Comparative culture and toxicity studies between the toxic ...directory.umm.ac.id/Data...

Journal of Experimental Marine Biology and Ecology255 (2000) 51–74

www.elsevier.nl / locate / jembe

Comparative culture and toxicity studies between the toxicdinoflagellate Pfiesteria piscicida and a morphologically

similar cryptoperidiniopsoid dinoflagellatea , a a a*Harold G. Marshall , Andrew S. Gordon , David W. Seaborn , Brian Dyer ,

b bWilliam M. Dunstan , A. Michelle SeabornaDepartment of Biological Sciences, Old Dominion University, Norfolk, VA 23529-0266, USA

bDepartment of Ocean, Earth, and Atmospheric Sciences, Old Dominion University,Norfolk, VA 23529-0266, USA

Received 10 July 2000; received in revised form 24 July 2000; accepted 28 August 2000

Abstract

A series of fish bioassays using cultures of the toxic dinoflagellate, Pfiesteria piscicida and acryptoperidiniopsoid dinoflagellate indicated various degrees of toxicity for Pfiesteria piscicidaand no toxicity by the cryptoperidiniopsoid. P. piscicida maintained toxicity in the presence of livefish, and this toxicity was perpetuated following a series of inoculations to other culture vessels.Differences in the onset and magnitude of the fish deaths occurred, requiring 16 days for the initialfish death when using P. piscicida from a culture that had previously been maintained on algalcells, to kills within hours when using a culture that had recently (previous day) killed fish.Autopsies of moribund fish from the test and control fish bioassays indicated a general lack ofbacterial infection, which ensued following death of other autopsied fish. Moreover, bacterialcomparisons of waters in the fish bioassay and control fish cultures indicated that similar bacterialconcentrations were present. Neither oxygen or ammonia levels were determined to be factors inthe fish death. Life stages of a cryptoperidiniopsoid dinoflagellate from Virginia estuaries werealso identified, including motile zoospore, gametes, planozygote, amoebae, and cyst stages. Thecryptoperidiniopsioid did not initiate fish deaths in bioassays conducted over a 14-week period at

21zoospore concentrations of ca. 700–800 cells ml . Elemental X-ray analysis of the scales fromcysts of this dinoflagellate and P. piscicida indicate that they both contain silicon. Overall, the datafrom this study demonstrate that the cryptoperidiniopsoid possesses several similar life stages andfeeding patterns as P. piscicida, but was not toxic to fish. 2000 Elsevier Science B.V. All rightsreserved.

Keywords: Pfiesteria piscicida; Cryptoperidiniopsis gen. nov.; Toxicity; Dinoflagellates

*Corresponding author. Tel.: 1 1-757-683-3595; fax: 1 1-757-683-5283.

0022-0981/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0022-0981( 00 )00288-4

52 H.G. Marshall et al. / J. Exp. Mar. Biol. Ecol. 255 (2000) 51 –74

1. Introduction

Estuarine and laboratory studies have identified dinoflagellates of the toxic Pfiesteriacomplex (TPC; thus far including Pfiesteria piscicida, Steidinger et al., 1996; and P.shumwayae sp. nov., Glasgow, 2000) with production of toxins associated with fish killevents and human illness (Burkholder et al., 1992; Glasgow et al., 1995; Burkholder andGlasgow, 1997; Grattan et al., 1998). The most frequent and extensive occurrences ofthese fish deaths have been in North Carolina’s Albemarle–Pamlico Estuarine System,with a lesser degree of incidence and fish mortality in tributaries of the eastern shore ofChesapeake Bay (Lewitus et al., 1995; Burkholder and Glasgow, 1997; Burkholder etal., 1999; Magnien et al., 1999). TPC species have also been found in other estuaries inbenign forms, from New York through the Gulf Coast (Burkholder and Glasgow, 1997;Rublee et al., 1999).

These studies indicate that Pfiesteria spp. exist as nontoxic (i.e., non-toxin producing)heterotrophs feeding on algae and other organisms, and that under certain environmentalconditions, the cells become toxic in the presence of fish. Pfiesteria spp. have beenfurther described by Burkholder and Glasgow (1995, 1997), Burkholder et al. (1999),and Burkholder (in press) as existing in several functional types based on their capabilityof toxin production (Woods Hole Oceanographic Institution (WHOI), 2000). Activetoxin-producing cells in the presence of live fish are considered the Toxic A functionaltype (TOX-A), which become temporarily nontoxic (TOX-B functional type) whenremoved from access to live fish and fed algae, or other prey. These TOX-B cells havethe potential for toxin production and become TOX-A, or actively toxic, when re-introduced to live fish. In addition, there are strains of Pfiesteria that do not have, orapparently have permanently lost, the toxin production response that is triggered by thepresence of live fish. This third functional type is called non-inducible (NON-IND), andon the basis of present understanding, cannot be induced to produce bioactive substancesthat cause fish disease and death (WHOI, 2000; Burkholder, in press).

Toxic strains of TPC species demonstrate the following traits: they show strongattraction toward live fish or their fresh tissues and secreta /excreta (attraction measuredusing motion analysis techniques as in Kamykowski et al., 1992; Burkholder andGlasgow, 1997); they produce bioactive substances or toxins that cause fish disease anddeath (Fairey et al., 1999); and they are stimulated to produce these substances in thepresence of live fish or their fresh tissues (separated from the live animal , 2 h;Burkholder and Glasgow, 1995, 1997). The two toxic Pfiesteria spp. known thus far(Glasgow, 2000) are heterotrophic and lack chloroplasts, but can be mixotrophic byretaining kleptochloroplasts from algal prey (Lewitus et al., 1999). If kleptochloroplastsare present, they are always contained within an epithecal food vacuole (Burkholder andGlasgow, 1995; Lewitus et al., 1999; Glasgow, 2000). Pfiesteria spp. also have acomplex life cycle with many stages or forms, which vary in response to the changingenvironmental conditions present in estuaries (especially prey availability; Burkholder etal., 1992, 1995b; Burkholder and Glasgow, 1995, Glasgow, 2000). These life formsinclude multiple amoeboid and cyst stages as well as flagellate stages (zoospores,anisogamous gametes, planozygote). Earlier reports of such diverse life stages indinoflagellates, with arrays of amoebae, include Pascher (1916), Bursa (1970a,b),

H.G. Marshall et al. / J. Exp. Mar. Biol. Ecol. 255 (2000) 51 –74 53

´ ´ ´Pfiester and Popovsky (1979), Pfiester and Lynch (1980), Popovsky (1982), Popovskyand Pfiester (1990), and Buckland-Nicks et al. (1990), among others.

The ecological and habitat relationships that stimulate Pfiesteria development andtoxin production have been discussed by Burkholder et al. (1992, 1995a, 1999),Burkholder and Glasgow (1997), Magnien et al. (1999), and others. The laboratory andfield studies that have associated fish mortality with P. piscicida have been reported overa broad salinity range and at various water temperatures. However, the toxic events havemost commonly occurred at low to mid-salinity values (5–15 psu) and temperaturesabove 268C. Most of these events have been in nutrient-enriched waters of shallow,poorly flushed estuaries (Burkholder and Glasgow, 1997; Burkholder et al., 1997;Magnien et al., 1999). These conditions also favor the development of the algal prey thatPfiesteria may utilize as a food resource in the absence of abundant live fish (Burkholderand Glasgow, 1995, 1997).

