The application of image cytometry to viability assessment in dual fluorescence-stained fish spermatozoa

The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by epifluorescence microscopy and image cytometry. Ten phase contrast and epifluorescent images were recorded per sample, corresponding images were overlaid, and the blended images were evaluated for live and dead spermatozoa, represented by green and red fluorescence signals. Live/dead proportions were assessed, after dual thresholding, by imaging software that counted absolute numbers of objects and computed their frequencies. All sperm heads were found to be labelled, emitting either green or red light. Mean numbers of spermatozoa per image were in the ranges 32-113, 61-105, 48-104 and 29-91 for Siberian sturgeon, common carp, tench and wels, respectively. The corresponding proportions of live spermatozoa were in the ranges 83.56-94.59%, 93.92-97.02%, 76.14-97.76% and 79.45-83.76%. Standard deviations did not exceed 5% of the means. The image cytometric system using dual staining with SYBR 14 and propidium iodide was clearly suitable for assessing the viability of freshwater fish spermatozoa.     

应用于中华鲟的Pop-up标志固定方法

简要介绍了应用于中华鲟(Acipenser sinensis)的pop-up标志固定方法,包括预埋体的制作、植入及pop-up标志悬挂等技术,并对其应用效果进行了评价。     

长江口中华鲟自然保护区底栖动物

根据2004年5、8、11月和2005年2月在长江口中华鲟自然保护区附近水域(31°19.58′—31°38′N;121°32.08′—122°11.65′E)4个航次的海洋综合调查资料,分析了该水域底栖动物种类组成、数量变动和优势种时空分布以及与中华鲟幼鱼食性的关系。结果表明:调查区共有底栖动物48种,种类组成和优势种均有明显的季节更替,四季皆为优势种的仅纵肋织纹螺(Nassariusvariciferus)和狭颚绒螯蟹(Eriocheirleptognathus)2种;夏季总生物量在四季中最高,春季次之,冬季最低;夏季总栖息密度大于其它3个季节,冬季次之,春季和秋季基本一致;夏季是底栖动物总生物量和总栖息密度较高的时期,也是中华鲟幼鱼在长江口停留觅食时期。与2002年的研究结果进行比较表明,虽然底栖动物优势种组成在深水航道工程后基本稳定,但生物量和栖息密度均呈下降趋势,这直接威胁到保护区内中华鲟幼鱼的饵料基础状况。     

Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the Acipenseriformes

The shortnose sturgeon Acipenser brevirostrum was revealed to have a larger number of chromosomes than previously reported for other sturgeon species. Its chromosome number ranged from 362 to 372 (of ten specimens examined), showing intraindividual variation. The karyotype of metaphase with the highest chromosome number (372) consisted of 89 pairs of macrochromosomes and 97 pairs of microchromosomes (fundamental number; NF=550). Although the microchromosomes were relatively shorter than the macrochromosomes, most of them had discernible arms and centromeres. Silver-stained nucleolar organizer regions (Ag-NORs) were localized on the telomeric regions of 5 pairs of chromosomes (Ag-NORs=10): 4 were made up of small meta/submetacentrics and 1 of acrocentrics. Polyploidy of A. brevirostrum should be hexaploid based on the karyotype, numerous chromosomes, Ag-NORs, and previously reported large genome size (ca. 13pg DNA/cell).Supplementary material to this paper is available in electronic format at http://dx.doi.org/10.1007/s10228-004-0257-z     

史氏鲟两种促性腺激素β亚基cDNA克隆及序列进化分析

从史氏鲟(Acipenserschrenkii)脑垂体中提取总RNA,利用GeneRacerTM技术,分别克隆得到促性腺激素Ⅰ(GTHⅠ)和Ⅱ(GTHⅡ)的β亚基cDNA全长。史氏鲟GTHⅠ-β亚基cDNA全长为1088bp,开放阅读框为384bp,编码128个氨基酸(Genbank序列登录号为:AY575920);GTHⅡ-β亚基cDNA全长为616bp,开放阅读框为408bp,编码136个氨基酸(Genbank序列登录号为:AY575921)。与其他脊椎动物进行了两种基因的氨基酸序列同源性比较表明:史氏鲟GTHⅠ-β与西伯利亚鲟相似性最高,为99·1%;与硬骨鱼类中鲈形目相似性最低,为34·5%。GTHⅡ-β与西伯利亚鲟相似性为98·3%,与其他硬骨鱼类除鲈形目外都超过60%,与鸟类的相似性最低,为42·8%。两个基因成熟肽构建的MP树显示:GTHⅠ与FSH的β亚基和GTHⅡ与LH的β亚基构成两个明显的聚类。用Mega2软件计算了所比对物种间开放读码框的核苷酸遗传距离并构建了NJ树和MP树,两个基因β亚基的NJ系统树和MP系统树拓扑结构相似,鲟鱼与真骨鱼类分别聚类,共同形成硬骨鱼纲聚类,与传统分类学一致。成熟肽相似性、遗传距离和系统树分支长度显示GTHⅠ-β亚基在真骨鱼类进化速率显著快于其他脊椎动物,而LH-β亚基在羊膜动物中进化比其他脊椎动物快。暗示这两种糖蛋白基因的进化伴随物种进化而显示其功能[动物学报52(2):362-375,2006]。     

