Carbon nanotube enhanced label-free immunosensor for amperometric determination of oocyte maturation-inducing hormone in fish

Maintaining high-quality fish eggs stably and efficiently is important for aquaculture. We developed a label-free immunosensor system for measuring 17,20β-dihydroxy-4-pregnen-3-one (DHP). DHP is suddenly secreted before ovulation as a maturation-inducing hormone in fish, and therefore, DHP levels are an indicator for predicting ovulation. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry (CV). For biomolecular immobilization on the surface of sensing electrode, Au electrode, we used self-assembled monolayers of thiol-containing compounds to fix anti-DHP immunoglobulin. In addition, we used a single-walled carbon nanotube to improve sensitivity. Using this electrode, we were able to determine the CV signal change caused by the antigen–antibody complex. The proposed immunosensor system showed a linear correlation (correlation coefficient: 0.9827) between the anodic peak current of the CV and the DHP level in range from 15.6 to 50,000 pg ml^?1. The sensor system was then applied to monitor DHP of goldfish (Carassius auratus). Blood plasma of fish was collected every 3 h after administering a DHP inducer. In the measurement, the anodic peak current of the CV showed distinct changes depending on DHP levels in the blood plasma. A good relationship was observed between DHP levels determined by our proposed system and the conventional method (correlation coefficient: 0.9351).     

Development of a label-free immunosensor system for detecting plasma cortisol levels in fish

Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. In the present study, we describe a novel label-free immunosensor for detecting plasma cortisol levels. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry. For the immobilization of the antibody on the surface of sensing electrode, we used a self-assembled monolayer of thiol-containing compounds. Using this electrode, we detect the CV signal change caused by the generation of antigen–antibody complex. The immunosensor showed a response to cortisol levels, and the anodic peak value linearly decreased with a correlation coefficient of 0.990 in diluted plasma. The specificity of the label-free immunosensor system was investigated using other steroid hormones, such as 17α, 20β-dihydroxy-4-pregnen-3-one, progesterone, estriol, estradiol, and testosterone. The specific detection of cortisol was suggested by a minimal change from ?0.32 to 0.51 μA in the anodic peak value of the other steroid hormones. The sensor system was used to determine the plasma cortisol levels in Nile tilapia (Oreochromis niloticus), and the results were compared with those of the same samples determined using the conventional method (ELISA). A good correlation was obtained between values determined using both methods (correlation coefficient 0.993). These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish plasma samples.     

Molecular endocrinology of oocyte growth and maturation in fish

Pituitary gonadotropins (GTHs) are of primary importance in triggering oocyte growth and maturation. However, the actions of GTHs are not direct, but are mediated by the ovarian production of steroidal mediators of oocyte growth (estradiol-17β) and maturation (maturation-inducing hormone, MIH; 17α,20β-dihydroxy-4-pregnen-3-one, 17α,20β-DP in salmonid fishes; 17α,20β,21-trihydroxy-4-pregnen-3-one, 20β-S in sciaenid fishes). It is established that production of estradiol-17β and 17α,20β-DP by salmonid ovarian follicles occurs via the interaction of two cell layers, the thecal and granulosa cell layers (two-cell type model). A distinct shift in the salmonid steroidogenesis from estradiol-17β to 17α,20β-DP occurs in the ovarian follicle layer immediately prior to oocyte maturation. It is possible that this shift is a consequence of dramatic changes in the expression of the genes encoding various steroidogenic enzymes. As an initial step to address this question, we have isolated and characterized the cDNAs encoding a number of ovarian steroidogenic enzymes including the rainbow trout cholesterol side-chain cleavage cytochrome P-450, 3β-hydroxysteroid dehydrogenase (HSD), 17α-hydroxylase/17,20 lyase cytochrome P-450, aromatase cytochrome P-450 cDNAS as well as the pig 20β-HSD cDNA. Estradiol-17β stimulates the hepatic synthesis and secretion of a yolk precursor, vitellogenin. Vitellogenin is then transported to the ovary where it is selectively taken up into the oocyte by a receptor-mediated process involving specific cell-surface receptors. Estradiol-17β was also shown to induce the synthesis of egg membrane proteins in the liver. The maturation-inducing action of 17α,20β-DP and 20β-S is through the binding to the oocyte plasma membrane. This initial MIH-surface interaction is followed by the formation of the major mediator of MIH, maturation-promoting factor (MPF). We have purified MPF from mature oocytes of carp. Carp MPF consists of two components: the homolog of the cdc2⁺ gene product of fission yeast (p34^cdc2) and cyclin B. The cdc2 kinase protein is present in immature oocytes as well as in oocytes induced to mature by 17α,20β-DP treatment, while cyclin B proteins can be detected only in mature oocytes. Addition of bacterially expressed goldfish cyclin B to the extracts of immature goldfish oocytes induced MPF activation. These results suggest that the appearance of cyclin B protein is a crucial step for 17α,20β-DP-induced oocyte maturation in fish.     

