除草剂草丁膦抗性基因bar作为三角褐指藻基因工程选择标记的研究

研究了三角褐指藻(Phaeodactylum tricornutum)对氯霉素、卡那霉素、青霉素、链霉素、潮霉素5种抗生素和一种除草剂草丁膦的敏感性,发现三角褐指藻对草丁膦非常敏感,用统计学分析法得出了其72h半致死质量浓度为22mg/L。通过基因枪法将携带草丁膦抗性基因bar的载体pSVB导入三角褐指藻细胞中,通过草丁膦筛选成功获得了抗性藻细胞,PCR检测获得阳性结果,提示草丁膦更适合用于构建安全、高效、廉价的三角褐指藻表达系统。     

Exudates of different marine algae promote growth and mediate trace metal binding in Phaeodactylum tricornutum

Phaeodactylum tricornutum was grown in filtered natural seawater enriched with nitrate, phosphate, and silicate only (control) or with exudates from itself, from Emiliania huxleyi (a coccolithophore micro-alga), Porphyra spp. (a red macro-alga) or Enteromorpha spp. (a green macro-alga). Cathodic (and anodic) stripping voltammetry (C(A)SV) were used to determine the concentrations of trace metals, both in the medium and in the algae, as well as total Cu-complexing organic ligands in the medium and, among these, some thiols (compounds identified as cysteine- or as glutathione by CSV). Exudates of different marine micro- and macro-algae could cause allelopathic effects in P. tricornutum cultures. Cell yield of P. tricornutum was increasingly promoted by exudates of E. huxleyi > Porphyra > Enteromorpha. Although exudates strongly complex Cu (and probably other metals), their presence promoted Cu uptake. Significant changes of Ni, Cd, Fe, Zn and Mn uptake by P. tricornutum were also observed in the presence of exudates of different algal species. In addition, both intensity of production and nature of exudates released by P. tricornutum were markedly influenced by the presence of exudates of other algae, the allelopathic effects being very specific (variable from one species to another). Allelopathy will probably also occur in the aquatic environment, although to a lesser extent than in cultures, particularly during bloom events and may have effects on both chemical speciation and bioavailability of chemicals to phytoplanktonic species. Such changes might cause the predominance of some species over other species. Therefore, in future in vitro culture studies with the purpose of using them as models of the real environment, more attention should be paid to the role of algal exudates, in order to improve the environmental relevance and significance of the results.     

三角褐指藻与东海原甲藻间的他感作用研究

本文主要探究了东海原甲藻(Prorocentrum donghaiense)与三角褐指藻(Phaeodactylum tricornutum)之间的他感作用。在实验室模拟条件下,分别研究了添加比例为10%、40%、60%、90%、100%的三角褐指藻无藻细胞滤液的培养基对东海原甲藻生长的影响,以及相同比例的东海原甲藻无藻细胞滤液培养基对三角褐指藻生长的影响。结果表明,低浓度三角褐指藻滤液对东海原甲藻(P.donghaiense)的生长表现为促进作用,高浓度则抑制其生长,因此,不同浓度的三角褐指藻藻液对东海原甲藻生长表现出两种相反的他感作用效果;而东海原甲藻滤液对三角褐指藻(P.tricornutum)的生长没有显著影响,说明东海原甲藻并未对三角褐指藻表现出他感作用。     

Sensitivity of three microalgae to crude oils and fuel oils

Four crude oils and five fuel oils have been tested for toxicity with three microalgae—a blue-green, a green and a diatom. The oils were absorbed on filter paper pads and the pads submerged in the growth medium. The crude oils were less inhibitory than equal amounts of fuel oils. Despite initial growth lags, the crude oils allowed growth at 30 μl/20 ml of culture medium (10⁵ cells/ml) while fuel oils were lethal at 10 μl/20 ml. The toxicity patterns of two of the whole fuel oils were different from that seen with their water soluble fractions (WSF); for example, the Baton Rouge fuel oil sample was very toxic to growth of the three test organisms whereas its WSF was relatively innocuous. Photosynthesis of a sensitive organism Chlorella autotrophica, strain 580 (10⁷ cells/ml), was only temporarily depressed by the crude oils (30 μl/20 ml). Four of the fuel oils inhibited photosynthesis, O₂ output decreasing to zero without recovery. When the fuel oils were heated in a stream of helium they were detoxified. Chemical analyses of two of the toxic fuel oils before and after heating were compared with analyses of the Montana sample, a largely non-toxic fuel oil, in an effort to determine what types of compounds might be involved. Classes of aromatic compounds which were not accountable for the toxicity observed here include naphthalene, methylnaphthalenes, dibenzothiophenes, phenanthrenes and compounds with volatilities greater than methylnaphthalenes. Paraffinic and asphaltic fractions of fuel oil were also non-toxic. The accumulated chemical data suggest that the toxicity of whole fuel oils is due to the less water soluble types of compounds in the higher boiling aromatic fraction.     

