**9. The oral cavity: completion of the eggs**

Cuttlefish eggs are large oocytes containing all the nutrient reserves required for embryo development. To withstand physical and microbial threats, mature oocytes are enclosed within a protective egg case produced by secretions of the female genital apparatus [53, 54]. This egg case is composed of two distinct envelopes. The inner layer is in direct contact with the chorion surrounding the oocyte; it is formed by secretions added, while the egg passes through the oviduct gland [53]. The oviduct gland secretes proteins and polypeptides. The main proteins secreted by this gland correspond to sex pheromones. Afterward, the oocyte is released inside the mantle cavity and embedded with an outer layer secreted by the two

The genital apparatus of *Sepia officinalis* contains two pairs of accessory reproductive glands partly involved in egg case formation (**Figure 11**). The main nidamental glands (MNGs) are related to the accessory nidamental glands (ANGs). The two paired glands are located on the ventral side of the visceral mass. The histological structure of these glands in cuttlefish is

The main nidamental gland and the oviduct gland both present a lamellar structure (**Figure 11C**). Each lamella consists of a central lamina of connective tissue covered with a glandular epithelium at the origin of the polysaccharides labeled by periodic acid-Schiff (PAS)-positive deposits (**Figure 11D**). The cells located at the free end of the lamellae produce particularly acid mucopolysaccharides and glycoprotein secretions revealed by alcian blue (**Figure 11E**), while the other cells secrete neutral mucopolysaccharides. During egg case formation, the secretions are released into the lumen and are led out through a duct opening onto the mantle cavity at the anterior end

The ANG is divided into four lobes attached to the anterior end of the MNG by conjunctive tissue. Histological observations of ANG reveal a tubular gland harboring symbiotic bacteria. These symbionts are enclosed in the lumen of tubular structures that nearly completely fill the gland (**Figure 11F**). The wall of each tubule appears to be composed of a single layer of ciliated epithelium with microvilli (**Figure 11G**). The role of this gland in reproduction is unclear. Some clues suggest its involvement in egg case formation at the spawning period. During sexual maturation, the ANG indeed increases in size and changes in color from white to bright orange at the time of spawning (**Figure 11A**). It also harbors a dense consortium of bacteria that secrete carotenoids at the origin of the intense orange color of ANGs in mature

Using 16S RNA gene sequencing, many bacterial taxa were identified in ANGs, including *Agrobacterium, Roseobacter, Sporichthya, Rhodobium, Xanthobacter*, and *Clostridium* [57]. The origin of the bacterial symbionts in cuttlefish remains undetermined. Although the presence of bacteria in the egg capsule suggests vertical transmission, we cannot exclude horizontal transmission as in *Loligo opalescens* [58]. In squid, ANGs develop only a few months after hatching from a

**8. Nidamental glands: a specificity in decabrachia cephalopods**

similar to the structure of squid (*Loligo forbesi*) nidamental glands [55].

of the gland (**Figure 11A**). MNG and ANG structures substantially differ.

single layer of cells containing many cilia and microvilli [58].

females [56].

nidamental glands and stained with ink (**Figure 11A**).

20 Biological Resources of Water

At the time of fertilization in the oral cavity of the female cuttlefish, the oocytes are already wrapped in the thick and complex egg case. The female's arms form a chamber to keep the freshly embedded oocytes near the oral copulatory pouch where spermatophores have been deposited by the male during mating (**Figure 1**). Fertilization of the oocytes by spermatozoa is facilitated by a diffusible chemoattractant factor: SepSAP (Sepia Attracting Sperm Peptide). This hexapeptide is expressed in the vitellogenic follicles and released by embedded oocytes through the various capsular envelopes to facilitate fertilization by increasing chances of gamete collision. SepSAP has an attractant effect on sperm from low concentrations around 10−17 M [64].

During fertilization, the eggs are also in contact with saliva. As early as 1934, Jecklin suggested that salivary secretions could protect the eggs during spawning [54]. A recent study of the transcriptome and proteome of *Sepia officinalis* posterior salivary gland seems to confirm this 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 protection or/and in improving fertilization.

(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

Egg-Laying in the Cuttlefish *Sepia officinalis* http://dx.doi.org/10.5772/intechopen.71915 23

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

**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.

hatching) begins once the organs are found in their final topology.

to its maximum (**Figure 13D**).

After 2 or 3 minutes in the oral cavity, the eggs are deposited by the female's arms on a natural structure like marine eelgrass (*Zostera marina*) or an artificial one like a rope.
