香鱼(Plecoglossus altivelis)CC型趋化因子受体3(CCR3)基因的分子鉴定及其对鳗弧菌感染响应的研究

CC趋化因子受体3(CCR3)是CC型趋化因子的受体,属于G蛋白耦联受体(GPCRs)家族成员,与过敏等多种炎性疾病密切相关。为探讨香鱼CCR3(PaCCR3)在响应鳗弧菌感染时的表达变化,从转录组测序数据中获得了PaCCR3基因全长cDNA序列。序列分析表明,PaCCR3具有7次跨膜结构,与胡瓜鱼CCR3氨基酸序列同源性最高(84.6%)。系统进化树分析表明,哺乳类、两栖类和鱼类CCR3单独成簇,鱼类CCR3形成一个大簇;PaCCR3与胡瓜鱼CCR3进化相关性最高。实时荧光定量PCR结果显示,PaCCR3基因mRNA在单核/巨噬细胞(MO/MФ)中表达量最高;鳗弧菌感染后肝、脾和头肾中PaCCR3基因mRNA表达量显著上调;且香鱼MO/MФ经鳗弧菌体外刺激后,PaCCR3基因mRNA的表达量显著上调。Western blot分析表明经鳗弧菌体外刺激后,香鱼MO/MФ中PaCCR3蛋白的表达量也显著增加。综上,PaCCR3基因mRNA及其编码蛋白响应于病原体感染而被诱导表达。研究结果首次揭示了香鱼CCR3基因mRNA及蛋白的表达响应于鳗弧菌侵染,它可能通过介导MO/MФ的功能活性参与免疫应答,可为深入探究鱼类CCR3的功能及作用机制提供新的切入点。

香鱼(Plecoglossus altivelis)致病性类志贺邻单胞菌的分离与生化药敏鉴定

香鱼(Plecoglossus altivelis)是名贵洄游性鱼类,由于高密度养殖等因素导致病害频发。2021年7月强台风“烟花”过境后浙江省宁海县养殖香鱼暴发体表溃烂病,死亡严重。病鱼皮肤、肌肉溃烂并伴有周围皮肤充血。组织切片观察显示病鱼患处肌纤维断裂,周围红细胞、炎性细胞浸润;鳃丝和鳃小片中红细胞增多;肝细胞核呈空泡,部分细胞核染色质边集;脾脏内红细胞数量减少,部分白细胞聚集;肠黏膜上皮细胞脱落。从患病香鱼肠中分离到一株菌(编号NH21),其菌落低突、边缘光滑表面湿润,该菌株为革兰氏阴性菌,菌体呈短杆状、端圆。经生化特性和16S rDNA序列分析鉴定NH21菌株为类志贺邻单胞菌(Plesiomonas shigelloides)。用该菌株对健康香鱼进行回归感染后,人工感染香鱼呈现与本次体表溃烂病香鱼相似的症状,经计算NH21菌株对香鱼的半致死剂量(LD_(50))为2.51×10^(7) CFU/mL。25种抗生素的药敏试验显示NH21菌株对阿奇霉素、头孢氨苄、新生霉素、多黏菌素B、头孢西丁、呋喃唑酮6种抗生素敏感。从NH21菌株中检出了毒力基因9个、耐药基因8个。综合分析表明,分离得到的类志贺邻单胞菌NH21菌株是本次宁海县香鱼体表溃烂病的病原菌,研究结果为香鱼出现类似病症的预防和治疗提供了基础资料。

Screening of stable internal reference genes by quantitative real-time PCR in humpback grouper Cromileptes altivelis

Humpback grouper Cromileptes altivelis is one commercial fish with considerable economic value.To determine the expression stabilities of six commonly used internal reference genes in C.altivelis challenged by Vibrio harveyi and viral nervous necrosis virus(VNNV)through quantitative real-time PCR(qRT-PCR),the expression levels of selected genes in five immune organs stimulated with pathogenic infection were carefully evaluated using algorithms of geNorm,NormFinder,and BestKeeper.The results show that the expre ssion stabilities of the six candidate inte rnal reference genes were diffe re nt.Under no rmal physiological conditions,RPL13 were identified as the most stably expressed genes among five different immune organs(liver,spleen,kidney,intestine,and gill).After V.harveyi stimulation,RPL13,RPL13,EF1 A,RPL13,and EF1 A were identified by geNorm,NormFinder,and BestKeeper as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.Combining these three algorithms suggested that under stimulation of VNNV,RPL13,EF1 A,Actin,RPL13,and Actin were as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.These results suggest that specific experiment conditions and tissue types shall be considered when selecting the reference genes in qRT-PCR analysis.This study provided a solid foundation for future studies on gene expression of C.altivelis under different conditions.

