THE PHYLOGENETIC RELATIONSHIP OF PFIESTERIA PISCICIDA, CRYPTOPERIDINIOPSOID SP. AMYLOODINOUM OCELLATUM AND A PFIESTERIA-LIKE DINOFLAGELLATE TO OTHER DINOFLAGELLATES AND APICOMPLEXANS

The taxonomic relationship between heterotrophic and parasitic dinoflagellates has not been studied extensively at the molecular level. In order to investigate these taxonomic relationships, we sequenced the small subunit (SSU) ribosomal RNA gene of Pfiesteria piscicida (Steidinger et Burkholder), a Pfiesteria -like dinoflagellate, Cryptoperidiniopsoid sp., and Amyloodinium ocellatum (Brown) and submitted those sequences to GenBank. Pfiesteria piscicida and Cryptoperidiniopsoid sp. are heterotrophic dinoflagellates, purportedly pathogenic to fish, and A. ocellatum, a major fish pathogen, has caused extensive economic losses in both the aquarium and aquaculture industries. The pathogenicity of the Pfiesteria -like dinoflagellate is unknown at this time, but its growth characteristics and in vitro food preferences are similar to those of P. piscicda. The SSU sequences of these species were aligned with the other full-length dinoflagellate sequences, as well as those of representative apicomplexans and Perkinsus species, the groups most closely related to dinoflagellates. Phylogenetic analyses indicate that Cryptoperidiniopsoid sp., P. piscicida, and the Pfiesteria -like dinoflagellate are closely related and group into the class Blastodiniphyceae, as does A. ocellatum. None of the species examined were closely related to the apicomplexans or to Perkinsus marinus, the parasite that causes "Dermo disease" in oysters. The overall phylogenetic analyses largely supported the current class and subclass groupings within the dinoflagellates.     

CRYPTOPERIDINIOPSIS BRODYI GEN. ET SP. NOV. (DINOPHYCEAE), A SMALL LIGHTLY ARMORED DINOFLAGELLATE IN THE PFIESTERIACEAE1

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

用分子遗传学及免疫学方法控制鱼类寄生性甲藻

寄生性甲藻,例如Amyloodiniumocellatum,给鱼类养殖带来严重的危害。虽然,近年来针对Amyloodinium的药物治疗有一些新的进展,如使用氯喹、过氧化氢和3,N泛影葡胺拉沙洛西等,然而,含铜类药物仍然是最有效的。近年来分子遗传学和免疫学的进展,使得我们能够更好地了解寄生性甲藻的流行病学及其防治方法。分子系统学研究认为某些寄生性甲藻,如Amyloodiniumocellatum,可以聚类为高度同源性的一支;而其它的如Piscinoodiniumpillulare,则可以认为不止一种或更高的分类阶元。这些分子分析也发展出一些高灵敏的检测技术,可以检测出环境中极少量的寄生性甲藻。通过对Amyloodinium的免疫学研究表明,鱼类能对寄生物的感染产生强烈的高度特异的保护性免疫应答,其中主要是抗体介导的免疫应答。鱼体皮肤和鳃也能表达内源非特异性多肽抗生素(类组蛋白),它们能对Amyloodinium造成致命的破坏。利用这些特异或非特异的免疫防御,将更有助于我们控制这些具有严重危害性的寄生性甲藻     

REPLY TO COMMENT ON THE LIFE CYCLE AND TOXICITY OF PFIESTERIA PISCICIDA REVISITED1

Free‐living, marine dinoflagellates are typified by a well‐defined, haplontic life cycle with relatively few stages. The most unusual departure from this life cycle is one reported for the heterotrophic dinoflagellate Pfiesteria piscicida Steidinger et Burkholder. This species is alleged to have at least 24 life cycle stages including amoebae and a chrysophyte‐like cyst form ( Burkholder et al. 1992 , Burkholder and Glasgow 1997a ) not previously known in free‐living marine dinoflagellates. Litaker et al. (2002) redescribed the life cycle of P. piscicida from single‐cell isolates and found only life cycle stages typical of free‐living marine dinoflagellates. The discrepancy between these observations and the life cycle reported in the literature prompted a rigorous study to resolve the life cycle of P. piscicida. Burkholder and Glasgow (2002) took exception to this study, arguing that Litaker et al. (2002) misunderstood the life cycle of P. piscicida and ignored recent publications. We present a rebuttal of their criticisms and suggest a simple way to resolve the discrepancies in the P. piscicida life cycle.     

