Evidence of plastics contamination and sewage-derived residues in a Brazilian Hope Spot for conservation of marine biodiversity - Cagarras Islands and surrounding waters.

Cagarras Islands Archipelago, a no-take MPA in Southeast Brazil, was designated as Natural Monument (MONA Cagarras) and, more recently, recognized as Hope Spot for biodiversity conservation. This study aimed to assess plastic contamination by analyzing marine litter and microplastics in MONA Cagarras and surrounding waters. Marine litter (34.12 kg) was caught by artisanal fishermen in MONA Cagarras proximities, and plastics represented ∼79 %. Personal hygiene items and strains of hair were found, suggesting sewage-derived contamination from Ipanema SSO. Microplastics were detected in MONA Cagarras surface waters. Fragments and black particle were the most frequently found microplastic shape and color, respectively. µ-FTIR analysis identified, in descending order of occurrence, polystyrene-PS, polyethylene-PE, polyvinyl chloride-PVC, polypropylene-P, and polyamide-PA. Our integrated results of macro and microplastic contamination highlight an issue of effective conservation and health of marine biodiversity in MONA Cagarras and surrounding waters and a concern for better management of Brazilian MPAs.

In vitro effects of the harmful benthic dinoflagellates Prorocentrum hoffmannianum and Ostreopsis cf. ovata on immune responses of the farmed oyster Crassostrea gasar.

Oyster culture is a sustainable solution to food production. However, this activity can be severely impacted by the presence and proliferation of harmful microalgae such as the benthic dinoflagellates Prorocentrum hoffmannianum and Ostreopsis cf. ovata. This study aimed to evaluate the in vitro effects of P. hoffmannianum and O. cf. ovata on immune system cells (hemocytes) of the native cultured oyster Crassostrea gasar. The direct toxicity of both dinoflagellates was first evaluated assessing hemocyte viability exposed to eight concentrations of each HAB species. No reduction in hemocyte viability was found with the exposure to cell culture or the crude extract of P. hoffmannianum, but O. cf. ovata culture induced hemocyte death in a concentration-dependent manner. Ostreopsis cf. ovata concentration that promoted half of maximal reduction in hemocyte viability (EC50) was 779 cells mL-1. Posteriorly, hemocytes were exposed to both dinoflagellate cells and crude extracts to investigate their effects on hemocyte functional parameters. Despite no direct toxicity of the dinoflagellate cells, P. hoffmannianum extract caused a threefold increase in ROS production and decreased the phagocytosis rate by less than half. Ostreopsis cf. ovata cells and crude extracts also triggered an increase in ROS production (two-fold), but the phagocytosis rate was reduced (by half) only in response to the two lower cell concentrations. These results indicate a harmful potential of both dinoflagellates through a direct toxicity (only for O. cf. ovata) and functional impairment of hemocytes (both species) which could expose C. gasar oyster to opportunistic infections.

Leachates of weathering plastics from an urban sandy beach: Toxicity to sea urchin fertilization and early development.

Plastic leachates have chemical and biological implications for marine environments. This study experimentally evaluated acute effects of weathering plastic leachates (0, 25, 50, 75 and 100 %) on fertilization and early development of the sea urchin Lytechinus variegatus. Fertilization, embryonic and larval development were drastically inhibited (~75 %) when gametes were exposed to intermediate and high leachate concentrations or delayed when exposed to the lowest concentration. Fertilization and first cleavage stages were highly affected by exposure to intermediate and high leachate concentrations. None of the cells incubated at concentrations from 50 % reached blastula stage, suggesting that embryonic development was the most sensitive stage. Abnormalities in embryos and larvae were observed in all leachate treatments. Chemical analysis detected high concentration of bisphenol A, which may induce these observed effects. Our results highlight the potential threats of plastic pollution to sea urchin populations, which may severely affect the structure and functioning of coastal ecosystems.

Phthalates contamination in the coastal and marine sediments of Rio de Janeiro, Brazil.

