A volatile inhibitor immobilizes sea urchin sperm in semen by depressing the intracellular pH

Sea urchin spermatozoa are normally immotile in semen, but motility can be initiated by increasing gas flow over the semen--for example, by blowing N2 gas over a thin layer of semen. This result indicates that sperm motility is not O2 limited and suggests that seminal fluid contains a volatile inhibitor of motility which is responsible for the paralysis of sperm in semen. This inhibitor might be carbon dioxide, which reversibly immobilizes sperm. 31P-NMR measurements of pH show that the sperm intracellular pH (pHi) increases by 0.36 pH unit upon dilution of semen into seawater. Since previous studies have shown that this magnitude of pH increase is sufficient to trigger sperm motility, we suggest that the volatile inhibitor is inhibiting sperm motility in semen by depressing the pHi. A simple hypothesis that explains these observations is that the volatile motility inhibitor is CO2, which could acidify pHi as a diffusable weak acid. In this regard, sperm diluted into seawater release acid, and this acid release is related to the pHi increase and motility initiation. In fact, nearly half of the acid released by sperm upon dilution is volatile and may therefore be due to CO2 efflux. Most of the acid, however, cannot be attributed to CO2 release because it is not volatile. Thus, when sperm are diluted into seawater, they raise their pHi by releasing CO2 and protons from the cytoplasm into the surrounding seawater.     

Carbon dioxide efflux accompanies release of fertilization acid from sea urchin eggs

“Fertilization acid” is released from sea urchin eggs upon fertilization and decreases the pH of the surrounding seawater. In bicarbonate-free artificial seawater flushed with nitrogen gas, the pH shift still occurs but returns to the original value in a few minutes, suggesting that the released acid is volatile. A likely candidate for a volatile acid is carbon dioxide released from the eggs. Therefore, the total CO₂ content of seawater was measured pre- and post-fertilization and was found to be correlated stoichiometrically with released proton equivalents, leading to the conclusion that fertilization acid is largely carbon dioxide. Manometric analysis of cell extracts and ashed eggs suggest that the carbon dioxide may be stored in the unfertilized egg as an inorganic carbonate.     

Genome sequence of the unclassified marine Gammaproteobacterium BDW918

The unclassified marine gammaproteobacterium BDW918, which utilizes volatile fatty acids but not most common carbohydrates and amino acids, was isolated from Dokdo seawater in South Korea. Here we present a draft genome of the strain BDW918, which encodes many putative genes related to fatty acid metabolism and aromatic hydrocarbon degradation.     

白木香内生真菌Aspergillus sp.A14的挥发性成分及其抗菌活性

通过溶剂萃取法提取白木香内生真菌A14(Aspergillus sp.)的挥发油,采用滤纸片琼脂扩散法分别测定了其对3种人体病原菌的体外抑菌活性。结果表明:A14挥发油对金黄色葡萄球菌(Staphylococcus aureus)、耐甲氧西林金黄色葡萄球菌(Methicillin-resistant S.aureus,MRSA)和白色念珠菌(Candida albicans)均表现出一定抑制作用。应用GC-MS技术,分析鉴定了内生真菌A14挥发油的14个化学成分,显示蜂蜜曲菌素是其中的主要成分,占挥发油峰面积的93.41%。     

Fertilization acid release in Urechis eggs: I. The nature of the acid and the dependence of acid release and egg activation on external pH

The amount of fertilization acid produced by eggs of Urechis caupo, monitored by automatically back-titrating egg suspensions with base, depends linearly on the pH of the seawater. Above pH 7.0, at which no acid is released (Paul, M., Dev. Biol.43, 299–312, 1975), acid release increased approximately 0.34 pmole/egg/0.1 pH unit. Activation (germinal vesicle breakdown) depended on the amount of acid release in natural seawater; it did not occur if eggs released <1.5 pmole acid/egg. When fertilization acid is released into HCO^?₃-free seawater and the pH permitted to decrease, the supernatant can be tested for the presence of a volatile acid, such as CO₂, by bubbling with N₂ and comparing the increase in pH as volatile acid is driven off with experiments in which HCl or CO₂ is substituted for fertilization acid. An increase in pH of <0.2 pH units occurred on N₂ bubbling when fertilization acid or HCl was used to acidify HCO^?₃-free seawater compared to an increase of >0.5 pH units when CO₂ was used. Therefore, most, if not all, of Urechis fertilization acid is not volatile, and since Paul (1975) showed that it is not a nonvolatile weak acid, it must be H⁺.     

