基于线粒体Cytb基因探讨我国日本囊对虾4个地理群体的遗传结构及种群分化

为了分析日本囊对虾两种表型差异类型的系统发育关系,实验系统比较了两种类型线粒体全基因组的基因结构、遗传距离和密码子偏好性等特征。结果显示,两种类型线粒体基因组的基因重叠区、基因间隔和密码子等方面均存在差异。两种类型的线粒体蛋白编码基因核苷酸序列的相似度为91.10%~95.00%,氨基酸序列的相似度为96.15%~100%。两种类型的A+T富集区的同源性只有82.60%,分歧度为15.40%,呈现高度遗传分化。日本囊对虾两种类型蛋白编码基因的核苷酸序列分歧度大于明对虾属和叉肢螯虾属,但氨基酸序列的分歧度小于后者。研究发现,基于cytb基因的种间遗传距离大于种内遗传距离的10倍;基于cox1基因的种间遗传距离分别为类型Ⅰ和类型Ⅱ的种内遗传距离的14.6倍和5.2倍。两种类型的nd1、nd4和nd5基因的Ka/Ks值大于1,表明受到正向选择。基于20种隶属9属的对虾线粒体基因组蛋白编码序列构建系统发育树,结果显示能有效区分各属对虾,其中日本囊对虾的两种类型首先聚类,再与宽沟对虾聚类。研究表明,日本囊对虾的两种表型差异类型基本达到物种水平,有必要进一步评估更多的性状差异,以提高两种囊对虾的特异性养殖技术。     

Sperm maturation and capacitation in the open thelycum shrimp Litopenaeus (Crustacea: Decapoda: Penaeoidea)

Transmission electron microscopy was applied to sperm removed from males and females belonging to Litopenaeus vannamei, L. stylirostris and L. occidentalis. It was discovered that a region named filamentous meshwork (FM), located between the nucleus and the hemispherical cap, develops differently in these three closely related species. In L. vannamei, the FM is synthesized in the male reproductive system, but seems to complete its formation after mating. In L. stylirostris, the FM region was not present in spermatophores collected from males or in sperm from the thelycum. In L. occidentalis, the FM region is fully developed in male sperm. It is suggested that completion of the FM is required for acrosome maturation, and the process continues after mating in some species of Litopenaeus. In vitro induction of the acrosome reaction in sperm from males and females of L. occidentalis demonstrated for the first time that reactivity is significantly superior in sperm cells that have been attached to the open thelycum for some hours, as compared to sperm in males (prior to transfer). This finding suggests that matured sperm cells of L. occidentalis become capacitated to react against egg water after mating.     

Postlarval growth of the freshwater prawn, Macro-brachium australiense Holthuis, 1950 (Crustacea: Decapoda: Palaemonidae)

Growth of newly metamorphosed Macrobrachium australiense was monitored in individually vidually held and group held laboratory animals over 28 weeks. A breeding population was also established in a farm dam and the growth of young juvenile F₁ progeny was recorded for a similar time. Up to 26 moults were recorded in this period and intermoult periods increased with age. Prawns held in groups grew better than those held individually. Reproductive maturity was attained by some females in group held laboratory and farm dam animals within the observation period. Length—weight regression equations were calculated for laboratory maintained prawns, i.e., log W = 0.44+ 2.44 log L; log W = 0.45 + 2.46 log L for individually held males and females, respectively; log W = 0.27 + 2.70 log L for group held males and females.     

Acrosome reaction of Chinese mitten-handed crab Eriocheir sinensis (Crustacea: Decapoda) spermatozoa: Promoted by long-term cryopreservation

