Molecular insights into the circadian clock in marine diatoms

The circadian clock is a fundamental endogenous mechanism of adaptation that coordinates the physiology and behavior of most organisms with diel variations in the external environment to maintain temporal homeostasis. Diatoms are the major primary producers in the ocean. However, little is known about the circadian clock in marine diatoms compared with other organisms. Here, we investigated circadian clock genes, their expression patterns, and responses to environmental stimuli such as light, nitrogen and phosphorus in two marine diatoms, Skeletonema costatum and Phaeodactylum tricornutum, using a combination of qRT-PCR and bioinformatic analysis. We identified 17 and 18 circadian clock genes in P. tricornutum and S. costatum, respectively. Despite significant evolutionary differences, these genes were similar to those of the higher plant Arabidopsis. We also established a molecular model for the marine diatom circadian clock comprising an input pathway, core oscillator, output pathway, and valve effector. Notably, the expression patterns of core clock genes (circadian clock associated 1 (CCA1), late elongated hypocotyl (LHY) and timing of cab 1 (TOC1)) in both species differed from those of terrestrial plants. Furthermore, the expression of these genes was influenced by variations in ambient light, nitrogen and phosphorus availability. Although marine diatoms and higher plants share common circadian clock components, their clock genes have diverged throughout evolution, likely as a result of adapting to contrasting environments.     

Nutrient limitation in two marine diatoms inhibits release of bromoform

Bromoform released from phytoplankton and kelp in the ocean is the largest known carrier of bromine to the atmosphere. The photoproducts of atmospheric bromoform catalyse ozone depletion. Laboratory investigations were conducted into the link between nutrient limitation and bromoform production using axenic cultures of two warm-water diatoms (Chaetoceros neogracile and Phaeodactylum tricornutum). During exponential growth the bromoform production was 2 000–3 000 nmol bromoform (g Chl a)?1 h?1, i.e. 10–100 times higher than earlier values for temperate and cold-water diatoms. Bromoform production decreased down to zero under CO2 and nitrate limitation for both species. These results suggest that the bromoform production could be directly related to bromoperoxidase activity (and irradiance) only during exponential growth, whereas compounds other than bromoform might be formed under nutrient limitation.     

不同无机氮浓度下中肋骨条藻和三角褐指藻生长对生长环境pH的影响

海洋酸化是当前一个严重的环境问题,而海水pH的变化与微藻生长时对溶解无机碳的吸收有关。在室内条件下采用生理生态学方法,研究中肋骨条藻Skeletonema costatum和三角褐指藻Phaeodactylum tricornutum在不同无机氮水平下生长到最大生物量时培养液pH的变化。结果表明,培养液无机氮浓度从10μmol.L-1上升到200μmol.L-1过程中,2种微藻的叶绿素浓度和最终pH均呈上升趋势。培养液pH的增加量与无机氮的消耗量呈正相关关系,培养液pH的增加量与溶解无机碳的消耗量也呈正相关关系。     

麻痹性贝毒对肝脏和肾脏氧化损伤的影响

为了探讨麻痹性贝毒(PSP)对肝脏和肾脏损伤的机制,采用膝沟藻毒素GTX-1,4对小鼠进行急性暴露,剂量为1μg/kg,时间分别为60、90、120min。结果发现,与对照组相比,肝脏染毒组中的还原型谷胱甘肽(GSH)含量没有显著变化;在肾脏中,60 min时染毒组GSH含量没有明显变化,90、120 min时GSH含量降低;两器官中抗氧化酶(GSH-Px、SOD)活性与对照组相比均无显著变化(P0.05)。从而推断,GSH参与了PSP对肾脏损害的毒性过程;肾中GSH含量有可能作为PSP早期检测的敏感指标。     

