Factors regulating the emergence of spontaneous and X-ray-induced variants in primary rat tracheal epithelial cell cultures

Summary A series of experiments have been carried out to identify those factors that affect the number of altered populations detected in control, nonexposed, and radiation-exposed primary cultures of rat tracheal epithelial cells. The number of colony forming cells per milliliter of culture medium and the frequency with which the culture medium is changed seemed to be the most critical factors regulating the emergence of induced and spontaneous variants. Increasing the number of cells plated so that of colony forming cells increase from 25 to 200 per ml, regardless of the dish size used, was associated with a 200-fold decline in the frequency of spontaneous variants and a 40-fold decline in X-ray-induced variants. Increasing the interval between medium changes from 3 to 7 days after the first week of culture was associated with a 10-fold decrease in the frequency of spontaneous variants. The frequency of spontaneous and induced variants is markedly less dependent on culture density at densities between 150 and 600 colony forming cells per ml. The type of medium used to establish primary cultures had little effect on the frequency of variants detected. Similarly, when assays were performed at densities in excess of 150 colony forming cells per ml the frequency of spontaneous and x-ray-induced variants was not affected by the absence of epidermal growth factor, increased levels of calcium (final concentration, 0.8 mM), or by removal of pyruvate from the selection medium.     

The role of despotism and heritability in determining settlement patterns in the colonial lesser kestrel

Avian colony size variation is an evolutionary puzzle in terms of unequal fitness payoffs. We used a long-term marked lesser kestrel (Falco naumanni) population, where individual fitness increases with colony size, to test whether subordinates are evicted despotically from the largest colonies. Yearlings were smaller and lighter, were more attacked than expected, and lost most disputes over nest holes with older birds. Agonistic interactions increased with colony size; consequently, most first breeders recruited in colonies smaller than those at which they first tried to settle. As expected when subordination is a transient state, birds dispersed to a larger colony as they got older even after breeding successfully. The population consequences of these behavioral processes were that the relative frequency of yearlings and first breeders decreased with colony size. At the same time, breeding colony size was repeatable within individuals, so we estimated the amount of heritable variation in this trait. Estimates of heritability derived from parent-offspring and full-sib analyses were consistently high (h2=0.53) when individuals reached asymptotic morphological values and presumably overcame subordinate transient states. Age-related dominance asymmetries masked resemblance among relatives in colony size, but both phenomena seem to coexist in this population and explain a considerable proportion of colony size variation.     

Habitat-related microgeographic variation of worker size and colony size in the ant Cataglyphis cursor

In social insects, colony size is a crucial life-history trait thought to have major implications for the evolution of social complexity, especially in relation to worker size polymorphism. Yet, little is known about how ecological factors can affect and constrain colony. Here, we explored the pattern of colony-size and worker-size variation in the Mediterranean ant Cataglyphis cursor, in relation to the type of habitats colonized (seaside vs. vineyard). The high level of the water table in the seaside habitat could constrain the depth of C. cursor underground nests and directly constrain its colony size. If worker size increases with colony size, as observed in other ant species, larger colony size and larger workers should be found in the vineyard populations. By comparing worker size among 16 populations, we verified that workers were significantly larger in the vineyard populations. We further determined that the morphological similarities detected among populations from the same habitat type were not due to geographic or genetic proximity. In two populations from each habitat type, the depth of nests was positively correlated with colony size and colony size with worker size. Using a type II regression approach, we further showed that the difference between the two populations in the depth of nest was sufficient to explain the difference in colony size, and similarly, variation in colony size was sufficient to explain variation in worker size. Our results suggest that a single proximate ecological factor could lead to significant variation in major life-history parameters.     

