中华绒螯蟹溞状幼体期复眼超微结构的观察

用电镜研究了中华绒螯蟹状幼体复眼的结构,结果显示出从状幼体Ⅰ期到状幼体V期复眼的形态发生了变化,其表面积从Ⅰ的15400μm2增加到V的0.4mm2,组成复眼的小眼由泡状突起发育成扁平六角形.小眼的折光部分的角膜、成角膜细胞、晶锥等均有一个发育成熟的过程.感光部分从Ⅱ起组成小眼的8个小网膜细胞分化成远端的1个细胞和近端7个细胞二部分,小眼的感杆束直径随幼体发育从3.6μm增加到5.2μm,同时组成的微纤毛数量增多.状幼体复眼结构中的一个特征是具有较多的高尔基体,随着幼体的发育小网膜细胞中的胞器种类和数量也逐渐增多,在V时小眼的结构基本上表现出与成体类似的光适应特征.从复眼的超微结构上看,1状幼体复眼的感光功能是逐渐完善的.     

漠斑牙鲆胚胎及仔稚鱼发育的形态学观察

对漠斑牙鲆(Paralichthys lethostigma)胚胎及胚后发育进行观察,描述各发育时期的发育时序和形态特征.在水温18.5±0.5℃,盐度32～33的条件下,受精卵历时49 h脱膜而出,孵化后仔鱼在水温20.5±0.5℃时,4日龄开口摄食,26日龄进入变态期,45日龄左右完成变态成为幼鱼.漠斑牙鲆的卵子为浮性卵,油球一个.刚孵化仔鱼的消化道细直,3日龄肛门开通,4日龄卵黄囊消耗殆尽,开口摄食.在原肠期胚体下包约1/2时,在视囊和胚盘上出现点状星状的黑色素细胞.孵化后体两侧菊花状黑色素均匀分布;进入变态期后,体两侧的幼体色素逐渐收缩消失,同时在有眼侧出现个体较小的圆形成体色素细胞.在早期发育过程中,伴随着冠状鳍条的发生、伸长和缩短吸收.无鳔器官的发生.     

甲状腺激素在变态中牙鲆组织发育中的作用

甲状腺激素在变态中牙鲆组织发育中的作用牙鲆变态期间各种器官会发生广泛的形态和功能变化。幼体肌肉嗜碱性肌浆丰富，肌原纤维量少，发育充分的成鱼肌肉肌原纤维丰富。伴随着形态变化，肌肉的蛋白质组成（包括肌钙蛋白Ｔ和肌球蛋白轻链）也发生变化。变态前幼体的红细胞...     

牙鲆中 Dicer 基因的鉴定与表达

通过PCR技术,从牙鲆(Paralichtys olvaceus)总RNA中克隆了牙鲆Dicer基因序列,该序列全长6 585bp,包括5 691 bp的开放阅读框,编码1 896个氨基酸。在多个物种间的蛋白序列对比中,牙鲆Dicer与其它物种的同源性平均高达78%,与尼罗罗非鱼(Oreochromis niloticus)同源性最高达到89%;在进化树分析中,牙鲆Dicer与其它鱼类Dicer聚集一支。荧光定量PCR显示,牙鲆Dicer基因在牙鲆大脑里的表达最高。同样的,在牙鲆胚胎和仔鱼早期发育阶段里也发现了相对高水平的Dicer mRNA表达。在用甲状腺激素(TH)处理过程中发现,牙鲆Dicer基因在用TH处理后17 dph的表达最高。本研究预测了牙鲆Dicer在牙鲆发育和变态过程中扮演的重要角色,为进一步阐明Dicer在牙鲆变态发育中的功能奠定了基础。     

甲壳动物复眼的研究

复眼是甲壳动物重要的光感受器,由几个到数万个小眼组成,对甲壳动物生理和行为有重要影响。介绍了甲壳动物成体复眼结构、胚胎和幼体复眼发育以及幼体复眼中Gq蛋白研究情况。     

