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.

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.

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.

Perspectives on Sampling and New Generation Sequencing Methods for Low-Biomass Bioaerosols in Atmospheric Environments.

Bioaerosols play essential roles in the atmospheric environment and can affect human health. With a few exceptions (e.g., farm or rainforest environments), bioaerosol samples from wide-ranging environments typically have a low biomass, including bioaerosols from indoor environments (e.g., residential homes, offices, or hospitals), outdoor environments (e.g., urban or rural air). Some specialized environments (e.g., clean rooms, the Earth's upper atmosphere, or the international space station) have an ultra-low-biomass. This review discusses the primary sources of bioaerosols and influencing factors, the recent advances in air sampling techniques and the new generation sequencing (NGS) methods used for the characterization of low-biomass bioaerosol communities, and challenges in terms of the bias introduced by different air samplers when samples are subjected to NGS analysis with a focus on ultra-low biomass. High-volume filter-based or liquid-based air samplers compatible with NGS analysis are required to improve the bioaerosol detection limits for microorganisms. A thorough understanding of the performance and outcomes of bioaerosol sampling using NGS methods and a robust protocol for aerosol sample treatment for NGS analysis are needed. Advances in NGS techniques and bioinformatic tools will contribute toward the precise high-throughput identification of the taxonomic profiles of bioaerosol communities and the determination of their functional and ecological attributes in the atmospheric environment. In particular, long-read amplicon sequencing, viability PCR, and meta-transcriptomics are promising techniques for discriminating and detecting pathogenic microorganisms that may be active and infectious in bioaerosols and, therefore, pose a threat to human health. Supplementary Information: The online version contains supplementary material available at 10.1007/s41745-023-00380-x.

Learning from mistakes: challenges in finding holobiont factors from environmental samples and the importance of methodological consistency.

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.

Protocol of living cell separation using the microfluidic dielectrophoresis integrated chip.

This protocol demonstrates the separation of living cells with the microfluidic dielectrophoresis chip, using the Jurkat cell as a model. The successful living cell separation lies in familiarity with the detailed tips, which are aided by this stepwise protocol. The knowledge of correct chip installation, sample and buffer filling, flow rate and cell concentration adjustments, and contamination sources increases the efficiency of target viable cell collection. Such instructions, although trivial, are critical for achieving cell separation. For complete details on the use and execution of this protocol, please refer to Oshiro et al. (2022).

Fabrication of a new all-in-one microfluidic dielectrophoresis integrated chip and living cell separation.

Microfluidic dielectrophoresis (DEP) technology has been applied to many devices to perform label-free target cell separation. Cells separated by these devices are used in laboratories, mainly for medical research. The present study designed a microfluidic DEP device to fabricate a rapid and semiautomated cell separation system in conjunction with microscopy to enumerate the separated cells. With this device, we efficiently segregated bacterial cells from liquid products and enriched one cell type from two mixed eukaryotic cell types. The device eliminated sample pretreatment and established cell separation by all-in-one operation in a lab-on-chip, requiring only a small sample volume (0.5-1 mL) to enumerate the target cells and completing the entire separation process within 30 min. Such a rapid cell separation technique is in high demand by many researchers to promptly characterize the target cells.

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.

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.

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.

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.

Distribution of dissolved iron and bacteria producing the photoactive siderophore, vibrioferrin, in waters off Southern California and Northern Baja.

Phytoplankton blooms can cause acute effects on marine ecosystems due either to their production of endogenous toxins or to their enormous biomass leading to major impacts on local economies and public health. Despite years of effort, the causes of these Harmful Algal Blooms are still not fully understood. Our hypothesis is that bacteria that produce photoactive siderophores may provide a bioavailable source of iron for phytoplankton which could in turn stimulate algal growth and support bloom dynamics. Here we correlate iron concentrations, phytoplankton cell counts, bacterial cell abundance, and copy numbers for a photoactive siderophore vibrioferrin biosynthesis gene in water samples taken from 2017 cruises in the Gulf of California, and the Pacific Ocean off the coast of northern Baja California as well as during a multiyear sampling at Scripps Pier in San Diego, CA. We find that bacteria producing the photoactive siderophore vibrioferrin, make up a surprisingly high percentage of total bacteria in Pacific/Gulf of California coastal waters (up to 9%). Vibroferrin's unique properties and the widespread prevalence of its bacterial producers suggest that it may contribute significantly to generating bioavailability of iron via photoredox reactions.

