Asexual reproduction and genetic determination of growth form in the coral Pavona cactus: biochemical genetic and immunogenic evidence

Summary Tissue grafting and electrophoresis were used to study the genotypic structure of a population of the scleractinian coral, Pavona cactus. Three growth forms were distinguished within one continuous population of this morphologically variable species. Both techniques provided evidence of localized asexual reproduction within each growth form, a result consistent with numerous field observations of naturally occurring fragments. A perfect association between clonal genotype and growth form was found in an electrophoretic survey of 80 colonies. 23 multi-locus genotypes were detected in the 80 colonies tested. All genotypically similar colonies had the same growth form, even where colonies were separated by 50 m. Although environmental gradients undoubtedly modify colony morphology, the high correlation between genotype and growth form suggests that major differences in colony morphology are genetically determined.Tissue grafting tests did not reliably identify all clones. Fusions developed between all electrophoretically indistinguishable colonies, consistent with the initial assumption that fusion between paired colonies would indicate selfrecognition. However, there was also one fusion in 20 pairings of electrophoretically different colonies. Although there was general agreement between the two techniques, the one inconsistent fusion suggests that caution should be exercised in the application of histocompatibility tests as bioassays for clonal population structure, and that electrophoresis is the more appropriate technique for this species.The ability of genotypes to dominate in intraspecific competitive interactions and to survive fragmentation was assessed. An intraspecific dominance hierarchy was identified among the 6 clones tested. Competition was highly asymmetrical between dominant and subordinate-ranking clones. Genotypes that were most successful in producing widespread clones were found to dominate intraspecific competitive interactions and had high rates of fragment survival. Offprint requests to: D.J. Ayre Contribution No 252 from the Australian Institute of Marine Science     

The Energetics of Asexual Reproduction and Colony Formation in Benthic Marine Invertebrates

There are several formsof individual and colon) organizationavailable to benthic marine invertebrates with indeterminategrowth In particular anthozoan coelenterates exhibit three suchforms of organization 1 solitary individuals without asexualreproduction 2 colonies or clonal groups with determinate polypsize and indeterminate colony size and 3 colonies or clonalgroups with both indeterminate polyp size and indeterminatecolonyor clone size The last group includes species where asexualreproduction is continuous and those where it is limited toa single season of the year A model is presented which definesthree optimal sizes for indeterminately growing organisms 1optimal size for a solitary individual 2 optimal size of a polypwithin a colony and 3 optimal size at asexual division The optimaare derived by maximizing the difference between energy intakeand energetic cost Simulations allow prey size and habitat qualityto vary and derive predictions of optimal size and organizationfor each case Individual and clonal growth rates are modelledfor the case where asexual reproduction is limited to a singleseason and poly p sizes within clones are compared The modelpredicts that the three torms of organization among anthozoanpolyps are related primarily to the size distribution of prey     

Variation in growth and competitive ability between sexually and clonally produced hydroids

In controlled laboratory experiments, colonies of Podocoryna carnea typically overgrow and kill colonies of Hydractinia symbiolongicarpus. Generally, these experiments have used colonies grown from tissue explants (clonal replicates) surgically removed from mature colonies taken from natural populations. In contrast, experiments involving interspecific bouts between small, sexually produced colonies reveal that both the characteristics and outcomes of competition differ from previous studies. During competition between small sexually produced colonies, H. symbiolongicarpus exhibits directional growth toward P. carnea and produces nematocyte-rich hyperplastic stolons more readily than P. carnea does. Nevertheless, P. carnea can still overcome H. symbiolongicarpus if it initially grows away from the contact zone and subsequently flanks H. symbiolongicarpus. Overall, sexually produced colonies of H. symbiolongicarpus destroyed their P. carnea counterparts in more than 35% of competitive bouts, whereas P. carnea dominated their H. symbiolongicarpus counterparts in all similar encounters between clonally produced colonies. In natural populations, competition between small sexually produced colonies of H. symbiolongicarpus may predominate, and these results support the hypothesis that this species is adapted to competition early in colony development. More generally, studies of competition between sexually produced colonies should complement similar studies of clonally produced colonies.     