大菱鲆早期变态发育和体表黑色素细胞形态学观察

对大菱鲆早期变态发育及体表黑色素细胞的形成过程进行了研究。绘制了1－60日龄大菱鲆鱼苗形态发育图，同时观察拍摄不同发育时期体表黑色素细胞的形态变化。14－17℃水温时，大菱鲆早期变态发育约需60d。1－14日龄主要为器官发育；14－40日龄主要为形态变化，25日龄左右开始变态；40－60日龄变态基本完成。变态前，鱼苗体表的幼体黑色素细胞对称分布，以素细胞密度先增加后减少。变态开始后，鱼体两侧体色出现不对称。有眼侧体表幼体黑色素细胞减少，逐渐由成体黑色素细胞替代，体色变深；无眼侧幼体黑色素细胞逐渐消褪，未出现成体黑色素细胞，体色逐渐变为白色。若有眼侧体表成体黑色素细胞的出现受阻，则形成白化。     

牙鲆dazl基因的克隆与表达分析

dazl(deleted in azoospermia-like)基因是多种物种精子发生的重要调控因子,为探讨该基因在牙鲆(Paralichthys olivaceus)性腺发育分化中的作用,本研究采用同源克隆和RACE技术克隆了牙鲆dazl基因的cDNA序列,利用生物信息学方法分析了其序列结构,并利用实时荧光定量PCR技术检测了该基因在牙鲆成体组织和早期发育阶段的表达情况。结果发现:牙鲆dazl cDNA序列全长2 031 bp,包括105 bp的5'端非翻译区,1 275 bp的3'端非翻译区和编码217个氨基酸残基的651 bp开放阅读框;氨基酸同源序列比对显示该序列存在一个高度保守的RRM(RNA recognition motif)结构域,且该结构域在鱼类中具有100%的序列一致性;荧光定量PCR结果显示dazl基因主要在牙鲆性腺中表达,且在精巢中的表达高于卵巢;在牙鲆早期发育阶段,dazl基因在未受精卵、受精卵及囊胚期均有较高的转录本,而在原肠胚期表达开始降低,至孵化后3 d一直保持较低的水平,这些丰富表达的母源性dazl mRNA可能参与了胚胎发育中原始生殖细胞的形成。本研究为进一步阐明dazl基因在牙鲆生殖发育中的功能奠定了基础。     

中华绒螯蟹Sou状幼体期复眼超微结构的观察

用电镜研究了中华绒螯蟹Sou状幼体复眼的结构，结果显示出从Sou状幼体Ⅰ期到Sou状幼体Ⅴ期复眼的形态发生了变化，其表面积从SouⅠ的15400μm^2增加到SouⅤ的0．4mm^2，组成复眼的小眼由泡状突起发育成扁平六角形。小眼的折光部分的角膜、成角膜细胞、晶锥等均有一个发育成熟的过程。感光部分从SouⅡ起组成小眼的8个小网膜细胞分化成远端的1个细胞和近端7个细胞二部分，小眼的感杆束直径随幼体发育从3．6μm增加到5．3μm。同时组成的微纤毛数量增多。Sou状体复眼结构中的一个特征是具有较多的高尔基体，随着幼体的发育小网膜细胞中的细胞种类和数量也逐渐增多，在SouⅤ时小眼的结构基本上表现出与成体类似的光适应特征。从复眼的超微结构上看，Sou状幼体复眼的感光功能是逐渐完善的。     

半滑舌鳎早期发育过程中体表色素变化的研究

对半滑舌鳎(Cynoglossussemilaevis Günther)早期发育过程中体表色素的分布及形态变化进行了连续观察,测量其生物学特征,同时拍摄不同发育时期体表黑色素的形态变化。在水温22～23℃,盐度31～33的条件下,半滑舌鳎完成早期变态发育约需要30d,1～15日龄主要是器官发育,16日龄进入变态期,30日龄左右变态完成。半滑舌鳎在原肠期开始出现幼体色素,以后逐渐密集,色素颗粒变大,幼体色素逐渐变成以树枝状和菊花状为主;仔鱼期体表的幼体色素对称分布,在躯干部形成相互间隔的5个色素带;在变态前期幼体色素发生至最发达时期。进入变态期后,幼体色素逐渐减少,同时有眼侧出现颗粒较小的成体色素,形状为点状为主,在身体有眼侧均匀分布。无眼侧色素完全消失,成为无色。个别个体有眼侧的成体色素形成受阻,幼体色素消失后,形成白化。     

