Environmental evaluation of the Reloncaví estuary in southern Chile based on lipophilic shellfish toxins as related to harmful algal blooms.

The Reloncaví estuary in southern Chile is famous for its aquaculture. However, recurring harmful algal blooms have adversely affected mussel production. Therefore, regular monitoring of algal toxins is urgently needed to better understand the contamination status of the estuary. In this study, we quantified 15 types of lipophilic shellfish toxins in Metri Bay in the Reloncaví estuary on a biweekly basis for 4 years. We identified algal species using microscopy and metabarcoding analysis. We also measured water temperature, salinity, chlorophyll-a, and dissolved oxygen to determine the potential relationships of these parameters with algal toxin production. Our results revealed the presence of a trace amount of pectenotoxin and the causal phytoplankton Dinophysis, as well as yessotoxin and the causal phytoplankton Protoceratium. Statistical analysis indicated that fluctuations in water temperature affected the detection of these toxins. Additionally, metabarcoding analysis detected the highly toxic phytoplankton Alexandrium spp. in some samples. Although our results suggest that the level of lipophilic shellfish toxins in Metri Bay during the study period was insignificantly low using our current LC-MS method, the confirmed presence of highly toxic algae in Metri Bay raises concerns, given that favorable environmental conditions could cause blooms.

Sources and profiles of toxins in shellfish from the south-central coast of Chile (36°â€’ 43° S).

The study of marine toxins in shellfish is of the utmost importance to ensure people's food safety. Marine toxins in shellfish and microalgae in the water column off the south-central coast of Chile (36°â€’43° S) were studied in a network of 64 stations over a 14-month period. The relative abundance of harmful species Alexandrium catenella, Alexandrium ostenfeldii, Protoceratium reticulatum, Dinophysis acuminata, Dinophysis acuta, Pseudo-nitzschia seriata group and P. delicatissima group was analyzed. The detection and quantification of lipophilic toxins and domoic acid (DA) in shellfish was determined by UHPLC-MS/MS, and for Paralytic Shellfish Toxins (PSTs) by HPLC-FD with post-column oxidation, while for a culture of A. ostenfeldii a Hylic-UHPLC-MS/MS was used. Results showed that DA, gonyautoxin (GTX)-2, GTX-3 and pectenotoxin (PTX)-2 were detected below the permitted limits, while Gymnodimine (GYM)-A and 13-desmethylespirolide C (SPX-1) were below the limit of quantitation. According to the distribution and abundance record of microalgae, DA would be associated to P. seriata and P. delicatissima-groups, PTX-2 to D. acuminata, and GTX-2, GTX-3, GYM-A, and SPX-1 to A. ostenfeldii. However, the toxin analysis of an A. ostenfeldii culture from the Biobío region only showed the presence of the paralytic toxins C2, GTX-2, GTX-3, GTX-5 and saxitoxin, therefore, the source of production of GYM and SPX is still undetermined.

Monitoring bacterial composition and assemblage in the Gulf of Corcovado, southern Chile: Bacteria associated with harmful algae.

Harmful Algal Blooms (HABs) have caused damage to the marine environment in Isla San Pedro in the Gulf of Corcovado, Chile. While rising water temperature and artificial eutrophication are the most discussed topics as a cause, marine bacteria is a recent attractive parameter as an algal bloom driver. This study monitored algal and bacterial compositions in the water of Isla San Pedro for one year using microscopy and 16S rRNA metabarcoding analysis, along with physicochemical parameters. The collected data were analyzed with various statistical tools to understand how the particle-associated bacteria (PA) and the free-living (FL) bacteria were possibly involved in algal blooms. Both FL and PA fractions maintained a stable bacterial composition: the FL fraction was dominated by Proteobacteria (α-Proteobacteria and γ-Proteobacteria), and Cyanobacteria dominated the PA fraction. The two fractions contained equivalent bacterial taxonomic richness (c.a. 8,000 Operational Taxonomic Units) and shared more than 50% of OTU; however, roughly 20% was exclusive to each fraction. The four most abundant algal genera in the Isla San Pedro water were Thalassiosira, Skeletonema, Chaetoceros, and Pseudo-nitzchia. Statistical analysis identified that the bacterial species Polycyclovorans algicola was correlated with Pseudo-nitzschia spp., and our monitoring data recorded a sudden increase of particle-associated Polycyclovorans algicola shortly after the increase of Pseudo-nitzschia, suggesting that P. algicola may have regression effect on Pseudo-nitzschia spp. The study also investigated the physicochemical parameter effect on algal-bacterial interactions. Oxygen concentration and chlorophyll-a showed a strong correlation with both FL and PA bacteria despite their assemblage differences, suggesting that the two groups had different mechanisms for interacting with algal species.

Interannual variability in mesoscale distribution of Dinophysis acuminata and D. acuta in Northwestern Patagonian fjords.

