2%) and dinoflagellates (35.6%). Phytoplankton abundance was dominated by the Raphidophyceae (73.72%) followed by dinoflagellates (11.75%), and nanoflagellates (11.53%). Algal blooms were formed by Thalassionema nitzschioides, Skeletonema costatum, Akashiwo sanguinea, Heterosigma akashiwo, Prorocentrum gracile, P. balticum, P. minimum, Scrippsiella acuminata, Dictyocha fibula, Leucocryptos marina and some nanoflagellates (< 20 µm). The highest phytoplankton abundance was observed in summer and autumn. Significant correlations were found between phytoplankton communities and physicochemical variables. Abiotic factors such as dissolved oxygen, temperature, salinity and pH favored the presence of algal blooms. Existence of potentially toxic / harmful phytoplankton species suggests a higher monitoring frequency of harmful algal blooms (FAN) at Pescadores Artesanales beach.

Este estudio examinó la variabilidad espacial y temporal de las principales comunidades del fitoplancton en tres estaciones de la playa de Pescadores Artesanales de Chorrillos, Lima, Perú, en relación con las variables fisicoquímicas. Se realizaron mediciones in situ de temperatura y pH todos los meses durante un año y salinidad y oxígeno disuelto durante medio año. Se colectaron muestras de agua superficial para determinar la composición del fitoplancton. Se identificcaron un total de 120 taxa de organismos fitoplanctónicos, pertenecientes a 46 familias y 66 géneros. Las especies de fitoplancton fueron dominadas por las diatomeas (59.2%) y dinoflagelados (35.6%). La abundancia del fitoplancton estuvo dominada por Raphidophyceae (73.72%), seguido de dinoflagelados (11.75%) y nanoflagelados (< 20 µm) (11.53%). Asimismo, se registraron floraciones algales originadas por las especies, Thalassionema nitzschioides, Skeletonema costatum, Akashiwo sanguinea, Heterosigma akashiwo, Prorocentrum gracile, P. balticum, P. minimum, Scrippsiella acuminata, Dictyocha fibula, Leucocryptos marina y nanoflagelados (< 20 µm) durante todo el periodo de estudio. La mayor abundancia de fitoplancton se observó en verano y otoño. Se encontraron correlaciones significativas entre las comunidades del fitoplancton y las variables fisicoquímicas. Se determinó que los factores abióticos como: oxígeno disuelto, temperatura, salinidad y pH favorecieron la presencia de las floraciones algales. La existencia de especies de fitoplancton potencialmente tóxico/nocivo sugiere un monitoreo frecuente de las floraciones de algas nocivas (FAN) en la playa de Pescadores Artesanales de Chorrillos.

RESUMEN En las últimas décadas, las floraciones de algas nocivas (FAN) y los eventos tóxicos, como la ciguatera, parecen haber aumentado en frecuencia e intensidad, afectando negativamente la salud humana, la economía y los ecosistemas marinos. En Colombia, estos eventos han causado una serie de consecuencias que van desde la muerte de peces y aves hasta efectos tóxicos en los seres humanos. Aunque algunas especies de dinoflagelados que producen toxinas son comunes en el Caribe, su dinámica es poco conocida, lo que dificulta la adopción de cualquier regulación. Para determinar la composición y abundancia de dinoflagelados asociados con los pastos marinos, recolectamos 18 muestras en Isla de Barú durante 2015. Encontramos diez géneros de diatomeas y tres géneros de dinoflagelados, Prorocentrum, Ostreopsis y Gambierdiscus, que incluyen especies toxigénicas relacionadas con la ciguatera y la intoxicación diarreica por mariscos. Prorocentrum lima fue el dinoflagelado más abundante, con densidades celulares promedio de 52 ± 48 células / g de peso húmedo de sustrato. La hipótesis de la temperatura gana fuerza como uno de los principales moduladores de la abundancia de dinoflagelados observados en el Caribe, especialmente con respecto a las especies de Prorocentrum y algunas diatomeas como Mastogloia corsicana y Actinocyclus normanii. Este supuesto está respaldado por la alta correlación positiva entre la Oscilación Sur de El Niño y la temperatura de la superficie del mar en el Caribe durante 2015.

