Indian J. Sci. Technol., 9 th ISRPF Issue           Proceedings of 9 th International Symposium on Reproductive Physiology of Fish, Cochin, India. August 9-14, 2011 98 MALE TILAPIA URINARY PHEROMONE INDUCES ENDOCRINE SEX RESPONSES IN CONSPECIFIC FEMALES Hubbard P.C ., Huertas M., Almeida, O.G. and Canário, A.V.M. Centro de Ciências do Mar, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal Fax: +351- 289 800 051; e-mail: phubbard@ualg.pt Introduction: The study of control of reproduction by sex pheromones in fish is mostly restricted to behavioural responses to sex cues. Our aim was to test whether male Mozambique tilapia (Oreochromis mossambicus) urinary pheromone modulates the steroidogenic - physiological - control of female reproduction. This hypothesis was addressed because: male tilapia can discern between olfactory stimuli of pre-ovulated and post-ovulated females [1]; male tilapia increase their urination rate in the presence of ovulated females [2]; female tilapia release eggs when exposed to male urine (unpublished). To test this hypothesis we used a non-invasive method, by measuring release of cortisol, testosterone, estradiol and 17,20βP by pre-ovulated and post- ovulated tilapia female into the water after exposure to pheromonal stimulus (male urine). Moreover, blood and urine samples were taken from the females at the end of the experiment for measurement of steroid concentration and plasma steroid binding properties to understand the dynamics of steroid transport and release in this species. Methods: A pre- or post-ovulatory female was placed in a tank with clean water (10 g fish.l -1 ). One litre of water was taken at 0h, 1h, 2h and 6h after male urine exposure (final dilution 1:10 000) or distilled water (control) and replaced with 1l clean water. At the end of the experiment (6h) urine and blood samples were taken. Steroid extraction from water and biological fluids and steroid quantification by radio-immuno assay (RIA) were carried out according Huertas et al. [3]. All steroid measurements were validated for recovery, specificity and identity following the guidelines of Scott et al. [4]. To characterize steroid binding affinity, capacity and specificity in female plasma, we followed the method of Scott et al. [5]. Results and Discussion: The exposure to male urine dramatically affected the release of 17,20βP to the water by female tilapia with a rapid response within 1h of treatment. The release rate of free 17,20βP rose to 113±24 pg.h -1 kg -1 fish after 1h. The release rate remained significantly higher than controls for both pre- and post-ovulatory females over 6h, and higher in post-ovulatory than pre-ovulatory females. The peaks of sulphated and glucoronidated 17,20βP were 61±9 and 171±45 pg.h -1 .kg -1 fish, respectively. The release of both conjugated forms returned to control values after 2h in all experimental groups. The release rate of free cortisol and estradiol (E 2 ) ranged from 5 to 98 pg.h -1 kg -1 fish and 10 to 200 pg.h -1 kg -1 fish respectively over 6h period and was unaffected by pheromone treatment or maturation state. Only E 2 showed significantly higher release of conjugated vs free. Free testosterone (T) release rate showed significant differences between pre-ovulatory (361±80 ng.h -1 kg -1 fish) and post-ovulatory females (99±23 ng.h -1 kg -1 fish). The increase of maturing inducing steroid 17,20βP after pheromone treatment strongly suggests a synchronization of spawning by inducing final maturation of the eggs [6]. As the number of immature follicles in post-ovulatory females is higher, this is consistent with the higher release, and presumably production, of 17,20βP in this group. The steroid concentrations in plasma or urine after 6h was unaffected by the treatment and only T was significantly different between pre- and post-ovulatory females. In plasma and urine, concentrations of 17,20βP (free and conjugated) were below 0.8 ng.ml -1 . Plasma had a specific binding protein for E 2 with high affinity (14nM) and moderate capacity (54nM). The relative binding affinity was E2 (100%) > T (35%) > 17,20 P (3.3%) > F and conjugated forms (<1%). Plasma sex steroid concentration is used as an indicator of reproductive status in fish. However, their concentrations in plasma are determined by the capacity and specificity of transport by steroid binding proteins (SBP). Thus, 17,20βP can be produced in significant amounts in the gonads but, due to its low affinity for SBP, it is rapidly metabolized and excreted (probably by the gills [6]). This leads to low concentrations in body- fluids but release rates as high as that of estradiol and suggests point measurement in the water as the most appropriate assay to evaluate changes. Conclusion: Urine from male tilapia contains a pheromone that dramatically affects 17,20βP metabolism in conspecific females, less than 1h after exposure. This pheromonal effect was only visible in non-invasive measurement of steroids in the water and can only be understood after evaluation of steroid binding proteins.