黑棘鲷早期色素细胞发育与体色变化

对黑棘鲷早期发育过程中体色和色素细胞的分布及形态变化进行了连续观察。结果表明,黑棘鲷幼体黑色素在原口关闭前期开始出现,黄色素在嗅囊期出现,彩虹色素出现较晚,孵化后6d(Day post hatching,dph)在眼球上端出现,而后密集于腹腔(28 dph)以及鳃盖骨附近(32 dph)。黑色素细胞在发育过程中,不仅数量增多,而且形状也由点状逐渐变成以树枝状和雪花状为主,为幼体黑色素的发育过程;28 dph幼体黑色素完全被成体黑色素替代,形态上再次变为小点状。黄色素几乎伴随着黑色素的分布,色素细胞几乎无形态变化,最终被黑色素覆盖而不明显。初孵仔鱼,尾端6～8体节躯干部具有黑色素丛,构成了该鱼的种属特征。在28 dph,体色发育进入到模式形成期,首先在躯干部形成相互间隔的3个色素带,至40 dph共7条色素带形成,完成体色的模式发育。     

牙鲆(Paralichthys olivaceus)仔稚幼鱼肠道菌群结构比较分析

采用MiSeq 16S rRNA高通量测序技术和生物信息学分析方法,构建了牙鲆(Paralichthys olivaceus)工厂化人工育苗模式下仔稚幼鱼阶段6个不同发育时期18个样品的16SrRNA基因测序文库,共获得7462个OTU (Operational Taxonomic Unit),分类为42个菌门972个菌属.对肠道菌群的形成过程及结构多样性变化分析显示,牙鲆初孵仔鱼的菌群组成多样性丰富,体内的优势菌为变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes);在9日龄和21日龄摄食轮虫(Rotifer)和卤虫(Artemia sp.)幼体样品中,肠道的优势菌群结构较单一,变形菌门成为此时期肠道的优势菌群;45日龄摄食配合饲料后,肠道中变形菌门的相对丰度显著降低,厚壁菌门和拟杆菌门的相对丰度明显增大,成为肠道菌群的优势菌群.在属水平的菌群结构中发现,牙鲆仔稚幼鱼肠道优势菌群的种类和数量都发生了较大变化,在9日龄和21日龄时期肠道中弧菌属(Vibrio)相对丰度最高,到45日龄后相对丰度锐减到最低水平;拟杆菌属(Bacteroides)和普氏菌属(Prevotella)在80日龄后达到较高水平,成为肠道优势菌属;厚壁菌门的8个菌属在80-115日龄时期均发展成为优势菌属,定植于牙鲆的肠道.本研究揭示了工厂化人工育苗模式下牙鲆仔稚幼鱼肠道菌群结构及演替规律.     

Characterization and expression of <Emphasis Type="Italic">lin-28a</Emphasis> involved in <Emphasis Type="Italic">lin28/let-7</Emphasis>signal pathway during early development of <Emphasis Type="Italic">P. olivaceus</Emphasis>

Heterochronic lin-28 is a conserved RNA-binding protein that plays a key role in the timing of developmental events in organisms. As a crucial heterochronic gene, the protein controls developmental events of the second of four larval stages in Caenorhabditi elegans. Heterochronic let-7 miRNAs are often present in various species and highly conserved in sequence and biological function and are required for various biological processes. Previous studies showed that ten let-7 miRNAs were identified in the Japanese flounder (Paralichthys olivaceus) and that they were primarily expressed during metamorphosis. In this study, we clone and characterize the lin-28a gene from P. olivaceus and exhibit its dynamic expression pattern at different developmental stages and various adult tissues. The results show that the P. olivaceus lin-28a gene has high sequence similarity with other species and is highly expressed in the embryonic stage but weakly expressed in the larval stage. In addition, lin-28a overexpression causes cell proliferation and significantly promotes the levels of pre-let-7a and pre-let-7d while markedly depressing let-7a and let-7d expression in FEC (Flounder Embryonic Cell), which indicate that lin-28 possibly blocks the maturation of let-7 miRNAs. Additionally, lin-28a is identified as a target gene of let-7 miRNAs, and let-7 miRNAs directly regulate lin-28a expression by targeting its 3′ UTR. Taken together, lin-28a along with let-7 miRNA participates in a lin-28/let-7 axis pathway that regulates cell division and timing of embryonic and metamorphic events in P. olivaceus.     

