CONDITIONS OF ILLUMINATION AND ZOOSPOROGENESIS IN KLEBSORMIDIUM FLACCIDUM1

The filamentous green alga Klebsormidium flaccidum will produce zoospores when cultured on a diurnal regime of 8-hr light and 16-hr dark. Zoosporogenesis is inhibited by interruption of the dark period with light of sufficient intensity and duration. The relationship between intensity and maximum time of interruption before total inhibition of zoosporogenesis is nonlinear.     

Microalgae as a raw material for biofuels production

Biofuels demand is unquestionable in order to reduce gaseous emissions (fossil CO₂, nitrogen and sulfur oxides) and their purported greenhouse, climatic changes and global warming effects, to face the frequent oil supply crises, as a way to help non-fossil fuel producer countries to reduce energy dependence, contributing to security of supply, promoting environmental sustainability and meeting the EU target of at least of 10% biofuels in the transport sector by 2020. Biodiesel is usually produced from oleaginous crops, such as rapeseed, soybean, sunflower and palm. However, the use of microalgae can be a suitable alternative feedstock for next generation biofuels because certain species contain high amounts of oil, which could be extracted, processed and refined into transportation fuels, using currently available technology; they have fast growth rate, permit the use of non-arable land and non-potable water, use far less water and do not displace food crops cultures; their production is not seasonal and they can be harvested daily. The screening of microalgae (Chlorella vulgaris, Spirulina maxima, Nannochloropsis sp., Neochloris oleabundans, Scenedesmus obliquus and Dunaliella tertiolecta) was done in order to choose the best one(s), in terms of quantity and quality as oil source for biofuel production. Neochloris oleabundans (fresh water microalga) and Nannochloropsis sp. (marine microalga) proved to be suitable as raw materials for biofuel production, due to their high oil content (29.0 and 28.7%, respectively). Both microalgae, when grown under nitrogen shortage, show a great increase (~50%) in oil quantity. If the purpose is to produce biodiesel only from one species, Scenedesmus obliquus presents the most adequate fatty acid profile, namely in terms of linolenic and other polyunsaturated fatty acids. However, the microalgae Neochloris oleabundans, Nannochloropsis sp. and Dunaliella tertiolecta can also be used if associated with other microalgal oils and/or vegetable oils.     

Production and analysis of secondary carotenoids in green algae

The microalgae Neochloris wimmeri, Scenedesmus vacuolatus, Scotiellopsisoocystiformis, Chlorella zofingiensis and Protosiphon botryoides were grown under secondarycarotenoid inductive conditions. The results indicatethat nitrogen deficiency and high light intensity arepotential inducers of astaxanthin formation in thefive microalgae studied. All these microalgaeaccumulate significant quantities of secondarycarotenoids, mainly as astaxanthin esters andcanthaxanthin. They also showed high resistance toenvironmental conditions. All these qualities makethese microalgae good candidates for successfulculture in open ponds.     

Regulation by light of ammonium transport systems in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is able to take up methylammonium/ammonium from the medium at different stages of its sexual life cycle. Vegetative cells and pre‐gametes mostly used a low‐affinity system (LATS) component, but gametes obtained after light treatment of N‐deprived pre‐gametes expressed both LATS and high‐affinity system (HATS) components for the uptake of methylammonium/ammonium. The activity of the LATS component was stimulated by light in only 5 min in a process independent of protein synthesis. By using the lrg6 mutant that produces sexually competent gametes in the dark, light effects on ammonium transport and gamete differentiation have been separately analysed. We have found light regulation of four Amt1 genes: Amt1; 1, Amt1; 2, Amt1; 4 and Amt1; 5. Whereas light‐dependent expression of Amt1; 1, Amt1; 2 and Amt1; 4 was independent of gametogenesis, and that of Amt1; 5 was activated in the lrg6 mutant, suggesting a connection between this transporter and the subsequent events taking place during gametogenesis.     