ABSTRACT In the last decades, harmful algal blooms (HAB) and toxic events such as ciguatera seem to have increased in frequency and intensity, negatively impacting human health, economy, and marine ecosystems. In Colombia, these events have caused a series of consequences ranging from the death of fish and birds to toxic effects on humans. Although some toxin-producing dinoflagellate species are common in the Caribbean, their dynamics are poorly understood, making the adoption of any regulations difficult. To determine the composition and abundance of dinoflagellates associated with seagrasses we collected 18 samples on Isla de Barú, during 2015. We found ten diatom genera and three dinoflagellate genera, Prorocentrum, Ostreopsis, and Gambierdiscus, that include toxigenic species related to ciguatera and diarrheic shellfish poisoning. Prorocentrum lima was the most abundant dinoflagellate, with average cell densities of 52±48 cells/g substrate wet weight. The temperature hypothesis gains strength as one of the main modulators of dinoflagellate abundance observed in the Caribbean, especially regarding Prorocentrum species and some diatoms such as Mastogloia corsicana and Actinocyclus normanii. This assumption is supported by the Since high positive correlation between El Niño Southern Oscillation and the sea surface temperature in the Caribbean during 2015.

RESUMEN Los estudios sobre dinoflagelados epibentónicos potencialmente tóxicos han incrementado en los últimos años debido al aumento en el número de eventos perjudiciales atribuidos a algunas de estas microalgas. Tales eventos constituyen un riesgo para la salud humana y para diversas actividades económicas. Con el objetivo de confirmar la presencia de estos dinoflagelados en el Parque Nacional Natural Tayrona (PNNT), Caribe colombiano, se recolectaron mensualmente muestras del pasto marino Thalassia testudinum entre enero de 2014 y diciembre de 2015 en dos sistemas costeros (bahía y laguna) ubicados en la Bahía de Chengue (11 ° 20' N y 74 ° 07' W). Las hojas del pasto marino se tomaron manualmente, se depositaron en bolsas plásticas y se trasladaron al laboratorio para el desprendimiento de los dinoflagelados epibentónicos, mediante agitación vigorosa. Para identificar las especies observadas, se utilizaron tres tipos de microscopía (óptica, invertida con epifluorescencia y microscopía electrónica de barrido). Se observaron 14 especies de dinoflagelados epifitos potencialmente tóxicos, distribuidos en cuatro géneros (Gambierdiscus, Coolia, Ostreopsis y Prorocentrum). Prorocentrum fue el género más representativo, siendo Ostreopsis cf. ovata, P. hoffmannianum y P. lima las especies más frecuentes en la bahía, mientras que en la laguna fueron Prorocentrum sp. 1 y P. rhathymum. Este estudio describe las características principales de las especies observadas, considerando que la identificación precisa a nivel de especie es requerida para cualquier estudio ecológico que busque proporcionar elementos para la gestión de riesgos contra los efectos tóxicos o perjudiciales causados por dinoflagelados epibentónicos.

