Longitudinal Flagella Research Articles

Evidence for resting cyst production in the cosmopolitan toxic dinoflagellate Karlodinium veneficum and the cyst distribution in the China seas

The naked dinoflagellate Karlodinium veneficum is a cosmopolitan and toxic species that frequently forms harmful algal blooms (HABs) in coastal waters. This species has been intensively studied from multiple aspects including toxicology, toxins, nutrition mode (e.g., mixotrophy, phagotrophy, etc.), blooming dynamics, allelopathy, and behavior, while the mechanisms accounting for its global distribution and possible invasion to new regions have not been investigated. Since the first report of a bloom of this species from the South China Sea in 2003, K. veneficum has been frequently detected in coastal waters of China. While resting cyst has been well documented to play vital roles in the initiation and decline of HABs and in facilitating geographical expansion of HABs species, whether or not K. veneficum forms resting cyst remains an open question. Here, we provide proofs for the resting cyst formation in K. veneficum based on both the observations on the life history of clonal cultures and cyst detections from field sediment. We microscopically observed the mating gametes, gametes in fusion, planozygotes (judged from the two longitudinal flagella and cell morphology such as a larger size), dark brown, thick-walled cysts with smooth surface, and cyst germination. The resting cyst was produced homothallically (i.e. from single clonal culture). We also determined the diploidity of cysts via measuring the copy numbers of the large subunit (LSU) rRNA gene in resting cysts and vegetative cells. The presence of K. veneficum cysts in field sediments was detected via fluorescence in situ hybridization (FISH) using species-specific probes, and further confirmed by single-cell PCR sequencing for the FISH-detected cysts. The distribution and abundance of K. veneficum cysts in the China Seas (Bohai Sea, Yellow Sea, East China Sea, and South China Sea) were mapped using a combined approach of real-time PCR and FISH, and quantified after measuring and taking into account the copy numbers of LSU rRNA gene in vegetative cells and cysts. We found a wide distribution of resting cysts of this organism in the seas of China, but generally with a low abundance in most of the samples (0 to 15 cysts per 32 g of wet sediment for FISH method; 0 to 25 cysts per 32 g of wet sediment for qPCR method). The confirmation of resting cyst production from both the laboratory cultures and field sediments and detection of a wide distribution of cysts in the China coasts in this study provide a possible mechanistic explanation for the frequent recurrences of blooms and the cosmopolitan distribution of K. veneficum. Our work also necessitates both a more intensive investigation on the life history (e.g. germination potential of cysts in the field) and an extensive cyst monitoring in coastal sediments, in order to better understand the general ecology and the bloom dynamics specific to this important species.

Proof of homothally of Pheopolykrikos hartmannii and details of cyst germination process

Resting cysts play crucial roles in the ecology of dinoflagellates, especially in wintering or surviving unfavorable conditions, seeding harmful algal blooms (HABs), and facilitating the geographic expansion. Encystment of dinoflagellates is tightly coupled with sexual reproduction in most cases, which can occur either through homothallism (self-fertilization) or heterothallism (intercrossing of +/− strains). The types of sexual reproduction have important ecological implications. The toxic and HAB-forming dinoflagellate, Pheopolykrikoides hartmannii has been previously reported to be heterothallic. Here, we provide visual confirmation of homothally of P. hartmannii and the first detailed visual recording of cyst germination based on the observations of a clonal isolate from Jiaozhou Bay, China. To document the homothallism, we first observed cell pairs in sexual mating, planozygotes with two longitudinal flagella, and cysts with typical morphology as described previously from the clonal culture. We then germinated a single cyst, established a new clonal culture from one of the two daughter cells after the first cell division of the germling (i.e. from the diploid germling to two haploid cells), and produced cysts again from the newly established clonal culture. For the observation of the germination time-series, we took micrographs and videos to show all germination processes, particularly with an interesting observation of the short amoeboid stage of the germling releasing from the archeopyle (~15 s), which was a landmark of the germination process and has not been reported elsewhere. This definitive evidence of homothallic sexuality and cyst production in P. hartmannii provides a new insight into the biology and ecology of the species, particularly a mechanism that may partly account for the population dynamics and ubiquitous distribution of the species.

