High commercial value – increased aquaculture effort.

1. Functional Asymmetry in the Olfactory System of a Flatfish, the Senegalese Sole (Solea senegalensis) Zélia Velez1,2,3, Peter C. Hubbard1, Kevin J. Weham3, Eduardo N. Barata1,2, Jörg D. Hardege3 and Adelino V.M. Canário1 1. Centro de Ciências do Mar, Faro, Portugal; 2. Departamento de Biologia, Universidade de Évora, Portugal; 3. University of Hull, UK

2. The Selenagese Sole (Solea senegalensis): High commercial value – increased aquaculture effort. Flatfish; left (lower) nostril does not migrate. Functional asymmetry in the olfactory system? Lower epithelium – prey location/identification Upper epithelium – chemical communication

3. Methods: Electro-olfactogram (EOG) recorded from the olfactory epithelium: Olfactory potency – identification of odorants Cross-adaptation – receptor specificity Pharmacological inhibition - transduction pathways Chromatography of biological fluids: Solid-phase extraction – crude separation of odorants HPLC – fine separation and purification of odorants LC-MS – identification of odorants

4. Methods: Food- and conspecific-related odorants Bile and intestinal fluid Ragworm-conditioned water Total; EOG C-18 Sep-Pak HPLC and EOG EOG Filtrate Eluate The ragworm Hediste diversicolor is one of the sole’s main prey. They live buried in the sand or mud (of estuaries). Bile acids are potent odorants in fish.

5. Methods: The electro-olfactogram (EOG) Velezet al. (2005) Physiol. Biochem. Zool.78: 756-765 EOG is a spacio-temporal summation of generator potentials of olfactory receptor neurones. L-cysteine is detected with equal sensitivity by the two olfactory epithelia; responses are normalised to that of 10-3 M L-cysteine.

6. Results I: HPLC fractionation of ragworm-conditioned water Fractions 1 and 2 contain the odorants of interest; active odorant identified using LC-MS

7. Results I: Identification of ragworm-related odorants: 1-methyl-L-tryptophan Active odorant identified as 1-methyl-L-tryptophan. Ragworms release this amino acid, along with L-phenylalanine and L-glutamine

8. Results I: Cross-adaptation - selectivity Olfactory sensitivity Lower epithelium has greater sensitivity to 1-methyl-L-tryptophan.

9. Results I: Olfactory sensitivity to conspecific odorants Bile Intestinal fluid Mucus Velez et al. (2007) Gen. Comp. Endocrinol. 153: 418-425 Conspecific body fluids are consistently better detected by the upper olfactory epithelium

10. Results I: Chromatographic separation of odorants in intestinal fluid Velez et al. (2009) J. Comp. Physiol. A 195: 691-698 Bile acids contribute about 40% of the olfactory potency of intestinal fluid. Bile acids in fraction 4 were identified by LC-MS.

11. Results I: Identification of odorants in intestinal fluid: Taurocholic acid Taurolithocholic acid Velez et al. (2009) J. Comp. Physiol. A 195: 691-698 Two major bile acids identified in the intestinal fluid (and bile fluid). A third, minor, bile acid was not identified.

12. Results I: Olfactory sensitivity to bile acids Taurocholic acid Taurolithocholic acid Sole release taurocholic acid – this is detected better by the upper olfactory epithelium

13. Conclusions I: Prey-related odorants – including 1-methyl-L-tryptophan – are better detected by the lower olfactory epithelium. Conspecific-related odorants – including taurocholic acid – are better detected by the upper olfactory epithelium. There is functional asymmetry in the olfactory system of this flatfish. Are the receptors and transduction pathways different?

14. Results II: Cross-adaptation 1-methyl-L-tryptophan Lower epithelium has greater specificity for the ragworm odorant 1-methyl-L-tryptophan.

15. Results II: Cross-adaptation taurocholic acid Upper epithelium has greater specificity for taurocholic acid.

16. Results II: Transduction U73122 – PLC inhibitor SQ-22536 – AC inhibitor L-cysteine L-cysteine L-cysteine is detected equally by the two epithelia The effects of the two drugs are the same in the two epithlia The transduction pathway for L-cysteine is mainly via PLC

17. Results II: Transduction 1-methyl-L-tryptophan U73122 – PLC SQ-22536 – AC The transduction pathway for 1-methyl-L-tryptophan is mainly PLC mediated There are differences between the two epithelia

18. Results II: Transduction taurocholic acid U73122 – PLC SQ-22536 – AC Both phospholipase C and adenylate cyclase are involved in transduction of taurocholic acid There are differences in the two epithelia

19. Conclusions II: There are specific receptors for 1-methyl-L-tryptophan in the lower epithelium. There are specific receptors for taurocholic acid in the upper epithelium. The transduction pathway for 1-methyl-L-tryptophan is mainly phospholipase C mediated. The transduction pathway(s) for taurocholic acid is/are mediated by both phospholipase C and adenylate cyclase.

20. Summary: Specific receptors in the lower olfacotry epithelium for prey-related odorants (1-methyl-L-tryptophan) Specific receptors in the upper olfactory epithelium for conspecific-related odorants (taurocholic acid) This suggests specialisation of the two epithelia – functional asymmetry Does the processing of the olfactory information in the olfactory bulb (and beyond) also show asymmetry?

21. The End: Thank you for your attention Funded by FCT grants SFRH/BD/16242/2004 and POCI/BIA-BMC/55467/2004