Molecular genetic delineation of Phaeocystis species (Prymnesiophyceae) using coding and non-coding regions of nuclear and plastid genomes

Sequence variation among 22 isolates representing a global distribution of the prymnesiophyte genus Phaeocystis has been compared using nuclear-encoded 18S rRNA genes and two non-coding regions: the ribosomal DNA internal transcribed spacer 1 (ITS1) separating the 18S rRNA and 5.8S rRNA genes and the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) spacer flanked by short stretches of the adjacent large and small subunits (rbcL and rbcS). 18S rRNA can only resolve major species complexes. The analysis suggests that an undescribed unicellular Phaeocystis sp. (isolate PLY 559) is a sister taxon to the Mediterranean unicellular Phaeocystis jahnii; this clade branched prior to the divergence of all other Phaeocystis species, including the colonial ones. Little divergence was seen among the multiple isolates sequenced from each colonial species complex. RUBISCO spacer regions are even more highly conserved among closely related colonial Phaeocystis species and are identical in Phaeocystis antarctica, Phaeocystis pouchetii and two warm-temperate strains of Phaeocystis globosa, with a single base substitution in two cold-temperate strains of P. globosa. The RUBISCO spacer sequences from two predominantly unicellular Phaeocystis isolates from the Mediterranean Sea and PLY 559 were clearly different from other Phaeocystis strains. In contrast, ITS1 exhibited substantial inter- and intraspecific sequence divergence and showed more resolution among the taxa. Distinctly different copies of the ITS1 region were found in P. globosa, even among cloned DNA from a single strain, suggesting that it is a species complex and making this region unsuitable for phylogenetic analysis in this species. However, among nine P. antarctica strains, four ITS1 haplotypes could be separated. Using the branching order in the ITS1 tree we have attempted to trace the biogeographic history of the dispersal of strains in Antarctic coastal waters.     

MORPHOLOGICAL AND GENETIC CHARACTERIZATION OF PHAEOCYSTIS CORDATA AND P. JAHNII (PRYMNESIOPHYCEAE), TWO NEW SPECIES FROM THE MEDITERRANEAN SEA

Two new Phaeocystis species recently discovered in the Mediterranean Sea are described using light and electron microscopy, and their systematic position is discussed on the basis of an analysis of their nuclear-encoded small-subunit ribosomal RNA gene (SSU rRNA) sequences. Phaeocystis cordata Zingone et Chrétiennot-Dinet was observed only as flagellated unicells. Cells are heart shaped, with two flagella of slightly unequal length and a short haptonema. The cell body is covered with two layers of thin scales. The outermost layer scales are oval, with a faint radiating pattern, a raised rim, and a modest central knob. The inner-layer scales are smaller and have a faint radiate pattern and an inflexed rim. Cells swim with their flagella close together, obscuring the haptonema, pushing the cell, and causing it to rotate about its longitudinal axis while moving forward. Phaeocystis jahnii Zingone was isolated as a nonmotile colony. It forms loose aggregates of cells embedded in a mucilaginous, presumably polysaccharide matrix without a definite shape or visible external envelope. The flagellated stage has the features typical of other Phaeocystis species. Cells are rounded in shape and slightly larger than P. cordata. The cell body is covered with extremely thin scales of two different sizes with a very faint radiating pattern toward their margin. Swimming behavior is similar to that of P. cordata, with the flagella in a posterior position as the cells swim. The SSU rRNA sequence analysis indicated that both species are distinct from other cultivated Phaeocystis species sequenced to date. Regions previously identified as specific for the genus Phaeocystis are not found in P. jahnii, and new genus-specific regions have been identified. P. cordata is more closely related to the colonial species P. globosa, P. antarctica, and P. pouchetii and has branched prior to the divergence of the warm-water P. globosa species complex from the cold-water species P. antarctica and P. pouchetii. These results are discussed within a framework ofthe available data on the evolution of the world’s oceans.     

