INFLUENCE OF SALINITY ON THE FORM OF ASTEROCYTIS IN PURE CULTURE1

The growth form of an isolate of Asterocytis ornata in pure culture changed from filamentous in a full-strength seawater medium to a unicellular or bicellular form closely resembling the genus Chroothece in a medium of quarter-strength seawater. The alga did not grow in media of lower salinities, or in media in which seawater was replaced by KCl, NaCl, or CaCl₂. The formation of filaments from single cells could not be attributed to osmolarity. The controlling factors seemed to be the concentrations of cations, notably Na⁺ and Mg⁺⁺. However, the filaments produced experimentally in a defined medium, were never as extensively developed as those occurring in seawater, an indication that further studies should be done. The occurrence of the Asterocytis form, in freshwater habitats remains to be explained. There may be distinct physiological races, if not true species, within the genus.     

PRODUCTION OF MALE GAMETES AND AUXOSPORES IN THE CENTRIC DIATOMS CYCLOTELLA MENEGHINIANA AND C. CRYPTICA12

Vegetative cells of Cyclotella meneghiniana Kützing form male gametes and anxospores following transfer from a synthetic freshwater medium to a modified artificial seawater. Both spermatogenesis and auxospore formation are correlated with an increase in the Na⁺ concentration in the medium. Spermatogenesis can also be induced in C. cryptica Reimann, Lewin, and Guillard in artificial seawater with an adjusted sodium level.     

A marine oomyceteAtkinsiella panulirata sp. nov. from philozoma of spiny lobster,Panulirus japonicus

A marine oomycete fungus was isolated from philozoma of the spiny lobster,Panulirus japonicus. The fungus exhibited slow growth, occasionally submerged, with a creamy white, raised moist colony. Hyphae were stout, arranged in radiating pattern, irregularly branched, 10–22 µm diam, occasionally separated by cross walls into subthalli. Thalli occasionally consisted of swollen features. Sporangia formed from the subthalli had 1–3 wavy or partly coiled discharge tubes at the terminal or subterminal area. Zoospores were pyriform or reniform, biflagellate, isokont and diplanetic. Encysted spores germinated as a hairlike filament with a globular enlarged tip in sterilized synthetic seawater, and directly as stout initial hypha in PYGS broth. This fungus was identified as belonging to the genusAtkinsiella and was designatedAtkinsiella panulirata sp. nov. The optimal temperature for the fungus was 25°C. Hyphal growth occurred in both sterilized synthetic seawater and NaCl in the range of 1.5–6.0% and 2.0–4.0%, respectively. The fungus could utilize all carbohydrates and derivatives tested as the carbon sources. From in vitro tests of five fungicides against the fungus malachite green was found to be the most effective.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Effects of temperature,nutrients, organic matter and coral mucus on the survival of the coral pathogen,Serratia marcescens PDL100

Serratia marcescens is an enteric bacterium that causes white pox disease in elkhorn coral, Acropora palmata; however, it remains unclear if the pathogenic strain has adapted to seawater or if it requires a host or reservoir for survival. To begin to address this fundamental issue, the persistence of strain PDL100 was compared among seawater and coral mucus microcosms. Median survival time across all conditions ranged from a low of 15 h in natural seawater [with a first‐order decay constant (k) = ?0.173] at 30°C to a maximum of 120 h in glucose‐amended A. palmata mucus (k = ?0.029) at 30°C. Among seawater and mucus microcosms, median survival time was significantly greater within Siderastrea siderea mucus compared with seawater or mucus of Montastraea faveolata or A. palmata (P < 0.0001). In seawater, the addition of phosphate and especially glucose resulted in significant improvements in survival (P < 0.001), while only the addition of glucose resulted in significant improvement in survival in A. palmata mucus (P < 0.0001). Increasing the temperature of seawater to 35°C resulted in a significantly slower decay than that observed at 30°C (P < 0.0001). The results of this study indicate that PDL100 is not well‐adapted to marine water; however, survival can be improved by increasing temperature, the availability of coral mucus from S. siderea and most notably the presence of dissolved organic carbon.     