Studies on woloszynskioid dinoflagellates VIII: life cycle, resting cyst morphology and phylogeny of Tovellia rinoi sp. nov. (Dinophyceae)

:Tovellia rinoi nov. sp. is described on the basis of light microscopic and scanning electron microscopic examination of motile cells and resting cysts and the phylogenetic analysis of partial sequences of the large subunit ribosomal rRNA gene. The species was isolated from a flooded area near Aveiro, Portugal, and followed in culture through all stages of the life cycle, including formation of vegetative cells and gametes, sexual fusion, planozygote division through division cysts and the production of resting cysts. Motile cells had an ovate-conical epicone and a truncated or slightly excavated hypocone and were a little flattened dorsoventrally. Cell length was mostly 9–19 μm, the smaller cells less pigmented and with the potential to act as gametes but also capable of vegetative multiplication. The amphiesma had three series of vesicles on the epicone, one in the cingulum and two on the hypocone and an apical line of plates (ALP) oriented almost dorsoventrally across the cell apex. One vesicle separated the ALP from the cingulum on the ventral side, two on the dorsal side. The resting cysts, showing an equatorial constriction and densely covered by hair-like spines, represented a new type within the family Tovelliaceae. The new species formed a well-supported sister group to the other Tovellia species in a LSU rDNA–derived phylogeny. Sexual fusion started with gametic contact within ‘dancing groups’ of small cells. The initial contact on the mid-ventral side of both gametes was often visible as a hyaline bridge, which apparently involved a globular structure protruding from the ventral ridge of one or both gametes. The two gametes in a fusing pair behaved differently, with one gamete rotating as it fused into its partner until gametic eyespots merged and the gametic longitudinal flagella assumed the position of the paired longitudinal flagella of the planozygote. Resting cysts developed from large cells morphologically identifiable as planozygotes, with cyst spines growing rapidly. Conversion of planozygotes into division cysts that produced motile cells was also observed. Germinating cysts produced regularly two motile cells, each apparently with a single longitudinal flagellum. Nuclear cyclosis was not detected at any stage of the life cycle.

The life history of the toxic marine dinoflagellate Protoceratium reticulatum (Gonyaulacales) in culture

Asexual and sexual life cycle events were studied in cultures of the toxic marine dinoflagellate Protoceratium reticulatum. Asexual division by desmoschisis was characterized morphologically and changes in DNA content were analyzed by flow cytometry. The results indicated that haploid cells with a C DNA content occurred only during the light period whereas a shift from a C to a 2C DNA content (indicative of S phase) took place only during darkness. The sexual life cycle was documented by examining the mating type as well as the morphology of the sexual stages and nuclei. Gamete fusion resulted in a planozygote with two longitudinal flagella, but longitudinally biflagellated cells arising from planozygote division were also observed, so one of the daughter cells retained two longitudinal flagella while the other daughter cell lacked them. Presumed planozygotes (identified by their longitudinally biflagellated form) followed two life-cycle routes: division and encystment (resting cyst formation). Both the division of longitudinally biflagellated cells and resting cyst formation are morphologically described herein. Resting cyst formation through sexual reproduction was observed in 6.1% of crosses and followed a complex heterothallic pattern. Clonal strains underwent sexuality (homothallism for planozygote formation and division) but without the production of resting cysts. Ornamental processes of resting cysts formed from the cyst wall under an outer balloon-shaped membrane and were fully developed in <1h. Obligatory dormancy period was of ∼4 months. Excystment resulted in a large, rounded, pigmented, longitudinally biflagellated but motionless, thecate germling that divided by desmoschisis. Like the planozygote, the first division of the germling yielded one longitudinally biflagellated daughter cell and another without longitudinal flagella. The longitudinal biflagellation state of both sexual stages and of the first division products of these cells is discussed.