The effects of three media, two temperatures, and fourteen durations of cryopreservation from 0 h to 450 d on in vitro acrosome reaction (AR) of spermatozoa in Chinese mitten-handed crab Eriocheir sinensis (Crustacea: Decapoda) were investigated. The spermatozoa of good quality were obtained from spermatophores by a glass homogenizer in an ice-bath and centrifugation at 4 °C. At 0 h, 2 h, 1 d, 3 d, 15 d, 30 d, 60 d, 90 d, 120 d, 150 d, 180 d, 270 d, 360 d, and 450 d of cryo-storage in Ca²⁺-free artificial seawater, 10% (v/v) glycerol, and 5% (v/v) dimethylsulfoxide at − 80 °C and in liquid nitrogen (− 196 °C), the changes in spermatozoal morphology, the time of beginning AR, and the time of maximum percentage of AR were observed. The relationships of the changes on AR presented with the different media, temperatures, and durations of cryopreservation were speculated. In this study, the cryopreserved spermatozoa all underwent AR in less than 1 h of settlement under room temperature while the percentage of AR in the control was only about 4.9%. Meanwhile, cryopreservation shortened both the time of beginning AR (from 30.11 min of the uncryopreserved spermatozoa to 0 min of cryopreserved spermatozoa on the 30th day) and the time of maximum percentage (from 59.88 min of the uncryopreserved spermatozoa to 0 min of cryopreserved spermatozoa on the 60th day). Whereas the effect of media on sperm cell AR was negligible (P > 0.05), the treatments of spermatozoa with short- and long-term cryopreservation resulted in extremely significant differences in the time of beginning AR as well as in the time of maximum percentage of AR (P < 0.01). The present data indicate that cryopreservation for long or short periods can promote the AR of sperm cells in E. sinensis and physiologically affect the ability to capacitate. It may be that the mechanism of AR in this study is the direct promotion of membrane fusion of the acrosomal cap, or destruction of the proteins inhibiting AR and activation of the proteins promoting AR, by cryopreservation. In addition, the results also show that cryopreservation can protect the spermatozoa because AR can occur in almost all sperm cells cryopreserved for less than 15 d.     

鲈幼鱼对饲料中苏氨酸的需要量

以初始体质量(8.00±0.20)g的鲈为实验对象,在海水网箱(1.5 m×1.5 m×2.0 m)中进行为期8周的摄食生长实验,研究鲈对饲料中苏氨酸的需要量。通过在半精制基础饲料中添加晶体L-苏氨酸使饲料中苏氨酸含量分别达到0.75%、1.03%、1.36%、1.67%、2.04%和2.31%,配制成6水平等氮等能饲料(41.83%粗蛋白质,19.68 kJ/g总能)。每种饲料设3个重复,每个重复随机放养30尾鲈。实验采用表观饱食投喂方式,每天投喂2次(06:00和17:30),实验期间水温为26～32℃,盐度为22～28,溶解氧含量在7 mg/L左右。实验结果表明,各饲料处理组成活率(94.4%～98.9%)无显著差异。随着饲料中苏氨酸含量的增加,鲈的增重率显著升高(150.3%～256.9%)(P<0.05),且在1.67%苏氨酸饲料组达到最大值(256.9%),然而,随着饲料中苏氨酸含量的进一步增加,增重率有下降的趋势。鲈的氮累积率随着饲料中苏氨酸含量的增加而显著升高(13.18%～26.36%)(P<0.05),且在1.67%苏氨酸饲料组达到最大值(26.36%),随着饲料中苏氨酸含量的进一步增加,氮累积率有下降的趋势。饲料苏氨酸含量对鲈鱼体粗蛋白、粗脂肪和灰分含量无显著影响。以增重率和氮累积率为评价指标经二次回归分析得出,鲈对饲料中苏氨酸的需要量分别为1.77%和1.88%,占饲料蛋白质的4.21%和4.47%。     

Development of hemolymph prostaglandins assay systems and their concentration variations during ovarian maturation in the kuruma prawn, Penaeus japonicus

Hemolymph prostaglandin (PGF₂ and PGE₂) measurement systems for the kuruma prawn Penaeus japonicus were developed and validated. In these systems, both PGF₂ and PGE₂ in hemolymph obtained from a single prawn were measured. PGs were extracted through reverse-phase C18 cartridge column, separated for isomers of each PG by high-performance liquid chromatography (HPLC), and quantified with a radioimmunoassay (RIA). Detectable range was 6.5–200 and 26–400 pg/tube for the PGF₂ and PGE₂ assays, respectively. Serial dilution of extracted and separated hemolymph PGs (RIA sample) produced a curve parallel to the standard curves in both assays. These assays were able to measure PGs in the RIA samples spiked with the authentic standard. Overall, results indicate that the presently established assay systems can accurately measure hemolymph PGs in female kuruma prawns.