In Virginia estuaries, intensive investigations over the past 3 years have documentednumerous ‘pfiesteria-like organisms’ (PLOs; Marshall, 1999; WHOI, 2000). Theseorganisms superficially resemble Pfiesteria spp. in general appearance under lightmicroscopy (LM), but they mostly have been tested as benign or incapable of producingbioactive substances that cause fish stress, disease or death (this study; Burkholder, inpress; J. Burkholder and H. Glasgow, North Carolina State University, Raleigh, NC,unpublished data). PLOs have similar size and morphological features as Pfiesteria spp.,and most cannot be distinguished from Pfiesteria spp. using only LM. Therefore, wehave used scanning electron microscopy (SEM) of both suture-swollen cells (Glasgow,2000), and membrane stripping protocols by K. Steidinger (pers. commun.) and Truby(1997) to determine the plate tabulation and plate characteristics of these PLOs. Thetoxin-producing capability is assessed by fish bioassays as in Burkholder et al. (1995b,1999); Burkholder and Glasgow (1997); and Burkholder (in press). Pfiesteria-likeorganisms in Virginia estuaries have included at least two cryptoperidiniopsoids(Cryptoperidiniopsis sp. [gen. nov.; Dr. K. Steidinger, Florida Department of En-vironmental Protection — Florida Marine Research Institute, St. Petersburg, FL, pers.commun.] and Cryptoperidiniopsis brodyi [gen. et sp. nov.; Steidinger, pers. commun.],along with certain Gymnodinium spp. and Gyrodinium spp. (e.g., Gyrodiniumgalatheanum Braarud [ 5 Gymnodinium galatheanum Braarud]). These PLOs are widelydistributed in Virginia estuaries and in other areas of the Chesapeake Bay (Marshall etal., 1999; Seaborn et al., 1999). Many of the smaller Virginia estuaries along thePotomac River and several of the smaller inlets along the mid-western shoreline of the

21Chesapeake Bay also have supported at times high concentrations ( . 200 cells ml ) ofthese cells (Marshall et al., 1999; H. Marshall, unpublished data).

Little is known about the comparative ecology of TPC species versus PLOs.Comparative growth studies with a TOX-B (temporarily nontoxic) P. piscicida, acryptoperidiniopsoid species, and G. galatheanum using Cryptomonas sp. and twodiatoms as a food source, showed that the cryptoperidiniopsoid had the highest growthrate (Seaborn et al., 1999). This characteristic may over time give this dinoflagellate acompetitive advantage in abundance over Pfiesteria during long term and/or seasonalperiods of development. G. galatheanum is an auxotroph and mixotroph, and photo-synthesizes with its own chloroplasts that are located in the cell cytoplasm (Tomas,


1996). Thus, it can be discerned from Pfiesteria spp. under light microscopy, especially4 21with epifluorescence. In high cell densities ( . 10 zoospores ml ), several isolates of

G. galatheanum have also been reported as toxic to fish (Steidinger, 1993; Nielsen,1993; Burkholder, 1998). However, isolates from North Carolina, Maryland, and Floridawaters thus far have shown neither attraction to live fish nor ichthyotoxicity in repeatedbioassays (Glasgow, 2000; Burkholder, in press). The objectives of the present studywere to compare P. piscicida and a cryptoperidiniopsoid dinoflagellate in terms of theirbasic stage morphologies and potential toxicity to fish, toward strengthening insightsabout distinguishing characteristics of co-occurring Pfiesteria versus pfiesteria-lookalikespecies.

2. Materials and methods

2.1. Cultures

Cultures of two pfiesteria-like species were developed from water (Gyrodiniumgalatheanum) and sediment (Cryptoperidiniopsis sp.) samples taken from Virginiaestuaries between 1997 and 1999 (Seaborn et al., 1999). Unialgal cultures of thesespecies were established after numerous individual cell isolations and subsequentdilutions. Scanning electron microscope examination of the cryptoperidiniopsoid speciesindicated that it was different from C. brodyi (gen. et sp. nov.; Steidinger, pers.commun.). This species ([DEQ002) and G. galatheanum were monitored daily androutinely checked for contaminants and other dinoflagellates through light microscopy.Subsequent DNA sequencing analysis by Dr. D. Oldach (U.MD) indicated thecryptoperidiniopsoid to be a Cryptoperidiniopsis sp. (gen. nov.) that was separate fromC. brodyi (gen. et sp. nov), but closely related to P. piscicida. The identity of G.galatheanum was confirmed by K. Steidinger (pers. commun.) with SEM and by D.Oldach through a Heteroduplex mobility assay (Oldach et al., 2000). These cultureswere maintained in falcon flasks in f /2-Si medium at 15 psu, under ambient light androom temperature, and were given Cryptomonas sp. (CCMP [767, Bigelow Labora-tory) as a food source. Prior to using the Cryptomonas culture, it was routinelyexamined for contaminants, with cells routinely isolated to establish a series ofsub-cultures.

A toxic Pfiesteria piscicida culture ([271A-1) was provided by Burkholder andGlasgow (NCSU). This culture had been isolated from the Neuse Estuary, NorthCarolina, cloned in uni-dinoflagellate culture with algal prey (cryptomonads) as a foodsource at NCSU. Pfiesteria spp. have not been cultured successfully without a preysource (e.g., Burkholder and Glasgow, 1995; Steidinger et al., 1996), and it has not beenpossible to induce toxin production unless live fish are added (Burkholder and Glasgow,1997). Thus, a clonal culture contains an isolate of (a uni-dinoflagellate) P. piscicida orP. shumayae (sp. nov.), with its associated endosymbiont bacterial consortium (Steiding-er et al., 1995) and its prey (Burkholder, in press). The culture was allowed to grazealgal prey to residual levels ( , 10 cells /ml), and then added to fish bioassays (see


Section 2.3, below) to test for ichthyotoxic activity (five fish per replicate culture vessel,n 5 3; Burkholder et al., 1995a,b; Burkholder and Glasgow, 1997; Glasgow, 2000;Burkholder, in press). The control fish remained healthy while the Pfiesteria-exposedfish all died, and accompanying tests indicated no difference between control and testculture vessels in presence /abundance of bacteria or other microflora / fauna that couldact as fish pathogens. Culture [271A-1 was thus evaluated as a toxic strain, wasidentified to species by H. Glasgow and J. Burkholder (SEM of suture-swollenzoospores; Burkholder and Glasgow, 1995; Glasgow, 2000), and made available for ourstudies.

