Effects of temperature and light on cyst germination and germinated cell survival of the noxious raphidophyte Heterosigma akashiwo

The effects of temperature and light on the germination of Heterosigma akashiwo cysts were examined using bottom sediments collected from Hakata Bay, Japan. In a suspension of mixed sediment and seawater in the temperature range of 5–30 °C, motile cells emerged within 3 weeks, but at ≤12 °C the cell numbers were markedly lower and the emergence of motile cells delayed. When suspension samples incubated at various temperatures were moved to 20 °C and incubated, only a few additional motile cells emerged. The number of motile cells germinated in the dark was significantly lower than under light conditions. When suspension samples incubated in the dark were exposed to light, only a few additional motile cells emerged. These results indicate that the initiation of germination in Heterosigma cysts suspended in seawater is not dependent on temperature and light conditions, although the speed of the germination process is affected by temperature, and cell survival just after germination is strongly affected by temperature and light.     

Astaxanthin biosynthesis enhanced by reactive oxygen species in the green algaHaematococcus pluvialis

The unicellular green algaHaematococcus pluvialis has recently attracted great interest due to its large amounts of ketocarotenoid astaxanthin, 3,3′-dihydroxy-β,β-carotene-4,4′-dione, widely used commercially as a source of pigment for aquaculture. In the life cycle ofH. pluvialis, astaxanthin biosynthesis is associated with a remarkable morphological change from green motile vegetative cells into red immotile cyst cells as the resting stage. In recent years we have studied this morphological process from two aspects: defining conditions governing astaxanthin biosynthesis and questioning the possible function of astaxanthin in protecting algal cells against environmental stress. Astaxanthin accumulation in cysts was induced by a variety of environmental conditions of oxidative stress caused by reactive oxygen species, intense light, drought, high salinity, and high temperature. In the adaptation to stress, abscisic acid induced by reactive oxygen species, would function as a hormone in algal morphogenesis from vegetative to cyst cells. Furthermore, measurements of bothin vitro andin vivo antioxidative activities of astaxanthin clearly demonstrated that tolerance to excessive reactive oxygen species is greater in astaxanthin-rich cysts than in astaxanthin-poor cysts or astaxanthin-less vegetative genesis and carotenogenesis, and the accumulated astaxanthin in cysts can function as a protective agent against oxidative stress damage. In this study, the physiological roles of astaxanthin in stress response and cell protection are reviewed.     

Application of real-time PCR assay for detection and quantification of Alexandrium tamarense and Alexandrium catenella cysts from marine sediments

The dinoflagellates Alexandrium tamarense (Lebor) Balech and Alexandrium catenella (Whedon and Kofoid) Balech (Dinophyceae) are believed to be the main species responsible for paralytic shellfish poisoning (PSP) all over the world. It is necessary to identify A. tamarense and A. catenella cysts and to monitor their distribution in sediment in order to minimize the damages caused by PSP to the economy and food quality because cysts are the seed population for blooms caused by motile vegetative cells. In this study, we developed an efficient DNA extraction method from the natural cysts present in marine sediments after they were size fractionated with a plankton net (mesh size of 20–150 μm). The 10–3000 cysts were added to the sediments collected from the Ariake Sea, and for which the primuline-staining method did not reveal any cysts. DNA was then extracted from each sample, and linear standard curves for A. tamarense and A. catenella cysts were obtained from the correlation between the Ct values by real-time PCR and the log of the initial densities of cysts. We monitored the A. tamarense and A. catenella cyst densities in the environmental samples. This assay was demonstrated to be a powerful tool for the identification, detection, and quantification of the cysts of the toxic dinoflagellates.     

