糙海参胚胎和幼体发育的形态观察

为提高糙海参育苗技术,研究描述了糙海参从受精卵发育到稚参的形态变化,在显微镜下测定了受精卵、胚胎和幼体的大小,确定了糙海参胚胎发育的过程。结果表明,通过利用阴干、流水刺激法对成熟亲参进行人工催产,得到大量的受精卵,其受精率为90%以上。糙海参胚胎和幼体发育可分为受精卵、卵裂期、囊胚期、旋转囊胚期、原肠期、初耳状幼体、中耳状幼体、大耳状幼体、樽形幼体和稚参等阶段;在平均水温29℃,平均盐度34条件下,糙海参受精卵经3 h发育形成囊胚,4 h进入旋转囊胚期,5 h进入原肠期,19 h完成胚胎发育变态为耳状幼体;并经过7 d的生长与发育进入樽形幼体;第15天变态为稚参。观察发现,多精入卵现象则会导致胚胎发育不正常,最终使胚胎发育停止并死亡。在糙海参幼体发育过程中,大耳状幼体期幼虫臂的大小及其球状体的形成均可作为判断其幼体发育健康状况的重要指标:幼虫臂越大,球状体出现率越高,其幼虫的变态率和成活率也越高。对比发现,位于糙海参尾部的突出结构——尾突,为仿刺参和新西兰刺参所没有,这一结构差异同时导致了其骨片位置也不同;并且,糙海参胚胎和幼体与其他种类的海参在发育时间上存在一定差异。     

黄河三角洲地区刺参胚胎及幼体发育形态观察

为提高刺参育苗技术,通过人工催产,对黄河三角洲地区养殖刺参的胚胎和幼体发育过程进行了研究,观察了刺参从受精卵发育到稚参的形态变化,在显微镜下测定了受精卵、胚胎和幼体的大小。试验结果:利用阴干流水刺激法对成熟亲参进行人工催产,其受精率达95%以上;在水温22℃、盐度30、pH 7.8的条件下,受精卵经7～9 h发育成囊胚,12 h进入旋转囊胚期,22 h进入原肠期,42～48 h完成胚胎发育,变态为耳状幼体,经10 d的生长发育进入樽形幼体期,经16 d变态为稚参。结果表明,刺参幼体的健康状况与其水体腔和胃部的生长发育状况密切相关,水体腔和胃部发育良好,有利于幼体的变态,提高存活率。     

花刺参胚胎和幼体发育的形态观察

为提高花刺参育苗技术,对花刺参自受精卵发育到稚参的形态变化进行了观察研究,详细描述了胚胎发育各个时期的形态特征。通过阴干、流水刺激法对成熟亲参进行人工催产,得到大量的受精卵。在水温27~30℃及盐度33.0~35.0室内水泥池进行花刺参幼体培育。花刺参受精卵经5h发育成囊胚,7h进入旋转囊胚期,9h进入原肠期,31h完成胚胎发育变态为耳状幼体,并经10d的生长发育进入樽形幼体,第20d变态为稚参。由结果可知,花刺参的幼体的健康状况与其水体腔和胃部生长发育状况密切相关,水体腔和胃部发育良好,花刺参的健康状况则佳,有利于幼体的变态与存活。     

几种单胞藻饵料对刺参耳状幼体烂胃的影响

刺参(Stichopus japonicus)属于楯手目(Aspido- chirota)、刺参科(Stichopodidae)、刺参属(stichopus)。刺参有明显的变态生活史,由受精卵卵裂经囊胚期→原肠期→小耳状幼虫→中耳状幼虫→大耳状幼虫→樽形幼虫→五触手幼虫→稚参→幼参→成参(廖玉麟, 2001)。其中小耳状幼体、中耳状幼体、大耳状幼体统称为耳状幼体。耳状幼体是刺参整个生命周期中短暂而唯一的一个浮游生活阶段,幼体浮游一般需10天左右,之后变态为樽形幼体改为底栖生活,浮游时期幼体     

