The Possible Involvement of Cyclic AMP and Volatile Substance (s) in the Development of a Macrocyst-Forming Strain of Dictyostelium mucoroides

A mutant MF1 previously isolated from Dictyostelium mucoroides -7 (Dm7) formed macrocysts with or without light when plated on agar at high cell dinsities. At lower cell densities, however, the MF1 cells formed only fruiting bodies. This failure to form macrocysts was shown to be due to the subthreshfold concentration of a volatile substance(s) required for macrocyst formation. Although ammonia is a volatile substance produced by both the Dm7 and MF1 cells, no evidence of its involvement in macrocyst formation was obtained. Mixing the Dm7 and MF1 in a one-to-one ratio resulted only in fruiting body formation suggesting that the Dm7 cells produced a factor which allowed MF1 cells to form fruiting bodies. This factor may be cyclic AMP (cAMP) since addition of cAMP to the medium directed development of MF1 cells to fruiting body formation. The effect of cAMP was exhibited most conspicuously when MF1 cells were exposed at the aggregation stage. Based on these results it is suggested that developmental pathway of the D. mucoroides macrocystforming strain Dm7 and its mutant MF1 may be determined by the relative concentrations of the volatile, macrocyst-inducing substance(s) and cAMP at the aggregation stage.     

MACROCYSTS IN THE LIFE CYCLE OF THE DICTYOSTELIACEAE. I. FORMATION OF THE MACROCYSTS

Macrocysts, a morphogenetic phase that is alternative to sorocarp formation in the life cycle of some cellular slime molds, are known for two genera and five species of the Dictyosteliaceae. In all of these macrocyst formation was found to be strongly affected by four factors: light, temperature, moisture, and the composition of the medium. In general, macrocyst formation was suppressed and sorocarp formation was enhanced by exposure to light, by incubation at temperatures lower than 20 C, by buffering nutrient media with phosphates, and by reducing atmospheric moisture through the use of clay covers on Petri dishes. The extent to which these environmental factors, singly or in combination, inhibited the production of macrocysts varied among the different strains and species.     

The developmental capacity of various stages of a macrocyst-forming strain of the cellular slime mold, Dictyostelium mucoroides.

Vegetative cells of certain strains of Dictyostelium mucoroides form fruiting bodies on an agar surface and macrocysts when placed under saline. This study sought to determine whether the aggregation and pseudoplasmodial stages of fruiting body formation could be induced to form macrocysts when placed under saline. Likewise, different stages in macrocyst formation were put on an agar surface to determine their potential to switch to fruiting body formation. It was found that 78% of the aggregates and 21% of the pseudoplasmodia placed under saline formed macrocysts indicating that as fruiting body development proceeds, there is a restriction of the capability of cells to respond to environmental conditions favoring macrocyst formation. Stages in macrocyst development prior to the formation of precysts always formed fruiting bodies when put on agar. Once precysts had formed, surrounded by their acellular sheath, they always developed as macrocysts on agar. Peripheral cells isolated from precysts and put on agar quickly aggregated; the aggregates became surounded by a sheath and developed as macrocysts. If isolated peripheral cells were allowed to proliferate on the agar surface, the resulting cells aggregated and formed fruiting bodies.     

Techniques and Marker Genes for Use in Macrocyst Genetics with POLYSPHONDYLIUM PALLIDUM

Previous work has shown that genetic exchange occurs in the macrocyst of Polysphondylium pallidum, as in species of Dictyostelium. These studies are extended here. Mutants resistant to six different poisons have been isolated for use as genetic markers. A replica-plating technique has been engineered whereby 14 progeny clones growing on a master plate may be simultaneously transferred to test plates containing individual poisons. Germination percent of macrocysts has been greatly increased by the presence of a growing fungus during the resting stage. These means have been used to analyze crosses showing that: (1) Vegetative amebae are haploid, at least at the three marker loci tested. (2) Amebae emerging from a single macrocyst are identical about 90% of the time. (3) Any single combination of parental markers may emerge from a given macrocyst, and all combinations appear in approximately equal frequencies. These findings suggest that normally one of four nuclei produced by meiosis survives in every macrocyst and that all markers examined are unlinked. (4) About 10% of the macrocysts germinate to give two or more classes of progeny. These may result from the presence of a second zygote in the macrocyst or from the survival of two nuclei after meiosis. (5) If genetic exchange occurs during spore formation or microcyst formation, its frequency is low (<0.01%).     

