5-azacytidine-induced re-expression of alleles on the inactive X chromosome in a hybrid mouse cell line

In order to test the hypothesis that DNA methylation is involved in mammalian X chromosome inactivation, cells of an HPRT-deficient Mus musculus × M. caroli line, having an inactive M. caroli X, were grown in 5-azacytidine, and HPRT⁺ reactivants selected in HAT medium. Recovery of colonies depended on azacytidine concentration, and on time between treatment and selection; the highest recovery frequency was 0.3%. All colonies re-expressed the M. caroli form of HPRT, showing that the Hpt⁺ allele on the inactive M. caroli X was reactivated by azacytidine treatment. About 6% of HPRT⁺ reactivants also re-expressed M. caroli G6PD, whereas none of the 56 azacytidine-treated unselected colonies did so; thus re-expression of the Hpt and Gpd loci appears to be co-ordinated to some extent. However, no HPRT⁺ reactivants, nor unselected colonies re-expressed M. caroli PGK-A.     

Chemically Defined Medium for Cultivation of Several Epiphytic and Phytopathogenic Spiroplasmas

A chemically defined medium, LD82, was formulated for in vitro cultivation of spiroplasmas. Medium LD82 supported good growth for four epiphytic and insect-pathogenic spiroplasmas, Spiroplasma floricola 23-6^T, Spiroplasma sp. strain SR3, Spiroplasma sp. strain brevi, and Spiroplasma sp. strain AS576, and of the phytopathogenic spiroplasmas Spiroplasma citri Maroc R8A2^T and PC1. Titers of all six strains grown in defined medium LD82 reached 2.0 × 10⁹ to 6.0 × 10⁹ CFU/ml of culture. All spiroplasma strains tested formed colonies readily on agar medium LD82. None of the spiroplasmas formed typical fried-egg colonies. All formed diffuse colonies, but the forms of colonies differed somewhat among the spiroplasma strains. In preliminary studies of nutritional requirements, phospholipids slightly enhanced the growth of the epiphytic and insect-pathogenic strains in medium LD82 and were found essential for good growth of S. citri.     

Interspecific variation in extracellular polysaccharide content and colony formation of <Emphasis Type="Italic">Microcystis</Emphasis> spp. cultured under different light intensities and temperatures

Single cells of five different Microcystis species (M. ichthyoblabe, M. viridis, M. flos-aquae, M. wesenbergii, and M. aeruginosa) were batch-cultured at different temperatures and light intensities: (a) 25 °C and 50 μmol photons m^?2 s^?1 (control culture); (b) 25 °C and 10 μmol photons m^?2 s^?1; and (c) 15 °C and 50 μmol photons m^?2 s^?1. The extracellular polysaccharide content was significantly higher in treatments b and c than in the control treatment. All Microcystis species existed as single cells under the control treatment but formed colonies in treatments b and c. All of the colonies were irregular with indistinct margins. M. ichthyoblabe, M. viridis, M. flos-aquae, and M. wesenbergii formed colonies with similar morphologies and their cells were loosely aggregated. In contrast, M. aeruginosa formed denser colonies with no distinct holes. The colony morphologies differed from the classic morphology of M. ichthyoblabe field-grown colonies but resembled that of small colonies found in Lake Taihu (Yangtze Delta Plain, China) during early spring. This indicates that field- and laboratory-grown colonies are governed by similar formation processes. We suggest that in laboratory and field environments, M. ichthyoblabe (or M. flos-aquae) colonies are representative of small colonies formed from single Microcystis cells, whereas the morphology of older colonies evolves to resemble M. wesenbergii and M. aeruginosa colonies.     

Coscinaraea marshae corals that have survived prolonged bleaching exhibit signs of increased heterotrophic feeding

