Nov 2, 2016

Sexual Reproduction in Volvox carteri

Overview of the Volvox
The spheroidal chlorophycean Volvox and its close relatives are suitable model organisms for addressing fundamental issues in the evolution of multicellularity and the development of a germ-soma dichotomy (Kirk 1998; Nozaki et al. 2000; Kirk and Nishii 2001; Nozaki 2003; Hallmann 2011). They form a group of genera closely related to the genus Volvox within the order Volvocales. This group ranges in complexity from unicellular organisms, such as Ch. Reinhardtii, to homocytic colonial organisms, such as Gonium pectorale, to heterocytic multicellular organisms with different cell types, as is found in Volvox, which is the most highly developed genus. Several patterns of sexual reproduction are exhibited by the different species of the genus. Some species are monoclonic, while others are diclonic. Monoclonic species are either monoecious or dioecious. All diclonic species are dioecious and produce male and female spheroids in separate clones. Volvox mostly reproduces asexually, although it is able to switch to the sexual pathway.

The involvement of sex pheromones in switching was first described by Darden who showed that asexual V. aureus (a monoclonic, monoecious species) cultures could be induced to change to the sexual pathway by supplementing the cell-free cultured medium of mature male spheroids (Darden 1966). The same phenomena were subsequently reported for other species of Volvox (Kochert 1981).

Life cycles of Volvox carteri
V. carteri, a dioecious species, is the most intensively studied Volvox species (Kirk and Nishii 2001). It is composed of only two cell types, 2000–4000 biflagellate Chlamydomonas-like somatic cells, which form a monolayer at the surface of a hollow sphere and 16 reproductive cells (gonidia) that lie just below the sheet of somatic cells (Starr 1969). During embryogenesis, the embryos first cleave symmetrically five times to form a 32-cell embryo with identical cells, and then 16 cells divide asymmetrically to each produce one large gonidial cell precursor and one small somatic cell precursor (Kirk and Kirk 2004). These gonidial precursors divide asymmetrically two more times and produce additional somatic precursors at each division. The gonidial precursors then temporarily stop cell division, while the somatic precursors divide symmetrically about three more times. At the end of embryogenesis, the volume of the gonidial precursors expands to about
30-times that of their somatic precursors.

Sexual reproduction is initiated by a mutation-like switch with a probability of 2 × 10–4, which leads to the formation of the first sexual male colony (Weisshaar et al. 1984). A pheromone, named “sex inducer” or “sex-inducing pheromone”, has been the focus of considerable attention (Starr 1970). The sex-inducing pheromone is produced and released by this sexual male colony and acts on the asexual gonidia of both sexes. It alters their developmental pathway such that sexual forms (egg- or sperm-bearing forms) are produced in the next generation. In sexually induced male embryos, asymmetric cell division is postponed from the sixth to the eighth division cycle (Starr 1969, 1970; Hallmann et al. 1998). At this point, somatic cell precursors no longer divide and the large gonidial precursors each symmetrically divide seven times to form sperm bundles containing up to 128 sperm cells. Thus, the male ends up with 128 sperm bundles and 128 somatic cells. In female embryos, the first asymmetrical cell division is also postponed from the sixth to the seventh division cycle. Asymmetrical cell division then occurs and the somatic cell precursors continue to further cleave, with the large gonidial precursors developing as about 32 eggs and about 2000 somatic cells. Sperm bundles generated on the male colony contact the female colony by chance rather than by directed swimming (Coggin et al. 1979; Kirk 1998), after which a specific transient binding to somatic cells occurs. The sperm bundles break up into individual sperm cells and the sperm penetrate the extracellular matrix (ECM) of the female to reach the eggs inside the spheroids. The fusion of gametes results in the formation of a dormant diploid zygote that survives the drought. Under favorable environmental conditions, the germination of the zygote occurs with meiosis to form only a single viable germling and three nonviable polar bodies. The germling will produce a haploid female or male and then reproduce asexually (Starr 1975; Fig below).


The Volvox carteri Sex-inducing Pheromone
Because the production of pheromone could not be detected in sexual females or the somatic cells of sexual males, it was believed that expression of the gene encoding the pheromone was tightly linked to sperm development (Starr 1970; Gilles et al. 1981). However, both asexual females and asexual males were able to produce the pheromone following exposure to a heat shock (Kirk and Kirk 1986). The participation of reactive oxygen species in both the production of the pheromone and its activity in triggering sexual development in gonidia has been suggested (Nedelcu and Michod 2003). Sex-inducing pheromones were independently purified from two isolates of V. carteri: one from Japan (V. carteri f. nagarensis) and the other from the United States (V. carteri f. weismannia) (Kochert and Yates 1974; Starr and Jaenicke 1974). Both were glycoproteins of about 30-kDa. The inducer of the V. carteri f. nagarensis was strictly competent for its own gonidia whereas that of V. carteri f. weismannia induced sexuality of both of them (Al-Hasani and Jaenicke 1992). The pheromone of V. carteri f. nagarensis is one of the most potent biological effector molecules known, exhibiting its full effectiveness below 10–16 M. As a result of successful largescale production, partial amino acid sequences were obtained, allowing the cloning of a genomic clone encoding the pheromone (Tschochner et al. 1987) and the cDNA (Mages et al. 1988).

Possible Mode of Action of the Pheromone
The pheromone seems to have at least two modes of action. One is that the pheromone molecules act directly on the receptors of the gonidial cells. For this, the pheromone molecules must pass through the ECM. The other is that the pheromone molecules exert the effect by binding to the receptors on somatic cells, surrounding the spheroid. The binding triggers the synthesis of extracellular proteins, generating signal amplification. Because the very first experimentally detectable cellular responses to the sex pheromone come from the somatic cells at the surface, but not from the gonidial cells, the latter mode of action seems to be more plausible (Hallmann 2003).

