A NEW SPECIES OF VOLVOX SECT. MERRILLOSPHAERA (VOLVOCACEAE,CHLOROPHYCEAE) FROM TEXAS1

Smith (1944) divided the familiar genus Volvox L. into four sections, placing seven species that lacked cytoplasmic bridges between adult cells in the section Merrillosphaera. Herein, we describe a new member of the section Merrillosphaera originating from Texas (USA): Volvox ovalis Pocock ex Nozaki et A. W. Coleman sp. nov. Asexual spheroids of V. ovalis are ovoid or elliptical, with a monolayer of 1,000–2,000 somatic cells that are not linked by cytoplasmic bridges, an expanded anterior region, and 8–12 gonidia in the posterior region. Visibly asymmetric cleavage divisions do not occur in V. ovalis embryos as they do Volvox carteri F. Stein, Volvox obversus (W. Shaw) Printz, and Volvox africanus G. S. West, so the gonidia of the next generation are not yet recognizable in V. ovalis embryos prior to inversion. Molecular phylogenetic analyses of the five chloroplast genes and the internal transcribed spacer (ITS) regions of nuclear rDNA indicated that V. ovalis is closely related to Volvox spermatosphaera Powers ( Powers 1908 , as “spermatosphara”) and/or Volvox tertius Art. Mey.; however, V. ovalis can be distinguished from V. spermatosphaera by its larger gonidia, and from V. tertius by visible differences in gonidial chloroplast morphology.     

BACTERIAL ENDOSYMBIONTS IN VOLVOX CARTERI (CHLOROP HYCEAE)1

The morphology of the bacterial endosymbiont of Volvox carteri Stein (Clone KA-1) was studied with the electron microscope. Endosymbionts were found in the cytoplasm of somatic cells, gonidia and sperm, but never in nuclei, chloroplasts or mitochondria. DNA preparations contained, an extra DNA species assumed to be endosymbiont DNA. Attempts to isolate the endosymbionts or to “cure” the alga with antibiotics were unsuccessful. All progeny from crosses of infected and noninfected strains contained the endosymbiont.     

Do Protoplasmic Connections Function in the Phototactic Coordination of the Volvox Colony During Light Stimulation?*

SYNOPSIS. The flagellar behavior of the colonial flagellates Volvox carteri Stein and Volvox perglobator Powers was examined by placing 1.01 μm polystyrene particles in solution with swimming colonies, and photographing these particle movements. When directional light stimulation was administered to individual colonies, a cessation of flagellar activity occurred in the anterior cells of the stimulated side in both species. Since Volvox perglobator possesses prominent intercellular connections and Volvox carteri does not, the results of these experiments suggest that the connections linking colony members in some species do not function in the coordination of flagellar activity associated with light orientation behavior.     

Characterization of the sex-inducer glycoprotein ofVolvox carteri f.weismannia

Summary Sexual inducer pheromones fromVolvox carten f.weismannia, strains 65–30(12) and 1B were purified and characterized as glycoproteins with apparent molecular weights of 27 kDa and 28.5 kDa, respectively. This subspecies yielded 20–40 times more pheromone based on weight per spheroid thanVolvox carteri f.nagariensis, but its specific activity (threshold dilution) is four to five orders of magnitude less (10^–12 to 10^–13 M). Gaschromatographic sugar analysis revealed quantitative differences in the composition of theO- andN-glucans compared with theV. carteri f.nagariensis inducer. TheV. carteri f.weismannia pheromones showed antigenic cross-reaction with an antiserum directed against chemically deglycosylated inducer fromV. carteri f.nagariensis. However, there is only unilateral biological cross-induction. TheV. carteri f.nagariensis inducer is strictly competent for its own gonidia only; the inducers fromV. carteri f.weismannia also cross-induceV. carteri f.nagariensis. This pattern of cross-induction suggests the existence of related pheromone receptors but with different ligand specificities.     