Colonies of Coscinaraea marshae corals from Rottnest Island, Western Australia have survived for more than 11 months in various bleached states following a severe heating event in the austral summer of 2011. These colonies are situated in a high-latitude, mesophotic environment, which has made their long-term survival of particular interest as such environments typically suffer from minimal thermal pressures. We have investigated corals that remain unbleached, moderately bleached, or severely bleached to better understand potential survival mechanisms utilised in response to thermal stress. Specifically, Symbiodinium (algal symbiont) density and genotype, chlorophyll-a concentrations, and δ¹³C and δ¹⁵N levels were compared between colonies in the three bleaching categories. Severely bleached colonies housed significantly fewer Symbiodinium cells (p < 0.05) and significantly reduced chlorophyll-a concentrations (p < 0.05), compared with unbleached colonies. Novel Symbiodinium clade associations were observed for this coral in both severely and moderately bleached colonies, with clade C and a mixed clade population detected. In unbleached colonies, only clade B was observed. Levels of δ¹⁵N indicate that severely bleached colonies are utilising heterotrophic feeding mechanisms to aid survival whilst bleached. Collectively, these results suggest that these C. marshae colonies can survive with low symbiont and chlorophyll densities, in response to prolonged thermal stress and extended bleaching, and increase heterotrophic feeding levels sufficiently to meet energy demands, thus enabling some colonies to survive and recover over long time frames. This is significant as it suggests that corals in mesophotic and high-latitude environments may possess considerable plasticity and an ability to tolerate and adapt to large environmental fluctuations, thereby improving their chances of survival as climate change impacts coral ecosystems worldwide.     

Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of Agrobacterium Biovars 1 and 2

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K₂TeO₃ was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K₂TeO₃ concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K₂TeO₃-amended medium. The bona fide agrobacterium colonies growing on media amended with K₂TeO₃ were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10³ to 10⁴ agrobacteria · g of dry soil⁻¹ in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.     

Distribution and Abundance of Fauna on Living Tissues of Two Brazilian Hermatypic Corals (Mussismilia hispida (Verril 1902) and Siderastrea stellata Verril, 1868)

This study describes the distribution and abundance patterns of the associate fauna on the living surface of the corals Siderastrea stellata Verril, 1868 and Mussismilia hispida (Verril 1902) using a non-destructive method, on the northern coast of Rio de Janeiro State. For each coral species, infestation density and proportions of infested colonies, colonies attached and unattached to the substrate were estimated. A total of 474 colonies of S. stellata and 452 colonies of M. hispida were examined. The barnacle Ceratoconcha floridana (Pilsbry, 1931) was the dominant coral associate found, followed by gall-crabs of the family Cryptochiridae Paulson, 1875 and the bivalve Lithophaga bisulcata (d’Orbigny, 1842). Both coral species presented similar patterns of infestation dominance. S. stellata colonies were more commonly infested and showed a greater mean infestation density of 0.62 ind/cm² at Armação dos Búzios, whereas M. hispida colonies had infestation densities of only 0.20 ind/cm². Infestation density does not appear to impact negatively on corals of Armação dos Búzios. A clear negative relationship between the number of associates in the coral colony and coral size was found. Evidently abundance and frequency of occurrence of associated fauna is highly related to coral community structure and composition and the results highlight the importance of local scale studies.     

Land-cover and climate factors contribute to the prevalence of the ectoparasitic fungus Laboulbenia formicarum in its invasive ant host Lasius neglectus

Understanding the distribution of parasites is crucial for biodiversity conservation. Here, we studied the distribution of the ectoparasitic fungus Laboulbenia formicarum in native and invasive Lasius ants in a 2000 km² area. We screened over 16,000 ant workers in 478 colonies of five different species. We found that Lab. formicarum was rare in native Lasius species but infected 58% of the colonies of the invasive species Las. neglectus. At landscape scale, Lab. formicarum presence could not be explained by geographic and genetic distances between Las. neglectus colonies but was associated with hotter and dryer climatic conditions and its prevalence in colonies increased with urbanization. Within infected colonies, fungal prevalence varied from 0 to 100 percent within meters and was negatively correlated with impervious ground cover. In a changing world, our findings emphasize the importance of land-use and climatic factors in shaping the distribution and prevalence of fungal parasites.     

Does exogenic food benefit both partners in an ant-plant mutualism? The case of Cecropia obtusa and its guest Azteca plant-ants

In the mutualisms involving the myrmecophyte Cecropia obtusa and Azteca ovaticeps or A. alfari, both predatory, the ants defend their host trees from enemies and provide them with nutrients (myrmecotrophy). A. ovaticeps provisioned with prey and then ¹⁵N-enriched food produced more individuals than did control colonies (not artificially provisioned). This was not true for A. alfari colonies, possibly due to differences in the degree of maturity of the colonies for the chosen range of host tree sizes (less than 3 m in height). Myrmecotrophy was demonstrated for both Azteca species as provisioning the ants with ¹⁵N-enriched food translated into higher δ¹⁵N values in host plant tissues, indicating that nitrogen passed from the food to the plant. Thus, the predatory activity of their guest ants benefits the Cecropia trees not only because the ants protect them from defoliators since most prey are phytophagous insects but also because the plant absorbs nutrients.     