黄海两种典型硅藻的磷胁迫生理研究

在实验室批次培养条件下,研究黄海两种浮游植物优势种玛氏骨条藻(Skeletonema marinoi)和旋链角毛藻(Chaetoceros curvisetus)在低磷条件下的生长特性、环境中不同形态磷浓度的变化以及磷饥饿状态下浮游植物的营养吸收动力学和碱性磷酸酶动力学。结果表明,玛氏骨条藻在磷胁迫情况下可利用自身较高的吸收速率和较小的细胞体积有效摄取环境中的无机磷快速生长并取得优势,而旋链角毛藻在无机磷的利用上并无优势,其比玛氏骨条藻需要更多的细胞颗粒磷去维持细胞活性,而且更容易诱导碱性磷酸酶分解利用有机磷,推测在有机磷丰富的环境下旋链角毛藻会在二者竞争中取得优势。这些生理特性有可能是二者在黄海季节性水华发生过程中大量存在的原因。     

多氯联苯在两种海洋微藻中毒性效应及富集效应的研究

通过分析2,2',3,4,4',5'-六氯联苯(PCB_(138))对牟氏角毛藻(Chaetoceros muelleri)和亚心形扁藻(Platymonas subcordiformis)生长的影响,以及PCB_(138)对两种微藻超氧化物歧化酶(Superoxide dismutase,SOD)活性的影响,研究多氯联苯对海洋微藻的毒性效应,并采用气相色谱法研究了两种微藻对PCB_(138)的生物富集效应。结果表明,PCB_(138)对牟氏角毛藻和亚心形扁藻均有明显的毒性效应作用,对2种藻类的96 h半抑制质量浓度(96hEC_(50))分别为0.057 mg/和0.354 mg/L,与亚心形扁藻相比,牟氏角毛藻对PCB_(138)更敏感,耐受性更差;低浓度的PCB_(138)对两种微藻SOD酶活性有诱导作用,但当PCB_(138)浓度过高时,SOD酶活性下降;两种微藻对PCB_(138)均有明显的富集作用,随着培养时间的延长,牟氏角毛藻中富集的PCB_(138)含量呈上升趋势,而亚心形扁藻中PCB_(138)含量呈现先上升后下降趋势。     

菲和Cd2+单一及复合污染对毛蚶氧化胁迫效应的比较

采用半静态染毒实验研究了菲和Cd²⁺单一及复合污染下胁迫9 d和清水释放2 d过程中毛蚶体内活性氧(ROS)、谷胱甘肽(GSH)含量和谷胱甘肽硫转移酶(GST)活性的变化。结果表明,菲和Cd²⁺单独胁迫能诱导毛蚶体内ROS含量的增加及GSH含量、GST活性的降低,且随染毒浓度的增加,对各指标影响愈明显,存在剂量-效应关系。毛蚶体内GSH和GST的变化趋势具有一致性。菲和Cd²⁺复合污染对毛蚶体内的氧化胁迫效应整体大于二者单独污染。在清水释放阶段,单一及复合污染胁迫组各指标均能恢复至对照组水平。表明毛蚶体内抗氧化系统适合作为监测海洋多环芳烃和重金属复合污染的生物标志物。     

Protective effects of Ecklonia cava extract on the toxicity and oxidative stress induced by hair dye in in-vitro and in-vivo models

Oxidative hair dyes containingρ-phenylenediamine(PPD)are reported to induce an allergic reaction by promoting oxidative stress when absorbed through the skin.Despite the associated risk,these hair dyes remain popular owing to their convenience and sharpness of color.This makes it important to minimize the cytotoxicity and oxidative stress induced by PPD-containing hair dyes.Ecklonia cava extract has been evaluated in different studies for its protective effects against external stress in fibroblasts and keratinocytes.Our study was aimed at using in-vitro and in-vivo models to investigate the extract’s effects on cytotoxicity of and oxidative stress induced by PPD-containing hair dyes.Analysis of CIEL*a*b*Color space was first used to determine the range of E.cava extract that would not interfere with the coloring ability of the dye upon addition.Subsequently,the set ranges of E.cava extract(5% and 7%)were added to the hair dye and their toxicity assessed by evaluating the viability of fibroblasts and keratinocytes.The effects on developmental phenotypes and induction of oxidative stress by hair dye were evaluated and compared with those of hair dyes containing different contents of E.cava extract using an in-vivo zebrafish model.Our results showed that E.cava extract in hair dye could significantly decrease the cytotoxicity and levels of oxidative stress caused by hair dyes containing PPD in both in-vitro and in-vivo models.These results suggest that the addition of 7% E.cava extract to 250μg/mL hair dye does not interfere with the coloring ability of the dye while showing significant protective eff ects against the hair dye.The study proposes that the use of E.cava extract as an adduct to hair dyes containing PPD reduces the cytotoxicity and oxidative stress induced by these hair dyes.     