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

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

IDENTIFICATION OF PFIESTERIA PISCICIDA (DINOPHYCEAE) AND PFIESTERIA‐LIKE ORGANISMS USING INTERNAL TRANSCRIBED SPACER‐SPECIFIC PCR ASSAYS1

The putative harmful algal bloom dinoflagellate, Pfiesteria piscicida (Steidinger et Burkholder), frequently co‐occurs with other morphologically similar species collectively known as Pfiesteria‐like organisms (PLOs). This study specifically evaluated whether unique sequences in the internal transcribed spacer (ITS) regions, ITS1 and ITS2, could be used to develop PCR assays capable of detecting PLOs in natural assemblages. ITS regions were selected because they are more variable than the flanking small subunit or large subunit rRNA genes and more likely to contain species‐specific sequences. Sequencing of the ITS regions revealed unique oligonucleotide primer binding sites for Pfiesteria piscicida, Pfiesteria shumwayae (Glasgow et Burkholder), Florida “Lucy” species, two cryptoperidiniopsoid species, “H/V14” and “PLO21,” and the estuarine mixotroph, Karlodinium micrum (Leadbetter et Dodge). These PCR assays had a minimum sensitivity of 100 cells in a 100‐mL sample (1 cell·mL^?1) and were successfully used to detect PLOs in the St. Johns River system in Florida, USA. DNA purification and aspects of PCR assay development, PCR optimization, PCR assay controls, and collection of field samples are discussed.     

Delimitation of the Thoracosphaeraceae (Dinophyceae), including the calcareous dinoflagellates, based on large amounts of ribosomal RNA sequence data

The phylogenetic relationships of the Dinophyceae (Alveolata) are not sufficiently resolved at present. The Thoracosphaeraceae (Peridiniales) are the only group of the Alveolata that include members with calcareous coccoid stages; this trait is considered apomorphic. Although the coccoid stage apparently is not calcareous, Bysmatrum has been assigned to the Thoracosphaeraceae based on thecal morphology. We tested the monophyly of the Thoracosphaeraceae using large sets of ribosomal RNA sequence data of the Alveolata including the Dinophyceae. Phylogenetic analyses were performed using Maximum Likelihood and Bayesian approaches. The Thoracosphaeraceae were monophyletic, but included also a number of non-calcareous dinophytes (such as Pentapharsodinium and Pfiesteria) and even parasites (such as Duboscquodinium and Tintinnophagus). Bysmatrum had an isolated and uncertain phylogenetic position outside the Thoracosphaeraceae. The phylogenetic relationships among calcareous dinophytes appear complex, and the assumption of the single origin of the potential to produce calcareous structures is challenged. The application of concatenated ribosomal RNA sequence data may prove promising for phylogenetic reconstructions of the Dinophyceae in future.     

Propagation of the marine dinoflagellate Amyloodinium ocellatum under germ-free conditions

Germ-free infections of Amyloodinium ocellatum were produced on both living fish and in organ cultures. Exposing gnotobiotic guppies (Poecilia reticulata) to 125 dinospores in multiwell tissue culture plates produced nonlethal infections that could be serially propagated. Exposure to 250 or more parasites killed the fish during the first infection cycle, but if the dead fish were incubated in a cell culture medium/seawater mixture, the parasites could survive and reproduce for up to 2 wk in these organ cultures. Organ cultures containing only seawater or those containing bacteria did not support the prolonged survival of Amyloodinium.     