Phoma sp. FOM-8108, a producer of gentisylquinones, isolated from sea sand

A fungal strain, FOM-8108, that was isolated from sea sand was found to produce gentisylquinone and chlorogentisylquinone, inhibitors of neutral sphingomyelinase. The fungus grew well under normal conditions, in the darkness, or on various agar media, but typical morphological characteristics were not observed to determine its taxonomy. Therefore, culture conditions were studied extensively, resulting in formation of a number of pycnidia by the fungus on the media containing seawater or on natural substrates such as hydrangea leaves, gardenia leaves, and rice straw under natural light or near-ultraviolet radiation exposure. Eventually, from the morphological characteristics of pycnidia, conidiogenous cells, and conidia, strain FOM-8108 was considered to belong to the genus Phoma. Culture studies showed seawater in the fermentation medium was necessary for the strain to produce gentisylquinones. Particularly, a full production of chlorogentisylquinone, which has a chloride ion in the structure, was performed in the medium with higher concentration (75%–100%) of seawater. Received: June 27, 2001 / Accepted: December 14, 2001     

Multiple allelopathic activity of the crustose coralline algaLithophyllum yessoenseagainst settlement and germination of seaweed spores

A study was made to investigate possible formation by the crustose coralline algaLithophyllum yessoenseof multiple allelopathic-related substances against the settlement and germination of spores of various seaweeds. Seven different solvents (n-hexane, diethyl ether, acetone, ethyl acetate, acetonitrile, methanol, distilled water) and seawater were used to obtain crude extracts and secretory exudates from the coralline alga. The extracts and the algal conditioned seawater were tested for inhibitory activity against the settlement and germination of spores from 17 species representing 15 genera. Spore settlement of 14 species was inhibited over 90% by one or more extracts of the six organic solvents and conditioned seawater. The germination of spores from 13 species was inhibited by one or more extracts of all seven solvents and conditioned seawater. The species where spore settlement was not significantly affected showed strong inhibition of germination, andvice versa.     

Recirculating Culture for Chondracanthus Exasperatus

The red alga Chondracanthus exasperatus is a source of the phycocolloid carrageenan as well as an ingredient referred to as ‘intralamellar gel’ in a recently developed cosmetic formula (US Patent 6136 329). The high value of the cosmetic product has sparked renewed interest in cultivation of this species. Previous cultivation methods for this species include open water culture on nets and immersed cultivation in tanks supplied with flow‐through pumped seawater. The installation of a high capacity seawater supply, pumping and drain system is a major cost for flow through systems. Recirculating or re‐use seawater systems that minimize seawater turnover may offer significant cost savings over single‐pass, flow through seawater systems. In this research several options for minimizing seawater use have been tested: recirculating batch culture in which nutrient replenished (carbon dioxide and mineral nutrients) natural or artificial seawater is used with minimal turnover and spray culture in which plants are suspended in air saturated with nutrient replenished natural or artificial seawater medium. Small volume (<2 L), single‐plant bioreactors and larger multiplant, 20, 80 and 320 L (sea water volume) immersion and spray systems have been developed and tested. Results from these systems will be presented. Research supported by Washington Sea Grant, Washington Biotechnology Center and Soliv International Corporation.     

A simple method to evaluate the potential for degradation of antifouling biocides

A simple method to measure the degradation of antifouling biocides is described which measures the loss of biocidal activity from seawater by bioassay. The bioassay employs either the ship‐fouling diatom Amphora or the brine shrimp Anemia. Loss of bioaclivity from sterile seawater indicates abiotic degradation whilst loss of bioactivity from natural seawater indicates biodegradation. Results are presented for three biocides, viz. the trihalomethylthio compound, N‐dichlorofluoromethylthio‐N’,N'‐dimethyl‐N‐phenyl‐sulphamide (Preventol A4S), di‐n‐octylamine, and the isothiazolone compound 4,5‐dichloro‐2‐n‐octyl‐4‐isothiazolin‐3‐one (Sea‐Nine 211).     

Leucine‐to‐carbon empirical conversion factor experiments: does bacterial community structure have an influence?