Molecular evidence identifies bloom-forming Phaeocystis (Prymnesiophyta) from coastal waters of southeast China as Phaeocystis globosa

Sequence data from the 18S small subunit ribosomal RNA gene have been used to identify the species of a Phaeocystis (Prymnesiophyta) that caused harmful algae blooms in the coastal waters of southeast China. This Phaeocystis has morphological and physiological features that differ from those previously described for either P. globosa Scherffel or P. pouchetii (Hariot) Lagerheim. However, the sequence comparison of the Phaeocystis 18S rDNA clearly showed that it was remarkably similar to several isolates of P. globosa. Thus, the species isolated from the southeast coast of China is identified as P. globosa rather than P. cf. pouchetii or another species. Our results also demonstrate that phenotypes of different members of the genus Phaeocystis are variable, apparently changing in response to environmental conditions. It is concluded that, on the basis of this phylogenetic analysis, the bloom forming southeast China coast species of Phaeocystis most likely originated from an endemic warm-water, rather than a foreign source.     

Living in a Phaeocystis colony: a way to be a successful algal species

Evidence is provided showing that in two species of Phaeocystis (P. globosa and P. pouchetii) the colonial cells possess a much higher growth rate than the single cells when grown under identical conditions. Based on the DNA-cell-cycle method gross growth rate of colony cells exceeded those of co-occurring single cells by a factor 1.5 up to 3.8. The dominance of colonies in blooms of Phaeocystis can therefore be primarily due to their significantly high growth rate allowing a rapid bloom formation.Both Phaeocystis species showed ultradian growth but differed in timing of the initiation of the second DNA replication phase. In both species the first DNA-replication period started at the end of the (local) light period and was completed in the early dark period. In P. globosa this was immediately followed by the second DNA-replication period (first half of the dark period). In P. pouchetii this process was delayed by ca. 12 h until the middle of the light period (local noon).Flow cytometric analysis of the cell size and chlorophyll fluorescence showed little variation in colony and single cells of P. pouchetii. In contrast, colonies of P. globosa showed often the presence of two cell morphs, co-occurring in the same colony. The size of both morphs was identical but they differed in chlorophyll fluorescence up to a factor 4. In general the high chlorophyll cell morph dominated (>70% of the total colony cells). Both colony cell morphs were observed in cultures, mesocosms differing in N/P ratio but also in the field.     

Methods used to reveal genetic diversity in the colony-forming prymnesiophytes Phaeocystis antarctica, P. globosa and P. pouchetii—preliminary results

Previous work on the genetic diversity of Phaeocystis used ribosomal DNA and internal transcribed spacer (ITS) sequence analyses to show that there is substantial inter- and intraspecific variation within the genus. First attempts to trace the biogeographical history of strains in Antarctic coastal waters were based on a comparison of ITS sequences. To gain deeper insights into the population structure and bloom dynamics of this microalga it is necessary to quantify the genetic diversity within populations of P. antarctica from different locations (i.e., each of the three major gyres in the Antarctic continental waters) and to calculate the gene flow between them. Here we describe methods to quantify genetic diversity and our preliminary results for P. antarctica in comparison to two other colonial species: P. globosa and P. pouchetii. For this study of genetic diversity, two fingerprinting techniques were used. First, amplified fragment-length polymorphisms (AFLPs) were established as a pre-screening tool to assess clone diversity and to select divergent clones prior to physiological investigations. Second, the more-powerful microsatellite markers were established to assess population structure and biogeography more accurately. Results show differences in the AFLP patterns between isolates of P. antarctica from different regions, and that a wide variety of microsatellite motifs could be obtained from the three Phaeocystis species.     

ISOLATION AND CHARACTERIZATION OF A VIRUS INFECTING PHAEOCYSTIS POUCHETII (PRYMNESIOPHYCEAE)1

A virus infecting the haptophyte Phaeocystis pouchetii (Hariot) Lagerheim was isolated from Norwegian coastal waters in May 1995 at the end of a bloom of this phytoplankter. The virus was specific for P. pouchetii because it did not lyse 10 strains of P. globosa Scherffel, Phaeocystis sp., and P. antarctica Karsten. It was a double-stranded DNA virus, and the viral particle was a polyhedron with a diameter of 130–160 nm. The virus had a main polypeptide of about 59 kDa and at least five minor polypeptides between 30 and 50 kDa. The latent period of the virus when propagated in cultures of P. pouchetii was 12–18 h, and the time required for complete lysis of the cultures was about 48 h. The burst size was estimated to be 350–600 viral particles per lysed cell.     

