MARINE ECOLOGY PROGRESS SERIES Published August 10 Mar Ecol Prog Ser
Fish kills linked to a toxic ambush-predator dinoflagellate: distribution and environmental conditions
JoAnn M. Burkholder, Howard B. Glasgow Jr, Cecil W. Hobbs
Department of Botany, Box 7612, North Carolina State University, Raleigh, North Carolina 27695-7612. USA
ABSTRACT. The toxic ambush-predator dinoflagellate Pfiesteria piscicida gen. et sp. nov. has been implicated as a causative agent of major fish kills in estuarine ecosystems of the southeastern United States. P, piscicida is stimulated by fresh fish secreta, and it was lethal to all 19 species of native and exotic flnfish and shellfish bioassayed in culture; thus far in field and aquaculture kills linked to the d~noflagellate.13 additional fish species have been affected. Field data in combination with confirming laboratory bioassays documented toxicity at temperatures ranging from 12 to 33OC, with most out- breaks occurring at 26°C or higher. P. piscicida also exhibits wide salinity tolerance; it was lethal to fish from 0 to 35 ppt in calcareous waters, with an optimum salinity for growth and toxic activity at 15 ppt. It was toxic to fish day or night (2250 toxic zoospores ml-l) without an apparent light optimum, in expenmental laboratory conditions ranging from 0.2 pEin m-2 S-' (darkness for all but 30 to 60 s at 20 pE~nm-2 S-' per 24 h period) to 200 pEin m-2S" (12:12 h 1ight:dark cycle). Moreover, f~eldfish kills have occurred in darkness and at light intensities up to 2400 pEin m-2S-'. Through direct microscope counts of water samples, confirmed identifications with scanning electron microscopy, and confirmed toxic activity in bioassays, P. piscicida was implicated as the causative agent of 52 * 7 % of the major fish kills (affecting 103 to 10' fish from May 1991 to November 1993) on an annual basis in North Car- ollna estuaries and coastal waters. Since their discovery in natural hab~tatduring 1991, Pfiesteria-like species also have been tracked to eutrophic sudden-death fish kill sites In estuaries, coastal waters, and aquaculture facihties from the mid-Atlantic through the Gulf Coast. Toxic ambush-predator dinofla- gellates likely are widespread in warm temperate/subtropical regions, acting as significant but often undetected sources of fish mortality and disease.
KEY WORDS: Fish kills . Estuaries . Pfiesterja . Toxic dinoflagellates
INTRODUCTION cicida to emerge from benthic cysts or amoeboid stages (Burkholder et al. 1995). As the chemosensory stimula- A toxic dinoflagellate with ambush-predator behav- tion increases, zoospores develop the capacity for toxin ior and a complex life cycle recently was implicated as a production. These toxic flagellated vegetative cells (or causative agent of major fish kills in estuaries of the toxic zoospores, TZs) maintain a morphology similar to southeastern United States (Burkholder et al. 1992). that of the nontoxic zoospores, but produce an exo- Pfiesteria piscicida (gen. et sp. nov.; taxonomy to be toxin(s) that narcotizes finfish, sloughs the fish epider- proposed by K. A. Steidinger et al.),which represents a mis, and causes formation of open ulcerative lesions new family, genus and species of dinoflagellate, was (Noga et al. 1995). The dinoflagellates consume bits of first observed as a contaminant of unknown origin in epidermal tissue and blood cells from affected fish, finfish cultures (Smith et al. 1988, Noga et al. 1993), and while also engulfing available phytoplankton (Burk- was discovered at a fish kill in estuarine habitat during holder & Glasgow 1995). In addition, they produce 1991 (Burkholder et al. 1992). gametes that complete sexual fusion in the presence of Detection of unknown substances secreted by dying fish. The gametes feed saprotrophically on pro- schools of finfish stimulates zoospores of Pfiesteria pis- tein hydrolysates and phagocytize bacteria (Glasgow &
O Inter-Research 1995 Resale of full article not permitted 44 Mar Ecol Prog Ser 124: 43-61., 1995
Burkholder 1993). Upon fish death, TZs and planozy- MATERIALS AND METHODS gotes form mostly nontoxic amoeboid stages, or encyst and descend back to the sediments. Alternatively, in Study area. With assistance from the North Carolina the absence of live fish, gametes and TZs revert to non- Division of Marine Fisheries (NC DMF), the North toxic zoospores that maintain activity in phosphate- Carolina Division of Environmental Management (NC enriched waters, especially when flagellated algal DEM), and concerned citizens, we sampled in- prey are abundant (Burkholder & Glasgow 1995, Burk- progress fish kills from several estuaries within the holder et al. 1995). Albemarle-Pamlico Estuarine System, North Carolina In maintaining an array of active benthic amoebae as (Fig 1). This shallow, turbtd, wind-mixed lagoon rep- well as planktonic flagellates, toxic ambush-predator resents the second largest estuary by aerial extent in dinoflagellates differ markedly from red tide dinofla- the United States (Epperly & Ross 1986). Two of its gellates and the bloom-forming 'hidden flora' that largest tributary rivers, the Neuse and the Pamlico appear in high abundance to cause massive fish death [mean depth of each system ca 2.5 to 3.0 m; flushing (Smayda 1989, 1992, Culotta 1992). By contrast, the rates 30 d (Neuse) to 100 d (Pamlico) under low-flow lethal stages of toxic ambush predators include both conditions in summer; Copeland et al. 1984, Epperly & flagellated and amoeboid forms that are usually Ross 1986, Stanley 19921, were the central focus of this ephemeral in the water column, with toxic outbreaks research. Both estuaries have been classified as nutri- lasting less than 24 h (Burkholder et al. 1992). Because ent-sensitive waters by NC DEM; by the early 1980s they often represent a minor component of Lhe phyto- some locations had sustained sufficient nutrient load- plankton commun~ty during toxic outbreaks, they ing to support nuisance algal blooms and oxygen rarely are detected by water discoloration (NC deficits, with chlorophyll a seasonally exceeding the DNRCD 1987, 1988, 1989, NC DEHNR 1990a, 1991, State standard (40 1-19 1-l; NC DEHNR 1990b). 1992, unpubl. records, Burkholder et al. 1992). Produc- Although the 2 estuaries are in close proximity, the tion of high quantities of lipophilic exotoxin(s) (D. Pamlico differs from the Neuse in that it lies on a geo- Baden, Univ. Miami, unpubl. data), nonetheless, logical phosphorus formation. Adjacent to the lower enables relatively low cell densities to be highly lethal Pamlico Estuary on South Creek, Texasgulf Chemical to fish. Company operates the largest phosphorus mine in the The discovery of toxic ambush predators has begun world and discharged ca 2500 metric tons of phospho- to alter paradigms about estuarine dinoflagellates, rus daily from the late 1960s to 1992. This point source even at the fundamental level of discerning the major has now been reduced by more than 90%, but it repre- players. Until we learned about the complex life cycle sented ca 50% of the total phosphorus loading to the of Pfiesteria-like species, multiple filose (filipodial or Pamlico Estuary during that period (NC DEHNR 'star'), lobose (lobopodial), and hellozoan amoebae in 1990b). In the So.uth Creek area, phosphate ranged estuarine habitat were not recognized as dinoflagel- from ca 300 to >600 pg Pod3--P 1-' during summer lates. These widespread forms likely were identified, low-flow conditions (Stanley 1987). Since phosphorus instead, as belonging among 3 or more genera of reductions were achieved by Texasgulf in late 1992, amoebae without dinoflagellate linkages (e.g. Bovee & phosphate in both the Pamlico and Neuse sampling Sawyer 1979). The overall function of ambush-preda- areas generally has ranged from ca 50 to 120 pg tor dinoflagellates - including both toxic and nontoxic POA3--PI-' (NC DEHNR unpubl. records, J.M.R. & forms - in the food webs of shallow eutrophic estuar- H.B.G. unpubl, data). The low flushing rates and shal- ies has not been determined. In the present research, low, wlnd-mixed character of the Pamlico and Neuse, our objectives were to (1) assess the potential signifi- and seasonally high activity of bottom-disturbing fish- cance of the representative toxic ambush predator ing practices such as crab trawling, suggest that sub- Pfiesteria piscicida as a causative agent of major estu- stantial supplies of phosphorus and other nutrients in arine fish kills, including examination of environmen- sediment deposits frequently are resuspended to sup- tal cond.itions during kill events; (2) experimentally port algal growth. determine optimal physical factors conducive to Culture techniques. Culture isolates of Pfjesteria pis- growth of dominant stages in its life cycle; (3) assay cicida were collected on 23 May 1991 from the Pamlico susceptibility of an array of finfish and shellfish species River Estuary near Channel Marker no. 9 at the mouth to its lethal toxin(s); and (4) examine its geographic of Blount Bay in Beaufort County, North Carolina, dur- range in the mid-Atlantic and southeastern United ing an active bloom of TZs while ca 10" Atlantic men- States. This information is co+ltr~butedtoward the ultl- haden Brevoortia tyrannus Latrobe, southern flounder mate goal of predicting outbreaks of toxic ambush- Paral~chthyslethostigrna Jordan & Gilbert, hogchokers predator dinoflagellates and mitigating their effects on Tnnectes maculatus Block & Schneider and spot Leios- coastal fisheries. tomus xanthuris Lacepede were dying (Burkholder et Burkholder et al.: Fish kills linked to a toxic ambush-predator dinoflagellate
