Disentangling the environmental processes responsible for the world's largest farmed fish-killing harmful algal bloom: Chile, 2016.

The dictyochophyte microalga Pseudochattonella verruculosa was responsible for the largest farmed fish mortality ever recorded in the world, with losses for the Chilean salmon industry amounting to US$ 800 M in austral summer 2016. Super-scale climatic anomalies resulted in strong vertical water column stratification that stimulated development of a dynamic P. verruculosa thin layer (up to 38 µg chl a L-1) for several weeks in Reloncaví Sound. Hydrodynamic modeling (MIKE 3D) indicated that the Sound had extremely low flushing rates (between 121 and 200 days) in summer 2016. Reported algal cell densities of 7000-20,000 cells mL-1 generated respiratory distress in fish that was unlikely due to low dissolved oxygen (permanently >4 mg L-1). Histological examination of salmon showed that gills were the most affected organ with significant tissue damage and circulatory disorders. It is possible that some of this damage was due to a diatom bloom that preceded the Pseudochattonella event, thereby rendering the fish more susceptible to Pseudochattonella. No correlation between magnitude of fish mortality and algal cell abundance nor fish age was evident. Algal cultures revealed rapid growth rates and high cell densities (up to 600,000 cells mL-1), as well as highly complex life cycle stages that can be easily overlooked in monitoring programs. In cell-based bioassays, Chilean P. verruculosa was only toxic to the RTgill-W1 cell line following exposures to high cell densities of lysed cells (>100,000 cells mL-1). Fatty acid profiles of a cultured strain showed elevated concentrations of potentially ichthyotoxic, long-chain polyunsaturated fatty acids (PUFAs) (69.7% ± 1.8%)- stearidonic (SDA, 18:4ω3-28.9%), and docosahexaenoic acid (DHA, 22:6ω3-22.3%), suggesting that lipid peroxidation may help to explain the mortalities, though superoxide production by Pseudochattonella was low (< 0.21 ± 0.19 pmol O2- cell-1 h-1). It therefore remains unknown what the mechanisms of salmon mortality were during the Pseudochattonella bloom. Multiple mitigation strategies were used by salmon farmers during the event, with only delayed seeding of juvenile fish into the cages and towing of cages to sanctuary sites being effective. Airlift pumping, used effectively against other fish-killing HABs in the US and Canada was not effective, perhaps because it brought subsurface layers of Pseudochattonella to the surface, or and it also may have lysed the fragile cells, rendering them more lethal. The present study highlights knowledge gaps and inefficiency of contingency plans by the fish farming industry to overcome future fish-killing algal blooms under future climate change scenarios. The use of new technologies based on molecular methods for species detection, good farm practices by fish farms, and possible mitigation strategies are discussed.

Unraveling the Karenia selliformis complex with the description of a non-gymnodimine producing Patagonian phylotype.

Karenia selliformis is a bloom-forming toxic dinoflagellate known for production of gymnodimines (GYMs) and causing mass mortalities of marine fauna. Blooms have been reported from coastal waters of New Zealand, Mexico, Tunisia, Kuwait, Iran, China and Chile. Based on molecular phylogeny, morphology, toxin production, pigment composition and cell growth of Chilean K. selliformis isolated in 2018 (CREAN_KS01 and CREAN_KS02), this study revealed a more complex diversity within this species than previously thought. A phylogenetic reconstruction based on the large sub-unit ribosomal nucleotide (LSU rDNA) and Internal Transcriber Spacer (ITS) sequences of 12 worldwide isolates showed that within the K. selliformis clade there are at least two different phylotypes with clear phenotypic differences. Morphological differences related to the dorsal-ventral compression, shape of the hyposome and the presence of pores on the left lobe of the hyposome. A comparison of pigment signatures among worldwide isolates revealed the existence of both acyl-oxyfucoxanthin and fucoxanthin-rich strains within the phylotypes. A LC-MS/MS screening on both Chilean 2018 K. selliformis strains showed for first time no GYMs production among cultured clones of this species. However, both CREAN_KS01 and CREAN_KS02 contained two compounds with the same mass transition as brevenal, a brevetoxin related compound. A fish gill cell-based assay showed that the CREAN_KS02 strain was highly cytotoxic but pure GYM standard did not exhibit loss of cell viability, even at cell concentrations equivalent or exceeding those reported in nature. The fatty acid profile of CREAN_KS02 included high levels of saturated (14:0; 16:0) and polyunsaturated (18:3ω6+18:5ω3; 22:6ω3) fatty acids but superoxide production in this strain was low (0.86±0.53 pmol O2- cell-1 h-1). A factorial T-S growth experiment using the CREAN_KS02 strain showed a µmax of 0.41±0.03 d-1 at high salinity and temperature, which points to its optimal environmental niche in offshore waters during the summer season. In conclusion, the present study provides evidence for significant genetic and phenotypic variability among worldwide isolates, which points to the existence of a K. selliformis "species complex". The massive fauna mortality during K. selliformis bloom events in the Chilean coast cannot be explained by GYMs nor brevetoxins, but can to a large extent be accounted for by the high production of long-chain PUFAs and/or still uncharacterized highly toxic compounds.

