Uptake of triphenyltin chloride by Enteromorpha intestinalis and Ulothrix pseudoflacca

Abstract. The uptake of triphenyltin chloride by Enteromorpha intestinalis and Ulothrix pseudoflacca zoospores and vegetative tissue was examined under various conditions affecting the metabolic state of the chloroplast. A synthesis for [¹¹³Sn]-triphenyltin chloride is presented. Results indicate that uptake of [¹¹³Sn]-triphenyltin chloride by vegetative tissue (and probably zoospores) is a passive process. Differences in the uptake of [¹¹³Sn]-triphenyltin chloride by zoospores of the two algae, in the dark and light, and by vegetative tissue of the two algae in the light, cannot be explained readily on the basis of surface area available for adsorption. Uptake studies suggest that differences in response to triphenyltin chloride exist between vegetative tissue and zoospores. Treatments which depress chloroplast membrane energization in vegetative tissue, e.g. lack of light, the presence of the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCP) or of DCMU prevent the exchange of triphenyltin chloride in pulse-chase experiments.     

Zoospore-specific antigen as a useful marker for molecular analysis of net formation in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)

In the green alga Hydrodictyon reticulatum zoospores are arranged in a regular fashion to form an intricate hexagonal network during the asexual reproductive cycle. A monoclonal antibody which was raised against a homogenate of zoospores recognized a single poly‐peptide in zoospores with a molecular mass of 31 kDa. The antigenic polypeptide, which was designated Amy1, was localized within the cytoplasm of zoospores. The accumulation of Amy1 occurred concomitantly with the transition from multinuclear vegetative cells to mononuclear zoospores, and the degradation of Amy1 occurred concomitantly with the further development of zoospores. Amy1 was constantly expressed during the period of mononuclear zoospores. Thus, we conclude that Amy1 is a zoospore‐specific polypeptide. Using the anti‐Amy1 monoclonal antibody, we could easily distinguish between mononuclear zoospores and multinuclear vegetative net‐cells. This provides an important tool for analysing the molecular mechanisms involved in the hexagonal net formation by zoospores.     

Attraction and attachment of zoospores of the parasitic chytridRozella allomycis in response to host-dependent factors

Summary This study examined the behavior of populations of zoospores of the obligately parasitic, endobiotic chytridRozella allomycis towards young, vegetative thalli of various saprophytic fungi, in order to identify host-dependent factors which control the development ofRozella. An inverted microscope was employed for continuous observation of parasite-host interaction in petri dishes of broth or agar medium. Two factors appear to control the initial stages of invasion byRozella of both susceptible and resistant species of the host genus,Allomyces: (i) a soluble exudate which attractsRozella zoospores, (ii) a receptor on the cell-wall surface which causesRozella zoospores to adhere, and to encyst and to germinate immediately thereafter. A related, nonsusceptible species,Blastocladiella emersonii, also attractsRozella zoospores, but supports very limited attachment.Rozella zoospores neither accumulate around, nor adhere to young thalli of non-blastocladialean fungi. This host-specific behavior pattern is compared with that of saprophytic and facultatively parasitic Phycomycetes, whose zoospores show nonspecific chemotactic responses and require no receptor for attachment, encystment and germination.     

AXILOSPHAERA AND HETEROTETRACYSTIS,NEW CHLOROSPHAERACEAN GENERA FROM TENNESSEE SOIL12

Two new chlorosphaeracean genera were isolated into axenic culture from soil collected in cedar glades in Cedars of Lebanon State Forest, Wilson County, Tennessee. The distinguishing characteristics of the new monotypic genus Axilosphaera include an axile (asymmetric) ckloroplast with at least 1 pyrenoid and Chlamydomonas-type (walled) zoospores. A. vegetata is the type species. Reproduction is by dissociation of daughter cells following vegetative cell division, by zoospores, and by aplanospores. The new polytypic genus Heterotetracystis, comprising 3 species, H. akinetos, H. macrogranulosa, and H. intermedia, is characterized by a parietal chloroplast with at least 1 pyrenoid and walled zoospores with flagella of unequal length. Reproduction is by dissociation of daughter cells following vegetative cell division and by zoospores. H. akinetos is designated as the type species.     

