Anthropogenic pressures like ocean warming, acidification, rising N:P ratios, and marine heatwaves are affecting eukaryotic plankton diversity, though their combined impacts are rarely studied. To address this, we conducted a mesocosm experiment on a North Sea plankton community, testing the influence of a marine heatwave under ambient and future environmental conditions. Using 18S rRNA amplicon sequencing, we found that global change generally reduced protist diversity, in particular that of phototrophic organisms. While heterotrophs were largely unaffected by heatwaves, phototrophic diversity declined especially during cooling and only recovered under ambient conditions. Global change shifted the community from nano- to pico-sized phototrophs and increased harmful algae bloom species and parasites, while heatwaves elevated marine ochrophytes. The coccolithophore Gephyrocapsa oceanica thrived under both stressors. Our findings suggest that changing baseline conditions and extreme events can differentially impact heterotrophic and phototrophic diversity, with potential consequences for the metabolic balance of eukaryotic plankton communities.

Toxin production in planktonic protists is widespread and comprises an effective mechanism to eliminate competitors or grazers. Toxins released into the water can mediate the immobilization of prey for subsequent consumption or can mediate the lysis and uptake of the released nutrients. For the worldwide-distributed ichthyotoxic and mixotrophic haptophyte Prymnesium parvum, the relationship between toxin production; impact on co-occurring species, and mixotrophy remains blurred. In the current study, we show that changes in salinities (5 vs 30), phosphorous (P) availabilities (P-replete vs P-deplete) and cell densities affect growth, toxicity and mixotrophy in P. parvum. Cell density positively affected cellular toxin content by a factor of up to 10. Low salinity resulted in a higher mortality of the cryptophyte prey Teleaulax acuta, with ~80% of cells being lysed after 2 h of incubation. However, phagotrophic rates were higher in P-deplete conditions, independent of the salinity. Transcriptomic analysis of the monocultures revealed the up-regulation of genes involved in endocytosis under either low salinity and phosphorous, suggesting that this process is evolutionarily conserved, triggered by environmental stressors and independent of prey presence. Polyketide synthase genes, potentially involved in toxin biosynthesis, exhibited distinct expression patterns, depending on the physiological status, toxicity and with generally higher expression under the high cell density conditions. Overall, our study contributes to a better understanding of the dynamics between the two critical processes of toxin production and mixotrophy, and has important implications for bloom formation and its maintenance in this ecologically important species.

High-throughput sequencing for analysis of environmental microbial diversity has evolved vastly over the last decade. Currently the go-to method for microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics datasets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.