The suitability of applying empirical conversion factors (eCFs) to determine bacterial biomass production remains unclear because seawater cultures are usually overtaken by phylotypes that are not abundant in situ. While eCFs vary across environments, it has not been tested whether differences in eCFs are driven by changes in bacterial community composition or by in situ environmental conditions. We carried out seawater cultures throughout a year to analyse the correlation between eCFs and bacterial community structure, analysed by catalysed reporter deposition fluorescence in situ hybridization. Gammaproteobacteria usually dominated seawater cultures, but their abundance exhibited a wide range (25–73% of cell counts) and significantly increased with inorganic nutrient enrichment. Flavobacteria were less abundant but increased up to 40% of cells counts in winter seawater cultures, when in situ chlorophyll a was high. The correlations between eCFs and the abundance of the main broad phylogenetic groups (Gamma‐, Alphaproteobacteria and Flavobacteria) were significant, albeit weak, while more specific groups (Alteromonadaceae and Rhodobacteraceae) were not significantly correlated. Our results show that the frequent development of the fast‐growing group Alteromonadaceae in seawater cultures does not strongly drive the observed variations in eCFs. Rather, the results imply that environmental conditions and the growth of specific phylotypes interact to determine eCFs.     

TETREUTREPTIA POMQUETENSIS GEN. ET SP. NOV. (EUGLENOPHYCEAE): A QUADRIFLAGELLATE,PHOTOTROPHIC MARINE EUGLENOID1

A new euglenoid genus and species, Tetreutreptia pomquetensis, is described from winter waters of Maritime Canada. This phototrophic species is characterized by four emergent heterodynamic flagella, two about the length of the cell and two less than one-half this length. Tetreutreptia pomquetensis has features in common with species of Eutreptiella while it differs in several respects from any of the described species of that genus. It could be assigned to the order Eutreptiales or Euglenamorphales sensu Leedale or the order Euglenales sensu Farmer. This new alga has a narrow range of temperature tolerance; it grows best from 0° to 7° C and dies at temperatures above 10° C. The optimum salinity for growth was near full-strength seawater. Growth conditions for the alga define the conditions whence this species was isolated.     

Adaptation of Salt-tolerant <Emphasis Type="Italic">Myxococcus</Emphasis> Strains and their Motility Systems to the Ocean Conditions

More and more studies have indicated that myxobacteria are able to live in seawater conditions, which, however, can decrease the fruiting body formation ability and also the adventurous (A) and social (S) motility systems of the myxobacteria. To learn the adaptation mechanism of the salt-tolerant myxobacteria to marine conditions, we analyzed 10 salt-tolerant Myxococcus strains of their fruiting body formation and motility. The isolates were from marine samples and possessed different levels of salt tolerance. They had the dual motility system and formed fruiting bodies in the presence of suitable seawater concentrations. Some high salt-tolerant strains even lost their fruiting abilities in the absence of seawater. In response to the presence of seawater, the S-motility was found to be increased in the high salt-tolerants but decreased in the low salt-tolerants. The A-motility, on the other hand, was observed in all the salt-tolerant Myxococcus strains, but increased or decreased in response to the presence of seawater. Perceived shifts of fruiting body formation abilities and motilities discovered in the salt-tolerant Myxococcus strains suggested an ecological adaptation of myxobacterial social behaviors to the marine environments.     

Characterization of marine microalga, Scenedesmus sp. strain JPCC GA0024 toward biofuel production

A marine microalga, strain JPCC GA0024 was selected as high amount of neutral lipid producers from marine microalgal culture collection toward biofuel production. The strain was tentatively identified as Scenedesmus rubescens by 18S rDNA analysis. The growth of strain JPCC GA0024 was influenced by artificial seawater concentrations. The optimum growth of 0.79 g/l was obtained at 100% artificial seawater. The lipid accumulation reached 73.0% of dry cell weight at 100% artificial seawater without additional nutrients for 11 days. Gas chromatography/mass spectrometry analysis indicates that lipid fraction mainly contained hydrocarbons including mainly hexadecane (C₁₆ H₃₄) and 1-docosene (C₂₂ H₄₄). Furthermore, calorimetric analysis revealed that the energy content of strain JPCC GA0024 was 6,160 kcal/kg (25.8 MJ/kg) of calorific value, which was equivalent to the coal engery. The strain JPCC GA0024, S. rubescens, will become a promising resource that can grow as a dominant species in the seawater for the production of both liquid and solid biofuels.     

The first methane-oxidizing bacterium from the upper mixing layer of the deep ocean:Methylomonas pelagica sp. nov.