The pheromone induces the synthesis of the deep zone hydroxyprolinerich glycoprotein (DZ-HRGP; Ender et al. 1999), chitinase/lysozyme, chitin-binding protein (Amon et al. 1998), and metalloproteinases (VMPs; Hallmann et al. 2001). Some of the ECM glycoproteins that are inducible by the sex pheromone are also inducible by mechanical wounding (Amon et al. 1998; Ender et al. 1999) but wounding does not cause the production of the sex pheromone itself. The majority of proteins synthesized shortly after the pheromone treatment in the ECM are part of a single family of glycoproteins: the pherophorins (Sumper et al. 1993; Godl et al. 1995, 1997). At least 34 different pherophorins occur in Volvox (Hallmann 2003, 2006; Prochnik et al. 2010). The carboxy-terminal domains of all pherophorins are similar to the pheromone. Pherophorin-II is considered to be responsible for the signal amplification mechanism of the pheromone (Sumper et al. 1993; Godl et al.
1995). Pherophorin-II is a glycoprotein that consists of three domains: the N-terminal domain, whose sequence is related to a motif of another ECM protein, SSG185; the polyproline spacer; and the carboxy-terminal domain, which is 30% identical to the sex-inducing pheromone. The carboxy-terminal domain is proteolytically liberated from the parent glycoprotein, after its pheromone induces synthesis. Because the inhibition of processing by protease inhibitors coincides with a suppression of sexual induction, and the induction of the gene expression of pherophorin-II by the pheromone could not be observed in all three independently isolated sterile mutants, the liberated domain may potentially act as an analog of the sex-inducing pheromone (Sumper et al. 1993; Godl et al. 1995). Transformed V. carteri expressing recombinant pherophorin-II, in which the carboxy-terminus had been fused with green fluorescent protein (GFP), indicated that the carboxy-terminal domain including GFP was cleaved proteolytically, as in the native protein (Ishida 2007). The GFP signal of the transformant was located at the ECM directly surrounding the gonidium, the final target of the sexual-induction signal. However, no sex-inducing activity of the domain has been experimentally demonstrated. Genes showing similarity with pherophorins have also been identified in other Volvocales (Ch. Reinhardtii, G. pectorale and Pandorina morum), although information regarding their expression conditions is not available (Hallmann 2006). These pherophorins contain a (hydroxyl-) prolinerich (HR) rod-like domain and are abundant within the extracellular compartment, in a similar manner to the extensins of higher plants. In addition, pherophorins show a striking general structural similarity with a special class of extensin: the solanaceous lectins. Pherophorins have been suggested to be used as the versatile building blocks for the ECM architecture. In view of the large number of pherophorins, the pheromone is considered to be a pherophorin paralog and might have evolved a new function over time (Hallmann 2006).

A small cysteine-rich extracellular protein, named VCRP, which was quickly synthesized by somatic cells in response to the pheromone, has been identified (Hallmann 2007). In addition, a VCRP-related protein, VCRP2, has also been found using genome information from V. carteri (Hallmann 2008). Both VCRPs are speculated to be candidates for the extracellular second messenger from somatic cells to gonidial cells.

Mating-type Loci of Volvox carteri

As indicated previously, sexual differentiation in Ch. Reinhardtii is largely controlled by the MID gene, encoding the RWP-RK family putative transcription factor. Nozaki et al. (2006) isolated an orthologous MID gene from the oogaomous volvocacean Pleodorina starrii. The gene, named PlestMID, is only present in the male genome and the protein is abundantly present in sperm nuclei. This finding strongly suggests that maleness was probably established from the minus mating type of its isogamous unicellular ancestor during the evolution of oogamy. The MID homolog has been identified in other volvocaceans, e.g., G. pectorale (GpMID in mt– genome; Hamaji et al. 2008). Recently, both alleles of the Volvox MT were sequenced. Only two sex-limited genes, MID and MTD1, located on the MT loci of Ch. Reinhardtii have recognizable homologs in the Volvox MT, and both are in the male MT. However, both Volvox MID (VcMID) and Volvox MTD1 (VcMTD1) are expressed constitutively (Ferris et al. 2010). The retinoblastoma-related protein1 (RBR1) gene, a homolog of Ch. Reinhardtii MATING-TYPE LINKED3 (MAT3), is located on the Volvox female MT and has a very different structure from the male MAT3 homolog (Kianianmomeni et al. 2008; Ferris et al. 2010). Both MAT3 homologs display sexually regulated alternative splicing and sex-specific selection. The predominant MAT3 splicing variant in sexual males includes an early termination codon. The downregulation of MAT3 in Volvox males may be linked to the production of small-celled sperm because mat3 mutants in Ch. Reinhardtii are known to produce tiny gametes (Umen and Goodenough 2001). However, MAT3 homologs from the five colonial species examined (isogamous G. pectoral and Yamagishiella unicocca, anisogamous Eudorina sp. and P. starrii, and oogamous Volvox africanus) had almost identical nucleotide sequences between the two sexes. The extreme gender-based MAT3 divergence observed in V. carteri species may not be directly related to the evolution of male and female dimorphism within the colonial Volvocales as a whole (Hiraide et al. 2013).