MACROCYSTS IN THE LIFE CYCLE OF THE DICTYOSTELIACEAE. II. GERMINATION OF THE MACROCYSTS

Macrocyst germination was demonstrated in the five species of the Dictyosteliaceae known to produce these structures. The morphological changes that occurred during germination appeared to be identical in all of the strains examined, showing the following stages: (1) swelling of the dark, contracted content of the dormant cysts, (2) gradual loss of color and reappearance of cells within what previously appeared as a homogeneous protoplasmic mass, and (3) rupture of the heavy cellulosic cyst wall to liberate the myxamoebae. The age of the macrocyst appeared to be the most critical factor in determining whether or not germination would occur, since the cysts in many of the strains needed to age for several weeks or months before germination could be demonstrated. In Dictyostelium mucoroides strain DM-7, upon which the current study was centered, light was necessary to stimulate germination of young macrocysts—a requirement that gradually diminished as the cysts aged. The rate of germination and the temperature permitting germination were also age dependent: older macrocysts germinated more rapidly and at considerably higher temperatures than did young cysts. Although light was not essential for germination in every strain, the results obtained with strain DM-7 seem to be generally applicable to the germination process.     

Genetic exchanges in the macrocysts of Dictyostelium discoideum.

Crosses were made between strains of Dictyostelium discoideum involving two drug resistance markers and the mating-type locus. Over 6000 progeny from 263 individual germinated macrocysts from four single-factor crosses, five two-factor crosses and one three-factor cross were characterized. In most cases the progeny from a single macrocyst were of one genotype, although in the population of macrocysts from any two-factor cross all possible parental and recombinant genotypes were recovered. There was no evidence of linkage between any of the markers examined. No selection against progeny carrying the methanol or the cycloheximide resistance markers was found in two-factor crosses, but selection against progeny carrying both resistance markers was found in the three-factor cross. Germination of macrocysts in all crosses was poor, only once exceeding 2.5% of the total macrocyst population. A variety of crosses and back-crosses with different parental strains indicated that germination might be influenced by both extrinsic (environmental) and multiple genetic factors. About 10% of the macrocysts yielded progeny spores that were ambivalent in their mating reactions. After extensive recloning these populations could be resolved to the normal matA (formerly A1) and mata (formerly A2) mating-types and might therefore have represented aneuploids. The results obtained with D. discoideum macrocysts differ from those obtained with other cellular slime moulds--Dictyostelium mucoroides, Dictyostelium giganteum and Polysphondylium pallidum--and are reminiscent of the results reported for germinated zygospores of Phycomyces blakesleeanus.     

Social amoebae mating types do not invest unequally in sexual offspring

Unequal investment by different sexes in their progeny is common and includes differential investment in the zygote and differential care of the young. The social amoeba Dictyostelium discoideum has a sexual stage in which isogamous cells of any two of the three mating types fuse to form a zygote which then attracts hundreds of other cells to the macrocyst. The latter cells are cannibalized and so make no genetic contribution to reproduction. Previous literature suggests that this sacrifice may be induced in cells of one mating type by cells of another, resulting in a higher than expected production of macrocysts when the inducing type is rare and giving a reproductive advantage to this social cheat. We tested this hypothesis in eight trios of field‐collected clones of each of the three D. discoideum mating types by measuring macrocyst production at different pairwise frequencies. We found evidence that supported differential contribution in only two of the 24 clone pairs, so this pattern is rare and clone‐specific. In general, we did not reject the hypothesis that the mating types contribute cells relative to their proportion in the population. We also found a significant quadratic relationship between partner frequency and macrocyst production, suggesting that when one clone is rare, macrocyst production is limited by partner availability. We were also unable to replicate previous findings that macrocyst production could be induced in the absence of a compatible mating partner. Overall, mating type‐specific differential investment during sex is unlikely in microbial eukaryotes like D. discoideum.     

