Preliminary investigation of feeding performance of larvae of early red-spotted grouper, Epinephelus coioides, reared with mixed zooplankton

Larvae of red-spotted grouper, Epinephelus coioides, were reared inoutdoor tanks with nauplii of copepods (mainly Pseudodiaptomus annandaleiand Acartia tsuensis) and/or rotifers, Brachionus rotundiformis. Grouperlarvae successfully started feeding on early stage nauplii even though theirabundance was as low as approximately 100 individuals l^–1 andshowed better survival and growth thereafter compared to those fed withrotifers only. Incidence of feeding reached 100% on day 4 whennauplii were available and only on day 9 when rotifers were given alone.Larvae seemed to be poor feeders at the onset of feeding, attempting tocapture any food organisms in the tank water. Selective feeding ability oflarvae started from day 4 and the larvae then preferred to feed on medium-and large-size nauplii rather than on rotifers as they grew. Larvae appearedto have a better chance at surviving in the presence of early stage nauplii,which were probably caught more easily than rotifers.     

Sphaerospora epinepheli N. Sp. (Myxosporea: Sphaerosporidae) Observed in Grouper (Epinephelus malabaricus)

Sphaerospora epinepheli n. sp. is described from grouper, Epinephelus malabaricus , in cage-cultured and wild fish collected from both coastal lines of southern Thailand. Subspherical to spherical spores and mono- or disporous pseudoplasmodia were observed in the lumen of kidney tubules. Pseudoplasmodia were round to elongate, size range 15.6–22.9 μm (length) × 8.4–21.6 μm (width). Spores were 7.8–10.0 μm (length) × 12.3–14.5 μm (thickness), and 7.0–9.5 μm (width) with two spherical polar capsules of equal size measuring 2.9–4.4 μm in diameter and containing polar filaments with six or seven windings. Two uninucleate sporoplasms showed iodine vacuoles. Blood stages, similar to C-blood protozoans observed from freshwater fish in Europe, were found from peripheral blood smears of grouper. Ultrastructural studies of blood stages showed a similar structure to unidentified mobile protozoans from the blood of carp. Electron dense bodies were observed in the cytoplasm of the primary cell blood stages. Infected proximal-tubular epithelial cells showed highly vacuolated cytoplasm and pycnotic nuclei.     

斜带石斑鱼(♀)×鞍带石斑鱼(♂)杂交子代(青龙斑)消化系统的早期发育

本文研究斜带石斑鱼(Epinephelus coioides)(♀)×鞍带石斑鱼(E.lanceolatus)(♂)杂交子代(青龙斑)仔、稚、幼鱼的消化系统发育,描述了其消化器官发育过程和组织学结构特征,充实青龙斑生物学研究文库,为其发育生物学研究和苗种培育提供技术支撑。青龙斑苗种培育于2012年6~8月期间进行。水温为(30±1)℃,盐度为28±1。利用形态学和连续组织切片技术,对出膜后0~40日龄幼鱼的消化系统进行了观察和研究。消化系统发育可划分为内源性营养、混合营养和外源性营养3个阶段:0至3日龄为内源性营养阶段,初孵仔鱼消化管为一简单的直形管,卵黄囊大,椭圆形,口和肛门尚未与外界相通;口腔中出现鳃弓的雏形,3日龄仔鱼食道由2~3层的复层立方上皮细胞组成,形成较低的褶皱;胃与小肠和食道的分界明显,上皮由单层柱状细胞组成;肠道分化,肛门开通体外,开始摄食;肝细胞团和胰腺细胞团形成。4~5日龄为混合营养阶段,6日龄之后进入外源性营养阶段,卵黄囊已经完全被吸收,前、中、后肠和直肠区分明显,肠黏膜上皮中出现少量的杯状细胞,由肠腔面向深层依次可以分为黏膜层、黏膜下层和浆膜层,肌层不明显。至25日龄,消化系统的结构和功能已经较为完善。38日龄时,胃、幽门盲囊、肠以及直肠各段分界明显,黏膜褶皱高度为前肠中肠后肠;肌层厚度为后肠前肠中肠;消化道和消化腺组织结构与成鱼基本相同。青龙斑的消化系统发育和分化是与其生理功能的逐步完善同步的。     

Allozyme and microsatellite loci provide discordant estimates of population differentiation in the endangered dusky grouper (Epinephelus marginatus) within the Mediterranean Sea

