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1 仿刺参骨片的种类和形态学研究张莉恒, 丁君, 韩昭衡, 常亚青, 宋坚, 田莹, 白雪秋, 丁文君 (, ) 摘要 : 以大连海洋大学农业部北方海水增养殖重点实验室培育的已明确年龄 (1 龄 2 龄 3 龄 ) 的仿刺参 (Apostichopus japonicus) 为实验材料, 采用原位压片法对仿刺参棘体壁触手管足纵肌呼吸树肠 7 种组织标本进行骨片形态学观察和 SPSS 统计分析, 获得了不同年龄组织仿刺参中各类型骨片的相对比例结果表明, 原位压片法得到的骨片较 NaClO 法结构损坏较小, 且能观察到骨片在组织中的分布仿刺参各组织中存在桌形体扣形体杆状体花纹状体复合盘状骨片和长孔状体 6 种主要骨片类型体壁中主要骨片类型为扣形体和桌形体 ; 棘和触手中主要骨片类型为扣形体桌形体和杆状体 ; 管足中主要骨片类型为特有的复合盘状骨片扣形体和桌形体 ; 呼吸树和肠中主要骨片类型为花纹状体 ; 纵肌不含任何类型骨片不同组织间相同类型骨片的形态结构一致, 无组织间特异性通过对不同年龄仿刺参各组织的骨片比较分析发现, 不同年龄仿刺参相同组织中骨片比例存在差异, 例如, 1 龄 2 龄 3 龄仿刺参触手中桌形体骨片的相对比例分别为 11.55%±2.69% 20.76%±2.34% 3.75%±0.72%, 2 龄仿刺参触手中桌形体骨片的相对比例显著高于 1 龄 3 龄仿刺参触手中桌形体骨片比例 (P<0.05); 1 龄 2 龄 3 龄仿刺参棘中扣形体相对比例分别为 92.55%±1.98% 65.45%±1.79% 88.36%±3.08%, 2 龄仿刺参棘中扣形体相对比例显著低于 1 龄和 3 龄仿刺参棘中扣形体骨片比例 (P<0.05) 本研究为仿刺参的基础生物学研究提供资料, 为仿刺参的年龄鉴别提供参考关键词 : 仿刺参 (Apostichopus japonicus); 骨片 ; 形态学中图分类号 : P735.2 文献标识码 : A 文章编号 : (2015) doi: /hykx (Echinodermate), (Holothuroidea) 160, 1400, 140 [1] [2-6] [7],,,, [8], [9] 134 ; Massin [10] (Holothuria scabra) ; Richard Rasolofonirina Michel Jangoux [11] (H. scabra), (H. scabra) ;, 赟 [12] 15 ; [13] 6,,, 8 / 2015 / 39 / 4, 1~3 (Apostichopus japonicus), SPSS,, 1 材料与方法 1.1 实验材料 1~3, 1 : ; : : 863 (2010AA10A401, 2012AA10A412); ( , ) : (1990-),,,,, : , shiqudeyoushang@ 126.com;,,, : , dingjun1119@dlou.edu.cn

54.7 mm±1.5mm; 2 81.7 mm±2.0mm; 3 102.3± 1.8mm 3, 5, 7 (6 ) (5cm) (4~8 ) (4~8 ) (6 ) (, 1cm 1cm 1cm) ( 4, 1cm)7 1.2 实验方法 NaClO, ; ; 1.2.1 NaClO,, 1.5mL, 0.

2 54.7 mm±1.5mm; mm±2.0mm; ± 1.8mm 3, 5, 7 (6 ) (5cm) (4~8 ) (4~8 ) (6 ) (, 1cm 1cm 1cm) ( 4, 1cm)7 1.2 实验方法 NaClO, ; ; NaClO,, 1.5mL, 0.5~1 ml30%, 20~60 s,,,, 3,, (Nikon ECLip5E 50i), 1.2.2,,,,,, ( ); (Nikon ECLip5E 50i), mol/L PBS NaClO, 5min,, 3, NaClO ;, (50% 70% 80% 90% 95% 100%), 3min ;, (JEOL JEM-1200EX),, 1.3 数据分析 Excel SPSS16.0, (One-Way ANOVA), P< 实验结果 2.1 NaClO 法和原位压片法制备仿刺参各组织骨片标本结果比较 NaClO ( 1) NaClO,,,,,,,,, NaClO Fig.1 1 The production of Apostichopus japonicus tentacle bone chips a. NaClO ; b. a. NaClO method; b. Situ compression method 2.2 仿刺参不同组织骨片类型分析, 6 ( 2) ;, ; ; ; ; ;, 3 C Excel SPSS16.0, 1 Marine Sciences / Vol. 39, No. 4 /

Fig. 2 图 2 仿刺参不同组织骨片的显微观察图 Ossicles of different tissues of Apostichopus japonicus observed under microscope A. 体壁中骨片(2 龄); B. 管足中骨片(2 龄, 显示复合盘状骨片); C. 管足中骨片(2 龄, 显示桌形体和扣形体); D. 棘中骨片(2 龄); E.