al. 1992, NC DEHNR 1992). Stock P. piscicida cultures the dinoflagellate tilapia Oreochromica mossambjca were maintained in isolated, quarantined facilities Peters (length 5 to 7 cm, rinsed thoroughly with deion- under 25 to 50 pEin m-' S-' (cool white fluorescent ized water; source Lake Geneva Fisheries, Geneva, lamps) at 18 to 20°C with a 12:12 h 1ight:dark (L:D) AL, USA) at a density of 9 to 12 fish d-l, and removed cycle in 40 l aerated, covered aquaria filled with artifi- all dead fish as live replacements were added. The cial seawater at 15 ppt salinity (standard medium; tilapia selected as the standard test species is not derived by adding Instant Ocean salts to deionized endemic but, nonetheless, is susceptible to the dinofla- water). Contact with culture water and associated neu- gellate's toxin. It offered the advantages of constant rotoxic aerosols has been associated with serious availability, wide salinity tolerance, and certainty of no human health effects (Glasgow et al. 1994, unpubl.). prior contamination by local P. piscicida populations. Hence, all work was completed using full-face respira- Stock tilapia were cultured in a separate building; to tors with organic acid filters; disposable gloves, boots avoid contamination, fish cultures were not visited and hair covers; and protective clothing that was after personnel had been in the dinoflagellate facility bleached (230% solution) after use to kill all dinofla- without shower and complete change of clothing. gellate stages. Using these precautions, we successfully prevented Pfiesterja piscjcida requires an unidentified sub- dinoflagellate contamination of stock fish. stance(~)in fresh fish excreta to initiate toxin produc- For all tests involving fish, each aquarium was filled tion (Burkholder et al. 1992); hence, it was necessary to with artificial seawater at 15 ppt salinity (except when maintain toxic cultures using live fish. We routinely fed testing salinity optima) that was continually filtered
PAMLICO ESTUARY
ATLANTIC OCEAN
KM
Fig. 1.Main study area (North Carolina. USA) showing the Neuse and Pamlico Estuaries and tributaries where toxic outbreaks of Pfjesteria piscicida have occurred (circles; large circles represent sites where fish kills were most frequent and affected a consid- erable area ). Sites: (1) below the city of Washington; (2) Hills Creek; (3)major 'hot spot' including Broad Creek. Blount Creek - Blount Bay, Ragged Point, Tripp Point, Bath Creek, Core Point; (4)area of Bayview, Hawkins Beach; (5-6) South Creek (location of Texasgulf phosphorus mine); (7) Flanners Beach - Cherry Point - Minnesott Beach segment; (8) Dawson Creek; (9) Coffee Creek; (10) Taylor's Creek; (11) Topsail Beach; (12) Wrightsville Beach. Note that sites with highest activlty were located about midway down the estuarine salinity gradient (mean salinity 12 ppt), a pattern that was corroborated by laboratory tests for TZ salinity optima (see text and Fig 4) The largest blackened area, on the Pamlico, indicates the area w~thh~ghest incidence of toxic outbreaks, whereas the large circle on the Neuse at Cherry Point - Minnesott Beach designates the site w~thhlghest known fish loss The latter kill was unusu.2l in that ~twas not short-term but, rather, occurred over a 6 wk period during autumn 1991 when schools of Atlantic menhaden were moving out to sea; more than 1 billion fish were affected, and bulldozers were used to clear the shorelines Also shown are locations (*) where amoebae and zoospores of P piscicida have been found at densities > 300 cells ml-l. Ulcerative f~shdiseases are common in the New kver Estuary as well as the Pamlico, suggestive of sublethal tox~cactivlty by this dinoflagellate 4 6 Mar Ecol Prog Ser
and aerated. The light regime was set at a 12:12 h L:D Argopecten irradians Lamarck; and eastern oysters cycle (30 pEin m-2 S-') except when testing light Crassostrea virginica Gmelin which tvere assayed only effects. Aquaria were maintained for 14 d before without finfish. Triplicate aquaria with 2 to 15 finfish (de- adding 3 tilapia to each Fish were acclimated 7 d to pending on the species) in 9 l of 15 ppt Instant Ocean ensure viability, and were fed daily with several flakes water were used for each species bioassay. The aquaria of Tetra Marin food unless otherwise indicated. The were each inoculated with active toxic culture to effect cultures also contained occasional small flagellates an initial density of 270 to 6-15 cells ml-l. Field concen- (e.g. Tetraselmis sp.), blue-green algae (Lyngbya sp. trations of active TZs typically range from 102to 103 cells and Gloeothece spp.), and protozoan ciliates (Sapro- ml-' (Burkholder et al. 1993). Behavior and survival of philus sp., Microthorax sp., and Stylonichia putrina animals in these aquaria versus in triplicate controls Dragesco & Njine; Lee et al. 1985). without the dinoflagellate were compared for 2 to 4 wk Bioassays to confirm toxic stages and response of under standard culture conditions. Adults were tested in fish species. In checking water samples for the pres- all cases except for bioassays with hybrid striped bass ence of this toxic dinoflagellate in the Neuse and Pam- Morone saxatilis X Morone chrysops Rafinesque, [both lico (May 1991 to December 1993), other coastal areas juveniles (length ca 25 cm) and adults] and oysters (pe- (May 1992 to September 1993), and aquaculture facili- diveligers and adults; G. Krantz, Oxford Cooperative ties (January 1992 to May 19941, aquarium bioassays Laboratory, MD, USA, with J.M.B. and H.B.G.). with fish were used to discern the small, nondescript In the shellfish bioassays without finfish, bay scallops TZs from other CO-occurring, nontoxic estuarine (3 per aquarium; shell width 4 to 5 cm) and blue crabs dilloflagellates that are similar in appearance under (1 per aquarium; carapace width 8 to 10 cm) were fed light microscopy. In the aquarium bioassays, tilapia dilute concentrations of TZs for 9 to 14 d. Blue crabs were exposed to field water samples under similar were also fed small tilapia and freeze-driedhehy- conditions as those used to maintain stock cultures. drated shrimp. Direct microscope counts of preserved Toxicity, where present, was confirmed in 514 d. We samples collected at daily intervals were used to assess preserved samples with acidic Lugol's solution (Vol- feeding actlvity as cell depletion. Oyster pediveliger lenweider 1974), and quantified abundance of toxic larvae (ca 100 per replicate; n = 3) tvere examined for stages (TZs and planozygotes, diameter 10 to 60 pm) feeding and settling/attachment behavior when added with an Olympus IMT-2 inverted microscope (phase, to vessels containing cleaned autoclaved adult oyster 600x) uslng the Utermohl technique from Lund et al. shells with TZs (ca 1000 cells ml-' d-' for 2 d), an equal (1958)in Burkholder & Wetzel (1989). At least 400 cells biovolume of nontoxic Pseudoisochrysis sp. (Prymne- were analyzed from each sample, with bioassays gen- siophyceae), or mixed toxic/nontoxic prey (15 ppt erally sampled at 1 to 2 d intervals. Stage identifica- salinity, 21°C, 30 pEin m-' S-', 12:12 h L:D cycle, 24 h). tions in water from fish kills, bioassays, and other Direct microscope counts of preserved samples col- field/aquaculture samples were confirmed using scan- lected at 2 h intervals (first 12 h) indicated that both ning electron microscopy (Burkholder et al. 1992). In pediveligers and adults consumed TZs; in the case of April 1994, we also received samples from a coastal pediveligers, this feeding activity was corroborated North Carolina aquaculture facility where juvenile with video imagery. Adult oysters were fed 3 times clams Mercenaria mercenaria Linne were dying, and daily with TZs from active stock cultures to maintain checked these samples for tox~cdlnoflagellate stages. available