Nile tilapia broodfish fed high‐protein diets: Digestive enzymes in eggs and larvae

The aim of this study was to assess the activity of gastric, pancreatic and intestinal digestive enzymes during the embryonic and larval development of Nile tilapia (Oreochromis niloticus) GIFT strain Aqua America^® 1 obtained from a broodstock fed two levels of crude protein (CP). A total of 72 females and 24 males, 10 hapas, two CP levels (32% and 38%) and six phases of embryonic (cleavage, blastula, gastrula) and larval (hatching, 7 and 10 days post hatch, dph) stages were used. The eggs were collected in cleavage, blastula and gastrula stages, 300 mg was collected, and kept in cryogenic tubes in liquid nitrogen. For the samples at larval stage, the remaining eggs were separated according to crude protein level and kept in hatcheries and samples were collected on 7 and 10 dph the same way as before. A total of 48 samples were collected: at each protein level (32% and 38% CP), four samples were collected in each phase of embryonic and larval development. Statistical differences were not observed during embryonic development for acid proteases, trypsin, amylase and lipase activity at both levels of crude protein (32 and 38% CP). Differences for acid proteases were noticed on 7 dph; trypsin and amylase on 7 dph and 10 dph. Significant differences on blastula and 7 dph for protease; as for aminopeptidase, there was significant difference on 7 dph. The data indicated early appearance of digestive enzymes in Nile tilapia broodfish receiving 32% CP taking into account the rapid growth and development of this species.     

牙鲆NR4A1基因在甲状腺素诱导仔鱼变态中的表达调控分析

NR4A1基因是一个重要的转录因子,在信号转导早期通过对靶基因的特异性转录调控发挥着重要的作用。从牙鲆(Paralichthys olivaceus)中克隆了NR4A1基因c DNA序列,检测了其在牙鲆变态中和成鱼各组织中的表达模式,分析了其在外源甲状腺素(TH)以及硫脲(TU)处理仔鱼中的表达变化。结果表明,牙鲆NR4A1 c DNA全长3264 bp,编码568个氨基酸;牙鲆NR4A1基因与其它物种具有很高的同源性,在系统进化树中与鱼类聚为一支;牙鲆NR4A1基因在成鱼心、肌肉和鰓中高表达;在变态过程中,NR4A1水平逐渐升高,且在25 dph达到最高,之后逐渐降低;TH组中的NR4A1基因水平在变态早期和高峰期显著低于正常组,TU组的水平在变态中后期和结束期显著高于正常组;TU组中变态被抑制的仔鱼在正常海水和0.1 mg·L~(-1)TH海水中饲养6 d后,能够顺利变态,且NR4A1基因在拯救组(TU抑制变态仔鱼在正常海水和0.1 mg·L~(-1)TH海水中饲养)中的表达水平与TU对照组相比显著降低,与正常组中同时期的水平一致。结果表明,牙鲆NR4A1基因可能在仔鱼变态调控中扮演着非常重要的角色。     