2,4-Diacetylphloroglucinol suppresses zoosporogenesis and impairs motility of Peronosporomycete zoospores

2,4-Diacetylphloroglucinol (DAPG) produced by Pseudomonas fluorescens, shows toxicity to many microorganisms including fungi, bacteria, and peronosporomycetes. Zoosporogenesis and motility of zoospores are critical for a complete disease cycle and pathogenicity of the peronosporomycete phytopathogens. The aim of this study was to test the effects of DAPG and its derivatives on zoosporogenesis and motility of zoospores of a downy mildew pathogen, Plasmopara viticola, and a damping-off pathogen, Aphanomyces cochlioides. In both cases, DAPG inhibited zoosporogenesis (5 μg/ml) and the motility of zoospores (10 μg/ml) in a dose-dependent manner. Generally, zoospores became immotile shortly after exposure to DAPG followed by lysis. However, a fraction of DAPG treated A. cochlioides zoospores formed round cystospores instead of lysis and then germinated with excessively-branched germ tubes. All derivatives of DAPG had similar inhibitory activities but at varying doses. Among them, 2,4-dipropylphloroglucinol exerted the highest inhibitory activity against both zoosporogenesis and motility of zoospores. This revealed that the degree of hydrogen atoms substitution in the benzene ring by acyl groups and the length of substituted acyl groups were related to the level of bioactivity. This is the first report of inhibitory activities of DAPG and its derivatives against zoosporogenesis and motility of zoospores of two important peronosporomycete phytopathogens.     

光质及种子大小对普洱地区14种植物种子萌发的影响

以普洱地区14种常见植物种子为材料,在实验室条件下研究了其在白光、黑暗、红光和蓝光条件下的萌发特性,并分析了种子大小与萌发率、萌发速率、萌发开始时间的关系。结果表明:光质对四方蒿、沙针、尖子木、藿香蓟种子萌发率和萌发速率均有显著影响(P0.05)。光质对大叶斑鸠菊、云南山枇花、臭灵丹、车桑子、光萼猪屎豆、葫芦茶、云南地桃花、西南宿苞豆、岗柃、中国宿苞豆10个物种的种子萌发率和萌发速率均没有显著影响(P0.05),以上物种中除中国宿苞豆外,其他物种种子萌发率均在20%以下,处于休眠状态。四方蒿种子在白光(89.9%)和红光(84.7%)下萌发率最高,红光下种子萌发最快(4.93),蓝光下种子萌发开始时间最晚(11.3 d);沙针种子在白光下萌发率最高(80.4%)、萌发速率最快(2.71),在黑暗和蓝光下萌发率较低(43.9%和38%)、萌发速率最慢(0.73和0.85),白光、红光下萌发开始最早(11 d),黑暗条件下萌发开始最晚(21.7 d);尖子木种子萌发率在白光、黑暗、蓝光下均在86%以上,而红光下仅32%且萌发速率最慢(1.29),在蓝光下萌发开始时间最晚(13 d);藿香蓟种子萌发率和萌发速率在红光下最高(分别为71.3%和6.46),黑暗条件下最低(分别为42.5%和2.62);大叶斑鸠菊萌发开始时间在黑暗条件下最早(6 d),其次是白光下(7 d),蓝光和红光下较晚,分别为8 d和7.7 d。14个物种种子的萌发率与种子大小间均有显著负相关关系;种子萌发速率、萌发开始时间与种子大小间也有负相关关系,但不显著;种子大小与萌发率、萌发速率和萌发开始时间的关系不会随着光质的变化而发生变化。     

THE LIFE CYCLE OF PEDIASTRUM SIMPLEX1

A sexual cycle and a newly discovered mode of asexual reproduction by azygotes arc described for Pediastrum simplex. In the sexual cycle isogametes from colony cells fuse in pairs, develop into zygotes, which grow and divide, producing motile cells which become angular polyeders. Polyeders may divide internally to form 4-celled colonies, or they enlarge, cleave, and produce zoospores or nonmotile cells. Zoospores are released in vesicles, swarm, adhere to each other, and form, flat colonies I cell thick. The nonmotile cells adhere to each other, enlarge, break out of the polyeder, and become spheroidal colonies. In the asexual cycle, nonfusing gametes, and motile cells derived from colony cells, become 4-spined azygotes which enlarge, divide internally, and release motile cells developing into polyeders. These polyeders develop further like those from zygotes.     

Cell Biology and Algal Taxonomy and Evolution Absolute Orientations of Basal Bodies in Green Algae Evaluated by Light Microscopy

Using high magnification Nomarski interference microscopy a series of optical sections has been obtained through flagellated cells of several green algae in an attempt to establish the absolute orientation of their basal bodies. Using this technique we have confirmed that in Spermatozopsis similis basal bodies are displaced into the 1/7 o'clock position, whereas in gametes of Enteromorpha linza, and zoospores of Trebouxia erici basal bodies are displaced into the 11/5 o'clock position. In addition we report for the first time that in zoospores of Microthamnion kuetzingianum basal bodies are also displaced into the 11/5 o'clock position. Basal body absolute orientations can thus be determined by light microscopy and do not require serial section electron microscopy. The method should be useful for class-level recognition of unknown green algae at the light microscope level.     