LIFE CYCLE OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellate Pfiesteria piscicida (Steidinger et Burkholder) has been reported to have an unusual life cycle for a free‐living marine dinoflagellate. As many as 24 life cycle stages were originally described for this species. During a recent phylogenetic study in which we used clonal cultures of P. piscicida, we were unable to confirm many reported life cycle stages. To resolve this discrepancy, we undertook a rigorous examination of the life cycle of P. piscicida using nuclear staining techniques combined with traditional light microscopy, high‐resolution video microscopy, EM, and in situ hybridization with a suite of fluorescently labeled peptide nucleic acid (PNA) probes. The results showed that P. piscicida had a typical haplontic dinoflagellate life cycle. Asexual division occurred within a division cyst and not by binary fission of motile cells. Sexual reproduction of this homothallic species occurred via the fusion of isogamous gametes. Examination of tanks where P. piscicida was actively feeding on fish showed that amoebae were present; however, they were contaminants introduced with the fish. Whole cell probing using in situ hybridization techniques confirmed that these amoebae were hybridization negative for a P. piscicida‐specific PNA probe. Direct observations of clonal P. piscicida cultures revealed no unusual life cycle stages. Furthermore, the results of this study provided no evidence for transformations to amoebae. We therefore conclude that P. piscicida has a life cycle typical of free‐living marine dinoflagellates and lacks any amoeboid or other specious stages.     

Massive infestation by Amyloodinium ocellatum (Dinoflagellida) of fish in a highly saline lake, Salton Sea, California, USA.

Persistent fish infestation by the parasitic dinoflagellate Amyloodinium ocellatum was found at a highly saline lake, Salton Sea, California, USA. The seasonal dynamics of the infestation of young tilapia was traced in 1997-1998. First appearing in May, it became maximal in June-August, decreased in October and was not detectable in November. Outbreak of the infestation and subsequent mortality of young fish was registered at the Sea at a water temperature and salinity of 40 degrees C and 46 ppt, respectively. Some aspects of the ultrastructure of parasitic trophonts of A. ocellatum and their location on the fish from different size groups are considered. The interactions of parasitological and environmental factors and their combined effect upon fish from the Salton Sea are discussed.     

A highly specific PCR assay for detecting the fish ectoparasite Amyloodinium ocellatum

Amyloodiniosis, caused by the dinoflagellate ectoparasite Amyloodinium ocellatum, is one of the most serious diseases affecting marine fish in warm and temperate waters. Current diagnostic methods rely entirely on the microscopic identification of parasites on the skin or gills of infested fish. However, subclinical infestations usually go undetected, while no method of detecting the free-swimming, infective (dinospore) stage has been devised. Targeting the parasite's ribosomal DNA region, we have developed a sensitive and specific PCR assay that can detect as little as a single cell from any of the 3 stages of the parasite's life cycle (trophont, tomont, dinospore). This assay performs equally well in a simple artificial seawater medium and in natural seawater containing a plankton community assemblage. The assay is also not inhibited by gill tissue. Sequence analysis of the internal transcribed spacer region of 5 A. ocellatum isolates, obtained from fish in the Red Sea (Israel), eastern Mediterranean Sea (Israel), Adriatic Sea (Italy), Gulf of Mexico (Florida), and from an unknown origin, revealed insignificant variation, indicating that all isolates were the same species. However, 3 of these isolates propagated in cell culture varied in behavior and morphology, and these differences were consistent during at least 2 yr in culture. Thus, our findings do not eliminate the possibility that different strains are in fact 'subspecies' or lower taxa, which may also differ in pathogenic and immunogenic characteristics, environmental tolerance, and other features.     