Iron uptake and storage in the HAB dinoflagellate Lingulodinium polyedrum.

The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood with two basic strategies being distinguished: Strategy I involves the induction of an Fe(III)-chelate reductase (ferrireductase) along with Fe(II) or Fe(III) transporter proteins while strategy II plants have evolved sophisticated systems based on high-affinity, iron specific, binding compounds called phytosiderophores. In contrast, there is little knowledge about the corresponding systems in marine, plant-like lineages. Herein we report a study of the iron uptake and storage mechanisms in the harmful algal bloom dinoflagellate Lingulodinium polyedrum. L. polyedrum is an armored dinoflagellate with a mixotrophic lifestyle and one of the most common bloom species on Southern California coast widely noted for its bioluminescent properties and as a producer of yessotoxins. Short term radio-iron uptake studies indicate that iron is taken up by L. polyedrum in a time dependent manner consistent with an active transport process. Based on inhibitor and other studies it appears that a reductive-oxidative pathway such as that found in yeast and the green alga Chlamydomonas reinhardtii is likely. Of the various iron sources tested vibrioferrin, a photoactive and relatively weak siderophore produced by potentially mutualistic Marinobacter bacterial species, was the most efficient. Other more stable and non-photoactive siderophores such as ferrioxamine E were ineffective. Several pieces of data including long term exposure to 57Fe using Mössbauer spectroscopy suggest that L. polyedrum does not possess an iron storage system but rather presumably relies on an efficient iron uptake system, perhaps mediated by mutualistic interactions with bacteria.

Evaluation of photo-reactive siderophore producing bacteria before, during and after a bloom of the dinoflagellate Lingulodinium polyedrum.

Evidence is increasing for a mutualistic relationship between phytoplankton and heterotrophic marine bacteria. It has been proposed that bacteria producing photoactive iron binding compounds known as siderophores could play an important role in such mutualistic associations by producing bioavailable iron utilizable by phytoplankton and in exchange receive autotrophically derived DOM. In order to understand the potential role photoactive siderophores might be playing in bacterial-algal mutualism or marine biogeochemistry in general, it is important to be able to detect and quantify their presence in various environments. One approach to accomplish that end is to make use of high sensitivity genomics technology (qPCR) to search for siderophore biosynthesis genes related to the production of photoactive siderophores. In this way one can access their "biochemical potential" and utilize this information as a proxy for the presence of these siderophores in the marine environment. In this report we studied the correlation of the presence of bacteria producing one of the three photoactive siderophores relative to total bacterial and dinoflagellate numbers from surface water at the Scripps Pier before, during, and after fall bloom of the dinoflagellate Lingulodinium polyedrum. We believe that these findings will aid us in gauging the importance of photoactive siderophores in the marine environment and in harmful algal bloom dynamics in particular.

Mutagenesis of lysine 62, asparagine 64, and conserved region 1 reduces the activity of human ecto-ATPase (NTPDase 2).

The human ecto-ATPase (NTPDase 2) contains conserved motifs including five apyrase conserved regions (ACRs) and four conserved regions (CRs) as well as conserved lysine and arginine residues that are also present in other cell surface E-NTPDases. Some of the positively charged amino acids may be involved in ATP binding. The protein also contains six potential N-linked glycosylation sites. Results obtained with seven lysine and six arginine mutants indicate the importance of K62 that is located in CR1, K182, which is downstream of ACR3, and R155, which immediately follows CR3. Mutation of asparagine at the six potential N-linked glycosylation sites individually to glutamine established the importance of N64 in CR1 and N443 in ACR5 in protein function and expression. Mutation of N64, which is conserved in all cell surface NTPDases, results in the expression of an unstable protein, the activity of which is only manifested in the presence of concanavalin A. Both K62 and N64 reside in CR1 that is conserved in all cell surface NTPDases. In the sequence of the CR1 of human ecto-ATPase, 58WPADKENDTGIV69, 65DTG67 is similar to the phosphate-binding motif (DXG) in ACR1 and 4. The D65A and G67A mutants have reduced protein expression and activity. Mutations of other residues in CR1 to alanine led to partial to complete loss of protein expression and activity except for P59. The alanine mutants of the three acidic amino acid residues, D61, E63, and D65, all have decreased affinity for divalent ions. D61 can be substituted by glutamate, but E63 appears to be invariable. Taken together, these results indicate that CR1, which follows ACR1 in the cell surface NTPDases, is an essential structural element in these enzymes.