Research paper
Crustacean cardioactive peptides: Expression, localization, structure, and a possible involvement in regulation of egg-laying in the cuttlefish Sepia officinalis

https://doi.org/10.1016/j.ygcen.2017.12.009Get rights and content

Highlights

Abstract

The cuttlefish (Sepia officinalis) is a cephalopod mollusk distributed on the western European coast, in the West African Ocean and in the Mediterranean Sea. On the Normandy coast (France), cuttlefish is a target species of professional fishermen, so its reproduction strategy is of particular interest in the context of stock management. Egg-laying, which is coastal, is controlled by several types of regulators among which neuropeptides. The cuttlefish neuropeptidome was recently identified by Zatylny-Gaudin et al. (2016). Among the 38 neuropeptide families identified, some were significantly overexpressed in egg-laying females as compared to mature males.

This study is focused on crustacean cardioactive peptides (CCAPs), a highly expressed neuropeptide family strongly suspected of being involved in the control of egg-laying. We investigated the functional and structural characterization and tissue mapping of CCAPs, as well as the expression patterns of their receptors. CCAPs appeared to be involved in oocyte transport through the oviduct and in mechanical secretion of capsular products. Immunocytochemistry revealed that the neuropeptides were localized throughout the central nervous system (CNS) and in the nerve endings of the glands involved in egg-capsule synthesis and secretion, i.e. the oviduct gland and the main nidamental glands. The CCAP receptor was expressed in these glands and in the subesophageal mass of the CNS. Multiple sequence alignments revealed a high level of conservation of CCAP protein precursors in Sepia officinalis and Loligo pealei, two cephalopod decapods. Primary sequences of CCAPs from the two species were fully conserved, and cryptic peptides detected in the nerve endings were also partially conserved, suggesting biological activity that remains unknown for the time being.

Introduction

In the cuttlefish Sepia officinalis, spawning begins with the release of mature oocytes into the genital coeloma, followed by their retention until mating. This blockage prevents oocyte emission if the female cuttlefish has not been fertilized. Mating triggers peristaltic contractions of the oviduct that restart oocyte transport through the genital tract. A first inner layer of the capsule is secreted by the oviduct gland (OvG), which is closely associated with the distal oviduct. The partially encapsulated oocyte is released into the mantle cavity where the outer capsule is secreted by the main nidamental glands (MNGs). This second capsule is made of egg-capsule proteins (Cornet et al., 2015) which make up a network with polysaccharides, bacteria, and melanin from the ink bag. The encapsulated oocyte quits the mantle cavity through the siphon, probably carried by the gill-current associated with siphon contractions, and remains approximately 3 min in a cavity formed by the arms and the buccal mass. At this stage, fertilization takes place with the spermatozoa stored by the female since mating. The eggs are finally laid on a supporting material near the bottom, e.g. seaweeds, to form the egg mass. The successive ovulation, oocyte transport, encapsulation and fertilization steps are orchestrated by neuropeptides (Henry et al., 2013, Henry et al., 1997, Henry et al., 1999), ovarian regulatory peptides (Bernay et al., 2006, Bernay et al., 2005, Bernay et al., 2004, Zatylny et al., 2000a, Zatylny et al., 2000b), and sex pheromones (Enault et al., 2012).

Although neuropeptides are not the only regulators involved in regulation of egg-laying mechanisms, they nevertheless play an important role in integrating environmental parameters such as the photoperiod, temperature, salinity or depth. Expressed and released by neurons, they make up a highly diversified category by their structure, their mode of action, and the physiological functions they regulate.

In cuttlefish, neuropeptides like APGWamide (Henry et al., 1997), FMRFamide (Henry et al., 1999) and sepiatocin (Henry et al., 2013) play a role in oocyte transport and capsular secretion. In other mollusks such as the Sydney Rock Oyster (Saccostrea glomerata), several neuropeptides induce egg laying in sexually mature individuals: egg-laying hormone (ELH), GnRH, APGWamide, buccalin, CCAPs (crustacean cardioactive peptides), and LFRFamide (In et al., 2016). In gastropods, ELH appears to be pivotal in egg-laying regulation, particularly in hermaphrodite snails such as Aplysia (Arch, 1972, Chiu et al., 1979, Chiu and Strumwasser, 1981) and Lymnaea (Ebberink et al., 1985, Geraerts et al., 1983, Vreugdenhil et al., 1985), whereas in gonochoric gastropods many neuropeptides also appear to be involved in addition to ELH. In the abalone Haliotis asinina, several genes encoding APGWamide, myomodulin, proctolin-like, FMRFamide, schistosomin-like, insulin and a halotid growth-associated peptide are differentially expressed during the two-week spawning cycle in both male and female abalone (York et al., 2012).

A recent study in cuttlefish (S. officinalis) identified the neuropeptidome as composed of at least 38 distinct families (Zatylny-Gaudin et al., 2016). On the basis of expression patterns and tissue localization, several neuropeptide families were strongly suspected to be involved in the control of the successive steps that lead to the formation and release of encapsulated eggs: APGWamide, CCAPs, clionin, FLGamide, PTSP-like peptides, SCP (small cardioactive peptide), insulin, myomodulin, sepiatocin and SPamide.

The present study is focused on SoCCAPs (for Sepia officinalis CCAPs), which are highly overexpressed in egg-laying females versus mature males and are detected by mass spectrometry in the nerve endings of mature female OvG.

