复方中草药对小丑鱼非特异性免疫、消化酶活性的影响及对刺激隐核虫杀灭效果分析

刺激隐核虫寄生感染引起的“白点病”不仅危害水族箱观赏鱼类,还会对水族馆大水体海水观赏鱼带来毁灭性打击,是海水观赏鱼养殖业危害最为严重的病害之一。本研究探讨了在基础饲料中添加不同剂量复方中草药“XCY-1”(苦参、地肤子、蛇床子、黄芪、甘草等按一定比例混合,超微粉碎后收集备用)对小丑鱼血清、肝脏中非特异性免疫指标和肠道消化酶活性指标变化及不同用药方式对防治刺激隐核虫病的效果。研究结果显示,投喂复方中草药“XCY-1”可使小丑鱼血清中溶菌酶(LZM)、超氧化物歧化酶(SOD)、碱性磷酸酶(AKP)、酸性磷酸酶(ACP),肝脏中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)、过氧化氢酶(CAT)、总抗氧化能力(T-AOC)活性均呈现先升后降趋势,第21 d各项非特异性免疫指标达到最高值,其中20 g/kg剂量组效果最好,能显著提高小丑鱼的免疫能力;肠道中淀粉酶(AMS)、脂肪酶(LPS)和胰蛋白酶在各取样点活性均高于对照组,20 g/kg剂量组在各取样点胰蛋白酶和AMS活性高于其他剂量组,20 g/kg和40 g/kg剂量组在各取样点LPS活性最高。复方中草药“XCY-1”药浴对刺激隐核虫幼虫的体外杀灭结果显示:4 h内,50 mg/L药物浓度即对刺激隐核虫幼虫具有一定的杀灭效果,400 mg/L药物浓度即可杀灭全部幼虫;当药物浓度达到800 mg/L时,3 h内幼虫全部死亡。不同用药方式防治小丑鱼刺激隐核虫病:实验分为4组,A组20 g/kg剂量组,口服无药浴;B组20 g/kg剂量组,口服且同时按50 mg/L进行药浴;C组20 g/kg剂量组,口服且同时按100 mg/L进行药浴;D组为对照组,仅投喂基础饲料。结果显示:实验第6 d,A、B、C三个药物组与对照组的存活率分别为:51.1%、53.3%、57.7%、0,药物组存活率显著高于对照组(P＜0.05);三个药物处理组每片鳃上仅有0~2个滋养体,对照组每片鳃上滋养体数量是用药组65倍。综上所述,饲料中添加复方中草药“XCY-1”可以提升小丑鱼血清、肝脏非特异性免疫指标和肠道消化酶活性,对刺激隐核虫病起到积极的预防和治疗效果。20 g/kg剂量组、投喂时间14~21 d时效果最佳。该研究为开发无公害、环保型天然植物源杀虫药提供理论参考和技术支撑。     

Breeding and mass scale rearing of clownfish Amphiprion percula: feeding and rearing in brackishwater

Breeding and mass scale larval rearing of clownfish Amphiprion percula is very limited in brackishwater. We designed an indoor program of A. percula culture in brackishwater with a salinity of 24±1, during which the impacts of feed type, water temperature, and light intensity, on the efficiency of its reproduction, were revealed. The fish were accommodated along with sea anemones in fibre glass tanks to determine the influence of brooder diet on breeding efficiency. Higher reproductive efficiency [number of eggs laid (276 22.3 eggs)] was observed when fish were fed live Acetes sp. rather than clam (204 16.4 eggs), trash fish (155 12 eggs) and formulated feed (110 10 eggs). The spawning rate was increased during September and October (water temperature, 28.74 0.55°C) on average of 2.4 spawning per month; and low spawning rate was in January (water temperature, 24.55 0.45°C) on average of 1 spawning per month. Among three light intensities (100, 500, and 900 lx) set to evaluate larval survival rate, larvae showed the highest survival rate (65.5%) at 900 lx. The breeding method specifically in brackishwater developed in the present study is a new approach, will help the people from the regions of estuary and backwater to enhance their livelihood and it will lead to reduce the exploitation from the wild habitat.     

