以虾肝肠胞虫(EHP)、致急性肝胰腺坏死病副溶血性弧菌(VpAHPND)、十足目虹彩病毒1(DIV1)和白斑综合征病毒(WSSV)4种常见病原为研究对象,将微流控芯片技术与LAMP技术有机结合,建立了相应的基因芯片联检系统,并开展了优化了基因芯片的反应体系及条件研究.结果表明,该联检系统可在30 min反应时间内同时检测上述4种病原,特异性好.各病原指标检出限分别为 EHP 0.57 copies·μL-1、DIV1 0.43 copies·μL-1、WSSV 2.12 copies·μL-1 和 VpAHPND 3.18 copies·μL-1,已达到甚至超越了 LAMP检测的敏感性,且该系统检测试剂盒具有操作简便,检测灵敏,特异性强,检测时间短,设备要求简单等特点,适合养殖全程中凡纳滨对虾EHP、VpAHPND、DIV1和WSSV的监测与筛查工作.

To evaluate the performance of microfluidic chip method in the joint detection of four common pathogens of Litopenaeus vannamei including Enterocytozoon hepatopenaei（EHP）, acute hepatopancreas necrosis disease caused by Vibrio parahaemolyticus（VpAHPND）, Decapod iridescent virus 1（DIV1） and white spot syndrome virus（WSSV）, primers for the amplification reactions of the above four pathogens were designed, new functional divisions were made for the existing chips, and the reaction system and constant temperature amplification conditions of the microfluidic chip were optimized. The results showed that, the optimized combined detection method could detect the above four pathogens simultaneously with good specificity, and the joint detection results could be obtained within 30 min reaction time. The detection limits of EHP, DIV1, WSSV and VpAHPND were 0.57 copies/μl, 0.43 copies/μl, 2.12 copies/μl and 3.18 copies/μl, respectively. Some of the limits had reached or even exceeded the testing sensitivity of loop-mediated isothermal amplification（LAMP） method. The detection kit used in the joint detection method of microfluidic chip for the four pathogens of L. vannamei in this test is characterized by simple operation, sensitive detection, strong specificity, short detection time and simple equipment requirements, and is suitable for the monitoring and screening of EHP, VpAHPND, DIV1 and WSSV of L. vannamei in the whole breeding process.

小型温棚养殖模式(下称小棚模式)是近年来凡纳滨对虾(Litopenaeus vannamei)养殖的热点模式.为阐明小棚模式养殖水体微生物群落的特点,探究该模式高产、高效的原因,基于16SrRNA基因的测序结果,对第2、第44、第69和第96天小棚模式养殖水体的微生物群落组成和功能进行了分析.结果表明:凡纳滨对虾小棚养殖后期,水体微生物群落的丰富度和多样性显著高于前期;在整个养殖过程中,门水平主要优势菌为变形菌门、拟杆菌门和放线菌门,其中变形菌门丰度在第69天显著增加,放线菌门丰度前期较高,后期下降;属水平优势菌属中Candidatus_Aquiluna的丰度在第2天最高(28.7％);海命菌属(Marivita)在中期富集,在第69天丰度最高(9.94％);黄杆菌属(Flavobacterium)的丰度随着养殖时间逐渐增加,在第96天达到最高(11.63％).通过PICRUSt2预测微生物群落的功能,丰度前20的代谢功能项在第69和第96天的丰度显著高于第2天,尤其萜类和聚酮类代谢、脂类代谢、异种生物降解和代谢等功能均高度富集,通过FAPROTAX鉴定得出,化能异养类菌的丰度在养殖后期显著增加.环境因子关联分析发现,总氮(TN)和化学需氧量(COD)对小棚模式水体微生物群落结构的影响最大,海命菌属、Candidatus_Aquiluna、红小梨形菌属(Rhodo-pirellula)等微生物发挥了固碳、降氮、降磷的作用.综上所述,在高密度、高氮磷的环境条件下,小棚模式的水体微生物在保持水环境稳定和增强对虾免疫与抗病能力方面发挥了重要作用.

