BACKGROUND: Harmful algal blooms (HABs), caused by the rapid proliferation or aggregation of microorganisms, are catastrophic for the environment. The Prymnesium parvum is a haptophyte algal species that is found worldwide and is responsible for extensive blooms and death of larval amphibians and bivalves, causing serious negative impacts on the ecological environment. For the prevention and management of environmental pollution, it is crucial to explore and develop early detection strategies for HABs on-site using simple methods. The major challenge related to early detection is the accurate and sensitive detection of algae present in low abundance.
RESULTS: Herein, recombinase polymerase amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats and Cas12a protein (CRISPR-LbaCas12a) systems, and the lateral flow dipstick (LFD) was used for the first time for early detection of P. parvum. The internal transcribed spacer (ITS) of P. parvum was selected as the target sequence, and the concentration of single-strand DNA reporters, buffer liquid system, reaction time, and amount of gold particles were optimized. The RPA-CRISPR-LbaCas12a-LFD approach demonstrated highly specificity during experimental testing, with no cross-reaction against different microalgae used as controls. In addition, the lowest detection limit was 10,000 times better than the lowest detection limit of the standalone RPA approach. The feasibility and robustness of this approach were further verified by using the different environmental samples. It also observed that P. parvum are widely distributed in Chinese Sea, but the cell density of P. parvum is relatively low (<0.1 cells/mL).
CONCLUSIONS: The developed approach has an excellent specificity and offers 10,000 times better sensitivity than the standalone RPA approach. These advantages make this approach suitable for early warning detection and prevention of HAB events in environmental water. Also, the outcomes of this study could promote a shift from traditional laboratory-based detection to on-site monitoring, facilitating early warning against HABs.

This study introduces a comprehensive method for quantifying mass mortality events in freshwater wildlife, exemplified by the ecological disaster in the Odra River in 2022. Our approach integrates field observations, statistical analysis, and ecological assessment to measure the impact of such events on various aquatic species. Key steps include systematic counting of deceased organisms, assessing population declines, and evaluating the ecological repercussions of invasive species. Utilizing the R programming language, we developed a framework that is adaptable to similar ecological crises in different aquatic environments. This methodology facilitates a detailed understanding of the scale and implications of mass mortality events, thereby contributing to effective environmental management and conservation efforts. •The analysis and modeling methods of the disaster are presented in the R programming language.•Exclusively open-source software was used for the analysis.•The analysis includes detailed data on the disaster\'s impact on various species.

Freshwater ecosystems are highly susceptible to harmful algal blooms (HABs), which are often caused by monospecific dense blooms. Effective preventive management strategies are urgently needed to avoid wide-ranging and severe impacts often resulting in costly damage to resources and unsustainable management options. In this study, we utilized SDM techniques focused on Prymnesium parvum, one of the most notorious HABs species worldwide. We first compare the climatic space occupied by P. parvum in North America, Europe and Australia. Additionally, we use MaxEnt algorithm to infer, for the first time, the potentially suitable freshwater environments in the aforementioned ranges. We also discuss the risks of invasion in reservoirs - prone habitats to persistent blooms of pests and invasive phytoplanktonic species. Our results show populations with distinctive niches suggesting ecophysiological tolerances, perhaps reflecting different strains. Our model projections revealed that the potential extent for P. parvum invasions is much broader than its current geographic distribution. The spatial configuration of reservoirs, if not sustaining dense blooms due to non-optimal conditions, favors colonization of multiple basins and ecoregions not yet occupied by P. parvum. Our models can provide valuable insights to decision-makers and monitoring programs while reducing the resources required to control the spread of P. parvum in disturbed habitats. Lastly, as impact magnitude is influenced by toxicity which in turn varies between different strains, we suggest future studies to incorporate intraspecific genetic information and fine-scale environmental variables to estimate potential distribution of P. parvum.

In the summer of 2022, the River Odra in Europe witnessed a significant ecological disaster, leading to an unprecedented mass mortality among fish, bivalves, and water snails. The disaster was attributed to toxins released by the haptophyte golden algae, Prymnesium parvum. This study primarily focused on the river\'s lower section, where the disaster\'s impacts were exacerbated by the downstream flow of deceased organisms. The Unionidae mussels\' mortality rate in this section was estimated at 65 million individuals, marking an 88 % decline in their population. The native mussel, Anodonta anatina, saw the steepest decline at 95 %, while the invasive Sinanodonta woodiana decreased by 15 %. Additionally, a minimum of 147 million dead water snails, predominantly Viviparus viviparus, were found ashore, indicating an 85 % population decline. An estimated 3.3 million fish, predominantly ruffe (Gymnocephalus cernua), bream (Abramis brama), and perch (Perca fluviatilis), were found deceased along the lower Odra, amounting to a biomass of 1025 tons. Across the entire 560 km affected stretch of the river, the estimated fish mortality was 1650 tons, a 60 % decline from pre-disaster levels. The swift deterioration of the river\'s ecosystem underscores the need for further studies on its adaptive capacity and potential recovery.

