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 effect of light on morphogenesis of Dictyostelium mucoroides

The effect of light on the production of macrocysts and sorocarps of Dictyostelium mucoroides, strain DM-7, has been studied with surface cultures grown on dilute lactose-peptone agar at 22 degrees C with Escherichia coli, strain B/r, as food bacteria. The production of sorocarps or macrocysts can be controlled by altering the light component of the environment. Far red light had no effect on macrocyst production, whereas visible light from 440 to 700 nm inhibited macrocyst production with production decreasing with increasing light intensity. Fluence response curves for macrocyst production were determined for twelve wavelengths of light between 400 and 700 nm. An action spectrum calculated from the fluence response curves shows a single major peak at about 425-430 nm.     

CONTROL OF GERMINATION OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) CYSTS FROM THE LOWER ST. LAWRENCE ESTUARY (CANADA)

Cysts of the toxic dinoflagellate Alexandrium tamarense (Lebour) Balech 1992 from the lower St. Lawrence estuary were used in a test of the following hypotheses: (1) cyst germination is triggered by a change in temperature, and (2) germination rate varies throughout the year and is controlled by a circannual internal biological clock. Results show that cyst germination was not affected significantly by temperature of incubation over the range 1°–16° C, and light showed no significant stimulation of germination. This is supported by the lack of effect of cyst incubation conditions during evaluation of the seasonal changes in germination rate (two temperatures: 4° and 15° C, and two light conditions: darkness and 150 μmol photons·m^?2·s^?1). Thus, direct environmental control through short-term increases in temperature and exposure to light has no effect on the germination of the cysts tested. The rate of germination, observed monthly over a 16-month period, showed low germination (<20%) over most of the period tested, except for a maximum reaching more than 50% germination in August to October of the second year of the experiment. This pattern was observed for cysts both from monthly field collections and from laboratory-stored cysts kept under constant environmental conditions (4° C, in the dark). The peak in germination observed under constant environmental conditions (in the laboratory), the almost coincidental increase in cyst germination observed for the field-collected cysts, and the absence of effects of temperature and light during incubation could be explained either by a temperature-controlled cyst maturation period (the time-temperature hypothesis of Huber and Nipkow 1923) or by the presence of an internal biological clock. However, the large decline in the rate of germination 2 months after the maximum provides strong support for the biological clock hypothesis. The ca. 12-month maturation (dormancy) period observed for the laboratory-stored cysts is the longest reported for this species to our knowledge; this might be related to the low storage temperature (4° C), which is close to bottom temperatures generally encountered in this environment (0° to 6° C). Similar field and laboratory storage temperatures could explain the coincidental increase in germination rate in the fall of the second year if cyst maturation is controlled by temperature. A fraction of the laboratory-stored cysts did not follow a rhythmic pattern: A rather constant germination rate of about 20% was observed throughout the year. This continuous germination of likely mature cysts may supplement the local blooms of this toxic dinoflagellate, as these often occur earlier than peak germination observed in late summer. It seems that two cyst germination strategies are present in the St. Lawrence: continuous germination after cyst maturation, with temperature controlling the length of the maturation period, and germination controlled by a circannual internal rhythm.     

ENZYMATIC DISSOCIATION OF NASCENT MACROCYSTS AND PARTITION OF THE LIBERATED CYTOPHAGIC GIANT CELLS IN DICTYOSTELIUM MUCOROIDES*

Nascent macrocysts of the cellular slime mold Dictyostelium mucoroides were dissociated enzymatically and the liberated cytophagic giant cells were partitioned by dextrin density gradient centrifugation. Enzymatic and cytochemical studies revealed that the primary wall is composed mainly of cellulose (β-1,4-glucan) associated with polysaccharides including hemicellulose, pectic substances and á-1,4-glucan. The buoyant density of the liberated cytophagic giant cells and peripheral cells was determined by density gradient centrifugation, and partitioning of the cells was possible due to the difference in this property. The process of macrocyst reconstitution was investigated using dissociated cells. The isolated cytophagic giant cell has a specific affinity for other cytophagic giant cells and predominantly ingests them by phagocytosis, while it retains the ability to ingest peripheral cells. The present study provides a clue for investigating the differentiation and development of sexual cells, since only the cytophagic giant cell gives rise to a zygote in macrocyst formation.     