Oct 30, 2016

Sexual Reproduction in Chlamydomonas reinhardtii

Overview of the Life Cycle of Chlamydomonas reinhardtii

Chlamydomonas (Ch.) reinhardtii was first isolated as a green soil alga and is used as a model organism in plants. It retains two flagella subsequently lost by most plants and a chloroplast being functionally equivalent to the chloroplasts of green plants. Its complete genome sequence is available (Merchant et al. 2007) and its life cycle is well characterized. Ch. Reinhardtii has two mating types: mating type plus (mt+) and mating type minus (mt–), which are controlled by a single complex mating type locus (MT+ or MT–) (Ferris et al. 2002). They proliferate asexually when an adequate source of nitrogen exists in the environment. After mitosis in the mitotic cell cycle, the newly formed daughter cells are liberated by the effect of sporangin (a subtilisin-like serine protease) acting on the breakdown of the sporangial cell walls (Matsuda et al. 1995; Kubo et al. 2009). When nitrogen levels fall below a certain threshold, vegetative cells having the MT+ locus differentiate into mt+ gametes and cells having the MT– locus differentiate into mt– gametes. Three hierarchically regulated gene expression programs are generally recognized as a response to nitrogen depletion: a program to adapt to nitrogen starvation, a gamete differentiation program, and a zygote formation program (Abe et al. 2004, 2005; Kubo et al. 2008). Within minutes of being mixed, gametes start to agglutinate. Gametes of opposite mating types pair with each other and fuse to form bi-nucleate quadriflagellated cells (QFCs). The two nuclei in the cell fuse, and a novel set of zygote-specific genes is expressed to form a dormant zygote that is resistant to both freezing and desiccation. When conditions improve, the dormant zygote initiates meiosis and the four recombinant haploid products resume vegetative growth (Fig. 1).
Fig 1

Sexual Adhesion
The sexual adhesion between the gametes is mediated by agglutinin molecules on their flagellar membranes. The plus and minus agglutinins are sex-specifically displayed by nitrogen-starved mt+ and mt– gametes, respectively. These molecules are encoded by different genes and possess complementary adhesive properties. They are both huge monomeric glycoproteins (>1000 kDa; Adair et al. 1983), possessing a large globular head and a fi brous shaft (Goodenough et al. 1985), and are members of the hydroxyproline-rich glycoprotein (HRGP) family (Cooper et al. 1983). Ferris et al. (2005) isolated the SEXUAL AGGLUTINATION1 (Sag1) and SEXUAL ADHESION1 (Sad1) genes encoding plus and minus agglutinins, respectively. Gene expression is restricted to gametes of one mating type.

The presence of the Minus-dominance (Mid) gene localized on the MT– locus suppresses the expression of Sag1 but induces the expression of Sad1. Both deduced proteins are organized into three distinct domains: a head (C-terminal), a shaft, and an N-terminal domain. The plus and minus heads are quite large domains (2006 and 2404 amino acids, respectively) having 12 and 14 putative N-glycosylation sites, respectively. Six of the putative N-glycosylation sites are in similar locations. They are poorly conserved in the amino acid sequence except for two regions of the predicted hydrophobic α-helix. The shafts contain numerous repeats of the PPSPX motif. Head–head interactions, heads–shafts interactions, and antiparallel shaft–shaft interactions may be involved in the sexual adhesion between two specific agglutinins (Ferris et al. 2005).

Signal Transduction after the Interaction of Agglutinins
After the interactions between plus and minus agglutinin molecules on the flagellar membranes, a gamete-specific flagellar adenylyl cyclase is activated via a protein kinase- and kinesin-II-dependent pathway (Saito et al. 1993;

Zhang and Snell 1994; Pan and Snell 2002) and the intracellular cAMP level is elevated nearly tenfold, triggering dramatic alterations in the cell (Pasquale and Goodenough 1987; Saito et al. 1993; Zhang and Snell 1994). The addition of a cell-permeable analog of cAMP, dibutyryl cAMP, can induce most of the cellular changes (Pasquale and Goodenough 1987). The mating-related effects of cAMP elevation include the following. First, flagellar motility is altered and the adhesiveness of the flagellar surface is increased by the translocation of inactive agglutinin molecules from the plasma membrane of the cell body onto the contiguous flagella membrane where the agglutinins become active (Saito et al. 1985; Goodenough 1989; Hunnicutt et al. 1990).

The process is mediated by the kinesin/dynein-mediated intra-flagellar transport system (Snell et al. 2004; Wang et al. 2006; Piao et al. 2009). Second, activated gametes secrete a serine protease (p-lysinase) that converts an extracellularly stored prometalloprotease into an active matrix-degrading enzyme (Buchanan et al. 1989; Snell et al. 1989; Kinoshita et al. 1992), and the cell wall (multilayered glycoproteinaceous extracellular matrix) surrounding each cell is degraded so that the gametes are able to fuse.
Third, mt+ gametes erect an actin-filled microvillus (“fertilization tube”) as a mating structure at the apical ends near the bases of the flagella and the mt– gametes also erect a small, dome-like, actin-free mating structure. The mating structures of both types of gametes display an extracellular coat of material referred to as fringe (Goodenough et al. 1982).

Molecules Required for the Fusion
Cell fusion is initiated by an adhesive interaction between the mt+ and mt– mating structures, followed by localized membrane fusion. Two proteins are known to be an essential for the membrane fusion reaction. The first is FUS1, which is a single transmembrane protein on the mating structure of the mt+ gamete (Ferris et al. 1996; Misamore et al. 2003). The FUS1 gene is sex-specifically expressed and is located in the MT+ locus (Ferris et al. 1996). FUS1 is an about 95-kDa protein and has domains related to the Ig-like domains of prokaryotic invasins and adhesins. It is essential for the adhesion of the mt+ mating structure to an unidentified receptor on the mating structure of mt– gametes (Misamore et al. 2002, 2003). The fus1-1 mutant undergoes normal flagellar adhesion and gamete activation, and produces an actin-filled fertilization tube in response to cAMP; however, the fus1-1 fertilization tube fails to fuse with the activated minus mating structure and the cells continue to agglutinate for several days. The mt+ fringe is encoded by the FUS1 gene (Misamore et al. 2003). Fertilization tubes on fus1-1 gametes do not contain the fringe (Goodenough et al. 1982).