dinoflagellate food at 500 to 1000 cells ml-' Each aquarium was covered with a tightly fitting or higher. Shellfish were considered dead if they polyethylene sleeve to minimize the potential for cross- ceased movement for 24 h after the suspected time of contamination, except for short periods (min) when death (pediveligers: no further motion or ciliary action; sampling, feeding fish, or exchanging dead with live additionally for bay scallops, open gaping shells with fish. For all bioassays, controls consisted of fish main- no further change for 48 h). tained without dinoflagellate inocula (from cultured or Distribution in the mid-Atlantic and southeastern field populations). Direct microscope counts of pre- United States. Toward determining the distnbution of served samples collected at 1 to 2 d intervals indicated toxic ambush-predator dinoflagellates along the Atlan- that controls did not become contaminated except tic and Gulf Coasts (1992 to 1994), water samples from during bioassays with blue crabs Callinectes sapidus known sites of sudden-death fish kills and ulcerative Rathbun from local habitat. disease in nutrient-enriched areas of the Indian River Similar tests were also completed with other fish spe- Inland Bay, Delaware (supplied by B. Anderson & R. cies to assess their susceptibility to the TZs. These bioas- Miller, Delaware Department of Natural Resources, says were performed as single-species (finfish) fall 1992), Charleston, South Carolina (early spring trials, or as species with/without tilapia (shellfish). Rep- 19931, the St. Johns River and Pensacola Bay, Florida resentative shellfish included blue crabs; bay scallops (collected in summer 1993 by colleagues K. Steidinger Burkholder et al.. Fish kills linked to a toxic ambush-predator dinoflagellate
and J. Landsberg of the Florida Department of Envi- tures. The illumination source consisted of VHO cool ronmental Protection, Marine Research Institute, with white fluorescent lamps (model F48T12/CW/VHO) J.M.B.), and h4obile Bay, Alabama (supplied by H. that maintained a constant intensity of 350 pEin m-2 Awetman, summer 1993) were also bioassayed with S-' at a 30 cm distance from the near side wall of each tilapia. Aquarium bioassays with tilapia were used by tank. Covered culture aquaria were wrapped with Lewitus et al. (1995) to test for the presence of Pfieste- neutral-density fiberglass screening to achieve 5 light ria piscicida in Chesapeake Bay (Jenkins Creek, tribu- treatments in triplicate (intensities of 0.2, 25, 50, 75, tary to the Choptank River, summer 1993; stage identi- and 200 pEin m-' ss'), and then were placed on fications confirmed by J.M.B. & H.B.G.). Preserved shelves with treatments randomly assigned by shelf samples from nutrient-enriched locations in Flamingo row. The selected irradiances were within the range Bay and the Indian River, Florida (summer 1994), and encountered by phytoplankton in moderately turbid Benedict Laboratory, Solomons, Maryland (in the latter estuarine waters such as the Neuse and Pamlico case, water from the Patuxent River added to fish (Mallin & Paerl 1992). Photosynthetically active radia- aquaria, with subsequent fish death; sampled in spring tion (PAR) was quantified using a LiCor data logger 1994 by K. Sellner, D. Britburg and C. Pacey, with (model 1000) connected to a submersible 4rc PAR quan- identifications confirmed by H.B.G. & J.M.B.) were tum sensor (model LI-193SA). Replicated controls (con- analyzed for flagellated and amoeboid stages of Pfies- taining fish without P. piscicida in each light regime) teria-like species. were also positioned at randomly assigned locations on Optimum physical conditions for toxic activity. the shelves. After setup, each aquarium was inocu- Under field and aquaculture conditions, toxic out- lated with 200 m1 of dinoflagellate stock culture con- breaks of Pfiesteria piscicida occurred across a wide taining ca 300 TZs ml-' (initial density ca 8 cells ml-'). temperature gradient from 6 to 31°C, with maximal Aquaria were monitored twice daily to exchange dead toxic activity from TZs during the warm season (May with live fish and to add fish food, exposing each through October). The most active toxic stage during aq.uarium to an additional 8 to 9 pEin m-2 S-' (i.e.min- colder periods was a large lobose amoeba (length ca 80 imal background PAR) for 30 to 60 S during each to 250 pm), with secondary involvement of TZs. In con- exchange. One 50 m1 sample was taken (in the same trast to temperature, influences of salinity (e.g. during 30 to 60 s period) at 5 d intervals, and was preserved estuarine stratification) and light were not clear from for quantifying dinoflagellate stages. field data; hence, the effects of these 2 variables on In the salinity and light experiments, triplicate aquaria toxic behavior were experimentally examined. The were also maintained with dinoflagellates (no fish; salinity experiment was conducted for 16 d as batch 'dinoflagellate-alone' control) to characterize the popu- cultures of dinoflagellates that were maintained under lation structure at each salinity or light level. As ex- conditions similar to those for stock cultures. Artificial pected, subsamples collected throughout the duration of seawater was adjusted to impose treatment salinities of each experiment revealed negligible or low densities 0, 5, 10, 15, 25 and 35 ppt by altering the quantity of (generally <50 cells ml-') of flagellated and amoeboid Instant Ocean salts added to wellwater from a source stages in the water column of these systems without fish, at North Carolina State University that was low in nutrients, or abundant algal/microfauna prey (note: ben- divalent cations (c4 mg Ca2++Mg2+I-'). Each treat- thic areas were not sampled). Hence, the dinoflagellate- ment was maintained in triplicate 10 1 aquaria that alone controls were not considered further. were randomly distributed on tables. The aquaria were To statistically analyze the experimental data, corre- aerated and filtered, with filters acclimated for 14 d lation analyses were performed initially by date to ex- before introducing 3 small tilapia. After 7 d with daily amine relationships between the abundance of various feeding of fish, the aquaria were each inoculated with stages of Pfiesteria piscicida with salinity or light. After 200 m1 of dinoflagellate culture containing approxi- testing for homogeneity of variance (Hartley's test; Gill mately 60 cysts or amoebae ml-' (initial density ca 2 1978), data were square-root transformed where ap- cells ml-l) from stock aquaria of P. piscicida that had propriate (SAS Institute, Inc. 1987). One-way ANOVAs been without live fish for ca 1 mo. During the 16 d were used to detect differences among treatments in salinity experiment, fish in triplicate controls (without abundances of TZs, gametes, planozygotes, cysts and the dinoflagellate) and treatments were not fed, and amoebae. Treatment means were compared using expired fish were replaced within 8 h of death. One Fisher's protected least significant difference test, with 50 m1 sample was taken from each aquarium at 2 d a comparisonwise error rate (a = 0.05; SAS Institute, intervals, and was preserved for analysis by the Uter- Inc. 1987, Day & Quinn 1989). Repeated-measures mohl technique. analysis was used to test for differences among treat- Experiments to test for light optima of toxic Pfiesteria ments over time in abundances of life cycle stages piscicida stages were conducted for 25 d as batch cul- (SAS Institute, Inc. 1987, Potvin & Lechowicz 1990). 48 Mar Ecol Prog Ser 124: 43-61, 1995
Table 1. Estuanne/coastal fish k~llslinked to the presence of flagellated stages (flag.) of Pfiesteria piscicida through sample identification with confirming scanning electron micrographs and bioassays for fish toxicity. na: not available