The dusky grouper, Epinephelus marginatus, inhabits coastal reefs in the Mediterranean Sea and Atlantic Ocean. A decline in the abundance of this long-lived protogynous hermaphrodite has led to its listing as an endangered species in the Mediterranean, and heightened management concerns regarding its genetic variability and population substructure. To address these concerns, we analysed genetic variation at seven microsatellite and 28 allozyme loci in dusky groupers sampled from seven areas (for microsatellites) and three areas (for allozymes) in the west-central Mediterranean. Levels of genetic variability were higher for microsatellites than for allozymes (mean H(E) = 0.78 and 0.07, respectively), but similar to those observed in other marine fishes with comparable markers. Both microsatellites and allozymes revealed significant genetic differentiation among all areas analysed with each class of marker, but the magnitude of differentiation revealed by allozymes over three locales (F(ST) = 0.214) was greater than that detected with microsatellites over seven areas, or over the three areas shared with the allozyme analysis (F(ST) = 0.018 and approximately 0, respectively). A large proportion of the allozyme differentiation was due to a single locus (ADA*) possibly influenced by selection, but allozyme differentiation over the three areas was still highly significant (F(ST) = 0.06, P < 0.0001), and the 95% confidence intervals for allozyme and microsatellite F(ST) did not overlap when this locus was excluded. There was no evidence of isolation by distance with either class of markers. Our results lead us to conclude that dusky groupers are not panmictic in the Mediterranean Sea and suggest that they should be managed on a local basis. However, more work is needed to elucidate genetic relationships among populations.     

An analysis of nucleotide and amino acid variability in the barcode region of cytochrome c oxidase I (cox1) in fishes

The 655 bp cytochrome c oxidase subunit I barcode region of single specimens of 388 species of fishes (four Holocephali, 61 Elasmobranchii and 323 Actinopterygii) was examined. All but two (Urolophus cruciatus and Urolophus sufflavus) showed different cox1 nucleotide sequences (99.5% species discrimination); the two that could not be resolved are suspected to hybridize. Most of the power of cox1 nucleotide sequence analysis for species identification comes from the degenerate nature of the genetic code and the highly variable nature of the third codon position of amino acids. Variation at the third codon position is bimodally distributed, and the more variable mode is dominated by amino acids with four or six codons, while the less variable mode is dominated by amino acids with two codons. The ratio of nonsynonymous to synomymous changes is much less than one, indicating that this gene is subject to strong purifying selection. Consequently, cox1 amino acid sequence diversity is much less than nucleotide sequence diversity and has very poor species resolution power. Fourteen of the 16 amino acid residues recognized as having important functions in the region of cox1 sequenced were completely conserved over all 388 species (and the bovine cox1 sequence), with one fish species varying at one of these sites, and three fish at another site. No significant differences in amino acid conservation were observed between residues in helices, strands and turns. Patterns of nucleotide and amino acid variability were very similar between elasmobranchs and actinopterygians.     

Establishment,Characterization, and Virus Susceptibility of a New Marine Cell Line from Red Spotted Grouper (<Emphasis Type="Italic">Epinephelus akaara</Emphasis>)

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25°C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with . A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10^8.5 TCID₅₀ ml⁻¹), while the viral titer of frog Rana grylio virus 9807 (RGV₉₈₀₇) reached 10^3.5 TCID₅₀ ml⁻¹. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.     

斜带石斑鱼TLR3基因的克隆及其在刺激隐核虫感染时的表达分析

TLR3(Toll like receptor 3)是Toll样受体家族的重要成员,通过识别病原相关分子模式,诱导宿主天然免疫应答。研究从斜带石斑鱼(Epinephelus coioides)中克隆得到TLR3 cDNA序列,全长为2937 bp,包括107bp的5′非编码区、100 bp的3′非编码区和编码909个氨基酸的2730 bp的开放阅读框。TLR3全长氨基酸序列包含1个信号肽、18个富含亮氨酸的重复序列(Leucine-rich Repeat LRR)、1个跨膜结构域和1个胞内TIR结构域(IL-R1 homologous region)。同源比对显示,斜带石斑鱼TLR3与其他已报道硬骨鱼类的TLR3具有较高的同源性(52%—67%)。组织表达分析显示,TLR3在健康斜带石斑鱼的组织中具有较广的表达分布,其中在前脑、体肾和脾脏中表达量较高。刺激隐核虫(Cryptocaryon irritans)感染斜带石斑鱼后:在皮肤中TLR3的表达量呈现先降低后升高的趋势,从感染后第7天开始上调,并在第10天达到高峰;而在脾脏中,TLR3的表达量在感染6h时就显著上调并达到峰值。结果表明斜带石斑鱼TLR3在抗刺激隐核虫的免疫应答过程中可能发挥重要作用。