中华羊茅内生真菌Epichlo? sp.挥发性物质的抑菌活性及成分分析

为测定中华羊茅内生真菌Epichloe sp.挥发性物质的抑菌活性和成分,依次采用挥发油提取器和乙酸乙酯萃取Epichloe sp.菌株的发酵产物,再采用琼脂扩散法、悬滴法和滤纸片扩散法测定其抑菌活性;基于顶空固相微萃取和气相质谱联用法分析挥发性物质成分。结果表明:挥发性物质、乙酸乙酯提取物、水相部分和发酵液可以抑制小麦根腐离蠕孢(Bipolaris sorokiniana)、新月弯孢(Curvularia lunata)和黑曲霉(Aspergillus niger)菌丝生长。高浓度的挥发性物质对四种真菌孢子萌发和芽管长度抑制效果显著高于低浓度(P<0.05)。结果表明,随着挥发性物质浓度的增加对金黄色葡萄球菌(Staphylococcus aureus)和大肠杆菌(Escherichia coli)的抑菌作用增强,同一挥发性物质浓度对金黄色葡萄球菌的抑制作用强于大肠杆菌。此外,从挥发性物质中鉴定出32种具有较高质谱匹配度的化合物,其中主要成分包括异山梨醇(16.26%)、甲基叔丁基醚(8.18%)、二氢甘露醇(3.75%)、羟基丙酮(2.99%)、乙酸(1.22%)、己醇(1.20%)、3-糠醛(0.92%)、5-羟甲基二氢呋喃-2-酮(0.86%)、5-羟基戊酸-2,4-二叔丁基苯酯(0.86%)和棕榈酸甲酯(0.85%)。总之,中华羊茅内生真菌Epichlosp.挥发性物质具有拮抗细菌和真菌的作用。     

不同海水浓度和培养时间对海洋真菌抗菌活性的影响

用滤纸片琼脂扩散法研究不同海水浓度和培养时间对海洋真菌抗菌活性的影响,旨在为大规模培养海洋真菌和提高获得抗菌物质的几率提供理论依据。结果显示：用不同海水浓度培养基发酵海洋真菌,在供试的10株海洋真菌中,5株海洋真菌的抗菌活性和抗菌谱有明显差异;培养时间不同,6株海洋真菌的抗菌活性差别较大。实验结果表明,不同海水浓度和培养时间对海洋真菌抗菌活性有显著影响。     

Radiotracer Experiments on Biological Volatilization of Organic Iodine from Coastal Seawaters

Biological volatilization of iodine from seawaters was studied using a radiotracer technique. Seawater samples were incubated aerobically in serum bottles with radioactive iodide tracer (¹²⁵I), and volatile organic and inorganic iodine were collected with activated charcoal and silver wool trap, respectively. Iodine was volatilized mainly as organic iodine, and inorganic iodine volatilization was not observed. Influence of light intensity on the volatilization was determined, but no significant differences were observed under light (70,000 lux) and dark conditions. The effect of the chemical form of iodine on the volatilization was determined, and the results suggested that volatilization preferentially occurs from iodide (I^?) but not from iodate (IO₃ ^?). Volatilization did not occur when the samples were autoclaved or filtered through a 0.22-μm pore size membrane filter. Incubation of the samples with antibiotics caused decreased volatilization. Conversely, enhanced volatilization was observed when the samples were incubated with yeast extract. Fifty-nine marine bacterial strains were then randomly isolated from marine environments, and their iodine-volatilizing capacities were determined. Among these, 19 strains exhibited significant capacities for volatilizing iodine. 16S ribosomal RNA gene comparisons indicated that these bacteria are members of Proteobacteria (α and γ subdivisions) and Cytophaga-Flexibacter-Bacteroides group. One of the strains, strain C-19, volatilized 1 to 2% of total iodine during cultivation, and the gaseous organic iodine was identified as methyl iodide (CH₃I). These results suggest that organic iodine volatilization from seawaters occurs biologically, and that marine bacteria participate in the process. Considering that volatile organic iodine emitted from the oceans causes atmospheric ozone destruction, biological iodine volatilization from seawater is of great importance. Our results also contribute to prediction of movement and diffusion of long-lived radioactive iodine (¹²⁹I) in the environment.     