The contribution of single and colonial cells of Phaeocystis pouchetii to spring and summer blooms in the north-eastern North Atlantic

Studies of the phytoplankton ecology in different localities in north-Norwegian fjords, the White Sea and the Barents Sea were carried out in spring and early summer to investigate the contribution of single and colonial stages of Phaeocystis pouchetii to phytoplankton abundance. Three different types of flagellated and four colonial cells were observed in all localities. P. pouchetii was rare under the ice of the Barents and White Seas, but their abundance increased rapidly during ice retreat. Single cell C dominated over colonial cell C, often by 50 times or more. The highest share of colonial cells was encountered in April in northern Norwegian fjords, in May in the Barents Sea and in May–June in the White Sea. At times the single cell dominated the total P. pouchetii biomass in Balsfjord (April 1999, 2001) with hardly any colonies present. In the White Sea colonies of P. pouchetii were less abundant than in the other regions. Cell carbon of P. pouchetii colonies appears never to be as dominating in the north-eastern North Atlantic as P. globosa blooms in coastal regions such as the southern North Sea. However, the lobal matrix of P. pouchetii colonies appears to be less solid than that of P. globosa and partly dissolution of the colony matrix during handling and storage of fixes samples induces uncertainty about the absolute numbers of P. pouchetii colonial cell counts. Despite of that, single cells of P. pouchetii seem to dominate significantly over colonial cell biomass at most sites and during some years and in some regions colonial cells seem rare. We speculate that top-down regulation of Phaeocystis spp. blooms possibly determines the ratio between single and colonial cells.     

MORPHOLOGY,PLOIDY, PIGMENT COMPOSITION,AND GENOME SIZE OF CULTURED STRAINS OF PHAEOCYSTIS (PRYMNESIOPHYCEAE)1

We examined cell morphology, ploidy level, cell size, pigment composition, and genome size in 16 cultured strains of Phaeocystis Lagerheim. Two strains originated from the Antarctic, 3 from the tropical Western Atlantic, and 11 from temperate regions (Eastern Atlantic, English Channel, North Sea, and Mediterranean Sea). Thirteen strains made colonies morphologically similar to P. glo-bosa Scherffel, whereas three never formed colonies under any circumstances. Five-rayed star-like structures with filaments were observed in 11 strains. In several strains, two ploidy levels were observed, one (haploid) linked to flagellates and one (diploid) linked to colonies. Cell size did not appear to be a very good criterion for distinguishing strains since size distributions overlapped. Pigment analysis by reversed-phase-high-performance liquid chroma-tography allowed the strains to be grouped into three clusters that differed from each other mainly by the relative proportions of three carotenoids: fucoxanthin, 19′-hex-anoyloxyfucoxanthin, and diadinoxanthin. All strains contained low levels of 19′-butanoyloxyfucoxanthin. Differences in genome size measured by flow cytometry delimited at least five groups. On the basis of both pigment composition and genome size, six clusters were defined, one corresponding to an Antarctic species (possibly P. antarc-tica), one to P. globosa, and the rest probably to several yet-undescribed species or subspecies. Two main conclusions emerge from this study. First, the taxonomy of the genus Phaeocystis needs to be clarified through a combination of morphological, biochemical, and molecular studies. Second, sexuality is a prevalent phenomenon in Phaeocystis, but controls of the sexual cycle are most likely strain-dependent.     