Effect of Tricarboxylic Acid Cycle Intermediates and Related Compounds on the Respiratory Rate of Urostyla cristata and Colpoda cucullus*

SYNOPSIS. The respiratory rates of vegetative forms of Urostyla cristata and vegetative and encysted Colpoda cucullus were measured in balanced salt solution and after addition of Na salts of various organic acids, including Krebs- and glyoxylic-cycle intermediates. The results point to some peculiarities in Urostyla metabolism; it was poisoned by succinate—an effect partly abolished by malonate; respiration was stimulated by malonate. Respiration of vegetative Colpoda was accelerated by Krebs- and glyoxylic-acid cycle intermediates. Most of these intermediates inhibited respiration of Urostyla. In experiments of short duration respiration of encysted Colpoda was not stimulated by these intermediates.     

Organotin-induced hemolysis,shape transformation and intramembranous aggregates in human erythrocytes

Organotin compounds examined in this study exhibited a relative order of potency for induction of in vitro hemolysis in human erythrocytes as follows: tri-n-butyltin > tri-n propyltin > tetra-n-butyltin > triphenyltin chloride > tri-n-ethyltin bromide > dibutyltin dichloride > stannous chloride > tri-n-methyltin chloride = butyltin chloride dihydroxide. All of the organotin compounds induced erythrocyte shape transformation from the normal discocyte to an echinocyte and, in addition, triphenyltin chloride, tetra-n-butyltin and tri-n-ethyltin bromide also elicited stomatocyte formation at higher concentrations. Select organotin compounds also formed tin-containing aggregates within the plasma membrane. The relative order of effectiveness for organotin induction of intramembranous aggregates was tri-n-butyltin > tri-npropyltin > tetra-n-butyltin > tri-n-ethyltin bromide, which was based upon the lowest concentration at which they were observed. These results support the previously suggested theory that organotins are membrane effectors because of their comparatively high hydrophobic, lipid partitioning properties. The relatively lipophilic compound, triphenyltin chloride, appeared to be anomalous because it did not readily promote hemolysis or induce the formation of intramembranous aggregates in human erythrocytes. A log-linear statistical model demonstrated an association of hemolysis with both tri-n-butyltin aggregate formation and shape transformation. Select organotin compounds should be useful probes in membrane studies because of their numerous effects.Abbreviations DBT dibutylin dichloride - MBT butyltinchloride dihydroxide - SnCl₂ stannous chloride - TBT tri-n-butyltin - TET tri-n-ethyltin bromide - TMT tri-n-methyltin chloride - TPhT triphenyltin chloride - TPT tri-n-propyltin - TTBT tetra-n-butyltin     

Real-Time Polymerase Chain Reaction Assays for Rapid Detection and Quantification of <Emphasis Type="Italic">Noctiluca scintillans</Emphasis> Zoospore

Application and availability of real-time polymerase chain reaction (PCR) assay to detect and quantify the Noctiluca scintillans zoospore were investigated seasonally. Specific primer set for N. scintillans 18S rDNA was designed and applied to real-time PCR assay using the serial dilutions of N. scintillans zoospores. The real-time PCR assays with Ns63F and Ns260R primers were applied to sea water samples collected weekly in Manazuru Port of Sagami Bay, Japan from April 2005 to June 2006. We developed effective DNA preparation steps for collecting the template DNA of N. scintillans zoospore: size fraction and filter concentration of the water samples, fixation with Lugol solution, cell lysis, and purification. This method is useful for the monitoring of the zoospores of N. scintillans, and can also be used for other small and physiologically fragile planktonic cell. Variation in the density of zoospore was successfully detected in the field samples. The peak density of N. scintillans zoospore was observed to occur just before or at the same time as the peak of the vegetative cells. Moreover, zoospores were detected in seawater even when the vegetative cells were not observed. The presence of zoospore was found all year round in the present study. In this regards, this information is essential for the study of the life cycle and seasonal variation of N. scintillans in the coastal waters.     

