Production of extracts with anaesthetic activity from the culture of Heterosigma akashiwo in pilot-scale photobioreactors

The shear-sensitive microalga Heterosigma akashiwo is known to produce brevetoxin-like compounds that are active in voltage-dependent sodium channels. In this work, we present a study on the production of anaesthetic extracts from H. akashiwo biomass obtained in low-shear bioreactors at different growth phases. The photobioreactors (PBRs) used had specific configurations and were operated in such a way as to avoid cellular damage by hydrodynamic stress. Cultures were developed in small static-control flasks and PBRs with volumes ranging from 1.5 L to 200 L. The bioactivity of the produced extracts was assessed in vitro (Neuro-2a cell-based assay) and in vivo (Zebra fish model). Bioactivity depended slightly on the growth phase and culture system, with greater toxicity with the Neuro-2a model when stationary-phase extracts were used. Interestingly, extracts were not cytotoxic against other human cell lines. Steady production of anaesthetic bioactives was observed. In vivo anaesthetic efficacy, assessed with the Zebra fish model, was similar to that of commercial products, and without any observed mortality at 24-h post exposure

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

Salinity-Growth Response and Ichthyotoxic Potency of the Chilean Pseudochattonella verruculosa

Despite salmon farmers suffering the worst damage from a harmful algal bloom in Chile’s history (US$800M) due to a massive outbreak of the dictyochophyte Pseudochattonella verruculosa in 2016 (∼7000–20,000 cells ml−1), the effect of environmental drivers and the potency of lytic toxins produced by the local clones of this species remain still largely unexplored. Based on the drastic oceanographic anomalies observed in the Chilean fjords during the 2016-El Niño “Godzilla” event, the role of salinity (15 to 35 psu) on Pseudochattonella cell growth and cytotoxicity was studied by culturing, scanning electron microscopy (SEM) and using a rainbow trout cell line RTgill-W1 assay to define: (1) vegetative growth rates, (2) cell taxonomy, (3) ichthyotoxicity of monoclonal cultures at 25 and 35 psu in salinity, (4) differences in toxicity of lysed cells and supernatant at different cell concentrations (from 10 to 100,000 cells ml−1), and (5) temporal stability of lytic compounds. This study formally confirms the presence of P. verruculosa in Chilean waters using the large subunit (LSU) of the nuclear ribosomal RNA gene. The Chilean P. verruculosa ARC498 strain showed maximum cell densities at 30 psu (max. 84,333 ± 4,833 cells ml−1) and maximum growth rates (μmax) at 20 psu (1.44 cells d−1). Cultures at 15 psu showed suppressed maximum cell density (max. 269 ± 71 cell ml−1) but high μmax were recorded at the beginning of the exponential growth (1.31 cells d−1). No significant differences were observed between lysed cells and supernatant treatments in the two salinity levels, suggesting that the most lytic portion is released into the cell-free media instead of remaining cell bound. Cytotoxicity was correlated to cell abundance, reducing gill cell viability down to 80 and 65% compared to controls at 10,000 and 100,000 cells ml−1, respectively. Unexpectedly, lytic compounds from P. verruculosa ARC498 at 35 psu showed to be less toxic than cultures at 25, where a noticeable presence of peripheral mucocysts were observed by SEM. Lytic compounds from in vitro experiments are weakly toxic even at high cell concentrations, highly unstable and rapidly degraded in the light after 5 days of storage at 15°C. Our results point to the important effect of salinity on growth and ichthyotoxic potency of Pseudochattonella species and highlight the need for a deeper insight into the role of mucocysts in fish gill damage, which would provide a greater understanding as to the harmful modes of action of this species

Production of extracts with anaesthetic activity from the culture of Heterosigma akashiwo in pilot-scale photobioreactors

The shear-sensitive microalga Heterosigma akashiwo is known to produce brevetoxin-like compounds that are active in voltage-dependent sodium channels. In this work, we present a study on the production of anaesthetic extracts from H. akashiwo biomass obtained in low- shear bioreactors at different growth phases. The photobioreactors (PBRs) used had specific configurations and were operated in such a way as to avoid cellular damage by hydrodynamic stress. Cultures were developed in small static-control flasks and PBRs with volumes ranging from 1.5 L to 200 L. The bioactivity of the produced extracts was assessed in vitro (Neuro- 2a cell-based assay) and in vivo (Zebra fish model). Bioactivity depended slightly on the growth phase and culture system, with greater toxicity with the Neuro-2a model when stationary-phase extracts were used. Interestingly, extracts were not cytotoxic against other human cell lines. Steady production of anaesthetic bioactives was observed. In vivo anaesthetic efficacy, assessed with the Zebra fish model, was similar to that of commercial products, and without any observed mortality at 24-h post exposure

New insight into the biological activity of Salmo salar NK-lysin antimicrobial peptides

NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig’s small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture

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)

Saxitoxin Modulates Immunological Parameters and Gene Transcription in Mytilus chilensis Hemocytes