Coastal and marine environments have been strongly influenced by anthropogenic activities, which may lead to high concentrations of different pollutants in sediments. Our study aimed to assess sediment contamination by polycyclic aromatic hydrocarbons (PAHs), phthalates (PAEs) and bisphenol A (BPA) in nine coastal and marine environments at Rio de Janeiro-Brazil. Physical and chemical water variables, grain-size parameters, moisture, and organic-matter content in sediments were assessed by sampling station. Multivariate analysis evidenced environmental differences between coastal lagoon and oceanic beaches, mostly influenced by marine waters. Differences among bay's beaches were mostly evidenced by sediment characteristics. PAHs and BPA were not detected in samples. For the first time, PAEs were found in sediments at Rio de Janeiro coast (South Atlantic). DEHP was detected in all coastal and marine environments, DBP was found in coastal lagoon and three marine environments. DnOP and DINP were solely found in the coastal lagoon.

The first use of LC-MS/MS proteomic approach in the brown mussel Perna perna after bacterial challenge: Searching for key proteins on immune response.

The brown mussel Perna perna is a valuable fishing resource, primarily in tropical and subtropical coastal regions. Because of their filter-feeding habits, mussels are directly exposed to bacteria in the water column. Escherichia coli (EC) and Salmonella enterica (SE) inhabit human guts and reach the marine environment through anthropogenic sources, such as sewage. Vibrio parahaemolyticus (VP) is indigenous to coastal ecosystems but can be harmful to shellfish. In this study, we aimed to assess the protein profile of the hepatopancreas of P. perna mussel challenged by introduced - E. coli and S. enterica - and indigenous marine bacteria - V. parahaemolyticus. Bacterial-challenge groups were compared with non-injected (NC) and injected control (IC) - that consisted in mussels not challenged and mussels injected with sterile PBS-NaCl, respectively. Through LC-MS/MS proteomic analysis, 3805 proteins were found in the hepatopancreas of P. perna. From the total, 597 were significantly different among conditions. Mussels injected with VP presented 343 proteins downregulated compared with all the other conditions, suggesting that VP suppresses their immune response. Particularly, 31 altered proteins - upregulated or downregulated - for one or more challenge groups (EC, SE, and VP) compared with controls (NC and IC) are discussed in detail in the paper. For the three tested bacteria, significantly different proteins were found to perform critical roles in immune response at all levels, namely: recognition and signal transduction; transcription; RNA processing; translation and protein processing; secretion; and humoral effectors. This is the first shotgun proteomic study in P. perna mussel, therefore providing an overview of the protein profile of the mussel hepatopancreas, focused on the immune response against bacteria. Hence, it is possible to understand the immune-bacteria relationship at molecular levels better. This knowledge can support the development of strategies and tools to be applied to coastal marine resource management and contribute to the sustainability of coastal systems.

First Record of Microplastic Contamination in the Non-Native Dark False Mussel Mytilopsis leucophaeata (Bivalvia: Dreissenidae) in a Coastal Urban Lagoon.

Microplastic contamination is a global concern due to its conspicuous presence in aquatic ecosystems and its toxic nature to environmental and human health. False mussels are among the most notable fresh- and brackish water invaders. The invasive Mytilopsis leucophaeata in Rodrigo de Freitas Lagoon-RFL (Rio de Janeiro, Brazil) is the most abundant macrofaunal invertebrate, widely established and distributed throughout the lagoon. This study aimed to assess microplastic contamination in this invasive filter feeder and evaluate its potential use as a bioindicator. Agglomerates (~100 mussels) were manually collected using a stainless-steel spatula in ten sampling areas distributed throughout the whole lagoon and kept frozen. In the laboratory, 60 individuals were sorted by area for soft-tissue digestion. Each pool of 10 soft-tissue mussels (n = 6 by area) was wet-weighted and then placed in a 150-mL decontaminated glass beaker with 50 mL of 10% KOH. Samples were heated (40 °C) for 48 h, and digested samples were filtered in glass-fiber membranes. Microplastics were found in all samples of mussels (n = 60) from RFL; the particles were mostly lower than 100 µm with a mean concentration (±SD) of 35.96 ± 47.64 MPs g wet-weight-1. Microplastics were distinguished in seven shapes with different occurrences in samples (%): fiber (43.3%); fragment (34.3%); film (16.3%); sponge/foam (4.9%); pellet (0.57%), rope/filaments (0.17%); and undefined (0.4%). Thirteen colors of microplastics were found, but transparent (54.94%), black (10.77%), and white (9.36%) were the most common. Mytilopsis leucophaeata were useful to assess microplastic contamination in RFL and might be preferentially used in other invaded brackish systems instead of native and often threatened bivalves. Our results confirm the effective application of bivalves as an indicator of coastal microplastic pollution.

