福建沿海部分地区织纹螺毒性消长及毒素成分分析

为了解福建沿海织纹螺毒性的动态变化,于2006年3月至9月,对福建省涵江、同安和霞浦三地的织纹螺毒性消长情况进行了跟踪监测,并对高毒性织纹螺样品中的毒素成分进行了分析.期间每周采样一次,采集的织纹螺经鉴定后,通过小鼠生物测试法分析其毒性,并选择毒性较高的织纹螺样品,应用高效液相色谱(HPLC)和高效液相色谱一质谱联用(LC-MS)技术分别对样品中的麻痹性贝毒(paralytic shellfish poison,PSP)和河豚毒素(tetrodotoxin,TTX)进行了分析.研究结果表明,三个采样点的织纹螺主要为半褶织纹螺,且均有阳性样品检出,涵江、同安和霞浦的织纹螺样品中阳性检出率分别为14%,43%和20%.采自福建三地的织纹螺样品总体毒性较低,毒性最高的样品为7月12日采自霞浦的半褶织纹螺,毒性为16.2 MU·g-1组织(湿重).对高毒性织纹螺样品的毒素分析结果表明,样品中不含麻痹性贝毒毒素,但是在样品中检测到了河豚毒素及其同系物三脱氧河豚毒素(trideoxy TTX).样品中河豚毒素含量为4.38μg·g-1组织(湿重),依据样品中河豚毒素含量计算得到的毒性与小鼠法测试结果基本相当,说明河豚毒素及其同系物是导致织纹螺毒性的主要毒素成分.涵江的阳性样品集中出现在3月份,而同安和霞浦的阳性样品则出现了3月份和6,7月份两个高峰时段,说明织纹螺的毒性变化具有一定的季节和地域特征.因此,建议福建地区在这两个时段加强对织纹螺毒性的监测.     

乌鳢抽出物成分的研究

分析了乌鳢(Channaargus)即杀后背肉、腹肉、尾部肉、肝脏、生殖腺中ATP关联化合物、游离氨基酸、多胺、糖元及糖酵解中间代谢物、有机酸等的含量。ATP关联化合物在肌肉中的总量为7.5～8.0μmol·g-1。ATP在背肉含量为3.9μmol·g-1,腹肉为4.1μmol·g-1,尾部肉为4.7μmol·g-1。运动激烈的尾部肉ATP占63%,含量相当高。肝脏中有少量的腺苷与肌苷酸一起被检出,据此可认为ATP的分解存在两个途径。游离氨基酸总量在背肉中为436.0mg·(100g)-1,腹肉中为405.0mg·(100g)-1,尾部肉中为356.3mg·(100g)-1。牛磺酸和甘氨酸为主要氨基酸,占68%～73%。丙氨酸和谷氨酸也相当高的含量检出。肌肉中的多胺检出为精胺和亚精胺,肝脏和生殖腺中有较高浓度的腐胺及亚精胺和精胺检出。即杀后糖元的量约占肌肉的0.5%,还有相当多的葡萄糖和6磷酸葡萄糖的糖酵解中间代谢物以及其最终产物乳酸的大量检出。     

多壁碳纳米管固相萃取-超高效液相色谱串联质谱法测定织纹螺中河豚毒素

建立了多壁碳纳米管(MWCNTs)固相萃取-超高效液相色谱串联质谱法(UPLC-MS/MS)测定织纹螺中河豚毒素(TTX)的方法,样品用1%乙酸甲醇溶液提取,调节pH至8.5~9.0后,采用碳纳米管净化处理后上机测定。以乙腈和0.1%甲酸水溶液为流动相,经Amide柱梯度洗脱,以电喷雾离子源正离子模式,多反应监测(MRM)测定,外标法定量。在0.3~50 ng·mL~(–1)质量浓度范围内,相关系数(R)大于0.999,加标回收率为83.7%~91.4%,相对标准偏差为2.3%~8.6%,检测限和定量限分别为0.3μg·kg~(–1)和1μg·kg~(–1)。该方法适用于织纹螺中TTX的检测。     