Month Location Salinity Temp Nutrients P. piscicida Affected taxa, number (PP~) ("c) (p9 I-') (flag. ml-')' affected, notes 1991 May Parnlico: Blount Bay, 4-6 24-26 TP 130-240 750 Atlantic menhaden, southern Core Point, Bath Creek P043--P 70 flounder, spot, hogchoker TKN 500-600 (1000 000) NH4+-N 10-20 June Pamlico: Tripp Pointb TP 130-140 1050 Atlantic menhaden, Pod3--P 40-50 others (15000) TKN 500-600 NO3--N Table 1 (continued) Month Location Salinity Temp. Nutrients P. piscicida Affected taxa, number (PP~) ("C) (p9 I-') (flag. ml-l)" affected. notes - -- ~ - - - Dec Atlantic, ca 0.5 km from 35 9 na 1400~ Atlantlc menhaden shore at Topsail Beach ('thousands') 1993 Jun Neuse: M~nnesottBeach 7 26 TP 70-110 830 Atlantic menhaden (ca 100); TKN 400- RESULTS fish had died (P. Tester unpubl. data). In about two- thirds of the fish kills, samples were taken while the Distribution and toxic outbreaks of ambush- 'sudden-death' kills (usually c48 h in duration, and predator dinoflagellates frequently much shorter) were in progress, and TZ densities were 2250 cells ml-l. In the remaining kills, Water samples were collected from 27 field fish kills samples were collected 2 to 4 d after fish death, when and 9 aquaculture kills (Tables 1 & 2). An additional the presence of common filose and lobose amoebae aquaculture kill was sampled by P. Tester [National suggested that dinoflagellate transformations from Marine Fisheries Service (NMFS), Beaufort, NCJ and toxic flagellated stages had already occurred. colleagues, and the water was found to contain the Toxic outbreaks of Pfiesteriapiscicida in local estuar- dinoflagellate at sublethal concentrations 1 to 3 d after ies occurred at mean temperature and salinity of 26 + 50 Burkholder et al.: Fish kills linked to a toxic ambush-predator dinoflagellate 2°C and 12 k 2 ppt [mean k 1 standard error (SE); n = 5 kills were available for analysis). Hence, our field 17 dates; authors' data in combination with NC data and supporting laboratory bioassays implicated P. DEHNR 1992 and NC DEHNR unpubl. records; Table piscicida as the causative agent of 52 * 7 % of the major l], indicating that this dinoflagellate is predominantly fish kills annually in the North Carolina estuaries and an estuarine, warm-temperate species. Lethal stages coastal waters examined from 1991 to 1993. During all were most active in nutrient-rich waters (status 3 years, open bleeding lesions were common on mori- inferred using information from Rudek et al. 1991, bund and dead fish observed at the lulls (Fig. 2E). Fur- Jaworski et al. 1992, Dennison et al. 1993, Mallin ther, in these locations between kills, sublethal densi- 1994), with mean total phosphorus (TP), ammonium, ties of P. piscicida flagellated and amoeboid stages and total nitrogen (TN) or total Kjeldahl nitrogen often were found in surface waters (Fig. 2A to D),coin- (TKN) at 275 * 30 pg TP 1-' (n = 16), 35 +- 10 pg NH, & ciding with ulcerations that were observed among fish N 1-' (n = 13), and 810 +- 135 pg TN or TKN 1-' (n = 16), species taken from pound nets by commercial fisher- respectively (data supplemented by NC DEHNR 1992 men. and NC DEHNR unpubl. records; Table 1). Aside from these field kill sites, analysis of water We obtained fresh (unpreserved) samples from 28 of samples collected in North Carolina estuaries between the 36 field and aquaculture kills after 1 to 2 d, and July and October 1993 confirmed the toxic dinoflagel- confirmed toxic activity of the dinoflagellate from lab- late's presence in the South River downstream from oratory bioassays in all of 26 samples tested; the Open Grounds Farm (with accompanying bioassays remaining 2 kills were sampled from aquaculture facil- that confirmed toxicity), the Newport River, the White ities during 1994, when laboratory redesign for Oak River near Swansboro, the New River (Northeast improved safety precautions prevented completion of Creek, Stones Bay, Hadnot Point, Wallace Creek, confirming bioassays for toxic activity. To conserva- Courthouse Bay and Sneads Ferry Manna), and the tively estimate the role of this toxic dinoflagellate in Cape Fear River (ca 100 m from the Wilmington rnunic- major fish kills from North Carolina's estuaries, we dis- ipal wastewater treatment plant). Finfish and shellfish counted other field kills in which Pfiesten'a piscicida kills associated with TZs or, more frequently, with was present but more than one-third of the water col- large lobose amoebae (Fig. 2C), also occurred in aqua- umn had low dissolved oxygen (14 mg 1-'). In contrast, culture facilities, especially within indoor tanks during 8 field kills (although with delayed sampling of 24 d) winter-spring, a short time (days) after incoming estu- yielded no evidence of P. piscicida stages in fresh or arine water was warmed to >20°C (Table 2). preserved material, nor in subsequent bioassays of live Fish culture bioassays of water or surficial sediment samples with tilapia, and hence were considered 'neg- samples collected from selected, known sudden-death ative' tests. The majority of those lulls were associated fish kill sites in eutrophic estuaries of Delaware, Mary- with anoxia or hypoxia (usually 13mg 1-l) in the lower land (Jenkins Creek, Chesapeake Bay; bioassay data of water column (NC DEHNR 1992, unpubl. records). Lewitus et al. 1995), South Carolina, Florida, and Al- During Year 1 (May to December 1991) in estuaries abama confirmed the presence of the dinoflagellate's and coastal waters, 8 of 15 major fish kills (defined as TZs (Fig. 3). Outbreaks of Pfiesteria piscicida TZs oc- affecting >103 fish) were linked to Pfiesteria piscicida curred in all cultures except those from Pensacola Bay (Table 1, in combination with data from NC DEHNR and Mobile Bay. A second Pfiesteria-like species 1992 and NC DEHNR unpubl. fish kill records). In con- (Landsberg et al. 1995) was found in preserved samples trast to the relatively warm conditions from spring to and in fish bioassays of fresh samples from the St. Johns fall 1991 (with water temperatures at 28 to 30°C by late River, the Indian River, Flamingo Bay, Pensacola Bay, May; NOAA 1992a), the summer of 1992 was the third and Mobile Bay (Fig. 3). P. piscicida was found in pre- coldest in a 50 yr record (NOAA 1993), and yielded served samples from a site on the York River, Virginia; fewer field kills but more numerous aquaculture kills and another site in the Chesapeake, the Patuxent River, relative to the previous year (Tables 1 & 2; but note: apparently provided an inoculum of the P. piscicida field and aquaculture fish kills prior to May 1991 were population that was implicated in an adjacent aquacul- not sampled and, thus, could not be considered). The ture kill (Table 2). dinoflagellate's TZs were implicated in 5 of 8 major field kills during Year 2 (data considered in combina- tion with fish kill records from NC DEHNR unpubl. Affected species of finfish and shellfish records; Fig. 2A, B). In Year 3 (1993), P. piscicida was found in lethal densities, with toxicity subsequently The TZs of Pfiesteria piscicida were lethal to all fish confirmed from laboratory bioassays, at 4 of 10 major observed during kills in estuarine habitat or aquarium fish kills (Table 1, considered with NC DEHNR unpubl. water when cell concentrations were 2ca 250 cells rnl-' fish kill records; but note: samples from only those (Tables 1 & 2), which is also the approximate density at Burkholder et al.: Fish kills linked to a toxic ambush-predator dinoflagellate 5 1 Table 2. Summary of aquaculture finfish and shellfish kills linked to the presence of Pfiesteria piscicida through sample identl- fication, confirming scanning electron n~icrographs,and bioassays for fish t~xicity.~NMFS: National Marine Fisheries Service; na: not available Month Location* Salinity Temp. P. piscicidaa Affected taxad, number (PPt) ("c) (cells ml-l) affected, notes 1992 Jan Aquaculture (Pamlico; 0 G nae.h Hybrid striped bass (25 000; Castle Hayne Aquifer) $90 000 loss); with lesions Feb NC Maritime Museum, 27 21 naah Sheepshead, others Beaufort (Newport River) (number na); with lesions Feb NMFS. Beaufort 24 19 120 TZs, Atlantic menhaden (ca 20); (Taylor's Creek) 80 gametes, with lesions 40 amoebaeb Mar NMFS, Beaufort 60 TZs, Atlantic menhaden (Taylor's Creek) 80 gametes, (>2000 larvae) 30 amoebaeb Nov NMFS, Beaufort 180 TZs, Southern flounder (Taylor's Creek) 80 gametes, (14);with lesions 10 amoebae Jan Dept of Zoology, NCSU 1260 TZs, Tilapia, white perch (Neuse) 1800 gametes, (ca 30); with lesionsC 40 amoebae May Dept of Zoology, NCSU 2700 TZs, Tilapia (75); (Charleston. SC) 240 gametes, with lesionsC 560 amoebae 1994 Feb-Mar Benedict, MD (Patuxent River, 9 1070 TZs, Naked goby (64); Chesapeake Bay) 1870 gametes, with lesion^^.