Characterization of a sperm lysin of Volvox carteri

Summary A cell-wall degrading enzyme has been isolated from mature sperm packets of the green flagellate Volvox carteri (Poona strain). This sperm lysin (S-lysin) is a Ca²⁺-dependent protease of 34 kDa with an essential serine group in its active centre. Neither SH group-blocking reagents nor transition metal chelators inhibit its action. S-lysin degrades the hydroxyproline-rich glycoprotein structures of the cell walls of sheath cells and gonidia (eggs) of vegetative and sexual spheroids in a characteristic manner. In asexual spheroids the somatic envelope is totally disintegrated, whereas in sexual spheroids pores are formed by local lysis at sites of adjacent eggs. Although S-lysin is very similar to the G-lysin of the closely related Chlamydomonads, it is species specific and does not attack the mother or daughter cell walls of Chlamydomonas reinhardtii. S-lysin resembles the aerosin of animal sperm cells in some aspects of its action.Dedicated to Professor Richard C. Starr on the occasion of his 65th birthday. He called the piper and gave the tune     

Spontaneous frequency of a developmental mutant in Volvox

Two types of mutants, those resistant to the base analog 5-bromo-2′-deoxyuridine (BrdU) and somatic regenerator (SR) mutants, have been analyzed in Volvox carteri. In somatic regenerator mutants, the somatic cells which are normally terminally differentiated dedifferentiate and regenerate gonidia [Sessoms, A., and Huskey, R. J. (1973). Proc. Nat. Acad. Sci. USA70, 1335–1338; Starr, R. C. (1970). Develop. Biol. Suppl.4, 59–100]. The SR phenotype allows recovery of SR mutations arising in somatic cells, since such somatic cells would regenerate gonidia and give rise to mutant clones. Mutants of any phenotype other than SR can only be recovered if the mutation first appears in a gonidium. Since the somatic cells are 100-fold more numerous than reproductive cells (gonidia), we have determined the spontaneous frequency of both somatic regenerator mutants and mutations to BrdU resistance in order to determine if the SR mutation exerts its effect in the gonidium or in the somatic cell. The two frequencies were found to be nearly identical, suggesting that the SR mutation must first appear in a gonidium in order to be expressed.     

FEULGEN MICROSPECTROPHOTOMETRIC STUDIES OF PANDORINA MORUM AND OTHER VOLVOCALES (CHLOROPHYCEAE)12

The nuclear DNA content during normal vegetative growth and division has been examined in three species of Volvocales, Chlamydomonas reinhardtii Dangeard, Pandorina morum Bory, and Volvox carteri f. nagariensis Iyengar. The results are consistent with the nuclear cycle reported in the literature for Eudorina. Nuclear DNA content does not increase during the prolonged cell growth phase. At the time of colony formation, nuclear DNA doubles, the nucleus divides, and this alternation continues until the final 2ⁿ complement of progeny nuclei is formed. The 4- and 8-nucleate stages of dividing gonidia of V. carteri have a nuclear DNA content in the same range as the somatic cells; they are not polyploid or polytene. Four normal clones of Pandorina, having 2, 5 or 12 chromosomes, all had similar amounts of DNA per nucleus, suggesting that the species has a nuclear genome of fairly constant size rather than consisting of many strains representing a polyploid series. One unique clone, a hybrid with double the chromosome number of either its parents, had twice as much DNA as the normal clones. The Feulgen spectrophotometric method is sufficiently sensitive to detect 2-fold differences in DNA content at the level of 2 × 10^?13 g of DNA /nucleus, and its use avoids the complications associated with the presence of organelle DNA.     

Sexual Differentiation in Volvox aureus*

SYNOPSIS. The complete life history of a homothallic, dioecious strain of Volvox aureus was studied in axenic culture. Asexual reproduction occurs by repeated division of specialized reproductive cells (gonidia), inversion of the resultant mass of cells to form daughter colonies, and subsequent morphologic differentiation of new gonidia in these daughters. Male colonies lack gonidia; however, the posterior 2/3 of the cells in a colony function as male initials each of which enlarges, divides, and undergoes rudimentary inversion to form a packet of 32 biflagellate sperm. Evidence was presented for the homology of eggs and undivided gonidia. The penetration of young vegetative colonies by sperm and the subsequent formation of apparent zygotes in these colonies was described and figured. Zygote germination, including division and inversion of the germling, was described; the cytological nature of the zygote divisions was not determined. A substance, MIS, in filtrates of sexual cultures induced differentiation of male colonies; a bioassay for it was perfected. Bioassay colonies show a differential susceptibility to male induction by MIS which is a function of the particular stage of development; colonies 48 hr after release from parentals are optimally susceptible. MIS was reasonably stable to heat, non-dialyzable, and Sephadex gel filtration indicated a molecular weight > 200,000. MIS activity is destroyed by trypsin and pronase but is unaffected by chymotrypsin. MIS was successfully concentrated ～37-fold by the Carbowax method. The patterns of differentiation in other species of Volvox are described and possibilities discussed for studies of cellular differentiation in the genus as a whole.     

