In vitro evaluation of the potential allelopathic and ichthyotoxic effect of the raphidophyte Heterosigma akashiwo and the dinoflagellate Alexandriumcatenella.

Ichthyotoxic algal blooms cause economic losses throughout the world. However, the mechanisms and molecules proposed so far fail to explain the massiveness of these events. In this research, the allelopathic effect of two bloom-forming species (the raphidophyte Heterosigma akashiwo and dinoflagellate Alexandrium catenella) was evaluated between them and with Rhodomonas salina bioassay. Mono- and co-cultures were carried out with the aim of providing evidence of the relation between allelopathy and ichthyotoxicity. The allelopathic inhibitory effect of the A. catenella's supernatant was significantly enhanced when supernatants were obtained from co-cultures with direct contact between these species. We could not observe any allelopathic response provoked by H. akashiwo. On the other hand, A. catenella was able to decrease the cell concentration of H. akashiwo and R. salina. Besides, allelopathy and ichthyotoxicity were found for A. catenella's supernant, being the allelopathic effect not related to saxitoxin. These results reinforce the hypothesis that the allelopathic effect being regulated by the presence of other microalgae and could be responsible for ichthyotoxicity.

Sublethal effect of the toxic dinoflagellate Karlodinium veneficum on early life stages of zebrafish (Danio rerio).

Dinoflagellates of the genus Karlodinium are ichthyotoxic species that produce toxins including karlotoxins and karmitoxins. Karlotoxins show hemolytic and cytotoxic activities and have been associated with fish mortality. This study evaluated the effect of toxins released into the environment of Karlodinium veneficum strain K10 (Ebro Delta, NW Mediterranean) on the early stages of Danio rerio (zebrafish). Extracts of the supernatant of K10 contained the mono-sulfated KmTx-10, KmTx-11, KmTx-12, KmTx-13, and a di-sulfated form of KmTx-10. Total egg mortality was observed for karlotoxin concentration higher than 2.69 µg L-1. For 1.35 µg L-1, 87% of development anomalies were evidenced (all concentrations were expressed as KmTx-2 equivalent). Larvae of 8 days postfertilization exposed to 1.35 µg L-1 presented epithelial damage with 80% of cells in the early apoptotic stage. Our results indicate that supernatants with low concentration of KmTxs produce both lethal and sublethal effects in early fish stages. Moreover, apoptosis was induced at concentrations as low as 0.01 µg L-1. This is of great relevance since detrimental long-term effects due to exposure to low concentrations of these substances could affect wild and cultured fish.

Physiological Response of Atlantic Salmon (Salmo salar) to Long-Term Exposure to an Anesthetic Obtained from Heterosigma akashiwo.

Despite the invaluable role of anesthetics as a tool for ensuring animal welfare in stressful situations, there is currently a lack of anesthetic drugs that meet the requirements of intensive aquaculture. In response to the growing interest in anesthetic substances of natural origin, this study evaluated the physiological and health impact of an anesthetic based on an extract of the microalga Heterosigma akashiwo on juvenile salmon (Salmo salar) exposed for a period of 72 h. To simulate a condition closer to reality where fish are subjected to stimuli (e.g., transport), the animals were exposed to 50 mg L-1 of algal extract and to physical stress. Functional, physiological, and histological parameters were evaluated in blood and tissues at different sampling periods (0, 24, and 72 h). There was no mortality and the induction and recovery times observed were within the established criteria for anesthetic efficacy. The anesthetic extract did not induce any side effects, such as stress or metabolic damage, indicating that this extract is a viable option for supporting fish welfare during deleterious events. This study provides information to support that the anesthetic extract tested, derived from H. akashiwo, is a promising candidate drug for operations requiring sedation (e.g., Salmonid transport).

Salmonid MyD88 is a key adapter protein that activates innate effector mechanisms through the TLR5M/TLR5S signaling pathway and protects against Piscirickettsia salmonis infection.

The membrane-anchored and soluble Toll-like Receptor 5 -TLR5M and TLR5S, respectively-from teleost recognize bacterial flagellin and induce the pro-inflammatory cytokines expression in a MyD88-dependent manner such as the TLR5 mammalian orthologous receptor. However, it has not been demonstrated whether the induced signaling pathway by these receptors activate innate effector mechanisms MyD88-dependent in salmonids. Therefore, in this work we study the MyD88 dependence on the induction of TLR5M/TLR5S signaling pathway mediated by flagellin as ligand on the activation of some innate effector mechanisms. The intracellular and extracellular Reactive Oxygen Species (ROS) production and conditioned supernatants production were evaluated in RTS11 cells, while the challenge with Piscirickettsia salmonis was evaluated in SHK-1 cells. Our results demonstrate that flagellin directly stimulates ROS production and indirectly stimulates it through the production of conditioned supernatants, both in a MyD88-dependent manner. Additionally, flagellin stimulation prevents the cytotoxicity induced by infection with P. salmonis in a MyD88-dependent manner. In conclusion we demonstrate that MyD88 is an essential adapter protein in the activation of the TLR5M/TLR5S signaling pathway mediated by flagellin in salmonids, which leads downstream to the induction of innate effector mechanisms, promoting immuno-protection against a bacterial challenge with P. salmonis.

Search for potential biomarkers for saxitoxin detection.

The Neuro-2a cell assay has been a promising in vitro alternative for the detection of saxitoxin (STX)-like toxins. However, its application is problematic in samples with complex matrices containing different toxins, whose mechanisms of action could be antagonistic. In the search of alternative procedures that reduce or avoid this interference, we evaluated the transcriptional modulation produced by a 24-h exposure to STX in Neuro-2a cells under three conditions: exposure to STX (33 nM), a mussel meat matrix (12.5 mg meat/mL) and a fortified sample (STX-fortified matrix). Differential gene expression was evaluated by RNA-seq after Illumina high-throughput sequencing, and data were analyzed to identify genes differentially expressed regardless of the matrix. From the 9487 identified genes, 213 were differentially expressed of these, 10 genes were identified as candidate markers for STX detection due to their regulation by STX regardless of the matrix interference. Expression dynamics of 7 of these candidate genes (Fgf-1, Adgrb2, Tfpt, Zfr2, Nup 35, Fam195a, and Dusp7) was further evaluated by qRT-PCR analysis of cells exposed to different concentrations of STX for up to 24 h. A downregulation of some markers expression was observed, namely Nup35 (involved in nucleo-cytoplasmic transporter activity) and Zfr-2 (involved in nucleic acids binding), whereas Fgf-1 (apoptosis signaling) was significantly upregulated. Markers' expression was not influenced by the matrix, suggesting that gene expression variations are directly related to STX response. These results support the potential of these genes as biomarkers for the development of an alternative STX-like toxins screening method.