Sexual resting cyst production by the dinoflagellate Akashiwo sanguinea: a potential mechanism contributing to the ubiquitous distribution of a harmful alga.

The dinoflagellate Akashiwo sanguinea is a well known, cosmopolitan harmful microalga that frequently forms harmful algal blooms (HABs) in marine estuaries from temperate to tropical waters, and has posed a severe threat to fish, shellfish, and sea birds. Therefore, it is important to understand the ecology of this species, particularly the mechanisms regulating its ubiquitous geographic distribution and frequent recurrence of. To date, the mechanisms regulating distribution and recurrence of HABs by this species have been poorly understood. While resting cyst production can play a central role in the geographic expansion and initiation of HABs, studies of the life cycle of this alga, including cyst production, have been lacking. Here, we demonstrate that A.sanguinea produces sexual resting cysts homothallically. We present evidence for cell pairs in sexual mating, biflagellated planozygote formation, and cysts of different morphologies, and we describe time series for germination of cysts to germlings with two longitudinal flagella, along with studies of possible factors affecting cyst production. Phylogenetic analysis of large sub-unit rDNA sequences revealed a monophyly of this species and thus possibly a recent common ancestor for all global populations. The discovery of resting cyst production by A.sanguinea suggests its frequent recurrence of blooms and global distribution may have been facilitated by the natural and anthropogenic transport of resting cysts.

The toxic dinoflagellate Cochlodinium polykrikoides (Dinophyceae) produces resting cysts

While harmful algal blooms (HABs) caused by the toxic dinoflagellate Cochlodinium polykrikoides have been known to science for more than a century, the past two decades have witnessed an extraordinary expansion of these events across Asia, North America, and even Europe. Although the production of resting cysts and subsequent transport via ships’ ballast water or/and the transfer of shellfish stocks could facilitate this expansion, confirmative evidence for cyst production by C. polykrikoides is not available. Here, we provide visual confirmation of the production of resting cysts by C. polykrikoides in laboratory cultures isolated from North America. Evidence includes sexually mating cell pairs, planozygotes with two longitudinal flagella, formation of both pellicular (temporary) cysts and resting cysts, and a time series of the cyst germination process. Resting cyst germination occurred up to 1 month after cyst formation and 2–40% of resting cysts were successfully germinated in cultures maintained at 18–21°C. Pellicular cysts with hyaline membranes were generally larger than resting cysts, displayed discernable cingulum and/or sulcus, and reverted to vegetative cells within 24h to ∼1 week of formation. A putative armored stage of C. polykrikoides was not observed during any life cycle stage in this study. This definitive evidence of resting cyst production by C. polykrikoides provides a mechanism to account for the recurrence of annual blooms in given locales as well as the global expansion of C. polykrikoides blooms during the past two decades.

Encystment and excystment under laboratory conditions of the nontoxic dinoflagellate Alexandrium fraterculus (Dinophyceae) isolated from the Seto Inland Sea, Japan

S. Nagai, G. Nishitani, Y. Takano, M. Yoshida and H. Takayama. 2009. Encystment and excystment under laboratory conditions of the nontoxic dinoflagellate Alexandrium fraterculus (Dinophyceae) isolated from the Seto Inland Sea, Japan. Phycologia 48: 177–185. DOI: 10.2216/08-43.1.The sexuality (i.e. encystment and excystment) of the nontoxic dinoflagellate Alexandrium fraterculus, isolated from the Seto Inland Sea, Japan, was clarified for the first time under laboratory conditions. Sexual reproduction was by conjugation of isogametes, and plasmogamy was completed in 25–60 min after the conjugation started and produced a planozygote with one transverse and two longitudinal flagella, then a cyst was formed. Cysts were round or elliptical. The cyst size was 37.5–50.0 µm in diameter. The surface of cysts was smooth, and there was no paratabulation. Encystment through sexual reproduction was observed in two of 28 pairs, which included seven self-crossings, by use of seven nonaxenic clonal strains. No planozygote formation or encystment was found in any of the self-crossings, indicating that this species is heterothallic. Successful excystment was observed when the cysts, which had been preserved at 10°C in the dark for 3 mo, were incubated under light conditions at 25°C, thus showing that this species has a similar life cycle as reported in A. tamarense/catenella/tamiyavanichii.

