This study investigated the growth performance and nutritional composition of scale artificially cultured cuttlefish Sepia pharaonis. Juveniles were cultured in an open-culturing cement pool system for 120 days. The body weight increased from 10.21 AE 1.44 g to 570.71 AE 126.32 g from 50 days old to 170 days old, and the average growth rate was 4.67%. The proximate, amino acid and fatty acid compositions of S. pharaonis muscles were analysed every 40 days to compare the quality. The cultured S. pharaonis were rich in essential amino acids (EAAs), functional amino acids (FAAs), docosahexaenoic acid (DHA) and polyunsaturated fatty acids (PUFAs), which accounted for 32%, 46%, 28% and 54% (dry weight) respectively.Total amino acids (TAAs) and EAAs exhibited a clear distinction between ages, and significant differences were observed among the levels of individual amino acids, including Pro, Ala, Asp and Lys, which were significantly higher at 130-170 days old than at 50 days old (p < 0.05). Although the total saturated fatty acids, monounsaturated fatty acids and PUFAs were statistically similar between ages, C17:0, C22:6 n-3 and PUFAs were higher at 130-170 days old than at 50 days old (p < 0.05). The results indicate that large-scale artificial culture of S. pharaonis can be achieved under the conditions of a cement pool. This study also provides new information regarding the growth performance and nutritional composition of cultured S. pharaonis, which will contribute to the development of aquaculture practices for this species. K E Y W O R D S age, artificial culture, biochemical composition, cuttlefish, growth, Sepia pharaonis wileyonlinelibrary.com/journal/are Aquaculture Research. 2018;49:2788-2798.

The ink gland in cephalopods is a modified part of the digestive tract that generates melanin used in defense activities against predators. In the present study, we document the histological structure of the ink gland of the cuttlefish Sepia pharaonis following light microscopy (LM) and transmission electron microscopy (TEM) examinations. Based on the observations, we infer the series of cellular events leading to the formation and secretion of melanin. LM observations revealed that the cuttlefish ink gland was cord‐like, and could be separated into two distinct regions with different functions, based on markedly different cellular and biochemical characteristics. The region with the function of secreting ink was the epithelial cell region (i.e., mature ink gland cells) and was mainly composed of melanosomes, rough endoplasmic reticulum, Golgi apparatus, and mitochondria. Conversely, the connective tissue region was the site of formation of cells and energy supply. In addition, the most probable sequence of activities from melanogenesis to the release, based on TEM observations, is as follows: (a) formation of a matrix of opaque electronic substances (melanin precursors); (b) melanization in the precursor matrix to form melanin‐like particles; (c) further melanin granule development and enlargement, and gradual intensifying of the hue; (d) production of melanin granules (melanosomes) in the precursor matrix; (e) binding of melanosomes to the cytoskeleton and migration to the cell surface; (f) fusion of melanosomes with the cell membrane at the apical pole, and discharge of melanin granules into intercellular spaces or lumen by exocytosis or cell fragmentation. We also investigated how the process of continuous ink release influences ink gland histology and ultrastructure. Inking stimulation induced the release of large amounts of ink by epithelial cells, and was associated with cell disorganization, and severe cellular vacuolization, in addition to severe organelle damage.

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Two scale trials were conducted to determine the optimum weaning protocols cuttlefish (Sepia pharaonis) juvenile for understanding the feeding habits of cuttlefish and the development of effective aqua‐cultural practices. Three experimental weaning protocols (i.e. cuttlefish size, feeding frequency and culture density) on the growth, survival and muscle proximate composition of cuttlefish juveniles were performed in a small‐scale trial. Then, the optimal weaning protocols were applied to a large‐scale trial for validation. The weaning period lasted 2 weeks. The results showed that larger individuals showed better growth performance than smaller individuals, especially in terms of juvenile survival and specific growth rate (SGR), indicating that larger individuals were more receptive to frozen prey. Increased feeding frequency did stimulate the appetite of animals raised during feeding conversion, and enhance survival, growth and protein deposition compared to low feeding frequency. Moreover, the weight gain (WG) and SGR in the 60 and 70 cuttlefish/m2 culture density groups were much higher than those in the 80 cuttlefish/m2 group; however, there was no significant difference in survival among the culture density groups. The validation results of optimal weaning protocols in the large‐scale trial indicated that, according to growth performance, the optimum culture density for juvenile cuttlefish weaning was 70 cuttlefish/m2. These results indicate that the optimal weaning protocols for cuttlefish juvenile are as follows: a dorsal mantle length of 24.0 mm or more, the feeding frequency of 4 meals/day and the culture density of 70 cuttlefish/m2. These findings have important implications for understanding the feeding habits of cuttlefish and for the development of effective aqua‐cultural practices.

Several marine mollusks, including cephalopods (cuttlefish, squid, and octopus) and gastropods (e.g., sea hares), can release a colored ink secretion when chased by predators or stimulated. Ink release is part of a defensive response, but the threshold for the biochemical responses caused by stimulation is unknown. The present study aimed to reveal antipredator responses of cuttlefish, such as escaping via inking and/or jetting, and to investigate its biological and biochemical responses to continuous ink release. Results showed that the behavioral responses to continuous ink release mainly manifested as blazing body pattern changes. Cuttlefish escaped from predators covered by jetting/inking and warned the potential threats by displaying a unique body pattern. Moreover, persistent inking in the presence of an overt stimulus caused uncontrollable ink release from the ink duct/anal canal (loss of control). This study first verified the characteristics of the cuttlefish ink solution, prepared a standard curve of ink solution concentrations, and fitted the relationship function between the release frequency and the released ink weight. Biological statistics indicated that cuttlefish has the ability to continuously release ink (releasing ∼90% of the ink from the ink sac) and that the individuals adapted well during the recovery period. However, re-releasing ink would result in “overexploitation” and high mortality. Hexokinase (HK), pyruvate kinase (PK), and superoxide dismutase (SOD) activities, as well as malondialdehyde (MDA) concentration increased or remained stable in different tissues after releasing ink. The expression of heat shock protein 90 and arginine kinase (AK) were upregulated by stimuli in all tissues. Biochemical changes indicated that continuous inking not only consumed considerable energy but also damaged the tissues. In summary, cuttlefish released almost 90% of their ink for active defense against predators, and it took ∼30 days for the ink sac to be refilled, but “overexploitation” resulted in serious physiological damage. These findings will be helpful to further study the defense and ink release mechanisms and to consider animal health and welfare when using cephalopods as experimental animals and for aquaculture practices.

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