Susceptibility and tolerance of spotted seatrout, Cynoscion nebulosus , and red snapper, Lutjanus campechanus , to experimental infections with Amyloodinium ocellatum

Amyloodinium ocellatum is a parasitic dinoflagellate that infects warm-water marine and estuarine fishes and causes mortalities in aquaculture. Its life cycle consists of 3 stages: a feeding trophont that parasitizes the gills and skin where it interferes with gas exchange, osmoregulation, and tissue integrity; a detached reproductive tomont; and a free-swimming infective dinospore. We compared the susceptibility and tolerance of juvenile spotted seatrout, Cynoscion nebulosus, and red snapper, Lutjanus campechanus, to this parasite by individually exposing fish in 3-L aquaria (at 25 C and 33 practical salinity units) to several dinospore doses over different time periods and quantified the size and number of resulting trophonts. We estimated the trophont detachment rate and trophont size at detachment, the 24-hr dinospore infection rate, the dinospore 48-hr median lethal dose (LD(50)), and the trophont lethal load at the 48-hr LD(50). There were no significant differences in dinospore infection rates or dinospore lethal doses between spotted seatrout and red snapper; however, trophonts remained attached longer and attained a larger size in red snapper than in spotted seatrout. The trophont lethal load was significantly higher in spotted seatrout than in red snapper. A proposed model simulating the trophont dynamics reflected our experimental findings and showed that A. ocellatum reproductive success is linked both to the number of dinospores and the size of the trophont, factors that, in turn, are linked to the time the trophont spends on the host and the number of trophonts the host can tolerate.     

Host site of activity and cytological effects of histone-like proteins on the parasitic dinoflagellate Amyloodinium ocellatum

Histone-like proteins (HLPs) are broad-spectrum, endogenously produced antibiotics which we have isolated from tissues of rainbow trout Oncorhynchus mykiss and hybrid striped bass (Morone saxatilis male x M. chrysops female). Here, we show that HLP-1, which has high sequence homology to histone H2B, equally inhibited both young and mature trophonts of the important ectoparasite Amyloodinium ocellatum. In addition to direct killing of Amyloodinium trophonts, there was evidence that HLP-1 from both rainbow trout and hybrid striped bass caused severe developmental abnormalities, including delayed development, in both the parasitic trophont stage as well as the reproductive tomont stage. The deleterious effects of HLP-1 also were manifested in what appeared to be 'delayed mortality', where parasites of normal appearance would die later in development. Similar serious damage was also seen with calf histone H2B and the unrelated peptide antibiotic magainin 2. A comparison of the antibiotic activity in mucus versus epidermis compartments of the skin of hybrid striped bass suggested that the majority of antibiotic (including HLP-1) activity resided in the epidermis, although some activity was present in the mucus. These data suggest that normal, nonimmune fish skin contains potent defenses against protozoan ectoparasites and that the effects of these defenses may extend beyond their transient interactions with the parasites, which has important implications for this host-parasite relationship.     

MIXOTROPHY AND NITROGEN UPTAKE BY PFIESTERIA PISCICIDA (DINOPHYCEAE)

The nutritional versatility of dinoflagellates is a complicating factor in identifying potential links between nutrient enrichment and the proliferation of harmful algal blooms. For example, although dinoflagellates associated with harmful algal blooms (e.g. red tides) are generally considered to be phototrophic and use inorganic nutrients such as nitrate or phosphate, many of these species also have pronounced heterotrophic capabilities either as osmotrophs or phagotrophs. Recently, the widespread occurrence of the heterotrophic toxic dinoflagellate, Pfiesteria piscicida Steidinger et Burkholder, has been documented in turbid estuarine waters. Pfiesteria piscicida has a relatively proficient grazing ability, but also has an ability to function as a phototroph by acquiring chloroplasts from algal prey, a process termed kleptoplastidy. We tested the ability of kleptoplastidic P. piscicida to take up ¹⁵N-labeled NH     , NO     , urea, or glutamate. The photosynthetic activity of these cultures was verified, in part, by use of the fluorochrome, primulin, which indicated a positive relationship between photosynthetic starch production and growth irradiance. All four N substrates were taken up by P. piscicida , and the highest uptake rates were in the range cited for phytoplankton and were similar to N uptake estimates for phagotrophic P. piscicida . The demonstration of direct nutrient acquisition by kleptoplastidic P. piscicida suggests that the response of the dinoflagellate to nutrient enrichment is complex, and that the specific pathway of nutrient stimulation (e.g. indirect stimulation through enhancement of phytoplankton prey abundance vs. direct stimulation by saprotrophic nutrient uptake) may depend on P. piscicida 's nutritional state (phagotrophy vs. phototrophy).     