Guano deposition and nutrient enrichment in the vicinity of planktivorous and piscivorous seabird colonies in Spitsbergen

The crucial role of seabirds in the enrichment of nutrient-poor polar terrestrial ecosystem is well-known. However, no studies have examined the potentially different impacts associated with piscivorous and planktivorous bird colonies on the surrounding tundra soils. Therefore, we compared guano deposition and physical and chemical parameters of soil near two large seabird colonies, one of planktivorous little auks (Alle alle) and the other comprising piscivorous Brunnich’s guillemots (Uria lomvia) and kittiwakes (Rissa tridactyla). The two colonies generated similar levels of guano deposition, with the intensity of deposition decreasing away from the colony. Guano deposition adjacent to both colonies was considerably higher than that in control areas. The increased guano supply around colonies significantly enhanced soil conductivity, nitrogen (NO₃ ^?, NH₄ ⁺), potassium (K⁺), and phosphate (PO₄ ^3?) ion concentrations and led to reduced pH values. Guano deposition explained 84 % (piscivorous colony) and 67 % (planktivorous colony) of the total variation in the tested soil parameters. Planktivore and piscivore colonies affected adjacent tundra in different ways. The phosphate content and pH value of soil influenced by piscivores were significantly higher than values measured in planktivore-influenced soil. The gradient of guano deposition and associated ion content in the soil decreased more rapidly with distance from the piscivore colony. Climate-induced changes in populations of planktivorous and piscivorous seabirds are expected in the study region and may therefore have substantial consequential effects on Arctic terrestrial ecosystems.     

Partitioning of CO2 Fixation in the Colonial Cyanobacterium Microcystis aeruginosa: Mechanism Promoting Formation of Surface Scums

Constraints on inorganic carbon (C_i) availability stimulated buoyancy in natural, photosynthetically active populations of the colonial blue-green alga (cyanobacterium) Microcystis aeruginosa. In nonmixed eutrophic river water and cultures, O₂ evolution determinations indicated C_i limitation of photosynthesis, which was overcome either by CO₂ additions to the aqueous phase or by exposure of buoyant colonies to atmospheric CO₂. Microautoradiographs of M. aeruginosa colonies revealed partitioning of ¹⁴CO₂ fixation and photosynthate accumulation between peripheral and internal cells, particularly in large colonies. When illuminated colonies were suspended in the aqueous phase, peripheral cells accounted for at least 90% of the ¹⁴CO₂ assimilation, whereas internal cells remained unlabeled. However, when ¹⁴CO₂ was allowed to diffuse into colonies 15 min before illumination, a more uniform distribution of labeling was observed. Resultant differences in labeling patterns were most likely due to peripheral cells more exclusively utilizing CO₂ when ambient C_i concentrations were low. Among colonies located at the air-water interface, internal cells showed an increased share of photosynthate production when atmospheric ¹⁴CO₂ was supplied. This indicated that C_i transport was restricted in large colonies below the water surface, forcing internal cells to maintain a high degree of buoyancy, thus promoting the formation of surface scums. At the surface, C_i restrictions were alleviated. Accordingly, scum formation appears to have an ecological function, allowing cyanobacteria access to atmospheric CO₂ when the C_i concentration is growth limiting in the water column.     

Response of foster Asian honeybee (Apis cerana Fabr.) colonies to the brood of European honeybee (Apis mellifera L.) infested with parasitic mite,Varroa jacobsoni oudemans

The response of Asian honeybee (Apis cerana Fabr.) colonies toward the introduced worker brood of the European honeybee (Apis mellifera L.) infested with the parasitic mite Varroa jacobsoni Oudemans was investigated. When no mites were present, 40% of the healthy open brood and 3% of the healthy capped brood of the European honeybees were rejected by the Asian honeybee colonies. When the brood was infested, brood rejection was significantly higher for open (P < 0.05) and capped broods (P < 0.01). The brood removal activity decreased with time. The quantity of brood removed was also correlated with mite infestation level for open (r² = 0.933) and sealed broods (r² = 0.918). The feasibility of using heterospecific colonies to control Varroa mite is unclear and is discussed from behavioral and ecological points of view.     