Impacts of the COVID-19 pandemic restrictions on solid waste pollution in the worldwide iconic Copacabana Beach (Rio de Janeiro, Brazil).

Total and partial restrictions to beach access during COVID-19 pandemic created an opportunity to evaluate its effect on coastal pollution. We aimed to determine the impact of access restrictions on solid waste pollution at Copacabana beach, Brazil. Solid waste amount was analyzed considering beach access restrictions: unrestricted, total closure, partial restriction, new normal. Relationships of atmospheric temperature and precipitation with access restrictions were assessed and confounding effects controlled for further analyses. Beach access restrictions significantly reduced solid waste pollution at Copacabana beach, beach closure reduced waste amount in 72 %. Partial restrictions and new normal periods have significantly reduced solid wastes amount on the beach in 60 % and 36.88 %, respectively. Qualitative data revealed that most of solid wastes were single-use plastics recently disposed at Copacabana beach, reflecting beachgoer's effects on waste left on the beach. A positive impact of COVID-19 pandemic restrictions was detected on solid waste pollution at Copacabana beach.

How does the brown mussel Perna perna respond to environmental pollution? A review on pollution biomarkers.

The brown mussel Perna perna (Linnaeus, 1758) is a valuable resource for aquaculture in tropical and subtropical coastal regions. It presents desirable characteristics for biomonitoring, including being sessile, widely distributed and abundant, and is a filter-feeder able to accumulate several classes of pollutants (e.g., metals, hydrocarbons, among others). Mussels' biological responses to pollution exposure can be measured as biomarkers, which include alterations ranging from molecular to physiological levels, to estimate the degree of environmental contamination and its effects on biota. This full review compiles two decades (2000-2020) of literature concerning biological effects on P. perna mussel caused by environmental pollutants (i.e., metals, hydrocarbons, and emerging pollutants), considering environmental and farm-based biomonitoring. Biochemical markers related to mussels' oxidative status were efficient for the biomonitoring of metals (i.e., antioxidant enzymes associated with oxidative damage in biomolecules). Genotoxicity and cytotoxicity indicators (i.e., comet, micronucleus, and neutral red assays) provided a depiction of hydrocarbon contamination. The neutral red assay gave a time-concentration cytotoxic response to a wide range of pollutants, including emerging pollutants (e.g., pharmaceuticals and biocides) and hydrocarbons. Perna perna hemocyte parameters provided a useful approach for biocide biomonitoring. This paper summarizes useful biomarkers from molecular to physiological levels in this mussel species used to identify and quantify the degree of coastal pollution. An integrated biomarker analysis may provide a way to overcome possible biomarker variations and assess multi-polluted sites. Nevertheless, it is necessary to investigate biomarker variations according to natural factors (e.g., season and gonad maturation stage) to standardize them for trustworthy biomonitoring.

Harmful algal blooms and shellfish in the marine environment: an overview of the main molluscan responses, toxin dynamics, and risks for human health.

Besides human health risks, phycotoxins may cause physiological injuries on molluscan shellfish and, consequently, damages to marine ecosystems and global fisheries production. In this way, this review aimed to present an overview of HABs impacts on marine shellfish by evaluating the effects of cultivated molluscs exposure to microalgae and cyanobacteria that form blooms and/or synthesize toxins. More specifically, it was assessed the main molluscan shellfish responses to harmful algae, trophic transfer and dynamics of phycotoxins, and the risks for human health. Of the 2420 results obtained from literature search, 150 scientific publications were selected after thorough inspections for subject adherence. In total, 70 molluscan species and 37 taxa of harmful algae were assessed from retrieved scientific publications. A significant positive correlation was found between the marine production of molluscs and the number of available studies by molluscan category. Molluscan responses to HABs and phycotoxins were categorized and discussed in three sub-sections: effects on grazing and behavior, metabolic and physiological reactions, and fitness consequences. The main histopathological injuries and toxin concentrations in molluscan tissues were also compiled and discussed. Bivalves often accumulate more toxins than gastropods and cephalopods, occasionally exceeding recommended levels for safe consumption, representing a risk for human health. Harmful algae impact on molluscan shellfish are complex to trace and predict; however, considering the perspective of increase in the occurrence and intensity of HABs, the intensification of efforts to expand the knowledge about HABs impacts on marine molluscs is crucial to mitigate the damages on economy and human health.

Mercury concentration in the sentinel fish species Orthopristis ruber: Effects of environmental and biological factors and human risk assessment.

