Three‐dimensional visualization of green fluorescence protein‐labelled Edwardsiella tarda in whole Medaka larvae

The invasive fish pathogen Edwardsiella tarda is common in aquatic environments and causes the environmentally and economically destructive emphysematous putrefactive disease called edwardsiellosis. In order to understand the organism's infection pathway, medaka larvae (Oryzias latipes) were immersion‐infected with E. tarda labelled with green fluorescence protein (GFP) and then visualized in three dimensions under confocal laser microscopy and light‐sheet fluorescence microscopy. Confocal microscopy revealed GFP‐labelled E. tarda in the mouth, head, gill bridges, gill cover, skin, membrane fin, gastrointestinal tract and air bladder, and in the caudal vein, somite veins, caudal artery and caudal capillaries. Light‐sheet microscopy additionally showed GFP‐labelled E. tarda in the pharyngeal cavity, muscle of the pectoral fin and cardiac atrium and ventricle. These findings suggest that during its infection of fish, E. tarda initially adheres to, and invades, the epithelial cells of the skin, gills and gastrointestinal tract (through the pharyngeal cavity); E. tarda then enters the blood vessels to access organs, including the air bladder and heart.     

基于BLAST与比较基因组学方法计算鉴定青鳉鱼的microRNA前体

microRNA（miRNA）是一类长度为18～25 nt的单链小分子RNA。它可以调控基因的表达调控。通过与目标mRNA的特定位点的结合,从而抑制蛋白质的翻译或诱导该mRNA的降解。目前鱼类miRNA方面以斑马鱼miRNA的研究较多。为发掘另一种鱼类模式生物——青鳉鱼（Oryzias latipes,medeka）的miRNA,利用BLAST同源搜索方法与比较基因组学方法,并根据成熟miRNA的序列保守性以及miRNA前体（premiRNA）的二级结构的特征,对该模式生物的pre-miRNA进行了计算鉴定与分析。通过与miRBase19中已经发布的miRNA进行比较,在青鳉鱼基因组中共找到了56条新序列。设定miRNA基因间距的阈值为5000nt,新发现的与已知的pre-miRNA共形成31个基因簇。同时,这56条新发现的pre-miRNA可以划分到33个已知的基因家族中,并分析了新发现的、保守miRNA的靶标与功能。     

钒对日本青鳉胚胎发育及仔鱼的毒性影响

为揭示钒对日本青鳉的生态毒理学效应,以日本青鳉受精卵和仔鱼为研究对象,进行了钒对日本青鳉胚胎的毒性和仔鱼的急性毒性试验。结果表明:随着钒浓度的升高,胚胎孵化率呈先升高后降低的趋势,当钒浓度大于10.13mg/L时,日本青鳉胚胎孵化率降低,胚胎畸形率增加;各钒暴露液均对日本青鳉胚胎孵化周期有较明显的迟滞效应。钒对日本青鳉仔鱼的72h、96h半致死浓度分别为0.85mg/L、0.43mg/L,安全浓度为0.0427mg/L。结论:钒对日本青鳉胚胎有一定的致畸和致死作用,对仔鱼有高毒性作用。     

3种杀菌剂对日本青鳉的急性毒性研究

[目的]探讨多菌灵及三唑类杀菌剂对水生生物的毒性.[方法]以不同鱼龄的日本青鳉(Oryzias latipes)为研究对象,采用半静态暴露法,测定了多菌灵、三唑醇、三唑酮3种杀菌剂对日本青鳉的急性毒性.[结果]多茵灵、三唑醇、三唑酮3种杀菌剂对日本青鳉成鱼、幼鱼、仔鱼的LC50(96 h)值分别为:0.749 8、0.4646、0.0512 mg/L;20.5415、14.4866、1.4735 mg/L;12.4707、11.0202、0.3536mg/L.单一杀菌剂对日本青鳉不同鱼龄毒性高低依次为:仔鱼＞幼鱼＞成鱼;3种杀菌剂之间毒性高低依次为:多菌灵＞三唑酮＞三唑醇.[结论]为相关部门制定3种杀菌剂的使用标准提供了理论依据.     

A Trial to Cryopreserve Immature Medaka (Oryzias latipes) Oocytes after Enhancing Their Permeability by Exogenous Expression of Aquaporin 3

Fish oocytes have not been cryopreserved successfully, probably because it is difficult to prevent intracellular ice from forming. Previously, we have shown in medaka that immature oocytes are more suitable for cryopreservation than mature oocytes or embryos, in terms of permeability. We have also shown in immature medaka oocytes that the exogenous expression of aquaporin 3 (AQP3), a water/cryoprotectant channel, promotes the movement of water and cryoprotectants through the plasma membrane. In the present study, we attempted to cryopreserve immature medaka oocytes expressing AQP3. We first examined effects of hypertonic stress and the chemical toxicity of cryoprotectants on the survival of the AQP3-expressing oocytes. Exposure to hypertonic solutions containing sucrose decreased the survival of oocytes, but the expression of AQP3 did not affect sensitivity to hypertonic stress. Also, AQP3 expression did not markedly increase sensitivity to the toxicity of cryoprotectants. Of the four cryoprotectants tested, propylene glycol was the least toxic. Using a propylene glycol-based solution, therefore, we tried to cryopreserve immature oocytes by vitrification. During cooling with liquid nitrogen, all intact oocytes became opaque, but many AQP3-expressing oocytes remained transparent. This indicates that the expression of AQP3 is effective in preventing intracellular ice from forming during cooling. During warming, however, all the AQP3-expressing oocytes became opaque, indicating that intracellular ice formed. Therefore, the dehydration and permeation by propylene glycol were still insufficient. Further studies are necessary to realize the cryopreservation of fish oocytes.