Characterization of colony morphology variants isolated from Streptococcus pneumoniae biofilms

In this study, we report the isolation of colony morphology variants from Streptococcus pneumoniae serotype 3 biofilms. The colony variants differed in colony size (large, medium, and small) and their mucoid appearance on blood agar. The small nonmucoid variant (SCV) emerged during the initial attachment stage of S. pneumoniae biofilm formation and dominated over the course of biofilm growth. Mucoid variants appeared at later biofilm developmental stages. The reduction in colony size/mucoidy correlated with a decrease in capsule production and an increase in initial attachment. The large mucoid variant formed flat unstructured biofilms, failed to aggregate in liquid culture, and adhered poorly to solid surfaces. In contrast, SCVs autoaggregated in liquid culture, hyperadhered to solid surfaces, and formed biofilms with significant three-dimensional structure, mainly in the form of microcolonies. The variants showed similar antibiotic resistance/susceptibility based on a modified Kirby-Bauer test and when grown as biofilms. However, antimicrobial treatment of S. pneumoniae biofilms altered the colony variant's distribution and mainly affected the most interior areas of biofilm microcolonies. To further explore the nature of the variants, the capsule biosynthetic operon (cps3DSUM) was explored in greater detail. The genetic analysis indicated that the emergence of nonmucoid variants was due to a deletion comprising cps3DSU as well as additional genes upstream of the cps3 operon. Overall, our findings suggest that in vitro biofilm formation of S. pneumoniae serotype 3 coincides with the emergence of colony variants with distinct genotypic and phenotypic characteristics.     

雄性菲菊头蝠夏季集群大小的变化及其影响因素

本研究以栖息于废弃防空洞的雄性菲菊头蝠(Rhinolophus pusillus)作为研究对象,探究其夏季集群大小变化及其潜在影响因素.应用红外热成像仪监测菲菊头蝠在6月到8月期间的集群大小.利用单因素方差分析和一般线性回归模型,检验菲菊头蝠集群大小的月变化及其与洞内微气候的关系.研究发现,菲菊头蝠集群大小从6月至8月...     

Biological attributes of colony-type variants of Candida albicans

Twenty 'commensal' oral or 'pathogenic' vaginal isolates of Candida albicans were examined for colony morphology on malt/yeast-extract and serum-based agar media. Diverse and variable colony morphology was seen on serum agar. In 17 strains, selective subculture of morphologically atypical colonies produced progeny which had reverted to the morphology of the majority of parental colonies. However, in one strain, a highly stable colony variant was isolated which did not revert on subculture. In two further strains, variants were isolated which could be maintained with at least 99% homogeneous colony type by selective colony subculture, but reversion to the parental type or switching to other morphologies occurred at rates of 10(-2) to 10(-4): a rapid switching phenomenon. The relative proportions of mycelial or yeast forms were the main determinants of colony morphology. The variants were biotyped using a selection of biochemical tests. The stable variant differed from its parent in several characters, including rate of production of a proteinase enzyme. The pathogenicity of variants was compared in mice, and both stable and switching variants differed in virulence from their parental strains. Colony-type variation on suitable media is thus a powerful tool in the isolation of mutants or variants of C. albicans which differ from 'isogenic' parents in significant biological properties. Such variants may aid identification and characterization at the molecular level of determinants of, for example, pathogenicity and morphogenesis.     

The Vlp system of Mycoplasma hyorhinis: combinatorial expression of distinct size variant lipoproteins generating high-frequency surface antigenic variation.

Isogenic populations of Mycoplasma hyorhinis undergo in vitro high-frequency phase variation in the expression of surface lipoproteins; these products also vary markedly in size through changes in periodic protein structure (R. Rosengarten and K.S. Wise, Science 247:315-318, 1990). In this report, we rigorously define three distinct translation products comprising the Vlp (variable lipoprotein) system of M. hyorhinis SK76 and establish parameters of Vlp structural diversity and expression that distinguish the Vlp system from previously described examples of antigenic variation. VlpA, VlpB, and VlpC are prominent amphiphilic membrane lipoproteins characterized by detergent-phase fractionation and metabolic labeling with [35S]cysteine and [3H]palmitate. VlpA is distinguished from VlpB and VlpC by its selective labeling with [35S]methionine; VlpB and VlpC are distinguished by specific epitopes defined by surface-binding monoclonal antibodies (MAbs); a third MAb defines a surface epitope shared by VlpB and VlpC (but absent from VlpA). Each Vlp displays 12 to 30 spontaneous size variant forms comprising a periodic ladder that could also be generated by partial trypsin digestion of individual Vlp size variants. Different periodic intervals within VlpB and VlpC further distinguish these two products structurally. Mycoplasma colony opacity correlates inversely with Vlp size. Each Vlp undergoes independent, oscillating high-frequency phase variation in isogenic populations and can be expressed individually or concomitantly with other Vlps in a noncoordinate manner. All seven possible combinations of these three products were observed; however, no variants were found that lacked a Vlp. High-frequency size variation of each Vlp superimposed on combinatorial diversity in Vlp expression yields greater than 10(4) possible structurally distinct Vlp mosaics, of which 104 were documented along with 24 of 42 possible transitions among the seven Vlp combinations. In addition to these features, VlpA, VlpB, and VlpC were specifically recognized by serum antibodies from swine with experimental M. hyorhinis SK76-induced arthritis, indicating expression and immunogenicity of Vlps in the natural host. The structure and variation of Vlps and their known involvement in MAb-mediated modulation of mycoplasma-infected host cell properties and mycoplasma killing are discussed in relation to the surface architecture and adaptive potential of the wall-less mycoplasmas.     