When mt+ gametes are incubated with the anti-FUS1 antibody, fusions with mt– gametes are blocked. The second protein required for the membrane fusion reaction is GCS1/
HAP2, which is expressed on the surface of the mt– mating structure as a single transmembrane protein (Liu et al. 2008). The gene has also been identified in other algae, protists, and higher plants (Mori et al. 2006; von Besser et al. 2006; Steele and Dana 2009; Wong and Johnson 2010). In Arabidopsis thaliana, the GCS1/HAP2 gene is specifically expressed in sperm cells, and the mutant fails to fuse with both egg and central cells (Mori et al. 2006). In the case of the malaria organism Plasmodium berghei, GCS1/HAP2 is required at a particular step in the membrane fusion reaction between gamete membranes (Liu et al. 2008). The knockout mutant shows male sterility (Hirai et al. 2008). In Ch. Reinhardtii, expression of the GCS1/HAP2 gene is confirmed in both mt+ and mt– gametes but is far stronger in mt– gametes (Mori et al. 2006). GCS1/HAP2 protein localizes at the fusion site of mt– gametes and mt– gcs1/hap2 mutant gametes can form tight perfusion membrane attachments with mt+ gametes, but they fail to fuse (Liu et al. 2008). Both FUS1 and GCS1/HAP2 proteins are degraded rapidly upon fusion, as would be expected to block polygamy (Liu et al. 2010).

Development of the Zygote
The zygote developmental program is triggered by the heterodimerization of two homeoproteins, Gamete specific plus1 (Gsp1) and Gamete specific minus1 (Gsm1), which are contributed by the mt+ gamete and mt– gamete, respectively (Lee et al. 2008). Gsp1 is distantly related to the BELL class homeoproteins and Gsm1 is an ortholog of the KNOTTED1-like homeobox (KNOX) class (Hake et al. 2004; Scofield and Murray 2006). The expression of the GSM1 gene is dependent on the expression of the MINUS DOMINANCE (MID) gene (detailed below) on the MT– locus, while GSP1 expression is inhibited by MID. GSP1 was identified as a gene expressed specifically in mt+ gametes (Kurvari et al. 1998). Ectopic expression of GSP1 in mt– gametes is responsible for the formation of the zygotic cell walls and the expression of several zygote-specific genes (Zhao et al. 2001). When both the GSP1 and the GSM1 genes were ectopically expressed in vegetative cells, a zygote developmental program was activated: they formed zygote-specific cell walls and expressed zygote specific genes, despite being in a nitrogen-supplemented medium. With ectopic expression of both GSP1 and GSM1 in a generated mt+ diploid background, the resulting zygotes undergo normal meiosis (Lee et al. 2008). GSP1 is also important for the uniparental inheritance of chloroplast and mitochondrial DNA. A mutant, biparental31 (bp31), having a deletion of about 60 kb on chromosome 2, including the GSP1 gene, impairs the uniparental inheritance of chloroplast and mitochondrial DNA. The mutant phenotype can be rescued by a cotrans-formation with both the GSP1 and INOSITOL MONOPHOSPHATASELIKE1 (INM1) genes (Nishimura et al. 2012).

Sex Determination in Chlamydomonas reinhardtii
As explained previously, mating types of Ch. Reinhardtii are controlled by a single complex mating type locus (MT+ or MT–) on linkage group VI (Ferris et al. 2002). Heterozygous mt+/mt– diploids, which are occasionally formed after mating, always mate as mt– gametes, indicating that MT– is dominant to MT+ (Harris 1989). The core of the two MT loci encompasses 200–300 kb (Ferris and Goodenough 1994; Ferris et al. 2002, 2010). The MT loci contain highly rearranged DNA sequences, characterized by several large inversions and translocations, which act to suppress the recombination. Some genes are specifically linked to either MT locus. The FUS1 gene on the MT+ locus, and the MT locus region d (MTD1) and MID genes on the MT– locus have been assigned mating type-specific functions in gametogenesis and mating.

Mt+ cells transformed with the MID gene differentiate as mt– gametes and the functional mutant in an mt– background differentiates into an mt+ gamete having all of the molecules required of an mt+ gamete, except for the FUS1 protein (Ferris and Goodenough 1997; Ferris et al. 2002). These results indicate that MID is necessary both to activate mt– gene expression and to prevent mt+ gene expression, allowing the conversion of wild-type mt+ gametes to mt– gametes. MID encodes a RWP-RK family putative transcription factor. Vegetative mt– cells express basal levels of MID. A pulse of upregulated expression (level 1, threefold increase to basal level) occurs at 30 min after nitrogen removal, followed by a return to the basal level at 1 h.


The expression is strongly upregulated (level 2, eightfold) at 4–6 h together with the acquisition of mating competency (Lin and Goodenough 2007).

Knockdown of MTD1 in mt– cells results in a failure to differentiate into gametes of either mating type after nitrogen removal. From the results, Lin and Goodenough (2007) proposed that the first increase of mid (level 1) is sufficient to activate MTD1 transcription and to repress mt+ gamete-specific genes, and that MTD1 expression in turn allows the second increase (level 2) that is necessary to turn on mt– gamete-specific genes.

The Windows Store – Tutorial 6

Today we will be focusing on the Windows Store in Windows 10.

Accessing the Windows Store


The new Windows Store can be accessed directly from the taskbar (see below).


You can also access the Windows Store from the new Start Menu.


Once you click on the Windows Store icon, the store opens up in a boxed Window.


The Windows Store previously looked, felt and was a Metro app.