The present study aimed to assess corocoro grunt use as bioindicator of Hg contamination in coastal marine systems by testing environmental (seasons) and biological effects (sexual maturity, size and weight) on THg accumulation and assessing human health risk. Fish was captured in winter and summer seasons at Vermelha Beach, Rio de Janeiro, Southeast Brazil. Adult O. ruber showed significantly higher THg than juveniles, and THg concentrations were positively correlated to fish length and weight. Fish THg concentrations did not differ between seasons and were below the accepted limit for human consumption. Human exposure risk by fish consumption was 0.31 µg MeHg kg-1 week-1 and the hazard quotient was 0.44 (0.11-1.84). Our results confirm the applied use of O. ruber as sentinel species for Hg contamination monitoring and highlight concern for its consumption, especially for local fishermen populations that highly consume this fish and may be more susceptible to Hg adverse effects.

Effects of the toxic dinoflagellates Prorocentrum lima and Ostreopsis cf. ovata on immune responses of cultured oysters Crassostrea gasar.

Oyster production in Brazil has been highlighted as an important economic activity and is directly impacted by the quality of the environment, which is largely the result of human interference and climate change. Harmful algal blooms occur in aquatic ecosystems worldwide, including coastal marine environments which have been increasing over the last decades as a result of global change and anthropogenic activities. In this study, the native oysters Crassostrea gasar from Northeast of Brazil were exposed to two toxic benthic dinoflagellate species, Prorocentrum lima and Ostreopsis cf. ovata. Their respective effects on C. gasar physiology and defense mechanisms were investigated. Oyster hemocytes were first exposed in vitro to different concentrations of both dinoflagellate species to assess their effects on hemocyte functions, such as phagocytosis, production of reactive oxygen species, as well as mortality. Results highlighted an alteration of hemocyte phagocytosis and viability in presence of O. cf. ovata, whereas P. lima did not affect the measured hemocyte functions. In a second experiment, oysters were exposed for 4 days in vivo to toxic culture of O. cf. ovata to assess its effects on hemocyte parameters, tissues damages and pathogenic Perkinsus spp. infection. An increase in hemocyte mortality was also observed in vivo, associated with a decrease of ROS production. Histopathological analyses demonstrated a thinning of the epithelium of the digestive tubules of the digestive gland, inflammatory reaction and a significant increase in the level of infection by Perkinsus spp. in oysters exposed to O. cf. ovata. These results indicate that oysters C. gasar seem to be pretty resilient to an exposure to P. lima and may be more susceptible to O. cf. ovata. Furthermore, the latter clearly impaired oyster physiology and defense mechanisms, thus highlighting that harmful algal blooms of O. cf. ovata could potentially lead to increased susceptibility of C. gasar oysters to parasite infections.

Acute toxicity of Bisphenol A (BPA) to tropical marine and estuarine species from different trophic groups.

BPA is chemical pollutant of very high concern due to its toxicity to the environment and risks for human health. Environmental concern consists in BPA entrance into aquatic ecosystems due to acute and chronic toxicity to invertebrates and vertebrates. This study aimed to determine acute BPA toxicity to tropical estuarine-marine species of four trophic levels and integrate BPA toxicity values using species sensitivity distribution (SSD) analysis. Our hypothesis is that BPA toxicity increases towards higher trophic levels. Microalga (Tetraselmis sp.), zooplanktonic grazer (Artemia salina), deposit-feeder invertebrate (Heleobia australis), and omnivorous fish (Poecilia vivipara) were chosen as experimental models. Tetraselmis sp. showed the highest BPA tolerance, without a concentration-dependent response. Species sensitivity have increased from A. salina (LC50,96h = 107.2 mg L-1), followed by H. australis (LC50,96h = 11.53.5 mg L-1), to P. vivipara (LC50,96h = 3.5 mg L-1). Despite the toxicity hierarchy towards trophic levels, which partially supported our hypothesis, SSD did not evidence a clear pattern among estuarine-marine trophic groups. Our study disclosed the sensitivity of not yet investigated species to BPA and, in an integrative way, highlighted BPA toxic effects at different trophic levels. Although estimated acute hazardous concentration (HC5 = 1.18 mg L-1) for estuarine and marine species was higher than environmentally relevant concentrations, sublethal adverse effects induced by BPA exposure may lead to unbalances in population levels and consequently affect the ecological functioning of tropical coastal systems.