双锯鱼(Amphiprion)胚胎不同发育阶段和成鱼不同组织中内参基因的筛选及应用

双锯鱼(Amphiprion)亦称为海葵鱼,又称为小丑鱼,是一类经济价值较高的海水观赏鱼。目前国内在双锯鱼的胚胎发育、人工饲养以及形态学观察等方面已经取得了有效成果,但在分子生物学水平上对其基因表达的研究较少。为了筛选出适用于双锯鱼胚胎不同发育阶段以及成鱼组织的内参基因,分析酪氨酸酶(TYR)基因的表达情况,以眼斑双锯鱼(A.ocellaris)和白条双锯鱼(A.frenatus)为材料,利用实时定量聚合酶链式反应(qPCR)对18SrRNA、GAPDH(甘油醛-3-磷酸脱氢酶基因)、Ef-1α(转录延伸因子基因)和β-actin(β-肌动蛋白基因)这4个候选内参基因的表达水平进行检测,同时通过geNorm和Norm Finder软件对其稳定性进行评估,最后以合适的内参基因作为参考,研究TYR mRNA的表达水平。结果表明,在双锯鱼胚胎的不同发育阶段,18S和β-actin的表达量相对于其他基因较为稳定;在双锯鱼的不同组织中,稳定性依次为18S>β-actin>Ef-1α>GAPDH。以18S作为内参基因时分析发现TYR基因的表达在双锯鱼胚胎发育过程中呈先上升后下降的趋势,在体节期表达量最高;在双锯鱼各组织中均有表达,且在眼、尾和红皮中表达量最高。     

短角异剑水蚤对克氏双锯鱼稚鱼成活、生长和脂肪酸组成的影响

为评估短角异剑水蚤(Apocyclops royi)作为海水鱼类仔稚鱼的饵料价值和投喂效果,比较分析了其与卤虫无节幼体(Artemia nauplii)的脂肪酸组成及2种生物饵料对克氏双锯鱼(Amphiprion clarkii)稚鱼存活、生长和脂肪酸组成的影响。结果显示,短角异剑水蚤DHA、EPA和ARA含量分别达21.185%、11.088%和3.250%,海水仔稚鱼必需脂肪酸(EFA)总含量高达37.417%,均显著高于卤虫无节幼体。投喂短角异剑水蚤的克氏双锯鱼稚鱼(5~20 d)成活率(80.71%±8.23%)与投喂卤虫无节幼体组(76.30%±7.00%)差异不显著;稚鱼体长(7.75 mm±1.18 mm)、体重(0.0163 g±0.0080 g)和体长特定生长率(3.46%±0.75%)均显著高于卤虫无节幼体投喂组;稚鱼体内DHA(21.843%)、EPA(6.914%)和ARA(2.725%)含量也均显著高于后者。研究表明,短角异剑水蚤适于作为海水鱼类仔稚鱼的生物饵料,在水产养殖中具有广阔的应用前景。     

眼斑双锯鱼(Amphiprion ocellaris)发育中体色花纹时序发生的色素细胞变化和控制基因表达的分析I.胚胎时期

眼斑双锯鱼(Amphiprion ocellaris)属于鲈形目、雀鲷科、双锯鱼属, 是热带珊瑚礁观赏鱼类的首选品种。不同体色和花纹与色素细胞相关基因的表达直接相关, 在其胚胎发育的过程中, 色素细胞的形态和数量一直在变化, 但不同发育时期各种色素细胞的动态变化及其控制基因表达情况等仍需明确研究。记录了眼斑双锯鱼胚胎时期色素细胞变化较为明显的6个发育时期, 观察了不同时期的体色及色素细胞的变化特点, 并利用荧光定量PCR检测了各发育时期的10个体色控制基因的表达情况。结果显示: 在胚胎发育阶段, 整个胚胎的颜色从橙黄逐渐转变为暗红、黑红、黑色到最终透明, 观察到卵黄囊表面幼体黑色素细胞到成体黑色素细胞的转变过程, 鱼体表面在翻转期出现成体黑色素细胞、在器官形成期出现红色素细胞, 眼睛及腹部在孵化期出现虹彩色素细胞。结合荧光定量PCR结果分析发现: 卵黄囊表面幼体黑色素细胞推测是外胚层中的神经嵴细胞直接发育而成, 不需要迁移过程; Ednrb、TYR、Tbx2b基因对幼体黑色素细胞的形成和发育有重要作用, Pax3、Dct、Aim1基因与成体黑色素细胞的分化、迁移、形成过程密切相关; 其中TYR基因的相对表达量在体节期出现显著增加, 说明TYR基因在胚胎发育初期就参与黑色素细胞的形成; Tbx2b基因不仅对黑色素细胞形成有影响, 对眼斑双锯鱼心脏的形成也有一定作用; Fms基因对红色素细胞形成有重要作用; Ltk基因的表达要早于虹彩色素细胞的出现; 相关性分析发现各基因之间存在相互作用。     

