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

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

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

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 females [56].

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

The conserved innate immune Toll/NF-κB pathway was described for the first time in *Sepia officinalis* ANG [59]. The transcriptomic analysis of ANG led to the identification of different constitutive elements of the Toll/NF-κB pathway. Five related Toll receptors (TLRs) have been characterized. Among them, TLRα shares 89% sequence identity with the unique TLR found in the light organ of *E. scolopes*. In addition, eight phosphorylation cascade elements have been demonstrated such as IRAK, TRAF6, and Rel/NF-κB. These immune pathway proteins (α2-macroglobulin-like protein, CD-63 antigen, transferrin) are probably involved in the establishment and maintenance of the bacterial symbionts like those in the light organ of *E. scolopes* [60]. Although several studies have been carried out about the subject, the real function of this ANG and its symbionts remains unknown. Several studies in squid suggest protection of the egg via the secretion of antimicrobial or antifouling compounds by ANG or its symbionts [61, 62], but no molecule has been characterized yet.

The function of the main nidamental gland (MNG) in egg case formation is clearer (**Figure 11A**). This white gland secretes the mucopolysaccharides and glycoproteins that form the egg case.

A recent unpublished analysis of the MNG proteome reveals the occurrence of proteins involved in glycolysis/gluconeogenesis (6-phosphofructo-2-kinase, type-2 Hexokinase, Pyruvate kinase, Glyceraldehyde 3-phosphate dehydrogenase, Fructose-1,6-bisphosphatase, fructosebisphosphate aldolase) and in glycogenolysis/glycogenesis (Glycogen phosphorylase, Glycogen synthase). These results indicate a large amount of energy production and consumption by the MNG due to an intense production and secretion of egg case components. Some of the identified proteins are also involved in the metabolism of polysaccharides or glycoproteins, like glycosyltransferases, which catalyze the transfer of oligosaccharide moieties from activated nucleotide sugars to nucleophilic glycosyl acceptor molecules or GDP-mannose pyrophosphorylase, involved in the production of N-linked oligosaccharides. Finally, the MNG secretes the main capsular components, the Egg Case Proteins, involved in the formation of a narrow mesh that provides elasticity and resistance properties to the egg case [63].