Pleiotropic effect of the mutation of streptomycin resistance in Micromonospora purpurea var. violacea]

Variation of different features of populations of streptomycin-sensitive and streptomycin-resistant forms of M. purpurea var. violacea, an organism producing gentamicin was studied. The population of the initial streptomycin-sensitive culture was characterized by high homogeneity with respect to the cultural, morphological and some physiological properties. The variation of the features, such as the colony size, pigment formation, auxotrophic mutations, antibiotic production significantly increased in populations grown on media with streptomycin. Mutants differing from the initial strain by a complex of cultural, morphological and physiological features and in particular the antibiotic production were isolated from populations of the streptomycin-resistant variants.     

Selection of colony, plasmid, and virulence variants of Staphylococcus epidermidis NRC853 during growth in continuous cultures exposed to erythromycin.

A continuous-culture system was developed to study changes in the structure of Staphylococcus epidermidis populations exposed to subminimum inhibitory concentrations of erythromycin. Continuous-culture experiments were carried out in a dextrose-free, tryptic soy broth medium supplemented with lactic acid and sodium lactate (MTSB-D). The multiresistant (penicillin-, tetracycline-, and erythromycin-resistant) S. epidermidis strain NRC853 was subjected to a series of experiments: (i) growth individually in continuous culture in the absence and presence of erythromycin and (ii) growth in mixed culture with the erythromycin-susceptible S. epidermidis strain NRC852 in the absence and presence of erythromycin. Strain NRC853 produced colony morphology variants during continuous culture in the presence of 0.05 and 0.1 microgram of erythromycin per ml. Variants (A, B, and C) were different from their wild-type parent on the basis of colony size, sector pattern, and/or the ability to transmit light. A variants rapidly lost a 2.7-MDa tetracycline resistance plasmid. B and C variants formed an ermC plasmid multimer series from unit size to a 16-mer and exhibited an approximately twofold increase in erythromycin MIC over that of the wild-type parent. They slowly lost the tetracycline resistance plasmid. The small-colony B variant demonstrated an increased virulence in the neonatal mouse weight gain test and an increase in fibronectin binding compared with the wild-type parent. The presence of a competing strain drastically increased the frequency of all variants.     

Selection of colony, plasmid, and virulence variants of Staphylococcus epidermidis NRC853 during growth in continuous cultures exposed to erythromycin