The fine structure of macrocyst germination in Dictyostelium mucoroides

The ultrastructure of macrocysts during germination is described from OsO₄ fixed material. The process begins with the splitting of the tertiary wall into two parts. This is followed by the cleavage of the protoplast into large uninucleate pro-amoebae. As these proamoebae become less electron dense most of the endocyte fragments are digested. Simultaneously the secondary wall and the outer part of the tertiary wall break, leaving only the thin inner part of the tertiary wall intact. The pro-amoebae divide several times to produce the myxamoebae that escape during germination by breaking through the inner layer of the tertiary wall. These myxamoebae multiply or form sorocarps or macrocysts, depending on conditions.     

Analysis of Dictyostelium mucoroides Macrocysts by Using a Simple Breakage and Fractionation Procedure

A method is described for the breakage and fractionation of Dictyostelium macrocysts. It involves vortexing with large glass beads to disrupt the macrocyst wall followed by differential low-speed centrifugation. It yields fractions containing purified wall material and intact endocytes. The endocytes can themselves be disrupted by using smaller glass beads.     

Cystein proteinase changes during macrocyst formation in Dictyostelium mucoroides

Cysteine proteinases were detected in vegetative myxamoebae of Dictyostelium mucoroides DM7 using chromogenic substrates and by electrophoretic analysis (gelatin-SDS-PAGE) which revealed three enzymes, dmCP30, dmCP35 and dmCP46 (a minor form). During the initial stages of macrocyst formation the cysteine proteinaes were secreted and disappeared almost completely from the cells. High extracellular levels of activity towards N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine 4-nitroanilide and of dmCP30 persisted throughout macrocyst development. Three new intracellular proteinases, dmCP31, dmCP36 and dmCP40, were produced as macrocysts formed but their activity was only detected by gelatin-SDS-PAGE. Their appearance was specific to the developmental pathway leading to macrocyst formation. This is the first direct evidence for the accumulation of cysteine proteinases during a developmental process in a cellular slime mould.     

Multiple light inputs control phototaxis in Synechocystis sp. strain PCC6803

The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a taxD1 mutant (Cyanobase accession no. sll0041; also called pisJ1) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A deltataxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.     

Involvement of cytoplasmic pH in the production of ethylene,a potent inducer of sexual development inDictyostelium mucoroides

Summary Dictyostelium mucoroides-7 (Dm 7) and a mutant MF 1 derived from it exhibit two developmental pathways: sorocarp formation occurs during the asexual process, and macrocyst formation during the sexual cycle. The two developmental pathways are mainly regulated by two chemical substances: 3,5-cyclic adenosine monophosphate (cAMP) and ethylene. Recently, we have demonstrated that cytoplasmic pH (pH_i) has a critical role for the choice of developmental pathways, higher pH_i being favourable to macrocyst formation. Thereupon, attention was riveted to the relation of pH_i to biosynthesis of cAMP and ethylene. Effect of pH_i on the production and release of ethylene, a potent inducer of macrocyst formation, was examined, using the two facing culture method. The result showed that lowered pH_i inhibits ethylene production, thus resulting in a failure of cells to form macrocysts. The accumulation of cAMP, an inhibitor of macrocyst formation, was found to vary depending on extracellular pH (pH_o), but diethylstilbestrol (DES) that is a proton pump inhibitor and also an inhibitor of macrocyst formation had no significant effect on the accumulation. Taken together these results indicate that higher pH_i may induce macrocyst formation through enhancement of ethylene production rather than inhibition of cAMP synthesis.Abbreviations cAMP 3,5-cyclic adenosine monophosphate - pH_i cytoplasmic pH - pH_o extracellular pH - ACC 1-1-aminocyclopropane-1-carboxylic acid     

Reverse genetic analyses of gamete-enriched genes revealed a novel regulator of the cAMP signaling pathway in Dictyostelium discoideum

Sexual development in Dictyostelium discoideum is initiated by the fusion of opposite mating type cells to form zygote giant cells, which subsequently gather and phagocytose surrounding cells for nutrition to form macrocysts. Here we performed the targeting of 24 highly gamete-enriched genes we previously isolated, and successfully generated knockout mutants for 16 genes and RNAi mutants for 20 genes including 6 genes without disruptants. In the knockout mutants of two genes, cell aggregation toward the giant cells was much less extensive and many cells remained around poorly formed macrocysts. We named these genes tmcB and tmcC. Although macrocyst formation of wild type cells was suppressed by the addition of exogenous cAMP, that of knockout mutants of tmcB was much less sensitive. The mRNA level of phosphodiesterase (pde) was higher and that of its inhibitor (pdi) was lower in the latter cells compared to the parental strains during sexual development. Thus, tmcB appeared to be a novel regulator of the cAMP signaling pathway specific to sexual development. Knockout mutants of tmcC were indistinguishable from the wild type cells with respect to the cAMP response, suggesting that this gene is relevant to other processes.     

