The circadian locomotor rhythm of Hemideina thoracica (Orthoptera; Stenopelmatidae): the circadian clock as a population of interacting oscillators

ABSTRACT. The behaviour of the circadian locomotor rhythm of the New Zealand weta, Hemideina thoracica (White), supports the model that the underlying pacemaker consists of a population of weakly coupled oscillators. Certain patterns of locomotor activity, previously demonstrated almost exclusively in vertebrates, are presented here as evidence for the above hypothesis. They include after-effects of various pre-treatments, rhythm-splitting and spontaneous changes in the rhythm. After-effects, which describe the unstable behaviour of free-running circadian rhythms following particular experimental perturbations, have been observed in Hemideina following single light pulses, constant dim light, and laboratory and natural entrainment. Period changes occurred in the activity rhythm after single light pulses of 8-h and 12-h duration (25 lx). Constant dim light (0.1 lx) increased the free-running period (τ) of the activity rhythm, but the after-effect of constant dim light was either an increase or a decrease in τ. After-effects upon both τ and the active phase length of the activity rhythm were found following non-24-h light entrainment cycles with 8-h and 12-h light phases of 25 lx. Qualitative measurements of these after-effects upon τ are presented which reveal a relationship between both the direction and amount of change in τ, and the difference between entrainment cycle length (T) and pre-entrainment free-running period. The after-effect of natural entrainment was an initial short-period free-run (τ < 24h) lasting 5–10 days, generally followed by a rapid period lengthening to τ= 25–26 h. Support for the population model was provided by spontaneous dampening, recovery, and period changes of the rhythm, together with the disruption of the active phase following critical light perturbations, and rhythm-splitting. These Hemideina results are compared with the simulations of the Coupled Stochastic System of Enright (1980).     

真鲷自然群体和人工繁殖群体的遗传多样性

采用RAPD技术对真鲷野生群体及人工繁殖群体各23个个体进行了DNA多态性检测。实验选取OPK组16个10 bp随机引物用于两群体的遗传多样性分析。在野生群体和人工繁殖群体中分别获得131和123条扩增片段,两群体的多态片段比例分别为62.60%和54.47%,平均杂合度分别为0.4786和0.3633,可见真鲷野生群体及人工繁殖群体的遗传多样性较为丰富,在选择育种和遗传改良方面具有较大的潜力。人工繁殖群体的多态片段比例和平均杂合度都低于野生群体,意味着在生产过程中要采取行之有效的管理保护措施以避免或减少遗传多样性水平的降低,确保真鲷增养殖业的可持续发展。     

Ontogenetic change in the relationship between metabolic rate and body mass in a sea bream Pagrus major (Temminck & Schlegel)

Ontogenetic changes in the relationship between resting rate of oxygen consumption and wet body mass were examined at 20° C with the sea bream Pagrus major ranging from 0.00020 g (weight just after hatching) to 270 g (weight at 530 days old). There was a triphasic relationship between oxygen consumption of an individual fish M (μl min⁻¹) and body mass W (g). During a very early stage (weight 0.00020–0.00025 g), corresponding to the pre-larval stage and with the transitional period to the post-larval stage, there was no substantial change in body mass. The mass–specific metabolic rate M/W (μl g⁻¹ min ¹) showed no clear relationship to body mass as expressed by the equation M/ W =4.86 + 1.47 D , where D is age in days. During the post-larval stage (weight 0.00031–0.005 g), M/W remained almost constant independent of body mass following the expression M = 12.5 W0 .949. During the juvenile and later stages (weight 0.005–270 g), M/ W decreased with increasing body mass following the expression M = 6.3 W ^0.821 which is significantly different from the expression for the post-larval stage ( P < 0.001). Ontogenetic changes in the metabolism-body mass relationship are discussed from the viewpoint of relative growth of organs with different metabolic activities.     