Growing Yeast into Cylindrical Colonies

Microorganisms often form complex multicellular assemblies such as biofilms and colonies. Understanding the interplay between assembly expansion, metabolic yield, and nutrient diffusion within a freely growing colony remains a challenge. Most available data on microorganisms are from planktonic cultures, due to the lack of experimental tools to control the growth of multicellular assemblies. Here, we propose a method to constrain the growth of yeast colonies into simple geometric shapes such as cylinders. To this end, we designed a simple, versatile culture system to control the location of nutrient delivery below a growing colony. Under such culture conditions, yeast colonies grow vertically and only at the locations where nutrients are delivered. Colonies increase in height at a steady growth rate that is inversely proportional to the cylinder radius. We show that the vertical growth rate of cylindrical colonies is not defined by the single-cell division rate, but rather by the colony metabolic yield. This contrasts with cells in liquid culture, in which the single-cell division rate is the only parameter that defines the population growth rate. This method also provides a direct, simple method to estimate the metabolic yield of a colony. Our study further demonstrates the importance of the shape of colonies on setting their expansion. We anticipate that our approach will be a starting point for elaborate studies of the population dynamics, evolution, and ecology of microbial colonies in complex landscapes.     

Growing Yeast into Cylindrical Colonies

Microorganisms often form complex multicellular assemblies such as biofilms and colonies. Understanding the interplay between assembly expansion, metabolic yield, and nutrient diffusion within a freely growing colony remains a challenge. Most available data on microorganisms are from planktonic cultures, due to the lack of experimental tools to control the growth of multicellular assemblies. Here, we propose a method to constrain the growth of yeast colonies into simple geometric shapes such as cylinders. To this end, we designed a simple, versatile culture system to control the location of nutrient delivery below a growing colony. Under such culture conditions, yeast colonies grow vertically and only at the locations where nutrients are delivered. Colonies increase in height at a steady growth rate that is inversely proportional to the cylinder radius. We show that the vertical growth rate of cylindrical colonies is not defined by the single-cell division rate, but rather by the colony metabolic yield. This contrasts with cells in liquid culture, in which the single-cell division rate is the only parameter that defines the population growth rate. This method also provides a direct, simple method to estimate the metabolic yield of a colony. Our study further demonstrates the importance of the shape of colonies on setting their expansion. We anticipate that our approach will be a starting point for elaborate studies of the population dynamics, evolution, and ecology of microbial colonies in complex landscapes.     

POPULATION STRUCTURE OF A CLONAL GORGONIAN CORAL: THE INTERPLAY BETWEEN CLONAL REPRODUCTION AND DISTURBANCE

Clonality is a common feature of plants and benthic marine organisms. In some cases clonal propagation results in a modest increase in population density, while in other cases dense populations may be generated by the propagation of only a few clones. We analyzed the population structure of the clonal gorgonian Plexaura kuna across several reef habitats with a range of disturbance regimes in the San Blas Islands, Panama, and the Florida Keys, U.S.A. Using multilocus DNA fingerprinting to distinguish clones, we estimated that clones ranged in size from single individuals to 500 colonies. The number of genotypes identified on nine reefs ranged from three to 25. Population density and clonal structure varied markedly among reefs with G_O:G_E ranging from 0.03 to 1.00. On some reefs vegetative reproduction transformed P. kuna from a rare species to the numerically most abundant gorgonian. The effect of clonal propagation on P. kuna population structure was dependent on interactions between fragmentation and the reef environment (disturbance regime, substratum). We present a generalized model relating population structure of clonal species to disturbance and the mode of vegetative propagation. Disturbance promotes colony propagation and skews the size-frequency distribution of clones among P. kuna and many species that propagate via fragmentation. Propagation of these species is promoted by disturbance (disturbance sensitive), and they tend to have clones that are dispersed across local sites. Species that fragment and have dispersed clones, have high genotypic diversity in habitats with low levels of disturbance. Genotypic diversity then decreases at intermediate disturbance and increases again at the highest disturbance levels. Clonal species that do not rely on disturbance for vegetative propagation (disturbance insensitive) generally do not disperse and form aggregated clones. Among these taxa disturbance has a greater affect on individual survival than on propagation. Genotypic diversity is directly related to the level of disturbance until very high levels of disturbance, at which time genotypic diversity declines.     