Temporal species distributions of planktonic protist communities in semi-enclosed mariculture waters and responses to environmental stress

In order to evaluate the relationships between temporal species succession of planktonic protists and physical-chemical parameters in semi-enclosed mariculture waters, species distributions in response to environmental stress were investigated in a shrimp-farming pond near Qingdao, China during a complete shrimp-culture cycle (May to October 2002). A clear temporal succession in species distribution was found over the complete farming cycle. For example, before the introduction of the shrimp larvae there was low-variability of species distribution in the protist communities whereas during the stages immediately after, there was higher variability in species composition. Multiple linear/logistic regression analyses demonstrate that 12 protist taxa (e.g., Gyrodinium spirale, Teleaulax acuta, Prorocentrum spp. and Mesodinium pupula) were related to the nutrients, in particular ammonia and phosphates, alone or in combination with water temperature. These results suggest that temporal variations in species distribution of planktonic protist communities might be used in assessing water quality of semi-enclosed mariculture waters.     

刺参(Apostichopus japonicus)幼参对高温和低盐协同胁迫的生理响应:生长和诱导型热休克蛋白基因表达

全球气候变化背景下的极端高温和强降雨频发天气导致的养殖池塘持续性的高温和低盐环境给刺参池塘养殖带来了严峻的挑战。为了研究刺参对高温和低盐环境的生理响应,本实验分析了高温和低盐协同胁迫对刺参幼参生长及诱导型hsp70和hsp90基因表达的影响。实验设置4个温度梯度（16℃、20℃、24℃和28℃）和3个盐度梯度（22、27和32）,共12个不同的胁迫组。经过40 d的长期胁迫,研究发现,随着温度的升高和盐度的降低,幼参的体重增加量减少,并在高温、低盐组出现体重负增长。长期胁迫提高了刺参幼参的诱导型hsp70和hsp90基因表达量,从而在一定程度上增强了对极端天气的抵抗能力。同时,高温下盐度22处理组刺参幼参诱导型hsp70和hsp90基因的表达量较盐度27条件下低。双因子方差分析结果显示,高温和低盐对幼参特定生长率和诱导型hsp70和hsp90基因表达量的交互作用均不显著,并且盐度对诱导型hsp70和hsp90基因表达量没有显著的影响。因此,相较低盐,高温对刺参幼参的影响更大,可作为刺参育种的选择压力。在高温期应该采取有效措施降低海水温度,并防止极端天气造成的养殖水体温度分层。该研究丰富了刺参生理生态学理论,可为刺参良种选育工作提供思路,并为指导极端天气下刺参生产实践活动提供一定的理论依据。     

Influence of nutritional stress on digestive enzyme activities in juveniles of two marine clam species, Ruditapes decussatus and Venerupis pullastra

The potential use of digestive activities as indicators of the nutritional status in bivalves is discussed in relation to the results obtained in two clam species exposed to starvation and refeeding. Activities of some digestive enzymes (amylase, laminarinase, cellulase, and protease) were measured in juveniles of two commercially interesting species of clams, Ruditapes decussatus and Venerupis pullastra. The specimens were fed normally, being after subjected to a 15-days starvation and a further refeeding period. Samples were obtained at different moments of such feeding schedule to evaluate enzymes as well as weight (live, dry and organic) and length, in order to calculate growth rates and feeding efficiencies. Starvation led to a major decrease in clam growth as measured by dry weight and a negative growth as measured by organic weight, this coinciding with a certain degree of growth of the shell and a consumption of soft tissue. This response occurred more rapidly in R. decussatus but was of a lower magnitude than in V. pullastra. Activity of carbohydrases decreased rapidly in both species with starvation, although protease activity was maintained in R. decussatus. Recovery after the end of starvation was not similar in both species; while R. decussatus attained similar growth rates and enzyme activities to those measured prior to nutritional stress, V. pullastra only recovered 50% of its initial values. For both species of bivalves it can be concluded that digestive enzymes, and more specifically amylase, could be used as indicative of their nutritional condition.     