The culture ([271A-1) was shipped to our laboratory as TOX-A or actively toxic P.piscicida, taken from the fish-killing cultures. However, since toxic strains of Pfiesteriaspp. are known to rapidly cease toxin production in the absence of live fish (withinhours; Burkholder and Glasgow, 1997), this strain was received in TOX-B (temporarilynontoxic) status. We initially maintained the culture for 4 weeks with Cryptomonas asthe food source. We also identified this isolate using SEM analysis (Leo model 435VPSEM) and, as had been done in the Burkholder and Glasgow laboratory, we againcross-confirmed the species identification as P. piscicida by two independent laboratories(Dr. P. Rublee of UNC-Greensboro, and Dr. D. Oldach of U. MD) using genesequencing techniques (Rublee et al., 1999; Oldach et al., 2000). This P. piscicidaculture was used in comparative growth studies (Seaborn et al., 1999) and in our initialfish bioassay studies (Fish bioassay I) with this species. A second TOX-A P. piscicidaisolate ([2200, Neuse Estuary) that had been similarly prepared was supplied by theBurkholder and Glasgow laboratory, and was used for additional fish bioassays andmorphological comparisons with the cryptoperidiniopsoid (Cryptoperidiniopsis sp. gen.nov.). Prior to the bioassays, the identity of this species was also cross-confirmed by thetwo laboratories mentioned above. This isolate was maintained with fish in culturefacilities in a TOX-A mode (see Section 2.3 fish bioassays, below), without the additionof algal prey.

2.2. Morphology of life stages

A Leo Model 435VP scanning electron microscope (SEM) at Old DominionUniversity was used for the plate tabulation of these dinoflagellates, following thesuture-swollen cell approach of Glasgow (2000). In addition, an environmental scanningelectron microscope (Phillips XL30 ESEM-FEG), at the University of Miami, RosenstielSchool of Marine and Atmospheric Science, Miami, FL, was also used to characterizeseveral life stages of Pfiesteria piscicida and the cryptoperidiniopsoid, and to conductelemental X-ray analysis on the cyst stages of these two species. Previous reports byBurkholder et al. (1992), Burkholder and Glasgow (1995, 1997), and Steidinger et al.(1996) have indicated P. piscicida is known to have chrysophyte-like scales. Our intentwas to compare scales we observed on the cysts of these species and the likelihood ofusing morphological differences among these scales as a basis for species identification.In addition, we wanted to determine if these scales were siliceous and/or organic, asfound in chrysophytes.


2.3. Fish bioassays

The Pfiesteria piscicida cultures used in these bioassays were previously identified asthe toxin producing source of fish deaths in the Burkholder and Glasgow laboratory.Their bioassay procedure to identify toxin producing P. piscicida has been brieflysummarized in various publications and used in their bioassay studies (e.g., Burkholderet al., 1995a,b, 1999; Burkholder and Glasgow, 1997; Glasgow, 2000), with a moredetailed description provided by Burkholder (in press). This approach evaluates thepresence of toxic agents in relation to fish deaths that may occur across a range ofenvironmental conditions (low to high nutrients, salinities, organics, toxic substances ofother types, bacterial concentrations, protozoan densities, etc.). The Pfiesteria culturesthat were provided for our studies by the Burkholder and Glasgow laboratory wereidentified as toxic strains based on bioassay results coming from the procedure outlinedin Fig. 1. This cause /effect relationship is based on the presence of toxic rather thaninfectious agents to determine ichthyotoxic activity of P. piscicida. The bioassay stepsinclude: (a) confirmation of the presence of Pfiesteria piscicida at potentially lethaldensities at an in-progress fish kill event, identified by SEM examination and genesequencing analysis; (b) isolation of P. piscicida from a water sample taken from thein-progress kill, in positive fish bioassays, and development of clonal culture(s) of thesecells (uni-dinoflagellate and axenic except for endosymbiont bacteria; containing residual

21axenic algal or other benign prey, such as ca. five to 10 cryptomonads ml ); (c)addition of the clonal Pfiesteria isolate to healthy fish cultures, resulting in fish deaths,while fish in control cultures remained healthy; and (d) re-isolation and re-cloning of theorganism from the second set of positive fish bioassays where deaths occurred, withsubsequent species identification verified by SEM and gene sequencing, plus cross-confirmation of toxicity by an independent laboratory experienced in culturing toxicPfiesteria.

Fish bioassays were used to test for ichthyotoxic activity of P. piscicida versus thecryptoperidiniopsoid dinoflagellate and G. galatheanum. The toxins of Pfiesteria spp. areincompletely characterized (Fairey et al., 1999). Thus, at present, properly conductedfish bioassays are the ‘gold standard’ — i.e., the only reliable technique available — forassessing toxin-producing capability of the known toxic Pfiesteria spp. and potentiallytoxic pfiesteria-like dinoflagellates (Burkholder and Glasgow, 1997; Burkholder et al.,1999, Glasgow, 2000; Burkholder, in press). The technique requires maintaining healthyfish in aerated culture vessels that are amenable to cell production and toxic activity ofTPC species, then adding the cloned dinoflagellate population to a subgroup of replicatefish cultures, while maintaining another set as replicate controls. Actively toxic,fish-killing strains of TPC species have been associated with serious human healthimpacts in laboratory and field exposures, apparently through production of aerosolizedneurotoxins (Glasgow et al., 1995; Grattan et al., 1998; Duke University Medical Centerrecords, Durham, NC). Therefore, we constructed a custom-designed biohazard IIIcontainment system at ODU (modified from the biohazard III containment system in theBurkholder and Glasgow laboratory at NCSU), and fish bioassays to detect and culturetoxic Pfiesteria were conducted within that system.

Hybrid tilapia (Oreochromis sp. (Aqua Safra, Bradenton, FL) total length, 3–6 cm)


Fig. 1. Standardized steps of the Burkholder /Glasgow fish bioassay procedure used to evaluate the role ofPfiesteria in fish kills, and grow toxic Pfiesteria (Burkholder et al., 1992, 1995a,b, 1999; Burkholder andGlasgow, 1995, 1997; Glasgow, 2000; modified from Burkholder, in press). These represent a modifiedapproach of Koch’s postulates (steps III, IV) regarding toxic rather than infectious organisms. The toxinproducing Pfiesteria piscicida zoospores (TOX-A functional type) were identified and isolated through thisprocedure by the Burkholder and Glasgow laboratory, with cultures of these zoospores provided for thebioassays used in this study.

were used as the standard test fish species. All fish were initially maintained in a holdingfacility at another ODU laboratory, separate from the biohazard III laboratory containingtoxic and potentially toxic Pfiesteria. Environmental conditions were similar to thosedescribed in Burkholder and Glasgow (1997) and Burkholder et al. (1995a,b). The fishbioassays were conducted at ca. 248C, under ambient light conditions, in 15 psu watermade with Instant Ocean salts. Each of the fish cultures was covered and aerated. When


Fig. 1. (continued)

fish died, they were replaced with live fish. No sediment was introduced into the culturevessels. Triplicate 1-ml water samples were taken from each series (I–IV) of bioassayand control vessels at 2–3-day intervals, preserved in acidic Lugols’s solution andexamined in a Palmer–Maloney counting cell at 3 400 magnification for Pfiesteriazoospore density (Marshall et al., 1999). Samples were also checked for discernabledifferences in other microflora / fauna in the control versus test fish culture vessels.Protozoan ciliates were rarely detected throughout the bioassays. Oxygen and ammoniawere measured at 2-day intervals. Oxygen was determined using an oxygen electrode(YSI model 5300 Biological Oxygen Monitor, Yellow Springs Instr., Yellow Springs,


OH). Ammonia was measured colorimetrically using an ammonia test kit (AquariumPharmaceuticals, Chalfont, PA).