Zooplankton grazing on Phaeocystis: a quantitative review and future challenges

The worldwide colony-forming haptophyte phytoplankton Phaeocystis spp. are key organisms in trophic and biogeochemical processes in the ocean. Many organisms from protists to fish ingest cells and/or colonies of Phaeocystis. Reports on specific mortality of Phaeocystis in natural plankton or mixed prey due to grazing by zooplankton, especially protozooplankton, are still limited. Reported feeding rates vary widely for both crustaceans and protists feeding on even the same Phaeocystis types and sizes. Quantitative analysis of available data showed that: (1) laboratory-derived crustacean grazing rates on monocultures of Phaeocystis may have been overestimated compared to feeding in natural plankton communities, and should be treated with caution; (2) formation of colonies by P. globosa appeared to reduce predation by small copepods (e.g., Acartia, Pseudocalanus, Temora and Centropages), whereas large copepods (e.g., Calanus spp.) were able to feed on colonies of Phaeocystis pouchetii; (3) physiological differences between different growth states, species, strains, cell types, and laboratory culture versus natural assemblages may explain most of the variations in reported feeding rates; (4) chemical signaling between predator and prey may be a major factor controlling grazing on Phaeocystis; (5) it is unclear to what extent different zooplankton, especially protozooplankton, feed on the different life forms of Phaeocystis in situ. To better understand the mechanisms controlling zooplankton grazing in situ, future studies should aim at quantifying specific feeding rates on different Phaeocystis species, strains, cell types, prey sizes and growth states, and account for chemical signaling between the predator and prey. Recently developed molecular tools are promising approaches to achieve this goal in the future.     

CURRENT KNOWLEDGE OF THE LIFE CYCLES OF PHAEOCYSTIS GLOBOSA AND PHAEOCYSTIS ANTARCTICA (PRYMNESIOPHYCEAE)1

Despite continuous efforts since the 1950s and more recent advances in culturing flagellates and nonflagellate cells of the prymnesiophyte Phaeocystis, a number of different life‐cycle models exist today that appear to apply for P. globosa Scherff. and P. antarctica G. Karst., both spherical colony formers. In one such model, this life cycle consists of three different flagellates and one nonmotile cell stage that is embedded in carbohydrate matrix‐forming colonies of different sizes and forms. Recently, noncolonial aggregates of diploid nonmotile cells attached to surfaces of diatoms were put forward as a new stage in the sexual life cycle of P. antarctica. However, it can be discussed that these “attached aggregates” (AAs) are an intermediate between motile diploid flagellates, with their well‐known tendency to adhere to surfaces, and the young spherical colony with its diploid nonmotile cells, which in nature is commonly found attached to diatoms. A life‐cycle model pertaining to both P. globosa and P. antarctica is presented.     

The colonization of two Phaeocystis species (Prymnesiophyceae) by pennate diatoms and other protists: a significant contribution to colony biomass

The association of Phaeocystis spp. with small pennate diatoms during three Phaeocystis-dominated spring blooms were investigated in the Eastern English Channel (2003 and 2004) and in coastal waters of Western Norway during a mesocosm experiment (2005). In each of these studies, colonization of the surface of large Phaeocystis spp. colonies by small needle-shaped diatoms (Pseudo-nitzschia spp.) were observed. In the English Channel the diatom Pseudo-nitzschia delicatissima colonized the surface of large (>100 μm) Phaeocystis globosa colonies. The abundance of Pseudo-nitzschia delicatissima reached 130 cells per colony and formed up to 70% of the total carbon associated with Phaeocystis cells during late bloom stages. In Norwegian waters, the surface of large (>250 μm) Phaeocystis pouchetii colonies were colonized by Pseudo-nitzschia cf. granii var. curvata and to a lesser degree by other phytoplankton and protist species, although the abundance of these diatoms was never greater than 40 cells per colony. Based on these observations we suggest that diatoms utilize Phaeocystis colonies not only as habitat, but that they are able to utilize the colonial matrix as a growth substrate. Furthermore, these observations indicate that a considerable fraction of biomass (chlorophyll) associated with Phaeocystis colonies, especially large colonies concerned with intense and prolonged blooms, are due to co-occurring plankton species and not exclusively Phaeocystis cells.     

