**10. The cuttlefish egg case**

During its development, the embryo is only secured by its egg case. The morphological evolution of the egg and its capsule from laying to hatching occurs in three phases during which the capsule undergoes major changes (**Figure 12**). The different steps described below correspond to embryonic development [66] first defined the different embryonic stages by performing a morphological study of the cuttlefish embryo during its development. The telolecithal egg presents a meroblastic discoidal cleavage (stages 1–9) associating blastomeres in central position and blastocones on its fringe. During epibolic gastrulation (stages 10–15), blastocones disappear under the ectoderm plate following the peripheral ring of blastula cells that will form the ring-shaped endo-mesoderm. At the end of gastrulation, the vitelline syncytium and extraembryonic ectoderm completely surround the yolk and internalize the vegetal pole to form the yolk sac. The cleavage period corresponds to the first phase (P1) of egg evolution. A few hours after laying, the egg cell is covered with a lamina propria and surrounded by a thick gelatinous capsule (1.4 mm, ±0.6 mm). In contact with seawater, the gelatinous and fluid capsule polymerize. This reduces the volume of the egg by about 30% (**Figure 12**) and its thickness by 50%.

After 15 days of incubation and following polymerization (**Figure 13A**), capsule thickness is down to 614 microns (±150 microns) (**Figure 13B**), and the outer and inner layers can be distinguished. Polymerization of the capsule proteins helps tighten the layers of coiled outer and inner envelopes, highlighting an increasing melanin gradient from the inner layers to the outer layers. The egg is then tightly wrapped by a hardened, strong yet elastic capsule. These morphological characteristics of the capsule define the second phase of egg evolution (P2), which lasts from the 7th day to the end of the first month and corresponds to gastrulation and the beginning of organogenesis. The embryo develops within the limits of a disk located at the animal pole, at the surface, or above the yolk mass (**Figure 12**), while the capsule size and thickness remain unchanged. The initiation of organogenesis marks the beginning of the last phase (P3) that ends with hatching. The embryo in early organogenesis does not yet fill the perivitelline space. However, the capsule has become permeable to let in water and solutes. Thus, the accumulation of fluids in the perivitelline space causes the capsule to stretch, and its thickness continues to decrease (437.9 (±104) μm). Organogenesis corresponds to 2/3 of the development period, and it follows after the closure of the yolk sac and ends with hatching and can be divided into three phases (**Figure 12**). (1) During discoid or early organogenesis (stages 15–20), the embryo forms a disk at the animal pole. The different embryonic territories build up above the yolk mass. (2) The second phase corresponds to an extension phase (stages 20–23). The brachial crown tightens on the yolk mass; the embryo straightens into the anteroposterior axis. Its rear end corresponding to the mantle gradually moves apart, leaving the brachial crown, mouth, and eyes toward the yolk. (3) The final growth phase (stage 23 to hatching) begins once the organs are found in their final topology.

hypothesis. In addition to enzymes and toxins such as cephalotoxins and CRISPs (Cysteine Rich Secreted Proteins), cuttlefish saliva contains many immune effectors like α-macroglobulin, lysozyme, Bactericidal/Permeability-Increasing proteins (BPIs), and Lipopolysaccharide-Binding Proteins (LBPs) [65]. These salivary proteins very likely play a role in gamete protec-

After 2 or 3 minutes in the oral cavity, the eggs are deposited by the female's arms on a natural

During its development, the embryo is only secured by its egg case. The morphological evolution of the egg and its capsule from laying to hatching occurs in three phases during which the capsule undergoes major changes (**Figure 12**). The different steps described below correspond to embryonic development [66] first defined the different embryonic stages by performing a morphological study of the cuttlefish embryo during its development. The telolecithal egg presents a meroblastic discoidal cleavage (stages 1–9) associating blastomeres in central position and blastocones on its fringe. During epibolic gastrulation (stages 10–15), blastocones disappear under the ectoderm plate following the peripheral ring of blastula cells that will form the ring-shaped endo-mesoderm. At the end of gastrulation, the vitelline syncytium and extraembryonic ectoderm completely surround the yolk and internalize the vegetal pole to form the yolk sac. The cleavage period corresponds to the first phase (P1) of egg evolution. A few hours after laying, the egg cell is covered with a lamina propria and surrounded by a thick gelatinous capsule (1.4 mm, ±0.6 mm). In contact with seawater, the gelatinous and fluid capsule polymerize. This reduces the volume of the egg by about 30% (**Figure 12**) and its thickness by 50%. After 15 days of incubation and following polymerization (**Figure 13A**), capsule thickness is down to 614 microns (±150 microns) (**Figure 13B**), and the outer and inner layers can be distinguished. Polymerization of the capsule proteins helps tighten the layers of coiled outer and inner envelopes, highlighting an increasing melanin gradient from the inner layers to the outer layers. The egg is then tightly wrapped by a hardened, strong yet elastic capsule. These morphological characteristics of the capsule define the second phase of egg evolution (P2), which lasts from the 7th day to the end of the first month and corresponds to gastrulation and the beginning of organogenesis. The embryo develops within the limits of a disk located at the animal pole, at the surface, or above the yolk mass (**Figure 12**), while the capsule size and thickness remain unchanged. The initiation of organogenesis marks the beginning of the last phase (P3) that ends with hatching. The embryo in early organogenesis does not yet fill the perivitelline space. However, the capsule has become permeable to let in water and solutes. Thus, the accumulation of fluids in the perivitelline space causes the capsule to stretch, and its thickness continues to decrease (437.9 (±104) μm). Organogenesis corresponds to 2/3 of the development period, and it follows after the closure of the yolk sac and ends with hatching and can be divided into three phases (**Figure 12**). (1) During discoid or early organogenesis

structure like marine eelgrass (*Zostera marina*) or an artificial one like a rope.

tion or/and in improving fertilization.

22 Biological Resources of Water

**10. The cuttlefish egg case**

After 72 days of incubation, a few days before hatching, the embryo completes its growth and has assimilated much of the yolk reserves. It now fills most of the available space in the egg and is surrounded by a large amount of perivitelline fluid (about 1 ml), stretching the capsule to its maximum (**Figure 13D**).

**Figure 12.** Evolution of *Sepia officinalis* egg size during embryogenesis at 16°C. Evolution phases of the egg case: P1, polymerization of the egg case; P2, stabilization of the egg case; P3, thinning and delamination of the egg case. Illustration of different stages of embryogenesis during cleavage, gastrulation and organogenesis. Yellow: vitellus, red: future eyes, blue: future mantle and shell, green: future arms; pf, perivitelline fluid.

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 ultrastruc-

Egg-Laying in the Cuttlefish *Sepia officinalis* http://dx.doi.org/10.5772/intechopen.71915 25

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

**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).

ture shows a narrow mesh composed of glycoproteins and polysaccharides.

**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. (photo credits: V. Cornet).

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

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

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