斑点叉尾肠道及其养殖环境菌群结构分析

为探究健康斑点叉尾(鱼回)肠道及其与养殖池塘水体中菌群结构的关系,采用16S rDNA高通量测序方法和生物信息学分析手段考察其肠道和养殖水体中微生物群落组成和多样性。多样性指数分析结果表明,斑点叉尾(鱼回)肠道菌群丰度和菌群多样性均较养殖水体低。斑点叉尾(鱼回)肠道中有69.57%的运算分类单元与水体中一致,水体中优势菌群主要为变形菌门、放线菌门和蓝藻门,而肠道中的优势菌群为拟杆菌门、梭杆菌门和厚壁菌门,说明肠道和养殖水体中的优势微生物种群的组成和丰度无明显的相关性,肠道中菌群组成有一定的独立性,其优势菌群结构几乎不受养殖环境菌群的影响。PICRUSt预测细菌表型发现,斑点叉尾(鱼回)肠道中以厌氧菌和革兰氏阳性菌为主;从生物膜形成和致病潜力表型可知,肠道和水环境中均存在大量条件致病菌。试验结果表明,池塘养殖健康斑点叉尾(鱼回)肠道微生物中的优势益生菌主要为鲸杆菌属和拟杆菌属,平均相对丰度高达36.60%和15.82%。本试验将为斑点叉尾(鱼回)的健康养殖和益生菌的筛选提供理论依据。     

应用PCR-DGGE和Q-PCR分析不同体重斑点叉尾鮰皮肤、鳃和胃肠道菌群结构

采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)和荧光定量-聚合酶链式反应(Q-PCR)技术分析高体重(high weight,HW)和低体重(low weight,LW)斑点叉尾鮰鮰(Ietalurus punetaus)皮肤、鳃和胃肠道菌群多样性,为斑点叉尾鮰微生态研究及筛选斑点叉尾鮰源益生菌提供理论依据。结果显示,斑点叉尾鮰菌群丰富度由低到高依次为鳃、皮肤、前肠、后肠和胃。肠杆菌科(Enterobacteriaceae)和气单胞菌属(Aeromonas)是皮肤的优势菌群;肠杆菌科(Enterobacteriaceae)、气单胞菌属(Aeromonas)和肠球菌属(Enterococcus)是水体、鳃和胃的优势菌群;肠杆菌科(Enterobacteriaceae)、拟杆菌属(Bacteroidetes)、气单胞菌属(Aeromonas)和酵母菌属(Saccharomyces)是肠道的优势菌群。HW斑点叉尾鮰鳃菌群的香农多样性指数、均匀度和丰富度及前肠菌群的丰富度显著高于LW斑点叉尾鮰(P0.05)。皮肤的黄杆菌属(Flavobacterium),胃的肠杆菌科(Enterobacteriaceae),前肠的拟杆菌属(Bacteroidetes)和双歧杆菌属(Bifidobacterium)及后肠的拟杆菌属(Bacteroidetes)、双歧杆菌属(Bifidobacterium)和酵母菌属(Saccharomyces)的拷贝数分别是101.97、107.69、106.19、103.83、106.13、103.92和104.26,均显著高于LW斑点叉尾鮰(P0.05)。结果表明,斑点叉尾鮰皮肤、鳃、胃肠道均形成独特的菌群结构,LW和HW斑点叉尾鮰菌群结构存在明显差异,HW斑点叉尾鮰菌群多样性增加。     

斑点叉尾(鱼回)鳃结构的扫描电镜观察

应用扫描电镜对斑点叉尾鮰（Ictaluruspunctatus）鳃的表面微细结构进行了观察。结果表明,斑点叉尾鮰鳃的表面结构和微细结构与其他硬骨鱼类基本相似,鳃弓和鳃耙表面被具环形微嵴的扁平上皮细胞所覆盖,鳃弓表面细胞之间有孔洞;鳃耙基部较粗壮;中部鳃丝上皮细胞表面的微嵴两侧有细小的横突,有些微嵴出现融合或间断;端部和基部鳃丝上皮细胞表面的微嵴消失,形成密集颗粒状突起;鳃小片的高度和片间距较大。讨论了斑点叉尾鮰鳃的形态结构与其生理功能的关系。     