摄食信息对球形棕囊藻和布氏双尾藻竞争结果的影响

浮游动物的摄食信息能增大棕囊藻囊体体积,囊体形成被认为是棕囊藻的诱导性防御机制。利用桡足类火腿伪镖水蚤和异养甲藻海洋尖尾藻释放的摄食信息,研究了诱导性防御对球形棕囊藻和布氏双尾藻的竞争的影响。结果表明,球形棕囊藻接收了火腿伪镖水蚤和海洋尖尾藻释放的摄食信息之后形成更大的囊体。防御启动后的球形棕囊藻比未接收摄食信息的球形棕囊藻更快地形成囊体,且囊体维持的时间更长。对照组和火腿伪镖水蚤摄食信息诱导的球形棕囊藻的生物体积比布氏双尾藻更高,且球形棕囊藻在竞争中占优势;而海洋尖尾藻摄食信息诱导的球形棕囊藻生物体积低于布氏双尾藻,且球形棕囊藻相对布氏双尾藻的竞争力下降。微型浮游动物海洋尖尾藻摄食信息导致球形棕囊藻相对硅藻布氏双尾藻的竞争力的下降,有利于解释硅藻先于棕囊藻发生藻华。     

Growth and mortality of flagellates and non-flagellate cells of Phaeocystis globosa (Prymnesiophyceae)

Two cell types of the same clone of Phaeocystis globosa, solitarynon-flagellate cells and flagellates, were grown in batch culturesunder identical conditions. The non-flagellate cells had a shorterlag phase (1.4 versus 2.8 days) and a higher growth rate (0.72versus 0.65 day^–1) than flagellate cells. The flagellateshad a longer stationary phase (15.6 versus 9.5 days) and a lowerdeath rate (0.07 versus 0.52 day^–1) than non-flagellatecells. All differences were statistically significant. Biomassyield did not differ between the two cell types. The short lagphase and high growth rate of nonflagellate cells correspondsto field observations of rapidly developing non-flagellate Phaeocystisblooms that are typically observed in nutrient-rich environmentssuch as temperate seas in spring. The flagellate cell type,with its longer stationary phase and lower death rate than non-flagellatecells, is better equipped for survival in oligotrophic environments.This explains why the flagellates of Phaeocystis are abundantafter the spring phytoplankton bloom in temperate seas and inother nutrient-poor environments such as the open ocean.     

Haemolytic activity of live Phaeocystis pouchetii during mesocosm blooms

Chemical defence is a potential mechanism contributing to the success of Phaeocystis species that repeatedly dominate the phytoplankton in coastal areas. Species within the genus Phaeocystis have long been suspected of imposing negative effects on co-occurring organisms. Recently a number of toxins have been extracted and identified from Phaeocystis samples, but it is not clear if they do enhance the competitive advantage of Phaeocystis species. In the present study the cytotoxic impact of live Phaeocystis pouchetii to human blood cells in close proximity, regardless of the nature of the responsible mechanism, was quantified using a bioassay. Haemolytic activity was measured during blooms of P. pouchetii in mesocosms. These environments were chosen to mimic natural conditions including chemically mediated interactions that could trigger defensive and/or allelopathic responses of Phaeocystis. Haemolytic activity correlated with P. pouchetii numbers and was absent during the preceding diatom bloom. Samples containing live P. pouchetii cells showed the highest activity, while filtered sea water and cell extracts were less haemolytic or without effect. Dose-response curves were linear up to 70% lysis, and haemolysis in samples containing live P. pouchetii cells reached EC₅₀ values comparable to known toxic prymnesiophytes (1.9 * 10⁷ cells l⁻¹). Haemolytic activity was enhanced by increased temperature and light. The results indicate that unprotected and thus presumably vulnerable cells present in a P. pouchetii bloom may lyse within days.     