培养法检测海滨浴场细菌多样性及耐药性研究

【目的】耐药基因细菌水平基因转移导致的耐药菌数量增加引发的公共安全问题日益引起人们关注,监测环境中耐药菌变得极为重要。【方法】采集湛江3处滨海浴场的水体、沙滩土样,通过平板稀释涂布和琼脂扩散法进行浴场微生物数量、多样性和抗生素耐药性分析。【结果】3处浴场水体无机氮含量偏高,浴场微生物数量随着客流量逐渐增加,沙滩中微生物数量显著高于水体。浴场细菌分布于3门12科18属,水体中变形菌门（Proteobacteria,49.64%）占优势,沙滩则是厚壁菌门（Firmicutes,54.74%）占优势。浴场细菌对β-内酰胺类耐药率较高,青霉素、万古霉素和头孢曲松耐药率分别达到23.25%、20.53%和17.42%,耐药菌株主要分布于芽孢杆菌属（Bacillus）、弧菌属（Vibrio）、假单胞菌属（Pseudomonas）、链霉菌属（Streptomyces）和肠杆菌属（Enterobacter）,水体中多重耐药细菌数量显著高于沙滩,集中于人流量多的浴场。【结论】滨海浴场环境中细菌耐药菌种类多,需持续监测以评估对当前地区公共卫生的潜在影响。     

Ketomethylthiobutyric acid formation from methylthioadenosine: A diffusion assay

Typical enzyme kinetics were observed when 5′-methylthioadenosine was used as substrate with extracts of malignant murine cells in a diffusion assay. The volatile product was measured after diffusion into a solution of the sulfhydryl reagent, 5,5′-dithiobis(2-nitrobenzoic acid), which it reduced to a yellow chromophore. Cysteine was required in the system. The volatile product was identified as H₂S derived from the cysteine. The yield of H₂S was similar to the amount of 2-keto-4-methylthiobutyric acid (KMTB) formed from methylthioadenosine when the KMTB was measured simultaneously in an ether extraction assay. KMTB could replace methylthioadenosine as a substrate capable of causing the formation of the diffusible product from cysteine. It is concluded that the following sequence of reactions takes place in the diffusion assay system: (1) 5′-methylthioadenosine + P_i → adenine + 5-methylthioribose-1-P, (2) 5-methylthioribose-1-P → KMTB, (3) KMTB + cysteine → methionine + 3-mercaptopyruvate, (4) 3-mercaptopyruvate + excess R-SH → pyruvate + H₂S, (5) H₂S + 5,5′-dithiobis(2-nitrobenzoic acid) → 5-mercapto-2-nitrobenzoic acid. Thus, the diffusion assay measures the amount of KMTB formed. The key enzyme, cysteine aminotransferase, EC 2.6.1.3, was partially purified from malignant cells and from liver and several of its characteristics are described. The diffusion assay using this enzyme is useful in measuring de novo synthesis of α-keto acids and it is applicable to crude enzyme preparations. The sensitivity is about 5 nmol of keto acid and the accurate range is 5 to 100 nmol.     

具抑菌活性海洋微生物的筛选

从上海郊区海域采集的海水、海泥样中分离出193株海洋细菌,对其进行抑菌活性菌株的筛选。对分离出的菌株分别利用琼脂块法和管碟法进行初筛和复筛,结果显示有29株海洋细菌具有抑菌活性,占总分离菌株的15%,其中抑菌能力较强的G4抑菌谱较广,同时能抑制革兰氏阳性和阴性指示菌。     

3-Decanol in the haemolymph of the hermit crab Clibanarius vittatus signals shell availability to conspecifics

Hermit crabs with poor fitting shells are chemically attracted to dying gastropods and conspecifics where a shell may become available. For land hermit crabs, the shell cue is a volatile compound found in the haemolymph. Based on this knowledge, we tested the hypothesis that shell investigation behavior in aquatic hermit crabs, the ancestral predecessors of terrestrial hermit crabs, is also triggered by volatile cues. Volatile compounds from haemolymph of Clibanarius vittatus and Pagurus pollicaris and brachyuran decapod crustaceans were purged from a water-haemolymph solution, trapped in seawater and tested for induction of shell investigation behavior with juvenile C. vittatus. Only volatiles from C. vittatus haemolymph stimulated shell investigation. Volatile compounds were isolated from haemolymph by headspace solid-phase microextraction (SPME) and analyzed by coupled gas chromatography-mass spectrometry (GC-MS). Two prominent compounds were identified, 3-decanol, which was unique to C. vittatus haemolymph, and 2-ethyl-1-hexanol, which was present in the haemolymph of all 4 crustacean species. In shell investigation bioassays, 3-decanol from C. vittatus haemolymph stimulated shell investigation behavior, while 2-ethyl-1-hexanol did not. In bioassays with synthetic 1-, 2-, 4-, and 5-decanol, shell investigation behavior was evoked by 1-decanol, 5-decanol and 3-undecanol. There was no response to 2- and 4-decanol. The response of C. vittatus to volatile shell cues supports the hypothesis that volatile cue detection evolved prior to the occupation of terrestrial niches by crustaceans.     