Rapid Colony Screening Method for Identifying Hydrogenase Activity in Rhizobium japonicum

A method has been developed for the rapid screening of Rhizobium japonicum colonies for hydrogenase activity based on their ability to reduce methylene blue in the presence of respiratory inhibitors and hydrogen. Hydrogen uptake-positive (Hup⁺) colonies derepressed for hydrogenase activity were visualized by their localized decolorization of filter paper disks impregnated with the dye. Appropriate responses were seen with a number of Hup⁺ and Hup⁻ wild-type strains of R. japonicum as well as Hup⁻ mutants. Its specificity was further confirmed in selected strains on the basis of comparisons with chemolithotrophic growth and the presence of other genetic markers. Utilization of the method in identifying Hup⁺ colonies among 16,000 merodiploid derivatives of the Hup⁻ mutant strain PJ17nal containing cloned DNA fragments of the Hup⁺ strain 122 DES has demonstrated its applicability as a screening procedure in the genetic analysis of the R. japonicum hydrogen uptake system.     

Factors Controlling the Production of Lysogenic Cultures of B. megatherium

(1) The proportion of infected B. megatherium cells which develop lysogenic colonies depends on the number and kind of infecting virus particles and on the culture medium in which the cells are growing. (2) Cells infected with 100 or more T virus particles (from megatherium 899) in yeast extract peptone disintegrate, produce very few virus particles, and less than one lysogenic colony per 10⁷ infected cells. Cells infected with one or a few particles produce 500 to 1000 virus particles each and about 30 lysogenic colonies per 10⁷ infected colonies. (3) T phage obtained from lysogenic magatherium KM cultures produces many more lysogenic cells than does the original megatherium 899 virus. (4) Cells infected with megatherium 899 T virus in peptone medium and then transferred to asparagine medium give rise to 10⁶ lysogenic colonies per 10⁷ infected cells and this transformation will occur even after the infected cells have been in peptone for 60 to 90 minutes and are beginning to produce virus particles. (5) Continued growth of KM strain with either C or T virus from megatherium 899 for several hundred generations in the steady state apparatus results in a lysogenic strain which produces several different types of virus.     

Biofilm Dispersal of Neisseria subflava and Other Phylogenetically Diverse Oral Bacteria

Polystyrene petri dishes containing liquid medium were inoculated with single-cell suspensions of a fresh clinical isolate of Neisseria subflava and were incubated under conditions of low vibration. N. subflava colonies grew firmly attached to the surface of the dish, while the broth remained clear. Growing colonies released cells into the medium, resulting in the appearance of 10² to 10⁴ small satellite colonies attached to the surface of the dish in an area adjacent to each mature colony after 24 h. Satellite colonies grew in patterns of streamers shaped like jets and flares emanating from mature colonies and pointing toward the center of the dish. This dispersal pattern evidently resulted from the surface translocation of detached biofilm cells by buoyancy-driven convection currents that were generated due to slight temperature gradients in the medium. Streamers of satellite colonies ranged from 2 to >40 mm in length. Satellite colonies in very long streamers were relatively uniform in size regardless of their distance from the mature colony, suggesting that mature colonies released single cells or small clusters of cells into the medium and that the detachment, surface translocation, and subsequent surface reattachment of released cells were a transitory process. Incubation of N. subflava single cells in a perfused biofilm fermentor resulted in a large spike of the number of CFU in the perfusate after 9.5 h of growth, consistent with a rapid release of cells into the medium. Biofilm colonies of several other phylogenetically diverse oral bacteria, including Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Streptococcus mitis, and a prevalent but previously uncultured oral Streptococcus sp., exhibited similar temperature-dependent dispersal patterns in broth culture. This in vitro spreading phenotype could be a useful tool for studying biofilm dispersal in these and other nonflagellated bacteria and may have physiological relevance to biofilm dispersal in the oral cavity.     

Different Mechanisms of Resistance to Bacillus thuringiensis Toxins in the Indianmeal Moth

Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of ¹²⁵I-labeled Cry1Ab and Cry1Ac has been examined in three Plodia interpunctella colonies, one susceptible (688^s) and two resistant (198^r and Dpl^r) to Bacillus thuringiensis. Toxicological studies showed that the 198^r colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dpl^r colony was 4-fold more resistant to protoxin versus toxin. Binding results with ¹²⁵I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac. Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways. Compared with the susceptible colony, insects from the Dpl^r colony showed a drastic reduction in binding affinity (60-fold higher K_d), although they had similar concentrations of binding sites. Insects from the 198^r colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higher K_d and ca. three-fold-lower R_t compared with the 688^s colony). No major difference in Cry1Ac binding was found among the three colonies. The fact that the 198^r colony also has a protease-mediated mechanism of resistance (B. Oppert, R. Hammel, J. E. Throne, and K. J. Kramer, J. Biol. Chem. 272:23473–23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab. It is noteworthy that the three colonies used in this work derived originally from ca. 100 insects, which reflects the high variability and high frequency of B. thuringiensis resistance genes occurring in natural populations.     