OBSERVATIONS ON CELL DEVELOPMENT IN CHLAMYDOMONAS SEGNIS (CHLOROPHYCEAE) AT LOW AND HIGH CARBON DIOXIDE TENSION1

Some cell cycle events were compared in Chlamydomonas segnis Ettl during its development in synchronous cultures (12:12 LD) supplied with air and air enriched with 5% CO₂. In cultures bubbled with air, growth resulted in production of 2 relatively small zoospores. In cultures provided with 5% CO₂, 4 large zoospores were formed but not released in darkness unless the cultures were bubbled with CO₂-free air and/or exposed to light. Respiration in zoospores was inhibited by high CO₂ tension. In cultures maintained under continuous illumination for one cell cycle, provision of 5% CO₂ led to enhanced growth, a relatively long S-phase and a 4 h delay of the second cell division. In such cultures, the DNA content of parental cells (12 h L) was insufficient to support two cell divisions. The RNA/DNA ratio of the resulting zoospores was 10 compared to 4 in air cultures. These results provided evidence that the delay of the second cell division was a consequence of the delay in DNA production.     

The effect of F0 inhibitors on the Vibrio alginolyticus membrane ATPase

The inhibition of membrane ATPase from the marine alkalotolerant bacterium Vibrio alginolyticus by DCCD, triphenyltin and venturicidin was studied. DCCD proved to be an irreversible inhibitor, while venturicidin and triphenyltin produced a reversible inhibitory effect. The DCCD-binding proteolipid was identified in the membrane preparations. The effect of the inhibitors on ATPase activity and ATP-dependent Na⁺-transport in V. alginolyticus subcellular vesicles is discussed.     

Studies on Antagonistic Effects of Trichoderma Isolates Against Phytophthora cactorum

Bioassays were used to demonstrate the antibiotic effect of Trichoderma isolates on P. cactorum. When both fungi were grown on benomyl-containing PDA medium, the mycelial growth of Trichoderma was suppressed. However, the production of antibiotics by this fungus remained active, leading to inhibition of the mycelial growth of P. cactorum. The antibiotic effect of Trichoderma on zoospores and cysts was tested on a PDA substrate precultured with Trichoderma on cellophane sheets. On the substrate of some Trichoderma isolates, lysis of zoospores, formation of extracellular vesicles, and hypertrophy of the water expulsion vesicle did occur, both resulting in the death of the zoospores. Conidial suspensions of Trichoderma isolates also induced zoospore lysis. It is presumed that membrane-active peptide antibiotics (peptaibols) are involved in zoospore lysis. The peptaibol paracelsin caused lysins of zoospores at a concentration of 2.5 × 10^?4 M. The effect on cysts depended on the Trichoderma isolate tested and the age of Trichoderma preculture. Old cultures (after beginning of sporulation) affected cysts more severely than young cultures (before sporulation) which usually were not lethal to the cysts but induced preferably microsporangium formation, inhibition of cyst germination, and retardation of germ tube growth.     

MORPHOLOGY AND THERMAL DEATH POINT OF OLPIDIUM BRASSICAE

The morphology of single-sporangial isolates of lettuce, tomato, mustard, and oat Olpidium brassicae (Wor.) Dang. growing in their respective hosts as well as in cowpea were compared in situ and after extraction from the roots. The sporangia, zoospores, and resting spores of all isolates were within the established limits of the species. Single exit tubes or pores predominated which means that these isolates should not be transferred to the genus Pleotrachelus. A satisfactory assay for the presence of resting spores was developed by air-drying of the roots for a week or longer. This treatment killed zoospores and vegetative sporangia, but not resting spores. Factors affecting resting spore formation were investigated unsuccessfully. The thermal death point of zoospores of mustard isolates that did not form resting spores was between 40 and 45 C for 10 min.     

Zoospore production in selected xanthophycean algae

The effects of incubation time and deficiencies of calcium and nitrate on zoospore production were investigated in Botrydiopsis arhiza Borzi, B. intercedens Vischer et Pascher, Bumilleriopsis peterseniana Vischer, Pseudobumilleriopsis pyrenoidosa Deason et Bold, and Ophiocytium maius Naegeli. Release of zoospores occurred after the transfer of stationary phase, vegetative cells into fresh Bold's basal medium. Maximum yields of zoospores were produced on the second day after transfer, 0·5–1·0 h after the start of the light period of a 12–12 h light-dark cycle. Lack of calcium or nitrate reduced the yield of zoospores in each taxon. Only O. maius produced large numbers of motile cells in nitrate-free medium.     

