**11. Egg case composition**

The capsule of *Sepia officinalis* eggs has a specific black color (**Figure 14A**). Only females belonging to the Sepiidae family include melanin granules into the egg capsule. Melanin is secreted by the ink bag and is integrated into egg case via secretions from the main nidamental gland (**Figure 11A**). Other compounds of the ink such as proteins may well also integrate the capsule.

Structural analysis of the egg capsule by photonic microscopy reveals a lamellar structure of the inner and outer envelopes (**Figure 13B**), with successive spirally wound layers. The outer envelope contains melanin deposits gathered in layers that become increasingly intense. Observations of the outer envelope by Transmission Electron Microscopy showed the presence of melanin deposits and revealed the occurrence of isolated or grouped structures whose size ranged between 0.4 and 1 μm, corresponding to bacterial structures (**Figure 13B** and **D**). These bacteria probably come from the accessory nidamental gland. The egg case ultrastructure shows a narrow mesh composed of glycoproteins and polysaccharides.

SepECP 1 and SepECP2 are cationic, cysteine-rich protein of 71 and 74 kDa, respectively (**Figure 15**). These two proteins were characterized as the main constituents of the cuttlefish egg case [16]. SepECPs are only secreted by females, mainly by the MNG and also by the oviduct gland. These two proteins are highly cationic, with 73 positively charged residues for ECP1 and 43 for ECP2. They exhibit bacteriostatic activity against a few pathogenic GRAMbacteria from the Vibrio genus. Their bacteriostatic activity could explain the occurrence of

At this stage, the embryo's features are similar to the adult's; the embryo enters a linear growth phase. All essential elements of the brachial device, the nervous system, the palleal, and visceral parts are now in place Organogenesis ends with the transfer of the outer yolk sac

**Figure 13.** Longitudinal sections of the egg after 15 days (A) and 72 days (D). ANG stained in Prenant-Gabe triple staining. Magnification of the egg case including capsule thickness after 15 days (B) and 72 days (C). C, capsule or egg case; ch, chorion; emb, embryo; il, inner layer; pf, perivitelline fluid; ol, outer layer; Ct, capsule thickness; v, vitellus.

The outer and inner capsule envelopes have now completely merged, and the outermost layers of the capsule including melanin appear to be delaminated (**Figure 13C**). Thus, at the time of hatching, the capsule has undergone significant changes: it has become extremely thin

At the time of hatching (Stage 30), 75–80 days after egg-laying and at 16°C, the release of enzymes by the Hoyle organ located on the end of the dorsal mantle facilitate the emergence

The capsule of *Sepia officinalis* eggs has a specific black color (**Figure 14A**). Only females belonging to the Sepiidae family include melanin granules into the egg capsule. Melanin is secreted by the ink bag and is integrated into egg case via secretions from the main nidamental gland (**Figure 11A**). Other compounds of the ink such as proteins may well also integrate the capsule.

(156.8 (±110) microns) and friable, so that it will break easily and release the juvenile.

of the juvenile [67]. Hatching is also facilitated by the thinning of the capsule.

**11. Egg case composition**

(photo credits: V. Cornet).

24 Biological Resources of Water

to the inner yolk sac, enabling faster assimilation of energy resources.

**Figure 14.** Photographs of the *Sepia officinalis* egg case and its components. (A) Freshly laid egg. (B and C) thin sections of the outer layer of the egg case in TEM. (D) Dividing bacteria and melanin granules. (C) Observation in TEM of SepECPs extracted from the egg case. White asterisks correspond to the protein network; b, bacteria; m, melanin. (Photo credits: C. Zatylny-Gaudin, V. Cornet, D. Goux).

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**Figure 15.** Amino acid alignments of the SepECP precursors. Yellow: signal peptide; orange: conserved cysteine domains; green: conserved motifs up to five amino acids. Red asterisks indicate conserved cysteines, and underlined sequences correspond to potential glycosylation sites.

bacteria in the egg case, corresponding to potential bacterial symbionts. The two SepECPs display 48 conserved cysteines grouped in three cysteine domains (**Figure 15**). These cysteines could be implied in intramolecular and intermolecular disulfide bonds involved in the formation of heterodimers. SepECPS are indeed involved in the formation of a network (**Figure 14C**) or dense matrix protecting the embryo against mechanical shocks and microbial infection during its development. No infection or biofilm is observed on cuttlefish eggs under controlled conditions or in natural environments. The capsule seems very effective: both antifouling and antibacterial coatings prevent pathogenic bacteria from proliferating [63].

During embryo development, the egg case becomes increasingly thin, but it retains elasticity to allow for embryonic growth. SepECPs are probably cleaved during the last phase (P3) to allow for hatching. During this phase, when the capsule seems more fragile, the embryo keeps developing without being affected by pathogens. When they are degraded, highly cationic SepECPs probably generate antibacterial cationic peptides. Last of all, a role of the perivitelline fluid in embryo protection should not be ruled out.