EVIDENCE FOR ASEXUAL RESTING CYSTS IN THE LIFE CYCLE OF THE MARINE PERIDINOID DINOFLAGELLATE,SCRIPPSIELLA HANGOEI1

Scrippsiella hangoei (Schiller) Larsen is a peridinoid dinoflagellate that grows during winter and spring in the Baltic Sea. In culture this species formed round, smooth cysts when strains were mixed, indicating heterothallic sexuality and hypnozygote production. However, cysts of the same morphology were also formed in clonal strains exposed to slightly elevated temperature. To better understand the role of cysts in the life cycle of S. hangoei, cyst formation and dormancy were examined in culture experiments and the cellular DNA content of flagellate cells and cysts was compared in clonal and mixed strains using flow cytometry. S. hangoei exhibited a high rate of cyst formation in culture. Cysts produced in both clonal and mixed strain cultures were thick‐walled and underwent a dormancy period of 4 months before germinating. The S. hangoei flagellate cell population DNA distributions consisted of 1C, intermediate, and 2C DNA, indicative of respective eukaryotic cell cycle phases G1, S, and G2M. The majority (>95%) of cysts had a measured DNA content equivalent to the lower 1C DNA value, indicating a haploid nuclear phase and an asexual mode of cyst formation. A small percentage (<5%) of cysts produced in the mixed strain culture had 2C DNA, and thus could have been diploid zygotes. These findings represent the first measurements of dinoflagellate resting cyst DNA content, and provide the first quantitative evidence for dinoflagellate asexual resting cysts. Asexual resting cysts may be a more common feature of dinoflagellate life cycles than previously thought.     

Cytology of lepidoptera. III. Giant cysts: A morphological trait of apyrene spermatogenesis in an Ephestia kuehniella strain

A comparative investigation of testicular eupyrene cysts (in larvae) and apyrene cysts (in pupae) of Ephestia kuehniella laboratory strains was conducted using light and electron microscopy. Eupyrene cysts in the first meiotic division contained 64 spermatocytes, which showed only moderate asynchrony. In one of the strains, a wild-type strain, L, normal-sized cysts occurred together with abnormally large cysts. These are called giant cysts in this article. One of the premeiotic cysts, early giant cysts, studied in detail, contained approximately a fourfold number of cells compared with the number in a eupyrene cyst of the same stage. In cysts harboring spermatocytes and spermatids, late giant cysts, cell differentiation was highly asynchronous. Failure in one of two control mechanisms in early cyst development may have caused the appearance of the cysts. Control of cell division might have been sloppy in apyrene spermatogonia. Hence, the spermatogonia within the cyst could have passed through additional division cycles. Alternatively, the giant cysts may have originated from more than one predefinitive gonial cell enclosed in a common envelope of sheath cells. As a third possibility, giant cysts could have arisen by fusion of normal cysts at a later stage. In either case, this is evidence that separation of eupyrene and apyrene pathways is earlier than was previously expected. In two other Ephestia strains, apyrene sperm development proceeded without formation of giant cysts. One was a mutant strain, a, and the other one was a recently established wild-type strain, Sbr. Apyrene sperm development is considered an example of degenerate evolution in which enhanced variability between species and even between populations of one species is a common phenomenon.     

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.     

ROLE OF ENCYSTMENT AND EXCYSTMENT OF PERIDINIUM BIPES F. OCCULATUM (DINOPHYCEAE) IN FRESH WATER RED TIDES IN LAKE KIZAKI,JAPAN1

The encystment flux of Peridinium bipes f. occulatum (Dinophyceae) was investigated with sediment traps from 1968 to 1990 in Lake Kazki. Cysts of P. bipes were formed throughout the blooms, Encystment flux of P. bipes in the pelagic zone was usually lower than those at shallow sites, and the density of P. bipes cysts in lake sediment was higher in the shallow region than in the pelagic zone. However, in the shallower region, The concentration of P. bipes cysts varied widely, possibly due to high rates of encystment and excystment. Peridinium bipes encystment occurred between 15° and 25° C in the laboratory, with very little cyst formation below 10°C. Though cyst formation was observed in continous darkness, the rate increased with irradiance. Under continuous darkness, no excystment was observed at any temperature from 5° to 25° C. Eighty-one percent of the cysts illuminated at 105 μE m^?2 s^?1 excysted after 13 days incubation at 15° C, and lower irradiances decreased germination success. Results from laboratory experiments suggest that light is a critical factor in the germination of P. bipes cysts. Bottom depth thus can have a significant effect on germination because cysts only can excyst from depths where light is sufficient. The shallow region of the lake is thus very important as a seed bed for P. bipes during early spring. Cyst deposited in deeper waters may not ever germinate unless they are resuspended and transported to shallow areas where light reaches the bottom.     