Variation in clone structure of fragmenting coral reef sponges

Populations of three branching Caribbean demosponge species are composed of clones produced by asexual fragmentation. Dispersal of the fragments before they become established as independent individuals scatters clone members widely and intermixes members of different clones, complicating study of the clone structure of these populations and contrasting with many other sessile clonal organisms. Clone structures of these populations were inferred using a combination of tissue-compatibility relationships and an analysis of variations in morphology and colour. Although tissue compatibility cannot be used for precise identification of sponge clones, in general, patterns of variation in morphological characters influencing fragmentation and patterns of fragment dispersal and recruitment suggest that, in these populations, tissue-compatibility relationships closely reflect clone structure. Conditions that must be met in order to use tissue compatibility for study of sponge clones are discussed, and previous results, from which conflicting conclusions have been drawn, reconciled in this context. Variations among clones in numbers of physiologically independent members and in size and shape of areal extent are discussed in the context of processes that may affect evolution of clonal characters in these populations and in other species that propagate by dispersing asexual fragments.     

Functional differentiation in bryozoan colonies: a proteomic analysis

Bryozoans are typical modular organisms. They consist of repetitive structural units, the zooids. Bryozoan colonies grow by zooidal budding, with the distribution pattern of the budding loci underlying the diversity of colony forms. Budding is usually restricted to the colony periphery, where a “growing edge” or local terminal growth zones are formed. Non-budding parts of the colony can be functionally subdivided, too. In many species colonies consist of regular, often repetitive zones of feeding and non-feeding modules, associated with a periodical degeneration and regeneration of the polypide retractile tentacle crown with a gut and the accompanying musculature. The mechanisms of functional differentiation in bryozoan colonies are unknown. Presumably, budding and/or polypide recycling are induced or inhibited by certain determinants of functional specialization in different colony parts. An effective tool of their identification is the comparison of proteomes in functionally different zones. Here we report the results of proteomic analysis of three bryozoan species from the White Sea with a different colony form: Flustrellidra hispida, Terminoflustra membranaceotruncata and Securiflustra securifrons. Using differential two-dimensional electrophoresis (2D-DIGE), we compared proteomes of the growing edge, the zone with polypides and the zone without polypides. We assessed the general level of differences between the zones and revealed proteins whose relative abundance changed gradually along the proximal-distal colony axis. These proteins might be involved in the determination of the functional differentiation of the colony.     

The characteristics of an anchorage-independent clonal agar assay for primary explanted bovine granulosa cells

Normal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage-independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4-fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells. Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self-renewal as assessed by the ability of cells from primary colonies to reclone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl-1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform in size.     

Determinate growth and modularity in a gorgonian octocoral

Growth rates of branches of colonies of the gorgonian Pseudopterogorgia elisabethae were monitored for 2 years on a reef at San Salvador Island, Bahamas. Images of 261 colonies were made at 6-month intervals and colony and branch growth analyzed. Branch growth rates differed between colonies and between the time intervals in which the measurements were made. Colonies developed a plumelike morphology through a pattern of branch origination and determinate growth in which branch growth rates were greatest at the time the branch originated and branches seldom grew beyond a length of 8 cm. A small number of branches had greater growth rates, did not stop growing, and were sites for the origination of subsequent "generations" of branches. The rate of branch origination decreased with each generation of branching, and branch growth rates were lower on larger colonies, leading to determinate colony growth. Although colonial invertebrates like P. elisabethae grow through the addition of polyps, branches behave as modules with determinate growth. Colony form and size is generated by the iterative addition of branches.     

Colony pattering ofAspergillus oryzae on agar media

To clarity the effects of nutrient concentration and diffusion on the pattern formation of fungal colonies, the colony patterning ofAspergillus oryzae at various nutrient and agar levels was studied experimentally and was summarized in a colony morphology diagram. Roles of the nutrient content and the relaxation of nutrient distribution on the colony patterning were discussed based on a computer model of the mycelial growth. The colony morphology changed from compact to ramified as the nutrient and agar levels were lowered. No clear boundary was found between these two morphologies. The deterioration of substrate around the growing colony was detected when the morphic switching from homogeneous into splitting patterns emerged in the growth of ramified colonies. In the mycelial growth model, dense compact colonies developed at low growth rates and high nutrient influx into the colonized area. Under low nutrient levels, splitting colonies appeared at high growth rates as compared with the nutrient influx.     