马氏珠母贝选群F1的表型性状的相关和通径分析

从7月龄的F1群体随机取样120个体,测量表型性状并进行相关和路径分析。所测量的表型性状包括壳长、壳高、壳宽、壳重和仝湿重共5个指标。结果表明：（1）壳长、壳高、壳宽、壳重和全湿重存在显著的相关性（P〈0．05）。全湿重与壳长、壳高、壳宽和壳重存在显著的正相关（P〈0．05）,相关系数分别为0．8970、0．6974、0．6521和0．5486。（2）壳重、壳高、壳宽和壳长对全湿重具有正直接影响,其值分别为0．6356、0．1872、0．1814和0．0599。结果说明了通过直接选择或间接选择可以改良马氏珠母贝选群F1的壳长、壳高、壳宽、壳重和全湿重等生长性状。     

鲈鱼胆汁中芘及代谢产物含量与水体中芘含量的关系

运用GC—MS的方法和荧光分光光度法，研究鲈鱼分别暴露于0．1、1．0和10．0μg／dm^3质量浓度芘溶液中7d，水体中芘及其鲈鱼胆汁中芘和1-羟基芘含量的变化,实验结果显示：(1)鲈鱼对水体中的芘具有非常显著的去除作用。(2)随着芘暴露浓度的增大，鲈鱼对芘的代谢去除作用增强。(3)随着暴露时间的延长和芘暴露浓度的增大，胆汁的1-羟基芘浓度递增。(4)鲈鱼胆汁具有较高的芘浓度，对海水中的芘具有较强的富集作用。(5)鲈鱼胆汁的1-羟基芘及芘的浓度与水体芘浓度均具有很好的相关性，对于指示水体的芘污染程度具有一致性；可作为指示水体芘污染程度的生物标志物。     

An in vitro procedure for evaluation of early stage oxidative stress in an established fish cell line applied to investigation of PHAH and pesticide toxicity

Oxidative stress by increased production of reactive oxygen species such as superoxide has been implicated in the toxicity of PCB's and non-target toxicity of many pesticides. We report the development of a microplate-based method for determination of early stage oxidative stress using an established cell line (EPC) from a skin tumour of carp Cyprinus carpio L. and 2',7'-dichlorodihydrofluorescein diacetate (H(2)-DCFDA) as a fluorescent probe for detection of reactive oxygen species (ROS) formation. Sublethal concentrations of the herbicide Paraquat, an established redox cycling agent and a crude PCB mixture, Arochlor 1254 elicited a linear increase in ROS formation over 2 h exposure which was some 45- and 10-fold higher, respectively, than attributable to basal respiration, confirming the suitability and response of the test system. Whilst in vivo studies in mammals have implicated early stage oxidative stress in the toxicity of pesticides, we did not observe an increase in ROS production after exposure of EPC cells to sublethal concentrations of Carbaryl, 2,4-DDT, Lindane or Malathion implying that this is not the causative mechanism of acute toxicity in this fish cell line. The apparent involvement of oxidative stress in their mammalian toxicity may therefore be an indirect effect or dependent upon compound metabolism.     