ABSTRACT Studies on potentially toxic epibenthic dinoflagellates have been increased in the last years due to growing number of harmful events attributed to some of these microalgae. These events represent a risk to human health, as well as to diverse economic activities. With the aim of confirming the presence of these dinoflagellates in Tayrona National Natural Park, Colombian Caribbean, monthly samples of the seagrass Thalassia testudinum were collected between January 2014 and December 2015 in two coastal systems (Bay and Lagoon) located in Chengue Bay (11°20´ N y 74°07´W). Grass leaves were manually collected, placed in plastic bags with seawater and transferred to laboratory for detachment of epibenthic microalgae by vigorous shaking. To identify the species, three types of microscopy were used (optical, inverted with epifluorescence and scanning electron microscopy). Fourteen species of potentially toxic epiphytic dinoflagellates, distributed in four genera (Gambierdiscus, Coolia, Ostreopsis and Prorocentrum) were identified. Prorocentrum was the most representative genus. Ostreopsis cf. ovata, P. hoffmannianum and P. lima were the most frequent species in the Bay, whereas Prorocentrum sp. 1 and P. rhathymum were the most frequent in the Lagoon. This study depicts the main features of the observed species, considering that precise identification at species level is required for any ecological study that seeks to provide elements for risk management against the toxic or harmful effects caused by epibenthic dinoflagellates.

Se estudió la abundancia espacial y temporal de dinoflagelados epífitos asociados a la ciguatera durante dos ciclos anuales (marzo 1999 a marzo 2000, y marzo 2001 a marzo 2002) en la costa del noroeste de Cuba. Recolectamos 14 especies de macroalgas (Phaeophyta, Chlorophyta y Rhodophyta) y obtuvimos 1340 muestras. Identificamos siete especies de dinoflagelados potencialmente nocivas, cinco de ellas nuevos registros para el fitobentos cubano: Prorocentrum belizeanum Faust, P. concavum Fukuyo, P. mexicanum Tafall, Coolia monotis Meunier, y Ostreopsis lenticulares Fukuyo. El análisis de ANOVA/MANOVA mostró diferencias espaciales significativas: la abundancia celular más baja se encontró cerca de la desembocadura del río y la más alta en el área más profunda. Prorocentrum lima (Ehrenberg) Dodge, se encontró principalmente sobre las Phaeophyta seguido de las Chlorophyta y Rhodophyta. Gambierdiscus toxicus se encontró principalmente sobre las Phaeophyta seguido de las Rhodophyta y Chlorophyta. Todas las especies halladas en el área del estudio estaban sobre Padina spp. (Phaeophyta). No se encontró ninguna especie de dinoflagelado sobre Acanthophora spicifera (Rhodophyta). Las condiciones ambientales en verano (temperatura, nutrientes, transparencia de agua y la baja velocidad del viento) favorecen el desarrollo de las macroalgas, siendo un substrato adecuado para el desarrollo de dinoflagelados potencialmente nocivos, y posiblemente el vector principal para la extensión de la ciguatera en la costa noroeste cubana.

The spatial and temporal abundance of epiphytic dinoflagellates associated with ciguatera was studied over two annual cycles (March 1999 to March 2000 and March 2001 to March 2002) in the northwestern coast of Cuba. From 14 species of macroalgae (Phaeophyta, Chlorophyta, and Rhodophyta), 1340 samples were obtained identifying seven species as potentially noxious; five of them are new reports for Cuba’s phytobenthos: Prorocentrum belizeanum Faust, P. concavum Fukuyo, P. mexicanum Tafall, Coolia monotis Meunier, and Ostreopsis lenticularis Fukuyo. ANOVA/MANOVA analysis showed significant spatial differences: lower cell abundance near the shoreline adjacent to a river inlet and higher cell abundance in the deepest area. Prorocentrum lima (Ehrenberg) Dodge 1975 was found mainly on Phaeophyta followed by Chlorophyta and Rhodophyta. Gambierdiscus toxicus was found mainly on Phaeophyta followed by Rhodophyta and Chlorophyta. All the species reported in the study area were mainly on Padina spp. (Phaeophyta). Acanthophora spicifera (Rhodophyta) did not host dinoflagellate species. Environmental conditions in summer (higher temperature, more nutrients, greater water transparency, and low wind intensity) are suitable for macroalgae development, which serves as a substrate for potentially harmful dinoflagellates, and possibly the main vector for spreading ciguatera along the coast of Cuba. Rev. Biol. Trop. 54(2): 299-310. Epub 2006 Jun 01.