Methane enrichment of twenty-three 100-ml portions of seawater from three stations in the Sargasso Sea yielded the same obligate type I methanotroph. It is pigmented white, requires NaCl, grows well in seawater with either methane or methanol, but not on other C₁ compounds nor on C–C bonded organic matter, and it uses either ammonia or nitrate but not dinitrogen as a nitrogen source. Formaldehyde is produced in marked amounts from methanol. Growth occurs at 20° and 30°C but not at 10°C and is inhibited in natural sunlight. Representative isolates from each hydrographic station assimilate one-carbon units via the ribulose monophosphate pathway for formaldehyde fixation, and have a DNA base composition of 49 mol% guanine plus cytosine. The type strain, NCMB 2265, has been namedMethylomonas pelagica sp. nov. This upper ocean methanotroph may obtain its C₁ substrates in situ from particles of algal debris that become anoxic, ferment, and accumulate in the thermocline to form a false benthos.     

Biodegradation of Petroleum Hydrocarbons in Seawater at Low Temperatures (0–5 °C) and Bacterial Communities Associated with Degradation

In this study biodegradation of hydrocarbons in thin oil films was investigated in seawater at low temperatures, 0 and 5 °C. Heterotrophic (HM) or oil-degrading (ODM) microorganisms enriched at the two temperatures showed 16S rRNA sequence similarities to several bacteria of Arctic or Antarctic origin. Biodegradation experiments were conducted with a crude mineral oil immobilized as thin films on hydrophobic Fluortex adsorbents in nutrient-enriched or sterile seawater. Chemical and respirometric analysis of hydrocarbon depletion showed that naphthalene and other small aromatic hydrocarbons (HCs) were primarily biodegraded after dissolution to the water phase, while biodegradation of larger polyaromatic hydrocarbons (PAH) and C₁₀–C₃₆ n-alkanes, including n-hexadecane, was associated primarily with the oil films. Biodegradation of PAH and n-alkanes was significant at both 0 and 5°C, but was decreased for several compounds at the lower temperature. n-Hexadecane biodegradation at the two temperatures was comparable at the end of the experiments, but was delayed at 0°C. Investigations of bacterial communities in seawater and on adsorbents by PCR amplification of 16S rRNA gene fragments and DGGE analysis indicated that predominant bacteria in the seawater gradually adhered to the oil-coated adsorbents during biodegradation at both temperatures. Sequence analysis of most DGGE bands aligned to members of the phyla Proteobacteria (Gammaproteobacteria) or Bacteroidetes. Most sequences from experiments at 0°C revealed affiliations to members of Arctic or Antarctic consortia, while no such homology was detected for sequences from degradation experiment run at 5°C. In conclusion, marine microbial communities from cold seawater have potentials for oil film HC degradation at temperatures ≤5°C, and psychrotrophic or psychrophilic bacteria may play an important role during oil HC biodegradation in seawater close to freezing point.     

Two newhalophytophthora species,H. tartarea andH. masteri,from intertidal decomposing leaves in saltmarsh and mangrove regions

Two new pythiaceous fungi were obtained from decaying leaves submerged in saltmarsh environments of the east coast of the U.S.A., or fringing mangroves in the Bahama Islands, and are described here asHalophytophthora tartarea andH. masteri. The two species have superficially similar zoosporangia whose dehiscence tubes have ragged-looking apices. However, differences in fine structures and development of the dehiscence tube and plug, characteristics of the dehiscence plugs, and presence or absence of vesicles clearly distinguish the two species.Halophytophthora masteri is the only species ofHalophytophthora that has a zoospore release mechanism involving both an extruded plug and a vesicle. Cultural properties concerning growth and asexual reproduction at various salinities and temperatures are also different between the two, probably reflecting adaptation to their respective habitats. Though zoospore release inH. masteri occurs spontaneously from mature zoosporangia, it is remarkably enhanced inH. tartarea and alsoH. masteri by mildly dehydrating mature zoosporangia followed by rewetting with seawater, which suggests a possible relation between the asexual reproduction of these oomycetes and the tidal rhythm in their natural habitats.     