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

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.

MECHANISM OF GAMETE FUSION IN DINOPHYSIS FORTII (DINOPHYCEAE, DINOPHYTA): LIGHT MICROSCOPIC AND ULTRASTRUCTURAL OBSERVATIONS1

A variety of studies have examined the sexual life cycle of species belonging to the genus Dinophysis Ehrenberg. Here, we used TEM to investigate the mechanism of cellular fusion during the sexual life cycle in Dinophysis fortii Pavillard. We observed that fusion always occurred between a normal‐sized cell and a small cell following attachment of their ventral margins. After cell attachment, the small cell moved toward the epitheca of the normal‐sized cell, and the cingular and sulcal lists of the small cell shrunk or were almost lost. The epitheca of the normal‐sized cell then opened between the cingulum plates and the upper cingular list, after which the small cell was gradually engulfed. This is the first ultrastructural observation in a dinoflagellate of a larger cell opening its epitheca to engulf the smaller gamete. In another case, the normal‐sized cell did not open the epitheca, the cell wall of the attached small cell underwent extensive extracellular digestion, and the cytoplasm appeared to flow into the normal‐sized cell via the periflagellar area. Inflow of the nucleus was not observed in this case, suggesting that it represented a failure of sexual fusion. In both cases, membranous separations between the cytoplasm of the two cells were not observed. At the beginning of the fusion process, the nucleus of the small cell was substantially deformed. The planozygote, formed upon completion of sexual fusion, sometimes had two longitudinal flagella, but was identical to a normal vegetative cell in its cellular shape, as already mentioned by previous authors.

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.

Observations of asexual and sexual processes in Protoperidinium depressum (Dinophyceae)

Life cycle stages of the heterotrophic dinoflagellate Protoperidinium depressum were determined for a culture isolated from the west coast of Ireland. Division, sexuality, DNA content, gamete morphology, and identity of morphologically distinct life‐history stages were revealed for the first time using a suite of morphological and molecular tools. Asexual division occurred by eleutheroschisis within a temporary, immotile cyst, yielding two daughter cells. Morphological features, including apical and antapical horns, were not fully formed upon emergence from the division cyst, but developed some time after cell division. Daughter cells were half to two‐thirds the size of parent cells and swam attached in an epitheca‐to‐hypotheca orientation before separating. Stages indicating a completed life cycle, including gametes, planozygotes, and nuclear cyclosis were observed. Small cells, believed to be gametes, formed in repeated clonal and non‐clonal re‐isolations of the culture. In addition to their small size (40–50 μm), blunt apical horn, small antapical horns, and lack of pink pigmentation, these putative gametes had a thecal plate morphology different from that described for P. depressum. The LSU rDNA sequence (D1–D3 region) of small cells was, however, the same as that of P. depressum. Gametes were never witnessed to feed, but did form fusing, isogamous pairs. When isolated to well plates, gamete pairs failed to complete fusion to form a planozygote. Thus, the morphology of the initial planozygote remains uncertain. Large cells believed to be planozygotes had “typical”P. depressum morphology, but possessed two longitudinal flagella and had 2C DNA based on DAPI. Nuclear cyclosis was documented in one such tri‐flagellated, motile cell. The cell remained motile after the completion of nuclear cyclosis, but did not progress to the next life stage. Dormant hypnozygotes were not observed.

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 &lt;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.

Encystment under laboratory conditions of the toxic dinoflagellate Alexandrium tamiyavanichii (Dinophyceae) isolated from the Seto Inland Sea, Japan

The encystment of the toxic dinoflagellate Alexandrium tamiyavanichii, isolated from the Seto Inland Sea, Japan, was clarified for the first time under laboratory conditions. Sexual reproduction was by conjugation of isogametes, and plasmogamy was completed 60–80 min after conjugation started, producing a planozygote with one transverse and two longitudinal flagella, and forming a cyst. Cysts were vertically compressed or spherical. Cysts were 45–75 μm long, 35–60 μm wide and 40–60 μm high. The surface of cysts was smooth, and there was no paratabulation. Encystment through sexual reproduction was observed in 54 pairs out of 136, which included 16 self-crossing by use of 16 nonaxenic clonal strains. No planozygote formation or encystment was found with any self-crossing, indicating the heterothallism of this species.