This is different and it looks and feels more like a Windows app.
Clicking on app categories brings up a full screen of apps, sorted into categories, such as:

Top Free Apps
Picks for You
Most Popular
Top Free Games


Oct 28, 2016

Reproduction in Haptophytes

The division Haptophyta is a lineage of unicellular algae that are widespread and often very abundant in diverse marine settings. Most haptophytes occur as solitary motile or non-motile forms, but a few form colonies or short filaments. Haptophyte cells are usually covered with one or several layers of organic scales of varying degrees of complexity, these being formed intracellularly in Golgi-derived vesicles. Haptophytes are characterized by the presence of a unique organelle called a haptonema (from the Greek Hapsis, touch, and Nema, thread), which is superficially similar to a flagellum but differs in the arrangement of microtubules and in function, being implicated in attachment or capture of prey. The haptonema is present in most species, sometimes in a reduced or vestigial form, but may rarely be absent.

The Haptophyta includes 2 classes: the Pavlovophyceae with only 13 described species and the Prymnesiophyceae which contains the vast majority of the known diversity of haptophytes and which comprises 2 orders of non-calcifying taxa, the Phaeocystales and the Prymnesiales, together with the calcifying coccolithophores making up a monophyletic clade (the sub-class Calcihaptophycidae) containing 4 orders (Isochrysidales, Coccolithales, Syracosphaerales, Zygodiscales).

There are some well-known non-calcifying haptophyte taxa, such as Phaeocystis, Prymnesium and Chrysochromulina, that form periodic harmful or nuisance blooms in coastal environments. However, the most familiar haptophytes are the coccolithophores, members of the Prymnesiophyceae that, in addition to a proximal layer of organic body scales, are covered with a distal layer of calcified scales (coccoliths) that are also formed intracellularly and that often have complex ornamentation. Coccolithophores are responsible for a large part of modern oceanic carbonate production and are thus key actors in global carbon cycling (Rost and Riebesell 2004).


Heteromorphic life histories have been documented in many members of the haptophyte class Prymnesiophyceae. These include alternations between non-motile and flagellated stages, between colonial and single cell stages, and between benthic and planktonic stages. The earliest studies on haptophyte life cycles focused mainly on members of the coccolithophores families Pleurochrysidaceae and Hymenomonadaceae (order Coccolithales), these being relatively easy to maintain in laboratory culture. Alternation of a non-calcifying (‘Apistonema’) stage with a coccolith-bearing stage has been reported in Ochrosphaera (Schwarz 1932; Lefort 1975), Hymenomonas (Fresnel 1994) and Pleurochrysis (Leadbeater 1970, 1971; Gayral and Fresnel 1983). In Pleurochrysis carterae (Rayns 1962) and Hymenomonas lacuna (Fresnel 1994), chromosome counting confirmed that the non-calcifying stage in these life cycles is haploid and the calcifying stage diploid, providing the first hard evidence of the existence of haplodiplontic life cycles in haptophytes (Fig. 6).

Figure 6

These life cycles were relatively easy to discern due to the presence of coccoliths (that are visible in light microscopy) in one of the phases. Two main types of coccoliths exist: heterococcoliths (formed of a radial array of complex-shaped interlocking crystals units) and holococcoliths (constructed of numerous small, similar sized and simple-shaped calcite elements). A culture study by Parke and Adams (1960) on the non-motile heterococcoliths bearing stage of Coccolithus braarudii (Coccolithales) demonstrated an alternation with Crystallolithus hyalinus, a motile stage bearing holococcoliths. Prior to this observation, heterococcolithophores and holococcolithophores had been considered as taxonomically discrete groups of species.

Reviewing these and other studies, Billard (1994) suggested that haptophyte life cycles typically include haploid and diploid phases, each capable of independent asexual reproduction (haplodiplonty), with distinct patterns of body scale ornamentation (and in some cases coccolith type) characteristic of each ploidy state. Prymnesiophycean body scales are composed of microfibrils and contain proteins and carbohydrates including cellulose (Leadbeater 1994 and references therein). The proximal (“body”) scales are composed of two layers with the proximal face (facing the cell membrane) having a radial pattern of microfibrils often arranged into quadrants, whereas the distal face either has a radial pattern or an interwoven spiral pattern of concentric rings. In this scheme, the body scales of the diploid cells have identical (radial) ornamentation on both sides, whereas those of the haploid stage have distinct patterns on the proximal and distal faces (radial and spiral, respectively). Billard (1994) predicted that the heterococcolith bearing phase of C. braarudii is diploid and the holococcolith-bearing phase haploid, by likening the patterns of body scale ornamentation in the known haplodiplontic life cycles of species in the Pleurochrysidaceae and Hymenomonadaceae with those in the life cycle of C. braarudii as illustrated by Manton and Leedale (1969). A number of other coccolithophores for which body scales have been illustrated in one phase only fi t this pattern, including the heterococcolithophores Syracosphaera pulchra (Inouye and Pienaar 1988), Umbilicosphaera hulburtiana (Gaarder 1970) and Jomonlithus littoralis (Inouye and Chihara 1983), and the holococcolithophores Calyptrosphaera sphaeroidea (Klaveness 1973) and Calyptrosphaera radiata (Sym and Kawachi 2000).

DNA quantification by flow cytometry later confirmed the haplodiplontic nature of the holococcolithophore-heterococcolithophore life cycle of Coccolithus braarudii as well as of two other species for which both phases were maintained in culture (Houdan et al. 2004). Further indirect evidence that Haplo-diplontic life cycles are widespread in coccolithophores comes from observations in field samples from various locations of ‘combination coccospheres’ bearing both heterococcoliths and holococcoliths, interpreted as capturing the instant of a life cycle phase change (Thomsen et al. 1991; Kleijne 1992; Alcober and Jordan 1997; Young et al. 1998; Cros et al. 2000; Cortes and Bollmann 2002; Geisen et al. 2002; Cros and Fortuno 2002). These observations indicate that life cycles with alternating heterococcolith-bearing (diploid) and holococcolith-bearing (haploid) stages span a large part of the diversity of coccolithophores.