Flow cytometry and ultrastructure of cryopreserved red seabream (Pagrus major) sperm

The objectives were to assess motility, fertilizing capacity, structural integrity, and mitochondrial function in fresh versus frozen-thawed (15% DMSO was used as a cryoprotectant) sperm from red seabream (Pagrus major). Mean (+/-S.D.) rates of motility, fertilization and hatching of frozen-thawed sperm were 81.0+/-5.4, 92.8+/-1.9, and 91.8+/-5.2%, respectively; for fresh sperm, they were 87.5+/-7.7, 95.8+/-2.4, and 93.8+/-4.2%. Although motility was lower in frozen-thawed versus fresh sperm (P<0.05), there was no effect (P>0.05) of cryopreservation on fertilization or hatching. Based on scanning and transmission electron microscopy, 77.8+/-5.6% of fresh sperm had normal morphology, whereas for frozen-thawed sperm, 63.0+/-7.2% had normal morphology, 20.6+/-3.1% were slightly damaged (e.g. swelling or rupture of head, mid-piece and tail region as well as mitochondria), and 16.4+/-4.2% were severely damaged. Sperm were stained with propidium iodide and Rhodamine 123 to assess plasma membrane integrity and mitochondrial function, respectively, and examined with flow cytometry. For fresh sperm, 83.9% had an intact membrane and functional mitochondria, whereas for frozen-thawed sperm, 74.8% had an intact membrane and functional mitochondria, 12.7% had a damaged membrane, 9.9% had nonfunctional mitochondria, and 2.6% had both a damaged membrane and nonfunctional mitochondria. In conclusion, ultrastructure and flow cytometry were valuable for assessment of frozen-thawed sperm quality; cryopreservation damaged the sperm but fertilizing ability was not significantly decreased.     

真鲷仔鱼用微粒子饲料中肽酶与大豆卵磷脂添加效果的研究

本研究以酪蛋白分解物为蛋白源配制三种微粒子饲料MD-S、MD-T和MD-U对真鲷开口仔鱼进行饲养试验。以MD-S的配方为基准,MD-T采用粉状大豆卵磷脂和麸质代替液状大豆卵磷脂;MD-U则另外添加0.1%的肽酶。结果表明,微粒子饲料在水中浸泡15min后,MD-T的溶出率(35.5%)低于MD-S(46.8%)和MD-U(45.8%);实验结束时(20日龄),仔鱼的成活率以生物饵料(轮虫)组为最高(86.3%),其次是MD-T组为20.7%,显著高于(P<0.05)MD-S组(13.3%)和MD-U组(13.6%);生物饵料组的仔鱼全长(6.14±0.49mm)显著大于微粒子饲料组(4.23±0.30mm~4.46±0.30mm),各微粒子饲料组之间仔鱼的全长并不存在显著差异(P>0.05)。在孵化后第12d,微粒子饲料组的仔鱼肠上皮细胞发育良好,但至孵化后第18d,仔鱼肠上皮细胞大部分萎缩、并发生脱落。鱼体的蛋白质、DNA与RNA日间增长率微粒子饲料MD-T组高于MD-S和MD-U组,但都低于生物饵料组。由此可见,微粒子饲料中添加肽酶并无助真鲷仔鱼对其消化吸收;可是,使用粉状大豆卵磷脂与麸质代替液状卵磷脂能增强微粒子饲料的黏合性,可减少其营养成分的溶出率,从而提高微粒子饲料的饲育效果。     

生物钟机制研究进展

由生物体内源性生物钟所产生的昼夜节律是近年来生命科学的研究热点之一。几种模型生物（蓝细菌、脉孢菌、拟南芥、果蝇、小鼠）的生物钟相关基因相继被克隆和鉴定,为理解昼夜节律的分子机制奠定了基础。振荡器蛋白对其编码基因的负反馈调控可能是不同生物的生物运作普遍机制,在此基础上,不同生物有不尽相同的调控方式;隐色素可能是高等生物的共同生物钟光受体。     

