七彩神仙鱼 BTN1A1 基因克隆及其在嗜水气单胞菌胁迫下的表达分析

【目的】克隆七彩神仙鱼（Symphysodon aequifasciatus）嗜乳脂蛋白 1A1（Butyrophilin 1A1, BTN1A1）基因,分析其在病原刺激下的表达模式,为了解七彩神仙鱼 BTN1A1 基因功能提供依据。【方法】利用生物信息学对七彩神仙鱼 2 个 BTN1A1 基因（BTN1A1-1 和 BTN1A1-2）进行结构和进化分析。根据前期获得的七彩神仙鱼皮肤转录组数据,选取 BTN1A1-1 和 BTN1A1-2 基因的 CDS 区设计引物进行克隆。采用 qRT-PCR 分析 BTN1A1 在各组织中的表达及嗜水气单胞菌刺激下的表达模式。【结果】BTN1A1-1 和 BTN1A1-2 的 ORF 序列长度为 894、1 275 bp,分别编码 298、424 个氨基酸。BTN1A1-1 和 BTN1A1-2 蛋白均具有 1 个信号肽、1 个 Ig 结构域、1 个 lg_like 结构域和 1个跨膜结构域的经典结构,BTN1A1-2 还具有 1 个胞质结构域。七彩神仙鱼 BTN1A1-1 与慈鲷科其他鱼类相似性较高,与尼加拉瓜湖始丽鱼（Archocentrus centrarchus）BTNIAI 对应氨基酸序列同源性最高、为 94.14%。但 BTN1A1-2 与其他鱼类发生分离,形成独特分支。BTN1A1-1 和 BTN1A1-2 在七彩神仙鱼各组织中均有表达,但在鳃、肠道、皮肤等与免疫相关的组织中表达量相对较高。七彩神仙鱼 BTN1A1-1 在嗜水气单胞菌胁迫后表达量先显著下降后上升,而 BTN1A1-2 表达量则先显著上升后下降。【结论】七彩神仙鱼 BTN1A1-1 基因相对保守,而 BTN1A1-2 基因在进化上较为特殊。二者在免疫刺激下的差异表达暗示其可能在七彩神仙鱼免疫防御中发生功能分化。     

5种七彩神仙鱼5S rDNA序列的比较

采集鳍条,设计引物,提取了5种(黑格尔、盖子红、鸽子红、天子蓝、红点绿)七彩神仙鱼(Symphysodon)的5S r DNA,分析了其序列。结果表明:5种七彩神仙鱼均具有201 bp和401 bp 5S r DNA结构单元,而黑格尔(野生)、盖子红和鸽子红分别具有300,299,338 bp 5S r DNA结构单元。其编码区高度保守,而非转录间隔区差异显著,特别是黑格尔(野生)300 bp、盖子红299 bp和鸽子红338 bp的非转录间隔区。5种七彩神仙鱼G+C碱基的含量很高。红点绿、盖子红、鸽子红和天子蓝4个人工品种与野生品种黑格尔的亲缘关系分别为97.53%、90.12%、88.89%和85.19%,其中红点绿与黑格尔的亲缘关系最近。     

Minimally invasive evaluation of the anaesthetic efficacy of MS‐222 for ornamental discus fish using skin mucus biomarkers

Skin mucus has been demonstrated to provide stress biomarkers for evaluating the physiological status, providing new convenient and non‐invasive methods to detect stress response in fish. Here, we investigated the anaesthetic efficacy of tricaine methanesulphonate (MS‐222; 75–115 mg/L) for discus Symphysodon aequifasciata (34.27 ± 4.46 g; 8.10 ± 0.59 cm) using skin mucus stress biomarkers. The induction time, recovery time and respiratory frequency were also determined. According to the criteria for anaesthesia and recovery, discus fish to reach stage A3 (deep anaesthesia) within 3 min and to reach stage R4 (full recovery of normal behaviour) within 5 min were observed at 95–105 mg/L MS‐222. Respiratory frequency increased first and then decreased during MS‐222 exposure and increased after recovery. At 10 min after deep anaesthesia, a lower mucus glucose was only observed at 115 mg/L MS‐222. No change in mucus cortisol and increased lactate were observed in all treatments. Increased mucus protein was observed at 75, 85 and 95 mg/L MS‐222. At 10 min after recovery, increased mucus glucose and decreased mucus protein were observed at 85, 95 and 115 mg/L MS‐222, but increased mucus cortisol only at 115 mg/L and lactate only at 75 and 105 mg/L MS‐222. At 24 hr after recovery, mucus glucose returned to the initial level only at 75, 95 and 105 mg/L MS‐222, while cortisol at 75 and 85 mg/L and protein and lactate at 75 mg/L respectively. Overall, the effective dose of MS‐222 for discus fish has been suggested to be 95–105 mg/L.     

