Leonard W. Haas

Pfiesteria shumwayae kills fish by micropredation not exotoxin secretion

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.

THE RECLASSIFICATION OF PFIESTERIA SHUMWAYAE (DINOPHYCEAE): PSEUDOPFIESTERIA, GEN. NOV.1

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′, 1a, 6′′, 6c, PC, 5+s, 5′′′, 0p, 2′′′′. 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′, four to five sulcal plates, and the conservative hypothecal tabulation of 5′′′, 0p, and 2′′′′. 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′, 0‐2a, 5‐6′′, 6c, PC, 5+s, 5′′′, 0p, and 2′′′′ 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.

Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteria shumwayae

The estuarine genus Pfiesteria has received considerable attention since it was first identified and proposed to be the causative agent of fish kills along the mid-Atlantic coast in 1992. The presumption has been that the mechanism of fish death is by release of one or more toxins by the dinoflagellate. In this report, we challenge the notion that Pfiesteria species produce ichthyotoxins. Specifically, we show that (i) simple centrifugation, with and without ultrasonication, is sufficient to “detoxify” water of actively fish-killing cultures of Pfiesteria shumwayae, (ii) organic extracts of lyophilized cultures are not toxic to fish, (iii) degenerate primers that amplify PKS genes from several polyketide-producing dinoflagellates failed to yield a product with P. shumwayae DNA or cDNA, and (iv) degenerate primers for NRPS genes failed to amplify any NRPS genes but (unexpectedly) yielded a band (among several) that corresponded to known or putative PKSs and fatty acid synthases. We conclude that P. shumwayae is able to kill fish by means other than releasing a toxin into bulk water. Alternative explanations of the effects attributed to Pfiesteria are suggested.

Phytoplankton Response to a Stratification-Mixing Cycle in the York River Estuary during Late Summer

As part of a larger multidisciplinary study of the lower York River estuary, phytoplankton response to a tidally related cycle of stratification-destratification was examined during August 1978. A “red water bloom” dominated by the dinoflagellate Cocchlodinium heterolobatum was initially observed in the lower York River coincident with the spring tide-induced water column destratification event. It is proposed that the dinoflagellates initiating the red tide were advected into the estuary in deep water during the preceding period of stratification or were derived from cysts in the sediments and that destratification provided access to the surface waters. The extent of the red water increased during the ensuing restratified period in the York River, and several lines of evidence indicated that C. heterolobatum migrated diurnally between ammonium enriched waters below the halocline (8–10 m) and the relatively nutrient-poor surface waters. Other estuarine systems in which phytoplankton blooms associated with alternating periods of stratification-destratification have been observed are noted. The results illustrate the close relationship between phytoplankton and hydrographic dynamics in this estuarine system and emphasize the necessity to include the study of hydrographic processes in the study of phytoplankton dynamics.

ELECTRON MICROSCOPY OF NANOPLANKTON FROM THE NORTH PACIFIC CENTRAL GYRE1

Nanoplankton was collected from the North Pacific Central Gyre during a cruise of R/V “Melville” in August 1985. Water samples from the surface and the deep chlorophyll maximum layer were fixed with glutaraldehyde and prepared for electron microscopy observation using formvar coated grids. The grids were examined first by epifluoresence microscopy under blue light to differentiate chloroplast and non‐chloroplast containing cells. The surface and deep samples were dominated strongly by members of the Prymnesiophyceae, accounting for 55% of the total number of organisms identified. Prymnesiales and Coccosphaerales were equally abundant although coccoliths‐bearing cells tended to dominate in the deep chlorophyll maximum layer. Heterotrophic choanoflagellates were also abundant at depth and were characterized by a high species diversity. Micromonas pusilla (Butcher) Manton & Parke was the major representative of the Prasinophyceae and was observed commonly within the deep chlorophyll maximum. Our observations reveal several new species, including autotrophic and heterotrophic specimens, and demonstrate the importance of knowing the structure of the planktonic community for ecological purposes. Indeed, the presence of a large number of heterotrophic organisms in the deep water suggests an active microbial food chain which may play an important role in regulating plankton processes in oligotrophic waters.