Copper complexing capacity in cultures of Phaeodactylum tricornutum: Diurnal changes

In batch cultures of the diatom Phaeodactylum tricornutum, organic materials capable of complexing ionic copper were released to the culture medium at all stages of growth; however, the largest proportion was released after the diatoms had reached the stationary phase. When the organisms were maintained in log phase growth in a cage culture turbidostat, a diurnal fluctuation in complexing capacity was found, similar to fluctuations measured in field populations.     

几种单胞藻对双刺纺锤水蚤雌体繁殖的影响

研究6种饵料微藻：微绿球藻（Nannochloropsis oculata）、小球藻（Chlorella．sp）、球等鞭金藻（Isochrysis galbana）、三角褐指藻（Phaeodactylum tricornutum）、亚心形扁藻（Platymonas subordi-formis）、中肋骨条藻（Skeletonema costatum）对双刺纺锤水蚤（Acartia bifilosa）雌体繁殖的影响,以探索适合培养双刺纺锤水蚤的藻类。应用解剖镜下计数方法对雌体存活率、产卵率、孵化率、排粪率几个指标进行检测。结果表明微绿球藻、小球藻和球等鞭金藻明显不适于用作雌体饵料;中肋骨条藻组产卵率最高,亚心形扁藻和三角褐指藻组的产卵率没有显著差异;亚心形扁藻组的排粪率明显低于中肋骨条藻和三角褐指藻组;摄食三角褐指藻的雌体所产的卵其平均孵化率要显著低于亚心形扁藻和中肋骨条藻;亚心形扁藻组雌体的产卵率尽管较中肋骨条藻组低,但却保持最高的雌体存活率,而且排粪率也要比中肋骨条藻低得多。综合比较得出结论：6种饵料微藻中,亚心形扁藻和中肋骨条藻是两种最适宜培养双刺纺锤水蚤雌体的优良饵料,二者混合使用效果会更好。     

N/P比失衡对两种硅藻元素组成及水体中氮磷组成的影响

近半个世纪以来,近海富营养化问题日益突出,微藻对C、N、P的吸收容易偏离Redfield比,自身元素组成受到影响。本文探究了在正常、低N/P和高N/P培养条件下,三角褐指藻(Phaeodactylum tricornutum)和中肋骨条藻(Skeletonema costatum)的元素(C、N、P)组成和其所在培养液营养盐含量的变化。结果表明:两种硅藻培养液的DIN、DIP和DIN/DIP在三处理组中,随时间的变化趋势不同;N/P比失衡的培养条件影响了两种硅藻的C/P、C/N和N/P值。高N/P比条件下,C/P值较对照组大幅度增加;低N/P比条件下,C/N值较对照组大幅度增加。而且,三角褐指藻各实验组的C、N、P化学计量比较中肋骨条藻表现出更强的稳定性;同种硅藻在N/P比失衡的培养条件下,P含量的变化幅度均大于N含量,表现出了更强的不稳定性。上述研究为营养限制对生物地球化学循环的影响的研究提供了重要的基础数据。     

Effects of different nitrate and phosphate concentrations on the growth and toxin production of an Alexandrium tamarense strain collected from Drake Passage