Trehalase expression during the germination of three resting stages of the dictyosteliaceae

Three resting stages (spores, microcysts, and macrocysts) of the Dictyosteliaceae were found to possess low levels of trehalase activity. A significant increase in the activity of trehalase was detected upon germination. The increase in activity appeared to be dependent on protein synthesis.     

THE LIGHT FACTOR IN THE GERMINATION ECOLOGY OF DRABA VERNA

The majority of the seeds of the winter annual Draba verna L. require light for germination, but light can be given before they are fully after-ripened and as long as 4 months before temperature and moisture conditions are favorable for germination. Seeds that are exposed to light in late spring, or in late spring and early summer, and then removed to darkness can germinate under favorable temperature and moisture conditions in autumn, even after passing through a long unfavorable (for germination) wetting and drying period at high summer temperatures in darkness. The light requirement for germination probably is not an important factor restricting D. verna to open, well-lighted habitats.     

Genetic control of the immune response

(I) The influence of the dose of antigen on the amount of antibodies produced was studied in two inbred strains of mice that were different with respect to the ability to produce antibodies top-aminobenzoic acid, i.e. well responding strain B10.LP and poorly responding CBA/J strain. Similar dependence between the dose of antigen and the antibody titre was demonstrated in both strains. (2) It was found that the type of reaction to the antigenic determinant (i.e. hapten) appeared to be a constant property of the inbred strain and that it did not change during the long period of the immunological maturity of the organism. (3) Antibodies of 19S type (2-mercaptoethanol sensitive) were formed in sera of both inbred strains, particularly in strain B10.LP, even after the third adjuvant immunization. Antibodies of 7S type appeared to be partially 2-mercaptoethanol sensitive, however, the major part was resistant to this agent. No 7S, 2-mercaptoethanol resistant antibodies were found in sera of the poorly responding strain CBA/J.     

Different excystment patterns in two calcareous cyst-producing species of the dinoflagellate genus Scrippsiella

Scrippsiella rotunda and Scrippsiella trochoidea var. aciculifera (order Peridiniales, subfamily Calciodinelloideae) are autotrophic orthoperidinioid dinoflagellates producing calcareous resting cysts which are at times abundant in coastal marine sediments. We have carried out laboratory experiments to investigate features of cyst germination in the two species, including dormancy length, germination pattern and germination success, over an annual cycle and under different light and temperature conditions. The maturation period for S. rotunda cysts was between 17 and 24 weeks, while that of S. trochoidea var. aciculifera was much shorter, ranging between 2 and 5 weeks. Both species required exposure to light for germination, while temperature shifts (from 14 to 20C) in the dark did not induce excystment of mature cysts. In both species, germination was not synchronous, but distributed over a variable time interval, suggesting a high physiological diversity within the cyst pool. Moreover, exposure to light of S. rotunda cysts that had not completed maturation impaired the germination of a great percentage of the cysts. Differences in dormancy length may partially explain the distinct cyst production patterns observed for the two species in the Gulf of Naples.      

BIOCHEMICAL COMPOSITION AND METABOLIC ACTIVITY OF SCRIPPSIELLA TROCHOIDEA (DINOPHYCEAE) RESTING CYSTS1

The composition and metabolic activity of cysts of the marine dinoflagellate Scrippsiella trochoidea (Stein) Loeblich were examined during dormancy, quiescence, and germination. On a per cell basis, newly formed cysts contained an order of magnitude more carbohydrate but significantly less protein and chlorophyll a than did exponentially growing vegetative cells. Loss of lipid and carbohydrate from cysts during the initial dormancy period reflected a respiration rate estimated to be 10% of the respiratory activity in vegetative cells. Among older, quiescent cysts the calculated respiration rate decreased further to approximately 1.5% of the vegetative rate and appeared to proceed largely at the expense of carbohydrate reserves. These estimated rates of respiration were in good agreement with direct measurements of cyst oxygen consumption. The transfer of quiescent cysts to conditions permissive for germination resulted in a rapid increase in respiration rate, as evidenced by carbohydrate loss and O₂ consumption. The increased respiratory activity was followed by an increase in protein content and, later, by an increase in chlorophyll a content and photosynthetic capacity. Just prior to germination the P/R ratio became greater than 1, and the estimated chlorophyll-specific photosynthetic activity reached 75% of the rate in vegetative cells. Complete restoration of photosynthetic and respiratory capacity apparently was not achieved until after excystment. These data confirm the common assumption that dinoflagellate cysts represent true “resting” cells, containing extensive energy reserves and displaying greatly reduced metabolic activity.     