Over time, culture studies and observation of combination coccospheres have demonstrated that diploid generations in coccolithophore life cycles always bear heterococcoliths, whereas the haploid generations are covered by either holococcoliths (Coccolithaceae, Calcidiscaceae, Helicosphaeraceae, Syracosphaeraceae), aragonitic coccoliths (Polycrater), nannoliths (Ceratolithaceae), a non-calcifying benthic stage (Pleurochrysidaceae, Hymenomonadaceae) or a non-calcifying motile stage (Noëlhaerhabdaceae) (Billard and Inouye 2004 and references therein).

Alternation between generations of different ploidy levels are known to occur in each of the other two non-calcifying orders within the Prymnesiophyceae. In the Phaeocystales, the most complete information on the life cycle has been obtained for Phaeocystis globosa (reviewed by Rousseau et al. 2007). A Haplo-diplontic life cycle has been described for this species, which includes diploid colonial cells (recorded either as free non-motile cells or within colonies), diploid flagellate cells without organic scales, and two types of haploid flagellates surrounded by organic scales, differing in size (meso- and microflagellates). P. Antarctica is thought to exhibit a similar life cycle (Zingone et al. 2011). In the Prymnesiales, Prymnesium parvum has been shown to be the diploid stage in a life cycle in which the haploid stage was originally described as a separate species, P. patelliferum (Larsen and Medlin 1997; Larsen and Edvardsen 1998) and Prymnesium polylepi (=Chrysochromulina polylepis) has also been demonstrated to have a Haplo-diploid cycle (Edvardsen and Vaulot 1996; Edvardsen and Medlin 1998). In each of these cases, body scale ornamentation fits the scheme of Billard (1994) and in some cases morphological differences are also evident in the distal organic scales (e.g., Probert and Fresnel 2007). The details of these non-calcified scales can only be observed by electron microscopy, generally making life cycles in these non-calcifying haptophytes difficult to identify.

In summary, dimorphic Haplo-diplontic life cycles appear to be widespread in the Prymnesiophyceae. To date, alternation of generations has not been demonstrated in members of the other haptophyte class, the Pavlovophyceae. The species within this distinctive clade do not possess the ornamented plate scales which have often proved indicative of ploidy state in the Prymnesiophyceae, and given the fact that different ploidy stages in many non-calcified members of the latter class can only be morphologically distinguished by this character, it is perhaps not surprising that the potential existence of Haplo-diplontic life cycles in the Pavlovophyceae has not been recognized. Transitions from non-motile to motile cells are common in the Pavlovophyceae (Billard 1994; Bendif et al. 2011), but relative motility is not often a good indicator of ploidy state.

There are few reports of cysts in the Haptophyta. Cysts of Prymnesium were described by Carter (1937) and Conrad (1941) and have been investigated by Pienaar (1980) who has shown that the walls of Prymnesium parvum cysts are composed of layers of scales with siliceous material on the distal surfaces. It is not known whether formation of these cysts is related to ploidy level.

It should be noted that in haptophyte life cycles the existence and place of sexuality, if applicable, generally remains unknown (Billard 1994). In the coccolithophores, sexuality has been revealed by direct observation of syngamy in only three species, Ochrosphaera neapolitana (Schwarz 1932), Pleurochrysis pseudoroscoffensis (Gayral and Fresnel 1983) and Coccolithus braarudii (Houdan et al. 2004). In O. neapolitana, meiosis, isogamete formation and syngamy were reported by Schwarz (1932). From light microscope observations on Coccolithus braarudii (Houdan et al. 2004) and Pleurochrysis pseudoroscoffensis (Gayral and Fresnel 1983), certain general inferences can be made about the meiotic process in coccolithophore life cycles. In Coccolithus braarudii, meiosis may occur within the heterococcosphere prior to production of the flagellar apparatus and subsequent liberation of the motile cell. Since only one viable cell emerges this would imply the redundancy of the other haploid nuclei formed by the meiotic divisions. Meiosis in the chlorophyte Spirogyra (Harada and Yamagishi 1984) is one of the best-known examples of this pattern. Alternatively, the motile cell that emerges from the heterococcosphere may still be diploid, with nuclear reduction occurring in subsequent divisions. The observation that holococcolith production does not commence immediately after formation of the flagellar apparatus may be interpreted as providing support for this second hypothesis (Houdan et al. 2004). In the life cycle of P. pseudoroscoffensis, four non-calcified motile haploid cells are formed within a heterococcosphere and following release, these cells remain motile for a short time before settling and dividing asexually to initiate the haploid non-calcified pseudofilamentous stage (Gayral and Fresnel 1983). Comparable ‘meiospores’ are formed in the life cycles of other members of the Pleurochrysidaceae (von Stosch 1967; Leadbeater 1971) and the Hymenomonadaceae (Fresnel 1994). In coccolithophores, the mode of initial gamete attraction and contact is not known, but once initiated, syngamy can clearly be completed within a very short period of time. The rapid initiation of heterococcolith production in the zygote observed in C. braarudii by Houdan et al. (2004) and reported for P. pseudoroscoffensis by Gayral and Fresnel (1983) indicates that the two haploid nuclei must fuse immediately following cytoplasmic fusion. In both species, a complete heterococcosphere was formed within 24 hours of the onset of fusion. From this limited evidence, it appears that in coccolithophores fusing gametes are isogamous and are morphologically indistinguishable from vegetative haploid cells, and that fusion can occur within a clone (homothallism). To our knowledge, crossing experiments between haploid coccolithophore strains have never been attempted.

