Sex pheromones of the cuttlefish Sepia officinalis identification

Sex pheromones of the cuttlefish Sepia officinalis: identification, structure and functions Maxime Endress 1, 2, Jérémy Enault 3, Valérie Cornet 1, 2, Céline Zatylny-Gaudin 1, 2, Benoît Bernay 1, 4, Joël Henry 1, 2, 4 1 Normandy University, Caen, France, e-mail: joel. henry@unicaen. fr , 2 Normandy University, UMR BOREA MNHN, UPMC, UCBN, CNRS-7208, IRD-207, F-14032 Caen, France, 3 Normandy University, Laboratoire de Microbiologie Signaux et Microenvironnement, LMSM EA 4312, F-27000 Evreux, France , 4 Post Genomic platform PROTEOGEN, Normandy University, SF ICORE 4206, Caen, France 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 k. Da (Enault et al, 2012). In addition, proteomic analysis of the oviduct gland 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 22 -26 k. Da 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. Sepia officinalis: a couple of spawners. Sepia officinalis Phylum: Mollusca Class: Cephalopoda Order: Sepiida Family: Sepiidae Genus: Sepia 2012: Identification of three pheromone precursors and a first cleavage process … 560 usable ESTs from oviduct glands: - 168 identified - 392 unknown Þ 260 unknown ESTs correspond to 3 related precursors 50% 37% 63% Candidates Unknown Identified by blast 13% Sex pheromone protein precursors and convertase cleavage products, from Enault et al. , Plos One (2012). By Enault et al. Bioactivity of synthetic β 2 and α 3 peptides: β 2 -induced contraction of (A) female gill and (B) penis from a threshold of 10 -8 M. No activity on (C) rectum. α 3 -induced contraction of (D) penis from a threshold of 10 -9 M, from Enault et al. , Plos One (2012). 2015: a second cleavage process releases high-molecular-weight pheromones. Experimental procedure 1. Purification of water extracts from water bathing the eggs Step 1 2. MALDI-TOF MS linear analysis of purified fractions 3. Sequencing of N-ter ends of purified fractions # 22811 Da: SP alpha-prime LLVSVRR FTYPYVQ GFGFRPS Step 2 # 22438 Da: SP beta # 25788 Da: SP gamma SP a’ measured mass: 22, 719 -22, 811 Da MDKPGKTSPLSWLVYVITALILVFFLARLEIRSNVQKAEFQHRAARLLVSVRRYYAAGPVFTRWGNNRCPYRSYRVYEGIMGGQDKTHRG GASNFLCLPRRPTWANLKGGSQLGGLIYGTQYKLYPSQVNGFGLFFQTHLKPPHNHDVPCAVCQVTKPATVLMIPGRKVCTPGWDLMYRG YLMSEKRNNAGRMTYVCVDKRPQVYWAGYLNENGATINHVESKCGSLPCPLYSNYKEVPCCVCSKCPI* p. Hi : 9, 33 Charge state at p. H 1 : +31 SP b measured mass: 22, 394 -22, 438 Da MMTSFDNKMPTSGKSKLRWFFYVFSLLALIFVFSRLEKKAEENKIQIQKKFTYPYVQVKIPGPGATYVIWGRKKCTSNDTRVYTGYTAGQ HYNHAGGGSNILCLPRFPVSDQITTSTENVASIFGVRYRIGSYNSLGTNGLFSTPNNISLNKRVVPCSVCLTNGSATIMVIPGRTHCYPG WYRQYSGYLMTAHYAHVGRSNHICVHRRPQRYFGTSPLQAFLYHVEAECGSLPCPPYCNGFELPCVVCRKCP* p. Hi : 9 Charge state at p. H 1 : +27 SP g measured mass: 25, 708 -25, 788 Da MFTPGKYGLYSWIGYLVAVSILVILVGRLENKFSANDVQLKKRGFGFRPSITHFYAPGSVYTRWGRTVCPRGSKVVYKGIMAAPKHTNTG GGSDYQCLPNNPVWGRRLPNFQSSGRLFGTEYKISLYLTQGVNGLFSNINARRVNFHNAPCAVCLTYMPRAVIMIPGRKRCDRGWKLEYS GYLMSERYTYRRTEYVCVDKDPEASESGNGWQHSAGLYHVEARCGSLPCPWYVQGFEMACAVCSRGPYPYYYKKIATQTSGSNNTSGNNA TIG* p. Hi : 9, 38 Charge state at p. H 1 : +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 Behavioral tests will consist of attraction experiments in open space and Y-maze aquaria (Jean Boal -1998). Conclusion • We validated a second precursor processing that releases 22 -25 k. Da polypeptides/proteins. • We characterized the full-length primary sequence of three related waterborne pheromones (40 -44% identity). • Size, hydrophobicity, structure, p. Hi 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. In vitro approach based on the contractions of organs involved in reproduction: oviduct, main nidamental glands, penis, etc. Dynamomete r Amplifier Organ Printer Sample Tank Infusate Three-way tap Peristaltic pump