SEXUALITY AND CYST FORMATION IN PACIFIC STRAINS OF THE TOXIC D1NOFLAGELLATE GONYAULAX TAMARENSIS1,2

Sexuality has been established for a culture of Gonyaulax tarnarensis Lebour (strain NEPCC–71). The addition of a thick inoculum to a nitrogen–deprived medium results in the occurrence of anisogamous sexual fusion within the first three days in the new culture. Planozygotes, large “lumpy” cells recognizable by their four flagella, may persist up to 2 wk before forming a smooth–walled, oval hypnozygote. The latter resembles cysts released asexually by ecdysis but has a slightly thicker wall. Viable cysts resembling hypnozygotes (zygotic cysts), but with reduced photosynthetic pigmentation, have been isolated from natural murine sediments in Hidden Basin, British Columbia, and a culture (strain NEPCC–254) was initiated from excysted individuals. Zygotic cysts of NEPCC–71 remained encysted in the light at 17 C for 8 wk before excysting. The presence of a ventral pen with toxicity in the latter strain indicates that the taxonomy of G. tamarensis-like organisms is still in a stale of flux and the criteria for recognition of G. excavata (Braarud) Balech as a separate species are not satisfactory as presently formulated.     

THE NINETY-YEAR PERIOD FOR DR. BEAL'S SEED VIABILITY EXPERIMENT

In 1879 Dr. W. J. Beal selected seeds of 23 different species of locally common plants, mixed 50 seeds of each species with moist sand in unstoppered one-pint bottles, and buried the bottles in a sandy knoll to be unearthed and the viability of the seeds tested periodically. The year 1970 marked the ninetieth year the seed had been buried, and the thirteenth bottle was recovered to test for seed viability. Of the three species which had germinated in the 1960 test (curly dock, Rumex crispus; evening primrose, Oenothera biennis; and moth mullein, Verbascum blattaria), only V. blattaria had viable seed with 20% germination. No other species germinated. All ten seedlings of V. blattaria were grown to maturity, and seeds were then harvested to study the possible deviations from normality and the requirements for seed germination. All seedlings emerging from the first progeny seed appeared normal. The most prominent requirement for germination was light, and this is a possible explanation of why the seeds had remained viable but dormant for so long a period. One-third of the freshly harvested seed germinated in darkness and, furthermore, redrying of dark-moistened seed in the absence of light induced additional germination. Germination of dark-moistened seed was not completely restored when the still moist seeds were subsequently exposed to light. However, when dark-moistened seeds were dried and then remoistened in the light, germination was about 95 %. About 5 % of the seed did not germinate under the conditions used. We find that 5 % of the population of V. blattaria seeds are dormant for unknown reasons, that 30 % will germinate if supplied only with moisture, and that 65 % are inhibited and require light and moisture simultaneously for germination. Supplying this 65 % of the population with moisture in darkness results in the development of a second type of inhibition which is no longer light reversible. It appears that the simultaneous requirement for light and moisture is an important factor in permitting V. blattaria seeds to remain dormant during prolonged burial.     

GROWTH AND THE PRODUCTION OF EXTRACELLULAR SUBSTANCES BY TWO STRAINS OF PHAEOCYSTIS POUCHETI1,2

Clones of Phaeocystis were isolated from winter surface waters off Woods Hole, Massachusetts, and from the tropical Atlantic near Surinam, South America. Northern strains survived only up to 14 C, while the tropical strain survived only as low as 17 C. Colony shapes of the northern and tropical clones differed somewhat, but the motile and non-motile single cells of both strains seemed identical in the light microscope. By current taxonomic criteria both strains belong to the species P. poucheti (Hariot) Lagerheim. When growing in the form of colonies, both strains excreted 16–64% of their photoassimilated carbon into the medium, mainly as carbohydrates of varying molecular weights. However, cultures predominantly in the form of single cells released only about 3% of their photoassimilated carbon. The qualitative composition of the carbohydrates released is similar for the 2 strains, consisting of some 8 sugars or sugar derivatives with glucose, mannose, and rhamnose as the dominant components. The production of acrylic acid was confirmed. We estimate that as much as 7 μg/liter of acrylic acid, and at least 0.3 mg/liter of polysaccharides can be liberated in a Phaeocystis bloom.     