Specificity of slide agglutination test for detecting bacterial fish pathogens

The usefulness of the slide agglutination assay for a rapid diagnosis of fish diseases was evaluated using a total of 80 pathogenic bacteria and environmental isolates belonging to the genera Vibrio (28), Pasteurella (5), Aeromonas (26), Yersinia (6), Edwardsiella (8), Pseudomonas (6) and Lactobacillus (1). Selected strains from each bacterial group were used as antigens for rabbit immunization.The specificity of the reactions between whole-cell antigens and the different whole-cell antisera varied according to the bacterial group analyzed. In the case of V. anguillarum, it was found that by using two sera against serotypes 1 and 2, it was possible to detect most of the strains causing vibriosis regardless of their origin. Cross-reactions were not detected between either both serotypes or with other pathogenic or environmental vibrios.In general, the whole-cell antisera from P. piscicida, A. salmonicida, Y. ruckeri and E. tarda detected all the strains within each species. However, a more heterogeneous pattern was exhibited by the motile Aeromonas species. The majority of A. hydrophila and A. sobria isolates did not agglutinate with the anti A. caviae serum, indicating that this antiserum is not adequate for identifying all motile Aeromonas strains.The antiserum against A. salmonicida subsp. masoucida displayed weak cross-reactions with some A. salmonicida subsp. salmonicida and A. hydrophila whole-cell antigens. Strong cross-agglutinations occurred also between types I and II of Y. ruckeri as well as between E. tarda and E. ictaluri. These cross-reactions were eliminated by using the respective thermostable somatic “O” antigens, which indicates that common thermolabile antigens are shared by these strains.The slide agglutination test, using anti whole-cell sera and two antigen preparations (whole-cells and “O” antigen) for each strain, is useful for a rapid detection of fish pathogens, with the additional advantage of its applicability for serotyping studies. Furthermore, some cross-reactions using the whole-cell antigens are reliable in identifying a wide range of strains causing similar diseases with a small number of anti whole-cell sera.     

All-fish gene constructs for growth hormone gene transfer in fish

In order to develop all-fish expression vectors for microinjection into fertilized fish eggs, we have prepared the following constructs: rainbow trout metallothionein a/b and the gilthead seabream growth hormone cDNA (ptMTa-gbsGHcDNA, ptMTb-gsbGHcDNA), carp β-actin gilthead seabream GH cDNA (pcAβ-gsbGHcDNA). The inducible metallothionein promoters a and b were cloned from rainbow trout, and the constitutive promoter β-actin was isolated from carp. The metallothionein promoters were cloned by using the PCR technique. The tMTa contains 430 bp, while the tMTb contains 260 bp (Hong et al. 1992). These two promoters were introduced to pGEM-3Z containing the GH cDNA of Sparus aurata to form ptMTa-gsbGH and ptMTb-gsbGH, respectively. The carp cytoplasmic β-actin gene was chosen as a source for isolating strong constitutive regulatory sequences. One of these regulatory sequences in pUC118 was ligated to GH cDNA of S. aurata to form the pcAβ-gsbGHcDNA. Expression of the constructs containing the metallothionein promoters was tested in fish cell culture and was found to be induced effectively by zinc. The ptMTa gsb-GH cDNA construct was microinjected into fertilized carp eggs, and integration in the genome of carp was detected in the DNA isolated from fins at the age of two months.

