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.     

ENCYSTMENT OF THE DINOFLAGELLATE GYRODINIUM UNCATENUM: TEMPERATURE AND NUTRIENT EFFECTS1

Sexual reproduction and encystment of the marine dinoflagellate Gyrodinium uncatenum Hulburt were induced in nitrogen and phosphorus-limited batch cultures. Sexuality did not occur under nutrient-replete conditions even when growth rate was reduced by non-optimal temperatures. Growth was optimal over a broader temperature range than encystment and virtually no cysts were produced at some low and high temperatures where growth occurred. Most cells initiated sexuality as intracellular pools of each limiting nutrient reached minimum or subsistence levels as much as four days after extracellular nutrients were exhausted. High nitrogen cell quotas during the phosphorus experiment indicate that sexuality was induced by a shortage of phosphorus and not by an indirect effect on nitrogen uptake. Total cyst yield corresponded to successful encystment of 9–13% of the motile populations, yet 60–85% of the plateau-phase motile cells were planozygotes (swimming zygotes formed from fusing gametes). Batch culture studies monitoring total cyst yield may thus seriously underestimate the extent of sexuality. More importantly, the number of cysts produced in a dinoflagellate population may be significantly reduced by environmental factors acting on the cells after sexual induction and fusion.     

SEXUAL PROCESSES IN THE LIFE CYCLE OF GYRODINIUM UNCATENUM (DINOPHYCEAE): A MORPHOGENETIC OVERVIEW1

Sexual processes in the life cycle of the dinoflagellate Gyrodinium uncatenum Hulburt were investigated in isolated field populations. Morphological and morphogenetic aspects of gamete production, planozygote formation, encystment, excystment, and planomeiocyte division are described from observations of living specimens, Protargol silver impregnated material and scanning electron microscope preparations. The sexual cycle was initiated by gamete formation which involved two asexual divisions of the vegetative organism. Gametes were fully differentiated following the second division and immediately capable of forming pairs. Either isogamous or anisogamous pairs were formed by the mid-ventral union of gametes. Gametes invariably joined with flagellar bases in close juxtaposition. Complete fusion of gametes required ca. 1 h, involved plasmogamy followed by karyogamy and resulted in a quadriflagellated planozygote. Planozygotes encysted in 24–48 h to yield a hypnozygote capable of overwintering in estuarine sediments. Hypnozygotes collected from sediment in late winter readily excysted upon exposure to temperatures above 15°C. A single quadriflagellated planomeiocyte emerged from the cyst and under culture conditions divided one to two days later. The four flagella were not evenly distributed at the first division and both bi- and tri-flagellated daughter cells were formed.     

ULTRASTRUCTURE AND LSU RDNA‐BASED PHYLOGENY OF ESOPTRODINIUM GEMMA (DINOPHYCEAE), WITH NOTES ON FEEDING BEHAVIOR AND THE DESCRIPTION OF THE FLAGELLAR BASE AREA OF A PLANOZYGOTE1

A small, freshwater dinoflagellate with an incomplete cingulum, identified as Esoptrodinium gemma Javornický (=Bernardinium bernardinense sensu auctt. non sensu Chodat), was maintained in mixed culture and examined using light and serial section TEM. Vegetative flagellate cells, large cells with two longitudinal flagella (planozygotes), and cysts were examined. The cells displayed a red eyespot near the base of the longitudinal flagellum, made of two or three layers of pigment globules not bounded by a membrane. Yellow‐green, band‐shaped chloroplasts, bounded by three membranes and containing lamella with three thylakoids, were present in both flagellate cells and cysts. Most cells had food vacuoles, containing phagotrophically ingested chlamydomonads or chlorelloid green algae; ingestion occurred through the ventral area, involving a thin pseudopod apparently driven by the peduncle. The pusule was tubular, with numerous diverticula in its distal portion, and opened into the longitudinal flagellar canal. Three roots were associated with each pair of flagellar bases, both in vegetative cells and in a planozygote. The longitudinal microtubular root bifurcated around the longitudinal basal body. The planozygote contained a single peduncle and associated structures, and a single transverse flagellar canal with the two converging transverse flagella. Using two ciliates as outgroup species, phylogenetic analyses based on maximum parsimony, neighbor‐joining and posterior probability (Bayesian analysis) supported a clade comprising Esoptrodinium, Tovellia, and Jadwigia.     

SEX‐SPECIFIC CELL SURFACE STRUCTURE OF ANISOGAMETES: MORPHOLOGICAL CHANGES DURING FERTILIZATION OF BRYOPSIS MAXIMA (ULVOPHYCEAE,CHLOROPHYTA) REVEALED BY ULTRA–HIGH‐RESOLUTION FIELD EMISSION SEM1

Cell surfaces of biflagellate gametes and their morphological changes during fertilization of Bryopsis maxima Okamura were observed using a high‐resolution field emission scanning electron microscope. Male gametes have broad and narrow faces, which are divided into at least five morphologically distinct regions: 1) the apical plate is a plate‐like structure that is approximately 380–530 nm long and approximately 190 nm wide, in the center of the papilla and slightly protruded from the plasma membrane; 2) strips are smooth materials on ridges that originate from the basal part of the papilla and extend downward; 3) the lateral belt is a belt‐shaped structure on the center of the narrower faces; 4) the flagellar surface; and 5) the other region of the cell body has a fine‐grained appearance. In contrast, the entire female gamete surface is rough because of many granular or amorphous cell coats on the plasma membrane. When both gametes were mixed together, the initial fusion proceeded between the broader face of the male gamete and the anterior side of the female one near the basal bodies. Morphology of the male gamete's cell surface changed gradually as fusion proceeded and was covered by the granular materials; that surface closely resembled those of female gametes except for the apical plate. It was present until the planozygote attached itself to the substrate by the papilla. It finally disappeared after settlement. Therefore, these results indicate that gametes of B. maxima have sex‐specific surface structures that change their morphology during fertilization and settlement.     