Physiological Studies in the Mucorales: III. THE ZYGOTROPISM OF ZYGOPHORES OF MUCOR MUCEDO BREFELD

Some previous work on zygotropism in Mucor is briefly reviewed,and further observations confirming earlier studies presented. In addition to positive zygotropism between (+) and (–)zygophores, there is claimed to be a corresponding ‘repulsion’between zygophores of the same mating type, (+) from (+) and(–) from (–). The stimulus appears to be transmittedby the diffusion of a volatile substance from one zygophoreto another. A tentative explanation of the phenomena describedis advanced, based on two postulated volatile hormones, eachevolved by zygophores of one mating type only, stimulating growthin zygophores of its own mating type and retarding it in thoseof the opposite type.     

Quantification of laminarialean zoospores in seawater by real‐time PCR

Laminarialean plants undergo heteromorphic alternation of generations between the macroscopic diploid sporophyte and the microscopic haploid gametophyte. The generations change through the formation and release of asexual or sexual reproductive cells. It is difficult to monitor the release and diffusion of zoospores into the environment. Furthermore, even if zoospores can be detected, species identification remains difficult. This study attempted to develop a sensitive and fast identification method for laminarialean zoospores using quantitative PCR. In addition, we aimed to estimate the density of zoospores in natural seawater. Specific primers for Saccharina japonica and Undaria pinnatifida were designed and used to estimate the quantity of zoospores in seawater. DNA samples (environmental DNA) were collected from seawater once or twice each month for 2 years at the same area, and seasonal variations in the release of zoospores was monitored. The estimated maturation period based on the number of released zoospores in this study was comparable with those of the previously reported maturation periods of S. japonica and U. pinnatifida sporophytes. This supports the validity of our method in estimating zoospore release from laminarialean plants. The method will be a useful tool for ecological studies on these commercially relevant species.     

Fertilization acid of sea urchin eggs: Evidence that it is H+, not CO2

The report of R. J. Gillies, M. P. Rosenberg, and D. W. Deamer (1981, J. Cell. Phys., 108, 115–122) that sea urchin fertilization acid is anaerobically produced CO₂, was reinvestigated by inseminating Strongylocentrotus purpuratus eggs in HCO^?₃-free seawater, then bubbling the seawater with N₂ to remove volatile acid. Fertilization acid production occurred in HCO^?₃-free seawater and with N₂-bubbling, the pH rose 0.28 ± 0.08 unit, significantly less than the rise of 0.63 ± 0.14 unit during N₂-bubbling of HCO^?₃-free seawater that had been acidified with CO₂ and similar to the rise of 0.18 ± 0.07 unit when acidification was with HCl. We conclude that most, if not all, of the sea urchin fertilization acid is nonvolatile and thus is not CO₂; since it is not a weak acid, it must be H⁺.     

Slow‐flow habitats as refugia for coastal calcifiers from ocean acidification

The pH of the oceans’ surface water is dropping, termed ocean acidification (OA), and the 0.4 unit reduction in pH by 2100 is projected to negatively impact benthic coastal organisms that produce calcium carbonate “skeletons.” Research has focussed on identifying species that are susceptible to OA, but there is an urgent need to discover refuge habitats that will afford protection to vulnerable species. The susceptibility of calcium carbonate skeletons to dissolution by OA depends on the pH at their surface, and this is controlled by the interaction between seawater velocity and organismal metabolism. This perspective considers how seawater velocity modifies the responses of calcifying organisms (seaweed, shellfish, and tropical corals) to OA through its action on controlling diffusion boundary layer thickness and thereby the pH and calcium carbonate saturation state (Ω) at the organisms’ surface. Evidence is presented to support the idea that slow‐flow habitats, such as wave‐sheltered bays or the within canopies of seaweed/seagrass beds, might provide inexpensive refugia from OA for vulnerable coastal calcifiers.     