When hemolymph PGF₂ and PGE₂ levels were determined and plotted against the gonado-somatic index (GSI), no correlation was found in either PGs. In terms of ovarian developmental stages, however, the concentration of PGF₂ significantly increased from 66.7 pg/ml at stage I (previtellogenic stage) to 303 pg/ml at stage II (primary vitellogenic stage) and decreased gradually thereafter. The concentration of PGE₂ also increased markedly (4037.7 pg/ml) at stage II and decreased at stage III (secondary vitellogenesis stage). These results indicate that PGs vary in concentration during ovarian development in kuruma prawns.     

Correction of black tiger prawn (Penaeus monodon fabricius) coloration by astaxanthin

After two months of feeding a group of juvenile black tiger prawn (Penaeus monodon) with a commercial feed (CF), their external pigmentation changed from its normal greenish-brown to a light blue. This condition was thought to be induced by a carotenoid deficiency in the feed. Nutritional improvement and hence correction of this condition was attempted by adding 50 ppm astaxanthin (Roche Ltd^®) into one feed (AF) and 5% addition of brown algae (Chnoospora minima) into a second feed (BF). After four weeks of feeding, the prawns fed on AF reverted to their normal greenish-brown pigmentation. The carotenoids present in the BF feed increased carotenoid content in the prawns, but the levels were not high enough to correct the blue coloration. Analysis of carotenoid content in the shells of the groups showed 318%, 57% and 14% increase for AF, BF, and CF respectively, with 70–90% of the total carotenoid being in the form of astaxanthin. Deposition of astaxanthin was higher in the shell than in the flesh, with deposition in the flesh plateauing after two weeks, whilst deposition in the shell increased steadily.

The importance of these findings to the marketing of cultured shrimps is discussed.     

仿刺参铁蛋白ferritin基因的序列分析及表达

通过构建仿刺参cDNA文库,获得铁蛋白全长cDNA序列。该基因序列全长792 bp,5′非翻译区长133 bp,开放阅读框长522 bp,编码173个氨基酸,3′UTR长137 bp;预测蛋白分子量为20 ku。5′UTR具有一个高度保守的铁离子应答元件。仿刺参ferritin氨基酸序列具备脊椎动物ferritin的亚铁氧化酶活性中心所特有的保守结构。该序列与海参的同源性最高,达84%,与其它无脊椎动物如:海星、鲍、牡蛎、海葵、线虫、小龙虾和果蝇的同源性为74%~34%;与脊椎动物ferritin重链亚基同源性高于轻链亚基。系统进化分析表明,仿刺参的ferritin与大部分无脊椎动物聚为一支。利用半定量RT-PCR检测,ferritin mRNA在仿刺参未受精卵、受精卵、多细胞期、囊胚期、原肠期、小耳状幼体、中耳状幼体、大耳状幼体、樽型幼体、五触手幼体、稚参11个发育阶段和幼参的体壁、体腔细胞、肠道和呼吸树中均表达。Quantitative real-time PCR结果显示, ferritin mRNA在未受精卵至原肠期表达量低,从小耳状幼体至稚参表达量显著增高;在幼参的不同组织中,ferritin mRNA在呼吸树中的表达量显著低于其他3种组织;幼参注射LPS后,4种组织中ferritin mRNA表达量与注射前无显著差异。     

褐藻羊栖菜繁殖生物学特征和生活史流程的补充研究

以温州市洞头区人工养殖羊栖菜和野生羊栖菜为研究样本,详细描述了羊栖菜成熟孢子体有性生殖,假根与侧生枝无性生殖的繁殖生物学特征和生活史流程。详细记录了羊栖菜卵、精子和受精卵的减数分裂和有丝分裂过程,假根、茎(主茎和侧生茎)、叶(气囊)和生殖托(雌托和雄托)等4类器官生长、发育和分化过程,胚、幼孢子体和成熟孢子体形态结构特征。在此基础上对现有羊栖菜繁殖生物学特征及生活史流程进行了补充和修正,增加了羊栖菜侧生枝无性生殖和羊栖菜幼孢子体期有性生殖的相关内容,并重新绘制了羊栖菜生活史流程图。该研究结果将为开展羊栖菜养殖生态学研究,深层次解析羊栖菜生殖节律、种群繁衍与环境关系奠定基础。     

