**4. Neuropeptides**

As mentioned above, the first neuropeptide identified in cuttlefish on the basis of its ability to modulate the contractile activity of the oviduct was characterized by Henry and collaborators [2] from the optic lobes of egg-laying females (**Figure 2**).

It is the GWamide, a dipeptide that belongs to the APGWamide family and is derived from enzymatic cleavage of the APGWamide by a CNS dipeptidyl aminopeptidase (DPAP).

DPAP activity appears to be an alternative mechanism for the maturation of precursors into bioactive proteins or peptides such as peptides produced by amphibian skin, precursors of lytic peptides in honeybee venom, bactericidal peptides secreted in the insect hemolymph, or extracellular proteases in yeasts [11].

In gastropods, another molluscan class, the APGWamide is involved in the control of male behavior. In the pond snail *Lymnaea stagnalis*, this tetrapeptide detected in the penile nerve regulates penis erection [12–14].

Besides, FMRFamide-related peptides (FaRPs) also occur in the nervous fibers of the female accessory sex glands. Their occurrence was demonstrated by immunohistochemistry. Perfused FaRPs induce strong modifications of the contractile activity of the distal oviduct [3]. The involvement of two neuropeptide families—APGWa-RPs and FaRPs—in the control of egg-laying suggests a complex regulation of the successive steps leading to the formation of the egg mass.

**Figure 2.** Decrease of the tonus, frequency, and amplitude of oviduct contractions following treatment with 10−8 M synthetic GWamide. A dose of 10−9 M GWamide did not induce contractions; therefore, the threshold for GWamide activity appears to be between 10−8 and 10−9 M [2].

The identification of the cuttlefish neuropeptidome by *de novo* RNAseq and mass spectrometry screening was the next step that provided an overview of the neuropeptidome *via* a deep structural and functional investigation [10].

Thanks to the sequencing of the neurotranscriptome, several neuropeptide families involved in the regulation of egg-laying were identified on the basis of expression pattern and tissue localization out of the 38 families composing the cuttlefish neuropeptidome [10]. Finally, the RNA sequencing of ovary tissue revealed that most of the ovarian regulatory peptides involved in oocyte release were cleaved from a single yolk protein (unpublished results).

As mentioned above, the first neuropeptide identified in cuttlefish on the basis of its ability to modulate the contractile activity of the oviduct was characterized by Henry and collaborators

It is the GWamide, a dipeptide that belongs to the APGWamide family and is derived from

DPAP activity appears to be an alternative mechanism for the maturation of precursors into bioactive proteins or peptides such as peptides produced by amphibian skin, precursors of lytic peptides in honeybee venom, bactericidal peptides secreted in the insect hemolymph, or

In gastropods, another molluscan class, the APGWamide is involved in the control of male behavior. In the pond snail *Lymnaea stagnalis*, this tetrapeptide detected in the penile nerve regulates

Besides, FMRFamide-related peptides (FaRPs) also occur in the nervous fibers of the female accessory sex glands. Their occurrence was demonstrated by immunohistochemistry. Perfused FaRPs induce strong modifications of the contractile activity of the distal oviduct [3]. The involvement of two neuropeptide families—APGWa-RPs and FaRPs—in the control of egg-laying suggests a complex regulation of the successive steps leading to the formation of the egg mass.

**Figure 2.** Decrease of the tonus, frequency, and amplitude of oviduct contractions following treatment with 10−8 M synthetic GWamide. A dose of 10−9 M GWamide did not induce contractions; therefore, the threshold for GWamide

enzymatic cleavage of the APGWamide by a CNS dipeptidyl aminopeptidase (DPAP).

[2] from the optic lobes of egg-laying females (**Figure 2**).

extracellular proteases in yeasts [11].

activity appears to be between 10−8 and 10−9 M [2].

**4. Neuropeptides**

6 Biological Resources of Water

penis erection [12–14].

Based on the filtering criteria applied to the 38 identified neuropeptide families—expression level, neuropeptide tissue mapping, and mRNA localization—seven neuropeptide families were finally selected: allatostatins, APGWamide, crustacean cardioactive peptides (CCAPs), FaRPs, FLGamide, myomodulins, and small cardioactive peptide (SCP).

