Isolation and characterization of the rbcS genes from a sterile mutant of Ulva pertusa (Ulvales,Chlorophyta) and transient gene expression using the rbcS gene promoter

We isolated two different genomic DNAs (UprbcS1 and UprbcS2) encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and portions of the 5′- and 3′-flanking regions from sterile Ulva pertusa Kjellman. The UprbcS1 and UprbcS2 genes had three introns in the coding region. Each predicted UprbcS polypeptide was a 180-amino-acid (AA) residue including a 38-AA transit peptide, although the 104th AA residue was replaced. The nucleotide sequences of UprbcS cDNAs isolated from a cDNA library corresponded to that of the UprbcS1 gene, suggesting that the UprbcS1 gene was predominantly expressed in sterile U. pertusa compared to UprbcS2. Southern blot analysis showed that each UprbcS gene was a single-copy gene in the sterile U. pertusa genome. Northern hybridization indicated that the expression of UprbcS was induced and repressed by dark and light treatments, respectively. When sterile U. pertusa cells were transformed with an expression vector containing the UprbcS1 promoter and terminator sequences fused with the green fluorescent protein (GFP) gene, GFP fluorescence was observed in the cells transformed. These results suggest that the UprbcS1 gene promoter is light regulated and highly active in the sterile U. pertusa cells and is available for genetic transformation system in the alga.     

孔石莼的同形世代交替

通过实验室培养和显微观察,描述了孔石莼同形世代交替各个阶段的发育情况。雌雄异体的配子体成熟后,放散两鞭毛的配子,雌雄配子接合成合子进而发育成孢子体,孢子体成熟后放散四鞭毛的游孢子,游孢子固着后,可直接发育成配子体。其同形世代交替是单倍体的配子体与二倍体的孢子体相互交替。     

干出胁迫对孔石莼生长及生理影响

通过人工模拟干出条件研究孔石莼(Ulva pertusa)在不同干出条件下的生理生化反应。以采自大连潮间带的孔石莼为材料,测定在不同的干出时间(0 h、0.5 h、1 h、2 h、4 h、6 h)下其叶绿素a(Chla)含量、可溶性糖含量、脯氨酸(proline,Pro)含量、丙二醛(MDA)含量和过氧化物酶(POD)活性的变化。结果表明:干出胁迫会对孔石莼的生理生化过程产生明显的影响。随着干出时间的延长,孔石莼中叶绿素a含量呈现下降的趋势;丙二醛和可溶性糖的含量呈现缓慢的升高趋势;过氧化物酶活性和脯氨酸含量呈现明显的升高趋势。这些结果为孔石莼的抗性生理研究提供了补充和参考。     

孔石莼在干出胁迫下上调表达基因消减cDNA文库的构建及分析

利用抑制消减杂交技术构建了潮间带绿藻孔石莼(Ulva pertusa)在干出胁迫状态下上调表达基因的消减cDNA文库.获得的阳性克隆经测序得到150条上调表达的EST片段,其中21条为冗余序列(RS),137条非冗余序列(NRS).片段中最长833 bp,最短106 bp,平均长度364 bp.参与比对的EST片段按照...     

cDNA cloning and expression analysis of two heat shock protein genes,Hsp90 and Hsp60, from a sterile Ulva pertusa (Ulvales,Chlorophyta)

Molecular chaperones are synthesized and accumulated under a variety of unfavorable conditions in all organisms. Heat shock protein 90 (Hsp90) and Hsp60, which are classified into the major classes of molecular chaperones, play important roles in cellular stress responses. In this study, we characterized sterile Ulva pertusa Hsp90 (UpHsp90) and UpHsp60 genes which may be involved in tolerance to thermal and heavy metal stresses in this alga. The UpHsp90 cDNA consisting of 2,118 nucleotides encoded a polypeptide of 705 amino acids (AA). On the other hand, the UpHsp60 cDNA consisting of 1,722 nucleotides encoded a protein whose predicted length was 573 AA. The AA sequence alignment and phylogenetic analyses showed that the UpHsp90 and UpHsp60 proteins were more similar to cytoplasmic Hsp90s and mitochondrial Hsp60s, respectively, than to other types of the respective Hsps. Southern blot analysis indicated that the sterile U. pertusa genome had at least two cytoplasmic Hsp90-encoding genes and two mitochondrial Hsp60-encoding genes. The UpHsp90 and UpHsp60 mRNA levels were significantly affected by diurnal and temperature changes, and slightly affected by exposure to heavy metals. These results suggest that UpHsp90 and UpHsp60 genes play particularly important roles in adaptation to diurnal and temperature changes.     