Regional size, age and growth differences of red grouper (Epinephelus morio) along the west coast of Florida

Red grouper (Epinephelus morio) were collected from the west coast of Florida, the central area of fishery harvest in U.S. waters, by fishery-dependent sources during 2000–2005. The west Florida shelf was divided into two regions: north (capture locations ≥28°N latitude) and south (capture locations <28°N latitude). Significant differences were found for age, length, and size-at-age by region and by gear; red grouper from the north were significantly younger and smaller on average than those from the south. Regional differences were also noted with respect to age progression; year class trends were only detected in the north. The 1996 year class dominated the landings in 2000–2001 (ages 4 and 5) and the 1999 year class dominated in 2004–2005 (ages 5 and 6). Regional data were fit to a size-modified von Bertalanffy growth model indicating smaller asymptotic length (L_∞) and faster growth rate (k) in the north (north: L_∞ = 800 mm, k = 0.23 mm year⁻¹, t₀ = 1.12; south: L_∞ = 863 mm, k = 0.15 mm year⁻¹, t₀ = 0.05). Mortality estimates derived from catch curves resulted in higher total and fishing mortality in the north for both gears. Alternative explanations of regional differences likely depend on nursery delineation and correlation to periodic environmental events such as red tides and hurricanes; all possibly important factors based upon anecdotal information. Nevertheless, our finding of regional demographic differences in red grouper from the west coast of Florida suggests a more complex population spatial structure for red grouper.     

Further evidences of deep-sea recruitment of Aristeus antennatus (Crustacea: Decapoda) and its role in the population renewal on the exploited bottoms of the Mediterranean

Data were collected during four trawl surveys (April, May, June and September 2006) in the north-western Ionian Sea (Eastern Mediterranean) with the aim of investigating the recruitment and population structure of Aristeus antennatus and evaluating whether population processes on bottoms deeper than 800 m could have a role to the renewal of the exploited population on fishing grounds, above 800 m. Samples were taken randomly between 400 and 1200 m using a professional fishing vessel equipped with an experimental otter trawl net, with 20 mm stretched mesh size in the cod-end. No variation in density index was shown across the whole depth range while the negative trend of biomass index with depth was due to the increasing proportion of small individuals and males. The recruitment was observed on a wide depth range with an increasing frequency of occurrence at greatest depths. It seems to occur as a discrete phenomenon. A significant bigger-shallower phenomenon was detected in the female population indicating upwards displacement from virgin grounds to fishing ones. The exploited population mostly consists of large females. Mating and reproduction also occur in deep waters. The contribution to the renewal of the fishing population by the virgin one seems to be mostly due to the recruitment on unexploited deep areas and displacement of individuals from these areas to fishing ones. The deep waters act as a refuge area mostly for the recruitment of A. antennatus and, to lesser extent, for spawners. On the basis of all these results the authors discuss the optimum exploitation conditions often detected for this shrimp throughout the Mediterranean highlighting the fact that, since the exploited population mostly consists of reproductive females, conditions of “recruitment overfishing” might occur in A. antennatus contrary to other Mediterranean demersal resources.     

Brachionus vs Artemia duel: Optimizing first feeding of Upogebia pusilla (Decapoda: Thalassinidea) larvae

Larval rearing of many marine organisms is dependent on the availability of live food. The aim of this study was to optimize larval first feeding for the mud shrimp Upogebia pusilla, by comparing the effectiveness of the two most commonly used live feeds: Brachionus plicatilis and Artemia sp. nauplii. Survival, larval duration, molt synchronism and megalop size were compared using five feeding treatments: Artemia from zoea I to IV (B0), Brachionus during zoea I and Artemia from zoea II to IV (B1), Brachionus during zoea I and II and Artemia during zoea III and IV (B2), Brachionus from zoea I to III and Artemia during zoea IV (B3) and Brachionus from zoea I to IV (B4). The proportion of larvae that reached the megalop stage was 0.00% in treatment B0, 3.33% in treatment B1, 33.33% in treatment B2, 66.67% in treatment B3 and 76.67% in treatment B4. Larvae fed on rotifers until zoea III or zoea IV stages had a higher survival but no differences were found either in time to reach megalop or in megalop size. This study demonstrates that rotifers are essential for the survival and development of U. pusilla early larval stages but that rotifers can be successfully replaced by Artemia nauplii in the zoea IV stage.     