夜光藻有性繁殖研究进展

夜光藻是全球最主要的赤潮生物之一,也是我国近海常见的浮游甲藻。根据营养方式分为异养的红色夜光藻和混合营养的绿色夜光藻,前者广泛分布于温带和亚热带近岸水域,后者仅分布于热带西太平洋、阿拉伯海、阿曼湾和红海。夜光藻的生活史包括无性繁殖和有性繁殖过程。少部分营养细胞自发转变为配子母细胞,启动了有性繁殖。每个配子母细胞可形成大量配子,具有横沟、纵沟和2根鞭毛,形态与裸甲藻接近。配子两两融合形成合子,合子不经过休眠孢囊阶段直接发育成新的营养细胞。目前,对配子母细胞形成的调控机制、合子发育的影响因素的认识还存在分歧。研究发现,营养细胞经过一定次数的二分裂后都会转变为配子母细胞,而配子的存在能够中止此过程,使营养细胞继续进行二分裂。因此,有性繁殖可能通过产生新个体对种群增长做出贡献,还可能通过释放配子维持无性繁殖,进而促进种群增长。配子在相模湾水域全年都有分布,其丰度峰值与营养细胞丰度峰值同步或提前出现,配子的大量出现可能是赤潮形成的必要条件。对有性繁殖的研究佐证了夜光藻在甲藻的系统进化中处于较为古老的地位。此外,还简单介绍了研究夜光藻有性繁殖的主要方法,回顾了国内的夜光藻研究,并对相关研究进行了展望。     

Notes on gamete production in Lanice conchilega (Annelida,Polychaeta, Terebellidae)

Summary

Male and female gametogenesis in Lanice conchilega are described and illustrated as seen by light microscopy. There is no evidence for size-selectivity in the uptake of male gametes by the hypertrophied reproductive nephromixia, since undissociated motile sperm platelets as well as spermatozoa appear to be taken up by the genital funnels, and such funnels show no sexual dimorphism. Maturation of the coelomic oocytes is marked by a change in shape and the appearance of a surface reticulum of ridges.     

Structure and reproduction of Hormidiella bharatiansis sp. nov.

The structure and reproduction of Hormidiella bharatiansis sp. nov. is described. Asexual reproduction is by zoospores: sexual reproduction is by morphologically similar gametes, some of which, after a brief period of swarming, settle down, round off, enlarge and act as females gametes, the others remaining active and functioning as male gametes. Fertilisation takes place about six to eight hours after liberation: one to two percent of the zygotes are abortive.     

EFFECTS OF SELECTED PHYSICOCHEMICAL FACTORS ON GROWTH AND ZOOSPOROGENESIS OF CLADOPHORA GLOMERATA (CHLOROPHYTA)1

The effects of vitamin B₁ and B₁₂, temperature, light intensity and photoperiod on dry weight production and zoosporogenesis of Cladophora glomerata (L.)Kütz. were investigated by factorial experiments. Statistical analysis showed all of the above factors to be significantly (P < 0.01) influenced by these two parameters. Zoosporogenesis appeared to be more sensitive to vitamin limitation than dry weight production. Dry weight production and zoosporogenesis exhibited significantly different maxima. Dry weight production was most strongly influenced by temperature whereas photoperiod exerted the strongest influence on zoosporogenesis. It was possible to experimentally separate the light intensity and photoperiod factors; analysis showed photoperiod to be the primary factor influencing zoosporogenesis. Among the conditions tested, an 8:16 h LD short day photoperiod elicited the greatest number of zoosporangia.     

Seasonal development of five Cephaleuros species (Trentepohliaceae,Chlorophyta) on the leaves of woody plants and the behaviors of their gametes and zoospores

The infection cycle of five Cephaleuros species – C. aucubae, C. biolophus, C. japonicus, C. microcellularis, and C. virescens – was clarified by investigating seasonal development and gamete and zoospore behavior at study sites in Matsue, Shimane Prefecture, Japan. Fresh thalli or lesions appeared on the leaves of various woody plants from August and continued to develop in general from April to September in the following year. Gametangia and zoosporangia matured on 2‐ and 3‐year‐old leaves. Gametes were released in C. microcellularis samples collected from late March to late May and in samples of the other four species collected from late April to late July. Zoospores were released in C. biolophus, C. japonicus, and C. virescens samples collected from mid‐May to early August. In the present study, gametes did not conjugate but germinated like zoospores to produce new plants. After inoculation with zoospores and gametes that behave as asexual spores, fresh thalli or lesions became evident in 2–5 months following inoculation and gametes and zoospores were produced on the developed thalli or lesions the following year; one cycle of the infection was completed per year in the study area.     