Phenotypic and Genetic Analyses of the Varroa Sensitive Hygienic Trait in Russian Honey Bee (Hymenoptera: Apidae) Colonies

Varroa destructor continues to threaten colonies of European honey bees. General hygiene, and more specific Varroa Sensitive Hygiene (VSH), provide resistance towards the Varroa mite in a number of stocks. In this study, 32 Russian (RHB) and 14 Italian honey bee colonies were assessed for the VSH trait using two different assays. Firstly, colonies were assessed using the standard VSH behavioural assay of the change in infestation of a highly infested donor comb after a one-week exposure. Secondly, the same colonies were assessed using an “actual brood removal assay” that measured the removal of brood in a section created within the donor combs as a potential alternative measure of hygiene towards Varroa-infested brood. All colonies were then analysed for the recently discovered VSH quantitative trait locus (QTL) to determine whether the genetic mechanisms were similar across different stocks. Based on the two assays, RHB colonies were consistently more hygienic toward Varroa-infested brood than Italian honey bee colonies. The actual number of brood cells removed in the defined section was negatively correlated with the Varroa infestations of the colonies (r² = 0.25). Only two (percentages of brood removed and reproductive foundress Varroa) out of nine phenotypic parameters showed significant associations with genotype distributions. However, the allele associated with each parameter was the opposite of that determined by VSH mapping. In this study, RHB colonies showed high levels of hygienic behaviour towards Varroa -infested brood. The genetic mechanisms are similar to those of the VSH stock, though the opposite allele associates in RHB, indicating a stable recombination event before the selection of the VSH stock. The measurement of brood removal is a simple, reliable alternative method of measuring hygienic behaviour towards Varroa mites, at least in RHB stock.     

Ecology of Pectinatella magnifica and associated algae and cyanobacteria

The freshwater bryozoan species Pectinatella magnifica was found in 6 sandpits and in 19 mostly extensively managed ponds in the Protected Landscape Area and Biosphere Reserve T?eboňsko (Czech Republic) from its first record (in 2003) to 2012. Mean fresh biomass and abundance of P. magnifica colonies were 0.6 ± 1.5 kg m^?2 and 0.7 ± 1.1 colony m^?2 (± SD), respectively, in the shoreline zone during the growing season 2006–2011. The maximum biomass was mostly recorded during the first half of August in all basins. Colonization of further localities was recorded rather than increasing of P. magnifica biomass or abundance in 2012. There were no correlations between water temperature or water transparency and biomass/abundance of P. magnifica during the growing season. P. magnifica colonies preferred to grow on the branches or roots (especially of Salix sp.) to aquatic macrophytes and stones. Most of the water bodies, where this bryozoan species occurred, had lower concentration of total phosphorus in the water when compared with the typical fishponds in the Czech Republic. Inner space of colonies of P. magnifica provided suitable higher trophic level substrate when compared with the water of the sandpits/fishponds especially for green coccal algae. A massive algal colonization was indentified in decomposing colonies at the end of the growing season.     

Carbon,nitrogen and O2 fluxes associated with the cyanobacterium Nodularia spumigena in the Baltic Sea

Photosynthesis, respiration, N₂ fixation and ammonium release were studied directly in Nodularia spumigena during a bloom in the Baltic Sea using a combination of microsensors, stable isotope tracer experiments combined with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorometry. Cell-specific net C- and N₂-fixation rates by N. spumigena were 81.6±6.7 and 11.4±0.9 fmol N per cell per h, respectively. During light, the net C:N fixation ratio was 8.0±0.8. During darkness, carbon fixation was not detectable, but N₂ fixation was 5.4±0.4 fmol N per cell per h. Net photosynthesis varied between 0.34 and 250 nmol O₂ h⁻¹ in colonies with diameters ranging between 0.13 and 5.0 mm, and it reached the theoretical upper limit set by diffusion of dissolved inorganic carbon to colonies (>1 mm). Dark respiration of the same colonies varied between 0.038 and 87 nmol O₂ h⁻¹, and it reached the limit set by O₂ diffusion from the surrounding water to colonies (>1 mm). N₂ fixation associated with N. spumigena colonies (>1 mm) comprised on average 18% of the total N₂ fixation in the bulk water. Net NH₄⁺ release in colonies equaled 8–33% of the estimated gross N₂ fixation during photosynthesis. NH₄⁺ concentrations within light-exposed colonies, modeled from measured net NH₄⁺ release rates, were 60-fold higher than that of the bulk. Hence, N. spumigena colonies comprise highly productive microenvironments and an attractive NH₄⁺ microenvironment to be utilized by other (micro)organisms in the Baltic Sea where dissolved inorganic nitrogen is limiting growth.     