^ 620 amoebae AP~ NMFS, Beaufort naah Atlantic menhaden (20; (Taylor's Creek) pathology consistent with toxin effe~ts)~ May-Jun Vicinity of Camp 980 TZs, Littleneck clam (8000 Leleune (White Oak River) 5700 gametes, larvae; $20 000 10s~)~ 780 amoebae 'Locations include the water source used in the facility. Dinoflagellate densities were rounded down to the nearest 10, affected fish were adults unless otherwise inbcated. Descriptions of facilities and fish involved during February-March 1994 were provided by D. Bntburg, C. Pacey & K. Sellner from Benedict Estuarine Research Laboratory. The April 1994 kill was sampled 1 to 3 d after fish had died, and flagellated cells were reported at S170 ml-' (P. Tester et al. unpubl. data) b~ delay of 2 to 4 d occurred between sampling and adding preservative, or between the time of the fish lulland sampling. In the January 1992 'sudden death' kill, hybrid striped bass died within 4 h; prior to death they exhibited symptoms that were consistent with those known to be induced by toxic P. piscicida. Bioassays of tilapia with field sample tested positive for toxic activity by the dinoflagellate 'Fish apparently carried P. piscicida into the facilities from the Neuse Estuary (January 1993) or from waters near Charleston, SC (May 1993) dConfirming bioassays could not be completed because our laboratory was being renovated to ensure safety when working with P. piscicida. Examination of the aquaculture facihties revealed that, despite passage of estuarine water through a series of diatomaceous earth filters, small stages of the dinoflagellate had remained in the filtrate used to grow large cultures of Isochrysis galbana Green (Prymnesiophyceae) that were maintained as a food supply for the clam larvae. These stock algal cultures were found to contain numerous zoospores of P. pjscicida that were thriving on I. galbana; thus, the unfortunate aquaculturist had been feeding his clams a robust population of P. piscicida mixed with smaller nontoxic algae which marine red tide dinoflagellates such as Gymno- increasingly frequent episodes, the fish suddenly strug- dinium breveDavis can be lethal to fish (Tester et al. gled to the water surface and gulped air, but was not 1991). The typical response followed this sequence: able to hold its position and slowly sank back down Upon initial exposure to water with high TZ activity, the where it landed on its side, head or tail and, having lost fish quickly darkened in color (initial pale brown its ability to maintain balance, leaned against the changing to dark brown/black mottling), became aquarium wall. The behavior appeared somewhat anal- lethargic, and settled on the bottom of the aquarium. In ogous to fish 'walks' reported by NC DMF biologists Mar Ecol Prog Ser 124: 43-61, 1995 Fig. 2. Pfiesteriapiscicida (acidic Lugol's-preserved) and appearance of sublethal toxin effects on fish. (A) Highly magnified. darkly stained TZ with reddish nucleus (arrow) and prominent longitudinal flagellum (f);scale bar = 5 pm; compare with scale of (B), the magnification of the origlnal micrograph. (B) Appearance of a water sample from a recent aquarium fish kill showing a darkly stained TZ (arrow), a '-' anisogamous gamete (g), filose amoebae from a TZ (t) and a planozgyote (p), and a small lobose amoeba likely transformed from a TZ (1); scale bar = 30 pm. (C) A larger lobose amoeba (colorless to light brown when alive), showing a reddish-stained nucleus (arrow) and tapering lobopodia with refractive appearance; scale bar = 25 pm. (D)A lobose amoeba of different shape and size, with prominent reddish nucleus (arrow); scale bar = 25 pm. (E)Moribund Atlantic menhaden with open bleeding ulcerations, collected while still swimming at the water surface (among ca 2 X 103flagellated and amoeboid cells rnl" of P. pisonda) in the Pamlico Estuary near Bath Creek (site 3 in Fig. 1). This area is noted for seasonally high abun- dance of P. piscicida,and high incidence of fish kills/disease; 98 % of the fish sampled on that date in mid-June, among all spe- cies present, were similarly affected during dinoflagellate- and hypoxia-related kills in nat- agent is unidentified neurotoxin(s) that is actively ural habitat, wherein affected finfish as well as blue released as exotoxin(s) when the dinoflagellate's TZs, crabs attempt to move from the water to drier shore- planozygotes, and lobose amoebae are stimulated by lines (Table 1). Lesions with sloughing of flecks or substance(s) in fresh fish secreta (Burkholder et al. patches of epidermal tissue, and/or subcutaneous hem- 1992). When we exposed fish to water with aging orrhaging developed as the exposure period length- dinoflagellate cysts (dormant >2 yr), lethal effects ened and the fish began to expire. This bleeding was often required about 6 to 8 wk. Cysts that had been most noticeable for hybrid striped bass in the lymphatic inactive for 1 to 2 mo induced fish death in 514 d. canal beneath the dorsal fin, and for southern flounder Recently formed cysts from highly active toxic popula- in sores that formed on the ventral surface by the tion~,however, were capable of producing TZs that mouth. Many of the tested fish attempted to reach the killed fish in hours, and TZs which had been given live well-aerated area by the aquarium filter and, if success- fish repeatedly for days to weeks killed fish in minutes. ful, remained there until death. Cultures that were maintained to provide high levels Among 19 finfish species bioassayed with Pfiesteria of toxin for biochemical characterization required 15 to piscicida,all were killed in <4 d, and 13 additional spe- 20 or more fish daily. In replicated bioassays with cies were involved in field or aquaculture kills linked exotic fish species (>280 TZs ml-'; Table 3), hybrid to the dinoflagellate (Table 3). The lethal ichthyotoxic striped bass were most susceptible (hemorrhaging and Burkholder et al.: Fish kills linked to a toxic ambush-predator dinoflagellate 53 death of juveniles within minutes) whereas adult gup- PFlES TERIA pies and mosquitofish generally survived several days. KNOWN DISTRIBUTION (1991-1994) Moreover, within a given species, susceptibility to the toxin(s) varied so that occasionally, some fish remained alive for hours or days after all other exposed fish had died. Subsequent addition of other live fish to the cul- ture, however, consistently led to death of the formerly resistant individuals. Bioassays with shellfish yielded similar results although blue crabs, bay scallops, and eastern oysters apparently did not strongly stimulate toxic activity. Blue crabs and scallops remained viable for 9 to 14 d test periods while filtering low concentrations of TZs (ca 60 cells m-'), although perceptible loss of the scal- lop closing reflex indicated a narcotizing effect of the toxin(s). When placed into aquaria with dying finfish (TZ densities 22100 cells ml-'; active cultures given 8 tilapia d-'), however, blue crabs were killed within hours to several days after numerous frenzied attempts MOBILE RAY . ST. 10m to leave the water. Efforts to maintain 4 of 6 blue crabs as controls failed, despite the fact that control tanks with crabs were each surrounded by control aquaria with unaffected tilapia (which consistently have tested GULP more sensitive to Pfiesteria piscicida than blue crabs), as an added precaution against contamination by dinoflagellate cultures. These crabs had been collected from the Newport River, and were suspected to have Fig. 3. Known geographic range of toxic ambush-predator carried small populations of encysted or amoeboid P. dinoflagellates including Pfjesteriapiscicida (circles; open cir- pisdcida on their carapaces. cle designates the Albemarle-Pamlico region) and a second, more subtropical Pfiesteria-like species (squares). The map In an analogous pattern, within 2 rnin of exposure to designates locations where TFVCs have been identified, and TZ cultures with dylng finfish, scallops exhibited an where potential toxicity has been verified using bloassays escape response (rapid propulsion backward until con- with tilapia; sites where confirming toxicity bioassays have tacting the aquarium walls) and then closed their shells not yet been completed are not shown, but lie within the indi- cated geographic range. These dinoflagellates have been tightly. The shells gaped open and death ensued tracked to known s~tesof fish kills and ulcerative disease from within 210 min (indicated as no further change for our northernmost sampling station to date (lnd~anRiver, 24 h). Oyster pediveliger larvae were narcotized after Delaware), throughout most estuaries of the Albemarle-Pam- consuming TZs for 22 to 3 h, indicated by depressed lico Estuarine System (open circle) to our most southwestern ciliary activity, premature settling behavior, and fail- site (Mobile Bay, Alabama), with apparent species overlap along the southern U.S.Atlantic Coast ure to attach to cleaned adult shells (Krantz et al. 1994). Approximately 50% of the tested pediveligers ap- peared moribund after 8 h of toxic exposure, and most we documented a hybrid striped bass kill in an aqua- died after 24 to 48 h with TZs. Adult oysters were also culture facility at ca 0 ppt (total hardness = 20 mg 1-'; narcotized as evidenced by a slower closing reflex Table 2). The water used in the aquaculture facility (Krantz et al. 19941, but continued to consume high originated from the calcium-rich Castle Hayne coastal concentrations of TZs over a 14 d test period (unpubl. aquifer. The dinoflagellate was believed to have been data of J.M.B.