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Résumé Afin de développer des vecteurs d'expression de poisson, entièrement homologues, destinés aux microinjections dans des oeufs fertilisés, les constructions suivantes ont été préparées: promoteurs de la metallothionine, a ou b, de truite arc-en-ciel d'une part, et promoteur de l'actine β de carpe d'autre part, associés à l'ADNc de l'hormone de croissance de daurade royale (ptMTa-gsbGH cDNA, ptMTb-gsbGH cDNA, et pcAβ-gsbGH cDNA). Les promoteurs de la metallothionine ont été clonés en utilisant la technique de la RCP. La tMTa comprend 430 pb. tandis que la tMTb en comprend 260 (Hong et al. 1992). Ces deux promoteurs ont été insérés dans pGEM-3Z qui contenait l'ADNc de GH de Sparus aurata, pour former, respectivement, ptMTa-gsbGH et ptMTb-gsbGH. Le gène de l'actine cytoplasmique β de carpe été choisi comme source d'isolement de séquences régulatrices fortement constitutives. Une de ces séquences régulatrices a été liguée à l'ADNc de GH de S. aurata dans pUC118, pour réaliser la construction pcAβ-gsbGH cDNA. L'expression des constructions contenant les promoteurs de la metallothionine a été tentée dans des cultures de cellules de poisson, où elle a été effectivement induite par le zinc. La construction ptMTa-gsbGH cDNA a été microinjectée dans des oeufs fertilisés de carpe. Son intégration dans le génome de carpe a pu être détectée dans l'ADN isolé à partir de nageoires d'animaux agés de 2 mois.

     

Development of a time-resolved fluoroimmunoassay for octopus gonadotropin-releasing hormone

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for octopus gonadotropin-releasing hormone (oct-GnRH) to determine the profiles of oct-GnRH peptide levels in cephalopods. The sensitivity and the intra-assay and inter-assay coefficients of variation were 4.9 pg/well and 6.8 (n = 10) and 2.7% (n = 5), respectively. Anti-oct-GnRH antibody was tested on all known forms of GnRH and found to cross-react with lamprey GnRH-II (27.1%), annelid GnRH (3.36%), tunicate GnRH-I (0.92%), dogfish GnRH (0.51%), and scallop Patinopecten yessoensis GnRH (0.05%). The displacement curve obtained for serially diluted brain extracts of three cephalopods, the spear squid Loligo bleekeri, swordtip squid Loligo edulis, and North Pacific giant octopus Octopus dofleini, paralleled the oct-GnRH standard curve. The presence of oct-GnRH in the central nervous system (CNS) of these cephalopods was further examined by a combination of reverse-phase high performance liquid chromatography and oct-GnRH TR-FIA. CNS extracts from these cephalopods showed peaks with retention times similar to that of synthetic oct-GnRH. These results indicate that this novel oct-GnRH TR-FIA is widely applicable for oct-GnRH measurement in cephalopods.     

鱼用常用催产剂的性能及使用剂量

一、鱼用绒毛膜促性腺激素（ＨＣＧ） （１）用途：促进各种养殖鱼类产卵、排精。 （２）使用剂量（以下均为雌亲鱼剂量，雄亲鱼减半）。 ①鲢、鳙鱼亲鱼：ＨＣＧ８００－１２００ＩＵ／ｋｇ． ②草鱼亲鱼：ＨＣＧ８００ＩＵ／ｋｇ＋垂体２ｍｇ／ｋｇ（１ｍｇ垂体相当于１个０．５ｋｇ重的鲤鱼垂体）。 ③青鱼亲鱼：分两次注射，第一次注射垂体０．５ｍｇ／ｋｇ，间隔２０小时再第二次注射ＨＣＧ２４００－３００ＩＵ／ｋｇ． ④团头鲂亲鱼：ＨＣＧ１６００—２４００ＩＵ／ｋｇ． ⑤泥鳅： ＨＣＧ２５０－５００ＩＵ／尾或ＨＣＧ１５～２０…     