物理法改善鱼肉蛋白功能特性研究概述

为探究超声预处理对鱼糜凝胶特性的影响,并进一步阐明凝胶特性变化与蛋白二级结构之间的相关性规律,本实验对鲢鱼糜进行超声预处理,探究超声过程中 (0~50 min)凝胶强度、色泽、水分分布、微观结构、蛋白分子量以及二级结构的变化。结果显示,超声波处理10 min,鱼糜凝胶强度显著提高,束缚水的能力增强,凝胶网络结构更为致密;但随超声时间的增加,凝胶劣化,不易流动水转变为自由水。凝胶电泳及红外光谱结果显示,随着超声时间增加,鱼糜中蛋白未发生降解或聚集;二级结构中α-螺旋相对含量降低,β-折叠、无规则卷曲和β-转角相对含量增加。通过皮尔逊相关性分析可知,超声预处理促进蛋白质结构展开,更利于鱼糜在加工过程中形成均匀的网络结构,提高凝胶强度以及保水能力。研究表明,适量超声预处理可以通过改变鱼糜蛋白结构,改善鱼糜凝胶特性。本研究可为超声波技术在鱼糜制品加工中的应用提供参考。     

Effect of prepared diet on the induction of precocious maturation in Pleoticus muelleri Bate (Crustacea, Penaeoidea)

Experiments were conducted comparing the reproductive performance of ablated female shrimp Pleoticus muelleri fed either a fresh frozen diet (ND) (clam, prawn, squid) and the same diet supplemented with 15% of an artificial diet (SD) (54% protein, 13% lipid, 7% moisture). The shrimp were maintained in 3500‐L tanks (12 h light‐12 h dark photoperiod, temperature 18–20°C, pH 7, salinity 31 g L^?1, ammonium <0.2 mg L^?1, and sea water exchanged at 100% per day) for 45 days. Significant differences in weight gain were determined between ablated females fed ND (37.2%) and SD (26.4%). No significant differences in the average duration of the intermolt period (19±2 days) were recorded. Females fed SD showed signs of maturation 20 days after ablation, with spawning occurring 1 day later, as long as ablated females fed ND reached maturation 4 weeks after did not occur. The number of eggs per spawn varied from 136 000 to 345 000; the percentage viability ranged between 39.3% and 99.3% with an average of 78.5%. The results indicated that a suitable formulated diet together with eyestalk ablation promotes maturation of P. muelleri in captivity.     

几种重要水产品活体运输技术研究

为研究虾苗运输中干露胁迫对克氏原螯虾(Procambarus clarkii)的影响,实验在(20±1) ℃和相对湿度 (relative humidity, RH) (50±5)% 的条件下,对不同干露时间(6 h、12 h、18 h和24 h)和再入水时长(1 h、6 h和12 h)幼虾的抗氧化应激能力和成活率进行了研究。结果显示,幼虾的干露时间不宜超过18 h,第24小时死亡率达到53.3%,入水阶段无幼虾死亡。干露阶段,总抗氧化能力(T-AOC)无显著变化(P>0.05);超氧化物歧化酶(SOD)活力显著降低;过氧化氢酶(CAT)活力随时间逐渐升高;丙二醛(MDA)质量摩尔浓度在干露第24 小时后达到峰值(P<0.05);血糖和肌肉乳酸浓度随干露时间显著升高(P<0.05)。再入水时,SOD和CAT呈波动性变化,无明显规律;入水12 h后MDA均恢复到对照组水平;T-AOC一直维持较高水平。6 h和12 h组入水1 h后乳酸即恢复到对照组水平,各处理组的血糖始终高于对照组(P<0.05)。研究表明,CAT和SOD对干露胁迫的反应更为敏感,可作为克氏原螯虾干露阶段的免疫指标。MDA、血糖和肌肉乳酸浓度可以反映幼虾再入水时的健康状况。