Induction of fruiting bodies in a macrocyst-forming mutant of the cellular slime mold, Dictyostelium mucomides

Some wild-type strains of Dictyostelium mucoroides exhibit dimorphism in development depending on culture conditions: on agar, fruiting bodies containing stalk and spore cells are formed, whereas under water, a thick-walled structure lacking spore and stalk cells (the macro-cyst) is formed. The mutant, MF-1, was derived from one of these wild-type strains. It forms macrocysts on an agar surfxe as well as under water. It was found that MF-1 could be induced to form fruiting bodies in two ways. First, when an aggregation center from the wild-type strain was grafted to an MF-1 aggregation center. MF-1 cells migrated to the center and formed a large aggregate that gave rise to many slugs that became fruiting bodies. This result, along with the observation that MF-1 aggregates have no tip, suggests that MF-1 normally produces an aggregation center that is unable to organize the aggregate to form a slug. Second, when MF-1 cells were allowed to develop on 1.2 mM ethionine (an analog of methionine), they formed aggregates with tips and developed into fruiting bodies with thick stalks instead of macrocysts. The effect of ethionine was blocked by the presence of 2.4 mM methio-nine. Two other methionine analogs were also tested, i.e., α-methylmethionine and norleucine. When cultured on the former at concentrations ranging from 1.2 to 9.6 mM, MF-1 cells still produced macrocysts; when cultured on norleucine at concentrations ranging from 2.4 to 9.6 mM, MF-1 cells aggregated into large clumps that formed numerous slugs, but these failed to continue development to fruiting bodies. In vertebrates, it is known that a major biochemical effect of ethionine is the inhibition of the methylation of nucleic acids, proteins, and phospholipids. Norleucine and a-methylmethionine inhibit methylation to a lesser extent. Thus, it can be speculated that the biological effects of ethionine on MF-1 cells may result from its interference with methylation reactions, suggesting that macrocyst formation may involve excess methylation as compared with the situation during fruiting-body development.     

Macrocyst genetics in Polysphondylium pallidum, a cellular slime mould.

The discovery of two mating types in the cellular slime mould Polysphondylium pallidum is reported. Two developmental mutants produced in strains of opposite mating type but which do not proceed past the aggregation stage of development are capable of producing macrocysts. These macrosysts were viable and 5 to 10% germinated after 6 weeks of storage. When the macrocyst progeny were cloned, several classes of non-parental phenotypes were recovered.     

Stress response in Caenorhabditis elegans caused by optical tweezers: wavelength, power, and time dependence

Optical tweezers have emerged as a powerful technique for micromanipulation of living cells. Although the technique often has been claimed to be nonintrusive, evidence has appeared that this is not always the case. This work presents evidence that near-infrared continuous-wave laser light from optical tweezers can produce stress in Caenorhabditis elegans. A transgenic strain of C. elegans, carrying an integrated heat-shock-responsive reporter gene, has been exposed to laser light under a variety of illumination conditions. It was found that gene expression was most often induced by light of 760 nm, and least by 810 nm. The stress response increased with laser power and irradiation time. At 810 nm, significant gene expression could be observed at 360 mW of illumination, which is more than one order of magnitude above that normally used in optical tweezers. In the 700-760-nm range, the results show that the stress response is caused by photochemical processes, whereas at 810 nm, it mainly has a photothermal origin. These results give further evidence that the 700-760-nm wavelength region is unsuitable for optical tweezers and suggest that work at 810 nm at normal laser powers does not cause stress at the cellular level.     