A continuous-culture system was developed to study changes in the structure of Staphylococcus epidermidis populations exposed to subminimum inhibitory concentrations of erythromycin. Continuous-culture experiments were carried out in a dextrose-free, tryptic soy broth medium supplemented with lactic acid and sodium lactate (MTSB-D). The multiresistant (penicillin-, tetracycline-, and erythromycin-resistant) S. epidermidis strain NRC853 was subjected to a series of experiments: (i) growth individually in continuous culture in the absence and presence of erythromycin and (ii) growth in mixed culture with the erythromycin-susceptible S. epidermidis strain NRC852 in the absence and presence of erythromycin. Strain NRC853 produced colony morphology variants during continuous culture in the presence of 0.05 and 0.1 microgram of erythromycin per ml. Variants (A, B, and C) were different from their wild-type parent on the basis of colony size, sector pattern, and/or the ability to transmit light. A variants rapidly lost a 2.7-MDa tetracycline resistance plasmid. B and C variants formed an ermC plasmid multimer series from unit size to a 16-mer and exhibited an approximately twofold increase in erythromycin MIC over that of the wild-type parent. They slowly lost the tetracycline resistance plasmid. The small-colony B variant demonstrated an increased virulence in the neonatal mouse weight gain test and an increase in fibronectin binding compared with the wild-type parent. The presence of a competing strain drastically increased the frequency of all variants.     

Ontogeny of worker body size distribution in bumble bee (Bombus impatiens) colonies

1. Bumble bees exhibit worker size polymorphisms; highly related workers within a colony may vary up to 10‐fold in body mass. As size variation is an important life history feature in bumble bees, the distribution of body sizes within the colony and how it fluctuates over the colony cycle were analysed. 2. Ten commercially purchased colonies of Bombus impatiens (Cresson) were reared in ad libitum conditions. The size of all workers present and newly emerging workers (callows) was recorded each week. 3. The average size of bumble bee workers did not change with colony age, but variation in body size tended to decrease over time. The average size of callows did not change with population size, but did tend to decrease with colony age. In all measures, there was considerable variation among colonies. 4. Colonies of B. impatiens usually produced workers with normally distributed body sizes throughout the colony life cycle. Unlike most polymorphic ants, there was no increase in worker body size with colony age or colony size. This provides the first, quantitative data on the ontogeny of bumble bee worker size distribution. The potential adaptive significance of this size variation is discussed.     

Genetic basis for colonial variation in Neisseria gonorrhoeae.

When the piliated colony types of Neisseria gonorrhoeae, which predominate in recent isolates, were nonselectively subcultured in vitro, they gave rise to large numbers of nonpiliated, avirulent colonial variants. Evidence is presented to show that most of this variation occurs after active growth has ceased and that the variation is sensitive to the action of deoxyribonuclease. We suggest that this variation is a result of transformation. A second variation in colonial morphology involved differing levels of "colony opacity-associated proteins" in the outer membrane. This variation was also inhibited by the presence of deoxyribonuclease, but the genetic basis for it is not as yet clear.     

Differences in membrane fluidity and fatty acid composition between phenotypic variants of Streptococcus pneumoniae

Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease. This study examined the relationship between membrane characteristics and colony morphology in a few selected opaque-transparent couples of S. pneumoniae strains carrying different capsular types. Membrane fluidity was determined on the basis of intermolecular excimerization of pyrene and fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH). A significant decrease, 16 to 26% (P < or = 0.05), in the excimerization rate constant of the opaque variants compared with that of the transparent variants was observed, indicating higher microviscosity of the membrane of bacterial cells in the opaque variants. Liposomes prepared from phospholipids of the opaque phenotype showed an even greater decrease, 27 to 38% (P < or = 0.05), in the pyrene excimerization rate constant compared with that of liposomes prepared from phospholipids of bacteria with the transparent phenotype. These findings agree with the results obtained with DPH fluorescence anisotropy, which showed a 9 to 21% increase (P < or = 0.001) in the opaque variants compared with the transparent variants. Membrane fatty acid composition, determined by gas chromatography, revealed that the two variants carry the same types of fatty acids but in different proportions. The trend of modification points to the presence of a lower degree of unsaturated fatty acids in the opaque variants compared with their transparent counterparts. The data presented here show a distinct correlation between phase variation and membrane fluidity in S. pneumoniae. The changes in membrane fluidity most probably stem from the observed differences in fatty acid composition.     

Growth kinetics by scanning of granulocytic cell colonies in glass capillaries.