Exposure to marine benthic dinoflagellate toxins may lead to mitochondrial dysfunction.

Even though marine dinoflagellates are important primary producers, many toxic species may alter the natural equilibrium of aquatic ecosystems and even generate human intoxication incidents, as they are the major causative agents of harmful algal blooms. In order to deepen the knowledge regarding benthic dinoflagellate adverse effects, the present study aims to clarify the influence of Gambierdiscus excentricus strain UNR-08, Ostreopsis cf. ovata strain UNR-03 and Prorocentrum lima strain UNR-01 crude extracts on rat mitochondrial energetic function and permeability transition pore (mPTP) induction. Our results, expressed in number of dinoflagellate cell toxic compounds tested in a milligram of mitochondrial protein, revealed that 934 cells mg prot-1 of G. excentricus, and 7143 cells mg prot-1 of both O. cf. ovata and P. lima negatively affect mitochondrial function, including by decreasing ATP synthesis-related membrane potential variations. Moreover, considerably much lower concentrations of dinoflagellate extracts (117 cells mg prot-1 of G. excentricus, 1429 cells mg prot-1 of O. cf. ovata and 714 cells mg prot-1 of P. lima) produced mPTP-induced swelling in Ca2+-loaded isolated mitochondria. The present study clearly demonstrates the toxicity of G. excentricus, O. cf. ovata and P. lima extracts at the mitochondrial level, which may lead to mitochondrial failure and consequent cell toxicity, and that G. excentricus always provide much more severe effects than O. cf. ovata and P. lima.

Are invasive species always negative to aquatic ecosystem services? The role of dark false mussel for water quality improvement in a multi-impacted urban coastal lagoon.

The dark false mussel Mytilopsis leucophaeata occurs as non-native species in Asia, Europe and South America. Despite the low population density usually found in its native range, M. leucophaeata forms dense clusters in newly invaded areas which lead to impacts on local organisms and environment. Some of the impacts of non-native species on newly colonized areas may be positive. However, despite the empirical evidences of increase in water transparency by freshwater dresseinids, the hypothesis that dark false mussel enhances the quality of brackish water has never been tested so far. Thus, the present study aimed to determine M. leucophaeata impacts on the water quality of a nutrient-enriched coastal lagoon by (1) analyzing a historical water dataset for pre- and post-invasion at Rodrigo de Freitas Lagoon (Brazil) and (2) experimentally testing dark false mussel direct effects on water quality. Historical field data evidenced significant lower phytoplankton abundance and chlorophyll a, and higher water transparency for the post-invasion period. These pattens were also supported by time-series analyses, which detected a decreasing trend for total coliforms density and an increased trend for dissolved oxygen over time. Moreover, time series indicated a gradual increase in lagoon water level with time, but none or minor changes were evidenced for floodgates operation routine, meteorological variables, and the frequency of sewage input. In microcosms, M. leucophaeata was effective to increase water transparency and decrease E. coli densities. The combination of field and laboratory data partially supported the hypothesis that M. leucophaeata invasion promoted an improvement in lagoon water quality, but increased phosphorus availability through excretion in microcosm assays. Management of dark false mussel populations seems to be a potential strategy for water quality improvement within already invaded systems where high sewage-enriched effluents are released. However, as non-native species often unbalance ecological relationships and functioning of the invaded ecosystems, new introductions of M. leucophaeata must be avoided.

High sensitivity of rat cardiomyoblast H9c2(2-1) cells to Gambierdiscus toxic compounds.

Ciguatera fish poisoning is a frequently reported non-bacterial food-borne illness related to the consumption of seafood contaminated with ciguatoxins, and possibly maitotoxins. These toxins are synthesized by marine dinoflagellate species of Gambierdiscus and Fukuyoa genera, and their abundance is a matter of great concern due to their adverse effects to aquatic life and human health. The present study aims to assess the sensitivity of rat cardiomyoblast H9c2(2-1) cells to Gambierdiscus toxic compounds using concentration- and time-dependent sulforhodamine B (SRB) colorimetric assays. Low concentrations of Gambierdiscus extracts (corresponding to 1.3-2.3 cells mL-1) induced a concentration-dependent response. Specificity in time-dependent response of H9c2(2-1) cells was demonstrated for G. excentricus after a 180 min exposure compared to both G. cf. belizeanus and G. silvae species, with EC50s obtained after 720 and 360 min, respectively. The sensitivity of H9c2(2-1) cells to dinoflagellate toxic compounds was also tested with other genera from benthic (Coolia malayensis, Ostreopsis cf. ovata, Prorocentrum hoffmannianum and P. lima) and planktonic (Amphidinium carterae and Lingulodinium polyedrum) habitats. Amphidinium, Coolia and Lingulodinium data did not present any concentration-response relationships, and EC50 values could only be obtained after 720 and 1440 min of exposure to both Prorocentrum species and O. cf. ovata, respectively. This study demonstrated that the H9c2(2-1) SRB assay represents a promising and sensitive tool for the detection of Gambierdiscus toxic compounds present in water samples, particularly of G. excentricus at very low cell abundances.