REPRODUCTION AND SEXUALITY IN PFIESTERIA SHUMWAYAE (DINOPHYCEAE)1

Pfiesteria shumwayae is a heterotrophic dinoflagellate with a widespread distribution in temperate‐subtropical estuarine waters. In this study, five clonal isolates from the eastern coast of North America, one from New Zealand, and a mixed composite of clones were cultured in aquaria and fed live fish. Division, sexuality, and phagotrophic feeding on fish were studied by LM, SEM, and flow cytometry. The development of reproductive cysts isolated from aquaria was followed. Synchronously excysted flagellate populations were examined for sexuality and then for feeding behavior and reproduction when given larval fish. Reproductive cysts varied in size and underwent protoplast division(s), most commonly producing two to eight biflagellated offspring. Fusing gametes, resulting planozygotes, and nuclear cyclosis were documented as evidence of sexuality. Gametes emerged from cysts, and fusions were approximately isogamous. Resulting planozygotes had two longitudinal flagella and one transverse flagellum and apparently fed before encysting. Distinct and lengthy chromosome movements (nuclear cyclosis) occurred in presumed zygotic cysts before nuclear division(s). These cysts did not exhibit dormancy in growing cultures and produced two or four biflagellated offspring. Flagellated cells fed on surficial fish tissues and then encysted for reproduction. Stages indicating a completed sexual cycle (fusion, planozygotes, and nuclear cyclosis) were uncommon or absent in clonal cultures but were relatively abundant in the mixed clone culture. Self‐sterility factors apparently influenced sexuality. Starved populations formed quiescent cysts that released swimming cells when food was provided. Pfiesteria shumwayae was similar in reproduction and sexuality to closely related species.

Vegetative and sexual reproduction in Pfiesteria spp. (Dinophyceae) cultured with algal prey, and inferences for their classification

Algal-fed clonal zoospore cultures of Pfiesteria piscicida and Pfiesteria shumwayae enabled description of certain conserved morphological and reproductive features. Common modes of reproduction (especially via division cysts) were documented in herbivorous P. piscicida and P. shumwayae using cultures fed algal prey, together with supporting photography and flow cytometric DNA measurements. Other cysts were characterized such as vacuolate cysts in starved P. piscicida cultures and temporary cysts in both species fed algal prey. This study also represents the first report of sexual reproduction in Pfiesteria spp. cultures fed algal prey rather than live fish; the first report of a technique for cell cycle synchronization for these heterotrophic dinoflagellates; and the first information on storage products of cells released from Pfiesteria reproductive cysts. Sexual reproduction in algal-fed P. piscicida clonal cultures was evidenced by fusing gametes, cells with two longitudinal flagella, and nuclear cyclosis. Both isogamous and anisogamous fusions were observed, and resulting cells with two trailing flagella (i.e., planozygotes and planomeiocytes) sometimes comprised ≥50% of the flagellated cells. These cells continued feeding activity and eventually (hours) lost their flagella and formed cysts. Nuclear cyclosis and a subsequent cell division were observed in thin-walled reproductive cysts prior to release of two flagellated cells. One gamete fusion event was also documented in 1 of 20 algal-fed clones of P. shumwayae, with an aplanozygote as the product. We obtained high cell synchrony (≥90% 1C) in the tested cultures using our preferential lysis technique and tracked the decline in lipid content of excysted zoospore populations over time. The data from this study were considered together with previous research to gain insights about relationships between Pfiesteria spp. and other heterotrophic dinoflagellates. Pfiesteria spp. should be regarded as free-living predators rather than parasites because they are prey generalists without demonstrated “host” specificity and their flagellated feeding stages are not morphologically distinct from swimming stages. Although they originally were placed within the Dinamoebales because amoebae can predominate, this study as well as other published research consistently has shown that the dominant stage varies depending on culture conditions, prey type/availability and strains. The peridinoid plate structure of each Pfiesteria species, which thus far has been conserved across culture conditions and strains, supports placement of Pfiesteria spp. within the Peridiniales. At the species level, plate structure (differing by one precingular plate) and molecular data (18S rDNA) indicate that the two Pfiesteria spp. are closely related in comparison to species grouped within other genera.