野生及人工养殖七彩神仙鱼肌肉成分的比较

对野生七彩神仙鱼、牛心汉堡养殖七彩神仙鱼和配合饲料养殖七彩神仙鱼的肌肉成分进行了分析比较。结果表明:野生七彩神仙鱼肌肉中粗蛋白最高,配合饲料养殖的七彩神仙鱼次之,牛心汉堡养殖的七彩神仙鱼最低(P0.05);野生七彩神仙鱼粗脂肪含量低于2种养殖七彩神仙鱼(P0.05),2种养殖七彩神仙鱼的粗脂肪含量没有显著差异(P0.05);3种不同摄食模式的七彩神仙鱼肌肉水分含量和灰分之间没有显著差异(P0.05)。3种不同摄食模式七彩神仙鱼的氨基酸组成基本一致,均含有17种氨基酸,含量最高的为谷氨酸(Glu),9种必需氨基酸中含量最高的为赖氨酸(Lys)。3种不同摄食模式的七彩神仙鱼肌肉中主要含有19种脂肪酸,6种饱和脂肪酸(SFA)、5种单不饱和脂肪酸(MUFA)和8种多不饱和脂肪酸(PUFA)中主要的脂肪酸分别为棕榈酸(C16∶0)、油酸(C18∶1)和花生四烯酸(C20∶4);19种脂肪酸中EPA(0.93%~1.86%)和DHA(1.57%~2.35%)的含量较低。为推断七彩神仙鱼的营养需求量及科学配制七彩神仙鱼配合饲料提供依据。     

盘丽鱼胚胎发育的研究

[目的]了解盘丽鱼的胚胎发育过程。[方法]对盘丽鱼胚胎发育过程进行观察,研究其胚胎发育的形态特征和发育特点。[结果]盘丽鱼的成熟卵子为粘性,呈椭球形,其纵轴和横轴分别为1.02~1.14 mm和0.75~0.79 mm。盘丽鱼的胚胎发育过程包括卵裂期、囊胚期、原肠胚期、神经胚形成期、器官形成期和孵化出膜期6个时期。在水温(30±1)℃的条件下,整个胚胎发育过程约需50 h。受精卵约50 min后进入卵裂期,3 h后进入囊胚期,9.50 h后进入原肠胚期,20 h后胚孔封闭,50 h后出膜。刚出膜的仔鱼全长为4.29 mm。盘丽鱼卵裂前期和原肠胚期发育较快,而器官形成期发育相对较慢。盘丽鱼孵化总积温为(1 500±50)℃.h。[结论]盘丽鱼与尼罗罗非鱼胚胎发育过程相似,说明它们具有较近的亲缘关系。     

Effects of dietary vitamin C and vitamin E on the growth,antioxidant defence and digestive enzyme activities of juvenile discus fish (Symphysodon haraldi)

A 56‐day experiment was carried out to investigate the effects of dietary vitamin C and vitamin E on the growth, antioxidant status and digestive enzyme activities of discus fish (Symphysodon haraldi; initial body weight: 7.96 ± 0.61 g and body length: 5.45 ± 0.65 cm). Animals were fed with 13 different diets including one control diet and 12 treatment diets containing four levels of vitamin C (magnesium‐L‐ascorbyl‐2‐phosphate; 40, 80, 120 and 160 mg/kg) crossed with three levels of vitamin E (DL‐α‐tocopheryl acetate; 40, 80 and 120 mg/kg). The results showed that the fish fed diets containing additional vitamin C (40 mg/kg) and vitamin E (80 mg/kg) showed higher specific growth rate, length growth rate, total antioxidant capacity and protease activity but had lower feed conversion ratio and total superoxide dismutase activity than those fed the control diet. Collectively, these findings suggest that the inclusion of additional 40 mg/kg of vitamin C and 80 mg/kg of vitamin E in the basal diet could have beneficial effect on the growth, antioxidant defence and digestion of S. haraldi.     