Mitochondrial impairment and cytotoxicity effects induced by the marine epibenthic dinoflagellate Coolia malayensis.

Mitochondria was used to clarify the effects of Coolia malayensis strain UNR-02 crude extract by studying mitochondrial membrane potential (ΔΨm) generation and the fluctuations of ΔΨm associated with the induction of mitochondrial permeability transition (MPT). The cytoxicity of C. malayensis was also determined using both HepG2 and H9c2(2-1) cells. C. malayensis extract significantly depressed the oxidative phosphorylation efficiency, as was inferred from the perturbations in ΔΨm and in the phosphorylative cycle induced by ADP. Increased susceptibility to Ca2+-induced MPT was also observed. At the cellular level, the extract significantly decreased cell mass of both cell lines.

Screening-level evaluation of marine benthic dinoflagellates toxicity using mammalian cell lines.

Complementary studies at different levels of the biological organization are fundamental to fully link environmental exposure to marine benthic dinoflagellate toxins and their effects. In order to contribute to this transdisciplinary evaluation, and for the first time, the present study aims to study the effects of Gambierdiscus excentricus, Ostreopsis cf. ovata, Prorocentrum hoffmannianum and Prorocentrum lima extracts on seven functionally different mammalian cell lines: HEK 293, HepG2, HNDF, H9c2(2-1), MC3T3-E1, Raw 264.7 and SH-SY5Y. All the cell lines presented cell mass decrease in a concentration-dependence of dinoflagellate extracts, exhibiting marked differences in cell toxicity. Gambierdiscus excentricus presented the highest effect, at very low concentrations with EC50,24h (i.e., the concentration that gives half-maximal response after a 24-h exposure) between 1.3 and 13 cells mL-1, followed by O. cf. ovata (EC50,24h between 3.3 and 40 cells mL-1), and Prorocentrum species (P. lima: EC50,24h between 191 and 1027 cells mL-1 and P. hoffmannianum: EC50,24h between 152 and 783 cells mL-1). Cellular specificities were also detected and rat cardiomyoblast H9c2(2-1) cells were in general the most sensitive to dinoflagellate toxic compounds, suggesting that this cell line is an animal-free potential model for dinoflagellate toxin testing. Finally, the sensitivity of cells expressing distinct phenotypes to each dinoflagellate extract exhibited low relation to human poisoning symptoms.

Ostreopsis cf. ovata (Dinophyceae) Molecular Phylogeny, Morphology, and Detection of Ovatoxins in Strains and Field Samples from Brazil.

: Recurrent blooms of Ostreopsis cf. ovata have been reported in Brazil and the Mediterranean Sea with associated ecological, and in the latter case, health impacts. Molecular data based on the D1-D3 and D8-D10 regions of the LSU rDNA and ITS loci, and the morphology of O. cf. ovata isolates and field populations from locations along the Brazilian tropical and subtropical coastal regions and three oceanic islands are presented. Additional ITS sequences from three single cells from the tropical coast are provided. Toxin profiles and quantities of PLTX and their analogues; OVTXs; contained in cells from two clonal cultures and two field blooms from Rio de Janeiro were investigated. Morphology was examined using both light and epifluorescence microscopy. Morphometric analysis of different strains and field populations from diverse locations were compared. Molecular analysis showed that six of the seven sequences grouped at the large "Atlantic/Mediterranean/Pacific" sub-clade, while one sequence branched in a sister clade with sequences from Madeira Island and Greece. The toxin profile of strains and bloom field samples from Rio de Janeiro were dominated by OVTX-a and -b, with total cell quotas (31.3 and 39.3 pg cell-1) in the range of that previously reported for strains of O. cf. ovata.