REGULATION OF GAMETOGENESIS IN SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE)1

The effects of nutrients, temperature and light on gametogenesis in Scenedesmus obliquus (Turp.) Kütz, were studied in culture. Concentrations of nitrogen in the medium employed showed a marked influence on gamete production. Gametogenesis is inhibited by N excess but is not a response to N starvation or depletion. A drop in N level from that of the growth medium is not required, nor does it per se trigger gametogenesis. The N concentration satisfying growth requirements insufficiently low to permit sexual differentiation. Nitrogen level in the growth medium has no effect on subsequent N to maintain a typical culture. Number of gametes present at maximum production time is inversely related to N concentration, but neither time of onset of gametogenesis, nor time of maximum gamete production is affected by N concentration. Cultures incubated at 15 C in medium lacking N take a minimum of 20—24 h to develop cells irreversibly committed to gamete formation. At the concentrations tested, no medium component other than the N-containing salt affected gametogenesis. Temperature influences both time of maximum production and numbers present at maximum production time. Time of maximum production is inversely related to incubation temperature; a 15 C incubation temperature yielded highest gamete production. Light enhances gametogenesis but gamete formation can occur in absence of light. Achievement of a light-saturated response is dependent upon illumination given at two critical periods: one occurs shortly after N withdrawal; the other occurs later, when cells are becoming irreversibly committed to gamete formation. Ability to produce gametes diminished with prolonged laboratory culture.     

OBSERVATIONS ON THE HABIT,MORPHOLOGY AND ULTRASTRUCTURE OF CEPHALEUROS PARASITICUS (CHLOROPHYTA) AND A COMPARISON WITH C. VIRESCENS1

Cephaleuros parasiticus Karsten, an endophyte of Magnolia grandiflora L. has been examined with light, and scanning and transmission electron microscopy. The discoid thalli are composed of filaments which ramify throughout the leaf tissues beneath the epidermis. Algal filaments do not penetrate host cells, but do produce black leaf spots which have been mistaken for those caused by the fungus Glomerella cingulate (Ston.) Spauld. and Schrenk. Two distinct thallus types occur, often simultaneously on a single leaf. One bears clusters of zoosporangiate branches which seasonally emerge through the ventral (and rarely, dorsal) surface of the leaf. In contrast, the other thallus type bears gametangia which break through the dorsal leaf surface. Zoosporangia and gametangia have never been found on the same thallus. The zoosporangia are smaller than, but almost identical in shape to, those of C. virescens Kunze. Simple plasmodesmata are present in crosswalls and acetolysis indicates that little or no sporopollenin is present in the cell walls. The ultrastructure of biflagellate gametes and quadriflagellate zoospores is virtually indistinguishable from that reported for C. virescens and similar to that reported for Phycopeltis and Trentepohlia. In both gametes and zoospores there are keeled flagella, overlapping and parallel basal bodies, two 3-layered multilayered structures with microtubular splines, and two medial compound microtubular roots. Pyrenoids, eyespots, flagellar and body scales, striated roots (or rhizoplasts), and distal bands are absent. Two presumptive mating structures are present in each biflagellate gamete, and flagellar collars occur in both types of motile cells. The extreme similarity in motile cell ultrastructure revealed in this interspecific comparison parallels that similarity revealed in intergeneric comparisons.     

THE SEXUAL AND ASEXUAL LIFE CYCLE OF GLENODINIOPSIS STEINII (DINOPHYCEAE)

The sexual and asexual portions of the life cycle of Glenodiniopsis steinii were examined at both the light and scanning electron microscopic levels. Asexual reproduction by cell division occurs every 2 to 3 d under optimal conditions. Gamete formation and sexual reproduction can be induced by either nitrogen deprivation or aging of cultures. Fusion of gametes peaks about 10 hr into the light cycle and requires about 10 hr for completion. Cells remain motile during fusion but the zygote loses motility. The cell then undergoes changes that give it a thick-walled, warty appearance.     