A NEW LARVAL FISH BIOASSAY FOR TESTING THE PATHOGENICITY OF PFIESTERIA SPP. (DINOPHYCEAE)1

Water quality, microbial contamination, prior fish health, and variable results have been major impediments to identifying the cause and mechanism of fish mortality in standard aquarium‐format Pfiesteria bioassays. Therefore, we developed a sensitive 96‐h larval fish bioassay for assessing Pfiesteria spp. pathogenicity using six‐well tissue culture plates and 7‐day‐old larval cyprinodontid fish. We used the assay to test pathogenicity of several clonal lines of Pfiesteria piscicida Steidinger and Burkholder and P. shumwayae Glasgow and Burkholder that had been cultured with algal prey for 2 to 36 months. The P. shumwayae cultures exhibited 80%–100% cumulative mortality in less than 96 h at initial zoospore densities of approximately 1000 cells·mL−1. No fish mortalities occurred with P. piscicida at identical densities or in controls. In a dose‐response assay, we demonstrated a strong positive correlation between dinospore density and fish mortality in a highly pathogenic culture of P. shumwayae, generating a 96‐h LD50 of 108 zoospores·mL−1. Additionally, we applied the assay to evaluate a 38‐L P. shumwayae bioassay that was actively killing fish and compared results with those from exposures of juvenile tilapia (Oreochromis niloticus) in a 500‐mL assay system. Water from the fish‐killing 38‐L assay was filtered and centrifuged to produce fractions dominated by dinoflagellates, bacteria, or presumed ichthyotoxin (cell‐free fraction). After 96 h, the larval fish assay exhibited 50%–100% cumulative mortality only in fractions containing dinoflagellates, with no mortalities occurring in the other fractions. The 500‐mL bioassay with tilapia produced inconsistent results and demonstrated no clear correlation between mortality and treatment. The new larval fish bioassay was demonstrated as a highly effective method to verify and evaluate dinoflagellate pathogenicity.

AN IMPROVED STRIPPING TECHNIQUE FOR LIGHTLY ARMORED DINOFLAGELLATES1

Identification of armored heterotrophic dinoflagellates relies, in part, on plate tabulations obtained by SEM. Currently, two methods are used to visualize plate morphology and develop plate tabulations: swelling the sutures between the cellulose plates of intact organisms or stripping off the outer membranes with ethanol to expose the underlying cellulose plates. Both approaches are problematic with lightly armored dinoflagellates because sutures do not consistently swell to enable visualization, and the outer membranes are not consistently stripped. Further, generic and species differences necessitate frequent modification of these protocols to obtain reliable results. We describe an improved membrane stripping technique using the detergent Triton X‐100. Our method provides a more consistent standardized approach to removing the outer membranes of lightly armored dinoflagellates, including Pfiesteria shumwayae Glasgow & Burkholder, a taxon that has, until now, proven very difficult to strip with currently published methods. This method allows visualization of the sulcus, a region previously difficult to observe, and will greatly facilitate taxonomic studies of the lightly armored forms.

SHORT TERM CHANGES IN THE VERTICAL SALINITY DISTRIBUTION OF THE YORK RIVER ESTUARY ASSOCIATED WITH THE NEAP-SPRING TIDAL CYCLE

A multidisciplinary investigation of hydrographic-nutrient-phytoplankton interactions was undertaken in the lower York River estuary of Virginia during August, 1978. The study centered on a spring tide-associated water column destratification event predicted to occur on or soon after August 19, the date of the maximum monthly spring tide. A station in the lower York River (depth 19 m) was occupied during four different periods, August 7–10, 16–17, 21–24 and 28–30, and temperature and salinity were measured periodically at 1 m depth intervals. During August 16–20 salinities were measured through the water column at seven stations in the York River extending from the mouth to 35 km upriver. During the first two sampling periods the water column was moderately to strongly stratified. Destratification was first observed 15–20 km upriver on August 18 and the lower river was destratified by August 21. Destratification persisted in the lower river for four days at which time increasing bottom salinities indicated the beginning of the restratification process. By August 28 restratification, resulting primarily from an increase in bottom salinities, was complete. The results illustrate the highly dynamic hydrographic nature of this estuarine system and the predictability of the stratification-destratification sequence. The effects of this hydrographic cycle on nutrient distributions, phytoplankton dynamics and benthic nutrient fluxes in this estuary are discussed in accompanying papers.

GENETIC VARIATION AMONG STRAINS OF PSEUDOPFIESTERIA SHUMWAYAE AND PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellates Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester and Pfiesteria piscicida Steid. et J. M. Burkh. have been implicated in massive fish kills and of having negative impacts on human health along the mid‐Atlantic seaboard of the USA. Considerable debate still remains as to the mechanisms responsible for fish mortality (toxicity vs. micropredation) caused by these dinoflagellates. Genetic differences among these cultures have not been adequately investigated and may account for or correlate with phenotypic variability among strains within each species. Genetic variation among strains of Ps. shumwayae and P. piscicida was examined by PCR–RFLP analysis using cultures obtained from the Provasoli‐Guillard National Center for Culture of Marine Phytoplankton (CCMP), as well as those from our own and other colleagues’ collection efforts. Examination of restriction digest banding profiles for 22 strains of Ps. shumwayae revealed the presence of 10 polymorphic restriction endonuclease sites within the first and second internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the rDNA complex, and the cytochrome oxidase subunit I (COI) gene. Three compound genotypes were represented within the 22 Ps. shumwayae strains. Conversely, PCR–RFLP examination of 14 strains of P. piscicida at the same ITS1, 5.8S, and ITS2 regions revealed only one variable restriction endonuclease site, located in the ITS1 region. In addition, a dinoflagellate culture listed as P. piscicida (CCMP 1928) and analyzed as part of this study was identified as closely related to Luciella masanensis P. L. Mason, H. J. Jeong, Litaker, Reece et Steid.