Development of vertebral column and appendicular skeleton in larval and juvenile of Platax teira
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摘要: 为掌握尖翅燕鱼 (Platax teira) 仔稚鱼脊柱及附肢骨骼系统的发育时序特征，采用软骨-硬骨双染色方法对尖翅燕鱼仔稚鱼 [0~30日龄 (dah)]、胸鳍、腹鳍、背鳍、臀鳍以及尾鳍进行染色观察与分析。结果表明，尖翅燕鱼有24枚脊椎骨，尾部骨骼由5枚尾下骨和2枚尾上骨构成，附肢支鳍骨发育的顺序为胸鳍、腹鳍、尾鳍、臀鳍和背鳍。尖翅燕鱼在初孵仔鱼已具备胸鳍支鳍骨原基和脊索，4 dah仔鱼胸鳍和腹鳍最先发育；7 dah仔鱼髓弓、脉弓开始出现；13 dah仔鱼骨骼形态和数量基本稳定；26 dah左右稚鱼开始骨化，脊柱和脊椎最先完成骨化；尖翅燕鱼30 dah进入幼鱼阶段，其外表形态已经固定，形态与成鱼无异，典型特征为头部、躯干部和尾部各有3条黑色条带，身体呈雪白银色，受到外界刺激时体色瞬间变为黑色。尖翅燕鱼脊柱及附肢骨的发育顺序与其他海水鱼基本一致，但骨骼发育时间节点和数量有所不同。研究结果对尖翅燕鱼苗种发育、环境适应和分类鉴定具有重要指导意义。Abstract: In order to understand the developmental time sequence characteristics of the spine and appendage skeletal system of Platax teira, we stained and analyzed the spine, pectoral fin, pelvic fin, anal fin and caudal fin of P. teira larvae (0–30 dah) by the method of cartilaginous and bony staining. The results show that there were twenty four vertebrae, and the tail bone was composed of 5 subcaudal bones and 2 upper caudal bones. The development sequence of appendage branch fin bones was pectoral fin, abdominal fin, caudal fin, anal fin and dorsal fin. The first hatched fish already had pectoral branch fin bone primordium and notochord. At 4 dah, the pectoral fin and pelvic fin developed first; at 7 dah, medullary arch and vein arch appeared; at 13 dah, the morphology and number of larval bones were basically stable; at about 26 dah, ossification began in juvenile fish, and the spine and spine were the first to complete ossification; at 30 dah, the larvae entered the juvenile stage, and their appearance has been fixed. The typical characteristics were three black bands on the head, trunk and tail, and the body was white silver. When stimulated or stressed by external stimulation, the body color changed to black instantly, and the shape had no difference from that of the juvenile. The developmental sequence of the spine and appendage bones was basically the same with that of other sea fish, but the time nodes and number of the development of bone were different. The results have important guiding significance for the development, environmental adaptation and classification of P. teira larvae.
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Key words:
- Platax teira /
- Bone staining /
- Larval and juvenile development /
- Spine /
- Appendicular skeleton
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图 1 尖翅燕鱼培养过程中的饲养方案和日龄与全长回归方程
Figure 1. Feeding scheme and regression equation of age and full length during P. teira culture
图 5 尖翅燕鱼背鳍、臀鳍、胸鳍和腹鳍的发育过程
Figure 5. Development of the dorsal, anal, pectoral and pelvic fins of P. teira
表 1 阿尔新蓝-茜素红S染色法
Table 1. Alcian blue and alizarin red S staining technique
染色步骤
Staining step试验过程
Experimental process样本处理
Sample treatment将仔稚鱼麻醉，测量体长和全长，注意不要采集外形畸形的样品，用10%的甲醛溶液浸泡样本，4 ℃条件下浸泡24 h或更长，完成样品的固定。 脱水
Dehydration取出固定样品后，用纱布包裹样品，蒸馏水浸泡至少24 h(中间多换几次蒸馏水)，或用自来水流水冲洗过夜，以清除固定剂。再用不同浓度的乙醇梯度脱水，乙醇溶液浓度依次为：50%、75%、90%和100%，脱水时间均为6 h。 软骨染色
Cartilage staining从脱水液中取出的样品，直接浸入软骨染色液 (将无水乙醇和冰醋酸1:1混合，加入 0.3 g阿利新蓝粉末，配置成1 000 mL溶液) 中，染色时间为24 h，期间可以更换染液。 漂洗
Rinsing在光线较强的环境下进行操作，观察到样品染色好后，从软骨染色液中取出样品放入漂洗液 [将30%的过氧化氢 (H2O2) 和蒸馏水按照1:5混合配制成1 000 mL溶液，并加入8 g氢氧化钾 (KOH)] 中，不间断用玻璃棒搅拌，直到样品表面颜色变淡。 中和
Neutralize将漂洗好的样品放入染色中和液 [蒸馏水中加入过量硼砂 (Na2B4O7)，得饱和硼砂溶液即可] 中进行中和，时间为20~24 h。 硬骨染色
Bone staining将样品放入硬骨染色液 (1 000 mL蒸馏水中加入8 g KOH完全溶解后，再加入0.3~0.5 g茜素红粉末) 中，每隔12 h取出样品观察染色情况，直至染色完成。 酶解
Zymolysis将样本浸泡在酶溶液 (将1.25 g重金属螯合剂EDTA-Na2和4.5 g胰蛋白酶溶解于1 000 mL饱和硼砂溶液中) 中，根据酶解程度调整消化时间，直至稍见骨骼即可，中间最好换酶解液。 保存
Preservation用蒸馏水清洗样本，然后依次移入体积比为3 : 1、1 : 1、1 : 3的1% KOH-甘油合剂中，最后保存于纯甘油中，并加入3~4 粒麝香草酚保存。 
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