蚂蚁岛海域刺参浮游幼体发育过程研究

该试验研究了蚂蚁岛海域周围水质环境因子和刺参(Apostichopus japonicus)浮游幼体的发育过程、丰度以及分布情况,结果表明:刺参浮游幼体的生长发育周期大约为15 d,大致变态过程可分为:受精卵—囊胚期—原肠期—小耳状幼体—中耳状幼体—大耳状幼体—樽形幼体—五触手幼体—稚参;刺参浮游幼体总丰度的最大值为每立方米8 756.50个;刺参浮游幼体的平均丰度为每立方米139.86～327.35个;东、西蚂蚁岛南部海域刺参浮游幼体明显高于北部。     

黄河三角洲刺参苗种繁育技术

为摸清黄河三角洲地区刺参繁育特性,提高刺参育苗技术,在显微镜下观察、测量了胚胎和幼体的大小及胚胎发育过程。结果表明,在水温22±0.5℃,盐度(30±0.5)‰、pH 7.8条件下,利用阴干、流水刺激法可成功进行人工催产。受精卵经42h完成胚胎发育变态为初耳状幼体,7d左右发育为大耳状幼体,体长为760.0~970.0μm;10d左右生长发育为樽形幼体,16d变态为稚参。     

刺参主要卵黄蛋白MYP1、MYP2在不同幼体发育阶段、成体组织及温度胁迫条件下的相对定量表达

为探讨温度胁迫对刺参基因表达的影响,从已构建的刺参耐寒基因筛选cDNA文库中选取差异基因主要卵黄蛋白(major yolk protein,MYP):MYP1和MYP2,利用实时荧光定量PCR研究了其在刺参胚胎发生和个体发育9个阶段(受精卵、囊胚期、原肠期、小耳状幼体、中耳幼体、大耳幼体、樽形幼体、五触手幼体和稚参)、成体7种组织(呼吸树、体腔液、肠、纵肌、体壁、雄性性腺和雌性性腺)、长时温度胁迫(20℃、4℃,30 d)和低温短时胁迫(7℃、4℃、1℃、-2℃,12 h)成体肠组织中的表达量。结果发现,MYP1和MYP2表达模式基本相同:(1)在胚胎发育阶段樽形幼体时期开始表达,随后的阶段持续表达,到稚参阶段表达量最高;(2)在7种组织中,肠中的表达量最高,在体腔液中基本不表达,其余组织中均有表达;(3)在长时(30 d)温度胁迫下,肠组织中MYP表达量由高到低依次为4℃、12℃、20℃,且在4℃的表达量为20℃的6倍左右;(4)在低温短时胁迫(1℃、-2℃,12 h)条件下,肠组织中MYP表达量受到显著抑制,约为常温条件下的一半。研究表明,MYP在刺参胚胎发育的樽形幼体阶段开始合成,成体合成部位主要为肠,水温过低会抑制其表达。     

不同发育期的幼体和不同规格刺参消化道中四种消化酶的活性

测定了刺参耳状幼体、樽形幼体、五触手幼体、稚参、3种规格幼参和成参[体质量分别为(82.349±1.532)g、(147.639±2.031)g和(179.866±1.609)g]消化道中蛋白酶、淀粉酶、褐藻酸酶和纤维素酶的活性。试验结果表明,耳状幼体至樽形幼体蛋白酶活力下降,之后,快速上升。稚参蛋白酶活性显著高于幼体。相反,从耳状幼体至稚参,淀粉酶的活力逐渐升高,至稚参达最大值。幼体期褐藻酸酶逐渐上升,至五触手幼体达峰值,然后下降,稚参降至最小值。幼体纤维素酶活性一直很低,变化不大,但稚参的纤维素酶活性增强。幼参和成参的蛋白酶和淀粉酶活力随着个体发育而逐渐上升,而褐藻酸酶的活性却随着个体的生长而缓慢下降,稚参达最小值。成体的纤维素酶活性低而稳。4种消化酶中,蛋白酶活力最高,纤维素酶最低。     

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。     

刺参樽形幼虫期高死亡率原因浅析

刺参樽形幼虫是刺参发育过程中继耳状幼虫以后的一个变态期。刺参在进入樽形幼虫期后,原来的纤毛运动逐渐减弱。个体的活动能力也因此而逐渐减弱。个体由原来的浮游生活逐渐过渡到底栖生活,这一时期由于幼体     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.