Several neuropeptides cleaved from the protein precursor of **allatostatin A1** and **A2** issued from alternative splicing were detected by nanoliquid chromatography tandem mass spectrometry (nLC-MS/MS) in the oviduct gland and the main nidamental glands, suggesting a role in egg capsule secretion. In insects, **FGLamide allatostatins** (also called **allatostatins A** or **buccalins**) are involved in reproduction [15] and feeding decisions interacting with the adipokinetic hormone (AKH) and insulin-like peptides [16]. In cuttlefish, **allatostatins A1** and **A2** were also detected in the hemolymph, in accordance with the status of neurohormones that could regulate the biosynthesis of egg capsule products during vitellogenesis.

The **APGWamide** was detected by nLC-MS/MS in the CNS, and the **GWamide** was characterized from the CNS [2]. Moreover, large amounts of mRNAs were found in the OGs, MNGs, and ANGs of egg-laying females. Similar observations were reported in the pond snail *Lymnaea stagnalis* by van Minnen and Bergman [17]. High amounts of mRNAs encoding the egg-laying hormone were detected in the nerve terminals after a stimulus, as well as polyribosomes, supporting that the translation of egg-laying hormone transcripts could occur in the axonal compartment. These data are supported by recent papers revealing the occurrence of rough endoplasmic reticulum, smooth reticulum, and Golgi apparatus in the axonal compartment [18]. Otherwise, Martin and Kim [19] used Aplysia as a model to show that netrin-1, already known to promote translation in axonal growth cones [20], increased translation of subcellular mRNAs localized at the level of dendrites or axons by binding the cytoplasmic domain of the netrin-1 receptor called DCC (for deleted colorectal cancer). The rapid reaction of female cuttlefish can be related to the state of readiness of the axons that innervate the ASGs.

The three neuropeptides predicted from the protein precursor of **CCAPs** were detected by nLC-MS/MS in the CNS, the oviduct gland and the main nidamental glands [10]. **CCAPs** are also overexpressed in the sub-esophageal mass of egg-laying females (as opposed to mature males), which is the only part of the CNS that innervates the genital apparatus. This neuropeptide family is strongly suspected to regulate egg capsule secretion.

**CCAPs** were initially described in crustaceans. They are usually C-terminally amidated neuropeptides in arthropods [21–24], as well as in mollusks [14, 25–27], whereas the cuttlefish preprohormone predicted three nonamidated peptides confirmed by nLC-MS/MS analysis.

The four neuropeptides predicted from the protein precursor of **FaRPs** were detected by nLC-MS/MS in the CNS, and the decapeptide ALSGDAFLRFamide was the only one detected in the neurohemal area connected to the sub-esophageal mass and in the oviduct gland and main nidamental glands, confirming the immunostaining results obtained by Henry and collaborators [3] in the ASGs of mature females. These neuropeptides are widely distributed in the animal kingdom and involved in many physiological regulation processes in mollusks such as heart activity [28], amylase secretion [29], feeding [30], and reproduction [31]. In cuttlefish, they are believed to regulate egg capsule secretion and oocyte transport in the oviduct [3] and also chromatophore control pathways [32].

of egg-laying females and detected in OG and MNGs; they stimulate the mechanical contraction of glands for egg capsule secretion when the oocyte reaches the distal oviduct embedded by the oviduct gland, just before being released into the mantle cavity; (3) **FLGamide** and **FMRFamide** neuropeptides are detected in OG and MNGs and also in the hemolymph; they modulate oviduct contractions and are probably involved in the mechanical secretion of the egg capsule. Moreover, as they are circulating peptides, they could also be involved in the

Egg-Laying in the Cuttlefish *Sepia officinalis* http://dx.doi.org/10.5772/intechopen.71915 9

**Figure 3.** Preprohormones of allatostatins A1 and A2, APGWamide, crustacean cardioactive peptide, FLGamide, FMRFamide-related peptides, myomodulin, neuropeptides F1, F2, F3, F4, F5, and small cardioactive peptide, that all encode neuropeptides detected in the nerve endings and/or in the CNS of *S. officinalis* egg-laying females or by *in silico* data mining. Predicted signal peptides are highlighted in yellow, convertase cleavage sites in red, cysteines are underlined, glycine residues predicted to be converted into C-terminal amides in blue-gray, as well as glutamine residues predicted to be converted into pyroglutamate. Neuropeptides are highlighted in gray, and the stop codons at

the end of the coding sequences are indicated by an asterisk [10].