七彩神仙鱼人工繁殖主要水质条件的探讨

报道了不同硬度、酸碱度的水质对七彩神仙鱼性腺发育、产卵、授精及哺育幼苗的影响.结果表明,七彩神仙鱼性腺发育、产卵、授精和分泌"乳汁”哺育幼苗的水质最佳硬度为2～3.5°DH,最佳pH值为5.5～6.5.雄鱼繁殖时对水质条件的敏感度比雌鱼繁殖时更高.水质硬度提高到5°DH以上或pH值提高到7.0以上时,雄鱼几乎失去了对鱼卵的授精能力,而雌鱼仍能正常产卵受精.     

饲料中脂肪含量对七彩神仙鱼幼鱼生长和消化的影响

在水温29.0±1.0℃下,将140日龄、体质量8～10g的七彩神仙鱼(Symphysodon aequifasciata)幼鱼放入室内54cm×40cm×28era的水箱中,投喂以鱼粉和豆粕为蛋白源(粗蛋白45.0%)、以豆油为脂肪源配制的5种脂肪水平(10.21%、12.02%、17.17%、21.0%,和22.14%)的饲料.90d的饲养表明,七彩神仙幼鱼的成活率变化在96.67%～100.0%之间,各组间差异不显著(P>0.05).随饲料中脂肪含量的增加,鱼的特定生长率(SGR)、蛋白质效率(PER)和增重率均先增加而后降低;摄食含12.02%脂肪饲料的鱼SGR、PER和增重率极显著地高于摄食含10.22%和22.14%脂肪饲料的鱼(P<0.001),而当鱼摄食含17.17%和21.05%脂肪的饲料时,SGR、PER和增重率与摄食含12.02%脂肪饲料的鱼差异不显著(P>0.05).当饲料中脂肪含量为12.02%时,鱼对饲料中蛋白质的消化率最高;摄食含脂肪为10.22%、12.02%和17.17%的饲料时,对饲料蛋白的消化率差异不显著(P>0.05).实验表明,七彩神仙鱼幼鱼饲料中适宜脂肪含量在12.02%～17.17%之间.     

黑格尔七彩神仙鱼摩氏摩根菌分离鉴定及耐药性分析

【目的】对患病七彩神仙鱼进行摩氏摩根菌(Morganellamorganii)分离鉴定及药敏试验,为七彩神仙鱼养殖过程中摩氏摩根菌感染的临床诊断和科学用药提供科学依据。【方法】从患病野生黑格尔七彩神仙鱼体表分离病原菌,然后对其进行形态观察、生理生化指标测定及16S rDNA序列分析,并采用纸片扩散法(K-B)进行药敏试验。【结果】从患病野生黑格尔七彩神仙鱼分离获得一株优势菌株(DS2015),该菌为革兰氏阴性杆菌,其生理生化测定结果与摩氏摩根菌的相似度达100%。菌株DS2015的16S rDNA序列1208 bp(GenBank收录号KR866070),由基于16S rDNA序列构建的系统发育进化树可知,菌株DS2015与摩氏摩根菌聚为一簇,Bootstrap检验值为99;Biolog微生物自动鉴定结果也显示,菌株DS2015最具可能性为摩氏摩根菌。药敏试验结果表明,菌株DS2015对庆大霉素、新霉素、卡那霉素、哌拉西林、哌拉西林/他唑巴坦、头孢曲松、氯霉素、诺氟沙星、氧氟沙星和复方新诺明等抗生素高度敏感,对米诺环素中度敏感,对青霉素G、头孢氨苄、头孢拉定、四环素、多西环素、红霉素、克林霉素、万古霉素和呋喃唑酮等抗生素已产生耐药性。【结论】摩氏摩根菌可引起七彩神仙鱼身体发黑、皮肤大量分泌黏液及鳍条溃烂等症状,严重者甚至引起死亡,生产中可选用庆大霉素、新霉素、卡那霉素、哌拉西林、哌拉西林/他唑巴坦、头孢曲松、氯霉素、诺氟沙星、氧氟沙星和复方新诺明等抗生素进行防治。