Nitrate (N) and phosphate (P) are believed to be two of the most important nutrients for the growth and toxin production for Alexandrium species. The study of the growth and toxicity characteristics of the Alexandrium spp. under the change of N and P can help us to understand the dynamics of algal bloom and toxification events in natural environments. A strain of Alexandrium tamarense (designated as Kci) was successfully isolated from the Drake Passage in 2001 and the clonal culture has been kept in our laboratory (Ho et al., 2003, 2012). In order to extend our understanding on the growth physiology and toxicity of this A. tamarense strain, growth and cellular toxin content were examined in unialgal batch cultures under different concentrations of N and P. The effects of variable N, P concentrations on growth, cellular toxicity (fg STXeq. cell⁻¹), and toxin composition (% molar) were determined in both exponential and stationary growth phases. The toxin profile, determined by high-performance liquid chromatography with fluorescence detection (HPLC-FD), was found to be remained relatively stable and was consistently dominated by the N-sulfocarbamoyl C-toxins (>90%) under different conditions and growth phases. There were also trace amounts of other carbamate gonyautoxins consistently expressed. The cellular toxicity varied under different N and P concentrations, as well as different growth stages. A positive correlation was observed between cellular toxicity and N concentrations, but the toxicity was enhanced when P was depleted. Both cell densities and growth rate of the cells were severely suppressed under N- or P-depletion. However, the biovolume of the cells tended to be larger at N- or P-depleted cultures. Results from the present study provide valuable insight for the ecophysiology of Alexandrium species in the coastal ecosystem of Drake Passage.     

A simple and reliable in vivo EROD activity measurement in single Fundulus heteroclitus embryo and larva

Dioxin-like compounds (DLC) induce toxic responses in early life stages of fish through activation of the aryl hydrocarbon receptor (AhR) which is frequently assessed by ethoxyresorufin-O-deethylase (EROD) activity. A novel spectrofluorimetric method was developed to quantitatively assess EROD activity in individual living embryos and prolarvae of a marine model fish species, the mummichog Fundulus heteroclitus. This in vivo method is based on the measurement of the production of resorufin by single live embryos or prolarvae after 5 h incubation with ethoxyresorufin. Freshly fertilized eggs were treated topically from 2.5 to 50 pg egg⁻¹, with 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), a prototypical DLC. EROD activity was assessed in embryos (7 days post-fertilization) and prolarvae (16 days post-fertilization). Resorufin was measured both in the culture medium (25‰ seawater) and in whole fish homogenates, to assess the percentage retained in the body. Approximately 95% and 17% of the resorufin produced in vivo was retained in embryos and prolarvae respectively. EROD activity in homogenates of embryos and in the culture medium of prolarvae increased linearly with dose. EROD activity measured by the in vivo method was highly correlated to that measured by a traditional in vitro technique using S9 fractions for both embryos and prolarvae. Both in vivo and in vitro EROD activity were higher in prolarvae than in embryos pretreated with PCB126. EROD induction measured in prolarvae by the in vivo and in vitro methods were similar whereas higher induction was measured in vivo than in vitro in embryos. The in vivo method was more sensitive and as reliable as the in vitro technique, and required a lower number of fish (4 compared to 3 pools of 5). This in vivo method is useful to link EROD induction in individual embryos or prolarvae to other organism-level responses. Further studies with other categories of xenobiotics should be performed to assess potential toxic effects on resorufin absorption/excretion processes which could affect in vivo measurements.     

防污剂Irgarol 1051对三角褐指藻生长及生理特性的影响

为研究防污剂Irgarol 1051对微藻生长及生理生化特性的影响,以三角褐指藻(Phaeodactylum tricornutum)为材料,分析了Irgarol 1051对P.tricornutum生长、叶绿素a含量、可溶性蛋白质含量、超氧化物歧化酶(Superoxide dismutase,SOD)活力及丙二醛(Malondialdehyde,MDA)含量等的影响。结果表明:Irgarol 1051可抑制P.tricornutum的生长,并诱导其产生耐受性;P.tricornutum中叶绿素a、可溶性蛋白质含量及SOD酶活力等均随Irgarol 1051质量浓度的增加而升高,以维持其生长和生理状态;随着Irgarol 1051质量浓度的升高,藻细胞中MDA含量显著增加,表明细胞中活性氧自由基(Reactive oxygen species,ROS)过量积累,这将破坏藻细胞的膜结构与功能。研究结果将为探讨Irgarol1051对微藻的效应机制及其环境风险评价提供数据资料和科学依据。     