Genes expressed in zoospores of<Emphasis Type="Italic"> Phytophthora nicotianae</Emphasis>

The genus Phytophthora includes many highly destructive plant pathogens. In many Phytophthora species, pathogen dispersal and initiation of plant infection are achieved by motile, biflagellate zoospores that are chemotactically attracted to suitable infection sites. In order to study gene expression in zoospores, we have constructed a cDNA library using mRNA from zoospores of Phytophthora nicotianae. The library was arrayed and screened using probes derived from mycelium or zoospore mRNA. More than 400 clones representing genes preferentially expressed in zoospores were identified and sequenced from the 5 end of the insert. The expressed sequence tags (ESTs) generated were found to represent 240 genes. The ESTs were compared to sequences in GenBank and in the Phytophthora Genome Consortium database, and classified according to putative function based on homology to known proteins. To further characterize the identified genes, a colony array was created on replicate nylon filters and screened with probes derived from four Phytophthora developmental stages including zoospores, germinating cysts, vegetative mycelium and sporulating hyphae, and from inoculated and uninoculated tobacco seedlings. Data from sequence analysis and colony array screening were compiled into a local database, and searched to identify genes that are preferentially expressed in zoospores for future functional analysis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by C. A. M. J. J. van den Hondel     

THE FINE STRUCTURE OF SPOROGENESIS IN THE XANTHOPHYCEAN ALGA PSEUDOBUMILLERIOPSIS PYRENOIDOSA1,2

The fine structure of vegetative cells, sporogenesis, and zoospores of the xanthophycean alga Pseudo-bumilleriopsis pyrenoidosa is described. Cleavage in sporogenesis closely resembles that of certain aquatic fungi.     

ÜberHydrianum viride (Characiaceae,Chlorophyceae), seine fortpflanzung und taxonomie

The little known epiphytic algaHydrianum viride was studied and pictured. The morphology and the variability of the vegetative cells are described in detail and compared with the observations of other authors. For the first time typical biflagellate zoospores of the chlorophycean-type have been recognized and the course of the asexual reproduction is studied. The residual protoplasm, that is the proximal half of the zoosporangium protoplast, growth up to the original size, typical for the genusHydrianum. The life cycle, especially the settling down of the zoospores, is described.Hydrianum viride belongs to the characteristic inhabitants of peaty waters; it belongs together with the host alga,Microspora loefgrenii, to the dominants of algal association of peat-springs. At last the taxonomic position and the synonyma of this organism are discussed.

     

Monoclonal antibodies to adhesive cell coat glycoproteins secreted by zoospores of the green alga Enteromorpha

Zoospores of Enteromorpha compressa (L.) Grev. secrete an adhesive cell coat which is involved in their attachment to various substrata. Two monoclonal antibodies (mAbs), designated Ent 1 and Ent 6, were raised against settled zoospores displaying secreted adhesive. Both antibodies labelled specifically the anterior region of the cell containing putative adhesive vesicles. During settlement the antigens recognised by both mAbs were secreted but whereas Ent 6 recognised a fibrillar material released within a few minutes of settlement, Ent 1 recognised components which were associated predominantly with the developing cell wall at later time points. Both mAbs also labelled a Golgi-rich region of settled spores, suggesting that these antigens are also synthesised after settlement. Both mAbs labelled the cell walls of vegetative tissue. Competitive enzyme-linked immunosorbent assay indicated that the two antibodies recognise separate, but overlapping epitopes. In spore settlement assays the Ent 6 immunoglobulin strongly reduced initial adhesion at low concentration whereas the inhibitory effects of Ent 1 occurred at later time points. On analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) both MAbs recognised a major buffer- and SDS-soluble, polydisperse 110-kDa antigen. The 110-kDa component was present in extracts of zoospores and sporulating tissue, but absent, in soluble form, from vegetative tissue. Deglycosylation of zoospore extract with anhydrous HF and peptide N-glycosidase digestion, showed that the major 110-kDa antigen is an N-linked glycan, and that the epitope is borne by the protein component. Time-course experiments showed that the Ent 6 antigen became progressively insoluble after zoospore attachment. Taken together, the data indicate that the two antibodies recognise separate but closely related antigens which have distinctive roles in adhesion and cell wall development. Received: 8 February 1999 / Accepted: 26 July 1999     