斑点叉尾鮰肝胆综合症继发烂鳃病的防治方法

斑点叉尾鱼回吃食出现异常,部分网箱的鱼出现闭口现象。解剖5尾病情严重的病鱼,症状相似。外消内服是治疗肝胆综合症继发细菌性鱼病的最好方法,但最佳的方法仍是投喂营养全面的饲料,避免药源性肝胆损伤,增强鱼的体质。     

红色和黑色斑点叉尾鮰人工繁殖试验

通过人工控制手段,促使红色斑点叉尾鮰与黑色斑点叉尾鮰自行配对、产卵、收集受精卵集中孵化,获得浅黑色的鱼苗;要获得稳定的红鮰鱼苗,必须采取红色斑点叉尾鮰自交的方法。     

红色和黑色斑点叉尾鱼回人工繁殖试验

通过人工控制手段,促使红色斑点叉尾鮰与黑色斑点叉尾鮰自行配对、产卵、收集受精卵集中孵化,获得浅黑色的鱼苗;要获得稳定的红鮰鱼苗,必须采取红色斑点叉尾鮰自交的方法。     

发酵饲料投喂方式对斑点叉尾鮰生长、肠道菌群及代谢组学的影响

本研究对斑点叉尾鮰(Ictalurus punctatus)的发酵饲料进行了合理饲喂,并确定了其对斑点叉尾鮰生长、肠道菌群和代谢组学的影响。实验设置3组：持续投喂组(A)(即在膨化饲料中持续添加发酵饲料),间隔投喂组即向膨化饲料中按周间隔添加发酵饲料(B),对照组仅投喂膨化饲料(C)。实验共持续6个月。实验结束后,对斑点叉尾鮰进行称重,并用16S rRNA扩增子测序技术和基于液相色谱的代谢组学技术分别检测斑点叉尾鮰肠道菌群组成以及代谢组学。结果显示,间隔投喂组斑点叉尾鮰终末体重(FBW)显著高于对照组和持续投喂组(P<0.05)。间隔投喂组的群落丰富度和多样性最高,持续投喂组群落丰富度最低(P>0.05)。厚壁杆菌门(Firmicutes)、放线菌门(Actinobacteriota)、蓝藻门(Cyanobacteria)、变形菌门(Proteobacteria)、梭杆菌门(Fusobacteriota)、绿弯菌门(Chloroflexi)和异常球菌门(Deinococcota)是优势细菌门。优势肠道菌属包括未命名的叶绿体科中的一个属(norank_f_norank_o_Chloroplast)、分枝杆菌属(Mycobacterium)、鲸杆菌属(Cetobacterium)、罗姆布茨菌属(Romboutsia)、微小杆菌属(Exiguobacterium)和狭义梭菌属(Clostridium_sensu_stricto_1)。代谢组学分析表明,持续投喂组主要通过N-乙酰-D-半乳糖胺显著上调影响肠道菌群半乳糖和磷酸转移酶系统代谢通路(P<0.05),进而影响鱼体对碳水化合物的消化吸收。而间隔投喂组差异代谢产物L-丝氨酸和L-苯丙氨酸显著上调(P<0.05),影响斑点叉尾鮰肠道菌群硫代谢、氨基酸代谢等,继而影响斑点叉尾鮰能量吸收、抗炎症和免疫等能力。本研究为斑点叉尾鮰发酵饲料投喂方式的探索和健康绿色养殖提供了理论依据。     

斑点叉尾生物学研究

斑点叉尾(鱼回)为原产于美洲的大型经济鱼类,美国自二十世纪60年代开始商业养殖,我国于1986年引进.我们对其进行了三年的研究,在观察解剖的基础上,系统总结了斑点叉尾(鱼回)的生物学特点.     