赤潮异弯藻对孔石莼生长及其某些生理特性的影响

通过实验生态学和生物化学的方法,研究了赤潮异弯藻[Heterosigma akashiwo(Hada)Hada]对孔石莼(Ulva pertusa Kjellman)生长及其叶绿素a(Chl-a)含量、丙二醛(MDA)含量、总抗氧化能力(T-AOC)、过氧化氢酶(CAT)活力、超氧化物歧化酶(SOD)活力和谷胱甘肽过氧化酶(GPX)活力的影响。结果表明:孔石莼的克生作用会明显抑制赤潮异弯藻的生长,同时赤潮异弯藻也会抑制孔石莼的生长,且孔石莼生理生化特性对其有明显的响应。赤潮异弯藻会使孔石莼CAT活力和MDA含量呈现升高的趋势;GPX活力呈现下降的趋势;T-AOC和T-SOD活力呈现先下降后升高趋势;Chl-a含量并无明显变化。推测赤潮异弯藻克生作用诱导孔石莼产生活性氧自由基,是藻体受损伤的主要原因。     

The advantages of Ulva (Chlorophyta) as an additive in sea urchin formulated feeds: effects on palatability,consumption and digestibility

The present study was performed to determine whether the palatability, consumption and digestibility of an artificial sea urchin diet could be improved by the addition of a highly preferred seaweed. Four species of macroalgae (Ulva rigida, Ecklonia maxima, Porphyra capensis and Gigartina polycarpa) were assessed by pairwise preference tests. Each macroalgal species was consumed at least once by Tripneustes gratilla and significant preferences always involved Ulva as the most preferred species. Four protein‐rich artificial diets supplemented with varying amounts of Ulva [0, 50, 150 and 200 g kg^?1 designated 0, 5, 15 and 20U, respectively] were then developed and fed to urchins over a 20‐day period. Inclusion of 200 g kg^?1 dried Ulva significantly improved the palatability of the artificial diet and mean dry feed consumption rates were higher in urchins fed the 20U and 15U diets compared with urchins fed diets with a lower Ulva content. Daily digestible protein (DP) intake also differed significantly, with urchins fed the 20U diet having a significantly higher DP intake compared with the 15U, 5U and 0U treatments. These results indicate that inclusion of the palatable seaweed Ulva acted as a feeding stimulant, increasing the acceptability of the diets and boosting protein intake.     

Osmoregulatory ability and stress responses during freshwater adaptation of black porgy (Acanthopagrus schlegeli) treated with exogenous prolactin

The effects of ovine prolactin (oPRL) on osmoregulatory ability (electrolyte balance, plasma osmolality and activity of gill chloride cells and gill Na⁺/K⁺‐ATPase) and stress responses (plasma cortisol, glucose, aspartate aminotransferase: AST and alanine aminotransferase: ALT) were investigated in black porgy transferred to freshwater (FW). Fish in seawater (SW) were injected twice at a 24 h interval with oPRL (at 1, 3, or 5 μg g^–1 body weight) or vehicle (0.9% NaCl) and then transferred to FW. They were sampled 3 days after the transfer. With oPRL at 5 μg g^–1, levels of plasma Na⁺ and Cl^? and osmolality were significantly higher than in saline‐treated fish, whereas gill CCs number and Na⁺/K⁺‐ATPase activity were lower. Also, the 5 μg g^–1oPRL treatment led to significantly lower plasma cortisol levels than did saline treatment. However, there were no significant differences in plasma AST and ALT between groups. These results support the positive osmoregulatory role of PRL in black porgy during FW adaptation.     

