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.

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.

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.

Microbiota associated with bivalves have drawn considerable attention because studies have suggested their relevance to the fitness and growth of marine bivalves. Although the mussel Choromytilus chorus is a valuable resource for Chilean aquaculture and fisheries, its microbiota is still unknown. In this study, the composition and predicted functions of the bacterial community in tissues of C. chorus specimens grown in an estuary (Nehuentue) and a bay (Hueihue) were investigated. Using 16S rRNA genes as targets, the bacterial abundance in tissues was estimated by quantitative PCR and sequenced via Illumina MiSeq. The abundances of bacteria ranged from 10³ to 10⁵ copies of 16S rRNA genes g⁻¹ tissue. In the Nehuentue estuary, the bacterial communities in the tissues were dominated by the Tenericutes phylum, whereas the Tenericutes and Proteobacteria phyla dominated in mussels from Hueihue Bay. Higher numbers of operational taxonomic units (OTUs) were observed in tissues from the Nehuentue Estuary than in those from Hueihue Bay. Differences in bacterial community compositions in tissues between both locations were confirmed by nonmetric multidimensional scaling (nMDS) and Venn diagram analysis. In addition, linear discriminant analysis effect size (LEfSe) revealed that the Mollicutes class and Actynomycetales order were key phylotypes in tissues from the Nehuentue Estuary and Hueihue Bay, respectively. Our analysis also predicted a high abundance of sequences assigned to heterotrophy; however, relatively high functional diversity was also found in tissues from Hueihue Bay. This work represents our first attempt to elucidate the C. chorus microbiota in contrasting Chilean aquatic environments.

The cause of harmful algal blooms has been a mystery, but research to elucidate its mechanism has progressed over the years thanks to genetic technologies. We have monitored toxic algae and its associated bacteria as a community, the so-called 'holobiont' in Chilean coastal waters for years from the perspective of bacteria as an algal bloom driver. This review describes the challenges of holobiont monitoring, specifically with respect to standardizing and compliance with the monitoring protocols to collect reliable and sustainable data. Further, we suggest adopting the high-throughput sequencing (HTS) standard operating procedure (SOP) by the International Human Microbiome to improve the quality and consistency of holobiont monitoring in the harmful algal world.