In our initial bioassay study (I), the TOX-B functional type of P. piscicida isolate[271-A was inoculated into three bioassay holding systems (initial concentration ca.

2150–60 zoospores ml ), each containing three tilapia. Three similar control fish systems(with three tilapia each) were also maintained.

In a second set of fish bioassays (II), we used TOX-A P. piscicida isolate [2200 andinoculated three replicate bioassay systems ([1, [2, [3) containing 10 tilapia each

2l(initial concentration ca. 50–60 zoospores ml ). Three similar systems each with 10tilapia were also maintained as controls without P. piscicida. These bioassays wereotherwise conducted similarly as the first set. When fish died, they were replaced tore-establish the original total of 10 fish. Actively toxic status of the P. piscicida isolatewas maintained by continual replacement of the dead fish with live fish. When fishdeaths occurred during the fourth day in replicate [2 of this series (II), a thirdexperimental series (III) of three bioassays ([4, [5, [6), plus three additional controls,was established, each containing10 tilapia. An inoculant from the surface waters ofreplicate [2 was introduced into [4, [5, and [6 (initial concentration ca. 50–75

2lzoospores ml ). A similar pattern of replacing dead fish with live fish, and recordingPfiesteria and bacterial concentrations was followed and the practice continued foranother 10 days. After 10 days, a similar inoculant was transferred from [6 to threeadditional replicate bioassay systems (series IV) with test fish ([7, [8, [9; initial

2lconcentration ca. 50–60 zoospores ml ). Three additional control fish bioassay systemswithout P. piscicida were also maintained.

The cryptoperidiniopsoid (Cryptoperidiniopsis sp. [gen. nov.] [CB002) and G.galatheanum, were introduced to separate bioassay facilities holding three to six tilapiaeach, with control sets with three to six fish also established to determine their toxicity tofish over time. These assays were continued for 10 and 14 weeks, respectively, for G.galatheanum and the cryptoperidiniopsoid species.

2.4. Fish autopsies

Autopsies were performed on fish in the test bioassay systems to assess the presenceand quantity of bacteria within the blood of the fish. These fish appeared stressed afterexposure to P. piscicida by their sluggish and spastic pattern of movement. Fish duringthe bioassays often rested on or near the bottom of the bioassay vessels prior to death.From each of the three replicate bioassays in experimental series II–IV, we randomlyselected 10 of these moribund fish and 10 dead fish for autopsy. The surface of each fishwas disinfected prior to autopsy with laboratory disinfectant (Conflikt, Fisher Scientific).Disinfectant was removed by rinsing with sterile Instant Ocean and wiping with a sterileswab, and an incision was made below the dorsal fin with a sterile scalpel. Blood wasremoved from the incision with a sterile swab and applied to bacteriological medium(TCBS, Difco, Detroit, MI) and incubated at room temperature for 24 h. The resultinggrowth was streak plated onto Trypticase Soy agar (Difco, Detroit, MI) for purification

´and identification using the API 20E identification system (Meriux Vitek, Hazelwood,


MD). This system consists of 23 standard biochemical tests for the identification ofEnterobacteriaceae and other Gram-negative bacteria. Triplicate 1-ml water sampleswere also taken from each series (I–IV) of bioassay and control vessels at 2–3-dayintervals, for determining bacterial abundance in the water by acridine orange directcount (Hobbie et al., 1977).

3. Results

3.1. Morphology of life stages

Seven life stage morphologies of Cryptoperidiniopsis sp. (gen. nov.) were identifiedduring this study. These included a cyst, a resting stage, zoospores, gametes,planozygotes, lobose amoebae, and a rhizopodal amoeba with slender, retractable,pointed pseudopodia (Fig. 2a–g). These pseudopodia were non-branching, but whenjoined by other similar amoebae, would form a network around algal prey (Fig. 2b). InP. piscicida we noted motile zoospores, rhizopodal amoebae, lobose amoebae, filose(star) amoebae, and a cyst stage. These P. piscicida life stages are similar to thosedescribed by Burkholder et al. (1992) and Burkholder and Glasgow (1995, 1997). In themotile zoospore stage, both P. piscicida and Cryptoperidiniopsis sp. (gen. nov.) used apeduncle to feed on microalgal prey. The peduncle was extended from behind a hingedplate on the ventral surface of these cells, and attached to the prey. A swarming actionaround the attacked cell by several zoospores was common, and resulted in the transferof the interior algal cell contents through the peduncle into the food vacuole located in

¨the zoospore epitheca (process called myzocytosis; Schnepf and Elbrachter, 1992). Thisfeeding process resulted in a swelling of the zoospore and extension of the food vacuoleinto the hypotheca sometimes occurred following high feeding activity. Examples of thistype of feeding behavior in dinoflagellates have been described earlier by Spero (1982),

¨Gaines and Elbrachter (1987), Glasgow et al. (1998), Lewitus et al. (1999), and others.Gyrodinium galatheanum also isolated and cultured from Virginia estuaries, exhibitedsimilar feeding behavior with extended peduncle when fed Cryptomonas

In the original description of the dinoflagellate that came to be formally named P.piscicida (Burkholder et al., 1992; Steidinger et al., 1995), it was noted that a cyst stageof this species possesses scales that resemble those found on chrysophytes. Burkholder(1999) also illustrated several types of Pfiesteria cysts that differ in their origin, size,and the type of scales present. The cyst we have identified from this Cryptoperidiniopsissp. (gen. nov.) has elongated spine-like projections resting upon a surface scale,somewhat similar to one of the P. pfiesteria cysts described by Burkholder (1999). Thisdinoflagellate has two types of scales covering the cyst. There is an inner layer ofsurface scales (Fig. 2f) external to the cell membrane that are flat, and slightlypanduriform (1.8 3 3.4 mm). They have dissimilar surfaces, containing small circularnodules, with a ridge on the outer surface and a corresponding depression on theirundersurface. The second type of scale is external to this first layer, with three to four of

H.G

.M

arshallet

al./

J.E

xp.M

ar.B

iol.E

col.255

(2000)51

–7461

Fig. 2. Cryptoperidiniopsis sp. life stages and scales: (a) lobose amoeba and a Cryptomonas cell; (b) rhizopodal amoebae with Cryptomonascells; (c) cyst; (d) amoebae and cyst; (e) spine-like scale from cyst; (f) surface scales from cyst; (g) motile zoospore.


these loosely attached to the surface of the other scales. They consist of a flattenedbottom portion that rests on these inner scales, with each possessing an elongated andbluntly pointed spine (Fig. 2e). The bottom portion of these scales is cordiform in shape(0.9–1.9 3 0.6–2.0 mm). The shaft of the spine is hollow (length of 6.6–10.7 mm, meandiameter 0.25 mm; n 5 15 spines measured). SEM X-ray analysis revealed that silicon isa component of both the cyst (Fig. 3a) and scale types. The examination of cysts derivedfrom the TOX-A P. piscicida using SEM X-ray analysis indicated these cysts alsocontained silicon (Fig. 3b).