Determination of optimal temperature(s) in juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) based on growth performance and stress responses

This study sought to determine the optimal temperature(s) for aquaculture of juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) (mean initial BW: 3.1 g). Growth performance, insulin‐like growth factor 1 (IGF‐1) expression and thermal stress responses (plasma cortisol, glucose, and hepatic heat shock protein 60 expression) were evaluated at three constant temperatures (24°C, 26°C and 28°C) in a 2‐week trial. At the end of the trial, final BW was significantly higher at 26°C and 28°C than at 24°C (p < 0.05); a quadratic regression analysis of final BW showed the optimum temperature for growth was 27.5°C (p < 0.05, R² = 0.806). The highest hepatic IGF‐1 expression was observed at 26°C (p < 0.05). On the other hand, hepatic heat shock protein 60 expression was highest at 28°C (p < 0.05), suggesting thermal stress. In conclusion, temperature optima, which support excellent growth but induce minimal thermal stress, was 26°C. This fine information within a narrow temperature range is expected to give empirical information for red‐spotted grouper farmers to sustain maximal production efficiency with avoiding thermal stress and to determine the future location of production, especially in consideration of arising seawater temperatures.     

Oxidative stress in juvenile chinook salmon, Oncorhynchus tshawytscha (Walbaum)

Juvenile chinook salmon, Oncorhynchus tshawytscha (Walbaum), were held in 8–11°C freshwater, starved for 3 days and subjected to a low‐water stressor to determine the relationship between the general stress response and oxidative stress. Lipid peroxidation (LPO) levels (lipid hydroperoxides) were measured in kidney, liver and brain samples taken at the beginning of the experiment (0‐h unstressed controls) and at 6, 24 and 48 h after application of a continuous low‐water stressor. Tissue samples were also taken at 48 h from fish that had not been exposed to the stressor (48‐h unstressed controls). Exposure to the low‐water stressor affected LPO in kidney and brain tissues. In kidney, LPO decreased 6 h after imposition of the stressor; similar but less pronounced decreases also occurred in the liver and brain. At 48 h, LPO increased (in comparison with 6‐h stressed tissues) in the kidney and brain. In comparison with 48‐h unstressed controls, LPO levels were higher in the kidney and brain of stressed fish. Although preliminary, results suggest that stress can cause oxidative tissue damage in juvenile chinook salmon. Measures of oxidative stress have shown similar responses to stress in mammals; however, further research is needed to determine the extent of the stress–oxidative stress relationship and the underlying physiological mechanisms in fish.     

缘管浒苔rbcL全长cDNA克隆与序列分析

对缘管浒苔光合作用第一关键酶Rubisco大亚基基因(rbcL)全长cDNA序列进行了克隆分离。首先应用RT-PCR方法获得了rbcL大片段cDNA序列,序列长度为1101bp,并进行了测序分析。然后分别应用5′RACE方法和3′RACE方法获得2个克隆序列,其序列长度分别为371bp和579bp,并进行了测序分析。在此基础上,将3个片段序列进行拼接,获得了Rubisco大亚基全长cDNA序列(1472bp),其中包括1425bp的编码区序列及47bp的前导序列(NCBI登录号:DQ813496),并推断出蛋白质序列(474个氨基酸)。对序列进行了相关生物信息分析和同源性比对,结果显示与已克隆出全长rbcL基因的7种绿藻比较具有较高同源性,核苷酸序列和蛋白质序列同源性分别达到了82.07%～85.78%和88.00%～94.11%,其中与蛋白核小球藻蛋白质序列同源性最高,为94.11%。在此基础上,使用生物信息学软件对缘管浒苔rbcL氨基酸序列与其它植物或藻类进行了多序列比对,并且进一步应用在线软件程序进行了蛋白质的二级结构分析和三级结构预测。     

Pigmentation of gilthead seabream, Sparus aurata (L. 1875), using Chlorella vulgaris (Chlorophyta, Volvocales) microalga