史氏鲟免疫球蛋白重链可变区序列及多样性

为了研究史氏鲟免疫球蛋白重链可变区基因的组织结构和多样性,采用RTPCR技术从史氏鲟(Acipenserschrenckii)脾脏总RNA中获得了免疫球蛋白重链可变区cDNA克隆,随机挑取31个阳性克隆进行测序。结果表明:所有序列相同率高于75%,前导肽相同率高于90%,应属于同1个VH家族。其变异主要存在于互补性决定区,特别是CDR3区。在D片段序列中发现大量保守的基因序列(motif)。并发现多个VH基因片段可以共用一个J片段的现象。在基因组DNA重排过程中,VH片段可以与任意的D和J片段结合。此外,史氏鲟免疫球蛋白重链可变区的VH,D和J片段的随机重排外,外切核酸酶作用,以及在重排位点大量N,P片段的插入现象,都大大增加了鲟鱼免疫球蛋白的多样性。     

Evaluation of Adult White Sturgeon Swimming Capabilities and Applications to Fishway Design

Concern over passage of sturgeon barriers, has focused attention on fishway design that accommodates its swimming performance. In order to evaluate swimming performance, regarding fish ladder type partial barriers, wild adult sturgeons, Acipenser transmontanus; 121–76m fork length, were captured in the San Francisco Bay Estuary and Yolo Bypass toe drain. Hydrodynamic forces and kinematic parameters for swimming performance data were collected in a laboratory flume under three flow conditions through barriers and ramp. The experiments were conducted in a 24.4 m long, 2.1 m wide, and 1.62 m deep aluminum channel. Two geometric configurations of the laboratory model were designed based on channel characteristics that have been identified in natural river systems. At a given swimming speed and fish size, the highest guidance efficiencies of successful white sturgeon passage as a function of flow depth, flow velocity, turbulence intensity, Reynolds number, Froude number and shear velocity observed in the steady flow condition, tested with the horizontal ramp structure, occurred at an approach velocity of 0.33 ms^-1. The guidance efficiency of successful sturgeon passage increased both with increasing flow velocity and Froude number, and decreased both with the flow depth and the turbulence intensity. This study also provides evidence that tail beat frequency increases significantly with swimming speed, but tail beat frequency decreases with fish total length. Stride length increases both with swimming speed and fish total length. The importance of unsteady forces is expressed by the reduced frequency both with swimming speed and fish total length. Regression analysis indicates that swimming kinematic variables are explained by the swimming speed, the reduced frequency and the fish total length. The results emphasize the importance of fish ladder type patchiness when a fishway is designed for the passage of sturgeon.     

中华鲟抗菌肽Hepcidin的克隆、表达及其抗菌活性分析

Hepcidin也称为铁调素,是在肝脏中特异表达的一种阳离子小分子抗菌肽。2000年,Krause,et al.首先从人血液中分离纯化得到了由25个氨基酸组成的LEAP-1抗菌肽[1]。2002年,Shike,et al.首次从杂交条纹鲈的鳃分离出鱼类Hepcidin,并从金眼狼鲈(Morone chrysops)克隆到     

Parasitic Attachments by Overwintering Silver Lampreys, Ichthyomyzon unicuspis, and Chestnut Lampreys, Ichthyomyzon castaneus

We present evidence that at least some parasitic-phase silver lampreys, Ichthyomyzon unicuspis, and chestnut lampreys, I. castaneus, remain attached to host fish during the winter. Lake sturgeon, Acipenser fulvescens, harvested through the ice by spearfishers in the Lake Winnebago system in Wisconsin may bear silver lampreys or fresh lamprey wounds, and sturgeon with lamprey marks were significantly larger than sturgeon without them. Silver lampreys collected on paddlefish, Polyodon spathula, in the Wisconsin River in March were not significantly longer than silver lampreys collected previously in late October, but they were significantly heavier, an indication that they were feeding to at least some extent during the intervening period. Other large fish species, including northern pike, Esox lucius, and flathead catfish, Pylodictus olivaris, have been collected or observed during the winter with silver or chestnut lampreys attached. Although energy and nutrient intake by parasitic lampreys may be reduced during the winter, lampreys attached to hosts may also benefit from the hosts' mobility and ability to avoid potentially harmful situations.