鲁氏耶尔森菌qPCR快速检测方法的建立和应用

鲁氏耶尔森菌(Yersinia ruckeri)是导致虹鳟(Oncorhynchus mykiss)肠炎红嘴病的病原。本研究以鲁氏耶尔森菌毒力因子rup A基因为靶标设计1对特异性引物,以含有rup A基因部分序列的重组质粒为标准品构建标准曲线,优化建立检测鲁氏耶尔森菌的SYBR Green I实时荧光定量PCR检测方法,并对腹腔注射鲁氏耶尔森菌菌悬液的虹鳟肝、肾、脾、血液样品进行检测。结果显示,设计的引物具有良好的种间特异性,标准曲线线性方程为y=–3.3766x+40.012(R~2=0.9958);最低检测限为57 copy/μL,较常规PCR的灵敏度高出约100倍。应用检测结果表明,本方法可准确检测被鲁氏耶尔森菌感染的虹鳟样品。研究表明,所建立的qPCR方法具有特异、灵敏、快速、定量的优点,可用于快速诊断虹鳟肠炎红嘴病早期病症及定量检测鲁氏耶尔森菌。     

用酶联免疫吸附受体法检测鱼类生长激素的生物活性

根据激素一受体反应的原理，结合酶联免疫吸附测定法（ＥＬＩＳＡ）和放射受体测定法（ＲＲＡ）的优点，应用我们纯化的Ａ昌鱼生长激素（ｇｃＧＨ）和大鳞大马哈鱼生长激素（ｓＧＨ）及其特异抗体，采用鱼类肝细胞膜受体制剂，首次建立了测定鱼类ＧＨ生物活性的酶联免疫吸附受体测定法（ＥＬＩＳＡ－ＲＡ）。此法检测草鱼ＧＨ的灵敏度达０．０６３￣０．１２５ｕｇ／ｍｌ，检测大马哈鱼ＧＨ与草鱼肝膜受体结合的灵敏度达０．２５ｕｇ     

鱼类铁调素（Hepcidin）的研究进展

铁调素是一类具有调节铁代谢功能的抗菌肽,在鱼类中广泛存在。文章介绍了Hepcidin的基本结构、生理功能,并总结了鱼类Hepcidin基因的克隆和表达、抗菌活性、铁代谢活性的研究进展,以期为鱼类Hepcidin的开发和利用奠定一定的基础。     

深水网箱多波束水声鱼群状态监测仪的改进设计

针对离岸深水网箱养殖的鱼群安全问题,采用多波束水声技术监测鱼群状态的方法.结合信号处理技术、通用分组无线业务(GPRS)无线传输技术及串口通信技术,开发了一套多波束水声鱼群状态监测仪.监测仪完成1次网箱扫描只需5s;实现了监测数据的即采即发功能,提高了系统的实时性;改进了电路结构,降低了系统复杂度,减小了系统体积,降低...     

气相色谱-质谱法检测鱼肉中MS-222残留

本文建立了鱼肉中MS-222残留GC-MS检测方法.鱼肉样品经乙腈提取,氮吹浓缩,盐酸溶液引导MS-222电离,Waters Oasis MCX固相萃取柱净化后,气相色谱-四极杆质谱检测.方法检出限为2.5 μg·kg-1、定量限为5.0 μg·kg-1;0.0025～1.0 μg·mL-1范围内线性关系良好（R≥0.9996）;MS-222浓度范围在5.0～ 100.0 μg·kg-1的鱼肉加标样,日内和日间平均回收率为78.4％～91.2％,相对标准偏差为3.62％ ～9.49％.结果表明,该检测方法适用于低浓度水平鱼肉中MS-222残留检测.     

Intensive rearing system for fish larvae research: I. Marine fish larval rearing system

Larvae nutrition and in general larvae culture is considered to be the ‘bottle neck’ for marine finfish culture. Fish larvae rearing experiments investigating nutritional factors or rearing protocols are carried out in various systems, from small beakers to very large commercial tanks, making it difficult to compare data across systems.

A continuous supply of live or dry feeds and a controlled environment, i.e. temperature, filtration, photoperiod, oxygen and pH, are essential for any experimental or commercial system. These environmental factors are best controlled automatically in order to minimize variations between tanks. However, only a few automatic systems have been developed for marine finfish hatcheries.