DESCRIPTION OF A NEW GENUS OF PFIESTERIA‐LIKE DINOFLAGELLATE,LUCIELLA GEN. NOV. (DINOPHYCEAE), INCLUDING TWO NEW SPECIES: LUCIELLA MASANENSIS SP. NOV. AND LUCIELLA ATLANTIS SP. NOV.1

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

A standardized method of propagating the marine fish parasite, Amyloodinium ocellatum

The peridinian dinoflagellate Amyloodinium ocellatum was propagated by serial passage in clownfish (Amphiprion ocellaris) and hybrid striped bass (Morone chrysops X Morone saxatilis). Each 25-50-mm fish was exposed to 4,000-6,000 dinospores in 400 ml of artificial seawater for 30 min. Two days after exposure, trophonts were harvested by immersing the fishes in fresh water. After encystment, tomonts were axenized by multiple washes with sterile distilled water and sterile artificial seawater containing penicillin and streptomycin, and then incubated in the antibiotic solution. High yields of both tomonts and dinospores of the same sizes and ages were obtained, and host mortalities were eliminated. Microbial growth in incubating cultures was inhibited until after dinospores had emerged from tomonts, and dinospores remained infective for at least 4 days at 26 C.     

Protective immunity in fish against protozoan diseases

The demand for and costs of producing land-based animal protein continues to escalate as the world population increases. Fish is an excellent protein, but the catch-fishery is stagnant or in decline. Intensive cage culture of fish is a viable option especially in countries with lakes/rivers and/or a long coastline; however, disease outbreaks will likely occur more frequently with cage culture. Hence protective strategies are needed, and one approach is to exploit the piscine immune system. This discussion highlights immunity (innate/natural and adaptive/acquired) in fish against three pathogenic protozoa (Amyloodinium ocellatum, Ichthyophthirius multifiliis and Cryptobia salmositica). Histone-like proteins in the mucus and skin of naturally resistant fish kill trophonts of A. ocellatum, and also may cause abnormal development of tomonts. Breeding of Cryptobia-resistant brook charrs is possible as resistance is controlled by a dominant Mendelian locus, and the parasite is lysed via the Alternative Pathway of Complement Activation. Production of transgenic Cryptobia-tolerant salmon is an option. Recovered fish are protected from the three diseases (acquired immunity). Live I. multifiliis theronts injected intraperitoneally into fish elicit protection. Also, a recombinant immoblizing-antigen vaccine against ichthyophthirosis has been developed but further evaluations are necessary. The live Cryptobia vaccine protects salmonids from infections while the DNA-vaccine stimulates production of antibodies to neutralize the disease causing factor (metalloprotease) in cryptobiosis; hence infected fish recover more rapidly.     

Tintinnophagus acutus n. g., n. sp. (Phylum Dinoflagellata), an Ectoparasite of the Ciliate Tintinnopsis cylindrica Daday 1887, and Its Relationship to Duboscquodinium collini Grassé 1952