Studies on woloszynskioid dinoflagellates VI: description of Tovellia aveirensis sp. nov. (Dinophyceae), a new species of Tovelliaceae with spiny cysts

A new species of Tovellia, T. aveirensis, is described on the basis of light (LM) and scanning electron microscopy (SEM) of motile cells and resting cysts, complemented with transmission electron microscopy (TEM) of flagellate cells and phylogenetic analysis of partial sequences of the large subunit ribosomal rRNA gene. Both vegetative cells and several stages of a life cycle involving sexual reproduction and the production of resting cysts were examined in cultures established from a tank in the University of Aveiro campus. Vegetative cells were round and little compressed dorsoventrally; planozygotes were longer and had a proportionally larger epicone. Chloroplast lobes were shown by TEM to radiate from a central, branched pyrenoid, although this was difficult to ascertain in LM. The amphiesma of flagellate cells had mainly 5 or 6-sided vesicles with thin plates, arranged in 5–7 latitudinal series on the epicone, 3–5 on the hypocone. The cingulum had 2 rows of plates, the posterior row extending into the hypocone and crossed by a series of small projecting knobs along the lower edge of the cingulum. A line of narrow amphiesmal plates extended over the cell apex, from near the cingulum on the ventral side to the middle of the dorsal side of the epicone. Eight or 9 narrow amphiesmal plates lined each side of this apical line of plates (ALP). Resting cysts differed from any described before in having numerous long, tapering spines with branched tips distributed over most of the surface. Most mature cysts showed an equatorial constriction. Neither cysts nor motile cells were seen to accumulate red cytoplasmic bodies in any stage of the cultures. The phylogenetic analysis placed, with high statistical support, the new species within the genus Tovellia; it formed a clade, with moderate support, with T. sanguinea, a species notable for its reddening cells.     

The significance of sexual versus asexual cyst formation in the life cycle of the noxious dinoflagellate Alexandrium peruvianum

Alexandrium peruvianum (Balech et Mendiola) is a noxious phototrophic marine dinoflagellate. During the life cycle of this species, two kinds of cysts are produced: resting cysts, which are long-lasting and double-walled, and temporary cysts, which are short-lasting and thin-walled. In addition, short-lasting, but resting-like cysts can also be formed. Although it is crucial to identify sexual events in a dinoflagellate population, sexual and asexual cysts are morphologically very similar in this species. Therefore, we studied the complete life cycle and the nature of the cyst-like stages formed after individual isolation of specimens and crossing of clonal cultures established from germination of wild resting cysts. Asexual division in A. peruvianum takes place either in the motile stage by sharing of the theca (desmoschisis), or inside a vegetative cyst (temporary cyst), from which two, or at times four or six naked daughter cells can originate. The daughter cells completely synthesize new cell walls (eleutheroschisis). Sexuality was confirmed by the presence of fusing gamete pairs and longitudinally biflagellated planozygotes after out-crossing of compatible clonal strains. However, the clonal cultures had low levels of self-compatibility, since a flow cytometry analysis showed that synchronized self-crosses produced few zygotes (<5%). After isolation of individual cells, it was proved that the fate of the planozygotes depended on the nutritional status of the isolation media. Most of the planozygotes isolated to replete medium (L1) divided, whereas in medium lacking nitrates (L-N) or phosphates (L-P) they formed temporary, thin-walled cysts. Temporary cysts formed in L1 were always uninucleated and gave rise to one cell, while those formed in L-N or L-P produced 1–6 small cells. In addition, resting cysts were formed in culture, but never after individual planozygote isolation. Resting cysts were uninucleated and needed maturation time before entering dormancy. The resting cysts were considered sexual products, since longitudinally biflagellate germlings were liberated after germination in all cases studied. Mature resting cysts (52.3 ± 3.0 μm) had a dormancy period of 1–3 months, whereas temporary asexual cysts (32.5 ± 5.4 μm) germinated in less than 7 days.     

Studies on woloszynskioid dinoflagellates IX: ultrastructure,cyst formation and phylogeny of the ‘red-snow’ alga Borghiella pascheri (Suchlandt) Moestrup (= Glenodinium pascheri,Woloszynskia pascheri,Gyrodinium nivalis)