A feeding experiment was conducted over 9 weeks with seven groups of 30 (fish per group) unpigmented gilthead seabream, Sparus aurata (L. 1875) (initial mean weight = 145.2 ± 12.3 g). Three experimental diets were prepared by adding to a basal diet free of carotenoid (final pigment content of around 40 mg per kg feed): (i) a biomass of the carotenogenic Chlorella vulgaris (Chlorophyta, Volvocales); (ii) a synthetic astaxanthin; and (iii) a mixture (1:1) of microalgal biomass and synthetic astaxanthin. At 3‐week intervals, five fish were sampled from each tank for total carotenoids analysis in skin and muscle. The carotenoid pigments (total amount = 0.4%) identified in the carotenogenic alga were lutein (0.3%), β‐carotene (1.2%), canthaxanthin (36.2%), astaxanthin, free and esterified forms (55.0%), and other pigments (7.3%). Carotenoid pigments were significantly deposited in the four skin zones studied during the feeding trial: the forefront between the eyes, the opercule, along the dorsal fin and in the abdominal area. In the muscle, regardless of the astaxanthin source, the amount of carotenoids measured was very low (less than 1 mg kg^?1) and differences not significant. Moreover, no muscle pigmentation was evident, and there was no variation in the amount of carotenoid analysed in skin tissue, through the trial, for each treatment. It was concluded that supplementing the feed with C. vulgaris would be an acceptable practice in aquaculture to improve the market appeal of the gilthead seabream.     

Physiological,ionoregulatory, metabolic and immune responses of Persian sturgeon,Acipenser persicus (Borodin, 1897) to stress

The physiological ionoregulatory, metabolic and immune responses of Persian sturgeon, Acipenser persicus, to acute stress were investigated. Water levels were lowered to the fish dorsal scutes, and fish were blood‐sampled before stress (pre‐stress), and 0, 6, 24 and 72 h after stress. Results showed that serum cortisol rapidly increased after stress, returning to initial levels at 24 h. Serum glucose significantly increased at 6 h, declining to the pre‐stress levels at 72 h. Serum triglyceride and cholesterol showed significant decreases at 0 h, then increasing to higher than the initial levels at 72 h. Serum T₃ and T₄ significantly decreased at 0 h and recovered at 72 h. Serum chloride levels showed no significant changes while serum calcium showed a significant increase at 0 h and a further increase until 72 h poststress. Serum total protein and alternative complement activity showed a significant initial decrease and recovery at 24 h with further increase at 72 h. Serum lysozyme activity increased significantly at 24 and 72 h after stress. Serum total immunoglobulin significantly increased at 0 h and peaked at 24 h. This is the first work showing thyroid hormone and immunological responses in Persian sturgeon subjected to stress, and the results show that this species follows a classical hormonal and energetic stress response, although, in the low range among the sturgeons. Although transient and moderate, confinement stress can induce significant changes in the innate immune response.     

Behavioural and metabolic responses of Unionida mussels to stress

1. The aim of this study was to assess the extent to which the behavioural traits of freshwater mussels provide suitable indicators of stress in individuals, towards the advancement of non-invasive, remote monitoring techniques to examine population condition.
2. Variation in the expression of particular behavioural metrics was examined in accordance with measurements of oxygen consumption, across environmental stressors (aerial exposure and high concentrations of total suspended solids), and between two freshwater mussel species (Margaritifera margaritifera and Anodonta anatina)
3. Aerobic metabolic rate was quantified using intermittent respirometry, and behaviour was observed using time-lapse footage. Comparisons of metabolic response and the occurrence of behavioural traits, across the two stressors, focused on differences between the 24 h pre-exposure period (pre-exposure), the first 3 h of post-exposure (immediate post-exposure), and the time following the initial 3 h of post-exposure until the end of the experimental run (extended post-exposure).
4. The results of this study demonstrated a relationship between the frequency of occurrence of behavioural responses to stress exposure, associated with valve activity, and significant changes in the metabolic functioning of A. anatina and M. margaritifera mussels. The findings from the study also highlighted substantial intraspecific variation across species and stressors.
5. Data from this research could assist in the development of novel biosensors that track mussel valve activity remotely in their natural environment. When coupled with real-time data examining alterations in environmental metrics, this technology could assist in the monitoring of population condition and aid conservation management.