Many harmful algae are mixoplanktonic, i.e. they combine phototrophy and phagotrophy, and this ability may explain their ecological success, especially when environmental conditions are not optimal for autotrophic growth. In this study, laboratory experiments were conducted with the mixotrophic and ichthyotoxic haptophyte Prymnesium parvum (strain CCAP 946/6) to test the effects of phosphorus (P) sufficiency and deficiency on its growth rate, phagotrophic and lytic activities. P-deficient P. parvum cultures were grown without or with addition of P in the form of inorganic phosphorus (nutrients) and/or living algal prey (i.e. the cryptophyte Teleaulax amphioxeia). The ingestion rate of P. parvum and prey mortality rate were calculated using flow cytometry measurements based on pigment-derived-fluorescence to distinguish between prey, predators and digesting predators. The first aim of the study was to develop a method taking into account the rate of digestion, allowing the calculation of ingestion rates over a two-week period. Growth rates of P. parvum were higher in the treatments with live prey, irrespective of the concentration of inorganic P, and maximum growth rates were found when both inorganic and organic P in form of prey were added (0.79 ± 0.07 d-1). In addition, the mortality rate of T. amphioxeia induced by lytic compounds was 0.2 ± 0.02 d-1 in the P-deficient treatment, while no mortality was observed under P-sufficiency in the present experiments. This study also revealed the mortality due to cell lysis exceeded that of prey ingestion. Therefore, additional experiments were conducted with lysed prey cells. When grown with debris from prey cells, the mean growth rate of P. parvum was similar to monocultures without additions of prey debris (0.30 ± 0.1 vs. 0.38 ± 0.03 d-1), suggesting that P. parvum is able to grow on prey debris, but not as fast as with live prey. These results provide the first quantitative evidence over two weeks of experiment that ingestion of organic P in the form of living prey and/or debris of prey plays an important role in P. parvum growth and may explain its ecological success in a nutrient-limited environments.

Bioassays using cultures of the toxic haptophyte Prymnesium parvum and the ciliate Cyclidium sp. as prey were conducted to test the effect of pH (range = 6.5 - 8.5), salinity (range = 1.50 - 7.50‰), and a combination of pH and salinity on the toxicity of P. parvum. pH had a significant effect on P. parvum toxicity. Toxicity was rapidly (within 24 hr) induced by increasing pH of the medium, or reduced by lowering pH. Conversely, lowering salinity reduced toxicity, albeit less effectively compared to pH, and P. parvum cells remained toxic at the lowest values tested (1.50‰ at pH 7.5). An additional effect between pH and salinity was also observed: low salinity combined with low pH led to not only decreased toxicity, but also resulted in lower P. parvum growth rates. Such effects of pH and salinity on P. parvum growth and toxicity provide insight into the environmental factors supporting community dominance and toxic blooms of the alga.

This study examined the association of air, land, and water variables with the first historical occurrence and current distribution of toxic Prymnesium parvum blooms in reservoirs of the Brazos River and Colorado River, Texas (USA). One impacted and one reference reservoir were selected per basin. Land cover and use variables were estimated for the whole watershed (WW) and a 0.5-km zone on either side of streams (near field, NF). Variables were expressed in annual values. Principal component and trend analyses were used to determine (1) differences in environmental conditions before and after the 2001 onset of toxic blooms in impacted reservoirs (study period, 1992-2017), and (2) traits that uniquely discriminate impacted from reference reservoirs (2001-2017). Of thirty-three variables examined, two positively aligned with the reoccurring appearance of blooms in impacted reservoirs (air CO2 and herbicide Glyphosate) and another two negatively aligned (insecticides Terbufos and Malathion). Glyphosate use was observed throughout the study period but a turning point for an upward trend occurred near the year of first bloom occurrence. While the relevance of the decreased use of insecticides is uncertain, prior experimental studies reported that increasing concentrations of air CO2 and water Glyphosate can enhance P. parvum growth. Consistent with prior findings, impacted reservoirs were of higher salinity than reference reservoirs. In addition, their watersheds had far lower wetland cover at NF and WW scales. The value of wetlands in reducing harmful algal bloom incidence by reducing nutrient inputs has been previously recognized, but wetlands can also capture pesticides. Therefore, a diminished wetland cover could magnify Glyphosate loads flowing into impacted reservoirs. These observations are consistent with a scenario where rising levels of air CO2 and Glyphosate use contributed to the establishment of P. parvum blooms in reservoirs of relatively high salinity and minimal wetland cover over their watersheds.