Impacts of the toxic benthic dinoflagellate Prorocentrum lima on the brown mussel Perna perna: Shell-valve closure response, immunology, and histopathology.

Prorocentrum lima is a widely distributed marine benthic dinoflagellate that produces diarrhetic toxins, okadaic acid (OA) and its analogs, that may promote damage on bivalve tissues and cellular responses. Cultivation of the brown mussel Perna perna represents an important economic activity in the tropical and subtropical regions, where mussels may co-occur with P. lima. This study aimed to assess the behavioral, cellular immune responses, and pathological condition of P. perna following a short-term experimental exposure to P. lima. The toxic dinoflagellate treatment was compared to a non-toxic exposure to the chlorophyte Tetraselmis sp. at similar concentrations. The prevalence of pathological conditions and parasites were assessed, and a pathological index was applied by scoring the prevalences into four levels. Reaction time and the number of stimuli necessary for shell-valve closure response significantly increased after 72 h of P. lima exposure. Circulating hemocyte concentration was significantly lower in P. lima exposed mussels than in control mussels at 48- and 96 h of incubation, while hemocyte relative size in exposed mussels was significantly higher than that in control mussels. Comparatively, phagocytic activity and ROS production by hemocytes was significantly higher in mussels exposed to P. lima at 48- and 96 h of incubation, respectively. In addition, exposed mussels significantly presented exacerbated hemocytic infiltration in digestive organs, higher prevalence of moderate to severe atrophy in digestive tubules, and higher pathological index which suggests an impairment of mussel immunologic responses. A lower prevalence of Rickettsia-like organisms (RLOs), trematodes and copepods in P. lima exposed mussels suggests a direct toxic effect of OA on parasites. The exposure of mussels to P. lima is likely to occur frequently and may lead to constraints on mussel behavior, physiology, and pathological condition.

Effects of the toxic benthic dinoflagellate Ostreopsis cf. ovata on fertilization and early development of the sea urchin Lytechinus variegatus.

Blooms of the benthic dinoflagellate Ostreopsis cf. ovata have been recorded with increasing frequency, intensity and geographic distribution. This dinoflagellate produces potent toxins that may cause mortality of marine invertebrates. Adults of sea urchins are commonly affected by O. cf. ovata exposure with evidence of spines loss and high mortality during periods of high dinoflagellate abundances. Here, we report on the effects of the toxic dinoflagellate O. cf. ovata on fertilization and early development of the sea urchin Lytechinus variegatus, a key ecological herbivore. Lytechinus variegatus eggs and sperm were experimentally exposed to different concentrations of Ostreopsis cf. ovata (4, 40, 400, and 4000 cells ml-1) to test the hypothesis that fertilization success, embryonic and larval development of the sea urchin are negatively affected by the toxic dinoflagellate even at low abundances. Reduced fertilization, developmental failures, embryo and larval mortality, and occurrence of abnormal offspring were evident after exposure to O. cf. ovata. Fertilization decreased when gametes were exposed to high O. cf. ovata abundances (400 and 4000 cells ml-1), but just the exposure to the highest abundance significantly reduced fertilization success. Sea urchin early development was affected by O. cf. ovata in a dose-dependent way, high dinoflagellate abundances fully inhibited the early development of L. variegatus. Ostreopsis cf. ovata significantly increased the mortality of sea urchin eggs and embryos in the first hours of exposure (∼1-3 h), regardless of dinoflagellate abundance. Abundances of 400 and 4000 O. cf. ovata cells ml-1 induced significantly higher mortality on sea urchin initial stages in the first hours, and no egg or embryo was found in these treatments after 18 h of incubation. The early echinopluteus larva was only reached in the control and in treatments with low Ostreopsis cf. ovata abundances (4 and 40 cells ml-1). The exposure to O. cf. ovata led to significantly higher occurrence of skeletal anomalies in the early larva of L. variegatus. Interactions of sea urchin gametes and Ostreopsis cells may naturally occur in coastal areas due to the match between O. cf. ovata blooms and L. variegatus reproductive period. Reduced larval density and increased larval abnormalities were observed even at low abundances (4 and 40 cells ml-1) frequently found in tropical environments all year round. The chronic exposure to O. cf. ovata could significantly impact larval fitness, thus compromising recruitment success, and highlight the negative effects of benthic HABs on sea urchin populations and its possible broader ecological implications.