MITFa及TYR基因在红色锦鲤体色发生不同阶段的表达分析

本研究描述了红色锦鲤从出膜到体色形成的过程,总结和归纳不同发育阶段体色变化异同点,筛选出6个体色变化较显著时期,分别为1、2、3、4、12、48日龄。利用荧光定量PCR分析MITFa及TYR基因在红色锦鲤6个体色变化时期的表达情况。结果显示,MITFa基因在1日龄时表达量最高,显著高于48日龄时(P0.05),极显著高于其他4个时期(P0.01)。48日龄时表达量次之,亦极显著高于2、3、4、12日龄4个时期(P0.01)。2、3、4、12日龄4个时期MITFa基因表达量较低且不存在显著差异(P0.05)。TYR基因在1日龄时表达量最高(P0.01),4日龄时表达量显著高于12、48日龄(P0.05),与2、3日龄不存在显著差异(P0.05)。TYR基因表达量在2、3、12、48日龄4个时期差异不显著(P0.05)。MITFa和TYR基因在红色锦鲤体色形成过程中,表达水平整体呈现降低的趋势,其中MITFa基因表达量表现为先降后升,TYR基因则先降后升再降。以上结果显示MITFa、TYR基因与锦鲤体色形成具有一定相关性,但MITFa和TYR基因在体色发生中的相互作用还有待进一步研究证实。     

Insulin-like growth factor binding protein-2 (IGFBP-2) in Japanese flounder, Paralichthys olivaceus: molecular cloning, expression patterns and hormonal regulation during metamorphosis

In this study, we cloned and characterized cDNA sequences of two insulin-like growth factor binding protein-2 (IGFBP-2a and IGFBP-2b) from Japanese flounder, Paralichthys olivaceus. The full-length cDNA of IGFBP-2a is 1,046 bp long and consists an open frame (ORF) of 876 bp, a 5′-untranslated region (UTR) of 125 bp and a 3′-UTR of 45 bp. IGFBP-2b is 1,067 bp, including a 5′-UTR of 53 bp, a 3′-UTR of 198 bp and an ORF of 816 bp. Real-time quantitative PCR results revealed that IGFBP-2a -2b mRNA was expressed in all detected tissues. Interestingly, the levels of IGFBP-2a mRNA in all detected tissues were higher in female than male, but IGFBP-2b was precisely the opposite. At different embryonic stages, the levels of IGFBP-2a mRNA were typically higher than IGFBP-2b. After hatching, IGFBP-2a mRNA was gradually decreased to a relatively lower level. However, the expression of IGFBP-2b mRNA was increased after hatching, including 3, 7, 10, 14, 17, 20 and 23 days post-hatching (dph), and it presents a higher level until 29 (metamorphic climax), 36 (post-climax) and 41 dph (the end of metamorphosis). In levothyroxine sodium salt (T4, the main form of thyroid hormone in animals)-treated and thiourea (TU)-treated larvae, the expressions of IGFBP-2a had not visibly changed, except in T4-treated 17 dph larvae. The expressions of IGFBP-2b mRNA were distinctly increased from 17 to 23 dph, but suddenly dropped to a lower level in and after 29 dph. However, the levels of IGFBP-2b mRNA during metamorphosis were greatly down-regulated after TU treatment. These results provided basic information for further studies on the role of IGF system in flatfish development and metamorphosis.     

大菱鲆胚胎和仔稚鱼的热刺激耐受能力

以培育大菱鲆Scophthalmus maximus耐高温品系为目的,研究了胚胎和仔稚鱼对热刺激的耐受力差别,为通过高温刺激选育大菱鲆耐高温品系提供指导和基础数据。实验分别对大菱鲆胚胎期(孵化前期)、卵黄囊期(初孵仔鱼,0dph)、前弯曲期(10dph)和弯曲期(25dph)的仔鱼、稚鱼(50dph)使用22～33℃范围内不同梯度的高温海水,进行突变热刺激,2h后转入原水体中培育,记录之后60～84h的死亡率、仔鱼孵化率、畸形率等数据。结果表明,上述5个发育期大菱鲆经受2h热刺激的亚致死温度为24、23、<22、28、28℃,半致死温度为26、28、22、29、29℃,致死温度为30、29、27、30、30℃。根据热刺激后24和48h死亡率,耐热能力由弱到强依次为10dph<胚胎<0dph<25dph<50dph,总体上呈现随生长发育逐渐增强的趋势。10dph左右可能为大菱鲆热刺激耐受力低谷,此时也是开鳔期,对外界环境反应灵敏,受到不良环境刺激易导致死亡。     