     

The effects of ozone and probiotics on the survival of black tiger shrimp (Penaeus monodon)

Total ozone production (TOP) from an ozonator, residual ozone concentration (ROC) in water, and the effects of ozone with or without probiotic supplemented feeds on bacterial growth, and shrimp (Penaeus monodon) survival were investigated. Minimal effective ROC to inhibit 3 log units of Vibrio harveyi D331 for 6 h and 2 log units of Bacillus S11 for 9 h was 0.38 g O₃/l of ROC from 5-min ozonation. Shrimp postlarvae exposed to 0.34–0.50 mg O₃/l ROC (8-h ozonation) caused loss of balance, immobility and destruction of gill lamellar epithelium. In vivo treatment of juvenile P. monodon reared on probiotic feed for 1 month revealed that 0.35 mg O₃/l ROC (30-min ozonation) effectively inhibited 3 log units of V. harveyi D331 for 24 h. At this ROC dosage, there was no effect either on shrimp or on intestinal probiotics (Bacillus S11). Shrimp survival from probiotic treatment, coupling with ozonation, increased significantly (P≤0.05) compared with controls.     

Sperm production in the red claw crayfish Cherax quadricarinatus (Decapoda, Parastacidae)

The objective of this study is to evaluate the effect of body size, temperature and annual cycle on sperm production in Cherax quadricarinatus. Sperm count and sperm mortality were estimated, the reproductive system was weighted, and macro and microscopical analysis of the testes and vasa deferentia were conducted. Sperm count and weight of the reproductive system are strongly related to male size, in contrast to sperm mortality. The spermatophore structure presented macro and microscopical differences between sizes. Males higher in size have more adherent spermatophores. This species has a reproductive cycle related to sperm production. Sperm count and weight of the vasa deferentia rise in summer, while the weight of the testes increases in winter. During the spring, the sperm cord presents a higher density than in other seasons. The temperature seems to affect sperm production being 27 and 29 °C the best assayed conditions. The present results are relevant information to obtain the best sperm viability selecting male size, season of sampling and the best temperature for the reproductive stock and future assays of spermatophore cryopreservation for this species aquaculture.     

仿刺参幼参对饲料中维生素B6需求量的研究

为研究仿刺参幼参对维生素B_6的最适需求量,配制维生素B_6实测含量分别为1.23、5.29、9.35、17.47、33.71和66.17 mg/kg的6组实验饲料D1、D2、D3、D4、D5和D6组,饲喂初始体质量为(12.23±0.11) g的仿刺参幼参12周。结果显示,①随着维生素B_6含量的增加,实验仿刺参的增重率、特定生长率均先升后降,在D5组达到最高值;体壁粗蛋白含量先升后降,D6组显著低于其他组;D1组粗脂肪含量显著低于其他组;②体腔液中葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、乙酰辅酶A羧化酶及一氧化氮合酶活性均先升后降,D6组显著低于其他组;③随着饲料中维生素B_6含量的增加,肠道蛋白酶及淀粉酶活性显著升高,纤维素酶活性显著降低,肠壁厚度及绒毛长度均显著升高。以增重率为评价指标,经一元二次回归分析得出仿刺参幼参饲料中维生素B_6的适宜需求量为45 mg/kg。     

Genetic characterizations of Mactra veneriformis (Bivalve) along the Chinese coast using ISSR-PCR markers

The genetic structure of seven different populations of the surf clam Mactra veneriformes along the coast of China was investigated by Inter-Simple Sequence Repeats (ISSR) fingerprinting of 210 individual clams. Of the 240 ISSR loci tested, 235 (97.9%) were polymorphic. We found a clear tendency for higher F_ST values and lower gene flow levels between the populations with increasing geographical separation. The seven different geographic populations can be divided into three subgroups: Liaoning, Qingdao/Lianyungang, and Ningbo based on the UPGMA dendrogram of Nei's genetic distance. These results indicated that isolation of geographic distance played an important role in population differentiation.