Identification of bacteria in potential mutualism with toxic Alexandrium catenella in Chilean Patagonian fjords by in vitro and field monitoring.

The dinoflagellate Alexandrium catenella is a well-known paralytic shellfish toxin producer that forms harmful algal blooms, repeatedly causing damage to Chilean coastal waters. The causes and behavior of algal blooms are complex and vary across different regions. As bacterial interactions with algal species are increasingly recognized as a key factor driving algal blooms, the present study identifies several bacterial candidates potentially associated with Chilean Alexandrium catenella. This research narrowed down the selection of bacteria from the Chilean A. catenella culture using antibiotic treatment and 16S rRNA metabarcoding analysis. Subsequently, seawater from two Chilean coastal stations, Isla Julia and Isla San Pedro, was monitored for two years to detect Alexandrium species and the selected bacteria, utilizing 16S and 18S rRNA gene metabarcoding analyses. The results suggested a potential association between Alexandrium species and Spongiibacteraceae at both stations. The proposed candidate bacteria within the Spongiibacteraceae family, potentially engaging in mutualistic relationships with Alexandrium species, included the genus of BD1-7 clade, Spongiibbacter, and Zhongshania.

Heterosigma akashiwo in Patagonian Fjords: Genetics, Growth, Pigment Signature and Role of PUFA and ROS in Ichthyotoxicity.

Heterosigma akashiwo is the only raphidophyte described for Chilean waters. A recent 2021 fish-killing bloom event of this raphidophyte ignited scientific research, but the ichthyotoxic mechanism and environmental conditions that promote its growth are still unclear. This is the first study confirming the occurrence of H. akashiwo in Chilean waters on the basis of the region D1/D2 of the 28S ribosomal gene. The pigment signature of the CREAN_HA03 strain revealed chlorophyll-a, fucoxanthin, and violaxanthin as the most abundant pigments, but profiles were variable depending on culture and field conditions. A factorial temperature−salinity growth experiment showed a maximal growth rate of 0.48 d−1 at 17 °C and 35 in salinity, but reached a maximal cell abundance of ~50,000 cells mL−1 at 12 °C and 25 in salinity. The fatty acid profile included high levels of saturated (16:0) and polyunsaturated (18:4 ω3; 20:5 ω3) fatty acids, but superoxide production in this strain was low (~0.3 pmol O2− cell−1 h−1). The RTgill-W1 bioassay showed that the H. akashiwo strain was cytotoxic only at high cell concentrations (>47,000 cells mL−1) and after cell rupture. In conclusion, salmon mortality during H. akashiwo bloom events in Patagonian fjords is likely explained by the high production of long-chain PUFAs at high cell densities, but only in the presence of high ROS production.

The effect of iron on Chilean Alexandrium catenella growth and paralytic shellfish toxin production as related to algal blooms.

The dinoflagellate Alexandrium catenella is a well-known paralytic shellfish toxin producer that forms harmful algal blooms (HABs) worldwide. Blooms of this species have repeatedly brought severe ecological and economic impacts to Chile, especially in the southern region, where the shellfish and salmon industries are world-famous. The mechanisms of such HABs have been intensively studied but are still unclear. Nutrient overloading is one of the often-discussed drivers for HABs. The present study used the A. catenella strain isolated from southern Chile to investigate how iron conditions could affect their growth and toxin production as related to HAB. Our results showed that an optimum concentration of iron was pivotal for proper A. catenella growth. Thus, while excess iron exerted a toxic effect, low iron media led to iron insufficiency and growth inhibition. In addition, the study shows that the degree of paralytic shellfish toxin production by A. catenella varied depending on the iron concentration in the culture media. The A. catenella strain from southern Chile produced GTX1-4 exclusively in the fmol cell-1 scale. Based on these findings, we suggest that including iron and paralytic shellfish toxin measurements in the fields can improve the current HAB monitoring and contribute to an understanding of A. catenella bloom dynamics in Chile.