The first CCAP was initially identified in Carcinus maenas on the basis of its cardio responsive activity (Stangier et al., 1987). Later, the same peptide was found to modulate oviduct activity in Locusta migratoria (Donini et al., 2001) and therefore thought be involved in egg emission. Furthermore, this neuropeptide is also involved in fertilization in L. migratoria since it increases the basal tonus and the frequency of spontaneous spermatheca contractions (da Silva and Lange, 2006).

In C. maenas, the CCAP protein precursor (Fig. 1A) encodes one copy of a single neuropeptide: PFCNAFTGCamide (Chung et al., 2006), also found in many arthropods such as Drosophila melanogaster (Adams et al., 2000), Manduca sexta (Loi et al., 2001), Periplaneta americana (Honegger et al., 2002), Euphausia crystallorophias (Toullec et al., 2013), Homarus americanus (Christie et al., 2017), and Chorismus antarcticus (Toullec et al., 2017). From a structural point of view, analysis of the S. officinalis precursor predicted the release of 3 neuropeptides with very similar sequences, subsequently characterized by mass spectrometry (Zatylny-Gaudin et al., 2016). A special feature of these neuropeptides lies in the fact that although they are not amidated at their C-terminal end they keep the characteristic disulfide bond, in contrast to CCAPs from arthropods and bivalves (In et al., 2016). We named these neuropeptides based on the order of their occurrence in the precursor: SoCCAP1 (VFCNSFGGCTNI), SoCCAP2 (VFCNSYGGCKSF), and SoCCAP3 (VFCNSFGGCQN). The neuropeptidome of the gastropod Deroceras reticulatum recently revealed 2 precursors able to release 3 CCAPs each, including one non-amidated peptide (Ahn et al., 2017). In other gastropods like Lottia gigantea (Veenstra, 2010) or Aplysia californica (XP_005103388.1), 3 amidated CCAPs are predicted to be generated from their precursor (Fig. 1B). In addition, as observed in arthropods, the primary sequence of molluscan CCAPs is well conserved (Fig. 1B), suggesting a major physiological role. In arthropods, a phylum with abundant physiological data, the involvement of CCAPs is well described in many main regulation pathways controlling the heart rate, ecdysis behavior, or oviduct contraction in insects such as Manduca sexta (Lehman et al., 1993), Tenebrio molitor (Furuya et al., 1993), D. melanogaster (Baker et al., 1999, Dulcis et al., 2005, Dulcis and Levine, 2003), L. migratoria (Donini et al., 2001), Anopheles gambiae (Estévez-Lao et al., 2013), or in crustaceans such as C. maenas (Stangier et al., 1987) and Cancer magister (McGaw et al., 1995).

The aim of the present study was to demonstrate the involvement of SoCCAPs in the regulation mechanisms of egg-laying in cuttlefish (S. officinalis). In this perspective, we investigated tissue expression of SoCCAPs and their putative receptor. The neuropeptides were localized by immunostaining using specific polyclonal antibodies, and characterized by mass spectrometry. The capacity of SoCCAPs to adopt a defined structure in a membrane mimicking their environment was evaluated by nuclear magnetic resonance (NMR). Biological activity was assessed by a myotropic bioassay performed on the ovarian stroma (oocyte release into the genital coelom), the distal oviduct (oocyte release into the mantle cavity and internal egg capsule secretion), and the MNGs outer egg-capsule secretion). Finally, expression of the receptor was studied in the different tissues by an in silico approach from the transcriptomes produced by Zatylny-Gaudin et al. (2016).

Section snippets

Animal and tissue collection

All mature cuttlefish were trapped in the Bay of Seine between April and June 2015, 2016 and 2017. They were maintained in 1000-liter outflow tanks at 15 ± 1 °C at the Marine Station of Luc-sur-Mer (University of Caen-Normandy, France) under a natural photoperiod. Organs were dissected on animals anesthetized with 3% ethanol according to (Gonçalves et al., 2012), and then immediately frozen in liquid nitrogen or stored in artificial seawater (Reef crystal®) containing 1 mM glucose and

Expression patterns of SoCCAPs and SoCCAP GPCR

Expression patterns revealed that SoCCAP transcripts were expressed in the three main parts of the CNS: the optic lobes (OL), the supraesophageal mass (SupEM), and the subesophageal mass (SubEM) (Fig. 2A). High levels of overexpression were observed in the SubEM, which is the part of the CNS that innervates viscera and the genital apparatus; it is connected to the only neurohemal area described in cuttlefish so far.

Expression of the SoCCAP receptor identified from the annotated S. officinalis

Discussion

In a previous paper, we showed that the SoCCAP precursor was expressed in different parts of the CNS and overexpressed in egg-laying females as compared to sexually mature males (Zatylny-Gaudin et al., 2016).

The present study addresses the putative involvement of SoCCAPs in the regulation of egg-laying through tissue expression patterns, tissue mapping of mature neuropeptides, identification and tissue mapping of receptor transcripts, and bioactivity of SoCCAPs on the genital tract. In

Acknowledgments

We thank Jean-Luc Blaie and Maxime Marie, captains of the professional fishing boats “Père Daniel” and “Bip-Bip”, and their crews for their valuable help in providing cuttlefish. We thank Beatrice Adeline for technical support in histological analysis, and Christophe Roger for technical support in the construction of specific equipments. We thank Drs Baptiste Legrand and Matthieu Simon for their help with the AMBER software program. This work was financed by the ANR “NEMO” and the Conseil

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