草鱼jam-a分子在胚胎幼鱼期及受GCRV感染PSF细胞的表达分析

草鱼呼肠孤病毒(Grass carp reovirus, GCRV)可引发草鱼(Ctenopharyngodon idellus)出血病，导致高死亡率。草鱼吻端成纤维细胞(Grass carp snout fibroblast cells, PSF)是GCRV的敏感细胞系。JAM-A (Junctional adhesion molecule A)为免疫球蛋白超家族成员，是多种病毒的细胞受体。本研究在前期克隆到草鱼3种jam-a基因，命名为gcjam-a1，gcjam-a2和gcjam-a3。在获取ORF序列的基础上，利用qRT-PCR分析了3种gcjam-a在草鱼胚胎及幼鱼不同发育时期及PSF细胞中受GCRV(GD108株)感染前后的表达模式。结果显示，检测的13个胚胎及幼鱼发育时期中，gcjam-a1在未受精卵中高表达，在受精卵至出膜前的胚胎表达水平均较低；从出膜后1~3 d表达量开始上升；出膜6~15 d均呈高水平表达。gcjam-a2与gcjam-a3在草鱼胚胎及幼鱼发育各阶段表达水平较低。在无病毒感染的PSF细胞中，gcjam-a只有少量表达。受GCRV-GD108感染后，病毒S7基因在PSF细胞中的拷贝数随时间呈显著上调趋势，gcjam-a的表达量也有不同程度的上调，mRNA上调水平为gcjam-a1>gcjam-a2>gcjam-a3。本研究证实了3种gcjam-a基因在PSF细胞中的表达均与GCRV-GD108感染相关，其中，gcjam-a1的表达水平受GCRV-GD108感染影响最大，同时，它在孵化出膜后表达上升，推测它可能与病毒的感染更相关。gcjam-a1可作为下一步GCRV与宿主互作研究中的候选分子。     

四指马泌尿系统胚后发育组织学研究

为了解四指马鲅(Eleutheronema tetradactylum)泌尿系统胚后发育的一般规律,采用石蜡组织连续切片和H-E染色技术,对1~50 dph(days post hatching)的四指马鲅泌尿系统胚后发育进行了研究。结果显示,四指马鲅前肾小管在3 dph已经形成,中间分布少量未分化干细胞;至7 dph前肾小管刷状缘明显,泌尿机能增强;15 dph前肾小管继续分化,管壁细胞结构清晰;20 dph观察到少量前肾小管退化,泌尿机能开始衰退;前肾在发育过程中未观察到肾小体形成。中肾肾小管原基在7 dph开始形成;20~25 dph中肾早期肾小体和肾小管芽形成;30 dph肾小管数量剧增,亚成熟肾小体形成,近端小管和远端小管开始区分;45 dph第一近端小管和第二近端小管开始区分,至50 dph观察到成熟肾小体;中肾有较多黑色素-巨噬细胞中心、淋巴细胞和红细胞分布,兼具造血、免疫及泌尿功能。中肾管为输尿管,始于中肾后端,并行向后合并膨大为输尿管膀胱。     

Ontogeny of immune system organs in northern bluefin tuna (Thunnus orientalis, Temminck and Schlegel 1844)

Serial sections, prepared from 0.5 to 30 days posthatch (dph) larval and juvenile Thunnus orientalis (Temminck & Schlegel 1844), were stained with haematoxylin and eosin and examined by light microscopy for immune organ development. The early kidney was present at 0.5 dph, undifferentiated stem cells began to appear at 2 dph, and by 7 dph occasional small lymphocytes were present. The thymus was first obvious at 5 dph, located above the fourth branchial arch, small lymphocytes appeared at 7 dph, and by 15 dph an outer thymocytic zone and an inner epithelioid zone were visible. The progenitor spleen was present at 2 dph, located close to the gut, and by 12 dph it consisted of a mass of sinusoids filled with red blood cells, and remained mainly erythroid throughout the period studied. These results suggest that development of immune organs in this species is precocious relative to other marine teleosts.