Investigation and comparison of one-dimensional (1-D) analytical models prediction for salt intrusion condition in two selected estuaries

Abstract

In this paper, two analytical models used worldwide to assess salinity variation in alluvial estuaries are applied to the Ashtamudi estuary, a Ramsar site, southwest coast of India; and Bouregreg estuary, in northwest part of Morocco. The estuaries’ bathymetry is described by an exponential function. Both models are quite similar and use a predictive equation for the dispersion in the estuary mouth (D₀). The major difference between the two models is the use of the constant value of K?=?0.5 for the Van der Burgh coefficient (K) and the introduction of the correction factor ζ, which is a function of damping (δ) and shape (γ). The performance of these two models was evaluated by comparing their results with field measurements; this revealed that both analytical models apply well to both the estimation of salinity distribution and the prediction of salt intrusion in the Ashtamudi and Bouregreg estuaries (Ashtamudi: RMSE = 0.60–1.22 ppt; Bouregreg: RMSE = 0.92–2.71 ppt). One model agrees more with the field measurements of salinity distribution along the estuaries axis; the second underestimate and overestimate some values of salinity distribution along the estuaries. Possibly, the constant value of K?=?0.5 for the Van der Burgh coefficient (K) has applicability limits for the estuaries under tidal conditions. The specifying of the parameterization may be a field of research.     

EROD activities of liver in Mugil so-iuy exposed to benzo (a)pyrene, pyrene and their mixture

Abstract-The effects on hepatic EROD (7-ethoxyresorufin O-deethylase) in Mugil so-iuy exposedto benzo(a)pyrene (BaP), pyrene and their mixtures of equal concentration were investigated, at con-centrations of 0.1, 1.0, 5.0, 10.0, 50.0 μg/dm~3, in experimental condition. Time-effects and dose-response of the biochemical indexs were observed. The results showed that the hepatic EROD activitieswere induced by the exposure of BaP, pyrene and their mixtures at high concentration. Dose-responseconnections were that the hepatic EROD activities were elevated with increasing concentration of the pol-lutants. The combined effect of BaP and pyrene at 1:1 concentration ratio on hepatic EROD activity wasantagonism.     

中国对虾“黄海1号”与野生群体F_1代生长发育规律比较

采用4种生长曲线模型对中国对虾"黄海1号"和野生群体F1代15项形态性状的生长规律进行了拟合,以三次函数模型的拟合优度(R2)最高;采用三次函数模型拟合的2个群体各形态性状的生长曲线、拐点月龄以及拐点体重(各形态性状长度)结果表明,中国对虾"黄海1号"的拐点月龄为2.87(拐点体重14.98 g),野生群体F1代的拐点月龄为4.05(拐点体重26.26 g);中国对虾"黄海1号"各形态性状的拐点月龄分布在0.51～3.07之间,达到最大生长速度的顺序分别为:头胸甲长>第1腹节宽>头胸甲高>腹1高>头胸甲宽>体长>全长>腹节5长>腹节3和4长>尾节长>腹节2长>腹节1长>腹节6长;野生群体F1代除第2腹节长的拐点月龄为0.45之外,其它性状的拐点月龄分布在2.38～3.08之间,达到最大生长速度的顺序分别为:腹节2长>腹节1长>腹节3长>腹节 4长>头胸甲高>腹节5长>头胸甲宽>全长>腹1高>腹1宽>尾节长＝头胸甲长＝体长> 腹节6长,除第1和第2腹节长2个性状外,野生群体F1代的其它性状均比中国对虾"黄海1号"发育迟缓了1个月左右.     

缢蛏(Sinonovacula constricta)氨氮胁迫应答miR-8245a-5p靶基因GOT验证及其表达特征分析

为鉴定缢蛏(Siaoraovacula constricta)氨氮胁迫应答相关miRNA靶基因及其表达特征,选择氨氮胁迫下缢蛏肝胰腺组织中表达水平差异显著的miR-8245a-5p进行研究。靶基因验证结果显示,缢蛏GOT基因表达量极显著高于未胁迫组(P<0.01),表明GOT是miR-8245a-5p的靶基因。缢蛏GOT基因的ORF长1227bp,编码408个氨基酸;氨基酸多重序列比对和系统进化树分析发现,缢蛏等贝类同源性较高,与其他物种同源性较低。基因时空特征表达分析结果显示,GOT基因在缢蛏肝胰腺中的表达量明显高于其余6个组织(P<0.01);140mg/L氨氮胁迫下缢蛏GOT基因在肝胰腺中的表达量1h内急剧上调,此后上调逐步减弱,60mg/L氨氮水平下第12h上调才达峰值;不同氨氮水平暴露下缢蛏肝胰腺中GOT活力整体均呈现先升后降趋势,但在较高氨氮浓度水平下同样表现出更快速的应答特征。这些结果为后续深入研究缢蛏miR-8245a-5p及GOT基因调控氨氮解毒机制提供了理论基础。     