Prymnesium parvum causes harmful algal blooms worldwide that are often associated with massive fish-kills and subsequent economic losses. Most of our knowledge of the toxicity of P. parvum derives from bioassays since methods for the identification and quantification of their toxins have been lacking. Recently, a quantitation method was developed for the causative lytic toxins, the prymnesins. Here, we for the first time present data on the influence of irradiance on cellular content and production of prymnesins under nutrient replete conditions in two P. parvum strains, which both produce B-type prymnesins. Large differences were observed between the two strains with regard to the influence of irradiance on prymnesin cell quota and production rates. At the highest irradiance level (550 µmol photons m-2 s-1), the cellular prymnesin quota was thirty times higher in strain K-0081 strain than in K-0374. The cellular prymnesin quota and production rates were closely linked to rates of growth and photosynthesis in strain K-0081, while this was not the case for K-0374. Yet, growth rate did explain the differences in prymnesin quota in the two strains. Consequently, the maximum prymnesin production rate (414 attomol cell-1 d-1) was only about three times higher in strain K-0081 than in K-0374, and revealed an optimum at the same irradiance of 200 µmol photons m-2 s-1 in both strains. At low irradiance levels, the difference in production rates between both strains became smaller, with 41 and 49 attomol cell-1 d-1 for K-0081 and K-0374, respectively. The carbon content of prymnesins made up for ∼3% and <1% of the total cellular carbon content in strains K-0081 and K-0374, respectively. The fraction of extracellular dissolved prymnesins was measured for strain K-0081, where it accounted for 14-30% of total prymnesin concentration in the cultures, irrespective of irradiance. The concentrations of prymnesins released to the water by the K-0081 strain were not significantly influenced by irradiance. Overall, we observed comparable responses in growth and photosynthesis of both tested strains toward changes in irradiance. However, the effects of irradiance on prymnesin quota and production rates were remarkably different between the two strains.

Prymnesium parvum is a toxin-producing microalga, which causes harmful algal blooms globally, frequently leading to massive fish kills that have adverse ecological and economic implications for natural waterways and aquaculture alike. The dramatic effects observed on fish are thought to be due to algal polyether toxins, known as the prymnesins, but their lack of environmental detection has resulted in an uncertainty about the true ichthyotoxic agents. Using qPCR, we found elevated levels of P. parvum and its lytic virus, PpDNAV-BW1, in a fish-killing bloom on the Norfolk Broads, United Kingdom, in March 2015. We also detected, for the first time, the B-type prymnesin toxins in Broads waterway samples and gill tissue isolated from a dead fish taken from the study site. Furthermore, Norfolk Broads P. parvum isolates unambiguously produced B-type toxins in laboratory-grown cultures. A 2 year longitudinal study of the Broads study site showed P. parvum blooms to be correlated with increased temperature and that PpDNAV plays a significant role in P. parvum bloom demise. Finally, we used a field trial to show that treatment with low doses of hydrogen peroxide represents an effective strategy to mitigate blooms of P. parvum in enclosed water bodies.

Harmful algal blooms are a significant environmental problem. Cells that bloom are often associated with intercellular or dissolved toxins that are a grave concern to humans. However, cells may also excrete compounds that are beneficial to their competition, allowing the cells to establish or maintain cells in bloom conditions. Here, we develop a yeast cell assay to assess whether the bloom-forming species can change the toxicity of the water environment. The current methods of assessing toxicity involve whole organisms. Here, yeast cells are used as a bioassay model to evaluate eukaryotic cell toxicity. Yeast is a commonly used, easy to maintain bioassay species that is free from ethical concerns, yet is sensitive to a wide array of metabolic and membrane-modulating agents. Compared to methods in which the whole organism is used, this method offers rapid and convenient cytotoxicity measurements using a lower volume of samples. The flow cytometer was employed in this toxicology assessment to measure the number of dead cells using alive/dead stain analysis. The results show that yeast cells were metabolically damaged after 1 h of exposure to our model toxin-producing euryhaline flagellates (Heterosigma akashiwo and Prymnesium parvum) cells or extracts. This amount was increased by extending the incubation time.