3 Fig. 2 图 2 仿刺参不同组织骨片的显微观察图 Ossicles of different tissues of Apostichopus japonicus observed under microscope A. 体壁中骨片(2 龄); B. 管足中骨片(2 龄, 显示复合盘状骨片); C. 管足中骨片(2 龄, 显示桌形体和扣形体); D. 棘中骨片(2 龄); E. 肠中骨片(2 龄); F. 触手中骨片(2 龄); G. 呼吸树中骨片(2 龄); H. 呼吸树中骨片(3 龄, 显示 C 形体); I. 肠中骨片(3 龄, 显示 C 形体) a. 扣形体; b. 桌形体; c. 长孔状体; d. 花纹状体; e. 杆状体; f. C 形体 A. ossicles of body wall(2 years); B. ossicles of tube feet(2 years, show composite discoid ossicles); C. ossicles of tube feet(2 years, show table-shaped ossicles and button-shaped ossicles); D. ossicles of spine(2 years); E. ossicles of intestine(2 years); F. ossicles of tentacle(2 years); G. ossicles of respiratory tree(2 years) ; H. ossicles of respiratory tree(3 years, show C-shaped ossicles); I. ossicles of intestine(3 years, show C-shaped ossicles) a. button-shaped ossicles; b. table-shaped ossicles; c. long hole-like body ossicles; d. rosste-shaped ossicles; e. rod-shaped ossicles; f. C-shaped ossicles 由表 1 可知, 仿刺参不同组织中桌形体骨片比触手中桌形体相对比例(P <0.05) 例是不一致的, 在 1 龄仿刺参中, 体壁和触手中桌形由表 1 可知, 仿刺参不同组织中扣形体骨片相体相对比例较高, 分别达到 12.15%±2.99% 和对比例不一致, 1-3 龄仿刺参棘和体壁中扣形体相对 11.55%±2.69%; 2 龄和 3 龄仿刺参中, 管足和棘中桌比例较高, 触手中扣形体比例最低, 触手中扣形体形体相对比例较高, 2 龄仿刺参管足和棘中桌形体比比例分别为 36.58%±6.13% 49.61%±2.46% 例分别为 21.83%±5.64%和 24.16%±0.93%, 3 龄仿刺 7.68%±2.89% 不同年龄仿刺参相同组织中扣形体骨参管足和棘中桌形体比例分别为 8.89%±4.84%和片相对比例具有显著差异性, 2 龄仿刺参棘中扣形体 11.36%±3.22% 不同年龄仿刺参相同组织中桌形体相对比例为 65.45%±1.79%, 显著低于 1 龄和 3 龄仿骨片的相对比例也存在差异, 2 龄仿刺参管足中桌形体骨片相对比例为 21.83%±5.64%, 显著高于 1 龄仿刺参管足中桌形体相对比例(P <0.05); 2 龄仿刺参棘和触手中桌形体骨片相对比例分别为 24.16%±0.93% 和 20.76%±2.34%, 显著高于 1 龄和 3 龄仿刺参棘和 10 刺参棘中扣形体(P<0.05); 3 龄仿刺参触手中扣形体相对比例为 7.68%±2.89%, 显著低于 1 龄和 2 龄仿刺参触手中扣形体(P<0.05) 2.3 仿刺参各组织骨片显微结构观察在扫描电子显微镜下观察仿刺参骨片的显微结海洋科学 / 2015 年 / 第 39 卷 / 第 4 期

4 表 1 不同年龄仿刺参不同组织各类型骨片分析 Tab. 1 The types of ossicles of different tissues in Apostichopus japonicus Selenka with different ages ( ) (%) C ±2.62 ax 12.15±2.99 ax 3.24± ±4.84 ax 7.31±2.36 ax 3.3± ±0.74 ax 7.42±0.95 ax 0.64± ±2.99 bx 3.57±0.69 bx 37.12± ±8.32 bx 21.83±5.64 by 17.33± ±17.29 ax 8.89±4.84 axy 22.83± ±1.98 ax 6.74±2.12 abx 0.71± ±1.79 by 24.16±0.93 by 10.39± ±3.08 ax 11.36±3.22 ax 0.28± ±6.13 cx 11.55±2.69 ax 51.87± ±2.46 bx 8.75± ±2.34 by 20.88± ±2.89 by 3.75±0.72 az 88.57± ± ± ± ± : a b c, x y z (p<0.05),, ( 3A),,,, 1, 4 1 ( 3B),,,,, 4 ( ), 4, ( 3C),, ( 3D), ( 3E), 8,,,,,,,, ( 3F) 3 讨论 3.1 实验方法 NaClO NaOH, NaClO NaClO,,,,,,, NaClO,,, NaClO 3.2 仿刺参各组织中骨片类型分析, Marine Sciences / Vol. 39, No. 4 /

