Effects of preservation methodology on multi-tissue stable isotope signatures of oceanic squid: a case study of jumbo squid (Dosidicus gigas)
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摘要: 碳、氮稳定同位素技术 (δ13C和δ15N) 是海洋动物摄食生态学研究的主要方法之一。目前对海洋动物各组织的保存方法尚无统一的标准，这可能造成测定结果出现偏差，并产生误导性结论。以大洋性头足类茎柔鱼 (Dosidicus gigas) 为研究对象，开展了冷冻保存 (−20 ℃)、乙醇 (体积分数75%) 与甲醛 (体积分数38%) 溶液保存对肌肉、角质颚和性腺组织δ13C、δ15N和碳氮比 (C/N) 测定的比较研究。结果表明，冷冻保存在第30天时仅对肌肉和性腺组织δ13C和C/N值产生了显著性影响 (P<0.05)，偏移量分别为(−0.89±0.30)‰和0.22±0.15，但相比于乙醇和甲醛方法偏移量较小。乙醇保存第30天时，肌肉δ13C、δ15N和C/N值均有显著性变化 (P<0.05)，偏移量分别为(−0.41±0.50)‰、(0.76±0.79)‰和0.34±0.05；性腺δ13C和和C/N值存在显著性变化 (P<0.05)，偏移量分别为 (−0.36±0.44)‰和 0.21±0.14；而角质颚的变化不显著 (P>0.05)。甲醛保存会导致3种组织的δ13C、δ15N和C/N值出现显著性变化 (P<0.05)，30 d后 δ13C 、δ15N和 C/N值总偏移量分别为 (−3.03±1.87)‰、(−0.48±0.72)‰和0.86±0.73，但保存至第180天时仅角质颚和性腺的δ15N和C/N值仍持续增大 (P<0.05)，可能与组织特异性有关。因此在分析稳定同位素时，应谨慎使用长时间保存后的软组织样品 (肌肉和性腺)，而硬组织 (角质颚) 可采用冷冻或乙醇保存。Abstract: Carbon and nitrogen stable isotope technology (δ13C and δ15N) is an important tool for the study of trophic ecology of marine organisms. However, there is no unified standard for the preservation methods in cephalopods' samples, which may cause biases in stable isotope values and easily lead to misleading conclusions. Thus, we assessed the effects of preservation method (Freezing, 75% ethanol solution and 38% formaldehyde solution) and duration (0, 30 and 180 d) on the stable isotope ratios of carbon and nitrogen of tissues (Muscle, beak and gonad) of jumbo squid (Dosidicus gigas). The results show that freezing affected δ13C and C/N values of muscle and gonad within 30 d (P<0.05), with offsets of (−0.89±0.30)‰ and 0.22±0.15, respectively, which were lower than the effects of alcohol and formaldehyde. After 30 days of storage in the alcohol solution, significant changes were observed for muscle [δ13C: (−0.24±0.42)‰ , δ15N: (0.17±1.07)‰ and C/N: (0.32±0.49)] and gonad [δ13C: (−0.36±0.44), C/N: (0.21±0.14)] (P<0.05), but no significant change was found in beak (P>0.05). Formaldehyde preservation induced a decrease in δ13C [(−3.03±1.87)‰] and δ15N [(−0.48±0.72)‰] but an increase in C/N (0.86±0.73) within 30 d (P<0.05). However, the δ15N and C/N values significantly increased in beak and gonad stored in formaldehyde on the 180th day (P<0.05). Therefore, it should be careful to use the preserved soft tissue samples during stable isotope analysis, while hard tissue, such as horny jaw, can be preserved by freezing or ethanol solution.
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Key words:
- Dosidicus gigas /
- Stable isotope /
- Freezing /
- Ethanol /
- Formaldehyde
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图 1 3种保存方法对茎柔鱼肌肉、角质颚和性腺组织δ13C、δ15N 和 C/N 的影响
Figure 1. Effect of three treatments on δ13C and δ15N and C/N of D. gigas muscle, beak and gonad tissues
表 1 茎柔鱼 3 种组织不同保存方式下的稳定同位素比值
Table 1. δ13C, δ15N values and C/N ratios of different preservation methods in three tissues of D. gigas
组织
Tissue对照
Control−20 ℃ 冷冻
Freezing75% 乙醇
Ethanol38% 甲醛
Formaldehyde第0天
Day 0第30天
Day 30第180天
Day 180第30天
Day 30第180天
Day 180第30天
Day 30第180天
Day 180肌肉 Muscle δ13C −17.97±0.30a −18.73±0.26b −19.31±0.33b −18.40±0.56b −18.47±0.37b −22.36±3.61b −21.66±1.31b δ15N 15.76±2.73a 16.06±2.80a 15.94±3.06 a 16.47±2.58b 16.62±2.71b 14.95±2.17b 16.09±2.60b C/N 3.22±0.07a 3.49±0.06b 3.51±0.12 b 3.57±0.14b 4.25±0.96c 4.79±1.15b 4.66±1.56b 角质颚 Beak δ13C −18.48±0.48a −18.54±0.53a −18.47±0.51a −18.50±0.52a −18.51±0.52a −20.50±2.29b −19.96±1.52b δ15N 10.61±0.98a 10.58±1.10a 10.25±1.14a 10.61±1.17a 11.39±1.02 a 9.85±1.04b 10.82±0.61a C/N 5.38±0.51a 5.56±0.32a 5.52±0.52a 5.47±0.53a 5.46±0.57 a 5.81±0.52b 6.36±0.40c 性腺 Gonad δ13C −17.61±0.79a −18.62±0.87b −18.94±0.56b −17.97±0.58b −18.94±0.56c −21.56±0.85b −21.40±0.80b δ15N 14.88±0.96a 14.82±1.03a 14.22±1.04 a 14.92±1.02a 14.65±1.01a 14.68±1.04b 15.02±1.17c C/N 3.21±0.16a 3.57±0.33b 3.55±0.35 b 3.42±0.13b 3.91±0.22c 3.97±0.36b 3.93±0.37c 注：同行数据不同上标字母表示有显著性差异 (P<0.05)。 Note: Values in each row followed by different superscript letters were significant differences (P<0.05). 
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