High yield and rapid growth of Neoparamoeba pemaquidensis in co-culture with a rainbow trout gill-derived cell line RTgill-W1

Neoparamoeba pemaquidensis is an ubiquitous amphizoic marine protozoan and has been implicated as the causative agent for several diseases in marine organisms, most notably amoebic gill disease (AGD) in Atlantic salmon. Despite several reports on the pathology of AGD, relatively little is known about the protozoan and its relationship to host cells. In this study, an in vitro approach using monolayers of a rainbow trout gill cell line (RTgill-W1, ATCC CRL-2523) was used to rapidly grow large numbers of N. pemaquidensis (ATCC 50172) and investigate cell-pathogen interactions. Established cell lines derived from other tissues of rainbow trout and other fish species were also evaluated for amoeba growth support. The amoebae showed preference and highest yield when grown with RTgill-W1 over nine other tested fish cell lines. Amoeba yields could reach as high as 5 x 10(5) cells mL(-1) within 3 days of growth on the gill cell monolayers. The amoebae caused visible focal lesions in RTgill-W1 monolayers within 24 h of exposure and rapidly proliferated and spread with cytopathic effects destroying the neighbouring pavement-like cells within 48-72 h after initial exposure in media above 700 mOsm kg(-1). Disruption of the integrity of the gill cell monolayers could be noted within 30 min of exposure to the amoeba suspensions by changes in transepithelial resistance (TER) compared with control cell monolayers maintained in the exposure media. This was significantly different by 2 h (P < 0.05) compared with control cells and remained significantly different (P < 0.01) for the remaining 72 h that the TER was monitored. The RTgill-W1 cell line is thus a convenient model for growing N. pemaquidensis and for studying host-pathogen interactions in AGD.     

Relationship between soak time and number of enmeshed animals in experimentally lost gill nets

In order to determine the effects of ghost fishing by lost gill nets, the relationship was examined between soak time and number of enmeshed animals in experimentally lost gill nets by using diving observations. Two experimental gill nets were set at 13 m depth in Tateyama Bay, Chiba Prefecture, Japan for approximately 200 days. One gill net was deployed in a small trough surrounded by artificial reefs, and the other was deployed on an adjacent open sandy bed. Twelve species of crustaceans, six species of gastropods, and five species of bony fish were enmeshed in the experimental gill nets. The number of enmeshed animals in the artificial reef gill net was substantially larger than that in the sandy bed gill net. The number of enmeshed animals in the experimental gill nets increased rapidly within one month after deployment, and then declined gradually showing fluctuations caused by the decrease in newly enmeshed animals, and the drop off from gill nets caused by the decomposition of dead animals. The decrease in the number of enmeshed animals was expressed by logarithmic equations, and based on these equations, the duration of capture function for the lost gill nets was calculated to be 284–561 days in the artificial reef gill net and 200 days in the sandy bed gill net. The duration of capture function for the lost gill nets for non-commercial by-catch species such as small crustaceans and gastropods was longer than for commercial species such as Japanese spiny lobster and bony fish.     

Production of salmonid amoebic gill disease by exposure to Paramoeba sp. harvested from the gills of infected fish

Atlantic salmon, S almo salar L., were exposed to different concentrations of Paramoeba sp. The lowest concentration which induced amoebic gill disease (AGD) was 230 Paramoeba sp. L^–1 and the severity of infection increased with increasing concentration. The concentration of Paramoeba sp. positively correlated with the number of gill lesions ( R ² > 0.7). This study provides evidence that Paramoeba sp. is the causative agent of AGD and describes an experimental model that enables the severity of the induced disease to be controlled.     