斑点叉尾(鱼回)肝胆综合症继发烂鳃病的防治方法

斑点叉尾(鱼回)吃食出现异常,部分网箱的鱼出现闭口现象.解剖5尾病情严重的病鱼,症状相似.外消内服是治疗肝胆综合症继发细菌性鱼病的最好方法,但最佳的方法仍是投喂营养全面的饲料,避免药源性肝胆损伤,增强鱼的体质.     

穿心莲对斑点叉尾(鮰)生长性能的影响

为研究穿心莲对斑点叉尾(鮰)生长性能的影响.设置8个试验组,每组3个重复,各试验组投喂穿心莲粉含量分别为0％,0.38％,0.75％,1.13％,1.88％,2.25％,2.63％和3.00％的饲料,试验周期为30 d.结果显示,各试验组鱼体的体质量增长率和特定生长率均随穿心莲添加量的增加呈先上升后下降的趋势,其中G4...     

斑点叉尾胚胎及卵黄囊期仔鱼发育的观察

试验对斑点叉尾的胚胎及卵黄囊期仔鱼形态发育的过程进行观察、描述,研究了其发育特征。结果表明：斑点叉尾受精卵在水温24±1℃时,出膜时间（胚胎期）约为147h 30min。水温25±1℃时,斑点叉尾鮰卵黄囊期约为134h。     

斑点叉尾鮰水库网箱无公害规模化养殖试验

2005年4月至2007年12月,在漳州市漳浦县马坪镇杨美水库淡水网箱养殖基地,进行斑点叉尾鮰无公害养成及示范推广试验.养殖期间,根据农业部无公害食品斑点叉尾鮰相关标准,分别经福建省渔业环境监测站及福建省海洋与渔业局进行环境检测,认定了斑点叉尾鮰无公害养殖产地.养殖产品分别经农业部农产品质量安全中心、农业部渔业产品质量监督检验测试中心(厦门)、福建出入境检验检疫局检验检疫技术中心检验,认定为无公害产品.形成规范化的斑点叉尾鮰无公害养殖模式,并示范推广.     

Encapsulation of Bolbophorus damnificus (Digenea: Bolbophoridae) Metacercariae in Juvenile Channel Catfish,Ictalurus punctatus,Is Linked to Delayed‐onset Mortality

Bolbophorus damnificus (Digenea: Bolbophoridae) causes significant economic losses in catfish aquaculture in the southeastern USA. To determine mortality trends associated with B. damnificus exposure, three separate disease challenges were conducted. The first challenge exposed channel catfish, Ictalurus punctatus, fingerlings (7–13 cm) to approximately 285 B. damnificus cercariae/L, while the second exposed fish to approximately 215 cercaria/L. Fish were monitored for morbidity and mortality for 28 d. In both studies, mortality first occurred 8 d postchallenge (PC) and peaked at 9 and 10 d PC, respectively. Mortality had subsided by 14 d PC, with only sporadic mortality occurring >15 d PC. Dead and moribund fish presented with lesions consistent with B. damnificus infection. A third challenge was performed to record histological changes associated with parasite development from initial exposure up to the cessation of mortality. Catfish (2–5 cm) were exposed as described above to a range covering 0, 35, 70, 100, 150, 175, and 215 cercariae/L. Each day, two fish were sampled from each treatment. Mortality trends were consistent with previous challenges. The mortality window coincided with parasite encapsulation, neovascularization, and development of the host‐derived cyst, suggesting that morbidity and mortality are linked to host pathophysiological changes in response to metacercariae encapsulation.     