Saxitoxin (STX) is a neurotoxin produced by dinoflagellates in diverse species, such as Alexandrium spp., and it causes paralytic shellfish poisoning (PSP) in humans after the ingestion of contaminated shellfish. Recent studies have suggested that the immune functions of bivalves could be affected by harmful algae and/or by their toxins. Herein, hemocytes are the main effector cells of the immune cellular response. In this study, we evaluated the response of hemocytes from the mussel Mytilus chilensis to STX exposure in a primary culture. Cell cultures were characterized according to size and complexity, while reactive oxygen species (ROS) production was evaluated using a dichlorofluorescein diacetate (DCFH-DA) assay. Finally, phagocytic activity was measured using both flow cytometry and fluorescence microscopy assays. Additionally, gene transcription of candidate genes was evaluated by qPCR assays. The results evidenced that exposures to different concentrations of STX (1–100 nM) for 24 h did not affect cell viability, as determined by an MTT assay. However, when hemocytes were exposed for 4 or 16 h to STX (1–100 nM), there was a modulation of phagocytic activity and ROS production. Moreover, hemocytes exposed to 100 nM of STX for 4 or 16 h showed a significant increase in transcript levels of genes encoding for antioxidant enzymes (SOD, CAT), mitochondrial enzymes (COI, COIII, CYTB, ATP6, ND1) and ion channels (K+, Ca2+). Meanwhile, C-type lectin and toll-like receptor genes revealed a bi-phase transcriptional response after 16 and 24–48 h of exposure to STX. These results suggest that STX can negatively affect the immunocompetence of M. chilensis hemocytes, which were capable of responding to STX exposure in vitro by increasing the mRNA levels of antioxidant enzymes

Polyphenols obtained from Didymosphenia geminata (Lyngbye) Schmith altered the viability and proliferation of salmonids cells lines SHK-1 and CHSE-214

Didymosphenia geminata (Lyngbye) Schmidt, also referred to as Didymo, is an invasive diatom that forms nuisance mats. Since it was first reported in our country in approximately 2010, Didymo has expanded and colonized different rivers in the Zona Austral region of Chile. Its biology and effects on ecosystems are still being studied because Didymo is an invasive algal mat that forms in a range of systems from oligotrophic austral rivers to more subtropical systems. We aimed to evaluate the viability of two salmonid cell lines, CHSE-214 and SHK-1 (somatic and embryonic cell lines, respectively), in dilutions of river water alone and in river water contaminated with Didymo or polyphenols extracted from Didymo under controlled conditions. We developed an artificial river system (2 aquariums/replicate) from five different rivers from the central area (Bio-Bio) and Patagonia area (Futaleufu) of Chile to maintain Didymo in the benthic phase. The Didymo populations were maintained for six months in the water from the rivers, after which samples were obtained. Following the extraction of polyphenols from the Didymo samples maintained in the artificial rivers, toxicity assays (10 assays) were performed to determine cell viability. Our results indicated that the CHSE-214 cells were highly sensitive to increasing concentrations of Didymo extracts. We observed a 50% reduction in cell viability after 24 h of exposure to a 0.01 V/V dilution, and this treatment further reduced the proliferative capacity by 70% after 120 h. The SHK-1 cells were less responsive, showing only a 20% decrease in viability at 24 h and a lower cell proliferation rate (45%) after 120 h, which remained higher than that of the CHSE-214 cells. We conclude that certain cell types are sensitive to Didymo in rivers, suggesting that there are chronic effects on several aquatic species following exposure to these diatom substances. These effects should be further studied using this laboratory model to understand the full impact of Didymo on river ecosystems

Study of the ichthyotoxic microalga Heterosigma akashiwo by transcriptional activation of sublethal marker Hsp70b in Transwell co-culture assays.

Despite the advance of knowledge about the factors and potential mechanisms triggering the ichthyotoxicity in microalgae, these remain unclear or are controversial for several species (e.g. Heterosigma). Neither typical toxicity tests carried out with cell extracts nor direct exposure to harmful species were proved suitable to unravel the mechanism of harm. Ichthyotoxic species show a complex harmful effect on fish, which is mediated through various mechanisms depending on the species. In this work, we present a method to study sub-lethal effects triggered by reactive oxygen species of a population of harmful algae in vivo over a fish cell line. To that end, Transwell co-cultures in which causative and target species are separated by a 0.4 μm pore membrane were carried out. This allowed the evaluation of the effect of the released molecules by cells in a rapid and compact test. In our method, the harmful effect was sensed through the transcriptional activation of sub-lethal marker Hsp70b in the CHSE214 salmon cell line. The method was tested with the raphidophyte Heterosigma akashiwo and Dunaliella tertiolecta (as negative control). It was shown that superoxide intracellular content and its release are not linked in these species. The methodology allowed proving that reactive oxygen species produced by H. akashiwo are able to induce the transcriptional activation of sub-lethal marker Hsp70b. However, neither loss of viability nor apoptosis was observed in CHSE214 salmon cell line except when exposed to direct contact with the raphidophyte cells (or their extract). Consequently, ROS was not concluded to be the main cause of ichthyotoxicity in H. akashiwo