Effects of preservation methodology on multi-tissue stable isotope signatures of oceanic squid: a case study of jumbo squid (Dosidicus gigas)

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 180^th 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.