施氏鲟、大杂交鲟和西伯利亚鲟卵及体腔液的生化组成比较

用生物化学方法测定施氏鲟Acipenser schrenckii、大杂交鲟A.schrencki i(♂)×Huso dauricus(♀)及西伯利亚鲟A.baeri成熟卵子及体腔液的酶、维生素、微量元素及氨基酸组成和含量。结果表明:施氏鲟与大杂交鲟的卵径显著大于西伯利亚鲟(P<0.05)。吸水后大杂交鲟的卵径显著大于施氏鲟和西伯利亚鲟(P<0.05)。大杂交鲟卵中谷草转氨酶(GOT,2.83U·g-1)、酸性磷酸酶(ACP,0.69 U·g-1)和碱性磷酸酶(AKP,0.81 U·mg-1)含量最高,均无显著性差异(P>0.05),仅西伯利亚鲟卵内琥珀酸脱氢酶(SDH)的活力显著升高(P<0.05)。3种鲟鱼卵与体腔液中蛋白质、微量元素及维生素组成差异显著。大杂交鲟体腔液内的ACP活力显著高于施氏鲟体腔液内的ACP活力(P<0.05)。西伯利亚鲟(0.51±0.16μmol·g-1)卵内的Fe含量显著高于施氏鲟(0.35μmol·g-1)和大杂交鲟(0.42±0.12μmol·g-1),而体腔液内均未检测到Fe和Zn,但体腔液中Vc(4.00±2.44μg·L-1)含量显著低于大杂交鲟(5.58±1.53μg·L-1)和施氏鲟(5.47±2.32μg·L-1)(P<0.05)。3种鲟鱼卵及体腔液内的酶、微量元素、维生素等组成相同,但SDH和ACP活力、Fe和Vc含量存在组织和种间差异,在亲鱼培育时应区别培育。     

HPLC-MS/MS法测定水产品中硫酸粘菌素、杆菌肽及维吉尼霉素M1的残留量

建立了水产品中硫酸粘菌素(CS)、杆菌肽(BTC)及维吉尼霉素M1(VBGMM1)3种多肽类抗生素残留量检测的HPLC-MS/MS法。样品经水溶液[V(甲醇)∶V(0.1%甲酸水溶液)=2∶5]提取,4%三氯乙酸乙腈除蛋白,乙腈饱和正己烷除脂,过OASIS HLB(60 mg)小柱净化后,利用HPLC-MS/MS法,以选择反应监测模式检测,外标法进行定量分析。CS和BTC在0.01～10.00 mg.L-1质量浓度范围内线性良好,VGMM1在0.002～2.000 mg.L-1质量浓度范围内线性良好,R2均大于0.995;3种多肽类抗生素的检出限分别为CS 10μg.kg-1、BTC 10μg.kg-1、VGMM12μg.kg-1,定量限分别为20μg.kg-1、20μg.kg-1和4μg.kg-1;选择3个不同浓度水平做加标回收,平均回收率在72.3%～103.9%,相对标准偏差为1.10%～10.92%。该方法具有操作简便、准确性高、灵敏度高和重现性好等优点,可为检测水产品中这3种药物的残留提供相关技术支持。     

雌二醇对日本沼虾肝胰腺的脂肪酸含量及组织结构的影响

日本沼虾(Macrobrachium nipponense)肝胰腺为卵巢发育提供脂类等营养,日本沼虾肝胰腺中含有与卵巢发育有关的雌二醇,日本沼虾卵巢发育过程中,肝胰腺中脂肪酸的含量及组织结构会发生相应的变化,本文研究雌二醇对日本沼虾肝胰腺中脂肪酸的含量及组织结构的影响。实验设3个试验组和1个对照组,试验组日本沼虾肌肉分别注射5、0.5和0.05μg·g-1体重的雌二醇,对照组注射生理盐水,5 d注射1次,共注射2次,10 d后测定肝胰腺中脂肪酸的含量及其组织结构的变化,从而研究雌二醇对日本沼虾肝胰腺中脂肪酸含量及组织结构的影响。测定结果表明,日本沼虾肝胰腺中主要脂肪酸为C16∶0、C18∶1n9和C18∶12n6(亚油酸),且单不饱和脂肪酸(MUFA)和多不饱和脂肪酸(PUFA)含量较高,分别为39.05%±1.16%和42.71%±2.56%。注射雌二醇主要影响肝胰腺中C18∶10、C18∶1n9、C18∶1n7、C18∶12n6、C18∶13n6、C18∶14n、C20∶14n6、C20∶15n3、C22∶14n6和C22∶15n3的含量,注射5μg·g-1和0.05μg·g-1雌二醇显著降低肝胰腺PUFA总含量(P0.05),其中对C18∶14n、C20∶15n3、C22∶14n6和C22∶15n3降低作用显著(P0.05);注射0.5μg·g-1雌二醇显著降低肝胰腺MUFA总含量(P0.05),其中对C18∶1n9、C18∶1n7和C20∶12n降低作用显著(P0.05);注射0.5μg·g-1雌二醇显著提高肝胰腺中PUFA总含量(P0.05),其中对C18∶13n6、C18∶14n、C20∶14n6、C20∶15n3(EPA)、C22∶14n6、C22∶15n3提高作用显著(P0.05),但是对C18∶12n-6和C20∶12n却有显著地降低作用(P0.05)。注射雌二醇会促使肝胰腺B细胞的体积增大和数量增加,其中0.5μg·g-1作用最明显。     