THE ULTRASTRUCTURE OF MEIOSPORES OF COLEOCHAETE PULVINATA (CHAROPHYCEAE)1

In view of the relative importance of reproductive cell ultrastructure in phylogenetic and systematic studies of green algae, we investigated the fine structure of germinating zygotes and meiospores of Coleochaete pulvinata Braun. Meiospores have a flagellar apparatus very similar to that of zoospores and spermatozoids of the same species. Meiospores differ from zoospores and spermatozoids of C. pulvinata in having pyramidal body scales similar to those present on zoospores of C. scutata. Meiospores of C. pulvinata had as many as twice the number of spline microtubules as zoospores, and four times the number present in splines of spermatozoids of the same species. Developing meiospores of C.pulvinata, like those of other Coleochaete species, are individually surrounded by chamber walls. These differed from vegetative cell walls in lacking plasmodesmata. Moreover, the chamber walls in germinating zygotes of C.pulvinata stained a cobalt blue color with resorcinal blue, and fluoresced yellow in the presence of aniline blue, thus exhibiting the staining characteristics of callose. In location, morphology and presence of callose, chamberwalls resemble “special walls” of land plants, they may represent a charophycean spore development preadaptation useful in the evolution of walled spores characteristic of land     

PSEUDOTETRACYSTIS,A NEW CHLOROSARCINACEAN ALGA12

Pseudotetracystis terrestris gen. et sp. nov., a member of the Chlorosarcinaceae, was isolated into axenic culture from dry surface soil collected near Texon in Reagan County, Texas. The characteristics of this presently monotypic genus include vegetative cells with a parietal chloroplast, a single pyrenoid, and polymorphic motile cells which are of the Protosiphon-type (naked with 2 equal flagella); P. terrestris is the type species. Vegetative cell division (desmoschisis) forms diads and tetrahedral or planar tetrads, sometimes aggregated in transitory complexes. Reproduction is by the polymorphic motile cells which may function either as gametes to form zygotes which develop without dormancy directly into vegetative cells, or they may function as zoospores and develop directly into vegetative cells without union.     

Some Observations on the Influence of Salinity on Photosynthetic Activity and Chloride Ion Loss in Several Seaweeds

The photosynthetic rates of Enteromorpha intestinalis, Fucus vesiculosus and Laminaria saccharina from the Baltic Sea were measured in different salinity range of Medium A (with NaHCO₃) and Medium B (without NaHCO₃). Photosynthetic activity in Medium A showed higher values than those in Medium B. The materials pretreated for one day showed a higher O₂ output than those untreated at almost every salinity range. In a chloride loss experiment the pretreated thalli of Porphyra umbilicalis, Fucus vesiculosus and Laminaria digitata were placed for a three minute period in distilled water. The excreted chloride ion content showed a higher value in sublittoral species after hypotonic and hypertonic treatment than in intertidal species.     

Chemotactic and chemokinetic activities of <Emphasis Type="Italic">Saprolegnia parasitica</Emphasis> toward different metabolites and fish tissue extracts

 The chemotactic and chemokinetic activities in zoospores of Saprolegnia parasitica NJM 8604 (= H2) were examined using various amino acids, carbohydrates, fatty acids, and fish tissue extracts to estimate one of the important factors for attachment of zoospores to their host. All the tested six amino acids showed strong chemotactic reactions whereas carbohydrates and fatty acids caused moderate or strong chemotactic reactions. The chemokinetic activities against amino acids and carbohydrates were moderate or weak, whereas almost all fatty acids showed negative chemokinetic responses. Almost all tested fish tissue showed moderate chemotactic response and weak or moderate chemokinetic responses. Generally, chemotactic activity was strong in the amino acids, and the strongest activity was observed in alanine. Based on these facts, we considered that zoospores may react against amino acids of the fish body to attach and establish their colonization. Received: January 10, 2001 / Accepted: December 12, 2002 Correspondence to:K. Hatai