ESTUARINE HETEROTROPHIC CRYPTOPERIDINIOPSOIDS (DINOPHYCEAE): LIFE CYCLE AND CULTURE STUDIES1

Cryptoperidiniopsoids are an unclassified group of delicately thecate heterotrophic dinoflagellates known to be common in eastern U.S. estuarine waters. Over the past 10 years cryptoperidiniopsoids were isolated from different geographical regions and cultured with cryptophyte algal prey. In the seven clonal isolates examined, reproduction was strongly linked to the availability of prey cells. The dinoflagellates phagocytized the contents of prey cells through a tube‐like peduncle, similarly as close relatives of Pfiesteria spp. and several other heterotrophic species. Cell division occurred while encysted, most commonly yielding two biflagellated offspring. Abundant fusing gametes, phagotrophic planozygotes, and cysts with a pronounced nuclear cyclosis characterized persistent sexuality. Cysts with nuclear cyclosis produced two flagellated offspring cells. The resistance of reproductive cysts to antimicrobial treatments was examined, and a simple high‐yield technique was developed for population synchronization while ridding the dinoflagellates of most contaminating vacuolar prey DNA and external contaminants. The DNA content and population DNA profiles of synchronously excysted cryptoperidiniopsoids from different isolates were measured using flow cytometry and were related to the life history of these and other dinoflagellates. Cryptophyte‐fed cultures with versus without extracellular bacteria were compared, and bacteria apparently promoted cryptoperidiniopsoid feeding and growth. Externally bacteria‐free dinoflagellates were cultured in media enriched with dissolved organic nutrients, and nutritional benefit may have occurred in some treatments. The potential for mixotrophic nutrition from maintenance of cryptophyte chloroplasts was examined using flow cytometrically sorted cells, but evidence of kleptoplastidy was not found in these isolates under the conditions imposed.     

Sexual and Asexual Processes in Protoperidinium steidingerae Balech (Dinophyceae), with Observations on Life-History Stages of Protoperidinium depressum (Bailey) Balech (Dinophyceae)

ABSTRACT. A suite of morphological, histological, and molecular techniques was used to reveal for the first time division, sexuality, mandatory dormancy period of hypnozygotes, and identity of life-history stages of any Protoperidinium spp. In both Protoperidinium steidingerae and Protoperidinium depressum , asexual division occurred by eleutheroschisis within a temporary cyst, yielding two daughter cells. Daughter cells were initially round and one-half to two-thirds the size of parent cells then rapidly increased in size, forming horns before separating. Gamete production and fusion was constitutive in clonal and non-clonal cultures, indicating that both species may be homothallic. Gametes were isogamous, approximately half the size and lacking the pink pigmentation of the vegetative cells, and were never observed to feed. Gamete fusion resulted in a planozygote with two longitudinal flagella. Planozygotes of P. steidingerae formed hypnozygotes. The fate of planozygotes of P. depressum is unknown. Hypnozygotes of P. steidingerae had a mandatory dormancy period of ca. 70 days. Germination resulted in planomeiocytes with two longitudinal flagella. Nuclear cyclosis occurred in the planozygotes of P. depressum , but in the planomeiocytes of P. steidingerae . The plate tabulation and gross morphology of gametes of P. steidingerae and P. depressum differed markedly from those of vegetative cells. Thus, misidentification of morphologically distinct life-history stages and incomplete examination of thecal plate morphology in field specimens has likely led to taxonomic confusion of Protoperidinium spp. in previous studies.     

THE SEXUAL LIFE CYCLES OF PFIESTERIA PISCICIDA AND CRYPTOPERIDINIOPSOIDS (DINOPHYCEAE)1

Sexual life cycle events in Pfiesteria piscicida and cryptoperidiniopsoid heterotrophic dinoflagellates were determined by following the development of isolated gamete pairs in single‐drop microcultures with cryptophyte prey. Under these conditions, the observed sequence of zygote formation, development, and postzygotic divisions was similar in these dinoflagellates. Fusion of motile gamete pairs each produced a rapidly swimming uninucleate planozygote with two longitudinal flagella. Planozygotes enlarged as they fed repeatedly on cryptophytes. In <12 h in most cases, each planozygote formed a transparent‐walled nonmotile cell (cyst) with a single nucleus. Zygotic cysts did not exhibit dormancy under these conditions. In each taxon, dramatic swirling chromosome movements (nuclear cyclosis) were found in zygote nuclei before division. In P. piscicida, nuclear cyclosis occurred in the zygotic cyst or apparently earlier in the planozygote. In the cryptoperidiniopsoids, nuclear cyclosis occurred inthe zygotic cyst. After nuclear cyclosis, a single cell division occurred in P. piscicida and cryptoperidiniopsoid zygotic cysts, producing two offspring that emerged as biflagellated cells. These two flagellated cells typically swam for hours and fed on cryptophytes before encysting. A single cell division in these cysts produced two biflagellated offspring that also fed before encysting for further reproduction. This sequence of zygote development and postzygotic divisions typically was completed within 24 h and was confirmed in examples from different isolates of each taxon. Some aspects of the P. piscicida sexual life cycle determined here differed from previous reports.