3.2. Fish bioassays

In the first set of fish bioassays (I), a strain of TOX-B Pfiesteria piscicida (potentiallytoxic, but previously fed algal prey, [271-A) was added to three replicate test fish

21bioassay systems (initial Pfiesteria density ca. 50–60 zoospores ml ). There was noimmediate or recognizable reaction from the fish for the first 15 days of exposure to P.piscicida. The first fish death occurred after 16 days. The dead fish were replaced withthree live tilapia which died within 24 h. Thereafter, fish deaths occurred on anintermittent basis in all three replicate bioassays. There were no fish deaths in thecontrols.

In the second set of fish bioassays (II), a strain of TOX-A P. piscicida (previouslygiven live fish, rather than algal prey, [2200) was added to the initial three test fish

21bioassay systems (initial Pfiesteria density ca. 50–60 zoospores ml ). After 72 h ofexposure to P. piscicida, six of 10 fish had died in replicate bioassay system [2, and

21zoospores had increased ca. 10-fold to ca. 600 cells ml (Fig. 4). The six dead fish andfour remaining live fish were removed and replaced with 10 live fish. Within 24 h, all 10

21fish had died, and the zoospore density had increased to ca. 1180 cells ml . Furtherdaily replacement of dead with live fish over the next 4 days resulted in death of all fish,

21with zoospore concentrations reaching ca. 5000 cells ml . Fish deaths in replicatebioassay system [1 did not occur until day 5 of exposure to P. piscicida (zoospores at

21 21ca. 460 cells ml ), with all fish dead by day 9 (zoospores at 10 000 cells ml ).In contrast to these results in the first two replicates, fish did not die in replicate

21bioassay [3 even when Pfiesteria concentrations were . 30 000 cells ml (day 9). Atday 14, the still-live fish in replicate [3 were transferred to replicate [2, and all fishwere dead within 24 h. When 10 replacement fish were added to replicate [3, no fishdeaths occurred over an additional 14-day period. In replicates [1 and [2, toxicity wasmaintained by replacing dead with live fish, with the shortest time to fish death occurringwithin 1.5 h of exposure to TOX-A P. piscicida. Throughout the experimental period,none of the 30 fish in the three replicate control bioassay systems (without exposure to P.piscicida) died, and all appeared healthy. These toxic P. piscicida have since beencontinuously maintained in our laboratory by providing them with live fish.

On the fifth day of the above bioassays, another series of inoculations (bioassay III)from replicate [2 with P. piscicida were transferred to three additional replicates, [4,

21[5, and [6 (initial P. piscicida density at 50–75 zoospores ml ). Fish deaths began tooccur on the fourth day of exposure to P. piscicida in replicate [4, on the seventh day inreplicate [5, and on the fifth day in replicate [6 (Fig. 5). Cell concentrations for P.


Fig. 3. Results of elemental X-ray analysis. Note presence of silicon: (a) Cryptoperidiniopsis sp. cyst; (b)Pfiesteria piscicida cyst.


Fig. 4. Results of fish bioassay II. Bars indicate percent of fish (10) deaths over time in reference to21concentrations of Pfiesteria piscicida zoospores ml in culture vessels [1–3.

21piscicida during the initial fish deaths were ca. 500–1200 zoospores ml . By the eighthday, death of all 10 fish had occurred in replicates [4 and [6, with P. piscicida at

218000–11 000 zoospores ml . Replicate [5 required a longer time interval for total fishdeath (by the 12th day). Toxicity was maintained in replicate test bioassays with P.


Fig. 5. Results of fish bioassay III. Bars indicate percent of fish (10) deaths over time in reference to21concentrations of Pfiesteria piscicida zoospores ml in culture vessels [4–6.

piscicida by replacing dead with live fish throughout an 18-day period. Over the testperiod, one of the 30 fish in the three replicate controls died, and examination revealedno dinoflagellates present in the water of that control vessel.

On the 18th day, fish bioassays [7, [8, [9 (10 fish per replicate) were inoculated21from replicate [6 (initial P. piscicida density ca. 50–60 zoospores ml ). Control


bioassay systems of fish without exposure to P. piscicida were maintained in triplicate aswell, with no deaths occurring in the controls over the 15-day experimental period. Incontrast, fish deaths occurred in the replicate bioassay systems containing P. piscicida at11 days ([9), 12 days ([8), and 15 days ([7) (Fig. 6), corresponding to zoospore

Fig. 6. Results of fish bioassay IV. Bars indicate percent of fish (10) deaths over time in reference to21concentrations of Pfiesteria piscicida zoospores ml in culture vessels [7–9.


21concentrations of 1000–16 000 cells ml . The results of the entire series (II, III, IV) areillustrated in Fig. 7.

In each of the test replicates with P. piscicida within all three of the experimentalseries, the fish exhibited noticeable stress prior to death. They moved with irregular,spastic motion, and had little forward advancement in their attempts to swim to thesurface, after which they slowly settled to the bottom of the bioassay systems. Thispattern was repeated until the fish died. All oxygen measurements taken during fishdeaths indicated there were comparable oxygen levels in both the bioassay and the

21controls ( . 4 mg dissolved oxygen l ), above levels generally considered to stress fish(Meyer and Barclay, 1990). Ammonia levels also were similar within the test bioassay

21systems and the controls. Ammonia was generally , 0.25 mg l . In one control21replicate, ammonia was 8 mg l on one date, but no fish died in this replicate; and all

replicate test bioassay systems with P. piscicida had ammonia concentrations , 0.05 mg21l .

Fig. 7. Comparison of serial transfer inoculations for fish bioassays (II–IV), indicating fish deaths in thebioassay and culture vessels, each containing 10 fish.


All fish bioassays conducted with the Cryptoperidiniopsoid sp. (gen. nov.) werenegative. This set of bioassays was conducted over a 14-week period, with cell

21abundance reaching ca. 600–750 zoospores ml . None of the fish died in either thebioassay, or the control vessels, and all appeared healthy. Similar results were found forthe fish bioassays using G. galatheanum. Over a 10-week period of exposure with cell

21concentrations reaching ca. 700–800 zoospores ml , no fish deaths occurred and allfish appeared healthy.

3.3. Fish autopsies and bacteria analysis

In nine of the 10 moribund fish taken from the fish bioassays (II–IV), the bloodcontained no bacteria-forming colonies on TCBS medium. Autopsy of the 10thpreviously moribund fish revealed the presence of oxidase-positive, non-fermentative,gram-negative rods in the blood. In contrast, the 10 fish that were randomly sampled forautopsy after death all contained bacteria in their blood that grew on TCBS medium,including several Vibrio and Aeromonas spp.