Effects of size and growth rate on vegetative reproduction in Typha

Summary The objective of this study was to separate the effects of plant biomass and growth rate on vegetative reproduction in two species of cat-tail, Typha latifolia and T. angustifolia. Replicate clones of both species were grown under conditions of 100%, 42%, 24%, and 9% full sunlight with harvests at 41, 70, and 91 days after shading. T. angustifolia produced most of its vegetative offspring before the first harvest and increased biomass over the remainder of the experiment by increasing the size of its ramets. In contrast, T. latifolia produced vegetative offspring gradually throughout the experiment adding new ramets only after existing clones were of mature size. As a result of these differences in the cloning process, T. angustifolia showed little correlation between vegetative reproduction and clone size while T. latifolia showed a strong correlation between gegetative reproduction and clone size at the three highest light intensities. Growth rates, average clone size and vegetative reproduction were all reduced by reductions in light intensity for both species. However, no effect of growth rate on the relationship between clone size and vegetative reproduction in T. latifolia could be detected. T. latifolia showed greater survivorship and more biomass production under 9% light than T. angustifolia indicating a greater shade tolerance.     

Clonal variation in colony morphology and growth of CHO cells cultured on agar

Single Chinese hamster ovary (CHO) cells plated on agar form macroscopic colonies with high efficiency. Colonies produced by cells from the uncloned cell line increase in diameter continuously for 10–12 days after plating to form mounds of cells about 1 mm in diameter. With further incubation, some of these colonies do not increase in diameter (arrested dome), some form an expanding annular monolayer of cells around the central mound (fried egg), and some grow by enlarging the central mound into a low multilayered disc (saucer).These colony types on agar appear to be clonal characteristics of the CHO cell line. Cloning the line gives two kinds of isolates: one forms a mixture of arrested dome and fried egg colonies in an inheritable ratio, and the other forms saucer colonies. Cells from saucer colonies form saucer colonies when replated on agar. Cells from all colony types replate with similar efficiency on plastic or agar, and exhibit the same growth rate and cell size in liquid suspension culture. On plastic substrate, all these CHO cells form colonies which increase continuously in diameter for as long as 21 days, and little clonal difference in the morphology of colonies or of single cells is observed.These observations reveal a previously unsuspected heterogenieity in an established line of cultured mammalian cells and provide a method for studying new classes of In vitro growth control phenomena. These control phenomena may help in the building an in vitro model for tumor growth.     

Colony formation by subpopulations of human T lymphocytes. VI. Further studies on colony phenotype, function, and cloning efficiency

Phytohemagglutinin (PHA)-induced colony formation in semisolid agar medium by human peripheral blood T lymphocytes showed an increasing cloning efficiency with decreasing numbers of cultured cells. Ninety percent of CD4+ cells (inducer/helper phenotype) and 20% of CD8+ cells (cytotoxic/suppressor phenotype) formed colonies when cultured at 10-200 cells/ml culture in the presence of sheep red blood cells (SRBC) and a source of interleukin-2 (IL-2). Probably all T-colony-forming cells, but none of the subsequent colony cells, expressed the Leu-8 antigen. The cloning efficiencies of FACS-sorted cells expressing the natural killer antigenic phenotypes Leu-7+ and CD16+ were found to be less than 1%. The costimulatory effect of red blood cells for colony formation was specific for SRBC and not observed in the presence of red cells obtained from seven other species including man. All T-lymphocyte colonies obtained from unseparated peripheral blood mononuclear cells expressed the CD25 antigen (IL-2 receptor) and colonies were always composed of either CD4+ or CD8+ cells. None of the colony cells expressed the Leu-8 or the CD16 antigens. By their specific morphology in agar culture the majority of colonies composed of CD4+ cells were easily recognized, but but approximately one-third of the CD4+ colonies could not be distinguished from colonies composed of CD8+ cells. On expansion of individual colonies in liquid subculture in the presence of interleukin-2, approximately 15% of the colonies developed natural killer (NK)-like cytotoxic activity, being capable of direct killing of K562 tumor cells. It is concluded that the present method for growing human T colonies exhibits the same cloning efficiency as the most efficient liquid culture systems. Individual T colonies are composed exclusively of T inducer/helper or T cytotoxic/suppressor cells, they are never of mixed phenotype, and they do not contain cells of natural killer phenotype. Regulatory mechanisms influencing colony formation are operating between and within the various subsets of T lymphocytes.     