APOLAR EMBRYOS OF FUCUS RESULTING FROM OSMOTIC AND CHEMICAL TREATMENT

Embryos of the brown alga Fucus vesiculosas L. were grown as populations in glass petri dishes in seawater at 15 C in continuous low-intensity unilateral fluorescent illumination for periods up to 2 weeks. A quantitative estimate of increase in nuclear number was made from acetocarmine squash preparations of samples taken at 12-or-24 hr intervals. Over the period of 2-6 days embryos showed a doubling time of about 12-18 hr. Under normal seawater culture conditions each embryo formed a single rhizoid. When grown in seawater supplemented with sugar concentrations above 0.4 m , Fucus embryos developed as multicellular spherical embryos lacking rhizoids. In 0.6 m sucrose-seawater, 97% of the embryos were apolar at 2 days; only 37% were apolar at 4 days, many having recovered from the sucrose inhibition. Some embryos remained apolar after growth in 0.6 m sucrose for 2 weeks. Nuclear counts showed that sucrose-seawater markedly inhibited the rate of cell division. Other sugars including D-glucose, D-fructose, D-galactose and the sugar alcohol D-mannitol were also effective. When apolar embryos grown in sucrose-seawater were returned to seawater, embryo growth resumed at the normal seawater rate, judged from nuclear counts. Such embryos formed multiple rhizoids, varying from two to eight rhizoids per embryo, which developed on the embryo quadrant or half away from the unilateral light. Each of the multiple rhizoids originated from a single small cell in the periphery of the multicellular spherica embryo. Thus the rhizoid-forming stimulus apparently had been subdivided among a number of the cells of the apolar embryos. The implications of this finding are discussed. Attempts to produce multiple rhizoids by treatment of embryos with indoleacetic acid or 2,4-dichlorophen-oxyacetic acid failed. However, embryos treated with 10⁻⁴ M or 5 × 10⁻⁵ m 2,3,5-triiodobenzoic acid formed 40 and 30% multiple rhizoids, respectively, suggesting that some chemical, perhaps hormonal, mechanism is involved in polarization and rhizoid initiation in Fucus embryogenesis.     

The Response of Prolactin,ACTH, and Growth Hormone Cells in the Pituitary Gland of the Three-Spined Stickleback,: Gasterosteus aculeatus L. form leiurus,to Increased Environmental Salinities

Benjamin, M. 1980. The response of prolactin, ACTH, and growth hormone cells in the pituitary gland of the three-spined stickleback, Gasterosteus aculeatus L. form leiurus, to increased environmental salinities. (Department of Anatomy, University College, Cardiff, Wales, U.K.) — Acta zool. (Stockh.) 61(1): 1–7. The time-sequence of response of the prolactin, ACTH and growth hormone cells in the pituitary gland of the leiurus form of the three-spined stickleback, Gasterosteus aculeatus L., to a transfer from freshwater to seawater, was studied by light microscopy. The appearance of the pituitary was compared with that of animals caught in brackish or sea water. The prolactin cells respond only slowly to seawater by cytological changes interpreted as signs of decreased secretory activity. It is thus suggested that prolactin is unlikely to be important for osmoregulation in this stickleback. The growth hormone cells are more active in all seawater-adapted animals, while the ACTH cells are less active—although only in laboratory experiments. The differing responses of the pituitaries of the leiurus and trachurus forms of G. aculeatus and Pungitius pungitius to high salinities are compared. Even in species whose pituitaries are virtually identical, the response to high salinites may differ widely.     

Changes in osmotic water permeability of the eel gills during seawater and freshwater adaptation

Summary Changes in osmotic water permeability of the isolated gills of the Japanese eel,Anguilla japonica were studied during transfer to seawater or to fresh water. The water permeability increased gradually during the course of seawater transfer and attained a maximal level after 2 weeks. The water permeability of the freshwater eel gills was reduced when calcium ions were added to the incubation medium at a concentration of 1 mM, where-as no effect of the ion was observed on the gills of the seawater-adapted eel even at a higher concentration (10 mM). In contrast to seawater transfer, the water permeability decreased to a low level almost immediately (3 h) after transfer from seawater to fresh water. The acute reduction of the water permeability was also seen in the gills of the hypophysectomized eel after transfer to fresh water.The gradual increase in the gill water permeability during seawater transfer is correlated with an increase in the number of chloride cells. In scanning electron microscopy, chloride cells of seawater-adapted eel gills exhibit a pit-like structure, which was larger than in the freshwater eel. On transfer from seawater to fresh water, the pit diameter became smaller within 6 h. Hypophysectomy did not affect the change in gill surface structures during transfer to fresh water. The junctions between the chloride cells of seawater eel gills are reported to be of the leaky type. The parallel change in osmotic water permeability and in pit size of the chloride cells during seawater or freshwater transfer or after hypophysectomy suggests that these cells could provide a major route of water as well as ion movement.This paper is a portion of a thesis presented to Hokkaido University by t. Ogasawara in partial fulfilment of the requirements for Doctor of Fisheries