Red snow caused by dinoflagellates is a phenomenon rarely reported, described from the European Alps from 1914 onwards, and subsequently observed outside Europe on several occasions in Ontario, Canada. Considerable taxonomic confusion exists regarding the identity of the organism(s) causing red snow, but the most recent occurrence in 2016 in Ontario has now allowed detailed studies, including LM, SEM, TEM and molecular sequencing of the causative species. We conclude that the two species originally described as the cause of red snow, Glenodinium pascheri and Gyrodinium nivale, are synonymous and that the appropriate name for the organism is Borghiella pascheri (syn. Woloszynskia pascheri) as suggested by Moestrup & Calado in the recent volume of the Süsswasserflora. The central part of Borghiella pascheri cells is tomato red and filled with numerous organelles, whose ultrastructure indicates modified chloroplasts. Lack of cultures has prevented chemical characterization of the red pigment. Formation of temporary cysts was common in the samples. Transformation of the motile cells into temporary cysts was followed in detail, and the cysts were shown to be surrounded by the fused inner membranes of the amphiesmal vesicles, which became the cell membrane of the cysts, covered by the fused pellicle precursors. The cell membrane from the motile cell was discarded together with the outer amphiesmal vesicle membrane and the thin thecal plates, and the temporary cysts were therefore not surrounded by any pattern of vesicles. Sexual reproduction resulted in the formation of hypnozygotes. Although the species possessed several unusual features, DNA sequencing showed it to belong to Borghiella. The culture established in 1965 from the Botanical Garden in Göttingen, Germany and generally identified as Woloszynskia pascheri belongs to a separate species of Borghiella, to be described separately.The occurrence of red snow caused by dinoflagellates is discussed.     

Scrippsiella hexapraecingula sp. nov. (Dinophyceae), a tida pool dinoflagellate from the Northwest Pacific

Specimens of dinoflagellate collected in tide pools along the Pacific coast of central and southern Japan are described as a new species,Scrippsiella hexapraecingula Horiguchi et Chihara, of the Peridiniaceae (Class Dinophyceae). The plate formula is pp, x, 4′, 3a, 6″, 6c, 5‴, 2″" and, 5s, the same as that of other species ofScrippsiella, except in lacking one precingular plate. The genus must be emended, therefore, as having either six or seven precingular plates. This dinoflagellate migrates diurnally. In the morning motile cells are released from non-motile cells attached to the substrate and in the evening the motile cells swim down to settle on the bottom of the tide pool. Attached non-motile cells form either motile mono- or bispores. Sexual reproduction was not observed.     

Atkinsiella dubia and its related species

Atkinsiella dubia, isolated from the mantle of abalone (Haliotis sieboldii), is described and illustrated as a new record from Japan. The fungus was also obtained from the gills of swimming crab (Portunus trituberculatus). Six other species of the genusAtkinsiella have hitherto been reported from various aquatic animals. The fungus is distinguished from the other six species by the morphology of its mycelia and the process of zoospore production. The most distinctive feature is that zoospores in the first motile stage ofA. dubia encyst in zoosporangia, unlike the other species. We therefore proposeHalocrusticida gen. nov. (Lagenidiales, Haliphthoraceae) for the other six species ofAtkinsiella. A key to species of the genusHalocrusticida is provided.     

Preliminary observations of cyst-theca relationships in Spiniferites ramosus and Spiniferites membranaceus (Dinophyceae)

Motile thecal cells derived from the hatching of single cysts identified as Spiniferites membranaceus and S. ramosus have been used to establish cultures. These cultures were examined in order to assess the cyst-theca relationships of these two taxa. The cultures produced two different motile Gonyaulax species belonging to Kofoid's Spinifera group. These cultures were then induced to form a new cyst generation under uniform conditions, and examination of large numbers of the resulting cysts has shown that process development is an extremely variable phenomenon although process morphologies display a continuum within a species. Process length (and to a certain degree, process morphology) requires careful interpretation when used to discriminate Spiniferites taxa, in both modern and ancient environments.     

Life history stages of Pyramimonas tychotreta (Prasinophyceae, Chlorophyta), a marine flagellate from the Ross Sea, Antarctica

