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.

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。