Taxonomy, ecology and biotechnological applications of thraustochytrids: A review.

Thraustochytrids were first discovered in 1934, and since the 1960's they have been increasingly studied for their beneficial and deleterious effects. This review aims to provide an enhanced understanding of these protists with a particular emphasis on their taxonomy, ecology and biotechnology applications. Over the years, thraustochytrid taxonomy has improved with the development of modern molecular techniques and new biochemical markers, resulting in the isolation and description of new strains. In the present work, the taxonomic history of thraustochytrids is reviewed, while providing an up-to-date classification of these organisms. It also describes the various biomarkers that may be taken into consideration to support taxonomic characterization of the thraustochytrids, together with a review of traditional and modern techniques for their isolation and molecular identification. The originality of this review lies in linking taxonomy and ecology of the thraustochytrids and their biotechnological applications as producers of docosahexaenoic acid (DHA), carotenoids, exopolysaccharides and other compounds of interest. The paper provides a summary of these aspects while also highlighting some of the most important recent studies in this field, which include the diversity of polyunsaturated fatty acid metabolism in thraustochytrids, some novel strategies for biomass production and recovery of compounds of interest. Furthermore, a detailed overview is provided of the direct and current applications of thraustochytrid-derived compounds in the food, fuel, cosmetic, pharmaceutical, and aquaculture industries and of some of the commercial products available. This review is intended to be a source of information and references on the thraustochytrids for both experts and those who are new to this field.

Screening of new British thraustochytrids isolates for docosahexaenoic acid (DHA) production.

Thraustochytrids isolated from hot tropical and sub-tropical waters have been well studied for DHA and biodiesel production in the last decades. However, little research has been performed on the oils of cold water thraustochytrids, in particular from the North Sea region. In this study, thraustochytrid strains from British waters showed high relative levels of omega-3 long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), including docosahexaenoic acid (DHA, 22:6ω3). The relative levels of DHA (as % of total fatty acids, TFA) in the different British strains are hitherto amongst the highest recorded from any thraustochytrid screening study, with strain TL18 reaching up to 67% DHA in modified Glucose-Yeast Extract-Peptone (GYP) medium. At this screening stage, low final biomass and fatty acid yield were observed in modified GYP and MarChiquita-Brain Heart Broth (MCBHB), while PUFA profiles (as % of PUFA) remained unaltered regardless of the culture medium used. Hence, optimizing the medium and culture conditions to improve growth and lipid content, without impacting the relative percentage of DHA, has the potential to increase the final DHA concentration. With this in mind, three strains were identified as promising organisms for the production of DHA. In the context of possible future industrial exploitation involving a winterization step, we investigated the recycling of the residual oil for biodiesel use. To do this, a mathematical model was used to assess the intrinsic properties of the by-product oil. The results showed the feasibility of producing primary DHA-rich oil, assuming optimized conditions, while using the by-product oil for biodiesel use.