During a summer cruise to the Ross Sea (Antarctica) areas of snow‐covered sea ice were red‐coloured due to high concentrations of the recently described Pyramimonas tychotreta Daugbjerg. Light microscopy of living material revealed that the population was comprised of quadriflagellate motile cells and thick‐walled cysts. The red colour was due to large numbers of secondary carotenoid‐containing granules, positioned in the periphery of motile cells and cysts. Mature cysts also contained numerous starch grains and lipid droplets. Cells from a red‐coloured field sample turned green overnight as the secondary carotenoids disappeared when cells were placed in low light conditions. The sample then exhibited the typical grass‐green colour of motile cells observed in water samples from the area. Under reduced light motile cells showed strong positive phototaxis. The encystment process involved the asexual transformation of quadriflagellate cells into cysts. A single type of square cyst scale, with perforated floors and walls, replaced the body scales of motile cells. A marked extension, often ending in a hook was at each corner of the cyst scales. Germinating cysts produced four motile cells. Electron microscopy showed the cyst wall to be tri‐layered, with a thin, electron‐dense inner layer, a thick middle layer and a thin outer layer. Sea ice samples with dense populations of motile cells and cyst stages also contained elongate uniflagel‐late cells. These cells were covered with box scales, foot‐print scales, an underlayer of pentagonal scales, limuloid scales and flagellar hair scales identical to those present on the quadriflagellate stage. We tentatively suggest that the uniflagellate stage represents a gamete and its presence implies the occurrence of sexual reproduction. Although, fusion of gametes was not observed, a biflagellate cell with a larger volume was seen which may have been a zygote. How this stage fits into of the life history remains to be explained.     

Relationship between the dinoflagellate cyst Spiniferites pachydermus and Gonyaulax ellegaardiae sp. nov. from Izmir Bay,Turkey, and molecular characterization

Here, we established the cyst‐motile stage relation‐ship for Spiniferites pachydermus through incubation of cysts with a characteristically microreticulate/perforate surface isolated from Izmir Bay in the eastern Aegean Sea of the eastern Mediterranean. The morphology of the motile stage was similar to Gonyaulax spinifera but had a different size, overhang, displacement and reticulations. Based on the distinct morphology of the cyst and morphological differences in motile cells, we assigned S. pachydermus from Izmir Bay to the new species Gonyaulax ellegaardiae. We elucidate the phylogenetic relationship of G. ellegaardiae through large and small subunit ribosomal DNA and show that it forms a clade with other species that belong to the G. spinifera complex.     

Laboratory culture and taxonomy ofHymenomonas coronata andOchrosphaera verrucosa (Class Prymnesiophyceae) from the Northwest Pacific

Hymenomonas coronata andOchrosphaera verrucosa, both members of the coccolithophorids, Class Prymnesiophyceae, have been studied by means of electron microscopy and with the aid of laboratory culture. Living specimens of these two species were collected in temperate and subtropical regions of Japan, including the Kii Peninsula and the Ryukyu Islands, and unialgal cultures were established in the laboratory. Their life histories are fundamentally identical, and consist of a non-motile vegetative stage that produces motile cells. The vegetative stage is either unicellular, or a packet consisting of a few cells. Both the non-motile cells and the motile cells are covered with two kinds of scales: these are thin scales of unmineralized nature and coccoliths. These two species differ from each other in the shape of the coccoliths and in the presence or absence of visible rudimentary haptonema, and they have been in separate families. The present study reveals that both species are fundamentally identical in the structure and the distribution of major organelles, especially with respect to two opposed pyrenoids which bulge from chloroplasts, each being traversed by two thylakoid bands, and a group of microtubules forming a flagellar root. On the basis of these characteristics, it would appear more logical to place these two species in the same family, namely the Hymenomonadaceae.     

CYST–THECA RELATIONSHIP,LIFE CYCLE,AND EFFECTS OF TEMPERATURE AND SALINITY ON THE CYST MORPHOLOGY OF GONYAULAX BALTICA SP. NOV. (DINOPHYCEAE) FROM THE BALTIC SEA AREA1

A new species of Gonyaulax, here named Gonyaulax baltica sp. nov., has been isolated from sediment samples from the southeastern Baltic. Culture strains were established from individually isolated cysts, and cyst formation was induced in a nitrogen‐depleted medium. Although G. baltica cysts are similar to some forms attributed to Spiniferites bulloideus and the motile stage of G. baltica has affinities with G. spinifera, the combination of features of cyst and motile stage of G. baltica is unique. The culture strains were able to grow at salinity levels from 5 to 55 psu and formed cysts from 10 to 50 psu. Cultures at each salinity level were grown at 12, 16, and 20° C. Temperature‐ and salinity‐controlled morphological variability was found in the resting cysts. Central body size varied with temperature and salinity, and process length varied with salinity. Cysts that formed at extreme salinity levels displayed lower average process length than cysts formed at intermediate salinity levels, and central body length and width were lowest at higher temperature and lower salinity. Models for the relationship between central body size and temperature/salinity and process length and salinity have been developed and may be used to determine relative paleosalinity and paleotemperature levels. Our results on salinity‐dependent process length confirm earlier reports on short‐spined cysts of this species found in low salinity environments, and the model makes it possible to attempt to quantify past salinity levels.     