Comparative transcriptome analysis on the alteration of gene expression in ayu （Plecoglossus altivelis）larvae associated with salinity change

Ayu （Plecoglossus altivelis） fish, which are an amphidromous species distributed in East Asia, live in brackish water （BW） during their larval stage and in fresh water （FW） during their adult stage. In this study, we found that FW-acclimated ayu larvae exhibited a slower growth ratio compared with that of BW-acclimated larvae. However, the mechanism underlying FW acclimation on growth suppression is poorly known. We employed transcriptome analysis to investigate the differential gene expression of FW acclimation by RNA sequencing. We identified 158 upregulated and 139 downregulated transcripts in FW-acclimated ayu larvae compared with that in BW-acclimated larvae. As determined by Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway mapping, functional annotation of the genes covered diverse biological functions and processes, and included neuroendo- crinology, osmotic regulation, energy metabolism, and the cytoskeleton. Transcriptional expression of several differentially expressed genes in response to FW acclimation was further confirmed by real-time quantitative PCR. In accordance with transcriptome analysis, iodothyronine deiodinase （ID）, pro-opiom- elanocortin （POMC）, betaine-homocysteine S-meth- yltransferase 1 （BHMT）, fructose-bisphosphate aldolase B （aldolase B）, tyrosine aminotransferase （TAT）, and Na＋-K＋ ATPase （NKA） were upregulated after FW acclimation. Furthermore, the mRNA expressions of b-type natriurefic peptide （BNP） and transgelin were downregulated after FW acclimation. Our data indicate that FW acclimation reduced the growth rate of ayu larvae, which might result from the expression alteration of genes related to endocrine hormones, energy metabolism, and direct osmoregulation.

Morphological Comparison between Chinese Ayu and Japanese Ayu and Establishment of Plecoglossus altivelis chinensis Wu&Shan subsp.nov.

The countable characters of Qingdao, Zhejiang, Liaoning and Japanese Ayu are compared. There are no substan- tial different characters among the former three Ayu, so the comprehensive values of them are taken as representative of Chinese Ayu, which is in turn compared with Japanese Ayu ( Plecoglossus altivelis altivelis and Plecoglossus altivelis ryukyuensis). By the coefficient of difference test, 3 and 4 characters are found to be beyond the subspecies level between Chinese Ayu and nominate subspecies (Plecoglossus altivelis altivelis) and between Chinese Ayu (Plecoglossus altivelis chinensis) and Ryukyus subspecies (Plecoglossus altivelis ryukyuensis), respectively, which shows that they belong to different subspecies. The China mainland Ayu is a new subspecies, Plecoglossus altivelis chinensis Wu & Shan, the establishment of which and its forming causes are discussed.

驼背鲈外周血细胞的形态学研究

利用Wright’s和Giemsa双重染色法和改良的血涂片染色法两种方式,通过光镜对驼背鲈(Cromileptes altivelis)的血涂片进行了观察。结果表明:驼背鲈的血涂片中可区分出6种细胞——红细胞、嗜中性粒细胞、嗜酸性粒细胞、单核细胞、淋巴细胞、血栓细胞,偶尔可见到分裂的红细胞、分裂的血栓细胞、核影红细胞和聚集的白细胞。此外,统计了红细胞数1.7462×106个/mm3,白细胞数4.7817×103个/mm3,并对白细胞进行了分类计数:淋巴细胞37.26%、血栓细胞30.82%、嗜中性粒细胞23.57%、单核细胞8.35%、嗜酸性粒细胞极少见。描述了以上各细胞的显微结构。单核细胞的体积最大,小淋巴细胞的体积最小;淋巴细胞的数量最多,嗜酸性粒细胞数量最少。

驼背鲈的胚胎发育

对驼背鲈（Cromileptes altivelis）的胚胎发育过程进行了观察，描述其发育各期的形态特征及发育速度。驼背鲈成熟卵呈浮性，透明，平均卵径为810μm，油球一个。在水温25～27℃，盐度30条件下，胚胎发育时间为24h 10min。