Growth Dynamics of the Threatened Caribbean Staghorn Coral Acropora cervicornis: Influence of Host Genotype,Symbiont Identity,Colony Size,and Environmental Setting

Background

The drastic decline in the abundance of Caribbean acroporid corals (Acropora cervicornis, A. palmata) has prompted the listing of this genus as threatened as well as the development of a regional propagation and restoration program. Using in situ underwater nurseries, we documented the influence of coral genotype and symbiont identity, colony size, and propagation method on the growth and branching patterns of staghorn corals in Florida and the Dominican Republic.

Methodology/Principal Findings

Individual tracking of> 1700 nursery-grown staghorn fragments and colonies from 37 distinct genotypes (identified using microsatellites) in Florida and the Dominican Republic revealed a significant positive relationship between size and growth, but a decreasing rate of productivity with increasing size. Pruning vigor (enhanced growth after fragmentation) was documented even in colonies that lost 95% of their coral tissue/skeleton, indicating that high productivity can be maintained within nurseries by sequentially fragmenting corals. A significant effect of coral genotype was documented for corals grown in a common-garden setting, with fast-growing genotypes growing up to an order of magnitude faster than slow-growing genotypes. Algal-symbiont identity established using qPCR techniques showed that clade A (likely Symbiodinium A3) was the dominant symbiont type for all coral genotypes, except for one coral genotype in the DR and two in Florida that were dominated by clade C, with A- and C-dominated genotypes having similar growth rates.

Conclusion/Significance

The threatened Caribbean staghorn coral is capable of extremely fast growth, with annual productivity rates exceeding 5 cm of new coral produced for every cm of existing coral. This species benefits from high fragment survivorship coupled by the pruning vigor experienced by the parent colonies after fragmentation. These life-history characteristics make A. cervicornis a successful candidate nursery species and provide optimism for the potential role that active propagation can play in the recovery of this keystone species.     

The growth and form of bacterial colonies.

A simple method is described for measuring the profile of bacterial colonies. Profiles were determined for colonies of Bacillus cereus, Escherichia coli and Staphylococcus albus of different ages. In spite of differences in cell morphology, the colony profiles had a common basic structure consisting of steeply rising leading edge connected by a ridge to an interior region where height also rose, though less steeply, to a flat or domed centre. The colony mass increased exponentially through part of the growth phase. It is suggested that net colony growth consists of a combination of leading edge growth, which is unrestricted and approaches the maximum specific growth rate of the organism, and diffusion-limited growth in the colony interior. Common elements of profiles from each species may be a consequence of such differences in growth rate.     

松嫩平原栽培条件下羊草无性系构件的结构

羊草是长根茎型禾草,是典型的无性系植物,在松嫩平原的生长季末期,栽培条件下羊草无性系分株由分蘖株和分蘖苗组成,在具有充分生长空间而又没有种间竞争的风沙土上,羊草分株的分蘖节在一个生长季内可以繁殖4个世代,按分蘖节的繁殖世代划分龄级,现实与潜在无性系构件的年龄谱均以1龄级比重最大,随着龄级的增加明显减少,呈增长型的年龄结构,羊草无性系分株的生产力主要与分株形成及生长的时间长短有关,形成时间越早、生长时间越长的分蘖株对无性系的物质生产和营养繁殖的贡献越大,羊草无性系在空间扩展与物质贮存上具有一定的可调节性。     

Blastocystis hominis: A simplified, high-efficiency method for clonal growth on solid agar

Colony growth of protozoan parasites in agar can be useful for axenization, cloning, and viability studies. This is usually achieved with the pour plate method, for which the parasite colonies are situated within the agar. This technique has been described for Giardia intestinalis, Trichomonas vaginalis, and Entamoeba and Blastocystis species. Extracting such colonies can be laborious. It would be especially useful if parasites could be grown on agar as colonies. These colonies, being exposed on the agar surface, could be conveniently isolated for further investigation. In this study, we report the successful culture of B. hominis cells as colonies on solid agar. Colonies were enumerated and the efficiency of plating was determined. It was observed that B. hominis could be easily cultured on agar as clones. The colonies were dome-shaped and mucoid and could grow to 3 mm in diameter. Flow cytometric analyses revealed that parasite colonies remained viable for up to 2 weeks. Viable colonies were conveniently expanded in liquid or solid media. Scanning electron microscopy revealed that each colony consists of two regions; a dome-shaped, central core region and a flattened, peripheral region. Older colonies possessed numerous strand-like surface coat projections. This study provides the first report of clonal growth of B. hominis on agar and a simple, effective method for cloning and expansion of B. hominis cells.