近年来,急性肝胰腺坏死病(Acute hepatopancreatic necrosis disease,AHPND)造成了凡纳滨对虾(Litopenaeus vannamei)养殖业巨大的经济损失,其病原为一类含有致病基因PirAB的弧菌(Vibrio),主要为副溶血弧菌(Vibrio parahaemolyticus,VPAHPND).本研究以VPAHPND为受试菌株,从江苏海域捕获的黄姑鱼(Nibea albiflora)体内分离筛选对VPAHPND具有抑制作用的拮抗菌株.通过形态学、生理生化及分子生物学方法进行鉴定,研究了其拮抗效果、拮抗活性物质,并对生物安全性和被动保护效果进行评估.结果表明,分离筛选出1株具有良好拮抗效果的菌株JSHY97,经鉴定该菌株为铜绿假单胞菌(Pseudomonas aeruginosa).该菌株对3株具有不同分型的VPAHPND均具有良好的拮抗效果,其抑菌圈直径达19.25 mm～21.80 mm,初步判断其活性物质来源于胞外产物.口服JSHY97的凡纳滨对虾的7 d保护率也达到80％,拮抗组的凡纳滨对虾体内的毒力基因PirA拷贝数显著低于对照组(P<0.05).安全性实验表明JSHY97对斑马鱼(Brachydanio reriovar)和凡纳滨对虾无毒力.由此表明,本研究得到的铜绿假单胞菌菌株JSHY97对VPAHPND具有较好的抑制作用,为AHPND的防治提供了新的菌株资源.

The quinolones and their use status were briefly described; The pretreatment and detection methods for the detection of quinolones residues are introduced, including enzyme-linked immunosorbent assay,colloidal gold immunochromatography, high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, chemiluminescence immunoassay, etc. It is pointed out that in the detection of quinolones residues in aquatic products, the solid phase extraction method and QuEChERS extraction method are used for sample pretreatment with low cost and high efficiency; High performance liquid chromatography will still be the most widely used method for the detection of quinolones residues for a long time in the future.

对近年来江苏沿海地区脊尾白虾养殖中常爆发的"僵尸病"进行了初步的防控药物筛选,试验采用纸片扩散法(K-B法)测定了二尖梅奇酵母MQ2101对18种抗菌类药物的敏感性,并采用微量稀释法测定了 8种优选药物及7种常见消杀制剂对二尖梅奇酵母MQ2101的最小抑菌浓度(MIC)和最小杀菌浓度(MBC).结果显示,在优选的8种抗菌药物中克霉唑、益康唑、制霉菌素、5-氟胞嘧啶、伊曲康唑、酮康唑对二尖梅奇酵母MQ2101具有较好的抑制效果,MIC均为0.125 μg.mL-1;伊曲康唑对二尖梅奇酵母的杀灭效果最好,MBC为0.25 μg·mL-1;在常见消杀制剂中高锰酸钾、甲醛对二尖梅奇酵母MQ2101的抑制效果最好,MIC为50 μg·mL-1,亚甲基蓝、甲醛对二尖梅奇酵母MQ2101的杀灭效果最好,MBC均为200 μg·mL-1.最后,本研究结合养殖过程中的实际情况提出了脊尾白虾"僵尸病"的防控建议.

于2020年对江苏省沿海脊尾白虾养殖区池塘的海水、沉积物和脊尾白虾肌肉组织中的重金属进行分析,同时采用单因子污染指数法和潜在生态风险指数法对其沉积物重金属进行评价.结果显示,养殖池塘水体中铜(Cu)、铅(Pb)、镉(Cd)和锌(Zn)的平均质量浓度分别为3.13,1.02,0.094和28.02μg/L;沉积物中的平均质量分数分别为22.88,19.93,0.18和140.69 mg/kg;虾肌肉组织中的平均质量分数分别为12.49,0.50,0.18和28.65 mg/kg.生态环境质量评价结果表明,参照《海水水质标准》(GB 3097—1997),水体中Cu、Pb和Cd的质量浓度达到一类标准,Zn的质量浓度达到二类标准,属于清洁水平;沉积物中重金属的质量分数均达到《海洋沉积物质量标准》(GB 18668—2002)一类标准,属于较清洁水平,潜在生态风险为Cd>Zn>Cu>Pb,整体为轻微生态风险等级,Cd为主要污染元素;虾肌肉组织中重金属Pb和Cd的质量分数均达到《无公害食品海水虾》(NY 5058—2006)标准.