哺乳动物昼夜节律生物钟研究进展

昼夜节律生物钟是一种以近似24小时为周期的自主维持的振荡器,在分子水平上,该振荡器是一个由9个基因组成的转录翻译反馈环路系统。它能受外界环境影响重新设置节律,使自身机体活动处于最佳状态。除了进行自我调节外,生物钟基因还能通过调节代谢途径中特定基因表达而影响机体生理生化过程。在过去的几年里,借用遗传学和分子生物学工具,我们对哺乳动物昼夜节律生物钟的分子基础有了新的认识,本文综述了这一进展,并展望了它们在研究人的昼夜节律行为异常领域的前景。     

Size-dependent mortality of red sea bream, Pagrus major, juveniles released with fluorescent otolith-tags in News Bay, Japan

Totals of 2^.67 x 10⁵ and 7^.56 x 10⁵ juvenile red sea bream of three size groups (10, 20 and 40 mm t.l.) marked with a fluorescent substance in the otolith were released in News Bay, Oita Prefecture, Japan, in July 1987 and June 1988, respectively; the aim was to estimate growth and mortality of different developmental stages.
Of fish released in 1987 and 1988, 10 618 and 4413, respectively were recaptured during those two years. Released fish remained in the bay until the end of summer, and afterwards migrated out towards open waters. Fish of the 40-mm group released in 1987 grew to over 200 mm t.l. in one year. Mean growth rate for 19 days after release was higher in the 40-mm group (0^.87 mm day⁻¹) than in the 20-mm group (0^.74mm day⁻¹). Survival rates over 19 days were 59^.0 and 10^.1 % for 40-mm and 20-mm fish, respectively, in 1987, and those over 30 days were 69^.2, 3^.3 and 0^.0% for 40-mm, 20-mm and 10-mm fish, respectively, in 1988.
Cannibalism was indicated by the presence of marked otoliths for 20-mm fish in the stomachs of a few 40-mm individuals recaptured 2 days after release. Size-dependent growth and size-selective mortality were both noted in juvenile red sea bream, i.e. the relative size differential between larger and smaller individuals was maintained in the period between marking and recapture, and mortality was inversely proportional to size.     

Effect of cryoprotectants on hatching rate of red seabream (Pagrus major) embryos

The objectives were to investigate the effect of cryoprotectants on the hatching rate of red seabream embryos. Heart-beat embryos were immersed in: five permeable cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), 1,2-propylene glycol (PG), and ethylene glycol (EG), in concentrations of 5-30% for 10, 30, or 60min; and two non-permeable cryoprotectants: polyvinylpyrrolidone (PVP), and sucrose (in concentrations of 5-20% for 10 or 30min). The embryos were then washed and incubated in filtered seawater until hatching occurred. The hatching rate of the embryos treated with permeable cryoprotectants decreased (P<0.05) with increased concentration and duration of exposure. In addition, PG was the least toxic permeable cryoprotectant, followed by DMSO and EG, whereas Gly and MeOH were the most toxic. At a concentration of 15% and 30min exposure, the hatching rate of the embryos immersed in PG was 93.3+/-7.0% (mean+/-S.D.), however, in DMSO, EG, Gly, and MeOH, it was 82.7+/-10.4, 22.0+/-5.7, 0.0+/-0.0, and 0.0+/-0.0%, respectively. Hatching rate of embryos treated with PVP decreased (P<0.05) with the increase of concentration and exposure time, whereas for embryos treated with sucrose, there was no significant decrease in comparison with the control at the concentrations used.     

真鲷肝脏解偶联蛋白2(UCP2)基因及其功能的探讨

从真鲷(Pagrus major)肝脏通过简并引物PCR克隆解偶联蛋白2(UCP2)cDNA部分序列。该片段长674bp,编码224个氨基酸残基。推测的此部分氨基酸序列包含线粒体载体蛋白的特征结构,并与其它脊椎动物UCP2氨基酸序列同源性在72．8％以上。对变温动物色类UCP2组织表达调控研究表明：与哺乳类UCP2基因不同,真鲷UCP2基因在肝脏大量表达,而在腹腔肠系膜脂肪组织则仅有痕迹量表达,两者表达水平相差20倍以上。饲料中添加10％绿鳕油或48h饥饿对真鲷肝脏UCP2基因的表达水平均无显著影响,表明UCP2基因在脂肪含量高的鱼类肝脏表达十分稳定,为维持其基本功能所必需。真鲷肝脏和腹腔肠系膜脂肪组织UCP2基因表达水平的强烈反差,与鱼类这两种贮脂器官完全不同的氧化活性相一致[动物学报49(1)：110—117,2003]。     