In Phaeocystis globosa, micro- and mesoflagellates (haploid stages) are produced (presumably by meiosis) within the colony and are eventually released and multiply vegetatively. The life cycle is completed by syngamy between a micro- and mesoflagellate that develops into a diploid macroflagellate that is believed to develop into a new colony. However, the formation of colonial stages was never observed in cultures of P. globosa containing only haploid cells (Vaulot et al. 1994). This could be explained either by assuming that conjugation only occurs between the two heteromorphic types of haploid flagellates (anisogamy) or that different mating types exist amongst haploid flagellates (heterothallism). A non-motile zygote linking the haploid unicellular stages and the diploid colonial stages has been documented in P. Antarctica (Gaebler-Schwarz et al. 2010). This zygote can divide vegetatively as a benthic palmelloid stage and not revert to the colonial stage at least in culture conditions.

Studies on the mechanisms of sexual reproduction in haptophytes are currently restricted by the limited number of cultures available and, moreover, by the lack of clear indications as to the factor(s) responsible for the induction of life cycle phase changes in this group. The transition between the life stages in haptophytes is presumably controlled by the interplay of endogenous and environmental factors, but the role and the relative importance of these factors are poorly known. A number of reports suggest that in the Pleurochrysidaceae factors such as temperature and light (Leadbeater 1970) and the addition of fresh medium (Inouye and Chihara 1979; Gayral and Fresnel 1983) may influence phase changes. In cultures of Calyptrosphaera sphaeroidea, Noel et al. (2004) demonstrated that exposure to selected vitamins and trace metals induced the transition to the heterococcolith-bearing phase, whereas a slightly higher concentration of components in the basic medium along with concomitant stresses of light and temperature induced formation of the holococcolith-bearing phase.

Houdan et al. (2004) suggested that concentrations of inorganic or organic trace elements in the medium may have played a role in phase change induction in three coccolithophore species, and also hypothesized that a biological clock may be involved in this process.

Life cycle transitions with each phase adapted to distinct ecological niches may also be an integral part of the ecological strategy of haptophytes. In ecological terms, a Haplo-diplontic life cycle is generally considered as an adaptation to an environment which is seasonally variable or that contains two different niches (see review by Valero et al. 1992). There is some evidence that heterococcolith-bearing and holococcolith-bearing or non-calcifying phases in the life cycle of certain coccolithophore species have differential in situ spatiotemporal distributions (Cros and Fortuno 2002; Frada et al. 2012). In general, holococcolith-bearing stages (which always possess flagella) may be adapted to warm stratified surface waters, whereas the more robust (and often non-motile) heterococcolith-bearing stages may be better suited to turbulent mixed-layer waters. Noel et al. (2004) extrapolated from growth medium preferences of the two stages of Calyptrosphaera sphaeroidea to propose a hypothetical ecological cycle in which the holococcolith-bearing stage occurs in offshore waters and the heterococcolith-bearing stage in coastal waters. Very few studies have experimentally compared the physiological characteristics of different ploidy stages in haptophytes, but in these studies differences in responses to physic-chemical and/or biotic parameters have consistently been found.

For example, in the extremely abundant coccolithophore Emiliania huxleyi, which has a life cycle with diploid non-motile heterococcolith-bearing cells alternating with haploid flagellate non-calcifying cells, the haploid stage appears to be relatively sensitive to high light (Houdan et al. 2005), but not susceptible to E. huxleyi specific viruses (EhVs) that routinely infect and kill diploid cells (Frada et al. 2008). Dramatic differentiation in gene expression between diploid and haploid phases of the coccolithophore E. huxleyi have been demonstrated, with greater transcriptome richness in diploid cells suggesting they may be more versatile for exploiting a diversity of rich environments whereas haploid cells appear to be intrinsically more streamlined (Von Dassow et al. 2009).

Oct 25, 2016

Reproduction in Dinoflagellates

Dinoflagellate sexual reproduction has long been disputed, and a 1973 textbook on protozoology contained no reference to this phenomenon (Grell 1973). The earliest documented report of sexual reproduction in dinoflagellates was Joseph’s (1879) description of pairing and fusion of swimming cells of Peridinium stygium, but careful studies by von Stosch in the 1960s (1965; 1969; 1972; 1973) have since transformed our understanding of dinoflagellate sexuality. With researchers’ increasing capacity to maintain laboratory cultures, sexuality has now been documented for some 100 species (Walker 1984; Wall and Dale 1968; Blackburn et al. 1989; Blackburn and Parker 2005). The reasons that sexual reproduction in dinoflagellates had so long been overlooked include: (1) gametes can look similar to vegetative cells; (2) gamete fusion is easily confused with cell division; (3) ‘warty’ zygotes have often been interpreted as aberrant cells (Pfister and Anderson 1987). To date sexual life cycles are increasingly elucidated using nuclear staining and flow cytometric techniques, not only in culture but also field surveys.