& H.B.G. with G. Krantz; however, note transported to the ponds either by waterfowl, on estuar- that neither oyster pediveligers nor adults were tested ine foam carried by wind, or from previous use of Pam- in the presence of dying finfish). lico Estuary water. Among the salinities tested, 15 ppt was optimal for growth and activity of TZs (significantly higher than at other salinities tested; p < 0.02; Fig. 4). Response to salinity and light gradients Fish death occurred at 10 ppt or higher in dinoflagel- late-exposed treatments during the 16 d experiment. Over a 16 d test period, Pfiesteria piscicida TZs did On Day 17 the fish were fed in all tanks, and 8 of 9 total not emerge from cysts that were placed in ca 0 ppt fish from the 5 ppt replicate aquaria died by Day 20, as salinity from the low alkalinity water source, although compared to no fish death in the 5 ppt controls. Mar Ecol Prog Ser 124: 43-61, 1995 Table 3. Species of finfish and shellfish that have been killed In natural habitat or culture bioassays containing active stages of Pficsteria piscicida. Asterisks (') indicate confirming aquarium bioassays on adults (A), pediveligers (P),or juveniles (J);mortal- ity of remaining species occurred during field or aquaculture kills in which P. piscicida was implicated as the causative agent (based on quantification of toxic stages at 2250 cells rn-' in water samples from the kills, and scanning electron microscopy to verify identification). The dinoflagellate has proven lethal to all tested finfish and shellfish Native estuarinelmarine species American eel Anguilla rostrata Lesueur A Atlantic croaker Micropogonias undulatus L. 'A Atlantic menhaden Brevoortia tyrannus Latrobe 'A, J Bay scallop Argopecten irradians Lamarck 'A Black grouper Mycteroperca bonaci Poey A Blue crab Callinectes sapidus Rathbun 'A Channel catfish Icatalurus punctatus L. A Eastern oyster Crassostrea virginica Gmelin 'Pd Hogchoker Trinectes maculatus Block & Schneider A Killifish (mummichog) Fundulus heteroclitus L. 'A Largemouth bass Micropterus sahoides Lacepede Mosquitofish Gambusia affinis Baird & Girard 'A Naked goby Gobiosoma bosc Lacepede A Northern quahog (littleneck clam) Mercenaria mercenaria Linne J, A Pinfish Lagodon rhomboides L. A Red drum Sciaenops ocellatus L. 'A Redear sunfish Lepomis microlophys L. Sheepshead Archosargus probatocephalus Walbaum A Southern flounder Paralichthys lethostigma Jordan & Gilbert 'A Spot Leiostomus xanthuris Lacepede 'A Spotted sea trout Cynoscjon nebulosus Cuvier A Striped bass Morone saxatilis Walbaum 'A Striped mullet Mugil cephalus L. A White perch Morone amen'cana Gmelin 'A Exotic (introduced) species Clownfish (clown anemonefish) Amphiprion percula Lacepede 'A Goldfjsh Carrasius auratus L. 'A Guppie Poecilia reticulata Peters 'A Hybrid str-lped bass Morone saxatilis X Morone chrysops Rafinesque 'A, J Tilapia Oreochromis aureus Steindachner, Oreochromis mossambica Peters, Tilapia nolotica L. 'A aConfirming bioassays on eastern oyster pediveliger larvae were completed by G Krantz with J.M.B. and H.B.G. (Krantz et al. 1994) I '~argemouth bass and redear sunfish are considered freshwater species, but they were found in slightly brackish waters I Pfiesteria piscicida also was capable of lethal activity from a 'borrowed' photosynthetic mode of nutrition. We across a broad gradient of light intensity. The dinofla- maintained cultures of TZs (c5000 cells rnl-')for several gellate showed comparable toxicity to fish across the days when supplied with microalgae (diatoms or small tested range of available light with no apparent optimum cryptornonad, green, or chrysophyte flagellates) as a (Fig. 5). Abundances of the 4 most common stages (TZs, food resource, but toxicity gradually decreased and re- gametes, amoebae and cysts) were highly variable versions to nontoxic zoospores or transformations to among replicates within each light treatment. The max- amoebae and cysts increased unless live fish or their imum for each active stage occurred at a different light fresh tissues were added (Burkholder et al. 1993, 1995). intensity (considering median cell densities among repli- cates: TZs, 25 pEin m-' S-'; gametes, 200 pEin m-* S-', the highest light level tested; amoebae, 0.2 pEin m-2 DISCUSSION S-'), suggesting a selective tendency for optimal light levels among stages. Recent documentation of 'clep- This research implicates toxic ambush-predator di- tochloroplasts' in the vegetative flagellated stage of P. noflagellates as major causative agents of fish kills piscicida (i.e. chloroplasts retained for use after digestion of the southeastern United States. Pfiesteria piscicida of photosynthetic algal prey; Steidinger et al. 1995) proved lethal to all 19 bioassayed species of finfish and points to a mechanism by which this alga could benefit shellfish. An additional 13 species were involved in Burkholder et al.: Fish kills linked to a toxic ambush-predator dinoflagellate 55 0 PP1 5 PP1 - 15 PP1 2500 i1--c- 25 ppr TIME (D) Fig. 4. Response of Pfiesteria piscicida to a salinity gradient imposed by mixing Instant Ocean salts with a (soft) wellwater source (alkalinity ca 4 mg I-'). Data are given as means i 1 SE (n = 3). The large error bars associated with the 15 ppt curve reflect the delayed response of the dinoflagellate in 1 of the 3 replicate aquaria for that treatment field or aquaculture kills associated with active TZ pop- Apart from the dinoflagellate's cryptic appearance ulation~.These data demonstrate that, unlike other and behavior, the extent of its involvement in kill known ambush predators (Spero 1982, Ucko et al. events likely has been underestimated because of dif- 1994), P. piscicida is a 'generalist' which targets a wide ficulty in reaching many kLls to obtain water samples array of finfish and shellfish species. It could be easily while fish were still dying, which is the optimal period overlooked in fish kills because the TZs closely resem- for detecting the lethal TZs (Burkholder et al. 1992). ble other small, nontoxic species (e.g. Campbell 1973, We believe that this problem has been compounded Spero 1982); they may be pigmented by retaining because the behavior of affected fish has sometimes chloroplasts from algal prey (Steidinger et al. 1995),but been mistaken as a response to low dissolved oxygen, otherwise they are colorless or light brown and, hence, rather than as suffocation from neurotoxin-induced readily blend with their turbid surroundings. Recogni- muscle paralysis. For example, from NC DMF and NC tion of ambush-predator dinoflagellates is further con- DEM records preceding the discovery of Pfiesteria founded because amoeboid stages in preserved mater- piscicida, kills of surface-dwelling Atlantic menhaden ial often resemble organic debris (Burkholder et al. commonly were attributed to low oxygen stress even 1995); in addition, the small flagellated forms tend to when dissolved oxygen was >5 mg 1-' in the upper preserve poorly in fixatives that are used routinely for water column (NC DEHNR unpubl. records). In other phytoplankton analysis (Spero 1982, Burkholder 1992). cases, kills were often sampled several days after fish TIME (D) Fig. 5. Toxicity of Pfiesteria piscicida to test tilapia across a light gradient from 0.2 pEin m-2 S-' (darkness except for all but 1 to 2 rnin at 20 *in m-2 S-', per 24 h period) to 200 pEin m-' S-' (12:12 h 1ight:dark cycle using halogen tubes, 20°C). Data are given as means i 1 SE of the fish killed (of 18 total fish added) in each replicate aquarium after the time indicated (n = 3). Note that the dinoflagellate became slightly more toxic at 25 pEin m-' S-' after 20 d (p < 0.045 for each date at 20 and 25 d) but, in general, lethal effects were comparable among treatments 56 Mar Ecol Prog Ser death; hence, hypoxic upper waters could have nontoxic species, and was recorded in fish kill records resulted from decomposition of fish remains, or could only if it comprised 8 % or more of the totalphytoplank- have acted synergistically with the dinoflagellate's ton biovolume. Analysis of NC DEM records revealed toxin(s) in contributing to addtional fish death. that 'G. aurantium' generally was low or subdominant Prior to this study, the overall role of Pfiesteria pisci- in abundance relative to other phytoplankters (< 10 % cida in fish kills from North Carolina waters also was of the total phytoplankton biovolume as zoospores) obscured by the lack of a central, organized data base except during fish kills, when this dinoflagellate (pre- of state records for estuarine and marine fish kills (cur- sumed TZ stage) contributed 18 * 11 % of the total bio- rently in handwritten files at various branch offices volume while representing only 9 & 9% of the total cell within 2 divisions of NC DEHNR, and