Harmful algal bloom species can persist in the environment, impacting aquatic life and human health. One of the mechanisms by which some harmful algal bloom species are able to persist is by consumption of organic particles. Methods to demonstrate and measure consumption can yield insight into how populations thrive. Here, we combine flow cytometry and real-time PCR to demonstrate consumption of a cryptophyte species (Rhodomonas sp.) by a toxic mixotrophic haptophyte (Prymnesium parvum). Using flow cytometry, the feeding frequency of a population of P. parvum cells was calculated using the phycoerythrin (PE) fluorescence signal from Rhodomonas sp. and the fluorescence of an acidotropic probe labeling the food vacuoles. Feeding frequency increased in the beginning of the experiment and then began to decline, reaching a maximum of 47.5% of the whole P. parvum population after 212 min. The maximum number of consumed Rhodomonas sp. cells was 0.8 per P. parvum cell, and occurred after 114 min corresponding to an ingestion rate of 0.4 Rhodomonas sp. cells/P. parvum/h. Cells from the feeding P. parvum population were sorted, washed, and subjected to a real-time PCR assay targeting the cryptophyte 18S locus. There was a correlation between cycle threshold (Ct) values and number of consumed prey cells calculated by fluorescence. Overall, this study shows that flow cytometric analysis, of the acidotropic probe and prey pigments, is an efficient and rapid tool in enumerating food vacuoles and the number of prey cells consumed. Furthermore, we suggest that real-time PCR can be applied to cells sorted by flow cytometry, thus allowing for the detection and potential quantification of the targeted prey cells.
Some microalgae are able to kill or inhibit nutrient-competing microalgae, a process called allelopathy. Inhibiting or killing competitors enable these species to monopolize limiting resources, such as nitrogen and phosphorus. Prymnesium parvum is known to produce such allelopathic compounds, substances that seem identical to the ichthyotoxins identified from this species. Biotic and abiotic environmental factors influence not only growth rates but also toxin/allelopathic compounds production by P. parvum cells. Toxin production, as well as allelopathy, including grazer deterrence, increases dramatically in light, temperature, or nutrient stressed P. parvum cells. Correspondingly, toxicity and allelopathy may decrease, or cease completely, if cells are grown with high amounts of N and P in balanced proportions. However, even under nutrient (N and P) sufficient conditions, P. parvum is able to produce toxins/allelopathic compounds, with negative effects on other phytoplankton species or grazers, if cells densities of P. parvum are high relative to other species. This negative effect might shift the plankton community to more toxin resistant species. Filtrates from nutrient-deficient P. parvum cultures have almost the same strong negative effect on grazers and other phytoplankton species as when Prymnesium cells are grown together with the target organisms. Eutrophication, the increased input of N and P to aquatic ecosystems, besides increasing nutrient concentrations, is usually provoking unbalanced N:P condition for the optimal growth of phytoplankton, deviating from the Redfield ratio, i.e., the phytoplankton cellular nitrogen to phosphorus ratio, N:P = 16:1 (by atoms) or 7.2:1 (by weight). Eutrophication thus both enhances P. parvum growth and increases production of toxins and allelopathic compounds. Supplying N-deficient or P-deficient P. parvum cells with the deficient nutrient reduces toxicity to less than half within 24 h after additions. As P. parvum is mixotrophic, uptake of dissolved or particulate organic N (DON or PON) can also reduce toxicity and allelopathy in the same manner as addition of inorganic N to N-starved cells. In conclusion, P. parvum, by increasing its toxicity and allelopathic ability under poor environmental conditions, outcompetes the co-occurring phytoplankton species.
Harmful algal bloom (HAB) control and mitigation is a complex problem in ecosystem management. Phytoplankton play an important role in aquatic ecosystems as primary producers and food sources for many commercially important shellfish and there are limited options for targeting just a single species within the community. Chemical treatments (e.g., algaecides), rotting barley straw, nitrogen and phosphorus manipulation, and clay and/or flocculants are but a few techniques tested or used to reduce fish kills or shellfish contamination during a HAB event. Prymnesium parvum control has focused on the use of chemicals, nutrient manipulation, and clay flocculation. However, many HAB control methods have been rejected due to their effects on ecosystems, high costs, or limited effects on target organisms. For example, rotting barley straw (Hordeum vulgare) is considered to be an environmentally friendly alternative, but has been found to have very different results on the phytoplankton community depending on the dominating taxa and is ineffective against P. parvum and dinoflagellate blooms. Clay flocculation is a useful control/mitigation technique during fish kills in marine aquaculture sites in South Korea and can be effective in freshwater if the correct combination of clay and flocculent is used. Toxins produced by P. parvum and Karenia brevis also bind to phosphatic clay, thereby removing and/or neutralizing the toxins, but there is concern that the clay will have a negative effect on sessile organisms. Some shellfish suffer high mortalities and significant impacts on somatic and reproductive tissue growth at high clay loads; however, benthic communities appear to be unchanged after five years of clay treatment in South Korea. There are likely site-specific and ecosystem-specific characteristics that make generalizations about control options difficult and require careful assessment of options at each location.