Rapid identification of Escherichia coli transformed by pBR322 carrying inserts at the PstI Site

An iodometric assay for β-lactamase has been employed for identifying colonies of Escherichia coli transformed to tetracycline resistance (Tc^r) by pBR322 carrying inserts at the PstI site. This assay is based upon the ability of β-lactamase produced by ampicillin-resistant (Ap^r) cells to convert penicillin to penicilloic acid which in turn binds iodine. Growth and selection of E. coli transformed to Ap^rTc^r or Ap^sT^r are obtained on Luria agar plates containing soluble starch and tetracycline. When indicator solution containing penicillin and iodine is added to the colonized plates, β-lactamase-producing (Ap^r) colonies rapidly clear the overlying indicator solution whereas non-β-lactamase-producing (Ap^s) colonies exhibit no clearing effect. This reaction persists and substantial numbers of viable cells remain well beyond the end of the 15-min observation period. In post-test assessment of phenotype, all nonclearing colonies exhibited the Ap^sTc^r phenotype while those that cleared the indicating solution exhibited the Ap^rTc^r phenotype. Application of this assay to an actual transformation experiment permitted rapid and unambiguous identification of the Ap^sTc^r phenotype.     

Experimentelle Untersuchungen zum Stockwachstum und zur Medusenbildung bei dem marinen HydrozoonEirene viridula

The development of both slide-grown and non-substrate bound colonies ofE. viridula (Thecata-Leptomedusae) ranging in size from 1 to 50 hydranths was investigated under various temperature conditions. The majority of slide-grown colonies reached a larger final size than non-substrate bound ones, in 20°, 25° and 29° C. Raising the temperature did not stimulate propagation of hydranths as expected. Most of the colonies transferred to 25° or 29° C finally were even smaller than those reared at 20° C. This was partially due to resorption of several hydranths about 9 days after the temperature rise; the influence of “physiological competition” between development of new hydranths and budding of medusae on colony growth is discussed. Transfer from higher to lower temperatures affected colony growth negatively. Raising the temperature from 20° to 25° or 29° C initiated formation of gonozooids from the distal part of hydranth stalks and development of medusa buds in both types of colonies. With the exception of slide-grown colonies transferred to 25° C, also young medusae were budded off. There was a remarkable coincidence in predominance of colony growth in slide-grown colonies and of medusa budding in non-substrate bound cultures. In the latter, medusa buds developed 1 to 2 days earlier. Most buds did not differentiate into liberated medusae, but were resorbed. Transformation of medusa buds into hydranths was not observed. In the clone ofE. viridula, onset of medusa budding did not depend on a “minimal colony size”. Even single hydranths were able to produce medusa buds after transfer to higher temperatures; budded off medusae were recorded from non-substrate bound colonies with an initial size of 3 hydranths. In slide-grown cultures, medusa buds developed into colonies with an initial size of only 3 hydranths. No hydranth propagation prior to medusa budding occurred in these cases. After raising temperature from 25° to 29° C medusa buds were observed in nonsubstrate bound colonies only; a small number of medusae were budded off from some of these colonies. Lowering the temperature from 29° or 25° to 20° C caused resorption of existing medusa buds. In several non-substrate bound colonies, transfer from 29° to 25° C induced development of gonozooids with medusa buds and, in some cases, of young medusae. Incubation with the alkylating cytostaticTrenimon and transfer from 20° to 25° C caused irreversible resorption of all hydranths when 4 × 10^?2 mg/ml were administered for 10 mins. Thereafter, only development of stolonial structures was observed. With one exception, the colonies treated with 4 × 10^?3 mg/ml, and all others submitted to 4 × 10^?4 mgTrenimon/ml were able to produce new hydranths and also medusa buds; some of the colonies first had to overcome a degressive phase. Treatment with 4 × 10^?2 mg destroyed all interstitial cells (I-cells). Incubation with 1 × 10^?3 or 1 × 10^?4 mg/ml left the I-cells at least partially intact. It is concluded that I-cells are indispensable for hydranth and medusan morphogenesis inE. viridula.