In experimental series II of the fish bioassays, the mean bacterial concentrations in thewater column of both the test replicates (fish bioassays [1, [2, [3, with P. piscicida)and the controls increased over the first 5 days before slightly decreasing by the seventhday (Fig. 8). Mean bacterial abundance in test fish bioassays were significantly differentfrom that of controls on only one of four dates analyzed (7 Feb., Student’s t-test,P 5 0.023). In analyses from experimental series II ([4, [5, [6 with P. piscicida),there were no significant differences in mean bacterial abundance from test fish bioassayversus control water. Of eight dates for series III ([7, [8, [9 with P. piscicida), thebacterial abundance was similar in the test bioassays and the controls, except for onedate (24 Feb., P 5 0.011). The bacterial cell concentrations in this last group were thelowest of the three sets, but these results were also the most comparable and extendedover a 16-day period. Overall, there were no significant differences in the bacterialabundance between the fish bioassays and the control vessels for 14 of the 16comparison dates.

4. Discussion

Dinoflagellates resembling Pfiesteria spp. are widely distributed in Virginia estuariesand in the lower Chesapeake Bay (Marshall et al., 1999). Among the more commonforms in these waters are the cryptoperidiniopsoid species that have morphologicalfeatures, life cycle stages, and some behavioral traits similar to those of Pfiesteriapiscicida. There are at least two cryptoperidiniopsoid species in Virginia estuaries andthe Chesapeake Bay. One is Cryptoperidiniopsis brodyi (gen. et sp. nov.), the other ispresently considered Cryptoperidiniopsis sp. (gen. nov.) that is, as yet, incompletelydescribed. A characteristic behavioral pattern shared by both of these Cryptoperidiniop-sis species and Pfiesteria can be observed with light microscopy. This behavior consistsof swarming activity around algal prey (e.g., Cryptomonas sp.), together with the use ofa peduncle during feeding. A variety of other algal species can also be a food source for


Fig. 8. Mean bacterial concentrations in each set of the experimental fish bioassays and the correspondingcontrol vessels (e.g., bioassays 1–3, series II; bioassays 4–6, series III; bioassays 7–8, series IV). *Dates whencell concentrations were significantly different.

these dinoflagellates (Burkholder and Glasgow, 1995; Glasgow et al., 1998; Seaborn etal., 1999).

This research corroborates reports of a complex life cycle with amoeboid stages in P.piscicida (Burkholder et al., 1992; Burkholder and Glasgow, 1995, 1997; Steidinger etal., 1996), and represents the first published report of a complex life cycle in the


cryptoperidiniopsoid, Cryptoperidiniopsis sp. (gen. nov.). These include the presence ofseveral amoeboid and cyst stages. Gradual transformation changes may be noted usinglong-term and/or intensive light microscopy. For example, we have observed dramatictransformations in overnight observations of unialgal cultures that initially consisted ofhigh zoospore densities of either P. piscicida or Cryptoperidiniopsis sp. (gen. nov.) thatwere food-depleted (without algal prey for 5–7 days). The next day, these cultures wereobserved to be devoid of most zoospores, comprised instead of high concentrations ofeither amoeboid or cyst stages. When cryptomonad algal prey were subsequently addedto these cultures, high densities of zoospores were re-established and the amoeboid andcyst stages were significantly reduced in abundance. In another approach, positivelinkages among these life stages can be confirmed from genetic sequencing of thevarious stages. For example, we have sent blind samples consisting of zoospores versusamoebae, taken from our cultures of Cryptoperidiniopsis brodyi (gen. et sp. nov.), to Dr.D. Oldach (UMD; Oldach et al., 2000). The gene sequencing results for both thezoospores and the amoebae were evaluated as positive for this species, and representstages of its life cycle. The Burkholder and Glasgow laboratory has confirmed variousstages in the life cycle of P. piscicida and P. shumwayae (sp. nov.) from molecularanalysis in independent laboratories (Dr. P. Rublee of UNC-Greensboro, and Dr. D.Oldach of UMD; e.g., Rublee et al., 1999). Similar action to cross-confirm life stagerelationships for other dinoflagellates with complex life cycles would be appropriate, andare recommended.

Various life stages are also identified in this study of Cryptoperidiniopsis (nov. gen.).These include a distinct cyst stage that is characterized by two types of scales that aredifferent than those noted for P. piscicida (Burkholder et al., 1992, 1995b; Burkholderand Glasgow, 1995, 1997). Burkholder and Glasgow (1995, 1997) and Burkholder(1999) have recognized multiple cyst forms in Pfiesteria that are produced fromdifferent life stages of the species, so it is likely that multiple cyst stages will also befound in the life cycle of this and other cryptoperidiniopsoid spp. When fullycharacterized, these species-specific morphological differences in the cyst scales mayprove to be of value in discerning the identification among pfiesteria-like species versesPfiesteria spp. The presence of silicon in the cysts of both Pfiesteria piscicida and theCryptoperidiniopsis sp. (gen. nov.) is of additional interest, since silicon is notcommonly found in dinoflagellates. Its presence here may indicate an early evolutionaryinfusion of this trait (scale formation) into the genome of these dinoflagellates by aprotozoan, or chrysophyte algal endosymbiont. Similarly, the ‘chrysophyte-like’ cyst ofP. piscicida zoospores that was reported by Burkholder et al. (1992), has fosteredspeculation that Pfiesteria may be an evolutionary ‘old’ dinoflagellate genus thatencompasses both chrysophyte and dinoflagellate characteristics.

Although the onset and initial strength of toxicity varied among the bioassayexperiments, Pfiesteria piscicida was directly associated with a series of fish deaths thatwere produced in this study. The presence of P. piscicida was linked to fish deaths in theinitial bioassay, and in eight of the nine subsequent bioassays. The P. piscicida culturepreviously associated with fish deaths, and then removed from fish and fed algal prey,took longer (16 days) to initiate fish mortality than the P. piscicida culture that was takendirectly from a fish killing culture. However, during the serial transfer of Pfiesteria


zoospores, there was also an increase in the duration before fish were affected. This wasclearly demonstrated in the third set of bioassays in which 11–15 days elapsed beforefish death occurred. However, once an actively toxic P. piscicida culture was established,the culture continued to be toxic as long as dead fish were replaced with live fish. Themost rapid time to fish death was 1.5 h of transfer into the actively toxic P. piscicidaculture. These results support those reported in previous studies by Burkholder et al.(1992, 1995a,b, 1999), Noga et al. (1993), Lewitus et al. (1995), Burkholder andGlasgow (1997), and Glasgow (2000) describing toxicity of Pfiesteria spp. to fish, andthe variability in the onset of toxicity by P. piscicida using TOX-A and TOX-Bfunctional types of Pfiesteria (Burkholder and Glasgow, 1997; Burkholder et al., 1999;WHOI, 2000; Burkholder, in press).

The serial transfer of P. piscicida zoospores from one set of bioassays through twoadditional bioassays perpetuated the toxicity in these dinoflagellate populations overtime in the laboratory, using different and multiple compliments of fish. Of note, duringthe bioassay experiments only one fish (out of 90) in the control experiments died, withnone of the deaths occurring within the toxic bioassays associated with depressedoxygen or high ammonia levels. Autopsies of live fish that exhibited symptoms ofPfiesteria toxicity showed absence of bacterial infection in nine out of 10 fish examined.In contrast, blood from dead fish contained bacteria. These results indicate that bacterialsepticemia was not the cause of fish mortality in these bioassays, although bacteriarapidly spread into the circulatory system after death. Further results of the bacterialconcentrations in this study show no significant differences in bacterial abundance in thewater from test fish bioassays with Pfiesteria versus the water from control fish cultures,in 14 of the 16 comparisons over the testing period. These findings indicate that bacterialabundance was not a factor related to the fish deaths. In addition, the systematicexamination of the water in all the bioassay series and the control facilities throughoutthese experiments indicated no perceptible differences in the presence of any microflora,or microfauna in the control or test fish bioassays throughout the study.