Differentiation of Reproductive Cells in Volvox carteri*

SYNOPSIS. The life cycle of Volvox carteri was studied in axenic culture using the NB-3 and the NB-7 strains isolated from Nebraska. Vegetative colonies of both strains contain 8–12 asexual reproductive cells (gonidia) which divide to form daughter colonies. During daughter colony formation, the reproductive cells of the daughters are delimited at an early stage of cleavage. Gonidia are delimited at the division from 16 to 32 cells, but eggs and male initial cells are not differentiated until the division of the 32-celled stage. In all instances the reproductive cells are the products of unequal cleavages. Male and female colonies are formed in separate clones. Female colonies contain approximately 20 eggs. Male colonies have approximately 50 male initial cells, each of which forms a sperm bundle containing 64 or 128 sperm. Sperm bundles penetrate female colonies and fertilize the eggs. Zygote formation, zygote germination, and the development of gone colonies is described. Sexual type was inherited in a 1:1 ratio. Male colonies appear spontaneously in the male strain, but female colonies were formed in the female strain only in the presence of a substance produced by colonies from male cultures. This female inducing substance is produced in male cultures primarily, if not exclusively, by male colonies rather than by vegetative colonies. The female inducing substance is heat labile and non-dialyzable. Activity is destroyed by Pronase, but not by trypsin, chymotrypsin or ribonuclease. Gonidia appear to be most susceptible to female induction during the early stages of their expansion prior to cleavage.     

A microspectrofluorometric analysis of nuclear and chloroplast DNA in Volvox

Nuclear division immediately follows nuclear DNA doubling in all stages of the life cycle examined in the green alga Volvox; fluorescence microfluorometry of individual cells revealed no evidence of prolonged accumulation of nuclear DNA prior to mitosis in reproductive cells. Somatic cell nuclear DNA quantity is unaffected by developmental events in gonidia of the same spheroid; it remains constant from the end of cleavage until the death of the cell. In reproductive cells, chloroplast DNA replication precedes nuclear replication. The sites of plastid DNA accumulation, made visible by use of the fluorochrome 4′,6-diamidino-2-phenylindole, increase in number during the prolonged growth phase of the V. carteri gonidium. Microspectrofluorometry of fluorochrome-stained DNA in situ shows that plastid DNA increases exponentially throughout this phase. The continuous plastid DNA accumulation during gonidial growth appears to represent a prokaryote-like instead of a eukaryote-like control of DNA synthesis. Most somatic cells contain plastid DNA, and this does not increase in amount during colony growth and reproduction. Most sperm cells also contain plastid DNA, although approximately 5% of somatic cells and up to 20% of sperm cells have no discernable plastid DNA. This is the second group of organisms in which DNA-free plastids have been observed.     

EFFECT OF HISTONE DEACETYLASE INHIBITORS ON TUBULIN ACETYLATION AND DEVELOPMENT IN VOLVOX CARTERI (VOLVOCALES)1

Volvox carteri f. nagariensis (Iyengar) possesses several thousand cells of just two types, gonida and somatic cells, that are set apart by asymmetric cell division. Because the division apparatus contains microtubules enriched in acetylated α‐tubulin, we wished to know whether acetylated tubulin plays any role in regulating division symmetry. Two different human histone deacetylases (HDACs) have been shown to deacetylate tubulin in vivo, thereby regulating cell motility. Here we set out to determine: (1) whether HDAC inhibitors that increase tubulin acetylation in animal cells have the same effect in V. carteri, (2) whether increasing acetylated tubulin affects microtubule stability, and (3) whether increasing acetylated tubulin affects division symmetry. Embryos exposed to two HDAC inhibitors, trichostatin A (TSA) and tubacin, accrued dramatically higher levels of acetylated tubulin (and more acetylated microtubules) and were significantly more sensitive to colchicine than controls. However, while TSA‐treated embryos cleaved aberrantly to produce adults with abnormal morphology, tubacin‐treated embryos developed normally. We conclude that increasing tubulin acetylation subtly alters microtubule stability, but does not appear to affect cell division in V. carteri.     

