Sex pheromones of the cuttlefish Sepia officinalis: identification, structure and functions Maxime Endress 1,2, Jrmy Enault 3, Valrie Cornet 1,2, Cline Zatylny-Gaudin 1,2, Benot Bernay 1,4, Jol Henry 1,2,4 The cuttlefish Sepia officinalis can be considered as a very relevant model to study the reproductive strategies associated with seasonal migrations. By combining transcriptomic, peptidomic and functional approaches, we identified sex pheromone peptides and polypeptides/proteins involved in the parents breeding behavior, i.e. aggregation in coastal spawning areas, mating, and egg-laying. Two successive transcriptomic approaches yielded 4 highly redundant transcripts from the oviduct gland, co-expressed and co-secreted with capsular proteins. We called them SP , SP ', SP and SP . Peptidomic analysis of the oviduct gland yielded some of the cleavage products predicted by the precursor structure analysis, ranging between 1 and 8 kDa (Enault et al, 2012). In addition, proteomic analysis of the oviduct gland and egg capsule evidenced high-molecular-weight polypeptides/proteins derived from SP precursors. These analytical results demonstrate the coexistence of two modes of cleavage of SP precursors that generate low-molecular-weight peptides (prohormone convertase cleavages), and also kDa polypeptides/proteins released by the eggs into the surrounding medium. Perfusing organs such as the penis, the oviduct or the gills with egg-bathing water induced a significant change in contractile activity. Synthetic and recombinant pheromones will allow us to carry out contractile in vitro tests (oviduct, nidamental glands, penis, gills, etc.) and in vivo behavioral tests in "open space" and "Y maze" aquaria to confirm that these sex pheromones actually attract and stimulate spawning, and cause massive aggregations of mates in the coastal mating and spawning areas of Normandy in spring. 1 Normandy University, Caen, France, 2 Normandy University, UMR BOREA MNHN, UPMC, UCBN, CNRS-7208, IRD-207, F Caen, France, 3 Normandy University, Laboratoire de Microbiologie Signaux et Microenvironnement, LMSM EA 4312, F Evreux, France, 4 Post Genomic platform PROTEOGEN, Normandy University, SF ICORE 4206, Caen, France Sepia officinalis Phylum: Mollusca Class: Cephalopoda Order: Sepiida Family: Sepiidae Genus: Sepia Sepia officinalis : a couple of spawners. Sex pheromone protein precursors and convertase cleavage products, from Enault et al., Plos One (2012). Bioactivity of synthetic 2 and 3 peptides: 2-induced contraction of (A) female gill and (B) penis from a threshold of M. No activity on (C) rectum. 3-induced contraction of (D) penis from a threshold of M, from Enault et al., Plos One (2012). Conclusion We validated a second precursor processing that releases kDa polypeptides/proteins. We characterized the full-length primary sequence of three related waterborne pheromones (40-44% identity). Size, hydrophobicity, structure, pHi and charge state appeared to be very similar in the three pheromones. Prospects Recombinant pheromones will be produced to perform: A structural approach by NMR Functional in vitro and in vivo approaches to elucidate the involvement of waterborne pheromones in the successive steps of egg-laying regulation and also in the reproductive behavior. Behavioral tests will consist of attraction experiments in open space and Y-maze aquaria (Jean Boal -1998). 2015: a second cleavage process releases high-molecular-weight pheromones. SP measured mass: 22,719-22,811 Da MDKPGKTSPLSWLVYVITALILVFFLARLEIRSNVQKAEFQHRAARLLVSVRRYYAAGPVFTRWGNNRCPYRSYRVYEGIMGGQDKTHRG GASNFLCLPRRPTWANLKGGSQLGGLIYGTQYKLYPSQVNGFGLFFQTHLKPPHNHDVPCAVCQVTKPATVLMIPGRKVCTPGWDLMYRG YLMSEKRNNAGRMTYVCVDKRPQVYWAGYLNENGATINHVESKCGSLPCPLYSNYKEVPCCVCSKCPI* pHi : 9,33 Charge state at pH1 : +31 SP measured mass: 22,394-22,438 Da MMTSFDNKMPTSGKSKLRWFFYVFSLLALIFVFSRLEKKAEENKIQIQKKFTYPYVQVKIPGPGATYVIWGRKKCTSNDTRVYTGYTAGQ HYNHAGGGSNILCLPRFPVSDQITTSTENVASIFGVRYRIGSYNSLGTNGLFSTPNNISLNKRVVPCSVCLTNGSATIMVIPGRTHCYPG WYRQYSGYLMTAHYAHVGRSNHICVHRRPQRYFGTSPLQAFLYHVEAECGSLPCPPYCNGFELPCVVCRKCP* pHi : 9 Charge state at pH1 : +27 SP measured mass: 25,708-25,788 Da MFTPGKYGLYSWIGYLVAVSILVILVGRLENKFSANDVQLKKRGFGFRPSITHFYAPGSVYTRWGRTVCPRGSKVVYKGIMAAPKHTNTG GGSDYQCLPNNPVWGRRLPNFQSSGRLFGTEYKISLYLTQGVNGLFSNINARRVNFHNAPCAVCLTYMPRAVIMIPGRKRCDRGWKLEYS GYLMSERYTYRRTEYVCVDKDPEASESGNGWQHSAGLYHVEARCGSLPCPWYVQGFEMACAVCSRGPYPYYYKKIATQTSGSNNTSGNNA TIG* pHi : 9,38 Charge state at pH1 : +33 Signal peptide in red N-terminal cleavage sites in green Blue boxes: N-terminal sequences determined by Edman degradation from purified HPLC fractions Full-length pheromones in yellow 3.Sequencing of N-ter ends of purified fractions LLVSVRR FTYPYVQ GFGFRPS 2.MALDI-TOF MS linear analysis of purified fractions # Da: SP alpha-prime # Da: SP beta # Da: SP gamma 1.Purification of water extracts from water bathing the eggs Step 1 Step : Identification of three pheromone precursors and a first cleavage process identified unknown 560 usable ESTs from oviduct glands: 260 unknown ESTs correspond to 3 related precursors By Enault et al. Dynamometer Amplifier Printer Sample Infusate Three-way tap Organ Tank Peristaltic pump In vitro approach based on the contractions of organs involved in reproduction: oviduct, main nidamental glands, penis, etc. Experimental procedure