in an incomplete number (means + 1 SE; Table 4). computerized data base), and by exclusion of data on From cursory sampling of sudden-death fish kill causative factors in the available records unless col- areas in Florida (July 1993 survey by colleagues K. lected by state personnel. Such lack of organized infor- Steidinger & J. Landsberg with J.M.B.), the presence of mation about fish kills, partly resulting from insuffi- Pfiesteria piscicida or a second Pfiesteria-like species cient funding to sustain innovative state programs or was established in 7 of 10 eutrophic, known fish kill even to support timely sampling efforts (e.g. Miller et sites visited over a 4 d period that was spent mostly in al. 1990), is widespread along U.S. coasts but worse in transit. Ths information, along with confirmation of North Carolina than in any other state (Lowe et al. Pfiesferia-like species from samples provided by col- 1991, Adler et al. 1993). Given projected exponential leagues in Delaware, Maryland, Virginia, South Car- population growth with associated water quality olina, and Alabama, indicates that toxic ambush- degradation in coastal areas (Holman 1993, Miller predator dino-flagellates are common in shallow 1994), and current severe declines in wild fish stocks eutrophic estuaries throughout the mid-Atlantic and (NOAA 1992b, NC DMF 1993, Food & Agriculture southeastern United States. The eurythermal, euryha- Organization 1994, Leavenworth 1994, NC DMF line character of P. piscicida increases the likelihood unpubl. records), this pervasive dearth of information for widespread distribution of this species and its close points to a critical need for higher regulatory staff relatives in other warm temperate and subtropical 'across' divisions and agencies to increase emphasis on regions. fish kill assessment programs. In North Carolina and Pfiesteria piscicida was implicated as the causative elsewhere, there is an equally pressing need for legis- agent of ca 50% of the major fish kills annually in the lators to recognize that funding for such agencies must Neuse and Pamlico Estuaries from 1991 through 1993. be increased to adequately support such programs Beyond the obvious acute effects manifested in fish (North Carolina Coastal Futures Committee 1994, kills, however, the role of toxic ambush-predator dino- Richissin 1994, World Resources Institute 1994). These flagellates in sublethal adverse impacts on fishery steps would enable sampling of major fish kills while in resources and other trophic levels merits serious con- progress so that pathogens, pollutants, and other con- sideration in future research. For example, within the tributory factors could be critically evaluated - infor- past decade the Albemarle-Pamlico Estuary has expe- mation that must be obtained before management rienced severe outbreaks of ulcerative skin and shell strategies can be developed to control these factors or diseases among fish species (e.g. Atlantic menhaden, mitigate their impacts. spot, Atlantic croaker, American eel, southern floun- At present, there is insufficient information to track der, blue crab; Mather 1988, Levine et al. 1990, Miller Pfiesteria piscicida through geological time, or to eval- et al. 1990). Among the most prevalent finfish diseases uate whether its toxic outbreaks are increasing in is ulcerative mycosis (Noga 1993), formerly attributed frequency or aerial extent. Species-specific molecular to fungal infection, wherein a mixed community of markers (work in progress) will enable us to. assess the opportunistic fungawbacterial pathogens partially col- dinoflagellate's long-term activity from cyst remains in onizes deep open sores from the epidermis inward but cores from estuarine sediments. Although its toxic generally not down to the lesion base (Noga et al. activity prior to May 1991 cannot be confirmed, some 1988). Fish species inhabiting low to moderate salinity unexplained sudden-death kills in the Pamlico and areas have been afflicted by such diseases (Levine et Neuse dating back to 1985 (first year of consistent state al. 1990). During late spring - early summer 1994, fla- records on phytoplankton abundance during fish kills) gellated stages of P. piscicida were common in the may have been Linked to it. During that time the TZ water column (ca 200 cells ml-l), and amoeboid stages stage was identified by state biologists (K.Lynch, NC formed a thin brownish ooze on fishermen's pound DEM, pers. comrn.). as 'Gymnodinium aurantium' nets (J.M.B. & H.B.G. unpubl. data). Many finfish sam- (nomen nudum), a name unofficially given by Camp- pled from these nets had open bleeding ulcerations be11 (1973). The alga was assumed to be an incidental (sore diameter 1 to 4 cm; H.B.G., J.M.B. & N. Deamer- Burkholder et al.: Fish kills linked to a toxlc ambush-predator dinoflagellate Table 4 'Historical record' of major fish kills potentially linked wlth the TZ stage of Pfiestena piscicida from September 1985 to early May 1991" Month Location Taxa affected TZs a 1985 Sep New River Flsh species 1400 ( "Data were provided by the NC DNRCD (1987, 1988, 1989) and by NC DEHNR (1990a, 1991), wherein TZs of P. piscjcida (then unknown) were ldentlfied as the incidental species 'Cymnodinjum aurantium'. Percent biovolume (BV): relative con- trlbution by TZs to the total phytoplankton community bThe klll was also associated with low dissolved oxygen (ca 3.0 my 1-l) in the bottom water Melia, from random sampling of 2 to 3 groups of 100 hosts' resistance. Repeated observations of damage to adult Atlantic menhaden with American eel, spot, fish epidermis by P. piscicida in aquarium cultures led Atlantic croaker, etc.; 30 + 2 % in early May, 60 * 3 % in to clinical research that corroborated the toxin's role in late May, 98 * 1 % in late June). In areas where lesions forming the lesions involved in ulcerative 'mycosis' of did not form over vital organs, they sometimes pene- Atlantic menhaden (Noga et al. 1995). trated completely through the body leaving large gap- Whether certain fishing practices might indirectly ing holes in the moribund but still swimming fish (also contribute to toxic Pfiesteria piscicida outbreaks observed by Noga et al. 1988). The opportunistic remains to be examined. In the mid-Pamlico Estuary, nature of the fungal/bacterial colonizers indicated that ulcerative disease incidence declined to 1 % and fish an unknown initial stressor could have weakened the kills ceased by mid-July 1994, shortly after intensive 58 Mar Ecol Prog Se trawling operations moved downstream to the lower Adverse effects can also be extended to humans. In a estuary and Pamlico Sound (H.B.G., J.M.B. & N. laboratory setting, several cases of narcosis (hours) and Deamer-Melia unpubl. data, NC DEM and NC DMF lingering symptoms such as reversible Alzheimer's- unpubl. records). Trawlers had fished the mid-Pamlico like short-term memory loss and cognitive dysfunction from late winter to early July. Moreover, a drought (days to weeks), suppressed immune system function, from May to early August allowed formation of an respiratory distress, elevated hepatic enzyme activi- unusually well-defined salt wedge with hypoxic bot- ties, slight renal dysfunction, and autonomic and tom water that extended upstream to the city of Wash- peripheral nervous system dysfunction (months to ington (NC DEM unpubl. records; Fig. 1, site 1). In the years) have occurred following chronic contact (6 to 8 early to mid-growing season, trawling activities in wk) with water containing low to moderate densities of some of the known dinoflagellate kill sites (P-enriched toxic stages (>2000 cells ml-'1, or exposure to neuro- at mid-range salinity) could have helped stimulate P. toxic aerosols from these cultures (Glasgow et al. piscicida by suspending benthic stages as well as other 1995). Apart from anecdotal information provided by microbial pathogens, nutrients, toxic substances, and local fishermen, carefully designed epidemiological wounded fish into the water column. Cessation of studies are lacking to determine whether humans who trawling would have allowed the salt wedge to more frequent estuaries with toxic outbreaks might be effectively isolate benthic stages from viable fish pop- adversely affected with subtle immune system sup- ulation~ in the upper water column. Dinoflagellate pression and other symtoms from chronic or acute cysts are known to survive several years of anoxia, and exposure to toxin(s) from Pfiesteria piscicida. excystment can occur in low-oxygen water (Anderson Aside from stimulation by finfish, nutrient enrich- & Keafer 1985, Anderson et al. 1987).Nonetheless, salt ment may contribute to maintenance of a water- wedge-imposed density/chemical gradients could