Fig.3 图 3 不同类型骨片的扫描电镜图 Scanning electron microscopy images of different types of ossicles A. 桌形体; B.扣形体; C.杆状体; D.长孔状体; E.花纹状体; F.复合盘状骨片 a.桌形体; b.扣形体 A. table-shaped ossicles; B.

5 Fig.3 图 3 不同类型骨片的扫描电镜图 Scanning electron microscopy images of different types of ossicles A. 桌形体; B.扣形体; C.杆状体; D.长孔状体; E.花纹状体; F.复合盘状骨片 a.桌形体; b.扣形体 A. table-shaped ossicles; B. button-shaped ossicles; C. rod-shaped ossicles; D. long hole-like body ossicles; E. rosste-shaped ossicles; F. composite discoid ossicles a. table-shaped ossicles; b. button-shaped ossicles 织间骨片类型具有差异性, 例如, 管足中存在独特片进化过程是由复杂到简单本研究发现随着仿刺的复合盘状骨片; 肠和呼吸树中存在花纹状体; 纵参年龄的增长发现, 不同年龄仿刺参不同组织各类肌中不含有任何类型骨片; 另外, 在 3 龄仿刺参肠和型骨片的比例是动态变化的, 例如, 桌形体骨片的呼吸树中发现了 C 形体, 而通常认为仿刺参中不存桌腿消失; 1 龄仿刺参体壁触手中桌形体的含同时比较了海参背脊量高于管足和棘, 但到了 2 龄 3 龄, 管足和棘中的部腹部触手口部和肛门部骨片的特征, 结果表桌形体的含量高于体壁和触手这种变化造成不同明, 海参不同部位骨片类型存在明显不同, 例如, 背年龄仿刺参不同组织中各类型骨片的相对比例存在部多为扣形体, 触手多为杆状体骨片, 与本研究结差异对于桌形体骨片, 1 龄 2 龄 3 龄仿刺参触手果有相同之处进一步比较不同组织中相同类型骨间桌形体骨片相对比例存在显著差异性(P <0.05) 对 [9] 在 C 形体骨片陈建伟等 [14] 片发现, 仿刺参不同组织中相同类型骨片的形态结于扣形体骨片, 2 龄仿刺参棘中扣形体骨片相对比例构一致, 无组织特异性, 如管足和体壁中的桌形体显著低于 1 龄和 3 龄仿刺参棘中扣形体相对比例骨片形态结构一致 Delboni[15] 研究了 Synaptula hydriformis 海参的骨片生长, 并记录了锚形体等骨片类型的生长变化过程本研究发现扣形体一面存在 4 个突起痕迹, 推测为桌形体退化形成, 是否能作为仿刺参骨片间类型转化依据还有待进一步探索本研究为仿刺参的基础生物学研究提供资料 3.3 (P<0.05) 通过仿刺参各组织不同类型骨片相对比例综合分析, 可为仿刺参的年龄判断提供参考参考文献: [1] 题与展望[J]. 水产科学, 2006, 25(4): [2] 不同年龄仿刺参不同组织各骨片相对比例分析国外学者 Rowe [16] 认为海参骨片类型的进化过 12 董颖, 高杉, 陈仲, 等. 仿刺参 profilin 基因全长 cdna 的克隆及表达分析 [J]. 中国农业科技导报, 2013, 15(2) : [3] 杜慧霞. 仿刺参(Apostichopus japonicus)转录组分析与遗传图谱构建[D].青岛: 中囯海洋大学, 程是由简单到复杂, 例如由杆状体和花纹状体进化到桌形体和扣形体, Levin 的结论与之相反, 认为骨常亚青, 隋锡林, 李俊. 刺参增养殖业现状存在问 [4] 鄢婧婧. 仿刺参(Apostichopus japonicus)分子标记开海洋科学 / 2015 年 / 第 39 卷 / 第 4 期