短期低盐度胁迫对驼背鲈(♀)×鞍带石斑鱼(♂)杂交子代幼鱼抗氧化及消化生理的影响

为探讨在不同程度的盐度降低下对驼背鲈(Cromilepptes altivelis♀)与鞍带石斑鱼(Epinephelus lanceolatus♂)的杂交品种(鼠龙斑幼鱼)摄食、抗氧化和消化生理的影响,本实验设置盐度突变组:5×10^(–3)、10×10^(–3)、15×10^(–3)、20×10^(–3)、25×10^(–3)、30×10^(–3)(对照组);盐度渐变组(每天盐度降低5×10^(–3))。在第0、3、7天时分别取样,测定血清和肝脏中的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)活力;胃组织中的胃蛋白酶、肠道中的脂肪酶、淀粉酶活性。结果表明:盐度突变组中,盐度降低幅度越大对酶活性的影响越大,尤其5×10^(–3)~15×10^(–3),3种抗氧化性酶活性在肝脏中的变化趋势相似,均呈显著升高再降低的趋势,血清中的SOD、CAT在第3、7天活性相似,均显著高于其他同时期各组,MDA在第7天显著升高;3种消化酶活性均显著下降,盐度越低,下降幅度越显著;其他各组的抗氧化性酶和消化酶活性与对照组相比无明显差异。盐度突变组各组的摄食量均呈下降趋势,尤其5×10^(–3)组最低。盐度渐变组SOD、CAT活性呈持续上升趋势,MDA先上升再下降,胃蛋白酶与脂肪酶活性均是呈先上升再下降的趋势,淀粉酶持续下降;其摄食量下降后又恢复至胁迫前。综上,鼠龙斑对盐度的适应范围较广,当盐度突变为较低的水平(<15×10^(–3))时,对鼠龙斑的抗氧化性及消化生理的影响较大,随着胁迫时间延长可能对鱼体肝脏抗氧化系统具有损害作用,从而影响其生长,因此在实际过程中可通过逐渐降低盐度的方式对鼠龙斑进行驯养,以达到降低应激伤害的作用。

A soluble FcγR homolog inhibits Ig M antibody production in ayu spleen cells

Classical Fc receptors (FcRs) mediate the binding to and recognition of the Fc portion of antibodies and play an important role during immune responses in mammals. Although proteins similar to soluble FcRs have been identified in fish, little is known about the role of such proteins in fish immunity. Here, we cloned a cDNA sequence encoding a soluble Fc receptor for an immunoglobulin G (FcγR) homolog from ayu (Plecoglossus altivelis)(PaFcγRl). The predicted protein was composed of two immunoglobulin C2-like domains but lacked a transmembrane segment and a cytoplasmic tail. The PaFcγRl transcripts were distributed at low levels in all tested tissues, but significantly increased after Vibrio anguillarum infection. The PaFcγRl protein was expressed in the head kidney, trunk kidney, and neutrophils. Recombinant PaFcγRl (rPaFcγRl) was secreted when transfected into mammalian cells and the native protein was also detected in serum upon infection. rPaFcγRl was also demonstrated to bind to ayu IgM, as assessed by cell transfection. Suppressive activity of the recombinant mature protein of PaFcγRl (rPaFcγRlm) on in vitro anti-sheep red blood cell (SRBC) responses was detected by a modified hemolytic plaque forming cell assay. In conclusion, our study revealed that PaFcγRl is closely involved in the negative regulation of IgM production in the ayu spleen.

浙江省景宁陆封型香鱼卵巢发育的组织学

采用常规石蜡切片,H.E染色法研究了浙江省景宁县陆封香鱼(Plecoglossus altivelis)各期卵巢发育和组织学结构特征。结果表明:陆封香鱼卵巢发育可分为6个时期。7月份部分性腺为细线状,属于Ⅰ期卵巢;7月份和8月份香鱼性腺为Ⅰ期或者Ⅱ期,7月份和8月份的平均成熟系数分别为1.28%和1.45%;9月份,性腺发育达到Ⅲ期和Ⅳ期,其中大多数解剖个体为Ⅳ期卵巢,平均成熟系数为2.59%;10月下旬达到Ⅳ期末—Ⅴ期,成熟系数也达到最高峰,平均成熟系数为7.66%。香鱼产卵类型为分批产卵类型。