哈氏仿对虾是我国重要的海洋捕捞虾类渔业资源之一,我国对该资源开发利用较早,近年,随着海洋捕捞强度的不断提高,以及海洋生态环境条件的日益恶化,哈氏仿对虾的资源量大幅缩减,作为海洋食物链的中间体,该资源量的减少会扰乱海洋生态网络,导致其时空生态位失衡,进而加速哈氏仿对虾资源的衰竭.哈氏仿对虾属于广温广盐的暖水虾种,对环境的适应能力较强,具有非常好的人工繁育和养殖前景,但目前关于哈氏仿对虾的研究还处于较低水平.本文拟对哈氏仿对虾的生物学及生态学特点、资源分布现状、遗传多样性、繁殖生物学和人工驯化养殖前景等进行综合概述,以期为哈氏仿对虾的资源保护和人工增养殖研究提供参考.

大部分池塘养殖尾水中总氮、总磷质量浓度偏高,测定过程中需要对样品进行稀释,耗时较长且影响准确度.实验室分析时,通常采用过硫酸钾对尾水样品进行消解,其配置过程对温度要求较高,易析出晶体,且耗时较长.而过硫酸钠易溶于水,受温度影响较小,易配制.因此,分别采用上述2种过硫酸盐消解液对样品进行消解并同时测定总氮、总磷的质量浓度,比较2种消解方式对校准曲线、加标回收率、养殖尾水实际样品测定的影响.结果表明,等质量或等物质的量的过硫酸钠消解液均能满足总氮、总磷的消解要求,且后者消解样品无须改变氢氧化钠的加入量和调整pH值,即可达到同等消解效果.因此,可采用等物质的量的过硫酸钠代替过硫酸钾同时消解养殖尾水中的总氮、总磷.

根据2011年5、8、10月及2012年2月对南黄海辐射沙脊群海域4个航次的调查,获取了全年浮游植物的种类、数量和优势种等数据.结果显示,辐射沙脊群海域在全年调查中共鉴定出浮游植物60种,其中硅藻门52种,占绝对优势.中肋骨条藻(Skeletonema costatum)、加氏星杆藻(Asterionella kariana)和有翼园筛藻(Coscinodiscus bipartitus)在4个季节均为优势种;不同季节藻类丰富度差异明显,以秋季最多,为40种,多样性和均匀度指数差异不明显.通过分析该海区浮游植物群落结构与环境因子间关系发现,相同属种类在典型对应分析(CCA)坐标中占据相似的生态位,且相对位置较近;与环境因子的关系也相似.

At the same time, the total nitrogen and total phosphorus in the off-shore sediments were simultaneously eliminated by two different methods of digestion, microwave digestion and high pressure digestion, and the results were compared. The results show that the relative standard deviation of total nitrogen in sample 1 and sample 2 were7.7％ and 7.4％ respectively, and those of total phosphorus were 3.9％ and 7.8％ under high pressure digestion; meanwhile the relative standard deviations of total nitrogen in sample 1 and sample 2 were respectively 9.4％ and 10.1％, and the standard deviations of total phosphorus were 6.9％ and 8.1％ under microwave digestion; the content of total phosphorus under the condition of two kinds of dispelling were similar (sample 1 total phosphorus content were 0.460 mg/kg and 0.454 mg/kg, sample 2 total phosphorus content were 0.449 mg/kg and 0.439 mg/kg), but total nitrogen content under the condition of high pressure digestion (sample 1 to 0.114 mg/kg, sample 2 was 0.122 mg/kg) were significantly higher than those of microwave digestion (sample 1 to 0.088 mg/kg, sample 2 was 0.092 mg/kg), and the relative standard deviation was less than that. Therefore, the high pressure digestion method is more precise than microwave digestion.

以脊尾白虾(Exopalaemon carinicauda Holthuis)为试验材料,以人工连续7 d投喂携带白斑综合征病毒(white spot syndrome virus,WSSV)的死虾为攻毒方式,得抗性试验组(Rm);以人工连续7 d投喂不携带WSSV的死虾为对照方式,得对照试验组(Vm),待存活率稳定后,分析比较抗性试验组和对照试验组的存活率及酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)和酚氧化酶(PO)活性的差异性;然后待Rm组中雌性脊尾白虾虾卵接近孵化时,挑选40~50尾体格较大的抱卵雌虾,分别放入事先准备好的桶中,让其自然孵化,以此得Rm1代;孵化完成后,收集各组亲虾并编号,用套式PCR法检测各组亲虾WSSV携带情况,将阴性组及虾卵孵化较少组淘汰,留下20组,重新编号Rm1代,然后将其喂养成成虾;以此方式连续选育得到Rm3代.用同样方法对Rm3代成虾和自然环境中WSSV携带成虾(Vn)进行攻毒试验,然后检测结果显示Rm3代成虾比Vn成虾存活率要高出很多且免疫相关酶活性之间存在显著性差异.

