Vegetative and sexual reproduction in Pfiesteria spp. (Dinophyceae) cultured with algal prey, and inferences for their classification

Algal-fed clonal zoospore cultures of Pfiesteria piscicida and Pfiesteria shumwayae enabled description of certain conserved morphological and reproductive features. Common modes of reproduction (especially via division cysts) were documented in herbivorous P. piscicida and P. shumwayae using cultures fed algal prey, together with supporting photography and flow cytometric DNA measurements. Other cysts were characterized such as vacuolate cysts in starved P. piscicida cultures and temporary cysts in both species fed algal prey. This study also represents the first report of sexual reproduction in Pfiesteria spp. cultures fed algal prey rather than live fish; the first report of a technique for cell cycle synchronization for these heterotrophic dinoflagellates; and the first information on storage products of cells released from Pfiesteria reproductive cysts. Sexual reproduction in algal-fed P. piscicida clonal cultures was evidenced by fusing gametes, cells with two longitudinal flagella, and nuclear cyclosis. Both isogamous and anisogamous fusions were observed, and resulting cells with two trailing flagella (i.e., planozygotes and planomeiocytes) sometimes comprised ≥50% of the flagellated cells. These cells continued feeding activity and eventually (hours) lost their flagella and formed cysts. Nuclear cyclosis and a subsequent cell division were observed in thin-walled reproductive cysts prior to release of two flagellated cells. One gamete fusion event was also documented in 1 of 20 algal-fed clones of P. shumwayae, with an aplanozygote as the product. We obtained high cell synchrony (≥90% 1C) in the tested cultures using our preferential lysis technique and tracked the decline in lipid content of excysted zoospore populations over time. The data from this study were considered together with previous research to gain insights about relationships between Pfiesteria spp. and other heterotrophic dinoflagellates. Pfiesteria spp. should be regarded as free-living predators rather than parasites because they are prey generalists without demonstrated “host” specificity and their flagellated feeding stages are not morphologically distinct from swimming stages. Although they originally were placed within the Dinamoebales because amoebae can predominate, this study as well as other published research consistently has shown that the dominant stage varies depending on culture conditions, prey type/availability and strains. The peridinoid plate structure of each Pfiesteria species, which thus far has been conserved across culture conditions and strains, supports placement of Pfiesteria spp. within the Peridiniales. At the species level, plate structure (differing by one precingular plate) and molecular data (18S rDNA) indicate that the two Pfiesteria spp. are closely related in comparison to species grouped within other genera.