尼罗罗非鱼表皮和鳃黏膜共生菌结构特征及其与鱼体健康状况相关关系

黏膜及其表面的共生菌群是鱼类抵御外界不利环境的第一道屏障。为探索养殖罗非鱼表皮和鳃黏膜共生菌菌群的结构特征是否与其健康状况间存在相关关系,本研究运用高通量测序技术,以无乳链球菌腹腔注射攻毒48 h后存活和濒死尼罗罗非鱼为检测对象,检测攻毒前后罗非鱼表皮和鳃黏膜共生菌群落结构差异。结果显示,健康尼罗罗非鱼表皮和鳃黏膜共生菌均存在优势菌群,主要为特吕珀菌属、硫杆菌属、弓形杆菌属、海单胞菌属和弧菌属。人工感染无乳链球菌后存活尼罗罗非鱼表皮和鳃黏膜共生菌菌群与感染前无显著差异;与存活组相比,濒死尼罗罗非鱼的表皮和鳃黏膜共生菌菌群多样性下降,其中弓形杆菌属、假交替单胞菌属、海单胞菌属、假单胞菌属和弧菌属等含量显著下降,链球菌属含量占总菌群的55.30%±1.24%,表明养殖尼罗罗非鱼表皮和鳃黏膜共生菌群落结构可能与其健康状况相关。     

急性氨氮胁迫及毒后恢复对团头鲂幼鱼鳃、肝和肾组织结构的影响

为进一步了解氨氮对团头鲂幼鱼的毒性毒理影响,以体质量为(14.27±0.01)g的团头鲂幼鱼为研究对象,研究了氨氮胁迫对其鳃、肝、肾组织结构的影响。实验首先进行96h的氨氮胁迫,得出96h LC50,在此基础上,设置对照组(0.472 mg/L)和实验组(25 mg/L)两个氨氮浓度处理组,进行0、6、12、24、48h的氨氮胁迫,取样后剩余团头鲂幼鱼移入曝气自来水进行96h的毒后恢复实验。结果表明：96h LC50为56.492 mg/L;三种组织观察表明,氨氮胁迫6h,鳃丝毛细血管扩张,上皮组织增生;肝细胞肿胀,细胞核肿大,肝细胞空泡化;肾小球萎缩,肾小囊腔膨大,肾小管管腔缩小;胁迫12h,泌氯细胞增生,呼吸上皮细胞出现部分脱落;肝细胞水样变性、血窦扩张、细胞轮廓模糊,形成点状病灶;肾小管上皮细胞肿大、水样变性、浊肿;胁迫24h,鳃小片融合、变短,呼吸上皮细胞大面积脱落;肝细胞水样变性、血窦扩张严重,形成局部病灶;肾组织淋巴细胞浸润严重,充血,肾小球坏死,肾小管坏死;胁迫48h,鳃小片卷曲,上皮细胞部分脱落;肝细胞部分溶解、血窦扩张,形成点状病灶;肾小管上皮细胞坏死,肾小球坏死;96h恢复后,泌氯细胞和上皮组织增生严重;肝组织大面积细胞核肿大,血窦扩张;肾组织淋巴细胞浸润严重,肾小管坏死,肾小球坏死。实验表明,不同的器官之间病症的损伤程度是不同的,肝组织的损伤最严重,然后依次是鳃和肾。随着胁迫时间延长,鳃、肝和肾组织受到的损害增加,同时鱼体也产生防御反应,但96h的恢复期不足以让团头鲂幼鱼在胁迫中完全恢复,而恢复能力最差的是肾组织。     

浅色黄姑鱼鳃结构及其呼吸面积的研究

应用扫描电镜技术对体重范围在4.75～35.51g的浅色黄姑鱼（Nibea coibor）鳃结构及鳃小片进行了观察与呼吸面积计算,研究体重、体长与呼吸面积之间的相关关系。结果表明,浅色黄姑鱼的平均相对呼吸面积为4.01±1.62cm^2·g^-1。体重与一侧鳃丝总数的相关性N=28.81＋158.45lnW（R^2=0.9670,P〈0.01）较体长与一侧鳃丝总数的相关性N=17.31L^1.4542（R^2=0.8241,P〈0.01）更为显著。单位mm鳃小片的数目随着体重的不断增加显著减少,N=49.971W^-0.3909（P〈0.01）。单个鳃小片的面积随体重的增大而显著增加,a=0.3199W^0.2528（P〈0.01）。总呼吸面积随体重的增大而显著增加,A=11.559W^0.7109（P〈0.01）。相对呼吸面积随体重的增大而显著减小,RA=11.561W^-0.2904（P〈0.01）。     

Immunohistochemical study on changes in gill Na+/K+-ATPase α-subunit during smoltification in the wild masu salmon, Oncorhynchus masou

