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Pfiesteria piscicida and P. shumwayae reportedly secrete potent exotoxins thought to cause fish lesion events, acute fish kills and human disease in mid-Atlantic USA estuaries. However, Pfiesteria toxins have never been isolated or characterized. We investigated mechanisms by which P. shumwayae kills fish using three different approaches. Here we show that larval fish bioassays conducted in tissue culture plates fitted with polycarbonate membrane inserts exhibited mortality (100%) only in treatments where fish and dinospores were in physical contact. No mortalities occurred in treatments where the membrane prevented contact between dinospores and fish. Using differential centrifugation and filtration of water from a fish-killing culture, we produced 'dinoflagellate', 'bacteria' and 'cell-free' fractions. Larval fish bioassays of these fractions resulted in mortalities (60-100% in less than 24 h) only in fractions containing live dinospores ('whole water', 'dinoflagellate'), with no mortalities in 'cell-free' or 'bacteria'-enriched fractions. Videomicrography and electron microscopy show dinospores swarming toward and attaching to skin, actively feeding, and rapidly denuding fish of epidermis. We show here that our cultures of actively fish-killing P. shumwayae do not secrete potent exotoxins; rather, fish mortality results from micropredatory feeding.

Pfiesteria shumwayae Glasgow et Burkholder is assigned to a new genus Pseudopfiesteria gen. nov. Plate tabulation differences between Pfiesteria and Pseudopfiesteria gen. nov. as well as a maximum likelihood phylogenetic analysis based on rDNA sequence data warrant creation of this new genus. The Kofoidian thecal plate formula for the new genus is Po, cp, X, 4 0 , 1a, 6 0 0 , 6c, PC, 5 þ s, 5 0 0 0 , 0p, 2 0 0 0 0 . In addition to having six precingular plates, P. shumwayae comb. nov. also has a distinctive diamond or rectangular-shaped anterior intercalary plate. Both Pfiesteria and Pseudopfiesteria gen. nov. are reassigned to the order Peridiniales based on an apical pore complex (APC) with a canal (X) plate that contacts a symmetrical 1 0 , four to five sulcal plates, and the conservative hypothecal tabulation of 5 0 0 0 , 0p, and 2 0 0 0 0 . These morphological characters and the life histories of Pfiesteria and Pseudopfiesteria are consistent with placement of both genera in the Peridiniales. Based on the plate tabulations for P. shumwayae, P. piscicida, and the closely related ''cryptoperidiniopsoid'' and ''lucy'' groups, the family Pfiesteriaceae is amended to include species with the following tabulation: 4-5 0 , 0-2a, 5-6 0 0 , 6c, PC, 5 þ s, 5 0 0 0 , 0p, and 2 0 0 0 0 as well as an APC containing a pore plate (Po), a closing plate (cp), and an X plate; the tabulation is expanded to increase the number of sulcal plates and to include a new plate, the peduncle cover (PC) plate. Members of the family have typical dinoflagellate life cycles characterized by a biflagellated free-living motile stage, a varying number of cyst stages, and the absence of multiple amoeboid stages.Pfiesteria and Pfiesteria-like species are morphologically similar thinly armored dinoflagellates with flagellated stages characterized by their distinct Kofoidian thecal plate formulae or plate tabulations. Currently, the plate formula of the genus Pfiesteria is characterized 1

A new genus of Pfiesteria-like heterotrophic dinoflagellate, Luciella gen. nov., and two new species, Luciella masanensis sp. nov. and Luciella atlantis sp. nov., are described. These species commonly occur with other small (<20 lm) heterotrophic and mixotrophic dinoflagellates in estuaries from Florida to Maryland and the southern coast of Korea, suggesting a possible global distribution. An SEM analysis indicates that members of the genus Luciella have the enhanced Kofoidian plate formula of Po, cp, X, 4¢, 2a, 6¢¢, 6c, PC, 5+s, 5¢¢¢, 0p, and 2¢¢¢¢. The two four-sided anterior intercalary plates are diamond shaped. The genus Luciella differs from the other genera in the Pfiesteriaceae by a least one plate in the plate tabulation and in the configuration of the two anterior intercalary plates. An SSU rDNA phylogenetic analysis confirmed the genus as monophyletic and distinct from the other genera in the Pfiesteriaceae. The morphology of Luciella masanensis closely resembles Pfiesteria piscicida Steid. et J. M. Burkh. and other Pfiesteria-like dinoflagellates in size and shape, making it easily misidentified using LM. Luciella atlantis, in contrast, has a more distinctive morphology. It can be distinguished from L. masanensis and other Pfiesteria-like organisms by a larger cell size, a more conical-shaped epitheca and hypotheca, larger rhombic-shaped intercalary plates, and an asymmetrical hypotheca. The genus Luciella is assigned to the order Peridiniales and the family Pfiesteriaceae based on plate tabulation, plate pattern, general morphology, and phylogenetic analysis.

A new genus and species of heterotrophic dinoflagellate, Cryptoperidiniopsis brodyi gen. et sp. nov., are described. This new species commonly occurs in estuaries from Florida to Maryland, and is often associated with Pfiesteria piscicida Steidinger et Burkholder, Pseudopfiesteria shumwayae (Glasgow et Burkholder) Litaker et al., and Karlodinium veneficum (Ballantine) J. Larsen, as well as other small (<20 μm) heterotrophic and mixotrophic dinoflagellates. C. brodyi gen. et sp. nov. feeds myzocytotically on pigmented microalgae and other microorganisms. The genus and species have the enhanced Kofoidian plate formula of Po, cp, X, 5′, 0a, 6″, 6c, PC, 5+s, 5″′, 0p, and 2″″ and are assigned to the order Peridiniales and the family Pfiesteriaceae. Because the Pfiesteriaceae comprise small species and are difficult to differentiate by light microscopy, C. brodyi gen. et sp. nov. can be easily misidentified.