**FLGamide** is a novel neuropeptide family never described so far in the animal kingdom although blastn revealed a similar precursor annotated "uncharacterized protein" (ELU03112) in the polychaete worm *C. teleta* [33]. Most of the eight neuropeptides predicted from the two protein precursors were detected by nLC-MS/MS in cuttlefish CNS, oviduct gland, main nidamental glands, and ovarian stroma [10]. It is the only neuropeptide family detected in the ovarian stroma, suggesting a putative involvement in the release of smooth oocytes into the genital coelom. But recent unpublished experiments show that these neuropeptides do not induce any modification on contractile activity when perfused into the ovarian stroma. As they are also detected in the hemolymph, they are thought to be involved in vitellogenesis regulation.

In the oviduct gland, recent unpublished experiments show that they regulate oocyte transport in the oviduct. They could also induce egg capsule biosynthesis and secretion.

**Myomodulins** were detected by nLC-MS/MS in the CNS, the oviduct gland and the main nidamental glands [10], and so were mRNAs, as described for **APGWamide** and **CCAPs**. They appear to be closely associated to accessory sex glands involved in egg capsule secretion.

In the tropical abalone *Haliotis asinine*, egg-laying is characterized by a dramatic increase in the expression of **APGWamide**, **myomodulins,** and **insulin** within 12 h of the spawning event. Expression strongly decreases 24 h after spawning, demonstrating that these neuropeptides have a regulatory role in the release of gametes [34]. In cuttlefish, the mRNAs of these three neuropeptides are recovered at the level of the oviduct gland and main nidamental glands, suggesting that they are involved in the rapid response of the genital apparatus after mating.

The detection of the **small cardioactive peptide (SCP)** by mass spectrometry proved very difficult and was restricted to the oviduct gland. As already described for **CCAPs**, **SCP** is also overexpressed in the sub-esophageal mass of egg-laying females (as opposed to mature males). This neuropeptide could be related to the secretion of the internal layer of the egg capsule and also in the release of oocytes into the mantle cavity. The preprohormones of all these neuropeptides are presented in **Figure 3**. The OG and MNGs are closely associated in egg capsule elaboration. Just before fertilization, oocytes are embedded into two layers of egg capsule proteins: the inner layer is secreted by the oviduct gland and the outer layer by the main nidamental glands. These glands synthesize and secrete most of the egg capsule constituents. The similar function of OG and MNGs could explain why they share so many common regulatory neuropeptides. Three categories can be distinguished among them (**Table 1**): (1) **APGWamide** neuropeptides and **myomodulins** whose mRNAs are recovered in OG and MNGs, probably located in the axon ends; they can be associated to a rapid responsiveness following mating; (2) **allatostatins A**, **CCAPs**, and **SCP** neuropeptides, overexpressed in the sub-esophageal mass of egg-laying females and detected in OG and MNGs; they stimulate the mechanical contraction of glands for egg capsule secretion when the oocyte reaches the distal oviduct embedded by the oviduct gland, just before being released into the mantle cavity; (3) **FLGamide** and **FMRFamide** neuropeptides are detected in OG and MNGs and also in the hemolymph; they modulate oviduct contractions and are probably involved in the mechanical secretion of the egg capsule. Moreover, as they are circulating peptides, they could also be involved in the

in the ASGs of mature females. These neuropeptides are widely distributed in the animal kingdom and involved in many physiological regulation processes in mollusks such as heart activity [28], amylase secretion [29], feeding [30], and reproduction [31]. In cuttlefish, they are believed to regulate egg capsule secretion and oocyte transport in the oviduct [3] and also chromatophore

**FLGamide** is a novel neuropeptide family never described so far in the animal kingdom although blastn revealed a similar precursor annotated "uncharacterized protein" (ELU03112) in the polychaete worm *C. teleta* [33]. Most of the eight neuropeptides predicted from the two protein precursors were detected by nLC-MS/MS in cuttlefish CNS, oviduct gland, main nidamental glands, and ovarian stroma [10]. It is the only neuropeptide family detected in the ovarian stroma, suggesting a putative involvement in the release of smooth oocytes into the genital coelom. But recent unpublished experiments show that these neuropeptides do not induce any modification on contractile activity when perfused into the ovarian stroma. As they are also detected in the hemolymph, they are thought to be involved in vitellogenesis

