A Complex Set of Sex Pheromones Identified in the Cuttlefish Sepia officinalis

Jérémy Enault,Benoît Bernay,Joël Henry,Benjamin Lefranc,Céline Zatylny-Gaudin,Michèle Baudy-Floc'H,Jérôme Leprince

doi:10.1371/journal.pone.0046531

Abstract

BackgroundThe cephalopod mollusk Sepia officinalis can be considered as a relevant model for studying reproduction strategies associated to seasonal migrations. Using transcriptomic and peptidomic approaches, we aim to identify peptide sex pheromones that are thought to induce the aggregation of mature cuttlefish in their egg-laying areas.ResultsTo facilitate the identification of sex pheromones, 576 5′-expressed sequence tags (ESTs) were sequenced from a single cDNA library generated from accessory sex glands of female cuttlefish. Our analysis yielded 223 unique sequences composed of 186 singletons and 37 contigs. Three major redundant ESTs called SPα, SPα′ and SPβ were identified as good candidates for putative sex pheromone transcripts and are part of the 87 unique sequences classified as unknown. The alignment of translated SPα and SPα′ revealed a high level of conservation, with 98.4% identity. Translation led to a 248-amino acid precursor containing six peptides with multiple putative disulfide bonds. The alignment of SPα-α′ with SPβ revealed a partial structural conservation, with 37.3% identity. Translation of SPβ led to a 252-amino acid precursor containing five peptides. The occurrence of a signal peptide on SPα, SPα′ and SPβ showed that the peptides were secreted. RT-PCR and mass spectrometry analyses revealed a co-localization of transcripts and expression products in the oviduct gland. Preliminary in vitro experiments performed on gills and penises revealed target organs involved in mating and ventilation.ConclusionsThe analysis of the accessory sex gland transcriptome of Sepia officinalis led to the identification of peptidic sex pheromones. Although preliminary functional tests suggested the involvement of the α3 and β2 peptides in ventilation and mating stimulation, further functional investigations will make it possible to identify the complete set of biological activities expected from waterborne pheromones.

Highlights

The cephalopod mollusk Sepia officinalis can be considered as a relevant model for studying reproduction strategies associated to seasonal migrations
A directional cDNA library was created from the accessory sex glands of three adult females
Redundant expressed sequence tags (ESTs) were assembled into overlapping contigs using CAP3 software, which yielded 223 unique sequences: 37 contigs and 186 singletons with a redundancy of about 66.7%

Summary

Introduction

The cephalopod mollusk Sepia officinalis can be considered as a relevant model for studying reproduction strategies associated to seasonal migrations. Using transcriptomic and peptidomic approaches, we aim to identify peptide sex pheromones that are thought to induce the aggregation of mature cuttlefish in their egg-laying areas. The French cuttlefish Sepia officinalis is a nectobenthic cephalopod that performs horizontal migrations [1]. The occurrence of chemical messengers released by the genital apparatus and by the egg mass has long been thought to explain the aggregation observed between April and June [4]. Ovarian regulatory peptides that stimulate oviduct and accessory sex gland contractions have already been identified in Sepia officinalis [5][6][7][8][9]

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