关于茭白黑粉菌的正名问题*

茭白黑粉菌在文献中出现Yenia esculenta(P.Henn.)Liou及Ustilago esculenta P.Henn.两个名字。现经形态发生学的研究以明确新名字是否有效。此菌冬孢子没有休眠期,孢子成熟后在适宜的环境中即可萌发。萌发的最适pH值为6;最适温度为25％;不需要外界光的诱导,在黑暗条件下也能正常萌发;培养基中的营养成份对孢子萌发的速率有一定影响。冬孢子在不同的环境条件下,其萌发形态及方式都是稳定的:孢子萌发时产生有隔的担子或先菌丝(promycelium)。该先菌丝起初可以是无隔的,但生长到一定程度,便产生分隔,分隔多为2—4个。初生的先菌丝和小孢子是单核的,但成熟的先菌丝、小孢子及短菌丝细胞内均是双核的。可以认为,该菌的双核期在整个生活史中占有相当长的时间,它只有短暂的单核期。没有发现先菌丝、小孢子彼此之间或相互间融合的现象;未见到双核的融合及减数分裂过程。茭白黑粉菌在人工组合培养基上生成厚壁的孢子,较自然界中的冬孢子为大,但萌发方式则相同。最后作者等认为茭白黑粉菌仍应保留在Ustilago属中,而Yenia esculenta(P.Henn.)Liou的名字是无效的。     

RESTING CYST FORMATION OF EUTREPTIELLA GYMNASTICA (EUGLENOPHYCEAE) IN THE NORTHERN COASTAL BALTIC SEA1

Resting cyst formation of Eutreptiella gymnastica Throndsen was observed during a mesocosm experiment, where nutrient enrichment had induced almost a unialgal bloom. Cells and resting cysts of E. gymnastica were examined in scanning (SEM) and transmission electron microscopy (TEM) and light microscopy. Mature cysts were spherical, with a smooth thick mucilaginous cover that appeared layered when observed with the TEM. Intermediate forms were spherical and lacking flagella and a mucilaginous cover; the euglenoid pellicular striation and canal opening were easily visible. The volume of these intermediate spherical cells and mature cysts was estimated to have increased threefold compared to flagellated cells and contained many paramylon grains. When the cells were grazed by zooplankton, the paramylon grains passed the gut intact and were packed into fecal pellets. Intact undigested paramylon grains were observed in SEM after the breaking up of the pellets.     

Differential utilization of pyrene as the sole source of carbon by Bacillus subtilis and Pseudomonas aeruginosa strains: role of biosurfactants in enhancing bioavailability

AIMS: Our goal is to compare the efficiency of utilization of pyrene as the sole source of carbon for growth and energy by two nonactinomycetous groups of bacteria viz., Bacillus subtilis DM-04 and Pseudomonas aeruginosa mucoid (M) and nonmucoid (NM) strains, isolated from a petroleum-contaminated soil sample of north-east India. METHODS AND RESULTS: Bacillus subtilis DM-04 and P. aeruginosa M and NM bacterial strains were capable of secreting biosurfactant in the culture medium while growing on pyrene and their pyrene utilizing efficiency was demonstrated by correlating the bacterial growth in the presence of pyrene as the sole source of carbon along with a concomitant decrease in pyrene content from the culture medium with respect to time. The biosurfactant secreted by the respective bacterial strains enhanced the apparent solubility of pyrene by factors of 5-7 and influenced the bacterial cell surface hydrophobicity resulting in higher uptake and utilization of pyrene by bacteria. The growth of B. subtilis DM-04 and P. aeruginosa M and NM strains at the expense of pyrene after 96 h showed an assimilation of about 48.0 +/- 1.1% (mean +/- SD) and 32.0 +/- 0.6% (mean +/- SD) of pyrene carbon, respectively, showing differences in metabolism of pyrene by these bacterial strains. CONCLUSIONS: Bacillus subtilis DM-04 strain exhibited higher utilization and cellular assimilation of pyrene compared with P. aeruginosa M and NM strains. Further, the biosurfactants produced by the bacteria under study are capable of enhancing the solubility of pyrene in aqueous media and can influence the cell surface hydrophobicity of the biosurfactant-producing strains that results in a higher uptake of pyrene. SIGNIFICANCE AND IMPACT OF THE STUDY: It may be suggested that the bacteria used in this study are suitable candidates for practical field application for effective in situ bioremediation of pyrene-contaminated sites.