斑点叉尾鮰胚胎及卵黄囊期仔鱼发育研究

探索研究了斑点叉尾鮰(Ictalurus punetatus)的早期牛活史及其发育生物学.运用解剖镜观察、记录、测量、描述、绘图等方法,连续规察了6个批次斑点叉尾鮰的胚胎及卵黄囊期仔鱼发育情况.结果表明:(1)在水温23～25℃时,斑点叉尾鮰胚胎期约需186 h,有效积温4 490～4 6lO qC·h;水温24～27℃时,班点叉尾鮰卵黄囊期约需6 d,有效积温3 620～3 710℃·h.(2)斑点叉尾鮰胚胎在出膜之前可见血液循环、胸鳍突起、眼球色素、直肠和肛门,卵黄囊耗尽期时各鳍鳍条明显且尾鳍已分叉.(3)斑点义尾鮰胚胎发育的温度上限是28～29℃.     

投喂频率对斑点叉尾鮰幼鱼生长、饲料利用和鱼体组成的影响

通过8周的生长试验,研究斑点叉尾鮰(Ictalurus punctatus)幼鱼(初始体重约4.5 g)的最适投喂频率。试验饲料为自制配合饲料,粗蛋白38%,总能19.0 kJ/g。设计了1～5次/d(M1-M5)5种投喂频率。结果表明:M2、M3、M4组摄食率均显著高于M1和M5组(P<0.05),其中M4组最大。M3、M4、M5组的特定生长率均显著高于M1组(P<0.05),其中M3组最大。M2、M3、M4、M5组的干物质表观消化率均显著高于M1组(P<0.05),M3和M4组的蛋白和能量表观消化率均显著高于M1和M2组(P<0.05)。M3组干物质含量显著高于M1和M2组(P<0.05),M2、M3、M4和M5组的粗蛋白、粗脂肪和能量含量均显著高于M1组(P<0.05)。因此,本研究建议,斑点叉尾鮰幼鱼养殖的最适投喂频率为3次/d。     

斑点叉尾鮰TICAM在细菌和病毒感染后的基因表达特征

采用实时定量RT-PCR方法研究斑点叉尾在不同病原(链球菌、嗜水气单胞菌、迟钝爱德华菌、斑点叉尾病毒)感染后TICAM基因在mRNA水平的组织和时空表达特征。感染嗜水气单胞菌可引起TICAM基因在肝脏和脾脏中的上调表达,在注射后24h和12h后上调最大分别为2.3倍和1.9倍;而在头肾和后肠中的表达则下调到感染后48h的0.15倍和24h的0.53倍;感染链球菌后则导致在肝脏、脾脏、后肠和头肾中TICAM基因表达的强烈上调,最大上调幅度为感染后7d肝脏中表达提高了23倍,其次,在脾脏和头肾中基因表达最大可上调到感染前的10倍左右;在感染迟钝爱德华菌后,TICAM基因在肝脏、脾脏、后肠和头肾中表达上调。其中,感染后7d脾脏中的表达提高到感染前的23.1倍。而感染叉尾病毒后,TICAM基因在肝脏、头肾和后肠的表达上调但幅度不大,基因表达在4种组织内表达变化量在1.5～3.7倍范围内波动,而在脾脏中TICAM表达下调,在感染24h后达到最低为感染前的0.13倍。以上实验结果显示,斑点叉尾TICAM基因表达变化与病原感染密切相关,暗示TICAM基因在斑点叉尾天然免疫中起重要作用。     

