Background: The urine of freshwater fish species investigated so far acts as a vehicle for reproductive pheromones affecting the behaviour and physiology of the opposite sex. However, the role of urinary pheromones in intra-sexual competition has received less attention. This is particularly relevant in lek-breeding species, such as the Mozambique tilapia (Oreochromis mossambicus), where males establish dominance hierarchies and there is the possibility for chemical communication in the modulation of aggression among males. To investigate whether males use urine during aggressive interactions, we measured urination frequency of dye-injected males during paired interactions between size-matched males. Furthermore, we assessed urinary volume stored in the bladder of males in a stable social hierarchy and the olfactory potency of their urine by recording of the electro-olfactogram.

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This paper is the consensus of a workshop that critically evaluated the utility and problems of video playbacks as stimuli in studies of visual behavior. We suggest that video playback is probably suitable for studying motion, shape, texture, size, and brightness. Studying color is problematic because video systems are specifically designed for humans. Any difference in color perception must lead to a different color sensation in most animals. Another potentially problematic limitation of video images is that they lack depth cues derived from stereopsis, accommodation, and motion parallax. Nonetheless, when used appropriately, video playback allows an unprecedented range of questions in visual communication to be addressed. It is important to note that most of the potential limitations of video playback are not unique to this technique but are relevant to all studies of visual signaling in animals.

Many species of freshwater fish with relatively simple mating strategies release hormonally derived sex pheromones in urine. However, it is not known whether species with more complex reproductive strategies use specialized urinary chemical signals. We addressed this by using the Mozambique tilapia (Oreochromis mossambicus Peters 1852), a lek-breeding species in which males establish dominance hierarchies and visiting females mate preferentially with territorial/dominant males. We measured urination frequency of territorial males in social isolation and in the presence of females that were either ready to spawn or had finished spawning. In groups of fish, we monitored the volume of urine stored in subordinate and dominant males to determine if urine volume and olfactory potency (by recording electro-olfactograms, EOG, in females) are related to the male's social rank. Dominant, territorial males stored more urine than subordinates and released it in short pulses, the frequency of which increased in the presence of females ready to spawn but not in the presence of post-spawn females. Urine from subordinate and dominant males was fractionated by liquid chromatography and fractions tested for olfactory potency by using the EOG, with the most potent fraction analyzed by mass spectrometry (MS). The olfactory system of females was sensitive to a urinary compound that was more abundant in the urine of dominant males than in that of subordinates. MS analysis suggested the compound is a sulfated aminosterol-like compound with a formula of C29H40N2O10S. Therefore, we suggest that dominant/territorial tilapia males dramatically increase urination frequency in the presence of females ready to spawn and that the urinary odorant acts as a pheromonal signal of dominance, thereby influencing female spawning.

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SUMMARY The current study investigated whether discrimination of sexual status of female tilapia by males is mediated by olfaction. Size-matched groups of female tilapia were assigned as pre- or post-ovulatory according to the time since their last ovulation (15-19 days pre-ovulatory, N=7; 1-3 days post-ovulatory, N=8). Female-conditioned water and body fluids(urine, bile, faeces and plasma) were assessed for olfactory potency in males by recording the electro-olfactogram (EOG). Water extracts, urine and faeces from pre-ovulatory females all evoked significantly larger amplitude EOGs in male fish (N=6), with correspondingly lower thresholds of detection,than those from post-ovulatory females. Plasma and bile evoked very large amplitude EOGs in males but with no differences between the two groups of females. Anosmic males (N=6) did not behave differently towards pre-or post-ovulatory females, while sham-operated males (N=6) showed a marked increase in urination rate towards pre-ovulatory females. We conclude that the ability of male tilapia to discriminate between females of differing reproductive status is mediated by odorants released into the water, probably via the urine and faeces, by pre-ovulatory females.

Knowledge of the chemical identity and role of urinary pheromones in fish is scarce, yet it is necessary in order to understand the integration of multiple senses in adaptive responses and the evolution of chemical communication [1]. In nature, Mozambique tilapia (Oreochromis mossambicus) males form hierarchies, and females mate preferentially with dominant territorial males, which they visit in aggregations or leks [2]. Dominant males have thicker urinary bladder muscular walls than subordinates or females and store large volumes of urine, which they release at increased frequency in the presence of subordinate males or preovulatory, but not postspawned, females [3-5]. Females exposed to dominant-male urine augment their release of the oocyte maturation-inducing steroid 17α,20β-dihydroxypregn-4-en-3-one (17,20β-P) [6]. Here we isolate and identify a male Mozambique tilapia urinary sex pheromone as two epimeric (20α- and 20β-) pregnanetriol 3-glucuronates. We show that both males and females have high olfactory sensitivity to the two steroids, which cross-adapt upon stimulation. Females exposed to both steroids show a rapid, 10-fold increase in production of 17,20β-P. Thus, the identified urinary steroids prime the female endocrine system to accelerate oocyte maturation and possibly promote spawning synchrony. Tilapia are globally important as a food source but are also invasive species, with devastating impact on local freshwater ecosystems [7, 8]. Identifying the chemical cues that mediate reproduction may lead to the development of tools for population control [9-11].

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