STUDY ON DOUBLE FLUORESCENCE MARKING OF SPINIBARBUS SINENSIS JUVENILE
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摘要: 为探究盐酸四环素(TCH)和茜素红S(ARS)对中华倒刺鲃(Spinibarbus sinensis Bleeker)的影响及标记效果, 丰富鱼类荧光标记模式, 研究使用TCH(100—500 mg/L)和ARS(100—300 mg/L)对中华倒刺鲃幼鱼进行双重浸染荧光标记, 实验共设置5个处理组和1个对照组, 浸染时间均为24h。结果显示, 经90d的养殖实验后, 标记鱼的耳石(包括矢耳石和星耳石)、倒刺、鳍条和鳍棘均能检测到双重荧光标记环, 且TCH产生的黄色荧光环比ARS产生的红色荧光环更接近骨质结构内部。较高浓度处理组的矢耳石(≥300 mg/L TCH和≥150 mg/L ARS)、星耳石(≥300 mg/L TCH 和 ≥200 mg/L ARS)和倒刺(400—500 mg/L TCH 和150—300 mg/L ARS)中均能检测到明显的标记环(n≥2), 但所有处理组的侧线鳞和非侧线鳞的荧光标记不明显(0≤n≤1)。经200—500 mg/L TCH和150—300 mg/L ARS处理的鳍条, 及经300—500 mg/L TCH和200—300 mg/L ARS处理的鳍棘中可以同时检测到明显的TCH和ARS的标记环(n≥2)。此外, 在整个实验中各处理组标记鱼与对照组相比, 在生长和存活率方面均无显著性差异(P>0.05)。结果表明, 使用TCH和ARS双重标记中华倒刺鲃幼鱼是可行的。双重荧光标记方法在水生生物标记回捕实验及实验性生物学研究方面具有潜在的应用前景。Abstract: In order to explore the effects of tetracycline hydrochloride (TCH) and alizarin red S (ARS) on Spinibarbus sinensis and their marker effects, and to enrich the fluorescent marking patterns of fish, TCH (from 100 to 500 mg/L) and ARS (from 100 to 300 mg/L) were used for double immersion marking juvenile Spinibarbus sinensis with 6 experimental groups for 24h. The results indicated that 24h double immersion produced detectable double marks in otoliths (sagittae and asteriscus), barbs, fin rays, and fin spines after 90 days in the laboratory growth experiment, with the exception of parts of scales. Yellow fluorescent rings produced by TCH were closer to the inside of the bony structures than red fluorescent rings produced by ARS. Sagittae (≥300 mg/L TCH and ≥150 mg/L ARS), asteriscus (≥300 mg/L TCH and ≥200 mg/L ARS), and barbs (400—500 mg/L TCH and 150—300 mg/L ARS) showed acceptable fluorescent marks at higher concentrations (n≥2). The fluorescence marking of lateral scales and non-lateral scales were not obvious in all treatment groups (0≤n≤1). Fin rays treated with 200—500 mg/L TCH and 150—300 mg/L ARS and fin spines treated with 300—500 mg/L TCH and 200—300 mg/L ARS simultaneously had both acceptable TCH and ARS marks (n≥2). In addition, double fluorescence labeling did not impact fish survival or growth (P>0.05) in the present study. The results suggest that double immersion with TCH and ARS is suitable for double mass-marking juvenile S. sinensis, and these double marks are useful in the experimental development of biological research or restocking methodologies.