Protein synthesis in a new system for the study of senescence

In asexual individuals of the green alga Volvox carteri, more than 99% of the cells are somatic cells which undergo synchronous programmed senescence and cell death every generation. Only a small number of reproductive cells survive to produce the next generation. The specific activity of pulse-labelled somatic cell protein preparations declines sharply during senescence, but no decline is seen in the nonageing reproductive cells. Two-dimensional polyacrylamide gel electrophoresis reveals that somatic and reproductive cells synthesize very different patterns of polypeptides. During the period when observable senescent changes are first evident in somatic cells, there is a change in the pattern of polypeptides being synthesized. Our results suggest that senescence in Volvox somatic cells is triggered by a change in the pattern of gene expression and are consistent with theories of programmed cell senescence.     

The relationship between cell size and cell fate in Volvox carteri

In Volvox carteri development, visibly asymmetric cleavage divisions set apart large embryonic cells that will become asexual reproductive cells (gonidia) from smaller cells that will produce terminally differentiated somatic cells. Three mechanisms have been proposed to explain how asymmetric division leads to cell specification in Volvox: (a) by a direct effect of cell size (or a property derived from it) on cell specification, (b) by segregation of a cytoplasmic factor resembling germ plasm into large cells, and (c) by a combined effect of differences in cytoplasmic quality and cytoplasmic quantity. In this study a variety of V. carteri embryos with genetically and experimentally altered patterns of development were examined in an attempt to distinguish among these hypotheses. No evidence was found for regionally specialized cytoplasm that is essential for gonidial specification. In all cases studied, cells with a diameter > approximately 8 microns at the end of cleavage--no matter where or how these cells had been produced in the embryo--developed as gonidia. Instructive observations in this regard were obtained by three different experimental interventions. (a) When heat shock was used to interrupt cleavage prematurely, so that presumptive somatic cells were left much larger than they normally would be at the end of cleavage, most cells differentiated as gonidia. This result was obtained both with wild-type embryos that had already divided asymmetrically (and should have segregated any cytoplasmic determinants involved in cell specification) and with embryos of a mutant that normally produces only somatic cells. (b) When individual wild-type blastomeres were isolated at the 16-cell stage, both the anterior blastomeres that normally produce two gonidia each and the posterior blastomeres that normally produce no gonidia underwent modified cleavage patterns and each produced an average of one large cell that developed as a gonidium. (c) When large cells were created microsurgically in a region of the embryo that normally makes only somatic cells, these large cells became gonidia. These data argue strongly for a central role of cell size in germ/soma specification in Volvox carteri, but leave open the question of how differences in cell size are actually transduced into differences in gene expression.     

microRNAs in a multicellular green alga Volvox carteri

microRNAs(miRNAs)have emerged as key components in the eukaryotic gene regulatory network.We and others have previously identified many miRNAs in a unicellular green alga,Chlamydomonas reinhardtii.To investigate whether miRNA-mediated gene regulation is a general mechanism in green algae and how miRNAs have been evolved in the green algal lineage,we examined small RNAs in Volvox carteri,a multicellular species in the same family with Chlamydomonas reinhardtii.We identified 174 miRNAs in Volvox,with many of them being highly enriched in gonidia or somatic cells.The targets of the miRNAs were predicted and many of them were subjected to miRNA-mediated cleavage in vivo,suggesting that miRNAs play regulatory roles in the biology of green algae.Our catalog of miRNAs and their targets provides a resource for further studies on the evolution,biological functions,and genomic properties of miRNAs in green algae.     

Phylogenetic relationships of the green algeVolvox carteri deduced from small-subunit ribosomal RNA comparisons

Summary The 1788-nucleotide sequence of the small-subunit ribosomal RNA (srRNA) coding region from the chlorophyteVolvox carteri was determined. The secondary structure bears features typical of the universal model of srRNA, including about 40 helices and a division into four domains. Phylogenetic relationships to 17 other eukaryotes, including two other chlorophytes, were explored by comparing srRNA sequences. Similarity values and the inspection of phylogenetic trees derived by distance matrix methods revealed a close relationship betweenV. carteri andChlamydomonas reinhardtii. The results are consistent with the view that these Volvocales, and the third green alga,Nanochlorum eucaryotum, are more closely related to higher plants than to any other major eukaryotic group, but constitute a distinct lineage that has long been separated from the line leading to the higher plants.     