The ethylene action in the development of cellular slime molds: an analogy to higher plants

Summary The cellular slime moldDictyostelium mucoroides-7 (Dm 7) and its mutant (MF 1) exhibit sexual or asexual development depending upon culture conditions. During the sexual cycle macrocyst formation occurs, whereas sorocarps containing spores and stalk cells are asexually formed. As previously reported, the macrocyst formation is marked by the emergence of true zygotes, and is induced by a potent plant hormone, ethylene. The concentration of ethylene required for macrocyst induction was determined to establish the similarity of ethylene action between this organism and higher plants. Macrocysts are induced by low (1 l/l) exogenous concentrations of ethylene. Higher concentrations (10–1,000 ul/l) also gave essentially the same inductive activity. Ethionine, an analogue of methionine, was found to inhibit zygote formation during sexual development through its interference with ethylene production by Dm 7 and MF 1 cells. In fact, the inhibitory effect of ethionine was mostly nullified by the application of ethylene, S-adenosyl-L-methionine, or 1-aminocyclopropane-1-carboxylic acid. Taken together these results suggest that both the effective concentration of ethylene and the pathway of ethylene biosynthesis inD. mucoroides may be similar to those in higher plants. Ethylene was also found to be produced in various species and strains of cellular slime molds, even during the asexual process. The possible functions of ethylene in the asexual development are discussed in relation to cell aggregation and differentiation.Abbreviations SAM S-adenosyl-L-methionine - ACC 1-aminocyclopropane-1-carboxylic acid - AOA (aminooxy) acetic acid - BSS Bonner's salt solution - DAPI 4,6-diamidino-2-phenylindole     

Action Spectra for Phycobiliprotein Synthesis in a Chromatically Adapting Cyanophyte, Fremyella diplosiphon

The action spectra for phycocyanin production by the cyanophyte Fremyella diplosiphon shows maxima at 463 and 641 nm. The action spectrum for phycoerythrin production includes maxima at 387 and 550 nm. The maxima are based on a relative response rate well within the linear ascending portion of the dose response curves; the positions of the maxima are independent of the relative response rates chosen for reference over a 3-fold range although the comparative effectiveness of light at pairs of wavelengths varies with the standard used for comparison. These action spectra differ from those reported previously for Tolypothrix tenuis by Fujita and Hattori (Plant Cell Physiol. 3: 209-220) and by Diakoff and Scheibe (Plant Physiol. 51: 382-385) in that blue light strongly promotes phycobiliprotein synthesis in F. diphosiphon but has been reported to have little or no effect on T. tenuis.     

Ammonia Determines the Alternative Pathways of Sexual or Asexual Development in the Cellular Slime Mold Dictyostelium discoideum

The sexual development, macrocyst formation, of Dictyostelium discoideum is initiated by sexual fusion of cells. The sexual fusion is only taken place under the culture conditions of excess water and darkness. Under these conditions, cells acquire the fusion competence, but lose it when cell density is high. The loss of the fusion competence is caused by accumulation of ammonia excreted by cells in a culture. Ammonia suppresses the fusion competence of cells at a certain concentration, and consequently inhibits formation of macrocysts and induces fruiting-body formation. Thus, excess water induces the sexual development by diluting ammonia and lack of water induces the asexual development.     

光谱和光强度对西花蓟马雌虫趋光行为的影响

利用行为学方法研究了光谱、光强对西花蓟马Frankliniella occidentalis (Pergande)雌成虫的趋、避光行为的影响。结果显示：（1）在340~605nm波谱内14个波长其光谱趋光行为反应为多峰型,峰间主次较明显。趋光行为反应中,蓝绿区498~524nm有一较宽峰,趋光率20.31%;其它各峰依大小次序分别位于紫光380nm、蓝光440nm;（２）避光行为反应中,蓝光440nm处略高,避光率17.19%;紫外340nm处亦有一峰,避光率15.63%;（３）随光强增强其趋光反应率增大,白光、380nm和524nm刺激时其光强趋光行为反应呈一倒“L”型式样,498nm为峰型,440nm时为一较缓的平直线型;光强最弱时仍均有一定趋光率 ,最强时均未出现高端平台;（４）随光强增强其避光反应率增大,440nm为较平缓直线;340nm刺激时为较缓波动线。结果表明：光谱对其趋光行为有很大影响,光强度的影响较大且影响大小与波长因素有关。