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图 1 双重标记后6个实验组中华倒刺鲃平均全长(a)和体重(b)
“0—30d”表示生长实验的第一阶段;“30—90d”表示生长实验的第二阶段; 误差线代表标准误, P>0.05
Figure 1. Mean total length (a) and body weight (b) of S. sinensis from six experimental groups after double fluorescent marking
“0—30d”represent the first stage of growth experiment; “30—90d”represent the second stage of growth experiment; Vertical bars represent standard error, P>0.05
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图 2 TCH和ARS荧光染料对中华倒刺鲃幼鱼矢耳石(a)和星耳石(b)的标记质量
Figure 2. The mark quality of TCH and ARS fluorochrome dyes on the sagittae (a) and asteriscus (b) of S. sinensis
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图 3 不同光源下中华倒刺鲃幼鱼矢耳石的双重荧光标记效果
a, b, c为处理组; d, e, f为对照组. 透射光(a, d); 蓝光(b, e); 绿光(c, f); 下同
Figure 3. The effect of double fluorescent marking in sagittae of S. sinensis larval observed with different light sources
a, b, c are treatment groups; d, e, f are control group. Transmitted light (a, d); blue light (b, e); green light (c, f); the same applies below
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图 4 不同光源下中华倒刺鲃幼鱼星耳石的双重荧光标记效果
Figure 4. The effect of double fluorescent marking in asteriscus of S. sinensis larval observed with different light sources
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图 5 不同光源下中华倒刺鲃幼鱼倒刺的双重荧光标记效果
Figure 5. The effect of double fluorescent marking in barb of S. sinensis larval observed with different light sources
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图 6 不同光源下中华倒刺鲃幼鱼背鳍的双重荧光标记效果
Figure 6. The effect of double fluorescent marking in dorsal fin ray of S. sinensis larval observed with different light sources
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图 7 不同光源下中华倒刺鲃幼鱼背棘的双重荧光标记效果
Figure 7. The effect of double fluorescent marking in dorsal fin spine of S. sinensis larval observed with different light sources
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图 8 TCH和ARS荧光染料对中华倒刺鲃幼鱼侧线鳞(a)和非测线鳞(b)的标记质量
Figure 8. The mark quality of TCH and ARS fluorochrome dyes on the lateral line (a) and non-lateral line scales (b) of S. sinensis
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图 9 TCH和ARS荧光染料对中华倒刺鲃幼鱼倒刺的标记质量
Figure 9. The mark quality of TCH and ARS fluorochrome dyes on the barbs of S. sinensis
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图 10 TCH和ARS荧光染料对中华倒刺鲃幼鱼背鳍(a)、胸鳍(b)、腹鳍(c)、臀鳍(d)和尾鳍(e)的标记质量
Figure 10. The mark quality of TCH and ARS fluorochrome dyes on the dorsal (a), pectoral (b), ventral (c), anal (d) and caudal (e) fin rays of S. sinensis
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图 11 TCH和ARS荧光染料对中华倒刺鲃幼鱼背棘(a)、胸棘(b)、腹棘(c)和臀棘(d)的标记质量
Figure 11. The mark quality of TCH and ARS fluorochrome dyes on the dorsal (a), pectoral (b), ventral (c) and anal (d) fin spines fin rays of S. sinensis
表 1 使用盐酸四环素和茜素红S双重浸泡标记的顺序和浓度
Table 1 The order of double immersion marking and concentration sets for TCH and ARS of experimental group
浸泡标记顺序
Marking sequence荧光染料
Fluorochrome dye实验组
Experimental groupⅠ Ⅱ Ⅲ Ⅳ Ⅴ Ⅵ 第一次The first TCH (mg/L) 0 100 200 300 400 500 第二次The second ARS (mg/L) 0 100 150 200 250 300 
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表 2 用于检测盐酸四环素和茜素红S标记的双色镜和滤光片组合
Table 2 Dichromatic mirror and filter combination wavelength for visualizing TCH and ARS marks
光源
Light source波长Wavelength (nm) 激发滤光片
Excitation
filter双色镜
Dichromatic
mirror抑制滤光片
Suppression
filter蓝光Blue light 450—490 510 515 绿光Green light 515—560 580 590 注: 蓝光用于检测盐酸四环素(TCH)标记, 绿光用于检测茜素红S(ARS)标记Note: Blue wavelength was used to detect TCH marks and green wavelength was used to detect ARS marks
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