红小丑人工繁殖和育苗的初步研究

为了了解红小丑鱼(Amphiiprion frenatus)在人工饲养条件下繁殖和仔鱼培养的条件,采用实验生物学的方法,在严格控制的条件下培养亲鱼、胚胎和仔鱼.结果表明,红小丑每次产卵500～1 100粒,为黏性卵,成片集中附着在靠近海葵的坚硬物体表面,亲鱼有护卵行为.受精卵呈长椭圆形,长径为2.6 mm±0.2 mm,短径为O.9 mm±O.1 mm,经过9～10 d孵出仔鱼.初孵仔鱼平均体长3.84 mm±0.21 mm,对不饱和脂肪酸的依赖非常高,最好的饲喂方式是立即投喂经小球藻(Chlorella sp.)强化的褶皱臂尾轮虫(Bra-chionus plicatiliis),密度在10个/mL左右,并保持到第7天.从第5天开始投喂经小球藻强化的卤虫(Ar-temia sp.)幼体.第6天进入稚鱼阶段,平均体长为6.32 mm±0.32 mm.到第18天,鱼苗已转入幼鱼阶段,平均体长为14.83 mm±1.62 mm.幼鱼期投喂用小球藻或其他单胞藻饲养了2 d的小卤虫.25 d后,饵料从卤虫逐渐过渡到鱼糜或虾糜.     

环境光色对白条双锯鱼(Amphiprion frenatus)幼鱼生长和体色的影响

本试验研究了循环水养殖条件下白条双锯鱼(Amphiprion frenatus)幼鱼在不同光照环境中的生长状况和体色变化特点。设置白色、红色、黄色、蓝色、绿色五个光照试验组,每组三个重复,试验周期120d。结果表明,各试验组幼鱼存活率和特定生长率均无显著性差异(P0.05),增重率在红光组和绿光组间差异显著(P0.05)。试验鱼红光组红色皮肤胡萝卜素含量显著高于蓝光、黄光和绿光组(P0.05);白光组红皮中胡萝卜素含量显著高于蓝光组(P0.05);在眼、肝、肌肉和白色皮肤组织中胡萝卜素含量在各试验组间无显著性差异(P0.05)。白光组肝组织中酪氨酸酶活性活性显著高于红光组(P0.05);在眼、皮肤、肌肉组织中各试验组酪氨酸酶活性无显著性差异(P0.05)。研究表明,在循环水养殖条件下,白条双锯鱼在红光环境下体表类胡萝卜素含量增加,而酪氨酸酶活性减弱,有提高鱼体色泽鲜艳度的作用,此研究结果可为人工养殖海水观赏鱼的体色改善提供理论依据。     

Embryology of Maldives clownfish, Amphiprion nigripes (Amphiprioninae)

This study investigated the embryonic development of Maldives clownfish Amphiprion nigripes under natural conditions (28 - 30℃) at a lagoon of Agatti Island, Lakshadweep, India. The newly deposited fish egg was capsule-shaped and orange-red, with a (0.73 ± 0.04) mm3 yolk containing 5 - 10 fat globules. The embryonic development of fertilized eggs was divided into 26 stages and the time elapsing for each stage was recorded. Results showed that the cleavage was rapid, with the first division observed 1 h 20 min after fertilization. Blastulation occurred 4 h later, followed by gastrulation 12 h after fertilization, with a yolk volume of (0.61 ± 0.06) mm3. The organogenesis process started 22 h after fertilization when the blastopores closed and notochord formation began. The embryonic stage was recorded 24 h later, with the appearance of forebrain, midbrain, hindbrain, melanophores on yolk-sac and 22 somites, and a decreased yolk volume of (0.54 ± 0.08) mm3. Other organs developed well 31 h after fertilization, whereas the heart started beating and blood circulation began 78 h later. Red pigmentation (erytrophores) appeared 96 h after fertilization, with a small yolk volume of (0.22 ± 0.02) mm3. Mouth developed well and eyes were noticeable 120 h later, with head, pectoral fin and tail frequently moving 144 h after fertilization. The embryo reached the pre-hatching stage 168 h later and started to hatch after 170 - 180 h incubation. This study first detailed the embryonic development and yolk absorption of A. nigripes under natural conditions.