STIMULATION OF LIGHT-SATURATED PHOTOSYNTHESIS IN LAMINARIA (PHAEOPHYTA) BY BLUE LIGHT1

The light-saturated rate of photosynthesis in blue light was 50-100% higher than that in red light for young sporophytes of Laminaria digitata (Huds.) Lamour., although photosynthetic rates were slightly higher in red than in blue light at low irradiances. Short exposures to low irradiances (e.g. 2 min at 20 μmol · m^?2· s^?1) of blue light also stimulated the subsequent photosynthesis of Laminaria sporophytes in saturating irradiances of red light but had little effect on photosynthesis in low irradiances of red light. The full stimulatory effect of short exposures to blue light was observed within 5 min of the blue treatment and persisted for at least 15 min in red light or in darkness. Thereafter, the effect began to decline, but some stimulation was still detectable 45 min after the blue treatment. The degree of stimulation was proportional to the logarithm of the photon exposure to blue light over the range 0.15-2.4 mmol · m^?2, and the effectiveness of an exposure to 0.6 mmol · m^?2at different wavelengths was high at 402-475 nm (with a peak at 460-475 nm) but declined sharply at 475-497 nm and was minimal at 544-701 nm. Blue light appears, therefore, to exert a direct effect on the dark reaction of photosynthesis in brown algae, possibly by activating carbon-fixing enzymes or by stimulating the uptake or transport of inorganic carbon in the plants.     

Nitrate uptake and extracellular alkalinization by the green alga Hydrodictyon reticulatum in blue and red light

Nitrate uptake and the medium alkalinization related to it were studied with nets of the coenocytic, giant cell, green alga Hydrodictyon reticulatum. A comparison of red, blue and white light irradiation showed no special control of nitrate uptake and of the corresponding alkalinization of the external medium by light quality, but rather a response as expected for the photosynthetic apparatus. In the dark, nitrate uptake rates amounted to one-fifth of those in saturating white light. This is in contrast to the chlorococcal microalga Monoraphidium braunii, where blue light specifically switched on nitrate uptake-dependent alkalinization and where uptake and reduction of nitrate strongly depended on blue light; the rates in pure red light and in the dark being very low. The stoichiometric ratio between nitrate taken up and extracellular alkalinization was close to 1 (0.86) in air with CO2 but close to 2 (1.84) in N2 for nitrate pre-loaded cells. In the absence of any carbon source, a high proportion of the absorbed and reduced nitrogen is released, most of it as ammonium which causes the excess alkalinization and some as nitrite, which lowers the ratio. Nitrite and ammonium release rates under anaerobic, CO2-free conditions were also independent of red or blue light and continued for several hours when the medium was buffered at pH 6. The data indicate that nitrate uptake, but less its reduction, is regulated differently in vacuolate, coenocytic algae from microalgae. In Hydrodictyon, nitrate uptake and reduction seems to be controlled by energy supply; in various microalgae, in addition, it is controlled specifically by blue light.     

SEXUAL REPRODUCTION OF PERIDINUM CINCTUM F. OVOPLANUM (DINOPHYCEAE)1,2

Sexual reproduction is induced in the dinoflagelate Peridinium cinctum f. ovoplanum Lindemann when exponentially growing cells are inoculated into nitrogen deficient medium. Small, naked vegetative cells, produced by division of the thecate cells, then act as gametes. The zygote remains motile for 12–13 days during which time it enlarges and the theca which it forms becomes warty. Thirteen to 14 day s following plasmogamy the zygote is nonmotile, the protoplast contracts, a large red oil droplet appears, the wall thickens and becomes chitinized. This hypnozygote germinates within 7–8 weeks at 20 c producing 1 post-zygotic cell retaining the large red oil droplet. The presence of 4 nuclei in these post-zygotic cells may be demonstrated by staining them with acetocarmine. Two of these nuclei are smaller than the other two and probably abort. One may infer that meiosis occurs immediately prior to or at the germinartion of the hypnozygote. This post-zygotic cell divides within 24 h into 2 daughter cells each with a promment red oil droplet. These daughter cells divide after 2–3 days into ordinary vegetative cells. Attempts to induce sexual reproduction by changes in temperature or light and by inoculation of cells into media deficient in a number of basic elements were unsuccessful.     

Vital role for Plasmodium berghei Kinesin8B in axoneme assembly during male gamete formation and mosquito transmission

Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin‐8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B‐gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal ‘9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.