In the oviduct gland, recent unpublished experiments show that they regulate oocyte trans-

**Myomodulins** were detected by nLC-MS/MS in the CNS, the oviduct gland and the main nidamental glands [10], and so were mRNAs, as described for **APGWamide** and **CCAPs**. They appear to be closely associated to accessory sex glands involved in egg capsule secretion. In the tropical abalone *Haliotis asinine*, egg-laying is characterized by a dramatic increase in the expression of **APGWamide**, **myomodulins,** and **insulin** within 12 h of the spawning event. Expression strongly decreases 24 h after spawning, demonstrating that these neuropeptides have a regulatory role in the release of gametes [34]. In cuttlefish, the mRNAs of these three neuropeptides are recovered at the level of the oviduct gland and main nidamental glands, suggesting that they are involved in the rapid response of the genital apparatus

The detection of the **small cardioactive peptide (SCP)** by mass spectrometry proved very difficult and was restricted to the oviduct gland. As already described for **CCAPs**, **SCP** is also overexpressed in the sub-esophageal mass of egg-laying females (as opposed to mature males). This neuropeptide could be related to the secretion of the internal layer of the egg capsule and also in the release of oocytes into the mantle cavity. The preprohormones of all these neuropeptides are presented in **Figure 3**. The OG and MNGs are closely associated in egg capsule elaboration. Just before fertilization, oocytes are embedded into two layers of egg capsule proteins: the inner layer is secreted by the oviduct gland and the outer layer by the main nidamental glands. These glands synthesize and secrete most of the egg capsule constituents. The similar function of OG and MNGs could explain why they share so many common regulatory neuropeptides. Three categories can be distinguished among them (**Table 1**): (1) **APGWamide** neuropeptides and **myomodulins** whose mRNAs are recovered in OG and MNGs, probably located in the axon ends; they can be associated to a rapid responsiveness following mating; (2) **allatostatins A**, **CCAPs**, and **SCP** neuropeptides, overexpressed in the sub-esophageal mass

port in the oviduct. They could also induce egg capsule biosynthesis and secretion.

control pathways [32].

8 Biological Resources of Water

regulation.

after mating.


**Figure 3.** Preprohormones of allatostatins A1 and A2, APGWamide, crustacean cardioactive peptide, FLGamide, FMRFamide-related peptides, myomodulin, neuropeptides F1, F2, F3, F4, F5, and small cardioactive peptide, that all encode neuropeptides detected in the nerve endings and/or in the CNS of *S. officinalis* egg-laying females or by *in silico* data mining. Predicted signal peptides are highlighted in yellow, convertase cleavage sites in red, cysteines are underlined, glycine residues predicted to be converted into C-terminal amides in blue-gray, as well as glutamine residues predicted to be converted into pyroglutamate. Neuropeptides are highlighted in gray, and the stop codons at the end of the coding sequences are indicated by an asterisk [10].

regulation of the synthesis of yolk proteins and/or egg capsule proteins. In addition to the neuropeptides directly involved in the regulation of egg-laying, RNAseq revealed a substantial overexpression of neuropeptides Y (NPY), also called neuropeptides F (NPF) in protostomes because of a C-terminal tyrosine amide substituted by a phenylalanine amide (**Figure 3**). The five transcripts of cuttlefish NPF are unequally overexpressed.

remains unknown despite *in silico* data mining from transcriptomic data associated to nLC-MS/MS screenings of the CNS of egg-laying females. Considering that cephalopods are the only class among the three main molluscan classes in which no ELH was ever identified, we can hypothesize that the reason is the loss of the ELH gene or more probably a low level of structural conservation leading to a failure of the data mining strategy or an insufficient depth of RNAseq. Further deeper RNA sequencing will probably allow for the identification of this

Egg-Laying in the Cuttlefish *Sepia officinalis* http://dx.doi.org/10.5772/intechopen.71915 11