ABSTRACT. The dinoflagellate Tintinnophagus acutus n. g., n. sp., an ectoparasite of the ciliate Tintinnopsis cylindrica Daday, superficially resembles Duboscquodinium collini Grassé, a parasite of Eutintinnus fraknoii Daday. Dinospores of T. acutus are small transparent cells having a sharply pointed episome, conspicuous eyespot, posteriorly positioned nucleus with condensed chromosomes, and rigid form that may be supported by delicate thecal plates. Dinospores attach to the host via a feeding tube, losing their flagella, sulcus, and girdle to become spherical or ovoid cells. The trophont of T. acutus feeds on the host for several days, increasing dramatically in size before undergoing sporogenesis. Successive generations of daughter sporocytes are encompassed in an outer membrane or cyst wall, a feature not evident in trophonts. Tintinnophagus acutus differs from D. collini in host species, absence of a second membrane surrounding pre‐sporogenic stages, and failure to differentiate into a gonocyte and a trophocyte at the first sporogenic division. Phylogenetic analyses based on small subunit (SSU) ribosomal DNA (rDNA) sequences placed T. acutus and D. collini in the class Dinophyceae, with T. acutus aligned loosely with Pfiesteria piscicida and related species, including Amyloodinium ocellatum, a parasite of fish, and Paulsenella vonstoschii, a parasite of diatoms. Dubosquodinium collini nested in a clade composed of several Scrippsiella species and Peridinium polonicum. Tree construction using longer rDNA sequences (i.e. SSU through partial large subunit) strengthened the placement of T. acutus and D. collini within the Dinophyceae.     

Development of an immunofluorescence technique for detecting Pfiesteria piscicida

While several DNA-based methods have been developed for the putatively toxic dinoflagellate Pfiesteria piscicida Burkholder et Steidinger, an independent detection method such as immunofluorescence can be a useful alternative. In this study, P. piscicida-specific antisera were developed, and an immunofluorescence (IF) procedure was optimized. A total of six antisera were raised using whole cells (WCA) and the insoluble cellular fraction (ICF) as antigens, respectively, and their titer and specificity were examined using dot blot analysis and whole cell IF. Results showed that the two antisera produced from the ICF antigen had a markedly higher titer (1500) than the other four yielded from the WCA (200). In addition, the two ICF-derived antisera exhibited much higher species specificity, showing no cross-reaction with P. shumwayae, Cryptoperidiniopsis sp., Karlodinium micrum, and other more distant algae tested, and very low background for field collected samples. In evaluation of the IF technique using a P. piscicida-specific polymerase chain reaction (PCR) technique, results from both methods generally agreed well for both field samples (from eastern Long Island Sound) spiked with cultured P. piscicida and those containing naturally occurring P. piscicida (from Chesapeake Bay tributaries).     

KLEPTOPLASTIDY IN THE TOXIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)

The ichthyotoxic dinoflagellate Pfiesteria piscicida Steidinger et Burkholder has a complex life cycle with several heterotrophic flagellated and amoeboid stages. A prevalent flagellated form, the nontoxic zoospore stage, has a proficient grazing ability, especially on cryptophyte prey. Although P. piscicida zoospores lack the genetic capability to synthesize chloroplasts, they can obtain functional chloroplasts from algal prey (i.e. kleptoplastidy), as demonstrated here with a cryptophyte prey. Zoospores grown with Rhodomonas sp. Karsten CCMP757 (Cryptophyceae) grazed the cryptophyte population to minimal densities. After placing the cultures in near darkness where cryptophyte recovery was restricted and further prey ingestion did not occur, the time-course patterns in growth, prey chloroplast content·zoospore⁻¹, and prey nucleus content·zoospore⁻¹ were followed. Ingested chloroplasts were selectively retained in the dinoflagellate, as indicated by the decline and, ultimately, near absence of cryptophyte nuclei in plastid-containing zoospores. Chloroplasts retained inside P. piscicida cells for at least a week were photosynthetically active, as indicated by starch accumulation and microscope-autoradiographic measurements of bicarbonate uptake. Recognition that P. piscicida can function as a phototroph broadens our perspective of the physiological ecology of the dinoflagellate because it suggests that, at least during part of its life cycle, P. piscicida 's growth and survival might be affected by photoregulation and nutritional control of photosynthesis.