Changes in immunoreactivity of Na⁺/K⁺-ATPase -subunit in gill sections of wild masu salmon (Oncorhynchus masou) during the parr-smolt transformation (smoltification) were compared with changes in gill Na⁺/K⁺-ATPase specific activity. Gill Na⁺/K⁺-ATPase specific activity increased from April and peaked in May. Immunohistochemical analysis, using an antiserum against a synthetic oligopeptide based on the conserved region of the Na⁺/K⁺-ATPase -subunit, revealed that immunoreactivity was confined to chloride cells in the surface layer of primary lamellae and the proximal end of secondary lamellae. The size and number of these cells increased gradually from February to May; however, the number of chloride cells of the secondary lamellae decreased in May. These data suggest that the synthesis of Na⁺/K⁺-ATPase and the proliferation of chloride cells occur prior to the elevation of enzyme activity. Moreover, it is likely the proliferation and hypertrophy of chloride cells on primary lamellae prepare smolts for entry into seawater and migration in the ocean.     

鲢滤食器官胚后发育生物学的研究

通过显微解剖、肉眼解剖、石蜡切片、光镜和扫描电镜观察等方法,系统观测全长5～395mm鲢标本730多尾,描述了鲢鳃耙和鳃耙网、腭褶、鳃耙管等滤食器官的形态学特点和数量性状在胚后发育过程中的变化规律;论述了鲢滤食器官发育与摄食方式的转化、摄食器官的数量性状发育与适口食物和食物组成的转变、鳃耙间距在胚后发育过程中的变化、鳃耙管的功能、鲢对食物的选择性、鲢滤食器官发育与苗种培育阶段的划分和饲养生物学技术措施。为制定培育鲢苗种和饲养食用鱼生物学技术措施提供了理论依据。     

凡纳滨对虾Toll受体和溶菌酶mRNA组织表达的差异研究

采用荧光定量PCR技术,检测了凡纳滨对虾不同组织(血淋巴、鳃、肌肉和肝胰腺)中Toll受体和溶菌酶mRNA的相对表达量。结果表明:凡纳滨对虾Toll受体和溶菌酶mRNA在不同组织间的表达量存在显著差异。Toll受体mRNA在各组织中的相对表达水平由高到低顺序依次为血淋巴>鳃>肌肉>肝胰腺。溶菌酶mRNA表达水平则依次为血淋巴>鳃>肌肉>肝胰腺,但血淋巴和鳃之间无显著差异。溶菌酶mRNA和Toll mRNA表达量重复测定间的变异度在血淋巴和鳃组织间无显著差异。表明鳃和血淋巴是检测凡纳滨对虾Toll受体和溶菌酶mRNA的相对表达量的理想实验材料。研究亮点:Toll受体能够将异物入侵的信号从细胞外传递到细胞内并引起胞内信号级联反应产生各种免疫效益因子,因此其表达水平一定程度上可以反映对虾对病原入侵的灵敏性。溶菌酶活性是评判对虾免疫抗菌能力的重要指标。研究Toll受体和溶菌酶基因表达的组织差异性,可以为后续开展对虾营养免疫学研究中免疫相关基因检测时实验材料的选取提供理论基础。     

pH对日本沼虾耗氧率及鳃组织CAT、SOD活力的影响

在水温24.0±0.2℃条件下,采用静水停食法开展了pH对日本沼虾存活、耗氧率及鳃组织CAT、SOD活力的影响实验。结果表明：（1）日本沼虾对碱性环境的耐受性略强于酸性环境,其pH耐受范围为5.5～8.5,该范围内日本沼虾组内耗氧率均呈夜均显著高于昼均（P〈0.05）,组间昼均和夜均耗氧率均无显著差异（P〉0.05）,且鳃组织SOD、CAT活力均呈先降后升走势;（2）日本沼虾pH适宜范围为6.5～8.5,该范围内日本沼虾鳃组织SOD低谷期均比pH 5.5实验组后延24 h,各观测时段CAT活力均显著低于pH 5.5实验组（P〈0.05）;（3）日本沼虾pH最适宜区间为6.5～8.5的中央区域,本研究所设实验梯度中pH 7.5实验组日本沼虾鳃组织SOD、CAT活力最为稳定。