采用不同蛋白酶对异育银鲫进行酶解,其产物的性质存在较大的差异.一方面,胰蛋白酶对异育银鲫的水解度最大,可以达到39.4％,而胃蛋白酶对异育银鲫的水解度最小,低于10％;中性蛋白酶、胰蛋白酶和风味蛋白酶酶解产物的产率较高;另一方面,胰蛋白酶和碱性蛋白酶酶解产物的分子量分布较为均匀;风味蛋白酶、胰蛋白酶和木瓜蛋白酶酶解产物的抗氧化活性相对较强;不同蛋白酶酶解产物的乳化性质均低于6 m2/g,但蛋白酶酶解产物的乳化稳定性之间差异明显;不同蛋白酶酶解产物的起泡性质和起泡稳定性存在较大差异,起泡性最强的是木瓜蛋白酶酶解产物,而起泡稳定性最高的是中性蛋白酶酶解产物.

A method for determination of inorganic arsenic in Porphyra yezoensis by high performance liquid chromatography and hydride generation atomic fluorescence spectrometry was developed.The samples were extracted with 10％HCL at 70 ℃,then the extracts were oxidized with hydrogen peroxide to transform As(Ⅲ) into As(Ⅴ).The conditions and parameters of both HPLC and AFS were optimized.The results showed that the linear range for As(Ⅴ) was all between 1 to 50 μtg/L with linear cofficients more than 0.999,instrument detection limit was 1.13μtg/L and the method detection limit was 0.023mg/kg for inorganic arsenic.The extraction efficiency of total arsenic was more than 87％ and the recoveries of standard addition were above 90％.The proposed method is simple,accurate and efficient and has been applied successfully in the determination of inorganic arsenic analysis in Porphyra yezoensis.

Gibel carp (Carassius auratus gibelio) protein was chosed as the raw material. It was independently hydrolyzed by trypsin, pepsin, flavourzyme, neutrase, alcalase, papain, bromelain and acid protease. The an-tioxidant capability was evaluated by the DPPH free radicals clearance rate. The results showed that the flavourzyme was the best proteolytic enzyme. The single factor and orthogonal experiment showed that the best hydrolysis conditions of the flavourzyme were:enzyme amount 3000 U/g, substrate concentration 7%, pH=7.5, T=55℃, reaction time 4 h. Under these conditions, the degree of hydrolysis (DH) was 46.78%, the DPPH free radicals clearance rate could achieve to 90.12 %, the scavenging rate of hydroxyl radical was 82.31%and the clearance rate of superoxide anion radical was 79.45%. The enzymatic hydrolysates were pro-cessed with ammonium sulfate salting-out, active carbon, ultrafiltration, dialysis, concentration and lyophiliza-tion, then antioxidant peptide with different molecular weights were got.

A digestion method which consists of microwave digestion and wet digestion for subsection by using hy-dride generation atomic fluorescence spectrometry was established to determine the total arsenic content in the shellfish and algae products. The effects of different acid systems and digestion processs in microwave digestion and different con-tents of perchloric acid and sulfuric acid in wet digestion were studied. The sample prepared by microwave digestion with 5 ml nitric acid and wet digestion with 1 ml perchloric acid and 2-3 ml sulfuric acid were digest to colorless solution. The pretreatment showed such advantages as high digestion efficiency, controllable operation process, well-judged reaction end point, less reagent consumption, environment friendly, and accurate and stable test data. The spiked recovery rate of arsenic was over 90%, which satisfied the requirement of total arsenic determination in shellfish and algae products.