Dynamic changes in microtubule organization during division of the primitive dinoflagellate Oxyrrhis marina

The marine dinoflagellate Oxyrrhis marina has three major microtubular systems: the flagellar apparatus made of one transverse and one longitudinal flagella and their appendages, cortical microtubules, and intranuclear microtubules. We investigated the dynamic changes of these microtubular systems during cell division by transmission and scanning electron microscopy, and confocal fluorescent laser microscopy. During prophase, basal bodies, both flagella and their appendages were duplicated. In the round nucleus situated in the cell centre, intranuclear microtubules appeared radiating toward the centre of the nucleus from densities located in some nuclear pores. During metaphase, both daughter flagellar apparatus separated and moved apart along the main cell axis. Microtubules of ventral cortex were also duplicated and moved with the flagellar apparatus. The nucleus flattened in the longitudinal direction and became discoid-shaped close to the equatorial plane. Many bundles of microtubules ran parallel to the short axis of the nucleus (cell long axis), between which chromosomes were arranged in the same direction. During ana–telophase, the nucleus elongated along the longitudinal axis and took a dumbbell shape. At this stage a contractile ring containing actin was clearly observed in the equatorial cortex. The cortical microtubule network seemed to be cut into two halves at the position of the actin bundle. Shortly after, the nucleus divided into two nuclei, then the cell body was constricted at its equator and divided into one anterior and one posterior halves which were soon rebuilt to produce two cells with two full sets of cortical microtubules. From our observations, several mechanisms for the duplication of the microtubule networks during mitosis in O. marina are discussed.

Life History of a Marine Dinoflagellate Pyrophacus steinii (Schiller) Wall et Dale

The life history of a marine dinoflagellate Pyrophacus steinii (Schiller)Wall et Dale was investigated using clonal cultures isolated from Tokyo Bay. The asexual reproduction is binary fission of the eleutheroschisis type. All processes of asexual and sexual cell division occur inside the theca of the mother cells. Sexual reproduction is anisogamous and heterothallic.Male gametes differ from female gametes and vegetative cells in size, shape and plate tabulation. The female gametes can not be differentiated from the vegetative cells. Cell fusion between the male and female gametes occurs within a few hours to several days after inoculation of the male gametes into a culture of a non-male clone. Zygotes are similar to the vegetative cells in shape except possessing two longitudinal flagella. One to two days after plasmogamy, the zygotes become non-motile, and their protoplasts contract. Within 1 to 3 days, following the condensation of protoplast, they transform to hypnozygotes (resting cysts). The specific features of the life cycle can be summerized as: i) the asexual reproduction is eleutheroschisis, ii) sexual fusion occurs after inoculation of the small round thecate cells, which are equivalent to male gametes, into a culture of a non-male clone, iii) male gametes are distinctive from vegetative cells in their smaller size, round shape, fewer plate number and pale chloroplasts, iv) female gametes can not be differentiated from the vegetative cells, v) the sexual life cycle is anisogamous and heterothallic.