梯度淹水胁迫下全日潮海区秋茄幼苗的生长和生理反应

以秋茄(Kandelia candel L.Druce)幼苗为材料,在全日潮海区广西英罗湾开展为期5个月的野外梯度淹水胁迫实验。3座平台作为重复,每座设置8个梯度,相邻高程组间的高度相差10cm。实验5个月后测定幼苗的生长状况和一些生理指标。结果表明:秋茄幼苗的生长高度、节数、叶数和大根数这4个外部形态特征指标的最大值均出现在360cm高程组。生长高度、节数、叶数的曲线较为一致,幼苗大根数的略有不同,但总体上可看出太低的高程均不利于这4个指标的增长。叶面积对淹水胁迫的反应与叶数一致,但高程组之间的差异更大。叶绿素含量则表现长时间淹水胁迫促进含量增高。在任一高程组,秋茄幼苗各新生器官生物量分配均表现为:茎>根>叶。各新生器官及全株生物量的最大值出现在360cm高程组,它们的干重曲线表现比较一致。幼苗根系中的活性氧清除酶类活性均高于叶片。根系中SOD酶和POD酶的分布规律较为一致,都以360cm高程组的活性最高。叶片中SOD酶和POD酶的变化则相反,叶片SOD酶随高程增大而降低,POD酶则随高程增大而增大。根系中活性氧清除酶类活性的水平与幼苗全株生物量的关系均呈显著相关,叶片中的则无此相关性。360cm及以上高程组幼苗全部存活,较低高程组有部分死亡。综合本文实验结果,可初步判断北部湾沿海秋茄造林的滩涂高程不宜低于当地平均海平面。     

低盐胁迫对脊尾白虾过氧化氢酶和硒谷胱甘肽过氧化物酶基因表达的影响

过氧化氢酶(CAT)和硒谷胱甘肽过氧化物酶(Se-GPx)在抗氧化防御体系中扮演重要的角色,在清除多余的活性氧自由基(ROS)防止机体氧化损伤方面具有重要的作用.本文利用RACE-PCR方法首次从脊尾白虾肝胰腺组织中克隆获得了CAT cDNA全长序列,该基因全长2649 bp,包括5′非编码区(UTR) 78 bp、开放阅读框(ORF)1554 bp和3′非编码区(UTR) 1017 bp,共编码517个氨基酸,预测分子量为58.46 kDa,理论等电点为6.64,利用PROSITE tools预测CAT基因的功能位点,发现该氨基酸序列包括酶活性中心序列“FDRERIPERVVHAKGAG”,亚铁血红素结合信号序列“RLFSYPDTH”和 3个催化位点残基His⁷¹,Asn¹⁴⁴和Tyr³⁵⁴.同源性分析表明,脊尾白虾CAT氨基酸序列与其他物种的相似性为68%-92%.荧光定量PCR分析结果表明,CAT基因在肝胰腺、血细胞、心脏、肌肉、卵巢、鳃、胃和眼柄均有表达,其中在肝胰腺中表达量最高.低盐胁迫后脊尾白虾鳃和肝胰腺中CAT和GPx基因分别在48 h和6 h达到最大值,且与对照组差异显著(P< 0.05),表明CAT和GPx基因参与了低盐胁迫反应,并表现出一定时间效应关系.综合分析结果指出脊尾白虾CAT基因是CAT家族主要成员之一,可能在低盐环境胁迫过程中具有重要的作用.