An experimental larval rearing system was developed to reduce variability amongst tanks (due to manual feeding and other parameters) and enhance control of environmental parameters while reducing the workload. The system includes 24 conical tanks with the option of either an up-welling or bottom draining flow through water delivery system. The inlet water passes through a gas exchange column that saturates the water with dissolved oxygen and stabilizes the pH. The system was originally designed for nutritional experiments using formulated feeds. The use of an up-welling water inlet method extends the suspension time of inert particles in the water column and also helps to suspend very small or passive swimming larvae. However, when the system is used to grow-on larvae or juvenile fish it can easily be switched to bottom draining to provide self-cleaning water dynamics for high organic loads.

A unique outlet filter was developed that eases the daily routine of replacing screens when enriched live food is used. This filter can be exchanged with a screened standpipe and outlet surface skimmer when the bottom draining flow characteristics are engaged.

The system is fully controlled by a single programmable logic controller (PLC). The PLC controls the light intensity, photoperiod, dimming time, live food and algae pumping intervals, substantially reducing labor requirements.     

淡水鱼类原良种场设施--水体自然净化系统的设计理念与建设实践

1 前言 20世纪80年代以来,我国建设了一批淡水鱼类的种质保存与繁育基地(即淡水鱼类原良种场).这对于保持淡水鱼类的遗传多样性状相对稳定,延缓退化和推广良种发挥了非常重要的作用,有力地推动了淡水渔业向前发展.     

鱼类补体系统的研究进展

鱼类补体大约由30余种蛋白裂解酶、酶抑制因子和受体构成的,是机体内最为复杂的限制性蛋白溶解系统。在鱼类免疫系统进化中,补体的出现比免疫球蛋白还要早。许多研究表明鱼类补体直接参与机体防御,其生物学活性影响机体抵抗微生物的能力、免疫反应细胞间的通讯联系、免疫复合物的形成和持续时间等。     

In vitro study of a putative role of gonad‐inhibiting hormone in oocyte growth stimulation in Penaeus monodon

Ovarian development in crustacean is controlled by several factors, among which a neuropeptide gonad‐inhibiting hormone (GIH) is known to inhibit vitellogenin (Vg) synthesis in the ovary. It has been postulated that GIH may control Vg synthesis by inhibiting the release of gonad‐stimulating factor (GSF) from brain and thoracic ganglia. To prove this hypothesis, this study was primarily aimed to investigate the influence of GIH on the release of GSF from thoracic ganglia of Penaeus monodon. Our result showed that GIH did not suppress the release of putative GSF from thoracic ganglia by calcium ionophore A23187 as the induction of oocyte growth in the ovary explants that were cocultured with thoracic ganglia in the presence of A23187 was not affected by the addition of recombinant GIH protein. In addition and interestingly, when the ovary explants were incubated with the recombinant GIH alone, the oocyte growth was increased at the rate comparable to that induced by A23187 in the presence of thoracic ganglia. Hence, our in vitro study demonstrated that the stimulation of GSF released from thoracic ganglia is independent of GIH, and that the GIH has a dual function in oocyte growth stimulation and inhibition of Vg synthesis in the early stage of ovarian development. This expands our knowledge on the regulation of ovarian development in shrimp by GIH. Further in vivo studies in this novel aspect of GIH function will be useful for the improvement of shrimp ovarian maturation in the future.     

High rate algal pond treatment for water reuse in a marine fish recirculation system: Water purification and fish health

Regardless of the degree of closure of a recirculation system, effluents are produced and replacement water is needed, which limits the possibility of locating a seawater production system away from the shoreline. At the Palavas Ifremer station, in the south of France, a High Rate Algal Pond (HRAP) was operated during several years to treat the effluent from a recirculating aquaculture system before reusing it. The effect of the HRAP-treated water on the recirculation system and on the fish was investigated and the optimal algae growing conditions were defined. The experiments were carried out in three rearing systems: one flow through, one recirculating and one recirculating with a HRAP. The water flow rate, temperature, pH and salinity conditions were similar in all systems.The effect of reusing the HRAP-treated water is very limited (1) on the functioning of the recirculation system and (2) on fish performance, but it allows a significant reduction of the dissolved inorganic nitrogen and phosphorus concentration in the rearing water. HRAP treatment reduced metal accumulation in muscle and liver of RAS fish, except for chromium and arsenic. All biomarkers presented no significant difference between systems, except for Superoxide Dismutase (SOD) and EROD, which showed a higher concentration in RAS and in both recirculating system respectively.