Mitigation of Marine Dinoflagellates Using Hydrogen Peroxide (H2O2) Increases Toxicity towards Epithelial Gill Cells.

Hydrogen peroxide (H2O2) has been shown to efficiently remove toxic microalgae from enclosed ballast waters and brackish lakes. In this study, in vitro experiments were conducted to assess the side effects of mitigating toxic and non-toxic dinoflagellates with H2O2. Five H2O2 concentrations (50 to 1000 ppm) were used to control the cell abundances of the toxic dinoflagellates Alexandrium catenella and Karenia selliformis and the non-toxic dinoflagellates Lepidodinium chlorophorum and Prorocentrum micans. Photosynthetic efficiency and staining dye measurements showed the high efficiency of H2O2 for mitigating all dinoflagellate species at only 50 ppm. In a bioassay carried out to test cytotoxicity using the cell line RTgill-W1, control experiments (only H2O2) showed cytotoxicity in a concentration- and time- (0 to 24 h) dependent manner. The toxic dinoflagellates, especially K. selliformis, showed basal cytotoxicity that increased with the application of hydrogen peroxide. Unexpectedly, the application of a low H2O2 concentration increased toxicity, even when mitigating non-toxic dinoflagellates. This study suggests that the fatty acid composition of toxic and non-toxic dinoflagellate species can yield toxic aldehyde cocktails after lipoperoxidation with H2O2 that can persist in water for days with different half-lives. Further studies are needed to understand the role of lipoperoxidation products as acute mediators of disease and death in aquatic environments.

Toxic Alexandrium catenella expanding northward along the Chilean coast: New risk of paralytic shellfish poisoning off the Bío-Bío region (36° S).

In Chile, the toxic dinoflagellate A. catenella shows an apparent oceanic range expansion from south to the north since its first detection in 1972 in the Magallanes Region (56° S). Until 2017, A. catenella detections were restricted to a geographic area between Magallanes to Los Rios Regions (40° S). The establishment of a monitoring program in the offshore Pacific coast allowed the detection of A. catenella between 2018 and 2019 in northern areas off the Bío-Bío Region (36°S). Monoclonal cultures established from the Bío-Bío coast were genetically identified, and PSTs screened. Phylogenetic analyses determined that the Bío-Bío isolates aggregated in Group I ribotype (previously A. tamarense or A. fundyense) and the presence of PSTs analogs were confirmed. It is the northernmost detection of the toxic dinoflagellate A. catenella in the Pacific coast of Chile. These results have important implications for species monitoring and governmental management in the Bío-Bío Region.

Development of an absolute quantification method for ribosomal RNA gene copy numbers per eukaryotic single cell by digital PCR.

Recent increase of Harmful Algal Blooms (HAB) causes world-wide ecological, economical, and health issues, and more attention is paid to frequent coastal monitoring for the early detection of HAB species to prevent or reduce such impacts. Use of molecular tools in addition to traditional microscopy-based observation has become one of the promising methodologies for coastal monitoring. However, as ribosomal RNA (rRNA) genes are commonly targeted in molecular studies, variability in the rRNA gene copy number within and between species must be considered to provide quantitative information in quantitative PCR (qPCR), digital PCR (dPCR), and metabarcoding analyses. Currently, this information is only available for a limited number of species. The present study utilized a dPCR technology to quantify copy numbers of rRNA genes per single cell in 16 phytoplankton species, the majority of which are toxin-producers, using a newly developed universal primer set accompanied by a labeled probe with a fluorophore and a double-quencher. In silico PCR using the newly developed primers allowed the detection of taxa from 8 supergroups, demonstrating universality and broad coverage of the primer set. Chelex buffer was found to be suitable for DNA extraction to obtain DNA fragments with suitable size to avoid underestimation of the copy numbers. The study successfully demonstrated the first comparison of absolute quantification of 18S rRNA copy numbers per cell from 16 phytoplankton species by the dPCR technology.