超高效液相色谱法测定黄颡鱼肌肉中叶黄素含量

本文建立检测黄颡鱼Pelteobagrus fulvidraco肌肉中叶黄素含量的反相超高效液相色谱分析方法。样品经95%乙醇提取,ACQUITY UPLC BEH C_18色谱柱(2.1mm×100mm,1.7μm)分离,以89%甲醇-1%乙腈-10%水为流动相等度洗脱,二极管阵列检测器检测,检测波长446nm,外标法定量。结果显示:叶黄素在0.04~0.8μg·mL~(-1)范围内线性良好,此方法检出时限0.03mg·kg~(-1)。添加量在0.5mg·kg~(-1)、1.0mg·kg~(-1)、5.0mg·kg~(-1)时,叶黄素的回收率为82.0%~84.2%,相对标准偏差在10%范围内。结果表明:该方法可用于黄颡鱼中叶黄素成分的定性和定量检测。     

用紫外和荧光检测器同时分析水产品中孔雀石绿和无色孔雀石绿含量的高效液相色谱法

建立了用紫外和荧光检测器同时分析水产品中有色孔雀石绿和无色孔雀石绿的高效液相色谱法。有色孔雀石绿检测采用紫外检测器,波长588 nm;无色孔雀石绿采用荧光检测器,其激发波长265 nm,发射波长360 nm,流动相0.1 mol/L(pH4.5)的乙酸铵溶液∶乙腈(20∶80),流速1.5 ml/min。采用ZOBAX C18(250 mm×4.6 mm,5μm)色谱柱。加标量为10μg/kg和250μg/kg(有色孔雀石绿),回收率分别为60.2%和56.6%,相对标准偏差分别为10.1%和5.6%;加标量为2μg/kg和50μg/kg(无色孔雀石绿)时,回收率分别为95.2%和92.3%,相对标准偏差分别为10.8%和11.3%。该方法的最低检测限1.1μg/kg(孔雀石绿和无色孔雀石绿总量),可满足欧盟对水产品中孔雀石绿的检测要求。检测方法稳定,简便,灵敏度高,无需柱后氧化柱,适合同时进行水产品中有色孔雀石绿和无色孔雀石绿含量的检测。     

河豚毒素胶体金免疫层析快速检测试剂盒的应用研究

在我国东南沿海地区,每年均有人因食用带有河豚毒素的河豚鱼或织纹螺引起中毒的事件。本文以河豚毒素标准品溶液分析河豚毒素胶体金层析快速检测卡的最低检测出限为0.5 MU/mL或100 ng/mL。根据河豚毒素限量值(10 MU/g)设计了织纹螺以及河豚鱼的样品前处理方法,进一步分析了20个织纹螺及河豚鱼实际样品,准确率为100%,单个样品测试时间为20 min。结果表明:河豚毒素胶体金层析快速检测卡具有灵敏度高、特异性好、操作简便、可通过肉眼直接判读结果的特性,尤其适合于基层检测机构、渔业管理单位及水产品加工企业对河豚鱼或织纹螺中河豚毒素的快速筛查与分析。     

液质联用法检测海水中氯霉素的含量

应用液质联用仪测定海水中氯霉素的含量。水样经乙酸乙酯萃取、浓缩,以1 mL50%的甲醇-水溶液定容,采用液相色谱-三重四级杆串联质谱仪、多反应监测扫描模式（MRM）检测和氘代氯霉素内标法定量。海水中氯霉素检测方法的线性范围是0.10～10.0ng.mL-1,检出限为0.04μg.L-1,定量限为0.10μg.L-1,加标浓度在线性范围内时,回收率大于90%。试验证明：该方法比较稳定,适用于检测海水中氯霉素的含量。     

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).     

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.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.