Physiological and physicochemical controls on foliar volatile organic compound emissions

Plant leaves emit a broad spectrum of organic compounds that typically play multiple roles in plant protection. Furthermore, most of these compounds actively participate in tropospheric chemistry. There has been rapid progress in understanding how the emission of volatiles is regulated, mostly focusing on the biochemical controls over compound production. However, physicochemical characteristics such as low volatility or diffusion can also control the emissions and interact with physiological limitations. In particular, non-specific leaf storage of less volatile compounds smooths the emission responses to fluctuating environmental conditions, and diffusion through stomata leads to conspicuous emission bursts after stomatal opening and modifications of diurnal emission time courses. Because natural conditions always fluctuate, both physiological and physicochemical controls exert a major influence over plant volatile emissions.     

Behavioral assay and chemical characters of female sex pheromones in the hermit crab <Emphasis Type="Italic">Pagurus filholi</Emphasis>

Males of the hermit crab Pagurus filholi perform assessment behavior toward females, as a preliminary step of precopulatory guarding, during the reproductive season. It is known that such behavior is elicited by female sex pheromones, but the compounds involved have never been characterized in this species. Several experiments were conducted to develop a reliable bioassay along with purification procedures to identify potential compounds with pheromonal activity in Pagurus filholi. We developed a bioassay protocol to assess pheromonal activity by using an empty shell with cotton containing either artificial seawater (control) or test water. We measured and compared the time duration of male assessment behavior toward each shell if the test water contained female sex pheromones. Ultra-filtering of seawater samples potentially containing pheromones showed that the compound was <1 kDa in molecular weight. Males showed precopulatory assessment behavior toward “female conditioned” water samples treated with open column purification and eluted with MeOH, suggesting that compounds triggering male behavior were low polar molecules. Molecules with pheromonal activity were not volatile after freeze drying, effective even after heating to 90 °C, and remained active in seawater at 12 °C even after 6 days from sample collection, which suggests a rather stable characteristic of the female sex pheromones of this species.     

Sensitive and Accurate Methodology for Measuring the Kinetics of Concentration-Dependent Hydrocarbon Metabolism Rates in Seawater by Microbial Communities

A method having sufficient sensitivity to resolve the kinetic constants for dissolved nonpolar substrate metabolism, together with the related rate constants in natural waters, is presented. The method is based on the rate of ¹⁴CO₂ recovery from radioactive dissolved substrate. Sensitivity is enhanced by using large seawater volumes, high-specific-activity isotopes, and by reducing background radioactivity. Before use, commercial isotopes are purified by mild alkaline hydrolysis followed by sublimation from base to remove ¹⁴CO₂ as well as interfering polar ¹⁴C-substrates. During sample analysis, chilled Tenax resin is used to remove volatile ¹⁴C-substrate from the nitrogen stream containing ¹⁴CO₂ recovered from substrate oxidation. Chromatographic evidence of purity, shown to be insufficient, is augmented by kinetic data from toluene utilization by mixed cultures and by rates in induced versus noninduced pure cultures. Accuracy is enhanced by using short (<10 h) incubation times and small hydrocarbon concentrations so that the metabolism rates in unamended natural water systems can be evaluated. Toluene metabolism rates in seawater as low as 1 pg/liter per h and at concentrations as low as 20 ng/liter have been determined.     

Thiosulfate elimination and permeability in a sulfide-adapted marine invertebrate.

Oxidation of hydrogen sulfide to thiosulfate is one of the best-characterized mechanisms by which animals adapted to sulfide minimize its toxicity, but the mechanism of thiosulfate elimination in these animals has remained unclear. In this study, we examined the accumulation and elimination of thiosulfate in the sulfide-adapted marine worm Urechis caupo. The coelomic fluid of U. caupo exposed to 50-100 micromol L-1 sulfide in hypoxic seawater (Po2 ca. 10 kPa) accumulated (mean+/-SD) 132+/-41 micromol L-1 thiosulfate after 2 h, reaching 227+/-113 micromol L-1 after an additional 4 h in aerated, sulfide-free seawater. In whole-animal thiosulfate clearance studies, the rate of thiosulfate elimination from the coelomic fluid followed a single exponential time course with a half-life of 6 h. The thiosulfate permeability coefficient of isolated preparations mounted in diffusion chambers was 7.6x10-5+/-7. 7x10-5 cm s-1 for the hindgut and 5.5x10-7+/-2.7x10-7 cm s-1 for the body wall. These rates were independent of the direction of net efflux (mucosal-to-serosal or serosal-to-mucosal). Using a simple mathematical model of U. caupo that incorporates the thiosulfate permeability coefficients, the thiosulfate half-life was calculated to be 23 h without hindgut ventilation but less than 1 h with normal hindgut ventilation. Based on this information, we propose that passive thiosulfate diffusion across the hindgut is adequate to explain the observed rates of thiosulfate elimination.