不同商品饲料对生长、体色及肝脏组织结构的影响

本试验评估了(Ictalurus punctatus)主产区4种商品颗粒饲料(分别记为S1、S2、S3和S4,其中S4为膨化饲料,其他均为颗粒饲料)和自行设计的颗粒饲料(记为S5)对试验阶段生长性能、体色变异、肝胰脏组织结构及形态指标的影响。7周的饲喂试验结果显示:饲料蛋白含量为38%的S4膨化饲料可明显的提高养殖鱼的体重,平均增重率为262.6%,但存活率较低,仅为72.3%;而喂养S5饲料组的增重率为168.89%,且存活率为92.4%,高于其他饲料组。养殖结束后,不同试验组的肥满度、脏体比有所差异,但均未达到显著性水平(P>0.05)。投喂S2饲料组养殖鱼体体色发白比较明显,体色异常比例高达91%;S4饲料组养殖后体色发白也比较明显,体色异常比例为72.3%;对背部黑色素扫描研究进一步证实饲喂S2饲料和S4饲料的黑色素细胞发生了变化,黑色素细胞边缘变得模糊,而自配S5饲料的黑色素细胞树突状分支较多;饲喂S4饲料组的背部斑点比例为12.15%,而S2饲料组的背部斑点比例为26.38%,自配S5饲料组的背部斑点比例为35.69%,S4饲料组的斑点比例明显较低。     

Effects of Feeding Diets with and without Fish Meal on Production of Channel Catfish,Ictalurus punctatus,Stocked at Varying Densities

ABSTRACT

Animal protein, generally fish meal, has traditionally been used in the diet of channel catfish. However, our previous research indicates that animal protein is not needed for growing stocker-size catfish to food fish when the fish are stocked at densities typical of those used in commercial catfish culture. Whether this holds when fish are stocked at high densities is not known; thus, we conducted an experiment to evaluate the effect of feeding diets with and without fish meal to channel catfish stocked in earthen ponds at different densities. Two 32% protein-practical diets containing 0% or 6% menhaden fish meal were compared for pond-raised channel catfish, Ictalurus punctatus, stocked at densities of 14,820, 29,640, or 44,460 fish/ha. Fingerling channel catfish with average initial weight of 48 g/fish were stocked into 30 0.04-ha ponds. Five ponds were randomly allotted for each fish meal level?×?stocking density combination. Fish were fed once daily to satiation for two growing seasons. There was a significant interaction between stocking density and fish meal for net production; net production increased in fish fed a diet containing fish meal compared with those fed an all-plant diet at the highest stocking density, but not at the two lower stocking densities. Net production of fish fed diets with and without fish meal increased as stocking density increased. Viewing the main effect means, weight gain decreased and feed conversion ratio increased for fish stocked at the two highest densities, and survival was significantly lower at the highest stocking density. Visceral fat decreased in fish at the two highest stocking densities. Body composition data were largely unaffected by experimental treatment except for a reduction in percentage filet fat in fish at the highest stocking density, and fish that were fed diets containing fish meal had a lower percentage fillet protein and a higher percentage fillet fat. It appears that at stocking densities two to three times higher than generally used, animal protein (fish meal) may be beneficial in the diet of channel catfish. In regard to stocking densities, high stocking results in higher overall production, but the average fish size decreased as stocking density increased.     

Development of an Indirect ELISA to Detect Humoral Response to Flavobacterium columnare Infection of Channel Catfish,Ictalurus punctatus

ABSTRACT

The humoral antibody response of channel catfish, Ictalurus punctatus, to experimental Flavobacterium columnare infection was measured in control (non-infected) and infected (intraperitoneal- and intramuscular-injected) fish by an indirect enzyme-linked immunoassay (ELISA). The antibody response of the experimentally infected fish was significantly higher (P<0.0001) than that of the non-infected channel catfish by both indirect ELISA and agglutination. The indirect ELISA utilized goat anti-channel catfish IgM and a commercially available rabbit anti-goat IgG enzyme conjugate. The antibody response was directed against a cell-free sonicated antigen preparation derived from live whole F. columnare cells. Indirect ELISA and agglutination results correlated (r² = 0.60) for anti-F. columnare antibody response in experimentally and non-infected fish. We were also able to correlate ELISA and agglutination results of 60 fish naturally infected with F. columnare(r² = 0.76). Indirect ELISA will allow for rapid monitoring of humoral antibody, following natural exposure or vaccination against F. columnare, with a small sample of serum.