In this study,we isolated strains MD5 and MD8 of nitrifying-aerobic denitrifying bacteria from intertidal sediments using microbial culture method,identified their biology taxonomy,and tested the ability of nitrogen removal.The morphology,16S rRNA sequencing and functional gene (nirS) results identified MD5 and MD8 as Halomonas and Alcaligenesx respectively with GenBank sequence number of KM362826 and KM406394.Both strains had nitrogen removal ability under aerobic condition.In the experiment with ammonium as the sole nitrogen resource,the nitrogen removal efficiency was up to 81％ in MD5 and 88.3％ in MD8.While in the experiment with nitrate as solo nitrogen resource,the efficiency reached 85.3％ and 92.1％ respectively.In experiment with the combination of ammonium and nitrate,the total removal rate reached 72％ and 76.8％ respectively.The pH,salinity and C/N ratio influenced to different degrees the removal ability of the two strains for NH4+-N,NO3--N and dissolved inorganic nitrogen (DIN).For instance,the removal abilities of MD5 and MD8 were significantly inhibited (P ＜ 0.05) at pH of 5.5-6.5,and the optimum pH was 7.5-8.5;the nitrogen removal ability of MD5 increased with the salinity within the range of 10-30,whereas MD8 reached its high removal ability within salinity of 20-25 g/L;MD5 and MD8 were significantly inhibited under C/N ratio of 6 (P ＜ 0.05),with the optimal C/N ratio ofMD5 (C/N =8) lower than that of MD8.The above results revealed that MD5 and MD8 were two strains of nitrifying-aerobic denitrifying bacteria,which could be applied for bioremediation of aquaculture effluents.

根据2011年5、8、10月以及2012年2月对南黄海辐射沙脊群海域4个航次海水水质的调查资料,分析了该海水营养盐的分布特征和季节变化,利用潜在富营养化评价模式评价水质营养状况.结果表明,无机氮年平均浓度为0.476 mg·L-1,春季最高,平均为0.636 mg.L-1.4个季节中溶解无机氮以硝态氮(NO3-N)为主要存在形式,占年平均含量的76.6％,其中以春季比例为最高,氨氮(NH4-N)占无机氮年平均含量的23.3％,亚硝氮(NO2-N)含量较低,全年基本未检出.磷酸盐的年平均浓度为0.164 mg.L-1,季节变化呈现春季至冬季递增的分布特征.氮和磷的空间分布基本符合由陆向海逐渐降低的趋势.本海域春季和夏季属于中度营养水平,秋季和冬季属于氮限制中度营养水平.

为了充分利用青蛤(Cyclina sineusis Gmelin)加工过程中产生的边下脚料制备抗氧化肽,对青蛤边下脚料进行了匀浆预处理.以酶解产物的1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率为评价指标,从胰蛋白酶、木瓜蛋白酶、胃蛋白酶、酸性蛋白酶、中性蛋白酶和碱性蛋白酶6种蛋白酶中筛选出木瓜蛋白酶作为最佳酶解用酶,通过单因素试验和正交试验确定木瓜蛋白酶酶解青蛤边下脚料制备抗氧化肽的最佳工艺条件为:加酶量2 500 U/g、底物浓度7％、反应pH 7.0、反应温度50℃、反应时间3h.在该条件下对青蛤边下脚料的水解率为31.56％,酶解产物的DPPH自由基清除率为74.62％,羟自由基清除率为59.29％,超氧阴离子自由基清除率为27.84％.

A denitrification-possessing bacterium strain was isolated and screened with enrichment culture method from low beach sediment in Jiangsu coastal area. Through morphological observation,sugar fermentation as well as 16S rDNA sequence analysis,the classification characterization,phylogenetic site,denitrifying performance,and oth-er aspects of the strain was studied,and named it as MD5. The results showed that the isolated strain from oceanic sediment by enrichment,isolation,screening possessed fairly strong denitrification reaction,under the conditions of 30 ℃ with initial concentration of nitrate at 1 500 mg/ L and after 144 h of cultivation,the nitrate in the medium could be removed by 90. 1% ;under the aerobic cultivation conditions the removal rate of concentration of nitrification acid mass declined to 89. 3% in a ability test tube by the strain. The results of MD5 in morphology and sugar fermentation were in accord with that in Halomonas,in addition,the homology of its gene sequence was as high as 99% with Halomonas sp. IW11-1(AB305262. 1),suggested that was a Halomonas strain. Strain MD5 obtained by isolation in this experiment had fairly strong denitrifying ability both under anaerobic and aerobic conditions,therefore,it could be applied in the oceanic sediment environment denitrification application study.