During a short life cycle of about 22 months, English Channel cuttlefish can perform four horizontal migrations from the Normandy coasts to the western part of the English Channel [35]. After a last migration to reach specific mating and spawning coastal areas, cuttlefish aggregate for mating and egg-laying between April and June on the Normandy coasts. This behavior suggests the occurrence of some kind of chemical communication *via* waterborne molecules that induce the aggregation of mates. Chemical communication in cuttlefish was first demonstrated by Boal and collaborators [36, 37] using y-mazes. They showed that recently laid eggs, ovary extracts and nidamental glands, induced an increase in ventilation rate and the attraction of sexually mature cuttlefish in the arm of the y-maze containing purified extract *versus* artificial sea water. In the same way, Cummins and collaborators [38] identified a 10 kDa protein in Loligo termed Loligo β-microsemino-protein (Loligo β-MSP) that immediately changes the behavior of male squid from calm swimming and schooling to extreme fighting. Loligo β-MSP is synthesized in the accessory sex gland of females—the oviduct gland, the main and accessory nidamental glands—and is secreted with the proteins of the outer tunic of egg capsules. When a male is attracted to the eggs visually, upon touching them and contacting Loligo

β-MSP, it immediately escalates into intense physical fighting with any nearby males.

Loligo β-MSP was originally discovered in human seminal plasma and prostatic fluids [39]. It is only described in other vertebrates [40–44] and in the basal chordate amphioxus [45]. It is a highly variable 91-amino-acid protein, with 10 spatially conserved cysteine residues than can potentially form five intramolecular disulfide bonds, giving resistance to proteolytic cleavage

In *Sepia officinalis*, Enault and collaborators [9] identified three major related transcripts encoding secreted peptides and expressed in the oviduct gland. RT-PCR and mass spectrometry analyses revealed that transcripts and expression products were co-localized in the oviduct gland. The two very similar protein precursors termed SPα and SPα′ (**Figure 4**) diverge by only four amino acids in the α3 and α3′ peptides. They yield seven putative expression prod-

All peptides except α1 contained at least one cysteine, and two of them, α3 and α3′, are

The third protein precursor, termed SPβ, shares 56.7% similarity with SPα and SPα′ (**Figure 4**) and yields five putative expression products ranging from 1.1 kDa (peptide β1) to 8.3 kDa

neuropeptide in cephalopods.

to prolong its activity on the egg surface.

ucts ranging from 1.3 kDa (α5) to 7 kDa (α3 and α3′).

C-terminally amidated like many bioactive peptides (**Figure 5A**).

**5. Sex pheromones**

The expression level of NPF 1, the most overexpressed of them, reaches 45-fold the expression level found in mature males in the same part of the CNS. NPFs are probably overexpressed in females to stimulate feeding in order to support gametogenesis and egg capsule synthesis between each spawning step. In this species, asynchronous gametogenesis allows females to resume egg-laying by replenishing their batch of mature oocytes and biosynthesizing egg capsule products until they die.

More generally, the overexpression of many neuropeptides found in the sub-esophageal mass of egg-laying females (as compared to males) could be due to the regulation associated to the production of several batches of oocytes that contain a large quantity of vitellus for embryonic development and to the mobilization of the energy needed to carry out gametogenesis. By contrast, in English Channel males, gametogenesis ends 6months before reproduction and produces a much smaller volume of gametes than in females. The energy required for male gametogenesis is probably very low compared with the energy required for female gametogenesis.


Finally, a 36-amino-acid neuropeptide called Egg-Laying Hormone (ELH) can induce egglaying following a single intramuscular injection in the foot of Gastropods. In cuttlefish, ELH

ANG: accessory nidamental gland; MNG: main nidamental gland; OG: oviduct gland; OS: ovarian stroma; SubEM: sub-esophageal mass.

**Table 1.** Tissue mapping of neuropeptides and mRNAs in the female cuttlefish.

remains unknown despite *in silico* data mining from transcriptomic data associated to nLC-MS/MS screenings of the CNS of egg-laying females. Considering that cephalopods are the only class among the three main molluscan classes in which no ELH was ever identified, we can hypothesize that the reason is the loss of the ELH gene or more probably a low level of structural conservation leading to a failure of the data mining strategy or an insufficient depth of RNAseq. Further deeper RNA sequencing will probably allow for the identification of this neuropeptide in cephalopods.