In contrast to these data supporting ichthyotoxicity of Pfiesteria piscicida at lower cell21densities (e.g., 250–300 zoospores ml ; Burkholder et al., 1995a), the experiments with

the Cryptoperidiniopsis sp. (gen. nov.) and G. galatheanum, indicate that neither of21these species is toxic to fish at the cell concentrations attained (ca. 700–800 cells ml )

over periods up to several months. These dinoflagellates did not increase cell productionto higher densities when grown with fish, with comparable cell densities present incontrol vessels without fish. Moreover, the fish in test bioassays with both speciesremained comparable in health to the control fish, showed no signs of stress or disease.As mentioned, other research has demonstrated no ichthyotoxicity of various strains andspecies of Cryptoperidiniopsis (gen. nov.) from the mid-Atlantic and southeastern US(Delaware to Florida: Burkholder et al., 1995a; Glasgow, 2000; Burkholder, in press). At

3 21extremely high cell densities (115 3 10 zoospores ml ), G. galatheanum has beenreported as a toxin producer associated with deaths of juvenile cod (Gadus morhua) byNielsen (1993), and with reduced growth rate in mussels (Mytilus edulis) by Nielsen and

3 21Stroemgren (1991) at concentrations above ca. 120 3 10 zoospores ml . If our strainof G. galatheanum is toxic, the cell densities attained in this study may have been toolow to cause adverse impacts on fish health. Overall, the findings from this research


indicated that the two ‘pfiesteria-lookalike’ species examined are strikingly distinct fromP. piscicida. Although they appear physically similar to Pfiesteria, they differ in theimportant trait of a toxic response toward fish. Further research will strengthen insightsabout the comparative ecology of toxic Pfiesteria complex species in comparison tothese and other dinoflagellates.

Acknowledgements

Financial support for various components of this study was provided by the VirginiaDepartment of Health, the Virginia Department of Environmental Quality, the Center forDisease Control and Prevention, and the University of Miami (grant ES05705).Appreciation is given to personnel from the Virginia Department of EnvironmentalQuality and Department of Health for providing the samples analyzed in this program.Special thanks and appreciation is given to JoAnn Burkholder, Howard Glasgow,Matthew Lynn, David Oldach, Parke Rublee, Karen Steidinger, and Ernest Truby fortheir contributions during various phases of this work. [SS]

References

Buckland-Nicks, J.A., Reimechen, T.E., Taylor, F.J.R., 1990. A novel associations between an endemicstickleback and a parasitic dinoflagellate. 2. Morphology and life cycle. J. Phycol. 26, 539–548.

Burkholder, J.M., 1998. Implications of harmful microalgae and heterotrophic dinoflagellates in managementof sustainable marine fisheries. Ecol. Appl. 8 (Suppl. 1), S37–S62.

Burkholder, J.M., 1999. The lurking perils of Pfiesteria. Sci. Am. 281, 42–49.Burkholder, J.M., in press. The toxic Pfiesteria complex. In: Hallegraeff, G., Blackburn, S., Bolch, C., Lewis,

R. (Eds.), Harmful Algal Blooms. IOC UNESCO, Paris.Burkholder, J.M., Glasgow, Jr. H.B., 1995. Interactions of a toxic estuarine dinoflagellate with microbial

predators and prey. Arch. Protistenk. 145, 177–188.Burkholder, J.M., Glasgow, Jr. H.B., 1997. Pfiesteria piscicida and other Pfiesteria-like dinoflagellates:

Behavior, impacts, and environmental controls. Limnol. Oceanogr. 42, 1052–1075.Burkholder, J.M., Noga, E.J., Hobbs, C.W., Glasgow, H.B. Jr., Smith, S.A., 1992. New ‘phantom’

dinoflagellate is the causative agent of major estuarine fish kills. Nature 358/360, 407–410, 768.Burkholder, J.M., Glasgow, Jr. H.B., Hobbs, C.W., 1995a. Fish kills linked to a toxic ambush–predator

dinoflagellate: distribution and environmental conditions. Mar. Ecol. Prog. Ser. 124, 43–61.Burkholder, J.M., Glasgow, Jr. H.B., Steidinger, K.A., 1995b. Stage transformation in the complex life cycle

of an ichthyotoxic ‘ambush–predator’ dinoflagellate. In: Lassus, P., Arzul, G., Erard, E., Gentien, P.,Marcaillou, C. (Eds.), Harmful Marine Algal Blooms, Technique et Documentation, Lavoisier. Intercept,Paris, pp. 567–572.

Burkholder, J.M., Mallin, M.A., Glasgow, H.B., Larsen, L.M., McIver, M.R., Shank, G.C., Deamer-Melia, N.,Briley, D.S., Springer, J., Touchette, B.W., Hannon, E.K., 1997. Impacts to a coastal river and estuary fromrupture of a large swine waste holding lagoon. J. Environ. Qual. 26, 1451–1466.

Burkholder, J.M., Mallin, M.A., Glasgow, Jr. H.B., 1999. Fish kills, bottom-water hypoxia, and the toxicPfiesteria complex in the Neuse river and estuary. Mar. Ecol. Prog. Ser. 179, 301–310.

Bursa, A., 1970a. Dinamoebidinium coloradense spec. nov. and Katodinium auratum spec. nov. in ComoCreek, Boulder County, Colorado. Arct. Alp. Res. 2, 145–151.

Bursa, A., 1970b. Dinamoebidinium hyperboreum spec. nov. in coastal plankton of Ellesmere Island, N.W.T.,Canada. Arct. Alp. Res. 1, 152–154.


Fairey, E., Edmunds, S., Deamer-Melia, N.J., Glasgow, Jr. H.B., Johnson, F., Moeller, P., Burkholder, J.M.,Ramsdell, J., 1999. Reporter gene assay for fish killing activity produced by Pfiesteria piscicida. Environ.Health Perspect. 107, 711–714.

¨Gaines, G., Elbrachter, M., 1987. Heterotrophic nutrition. In: The Biology of Dinoflagellates. BotanicalMonographs, Vol. 31. Blackwell Scientific, Oxford, pp. 224–268.

Glasgow, H.B. Jr., 2000. The Biology and Impacts of Toxic Pfiesteria Complex Species. Ph.D. Dissertation,Department of Marine, Earth and Atmospheric Sciences, North Carolina State University, Raleigh, NC.

Glasgow, Jr. H.B., Burkholder, J.M., Schmeche, D.E., Fester, P., Rublee, P.A., 1995. Insidious effects of atoxic estuarine dinoflagellate on fish survival and human health. J. Toxicol. Environ. Health 46, 501–522.