column inoculum of nontoxic precursor stages to TZs. have slowed the excystment process, minimized detec- These stages would be positioned to respond rapidly tion of fish secreta, and impeded dinoflagellate move- to the presence of feeding schools of fish under calm ment from the sediment to surface waters. Further conditions in poorly flushed upper tributaries, when study will help clarify whether the timing of reduced wind and wave action were not available to suspend fish disease/kills during a season when P. piscicida has the benthic amoebae and cysts. Approximately 75 % usually been active, and shortly after trawlers moved of the kills (14 of 19, from previous state records 1985 to less favorable areas for the dinoflagellate (higher to early 1991) that were associated with potentially salinity, lower background nutrients), was only coinci- lethal densities of 'Gymnodinium aurantium' (2300 dental. Fish kills linked to P. piscicida did not recur in cells ml-') occurred in nutrient-enriched areas of the the mid-Pamlico until late August 1994 (H.B.G., J.M.B. Pamlico (naturally P-rich sediments, with water-col- & N. Deamer-Melia unpubl. data; NC DEM and NC umn and sediment P augmented in some kill sites by DMF unpubl. records). P mining, sewage, and other sources) and the Neuse Toxic ambush-predator dinoflagellates likely cause (Cherry Point region). Similarly, between 1991 and other chronic, insidious effects on fish recruitment and 1993, 74% of the field kills occurred in the Parnlico food resources. Our preliminary data indicate that (10 kills) or the Cherry Point area of the Neuse (4 Atlantic menhaden eggs do not hatch when TZs are lulls). Laboratory bioassays have indicated a direct abundant (unpubl. data of W. Hettler, National Marine stimulatory effect of phosphourus enrichment on Fisheries Service, Beaufort, NC, with H.B.G. & J.M.B.), growth of Pfiesteria piscicida's nontoxic zoospores and that juvenile hybrid striped bass are more suscep- (reverted from gametes) and amoebae, especially if tible to the toxin(s) of Pfiesteria piscicida than adults supplemented with smaller flagellated algal prey (i.e. juveniles die more rapidly, and are affected by (Burkholder et al. 1992, 1995, Burkholder & Glasgow lower TZ densities). These observations suggest that a 1995). The dinoflagellate's apparent preference for school of fish which successfully leaves a toxic out- nutrient-rich waters suggests that precursor stages to break area may have only escaped on a short-term lethal TZs might be expected to proliferate in areas basis. The narcotizing effects induced by the toxin on where nutrient loading directly stimulated their the closing reflex of adult bay scallops and oysters, and metabolism and/or indirectly enhanced growth by on the activity of oyster pediveliger larvae (Burkholder supporting blooms of smaller algae. Additional data in et al. 1992, Krantz et al. 1994), could result in greater support of this hypothesis would present a powerful susceptibility of these animals to predators. The toxin's argument in favor of improved regulations to reduce sporadic 'speed' effect on zooplankton (more active, nutrient loading to shallow, poorly flushed estuanes in erratic swimming that includes head-on collisions with warm temperate and subtropical regions that serve as the culture vessel walls; Mallin et al. 1995) and blue optimal habitat for these widespread cryptic predators crabs could enhance detection by predators, as well. of targeted fish and other prey. Burkholder et a1 : Fish kills linked to a toxic ambush-predator dinoflagellate 59 Acknowledgements. Funding support for this research was Burkholder. JM (1992) Phytoplankton and episodc sus- provided by the National Science Foundation (grant OCE- pended sediment loading: phosphate partitioning and 9403920); the Office of Sea Grant, NOAA, the U.S. Dept of mechanisms for survival. Limnol Oceanogr 37: 974-988 Commerce (grant NA90AA-D-SG062), the UNC Sea Grant Burkholder JM, Glasgow HB Jr (1994) The enriched life of an College (projects R/MER-17 and R/MBT-1A); the Albemarle- ambush predator: a new ichthyotoxic dinoflagellate in Pamlico Estuarine Study (U.S. EPA National Estuary Program eutrophic estuaries (Abstract). In: Proceedings, 12th Bien- and NC DEHNR) through the UNC Water Resources nial International Esturarine Research Federation Confer- Research Institute; the U.S. Manne Air Station at Cherry ence, H~ltonHead, SC, p 16 Point; the NC Agricultural Research Foundation (project Burkholder JM, Glasgow HB Jr (1995) Interactions of a toxic 06034), the NC Agricultural Research Service (project 06188), estuarine dinoflagellate with microbial predators and the NCSU College of Agriculture & Life Sciences, and the prey. Archiv Protistenk 145:177-178 NCSU Department of Botany (E. Seneca). A. G1asgom~'s Burkholder JM, Glasgow HB Jr, Noga EJ, Hobbs CW (1993) understanding and faith in the investigators greatly enriched The role of a newly discovered toxic dinoflagellate in this project. We are grateful for the help of many staff from finfish and shellfish kills in the Neuse and Parnlico Estuar- NC DMF and NC DEM in providing algal samples from fish ies. Albemarle-Pamlico Estuarine Study Rep No 93-08. NC kills in estuarine habitat including K. Miller. K. Lynch, J. DEHNR and US EPA National Estuary Program, Raleigh, Hawkins, K. West, J. Mulligan, R. Carpenter, V. Coleman, B. p 1-58 Adams, A. Hodge, M. Yount, M. Street, R. Tankard, and P. Burkholder JM. Glasgow HB Jr. Steidinger KA (1995) Stage Fowler. We thank B. J. Copeland, F. Cross. R. Dove, R. Hod- transformations in the complex life cycle of an 'ambush- son, B. Hettler, N. McNeil, M. Mallin, R. Mays, A. Powell, C. predator' estuarine dinoflagellate. In: Lassus P, Arzul G, Sullivan, and P. Tester for providing information or samples Erard E, Gentien P, Marcaillou C (eds) Harmful marine that led to confirmation of Pfiestena activity during estuarine algal blooms. Elsevier, Amsterdam, p. 567-572 and aquaculture fish kills. R. Dove, D Phelps, B. Fritz, G. Burkholder JM, Noga EJ, Hobbs CW, Glasgow HB Jr, Smith Evans, Y. Barber, and T. Quay helped establish volunteer SA (1992) New 'phantom' dinoflagellate is the causative efforts to sample lulls, such as the Neuse River Foundation's agent of major estuarine fish kills. Nature 358:407-410; Creek Keepers. D. Ormond offered h~stime and his boat to 360:768 help obtain Pfiesteria samples and pound net data for fish Burkholder JM, Wetzel RG (1989) Epiphytic mlcroalgae on ulcerations. K. Steidinger, J Landsberg. B. Anderson, R. natural substrata in a hardwater lake: seasonal dynamics Miller, H. Awetman, K. Sellner, D. Britburg. C. Pacey. C. Sul- of community structure, biomass and ATP content. Arch livan, and B. Lapointe helped in our effort to document the Hydrobiol (Suppl) 83:l-56 presence of Pfiesteria-like species outside North Carolina. D. Campbell, PH (1973) Studies on brackish water phytoplank- Rainer and B. MacDonald contributed sound counsel in labo- ton. Sea Grant Publication UNC-SG-73-07. UNC Sea ratory safety protocols. J. Sauber provided NC DEHNR data Grant Program, Chapel Hill to strengthen our nutrient data in the vicinity of field fish kills; Copeland BJ, Hodson RG, Riggs SR (1984) The ecology of the and K. Lynch, K. Miller, and V. Colenian helped provide a Pamlico River, North Carolina: an estuarine profile. US complete set of NC DEHNR data for estuarine fish lulls and F~sh& Wildlife Service Rep FWS/OBS-82/06. US Fish & dinoflagellate abundance. Research assistance was con- Wlldlife Servlce, Washington, DC tributed by V. Coleman, J. Compton, D. Bnley, F. Johnson, L. Culotta E (1992) Red menace in the world's oceans. Science (Taggett) Taylor, C. Harrington, M. McAuley and G. Morgan. 257:1476-1477 C. Brownie counseled us on statistics; U. Blum reviewed pho- Day RW, Quinn GP (1989) Coniparison of treatments after an tos; and B. J. Copeland, J. Hawkins, K. Miller. K. West, A. analysis of variance in ecology. Ecol Monogr 59:433-463 Lewitus. M. Mallin, P. Tester, S. Shumway, N. Deamer-Melia, Dennison WC, 01th RJ, Moore KA, Stevenson JC, Carter V, M. Larsen. B. Touchette, E. Fensin, D. Briley, J. Compton, and Kollar S. Bergstrom PM', Batiuk RA (1993) Assessing water E. Hannon kindly critiqued the manuscript. Finally, we thank quality with submersed vegetation. BioSci 43:86-94 B. J. Copeland, R. Waite, J. Wynne, C. Moreland, B. Graham, Epperly SP, Ross SW (1986) Characterization of the North F. Cross, D. Moreau, R. Holman. B. Hogarth, R. Dove, M. Carolina Pamlico-Albemarle estuarine complex. NOAA Smith, B Leslie, T Miller, and R. Mason, whose interest and Tech Memo NMFS-SEFC-175 support helped make this research possible. 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