Characterization of microsatellite loci for the redbanded seabream,Pagrus auriga (Teleostei,Sparidae)

The redbanded seabream Pagrus auriga (Teleostei, Sparidae) is a species of a high commercial value in Spain. There is currently little information available about the genetic characteristics of both wild and cultured populations. In this survey, we have developed eight polymorphic microsatellites for the redbanded seabream using an enriched genome library protocol. All of them were polymorphic in the 64 individuals tested, 22 of which were wild specimens, and 42 were individuals from a captive reproductive broodstock. These markers can potentially be useful tools for use in population genetic studies.     

Dexamethasone induces high-amplitude rhythms in preadipocytes,But hinders circadian expression in differentiated adipocytes

Glucocorticoids induce circadian gene expression in cultured cells and change the phase of circadian gene expression in vivo. In addition, glucocorticoids induce differentiation of preadipocyte to adipocytes. We set out to test the effect of dexamethasone, a glucocorticoid receptor agonist, on circadian rhythms in 3T3-L1 differentiated adipocytes. Our results show that differentiated adipocytes exhibit robust circadian rhythms without dexamethasone. Dexamethasone induces phase changes and increases the amplitude of circadian gene expression in nondifferentiated 3T3-L1 preadipocytes. However, dexamethasone had an opposite effect on differentiated adipocytes, leading to low-amplitude circadian expression. In conclusion, although glucocorticoids reset circadian rhythms, once rhythms are reset, glucocorticoid administration hinders circadian expression.     

Larval-rearing and growout of the red porgy (Pagrus pagrus) in the Riopesca hatchery (Greece)

Juveniles of red porgy (Pagrus pagrus 1758 L.) were experimentallyproduced in RIOPESCA hatchery during Spring 1992–95. Wild broodstock,weighing from 500 to 1000 g, was caught and brought to the hatchery insummer 1991. They were placed in a 30 m³ rectangular outdoorbroodstock tank with a sea-water flow of 5 m³ per hour.Salinity ranged from 3.9 to 4.1%, temperature from 13 to25°C , the stocking density was 4 kg m^–3,and the sex ratio was 1:1. The females spawned spontaneously in captivity producing approximately 200 000 eggs per kg of body weight. The percent offertilization was between 85–95%. Hatching was completed 85 hours later at 18 °C with a hatching percent of80–90%. The larvae were introduced into a 15 m³tank using green water (Nannochloropsis gaditana & Isochrysis galbana)and fed rotifers (Brachionus rotundiformis), Artemia salina nauplii andmetanauplii, and artificial food. After the end of the weaning period (day80 posthatch) the juveniles reached a weight of 1.4 g with a mean percentof survival of 10%. The fry were transferred to a cage site in which the growout proceeded without particular difficulties. The onlyproblem during growout was the skin coloration, that was darker than thatof wild porgies. Red porgies growth in our installations is satisfactory,with fry reaching 360 ± 12 g within 19 months, with a foodconversion rate of 1.8:1 , and a 6% mortality.     

Genome-wide identification of major protein families of cyanobacteria and genomic insight into the circadian rhythm