高效分解水产原料的菌株分离鉴定和组合发酵初探

为从一种组成未知的商用复合菌中分离并筛选出优势发酵菌株,本实验采用稀释平板培养法,对菌种进行分离纯化,同时参照《真菌鉴定手册》和《常见细菌鉴定手册》,根据其形态学特征、生理生化特性,并分别结合16S r DNA和18S r DNA序列的比对分析对菌种进行鉴定。另外,本研究通过生长曲线测定,单菌种发酵试验以及多菌种混合发酵发酵试验,以游离氨基酸态氮为指标,以期筛选出发酵低值水产品制备菌肥的最佳菌株组合。结果显示,共分离得到4株菌,分别为a热带假丝酵母、b罗伦隐球菌、c枯草芽孢杆菌、d蜡样芽胞杆菌。且菌种组合acd发酵后游离氨基酸态氮的含量较对照组上升最显著,为8.276g/L。故可确定最佳发酵菌株配方为acd(热带假丝酵母+枯草芽孢杆菌+蜡样芽胞杆菌)。     

Toxicity evaluation of diethylene glycol and its combined effects with produced waters of off-shore gas platforms in the Adriatic Sea (Italy): Bioassays with marine/estuarine species

Diethylene glycol (DEG) is commonly used to dehydrate natural gas in off-shore extraction plants and to prevent formation of gas hydrates. It may be released into the sea accidentally or in discharged produced waters (PWs). PWs samples from off-shore gas platforms in the Adriatic Sea (Italy) have been used in this study. The objectives of the study were: a) to evaluate the toxicity of DEG for marine organisms; b) to evaluate if a high DEG content in PWs may alter their toxicity; c) to verify whether the DEG threshold concentration established by the Italian legislation (3.5 g/l) for PWs discharged at sea is safe for marine environment. Ten different species (Vibrio fischeri, Phaeodactylum tricornutum, Dunaliella tertiolecta, Brachionus plicatilis, Artemia franciscana, Tigropus fulvus, Mytilus galloprovincialis, Crassostrea gigas, Tapes philippinarum and Dicentrarchus labrax) have been exposed to DEG; four of these species were also exposed to PWs in combination with DEG. The results showed that: a) DEG is not toxic at levels normally detected in Adriatic PWs; b) DEG in combination with PW showed mainly additive or synergistic effects; c) short-term bioassays showed that the DEG limit of 3.5 g/l could be acceptable.     

Sensitivity of two psychrophilic diatoms to crude oil and fuel oil

Two pure cultures of diatoms, a Nitzschia sp. and a Chaetoceros sp., were grown at 0°C or 10°C in the presence of two crude oils and two fuel oils. The petroleum samples were absorbed onto filter paper discs and added directly to the algal cultures. In all cases the organisms were much more sensitive to the crude oils or fuel oils when growing at 0°C than at 10°C. The rate of photosynthesis measured by an oxygen electrode for Nitzschia sp. grown and tested at 0°C was not affected during 3 hours' incubation with some four times the amount of a crude oil or fuel oil that blocked growth. The experiments lead to the cautious suggestion that psychrophilic algae will prove some fivefold more sensitive than mesophilic algae to petroleum pollution.     

Thermal effects on the growth and fatty acid composition of four harmful algal bloom species: Possible implications for ichthyotoxicity

Little is known regarding how harmful algal bloom species respond to different temperatures in terms of fatty acid production. This study examined the effects of temperature on the growth rates, cell volumes, and fatty acid concentrations and compositions of four harmful algal bloom species (HABs), Akashiwo sanguinea, Alexandrium tamarense, Chattonella ovata, and Prorocentrum minimum. The HABs species were cultured at 15, 20, 25, and 30°C in a nutrient-enriched medium. Three of the species maintained optimal growth rates over a wide range of temperatures, but A. tamarense did not. The cell volumes of each species showed little change over the temperature range. The total fatty acid concentrations in A. sanguinea, A. tamarense and C. ovata decreased as the temperature increased, but P. minimum showed no trend in this respect. Polyunsaturated fatty acids (PUFAs), the key biochemical components that maintain cell membrane fluidity and which are associated with toxicity, decreased in both concentration and proportion of total fatty acids as temperature increased, except in A. sanguinea, in which the proportion of PUFAs to the total fatty acids increased. These reductions in PUFA concentration and proportion could reduce cell membrane fluidity and toxicity in HABs; however, enhanced growth and/or ruptured cells, which are considered more toxic than intact cells, could compensate for the reduced per-cell toxicity. This phenomenon might impact on the marine ecosystem and aquaculture industry.     