Stable nuclear transformation of Gonium pectorale

Background  

Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. The volvocine alga Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of ~16 biflagellate cells arranged in a flat plate. The detailed molecular analysis of any species necessitates its accessibility to genetic manipulation, but, in volvocine algae, transformation procedures have so far only been established for Chlamydomonas reinhardtii and Volvox carteri.     

Genetic basis for soma is present in undifferentiated volvocine green algae

Somatic cellular differentiation plays a critical role in the transition from unicellular to multicellular life, but the evolution of its genetic basis remains poorly understood. By definition, somatic cells do not reproduce to pass on genes and so constitute an extreme form of altruistic behaviour. The volvocine green algae provide an excellent model system to study the evolution of multicellularity and somatic differentiation. In Volvox carteri, somatic cell differentiation is controlled by the regA gene, which is part of a tandem duplication of genes known as the reg cluster. Although previous work found the reg cluster in divergent Volvox species, its origin and distribution in the broader group of volvocine algae has not been known. Here, we show that the reg cluster is present in many species without somatic cells and determine that the genetic basis for soma arose before the phenotype at the origin of the family Volvocaceae approximately 200 million years ago. We hypothesize that the ancestral function was involved in regulating reproduction in response to stress and that this function was later co‐opted to produce soma. Determining that the reg cluster was co‐opted to control somatic cell development provides insight into how cellular differentiation, and with it greater levels of complexity and individuality, evolves.     

VOLVOX POCOCKIAE,A NEW SPECIES WITH DWARF MALES1

Volvox pocockiae is described as the second species in the section Janetosphaera. The somatic protoplasts are connected by cytoplasmic strands approximately the same diameter as flagella, and the construction of the spheroid is identical to that of V. aureus. Asexual reproduction by the division of gonidia differs from that in V. aureus in the enlargement of the gonidium prior to its division to form the embryo. Sexual reproduction is very similar to that in V. spermatosphaera, a species in the section Merrillosphaera without cytoplasmic connections. Dwarf males are formed in the posterior end of the parental spheroid, and, as in V. spermatosphaera, the dwarf males are composed exclusively of androgonidia with no sterile somatic cells. Females are facultatively asexual spheroids, the gonidia of which function as eggs. The single biflagellate zoospore produced by the germinating zygote undergoes cleavage to form a germling spheroid. The differentiation of gonidia in the asexual embryo and in the germling spheroid is evident only after inversion and enlargement of the spheroid have begun.     

VCRPs,small cysteine-rich proteins,might be involved in extracellular signaling in the green alga Volvox

The sex-inducer of the spherical green alga Volvox carteri is one of the most potent biological effector molecules known: it is released into the medium by sexual males and triggers the switch to the sexual cleavage program in the reproductive cells of vegetatively grown males and females even at concentrations as low as 10^-16 M. In an adult Volvox alga, all cells are embedded in an extensive extracellular matrix (ECM), which constitutes >99% of the volume of the spheroid. There exist no cytoplasmic connections between the cells in an adult alga, so any signal transduction between different cells or from the organism''s environment to a reproductive cell must involve the ECM. Recently, a small cysteine-rich extracellular protein, VCRP, was identified in Volvox and shown to be quickly synthesized by somatic cells in response to the sex-inducer. Due to its characteristics, VCRP was speculated to be an extracellular second messenger from somatic cells to reproductive cells. Here a related protein, VCRP2, is presented, exhibiting a 56% amino acid sequence identity with VCRP. Two possible scenarios for signal transduction from the sex-inducer to the reproductive cell are discussed.Key words: cell wall, extracellular matrix, extracellular second messenger, green algae, sex-inducer, sex inducing pheromone, sexual development, stress response, Volvocaceae, wounding     

Mitosis and cytokinesis in androgonidia ofVolvox carteri f.weismannia

Summary Reproductive cells (androgonidia) ofVolvox carteri f.weismannia divide to form packets of 64 or 128 sperm cells. The androgonidium morphology, stages of mitosis, and cytokinesis were examined by electron microscopy. The biflagellate androgonidium loses its flagella before mitosis but the flagellar bases at the anterior end of the cell are retained. Two additional basal bodies are formed and the nucleus migrates from its central position to the area of the basal bodies before mitosis begins. A five-layered kinetochore is present on the chromosomes and remnant nucleolar material persists during mitosis. A furrow at the chloroplast end of the cell and the formation of phycoplast microtubules and vesicles signal the beginning of cytokinesis at early telophase. The cells maintain cytoplasmic connections until after the packet of sperm cells completes its development.