Differentiation betweenPhaeocystis pouchetii (Har.) Lagerheim andPhaeocystis globosa Scherffel

An Arctic clone ofPhaeocystis pouchetii LAGERHEIM was compared toPhaeocystis globosa SCHERFFEL isolated from the southern North Sea with regard to temperature tolerance and colony shapes. Already youngP.pouchetii colonies (<100 m) show the typical distribution of the cells in groups, separated from each other by wide zones of cell-free mucilage; the maximum colony size is ca 2 mm in diameter.P.pouchetii colonies form clouds with bubble-like vesicles, spherical colony-shapes are seldom found.P.globosa colonies are spherical up to a size of 2 mm; the cells are distributed homogeneously over the periphery of the colonies. A pouchetii-like distribution of cells never occurs either in the spherical young colonies or in the pear-shaped old colonies (size up to 8 mm). A development from the colony shape of the globosa-type to the pouchetii-type or vice versa was never found. Therefore the colony shape has to be considered a constant distinctive character. Single cells ofP.pouchetii andP.globosa cannot be separated from each other by using the light microscope; this also holds for the flagellates and the non-motile cells.P.pouchetii grows well between 0°C and 14°C,P.globosa between 4°C and 22°C, respectively. Because of the distinctive differences in the morphology of the colonies and the differences in temperature tolerances we propose thatPhaeocystis globosa should no longer be considered conspecific withPhaeocystis pouchetii.     

A comparative analyses of morphological variations,phagocytosis and generation of cytotoxic agents in flow cytometrically isolated hemocytes of Indian molluscs

A comparative analyses of hemocytes of molluscs, Pila globosa (Gastropoda: Prosobranchia), Bellamya bengalensis (Gastropoda: Prosobranchia) and Lamellidens marginalis (Bivalvia: Eulamellibranchiata) were carried out for morphotype and subpopulation identification, analyses of phagocytosis and generation of cytotoxic agents. Flow cytometry and microscopic analyses of hemocytes revealed the existence of agranulocytes (blast like cells, round hyalinocytes and spindle hyalinocytes), semigranulocytes (semigranular asterocytes and round semigranulocytes) and granulocytes (round granulocytes, spindle granulocytes and granular asterocytes) as three morphotypes. In P. globosa, granulocytes and semigranulocytes and in B. bengalensis granulocytes and agranulocytes are the chief phagocytes and major producers of superoxide anion and nitric oxide. In L. marginalis, granulocytes were identified as principal phagocytes with prominent activity of superoxide anion and nitric oxide. Highest activity of phenoloxidase was recorded in the agranulocytes of P. globosa with moderate activities among other morphotypes of all three species. Differential result may be due to species specific response, non-identical habitat preference and related adaptation of the species to their different ecological niches.     

PLOIDY ANALYSIS OF THE TWO MOTILE FORMS OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYCEAE)1

In some cultures of the flagellate Chrysochromulina polylepis Manton et Parke, established from cells isolated from the massive bloom in Skagerrak and Kattegat in 1988, we observed, two motile cell types. They were termed authentic and alternate cells and differed with respect to scale morphology. To investigate whether or not the two cell forms were joined in a sexual life cycle, the relative DNA content per cell and relative size of cells of several clonal cultures of C. polylepis were determined by flow cytometry. Percentages of authentic and alternate cells in the cultures were estimated by transmission electron microscopy. Pure authentic cultures (α) contained cells with the lowest level of DNA and were termed haploid. Two pure alternate cultures (β) contained cells with double the DNA content of authentic cells and were termed diploid. Other pure alternate cultures contained haploid cells only, or both haploid and diploid cells. Three cell types were observed, each capable of vegetative propagation: authentic haploid, alternate haploid, and alternate diploid cells. Both the haploid and diploid alternate cells were larger than the haploid authentic cells. Cultures containing diploid cells appeared unstable: cell type ratio and ploidy ratio changed during the experiment where this cell type was present, particularly when grown in continuous light. In contrast, cultures with only haploid cells remained unchanged at all growth conditions tested. Light condition may influence cell type ratio and ploidy ratio. Our attempt to induce syngamy by mixing different authentic haploid clones did not result in mating. Assuming that the authentic and alternate cell types are of the same species, the life cycle of C. polylepis includes three flagellated scale-covered cell forms. Two of the cell types are haploid and may function as gametes, and the third is diploid, possibly being the result of syngamy.