Benthic foraminifera for heavy metal pollution monitoring: A case study from the central Adriatic Sea coast of Italy

Benthic foraminifera are increasingly used as environmental bio-indicators of pollution in coastal and marginal marine settings. Their community structure provides information on the general characteristics of the environment and some species are sensitive to specific environmental parameters. Among various criteria, the occurrence of test abnormalities may represent a useful bioindicator for monitoring environmental impacts in coastal regions. A study of living benthic foraminifera was carried out in 42 sediment samples collected from the central Adriatic coast of Italy. Benthic foraminiferal assemblages from this area are rich, well preserved, and dominated by Ammonia parkinsoniana, and subordinately by Ammonia tepida, Aubignyna perlucida, Eggerella scabra, and Nonionella turgida. Heavy metal concentrations have been analysed which indicate low polluted environmental conditions. Foraminiferal species and heavy metal concentrations were investigated both with bivariate (correlation matrix) and multivariate techniques of principal component analysis (PCA) and cluster analysis. Statistical analysis shows a possible control of these pollutants both on the taxonomic composition of the benthic foraminiferal assemblages and the development of test malformations. Increasing heavy metal contents lead to an increase in relative abundance of A. tepida A. perlucida, N. turgida and E. scabra, and a relative concurrent decrease in relative abundance of A. parkinsoniana and higher percentages of deformed specimens (FAI) and species (FMI). Our results confirm that A. parkinsoniana prefers clean to low polluted environments and show that it is a very sensitive and un-tolerant species to heavy metal pollution being deeply affected by heavy metal content even at low concentrations. Our findings also confirm the capacity of the A. tepida to tolerate increasing heavy metal concentrations, and highlights that A. perlucida, N. turgida and E. scabra can be considered as tolerant species at least in low polluted environments. Following this, A. parkinsoniana and A. tepida can be reciprocally considered good bioindicator of heavy metal pollution over the surveyed area. The development of test abnormalities with a variety of malformations is a noticeable feature over the study area where the living deformed assemblages are largely dominated by a few species. The low percentages of deformed specimens (Foraminiferal Abnormality Index up to 4.7, with 2 on average) match well with the low concentrations of heavy metals that lead to low polluted environmental conditions. This study confirms and supports the suitability of studying benthic foraminifera as a technique for the in situ continuous bio-monitoring of heavy metal pollution of coastal marine sediments.     

Ecotoxicological evaluation of sewage sludge using bioluminescent marine bacteria and rotifer

Bioassay using the marine bacteria,Vibrio fischeri and rotifer,Brachionus plicatilis, and chemical analyses were conducted to assess the toxicity of the various sewage sludges, one of the major ocean dumped materials in the Yellow Sea of Korea. Sludge elutriates extracted by filtered seawater were used to estimate the ecotoxicity of the sludge. Chemical characterization included the analyses of organic contents, heavy metals, and persistent organic pollutants in sludge. Bacterial bioluminescent inhibition (15 min), rotifer mortality (24 hr) and rotifer population growth inhibition (48 hr) assay were conducted to estimate the sludge toxicity. EC50 15 min (inhibition concentration of bioluminescence after 15 minutes exposed) values by Microtox® bioassay clearly revealed different toxicity levels depending on the sludge sources. Highest toxicity for the bacteria was found with the sludge extract from dyeing waste and followed by industrial waste, livestock waste, and leather processing waste. Clear toxic effects on the bacteria were not found in the sludge extract from filtration bed sludge and rural sewage sludge. Consistent with Microtox® results, rotifer neonate mortality and population growth inhibition test also showed highest toxicity in dyeing waste and low in filtration bed and rural sewage sludge. High concentrations of persistent organic pollutants (POPs) and heavy metals were measured in the samples from the industrial wastes, leather processing plant waste sludge, and urban sewage sludge. However, there was no significant correlation between pollutant concentration levels and the toxicity values of the sludge. This suggests that the ecotoxicity in addition to the chemical analyses of various sludge samples must be estimated before release of potential harmful waste in the natural environment as part of an ecological risk assessment.     