The life cycle of Gyrodinium instriatum (Dinophyceae) in culture*

SUMMARYThe life cycle events of an unarmored dinoflagellate Gyrodinium instriatum Freudenthal et Lee has been investigated using clonal cultures. After the inoculation of vegetative cells into fresh medium, clumping of gametes was observed after a period of 10 days. In the clumps, a number of gametes were observed to be swimming in close contact with each other, pairing successively and then forming a plano‐zygote. This clumping behavior is considered to be useful in the performance of sexual reproduction, particularly in the event of low cell density, because it increases the chance of fusion. Gametes of this species were most often isogarnous, although apparent amsogamous fusions were occasionally observed. When planozygotes were isolated and placed in fresh medium, they enlarged their size and finally divided into two cells. The daughter cells continued to multiply by binary fission and produced vegetative cells. Thus, G. instriatum has an alternative cycle between vegetative cells and zygotes without a hypnozygote stage. However, cysts of this species were transformed from large motile cells (pre‐cyst cells) which are oblong and dorso‐ventrally flattened in shape. These mottle pre‐cyst cells have two longitudinal flagella, which may indicate that cysts of this species are of zygote origin. On the basis of these results, the relationship between zygotes and pre‐cysts of G. instriatum is discussed. Excystment was enhanced by dark and cold treatments prior to the incubation for germination experiments with a germination success rate of 26–64%. Encystment was greatly inhibited by the lack of dark and cold treatments.

Quantified small-scale turbulence inhibits a red tide dinoflagellate, Gonyaulax polyedra Stein

The development of marine dinoflagellate red tides off southern California requires optimal temperature and light regimes, a source of nutrients that may be supplied by wind-induced upwelling and upmixing, and vertical migration by cells to this source. Red tides occur after the winds decrease and the water becomes highly stratified with a shallow mixed layer. This implies low turbulence levels may be an additional requirement for red tide development. Because dinoflagellates with sizes about 35 μm are much smaller than the inertial-viscous, or Kolmogorov scales L κ ≡ (ν 3/ϵ) 1 4 = (ν/γ) 1 2 of oceanic turbulence, the important flow parameters are the viscous dissipation rate per unit mass ϵ (cm 2s −3 or ergs g −1s −1), the rate-of-strain γ ≡ (ϵ/ν) 1 2 ( rad s −1) , and the stress τ ≡ μγ (dyne cm −2), where ν is the kinematic viscosity and μ is the dynamic viscosity. In the present work we have cultured the red tide dinoflagellate, Gonyaulax polyedra Stein, under conditions of known ϵ, γ and τ. Growth was inhibited at ϵ values from 0.18 to 164 cm 2 s −3 (γ from 4.4 to 132 rad s −1) but not at 0.05 ( γ = 2.2) so the threshold stress τ for growth inhibition was 0.02–0.04 dyne cm −2 (0.002–0.004 Pa). This is in the expected range for light winds at the sea surface, suggesting small-scale scale turbulence is the reason higher winds inhibit red tides. Threshold turbulence levels are related to calculated surface levels at various wind speeds and to postulated subsurface euphotic zone levels under incipient red tide conditions. Motile cells in shear-inhibited cultures lost their ability to swim forward vigorously but rather spun in place due to the loss of longitudinal (trailing) flagella, observed microscopically, without loss of girdle flagella.

Effects of small-scale turbulence on microalgae

Turbulence flows are characterized by their viscous dissipation rates ɛ and the kinematic viscosity of the fluid ν, but the effects of turbulence on organisms such as microalgae smaller than the Kolmogorov inertial-viscous length scale LK ≡ (ν3/e)/14 depend on the stress τ ≡ µγ, where µ = ϱν is the dynamic viscosity, ρ is the density, and the rate-of-strain γ ≡ (e/ν)/12. While various workers have shown qualitatively that turbulence affects several microalgal physiological processes, these effects have not been quantified in terms of e, τ or γ. Various microalgal groups seem to have different sensitivities to inhibition by turbulence. The relative sensitivities aregreen algae 0.18 cm2s−3, τ > 0.04 dynes cm−2 (0.002 N M−2 or Pa), γ > 4.4 rad s−1, cell numbers and chlorophyll fluorescence declined, and cells lost their longitudinal flagella and the ability to swim forward. At lower e, τ and γ values growth rates and cell morphology were the same as in unsheared control cultures. High turbulence may affect other algae, such asSpirulina, which are commonly mass cultured.