6 [D]. : 囯, [5]. (Apostichopus japonicus) [D]. : 囯, [6]. DNA [D]. :, [7]. [D]. : 囯, [8],,,. [M]. :, 2004: [9]. [M]. :, [10] Massin C, Mercier A, Hamel J F. Ossicle change in Holothuria scabra with a discussion of ossicle evolution within the Holothuriidae (Echinodermata)[J]. Acta Zoologica (Stockholm), 2000, 81: [11] Richard Rasolofonirina and Michel Jangoux. Appearance and development of skeletal structures in Holothuria scabra larvae and epibiont juveniles[j]. SPC Beche-de-mer Information Bulletin, 2005, 12: [12] 赟,,,. [J]., 2008, 38: [13],,,. 6 [J]., 2009, 31(2): [14],,,. [J]., 2002, 27 (4) : [15] Delboni C. Description of calcareous structures of the apoda holothurian Synaptula hydriformis (Lesueur 1824)[J]. SPC Beche de Mer Information Bulletin, 2008, 27: [16] Kerr M A, Janles A D, Clouse M R, et a1 Molecular phylogeny of coral reef sea cucumbers(holothuriidae: Aspid0chir0tida) based on 16S mitochondrial ribosomal DNA sequence[j] Marine Biotechnology, 2005, 7: Classification and morphology of ossicles of Sea cucumber (Apostichopus japonicus) ZHANG Li-heng, DING Jun, HAN Zhao-heng, CHANG Ya-qing, SONG Jian, TIAN ying, BAI Xue-qiu, DING Wen-jun (Dalian Ocean University, Key Laboratory of Mariculture & Stock Enhancement in North China, s sea, Ministry of Agriculture, Dalian , China ) Received: Jul., 7, 2014 Key words: sea cucumber (Apostichopus japonicus); ossicle; morphology Abstract: In this study, the sea cucumbers (Apostichopus japonicus cultivated by Dalian Ocean University, Key Laboratory of Mariculture & Stock Enhancement in North China s Sea, Ministry of Agriculture, with clear ages (1~3 years old) were used as experimental materials. The proportions of various types of ossicles in different tissues including spine, body wall, tentacle, tube feet, longitudinal muscle, respiratory tree and intestine were obtained and analyzed with in-situ compression method. The results showed that in-situ compression method was superior to NaClO method. The in-situ compression method created less damage to the structure of ossicles. And the distribution of ossicles can be observed clearly by this method. Six types of ossicles, including table-shaped ossicles, button-shaped ossicles, rod-shaped ossicles, rosste-shaped ossicles, composite discoid ossicles and long hole-like body ossicles were observed. The button-shaped ossicles and table-shaped ossicles were observed in body wall; the button-shaped ossicles, table-shaped ossicles and rod-shaped ossicles were observed in spine and tentacle; unique composite discoid ossicles, button-shaped ossicles, and table-shaped ossicles were observed in tube foot; Marine Sciences / Vol. 39, No. 4 /

7 rosste-shaped ossicles were observed in respiratory tree and intestine; no ossicles were observed in longitudinal muscle. The structures of the same type ossicles in different tissues were same. There is no specific structure in different organizations. Furthermore the proportions of different types or ages were different in the same tissue, the proportion of table-shaped ossicles in the tentacle of A. japonicus(1, 2 and 3 years old)was 11.55% ±2.69%, 20.76%±2.34% and 3.75%±0.72%, respectively. The proportion of table-shaped ossicles in the tentacle of 2 years old A. japonicus was significantly higher than that of table-shaped ossicles in the tentacle of 1 and 3 years old (P <0.05). The proportion of button-shaped ossicles in the spine was 92.55%±1.98%, 65.45%±1.79% and 88.36%±3.08%, respectively. The proportion of button-shaped ossicles in the spine of 2 years old A. japonicus was significantly lower than that of button-shaped ossicles in the spine of 1 and 3 years old (P <0.05). This reseach provided information for basic biological research of sea cucumber, and also a reference for identification of sea cucumber ages. ( 本文编辑 : 梁德海 ) 14 / 2015 / 39 / 4

s 15 contact s W Si 3 N μm N / m 4 AFM 95% AFM WPOJ UPOJ WPOJ UPOJ 260 ~ 280 nm WPOJ-DS UPOJ-DS 1 cm 1 cm DEAE-ce

s 15 contact s W Si 3 N μm N / m 4 AFM 95% AFM WPOJ UPOJ WPOJ UPOJ 260 ~ 280 nm WPOJ-DS UPOJ-DS 1 cm 1 cm DEAE-ce 32 1 2013 2 Journal of Chinese Electron Microscopy Society Vol. 32 No. 1 2013-02 1000-6281 2013 01-0054-08 1 1* 2 1 1 1. 2. 710062 AFM SEM WPOJ-DS UPOJ-DS WPOJ-DS β- UPOJ-DS AFM WPOJ-DS UPOJ-DS SEM UPOJ-DS