Life Cycle

The life cycle of Gymnodinium catenatum is representative of those observed in many dinoflagellates (Fig. 4). Motile vegetative cells divide vegetatively (by mitosis) to form chains. With the onset of sexual reproduction, vegetative division results in single cell gametes. Usually two types of gametes from different clonal strains (heterothallism) are required for sexual reproduction, pairs of which fuse to give a planozygote. This cell loses motility to form a benthic resting cyst (hypnozygote). Excystment produces a planomeiocyte, similar to a planozygotes, which divides (by meiosis) to re-establish the planktonic vegetative stage (after Blackburn et al. 1989). In Spanish cultures studied by Figueroa et al. (2006a, 2008) (dotted lines in Fig. 4), most planozygotes divided by binary fission to produce vegetative cells but without undergoing a cyst stage. Furthermore, some fusing gamete pairs did not form planozygotes but went through a division process before completing cytoplasmic fusion.
Figure 4
Patterns of Sexual Reproduction
All dinoflagellates studied to date, but one, exhibit the haplontic type of life cycle, the dominant vegetative stage (the one undergoing vegetative growth) being haploid. The single exception is Noctiluca which is claimed to have a diplontic life cycle (Zingmark 1970). Asexual reproduction can happen much more quickly, and therefore is the predominant manner of reproduction during optimal environmental conditions, but sexual reproduction is essential for species adaptation and allows for genetic recombination. Under appropriate conditions, dinoflagellate gametes are produced and fuse to form a diploid planozygote. Often gametes swim faster, can be paler in color and collect in ‘dancing groups’. Fusing pairs of gametes can be distinguished from dividing vegetative cells because their cingula are perpendicular. Dinoflagellates often produce gametes that do not differ morphologically from vegetative cells (a condition called hologamous). Fusing gametes can be identical to each other (isogamous) or be different from each other (anisogamous, e.g., Ceratium cornutum, C. horridum, Alexandrium tamarense). As for diatoms, species are monoecious or homothallic, i.e., sexual reproduction can occur within a clone (e.g., Alexandrium taylori), or dioecious or heterothallic, i.e., two different mating types (designated plus or minus) must be combined. Sexual compatibility can comprise only two different mating types (simple heterothallism), such as in Lingulodinium polyedrum (Figueroa and Bravo 2005b) or more complex heterothallism, such as in Alexandrium minutum (Figueroa et al. 2007). Sexual mating compatibility has been used to elucidate species synonymy but also genetic affinities between geographic populations of the same species (Blackburn et al. 2001).

Three types of zygotes have been reported: (1) planozygote motile throughout and meiosis is completed without cyst formation (e.g., Ceratium horridum); (2) planozygote loses motility and forms a temporary cyst (e.g., Helgolandinium subglobosum); (3) planozygote forms a resting cyst or hypnozygote. The planozygote can be identified by two (‘ski track’) longitudinal flagella. This stage often develops into a resting cyst with a thick resistant cell wall and often requires a period of dormancy before germination is possible. Studies on freshwater dinoflagellates in particular have indicated that the timing of meiotic division in the sexual phase is variable (Pfister 1975, 1976, 1977). Meiosis occurs (1) within the planozygote; or (2) after excystment to release an appropriate number (2–4) of daughter cells; or (3) by subsequent divisions of the single meiocyte released by excystment. In a growing number of dinoflagellate zygotes, the nucleus has been observed to enlarge further and rotate rapidly within the cell (so called ‘nuclear cyclosis’ first described by Pouchet 1883) which von Stosch (1973) associated with the onset of meiosis.

Until recently the most common pathway observed was the transition of planozygote to resting cyst but it is now thought that the planozygote can also skip cyst formation (Figueroa and Bravo 2005a,b). Other possible pathways are: (1) gametes can revert to an asexual phase and undergo binary fission rather than fusion (e.g., Gymnodinium nolleri, G. catenatum,

Alexandrium taylori or Lingulodinium polyedrium); and (2) planozygotes undergo meiosis and division without the production of a hypnozygote (Figueroa and Bravo 2005a, b; Figueroa et al. 2006a, b). In some species for which a sexual cycle has been reported no resting cyst is known (e.g., Karlodinium venefi cum, Karenia brevis; Walker 1982). Asexual resting cysts are also known, e.g., in Scrippsiella hangoei (Kremp and Parrow 2006). Another type of quiescent stage is what is variably called temporary, pellicle or ecdysal cysts with a thin wall and limited capacity to withstand adverse environmental conditions, either produced sexually or asexually.

Cyst as Survival Strategies
Interest in dinoflagellate sexual reproduction was triggered in the early 1960s with the recognition that many fossil cysts (first described by Ehrenberg 1838 from Cretaceous flints as ‘hystrichosphaeres’) are in fact dinoflagellate hypnozygotes. Wall and Dale (1968) conducted the first experiments incubating living ‘cysts’ from Woods Hole bottom sediments.

Excellent cyst preservation in the fossil record is due to the presence of sporopollenin in the wall of many (but not all) species (Fig. 5). Currently more than 80 marine and 15 freshwater species of modern dinoflagellates are known to produce resting cysts. This number of cyst-producing species is small however compared with the total number of extant dinoflagellates (more than 2000). Resting cysts can survive harsh environmental conditions and thus play an important ecological role as the inoculum for recurrent blooms. Dinoflagellate cysts can remain viable in sediments for up to 100 yrs. They also facilitate expansion of the geographical distribution of a species through cyst dispersal via ocean currents and even ship ballast water discharge.

Factors Triggering Sexual Reproduction
Sexuality has been traditionally achieved in culture through nutrient depletion, with temperature and light being important modulators of the cyst yield. However, gamete pairing and planozygote formation in nature may not be always linked to nutrient shortage, since sexuality has been observed either at termination of blooms or during active growth, or when reaching a cell abundance threshold (Garcés et al. 2002).
Figure 5 Sexually Produced Dinoflagellate Resting Cysts from Top Left to Bottom Right
 The conditions that trigger cyst formation and cyst germination have become the key focus of predicting blooms of cyst forming toxic dinoflagellates such as Alexandrium tamarense, Gymnodinium catenatum and Pyrodinium bahamense (Anderson and Wall 1978; Dale 1983). The dormancy period is a maturation time during which biological activity is suspended; this can last from hours to days (Kryptoperidinium foliaceum), weeks (Gymnodinium catenatum) to months (Alexandrium tamarense). Germination cannot be induced during dormancy, but once completed the cyst enters quiescence, during which germination can occur if environmental conditions are suitable. A genetic (endogenous) control of dormancy has been documented in some species (Anderson and Kiefer 1987), but exogenous (abiotic and physiological) modulators and internal clocks (endogenous rhythms) also play a role. Germination patterns thus can drive bloom strategies and seasonal species succession. Once germination occurs, the size of the inoculum will be influenced not only by the number of germinating cysts but also by their viability.