Cyanobacteria are among the most abundant organisms present on earth and are considered to be one of the oldest known clades. Cyanobacteria are oxygenic photosynthetic bacteria and are well known as promising renewable sources of energy; therefore, it is important to understand aspects of their genomes in detail across species. Advances in sequencing technology and the availability of several cyanobacterial genomes have provided an excellent opportunity to understand the diversity and evolution of the cyanobacterial genome. Here, we compared the genomes of 62 different phototrophic cyanobacteria. Evaluation of genetic diversity of all the cyanobacteria species studied revealed that evolution from their common ancestors was polyphyletic. In addition, the genomes were very diverse and varied among species, and significant genomic diversity was observed at the species and strain level. Overall, we identified 56 different protein families of cyanobacteria species/strains and found that they varied significantly among strains of a species. The circadian clock proteins KaiA, KaiB and KaiC (KaiABC complex proteins) of cyanobacteria were found to be present and consistent in the majority of cyanobacterial species while absent in a few others. Evolutionary analysis of the KaiABC protein complex showed that the KaiA protein has a high frequency of polymorphism, and multiple alleles were found to be present at high frequency. These results demonstrated that evolution of phosphorylation events occurred via KaiA in the KaiABC complex. Furthermore, multiple sequence alignment showed that KaiA, KaiB and KaiC proteins are highly conserved in nature. Our results provide direct information regarding the presence of different protein or protein families in cyanobacteria. The information presented here will serve as a starting point to explore the genetic diversity of cyanobacteria with the potential to play important roles in biotechnological applications.     

海水鱼真鲷脂蛋白脂肪酶基因cDNA序列与组织表达

为研究脊椎动物真鲷脂蛋白脂肪酶 (LPL)结构与功能关系以及探讨动脉粥样硬化形成机理 ,通过构建cDNA文库 ,克隆对动脉粥样硬化表现抗性的海水鱼真鲷LPL基因cDNA全序列 .再通过PCR方法扩增基因组DNA ,获取内含子 9及其两侧序列以确定外显子 10的大小 ,最后通过RT PCR ,以 β肌动蛋白为外参照 ,比较真鲷在食用两种脂肪含量不同饲料和摄食状态不同的处理条件下 ,肝脏和腹腔肠系膜脂肪组织LPLmRNA的相对水平 .从腹腔肠系膜脂肪组织cDNA文库中克隆出LPLcDNA序列 ,其完整的开放阅读框架由 15 36bp组成 ,编码 5 11个氨基酸残基 .与哺乳类不同 ,真鲷LPL基因外显子 10的开始部分是翻译的 .LPL的催化位点、二硫键位点、N 糖基化位点、肝素结合区、脂质结合位点、介导脂蛋白与低密度脂蛋白受体结合位点、二聚体形成位点等主要功能域在真骨鱼类真鲷与其它脊椎动物间基本保守 ,但肝素结合区的碱性氨基酸残基含量较人类减少 ,并在结合脂质底物的疏水环套中出现插入片段 .与哺乳类不同 ,真鲷LPL基因在成体肝脏存在诱导性表达 ,而在其腹腔肠系膜脂肪组织则存在与哺乳类相似的组成性表达 .当真鲷喂食高脂饲料时 ,其饱食状态下肝脏LPLmRNA水平升高 ,但对其腹腔肠系膜脂肪组织LPL表达没有影响 .当真鲷喂食标准商业饲料时 ,     

Immunogenicity, retention and protective effects of the protein derivatives of formalin-inactivated red seabream iridovirus (RSIV) vaccine in red seabream, Pagrus major

A formalin-inactivated virus was previously found to be efficient in protecting fish against challenge with red seabream iridovirus (RSIV), a DNA virus belonging to the Iridoviridae family. In the present study, we determined the amount of the virus in the vaccine in terms of the number of copies of the gene for the major capsid protein (MCP) gene by quantitative real-time PCR and examined the longevity and types of immune response generated after intramuscular vaccination. We also tested whether the protein components of the vaccine are able to mount a protective immune response in fish. The vaccine contained 10(7) MCP copies per microliter of vaccine, and was detected in blood, kidney and spleen of vaccinated fish up to 15 days post-vaccination. Fish vaccinated with either the intact formalin-inactivated vaccine or its protein derivatives had increased serum neutralization antibodies and enhanced expression of MHC class I, although the kinetics of expression varied among groups. However, only those vaccinated with the intact vaccine survived the virus challenge, and this indicates that serum neutralization antibodies have scarce role in protecting the fish against RSIV. We hypothesize that the cell-mediated immunity, particularly the MHC class I pathway is responsible for such protection.