Toxic effects of harmful benthic dinoflagellate Ostreopsis ovata on invertebrate and vertebrate marine organisms

Harmful benthic microalgae blooms are an emerging phenomenon causing health and economic concern, especially in tourist areas. This is the case of the Mediterranean Sea, where Ostreopsis ovata blooms occur in summer, with increasing regularity. Ostreopsis species produce palytoxin (PTX) and analogues, and a number of deaths directly associated with the ingestion of PTX contaminated seafood have been reported. PTX is considered one of the most toxic molecules occurring in nature and can provoke severe and sometimes lethal intoxications in humans. So far in temperate areas, O. ovata blooms were reported to cause intoxications of humans by inhalation and irritations by contact.In addition, invertebrate mass mortalities have been reported, possibly linked to O. ovata blooms, although other causes cannot be ruled out, such as oxygen depletion or high seawater temperature. In order to improve our knowledge about the direct toxicity of this species on invertebrate and vertebrate marine organisms, we performed an ecotoxicological screening to investigate the toxic effects of different concentrations of O. ovata (cultured in the laboratory and sampled in the field during blooms) on crustaceans and fish as model organisms. Artemia salina, Tigriopus fulvus, and Amphibalanus amphitrite larvae and juveniles of the sea bass Dicentrarchus labrax were used as model species. Toxic effects associated with cultured O. ovata cells were investigated using a crossed design: testing two different temperatures (20 and 25 °C), four different cell concentrations, and four treatments (untreated O. ovata culture, filtered and resuspended algal cells, growth medium devoid of algal cells, and sonicated algal cells). The results indicate that the toxicity of cultured O. ovata is related to the presence of living O. ovata cells, and that this effect is amplified by temperature. Furthermore, both tests with laboratory cultured algae and field sampled cells pointed out that A. salina is the most sensitive species even at concentrations below the Environmental Alarm Threshold set by the Italian Ministry of Health.Some possible explanations of such sensitivity are discussed, taking into account evidence of O. ovata cells ingestion and the activity of its toxins on the Na⁺/K⁺-ATPase.     

Growth and physiological state of the microalgae <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> Bohlin (Bacillariophyta) in the water taken from peter the Great Bay

Bioassay of water from different parts of the Peter the Great Bay using microalgae P. tricornutum revealed that water quality influences not only the growth but also the physiological state of the microalgae. There was no difference in the cell size between control and experimental cultures. Inhibition of microalgae growth was revealed in water with low phosphates concentration, for example, in the Marine Reserve. High phosphates concentration and moderate organic pollution caused the stimulation of miñroalgae growth in such sites as the Tumen, the Partizanskaya and the Vtoraya rechka rivers mouths.     

Application of indigenous benthic amphipods as sediment toxicity testing organisms

A series of experiments were conducted to develop standard test organisms and test protocols for measuring sediment toxicity using candidate amphipods such asMandibulophoxus mai, Monocorophium acherusicum, Haustorioides indivisus, andHaustorioides koreanus, which are indigenous to Korea. The relevant association of test species with sediment substrates was one of the important factors in sediment bioassay. The indigenous amphipodsM. mai andM. acherusicum were well associated with test sediments when they were exposed to various sediment substrates from sand to mud. The tolerant limits to various physico-chemical factors affecting bioassay results such as temperature, salinity and ammonia, as well as sensitivities to reference toxicant and contaminated sediments, were investigated usingM. mai andM. acherusicum in the present study. These amphipods were tolerant to relatively wide ranges of salinity (10~30 psu) and ammonia (<50 ppm), and displayed relevant sensitivity to temperature as well. They are more sensitive to Cd, the reference toxicant, when compared to the standard test species used in other countries. Field-sediment toxicity tests revealed thatM. mai would be more sensitive to sediment-associated pollutants thanM. acherusicum, while the sensitivity ofM. acherusicum was comparable to that ofLeptocheirus plumulosus, which has been used as a standard test species in the United States of America. Overall results of this first attempt to develop an amphipod sediment toxicity test protocol in Korea indicated thatM. mai andM. acherusicum would be applicable in the toxicity assessment of contaminated sediments, following the further evaluation encompassing various ecological and toxicological studies in addition to test method standardization.