The effect of penicillin on the encystment of Bdellovibrio sp. strain W

When penicillin, and other inhibitors of peptidoglycan synthesis were added to encysting cultures of Bdellovibrio strain W, the encysting process continued, resulting in the production of cysts which were spherical in shape. Transmission electron micrographs of these spherical bdellocysts revealed the absence of an outer cyst wall. These cysts, devoid of cyst wall, were capable of germination under appropriate condition with the emergence from the prey ghost of highly motile spheroplasts. Withdrawl of the antibiotics after encystment had begun led to the production of spherical cysts that were surrounded by an outer cyst wall.     

Ravenelia cenostigmatis, a new rust fungus from Brazil

Ravenelia cenostigmatis is described as a new species of plant rust (Uredinales) onCenostigma gardnerianum (Caesalpiniaceae) from Brazilian Cerrado. Teliospore heads ofR. cenostigmatis exhibit an unusually complex structure with four different kinds of cells: teliospore cells, sterile apical cells (intercalary between pedicel hyphae and teliospores), sterile hygroscopic cysts, and morphologically distinct border cells. The last are flattened, elaborately ornamented and probably sterile cells that are wedged between the teliospores and the cysts.Ravenelia cenostigmatis probably belongs to a natural assemblage of ravenelias that occur on caesalpiniaceous hosts and exhibit similar morphological traits. Part 179 of the series “Studies in Heterobasidiomycetous Fungi” of the Botanical Institute, University of Tübingen     

Motile iridophores of a freshwater goby,Odontobutis obscura

Summary Reflecting chromatophores in the dermis of the skin of a freshwater goby, Odontobutis obscura, are of an iridophore type. These chromatophores contain numerous reflecting platelets, which are similar to those in iridophores of other fish and amphibian species. It was found that these iridophores are motile, i.e., these cells respond to certain stimuli with translocation of the platelets within the cells. K⁺ ions induced dispersion of the platelets in excised scale preparations, but not in excised scales from chemically denervated fish. Norepinephrine and melatonin also induced dispersion of the platelets. Alpha-MSH was effective in aggregating these organelles into the centrospheres of the cells. The conclusions reached are: (1) iridophores of O. obscura are motile; (2) the movement of the iridophores is under nervous and hormonal control.     

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

The 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.     

Development,morphological characteristics and viability of temporary cysts of Pyrodinium bahamense var. compressum (Dinophyceae) in vitro

Pellicle or temporary cysts of Pyrodinium bahamense var. compressum (Böhm) Steidinger, Tester & F.J.R. Taylor and their role in bloom dynamics have not yet been adequately characterized and understood. We investigated the role of temperature- and nutrient-mediated stress as factors that could induce pellicle formation in batch cultures. Cellular features and their implications for temporary cyst viability were examined using confocal laser scanning microscopy (CLSM). Our data suggest that temperature change is one of the key factors influencing pellicle formation, preserving viability at low temperature (i.e. 13°C). Hypnocysts (resting cysts) were not observed. During pellicle formation, motile cells generally undergo ecdysis, extrusion of cytoplasmic materials and bacteria, compaction of the nucleus and non-motility. The outermost covering of the temporary cysts shows red autofluorescence and it contains lower concentrations of chlorophyll (chl) a and no detectable chl c. The nuclear region is surrounded by transitional red bodies and other unidentified cellular structures. Temporary cysts can immediately revert back to the motile state upon exposure to optimum conditions. This is accompanied by the expansion of the nuclear region, regeneration of the chloroplasts and enlargement of the cell. Developmental changes during reversal of temporary cysts to motile forms were also observed to cause breaks in the cell covering that could serve as sites for bacterial entry. Though observed in vitro, such behaviour may also be occurring in nature especially as a response to drastic short-lived environmental changes. This is the first detailed report on the characteristics of temporary cysts of P. bahamense var. compressum.