Glasgow, Jr. H.B., Burkholder, J.M., Lewitus, A.J., 1998. Feeding behavior of the ichthyotoxic estuarinedinoflagellate, Pfiesteria piscicida, on amino acids, algal prey, and fish vs. mammalian erythrocytes. In:Reguera, B., Fernandez, M.L., Wyatt, T. (Eds.), Harmful Microalgae. UNESCO, Paris, pp. 394–397.

Grattan, L., Oldach, D., Perl, T., Lowitt, M., Matuszak, D., Dickson, C., Parrott, C., Shoemacher, R.,Wasserman, M., Hebel, J., Charache, P., Morris, J., 1998. Problems in learning and memory occur inpersons with environmental exposure to waterways containing toxin-producing Pfiesteria or Pfiestera-likedinoflagellates. Lancet 352, 532–539.

Hobbie, ley J., Daley, R., Jasper, S., 1977. Use of Nucleopore filters for counting bacteria by fluorescencemicroscopy. Appl. Environ. Microbiol. 33, 1255–1258.

Kamykowski, D., Reed, R., Kirkpatrick, G., 1992. Comparison of sinking velocity, swimming, rotation andpath characteristics among six marine dinoflagellate species. Mar. Biol. 113, 319–328.

Lewitus, A.J., Jesien, R., Kana, T., Burkholder, J.M., Glasgow, Jr. H.B., May, E., 1995. Discovery of the‘phantom’ dinoflagellate in Chesapeake Bay. Estuaries 18, 373–378.

Lewitus, A.J., Glasgow, Jr. H.B., Burkholder, J.M., 1999. Kleptoplastidy in the toxic dinoflagellate, Pfiesteriapiscicida. J. Phycol. 35, 303–312.

Magnien, R., Goshorn, D., Michael, B., Tango, P., Karrh, R., Nelson, R., 1999. Findings for water quality,habitat and Pfiesteria related comprehensive assessment and response efforts in 1998. Special Reportprepared by the Maryland Pfiesteria Study Team, Annapolis, MD, February 1999, pp. 13–15.

Marshall, H.G., 1999. Pfiesteria piscicida and dinoflagellates similar to Pfiesteria.Virginia J. Sci. 50, 281–286.Marshall, H.G., Seaborn, D., Wolny, J., 1999. Monitoring results for Pfiesteria piscicida and Pfiesteria-like

organisms from Virginia waters in 1998. Virginia J. Sci. 50 (4), 287–298.Meyer, F., Barclay, L., 1990. Field Manual for the Investigation of Fish Kills. US Fish & Wildlife Service

Resource Publication 1777. US Department of the Interior, Washington, DC.Nielsen, M., 1993. Toxic effect of the marine dinoflagellate Gymnodinium galatheanum on juvenile cod Gadus

morhua. Mar. Ecol. Prog. Ser. 95, 273–277.Nielsen, M., Stroemgren, T., 1991. Shell growth response of mussels (Mytilus edulis) exposed to toxic

microalgae. Mar. Biol. 108, 263–267.Noga, E.J., Smith, S.A., Burkholder, J.M., Hobbs, C.W., Bullis, R.A., 1993. A new ichthyotoxic dinoflagellate:

cause of acute mortality in aquarium fishes. Vet. Rec. 133, 48–49.Oldach, D., Delwiche, C., Jakiobsen, K., Tengs, T., Brown, E., Kempton, J., Schaefer, E., Bowers, H.,

Glasgow, Jr. H.B., Burkholder, J.M., Steidinger, K.A., Rublee, P.A., 2000. Heteroduplex mobility assayguided sequence discovery: elucidation of the small subunit (18S) rDNA sequence of Pfiesteria piscicidafrom complex algal culture and environmental sample DNA pools. Proc. Natl. Acad. Sci. USA 97,4304–4308.

¨ ¨ ¨Pascher, A., 1916. Uber eine neu Amoeba (Dinamoebe varians) mit dinoflagellatenartigen Schwarmen. Arch.Protistenk. 36, 118–136.

Pfiester, L., Lynch, R., 1980. Amoeboid stages and sexual reproduction of Cystodinium bataviense and itssimilarity to Dinococcus (Dinophyceae). Phycologia 19 (3), 178–183.

´Pfiester, L., Popovsky, J., 1979. Parasitic, amoeboid dinoflagellates. Nature 279, 421–424.´Popovsky, J., 1982. Another case of phagotrophy by Gymnodinium helveticum Penard f. achroum Skuja. Arch.

Protistenk. 125, 73–78.´Popovsky, J., Pfiester, L., 1990. In: Dinophyceae (Dinoflagellida). Gustav Fischer, Jena, Stuttgart, p. 272.

Rublee, P.A., Kempton, J., Schaefer, E., Burkholder, J.M., Glasgow, Jr. H.B., Oldach, D., 1999. PCR and FISHdetection extends the range of Pfiesteria piscicida in estuarine waters. Virginia J. Sci. 50, 325–336.


¨Schnepf, E., Elbrachter, M., 1992. Nutritional strategies in dinoflagellates. A review with emphasis on cellbiological aspects. Eur. J. Protistol. 28, 3–24.

Seaborn, D., Seaborn, A., Dunstan, W., Marshall, H., 1999. Growth and feeding studies on the algal feedingstage of a Pfiesteria-like dinoflagellate. Virginia J. Sci. 50, 337–344.

Spero, H., 1982. Phagotrophy in Gymnodinium fungiforme (Pyrrhophyta): the peduncle as an organelle ofingestion. J. Phycol. 18, 356–360.

Steidinger, K.A., 1993. Some taxonomic and biologic aspects of toxic dinoflagellates. In: Falconer, I. (Ed.),Algal Toxins in Seafood and Drinking Water. Academic Press, London, pp. 1–28.

Steidinger, K.A., Truby, E., Garrett, J., Burkholder, J.M., 1995. The morphology and cytology of a newlydiscovered toxic dinoflagellate. In: Lassus, P., Arzul, G., Erad, E., Gentien, P., Marcaillou, C. (Eds.),Harmful Marine Algal Blooms, Technique et Documentation, Lavoisier. Intercept, Paris, pp. 83–88.

Steidinger, K.A., Burkholder, J.M., Glasgow, Jr. H.B., Hobbs, C.W., Garrett, J., Truby, E., Noga, E.J., Smith,S.A., 1996. Pfiesteria piscicida gen. et sp. nov. (Pfiesteriaceae Fam. nov.), a new toxic dinoflagellate with acomplex life cycle and behaviour. J. Phycol. 32, 157–164.

Tomas, C.R., 1996. In: Identifying Marine Diatoms and Dinoflagellates. Academic Press, New York, p. 598.Truby, E.W., 1997. Preparation of single-celled marine dinoflagellates for electron microscopy. Microsc. Res.

Tech. 36, 337–340.WHOI (Woods Hole Oceanographic Institute), 2000. Pfiesteria Interagency Coordination Workgroup, Glossary

of Pfiesteria-Related Terms. http: / /www.redtide.whoi.edu/pfiesteria / 7 pp.

Comparative culture and toxicity studies between the toxic ...directory.umm.ac.id/Data...

Documents

Transcript of Comparative culture and toxicity studies between the toxic ...directory.umm.ac.id/Data...