     

干露对三疣梭子蟹抗氧化和应激能力的影响

在实验室条件下,研究了干露时间(0.5、1.5和3 h)胁迫对三疣梭子蟹抗氧化能力和应激能力的影响,初步探讨了能够较灵敏地指示梭子蟹健康状况的胁迫指标。结果显示:(1)干露时间胁迫对超氧化物歧化酶(SOD)活力和丙二醛(MDA)含量均无显著影响(P0.05);但干露时间显著影响肝胰脏总抗氧化能力(T-AOC)水平、热休克蛋白70(HSP70)表达量和肌肉乳酸含量(P0.05)。(2)0.5 h干露胁迫后,SOD活力在入水恢复4 h时达到峰值;MDA含量在入水恢复2~10 h内显著高于对照组水平(P0.05);T-AOC水平在恢复阶段基本维持在对照组水平。1.5和3 h干露后,SOD活力在入水恢复阶段一直维持在对照组水平,且各时间点与对照组差异不显著(P0.05)。1.5 h干露后MDA含量在入水恢复阶段呈先升高后降低趋势,各时间点与对照组差异不显著(P0.05);而3 h干露后入水恢复10 h时显著增加(P0.05)。1.5 h干露后入水恢复4 h时,T-AOC水平显著高于对照组(P0.05);3 h干露后入水恢复0.5 h时显著升高(P0.05),2~10 h内基本恢复到对照组水平。干露时间胁迫后,乳酸含量均在入水恢复0.5 h时达到最大值;HSP70表达量均在入水恢复2~4 h内达到峰值。研究表明,相比于其他所选指标,T-AOC水平能够较敏感地反映干露时间胁迫对机体的生理状态的影响;SOD和MDA相互配合能够较灵敏地反映不同干露时长后机体在恢复阶段的免疫水平。     

Critical Thermal Maximum of Juvenile Spotted Seatrout (Cynoscion nebulosus) Reared for Summer Stocking in Texas

The critical thermal maximum of juvenile spotted seatrout (SL range 18–33 mm) was determined using a temperature increase of +0.26°C per hour. The critical thermal maximum (water temperature that was lethal to 50% of the test fish [LT₅₀]) for trial 1 was LT₅₀ = 38.8°C, LT₅₀ = 39.4°C for trial 2, and LT₅₀ = 38.9°C for trial 3. Critical thermal maximums differed significantly (P < 0.05) between trials 2 and 3, whereas trial 1 did not differ among trials. This difference correlated with body size, where fish in trial 2 were significantly larger (P < 0.05) (mean = 27.6 ± 2.0 mm in SL) (mean ± SE) than the fish of trials 1 (mean = 23.1 ± 0.5 mm in SL) and 3 (mean = 21.5 ± 0.7 mm in SL), suggesting positive size dependence in the critical thermal maximum.     

Growth compensation in juvenile tongue sole,Cynoglossus semilaevis (Güther, 1873): responses to thermal stress and feed restriction

Responses of compensatory growth to high temperature and feed restriction in juvenile tongue sole, Cynoglossus semilaevis (Güther, 1873), were investigated during a 56‐days experiment. Fish were divided into seven groups including three high‐temperature treatments which were reared at 28°C for 1, 2 and 3 weeks, respectively, then returned to 22°C (recorded as T1, T2 and T3), three feed‐restricted treatments which were fed 25% satiation for 1, 2 and 3 weeks, respectively, then fed ad libitum (recorded as R1, R2 and R3) and a control (continuously reared at 22°C). The results showed that juvenile tongue sole exposed to thermal stress for 1 week exhibited overcompensatory growth. However, complete compensation occurred in T2, T3 and R1 fish, while partial compensation occurred in R2 and R3 fish. The body composition and energy content of fish were not significantly different among all treatments (P > 0.05). The specific growth rate and feed efficiency of fish in T1 were significantly higher than other treatments during the whole experiment period (P < 0.05). Higher feed efficiency and apparent digestion rate during recovery might account for the compensatory growth in tongue sole in the present study. Hyperphagia played an important role in compensatory growth for feed‐restricted fish, but was not involved in the compensatory response in thermal stressed fish in the present study. The results from the present study suggested that a suitable thermal stress could be more feasible to elicit compensatory growth than feed restriction in juvenile tongue sole.