Dinophysis acuminata and D. acuta, which produce diarrheogenic toxins and pectenotoxins in southern Chile, display site-specific differences in interannual variability (2006 - 2018) in Reloncaví, Pitipalena and Puyuhuapi fjords (41 - 46 °S), Chilean Patagonia. Linear Models show decreasing trends in rainfall and river discharge. Latitudinal decreasing gradients in SST temperature and vertical salinity gradients were observed. A brackish water layer (FW salinity <11 psu), permanently present in Reloncaví, decreased in thickness with time in Pitipalena and was usually absent in Puyuhuapi, the only fjord where D. acuta reached bloom (>103 cells L‒1) densities every season. Dinophysis acuminata, associated with toxin profiles in shellfish that include only pectenotoxins, bloomed everywhere with a poleward increasing gradient. Absence of the FW layer provides a possible index of risk for D. acuta blooms. An apparent poleward shift of D. acuta populations, responsible for DSP outbreaks in Reloncaví in the 1970s, and the recent EU deregulation of pectenotoxins will have a positive impact on the mussel industry in Los Lagos Region. Changes to ongoing monitoring protocols to improve risk assessment capabilities are suggested.

A Standardized Procedure for Monitoring Harmful Algal Blooms in Chile by Metabarcoding Analysis.

Harmful algae blooms (HABs) monitoring has been implemented worldwide, and Chile, a country famous for its fisheries and aquaculture, has intensively used microscopic and toxin analyses for decades for this purpose. Molecular biological methods, such as high-throughput DNA sequencing and bacterial assemblage-based approaches, are just beginning to be introduced in Chilean HAB monitoring, and the procedures have not yet been standardized. Here, 16S rRNA and 18S rRNA metabarcoding analyses for monitoring Chilean HABs are introduced stepwise. According to a recent hypothesis, algal-bacterial mutualistic association plays a critical synergetic or antagonistic relationship accounting for bloom initiation, maintenance, and regression. Thus, monitoring HAB from algal-bacterial perspectives may provide a broader understanding of HAB mechanisms and the basis for early warning. Metabarcoding analysis is one of the best suited molecular-based tools for this purpose because it can detect massive algal-bacterial taxonomic information in a sample. The visual procedures of sampling to metabarcoding analysis herein provide specific instructions, aiming to reduce errors and collection of reliable data.

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.

Suitcase Lab: new, portable, and deployable equipment for rapid detection of specific harmful algae in Chilean coastal waters.

Phytoplankton blooms, including harmful algal blooms (HABs), have serious impacts on ecosystems, public health, and productivity activities. Rapid detection and monitoring of marine microalgae are important in predicting and managing HABs. We developed a toolkit, the Suitcase Lab, to detect harmful algae species in the field. We demonstrated the Suitcase Lab's capabilities for sampling, filtration, DNA extraction, and loop-mediated isothermal amplification (LAMP) detection in cultured Alexandrium catenella cells as well as Chilean coastal waters from four sites: Repollal, Isla García, Puerto Montt, and Metri. A LAMP assay using the Suitcase Lab in the field confirmed microscopic observations of A. catenella in samples from Repollal and Isla García. The Suitcase Lab allowed the rapid detection of A. catenella, within 2 h from the time of sampling, even at a single cell per milliliter concentrations, demonstrating its usefulness for quick and qualitative on-site diagnosis of target toxic algae species. This method is applicable not only to detecting harmful algae but also to other field studies that seek a rapid molecular diagnostic test.

Protocols for Monitoring Harmful Algal Blooms for Sustainable Aquaculture and Coastal Fisheries in Chile.

Harmful algae blooms (HABs) cause acute effects on marine ecosystems due to their production of endogenous toxins or their enormous biomass, leading to significant impacts on local economies and public health. Although HAB monitoring has been intensively performed at spatiotemporal scales in coastal areas of the world over the last decades, procedures have not yet been standardized. HAB monitoring procedures are complicated and consist of many methodologies, including physical, chemical, and biological water sample measurements. Each monitoring program currently uses different combinations of methodologies depending on site specific purposes, and many prior programs refer to the procedures in quotations. HAB monitoring programs in Chile have adopted the traditional microscopic and toxin analyses but not molecular biology and bacterial assemblage approaches. Here we select and optimize the HAB monitoring methodologies suitable for Chilean geography, emphasizing on metabarcoding analyses accompanied by the classical tools with considerations including cost, materials and instrument availability, and easiness and efficiency of performance. We present results from a pilot study using the standardized stepwise protocols, demonstrating feasibility and plausibility for sampling and analysis for the HAB monitoring. Such specific instructions in the standardized protocol are critical obtaining quality data under various research environments involving multiple stations, different analysts, various time-points, and long HAB monitoring duration.