Pure granulocytic colonies were cultivated from mouse bone marrow cells in agar contained in glass capillary tubes using mouse embryo conditioned medium as colony stimulating activity. A random distribution of colonies along the agar gels was achieved under controlled conditions. Only 3 capillaries were needed for a coefficient of variation around 5% provided at least 104 cells were seeded per capillary. The daily growth of single colonies within an agar capillary was followed, using the light scattering properties of the colonies for automatic scanning. The position of the colonies in the capillary greatly affected the scan signal; the consequences of positional changes were studied in detail. Using the mean peak height as growth parameter, the onset of measureable granulocytic colony growth was found between day 2 and 3, the maximum colony size was reached between day 7 and 9, after which the colonies decayed. Other parameters such as colony count and total peak are were determined and their sinificance discussed.     

Egg‐size plasticity in Apis mellifera: Honey bee queens alter egg size in response to both genetic and environmental factors

Social evolution has led to distinct life‐history patterns in social insects, but many colony‐level and individual traits, such as egg size, are not sufficiently understood. Thus, a series of experiments was performed to study the effects of genotypes, colony size and colony nutrition on variation in egg size produced by honey bee (Apis mellifera) queens. Queens from different genetic stocks produced significantly different egg sizes under similar environmental conditions, indicating standing genetic variation for egg size that allows for adaptive evolutionary change. Further investigations revealed that eggs produced by queens in large colonies were consistently smaller than eggs produced in small colonies, and queens dynamically adjusted egg size in relation to colony size. Similarly, queens increased egg size in response to food deprivation. These results could not be solely explained by different numbers of eggs produced in the different circumstances but instead seem to reflect an active adjustment of resource allocation by the queen in response to colony conditions. As a result, larger eggs experienced higher subsequent survival than smaller eggs, suggesting that honey bee queens might increase egg size under unfavourable conditions to enhance brood survival and to minimize costly brood care of eggs that fail to successfully develop, and thus conserve energy at the colony level. The extensive plasticity and genetic variation of egg size in honey bees has important implications for understanding life‐history evolution in a social context and implies this neglected life‐history stage in honey bees may have trans‐generational effects.     

Fitness in invasive social wasps: the role of variation in viral load,immune response and paternity in predicting nest size and reproductive output

Within any one habitat, the relative fitness of organisms in a population can vary substantially. Social insects like the common wasp are among the most successful invasive animals, but show enormous variation in nest size and other fitness‐related traits. Some of this variation may be caused by pathogens such as viruses that can have serious consequences in social insects, which range from reduced productivity to colony death. Both individual immune responses and colony‐level traits such as genetic diversity are likely to influence effects of pathogen infections on colony fitness. Here we investigate how infections with Kashmir Bee Virus (KBV), immune response and intracolony genetic diversity (due to queen polyandry) affect nest size in the invasive common wasp Vespula vulgaris. We show that KBV is highly prevalent in wasps and expression of antiviral immune genes is significantly increased with higher viral loads across individuals. Patriline membership within a nest did not influence KBV susceptibility or immune response. A permutational MANCOVA revealed that polyandry, viral load and expression of the immune gene Dicer were significant predictors of variation in nest size. High intracolony genetic diversity due to polyandry has previously been hypothesized to improve colony‐level resistance to parasites and pathogens. Consistent with this hypothesis, we observed genetically diverse colonies to be significantly larger and to produce more queens, although this effect was not driven by the pathogen we investigated. Invasive wasps clearly suffer from pathogens and expend resources, as indicated here by elevated immune gene expression, toward reducing pathogen‐impact on colony fitness.     

Mean body size predicts colony performance in the common eastern bumble bee (Bombus impatiens)

1. Prior studies suggest that both the mean and variation of worker size predict the performance of bumble bee colonies. The ‘variation hypothesis’ posits that colony performance increases with variation of worker body size due to more efficient division of labour within colonies. The ‘mean size hypothesis’ posits that colony performance increases with mean bumble bee size, as each individual's efficiency tends to increase with body size. 2. The present study tested these non‐mutually exclusive hypotheses by establishing 62 Bombus impatiens Cresson (Hymenoptera, Apidae) nest boxes in 32 semi‐natural savanna fragments within large‐scale experimental landscapes in South Carolina (U.S.A.). 3. Based on measurements of > 24 000 individuals and on colony growth over ～7 weeks, our results support the mean size hypothesis, not the variation hypothesis.