Olfactory transduction pathways in the Senegalese sole Solea senegalensis.

This study tested whether differences in sensitivity between the upper and lower olfactory epithelia of Solea senegalensis are associated with different odorant receptors and transduction pathways, using the electro-olfactogram. Receptor mechanisms were assessed by cross-adaptation with amino acids (L-cysteine, L-phenylalanine and 1-methyl-L-tryptophan) and bile acids (taurocholic acid and cholic acid). This suggested that relatively specific receptors exist for 1-methyl-L-tryptophan and L-phenylalanine (food-related odorants) in the lower epithelium, and for taurocholic acid (conspecific-derived odorant) in the upper. Inhibition by U73122 [a phospholipase C (PLC) inhibitor] suggested that olfactory responses to amino acids were mediated mostly, but not entirely, by PLC-mediated transduction (IC50 ; 15-55 nM), whereas bile acid responses were mediated by both PLC and adenylate cyclase-cyclic adenosine monophosphate (AC-cAMP) (using SQ-22536; an AC inhibitor). Simultaneous application of both drugs rarely inhibited responses completely, suggesting possible involvement of non-PLC and non-AC mediated mechanisms. For aromatic amino acids and bile acids, there were differences in the contribution of each transduction pathway (PLC, AC and non-PLC and non-AC) between the two epithelia. These results suggest that differences in sensitivity of the two epithelia are associated with differences in odorant receptors and transduction mechanisms.

The effect of nest aggregation on the reproductive behaviour of the peacock blenny Salaria pavo.

The effect of nest aggregation in courtship behaviour was tested experimentally in an ecologically constrained, sex-role reversed population of the peacock blenny Salaria pavo. Mixed sex groups of eight males and eight females were tested in experimental tanks, containing eight potential nests either aggregated or dispersed. In the aggregated treatment, males spent more time inside their nests and monopolized other potential nests, causing a female-biased operational sex ratio (OSR). In the aggregated treatment, females also expressed more courtship behaviour. The results in general support the prediction that the aggregation of nests promotes male monopolization of potential nests, resulting in fewer nest-holding males and therefore a female-biased OSR that leads to the reversal of sex roles.

Olfactory discrimination of female reproductive status by male tilapia (Oreochromis mossambicus).

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.

Olfactory sensitivity of the gilthead seabream (Sparus auratus L) to conspecific body fluids.

The potential for intraspecific chemical communication in the gilthead seabream (a marine perciform) was investigated by assessing the olfactory sensitivity to conspecific body-fluids (water occupied by conspecifics, intestinal fluid, urine, semen, egg fluid) by multiunit electrophysiological recording from the olfactory nerve. The olfactory system was responsive to water previously occupied by conspecifics, and the active compound(s) could be extracted by solid-phase extraction. The olfactory system was extremely sensitive to body fluids of sexually mature conspecifics: thresholds of detection were 1:10(7.4) (intestinal fluid), 1:10(6.1) (gametes), and 1:10(4.2) (urine). The olfactory system was also sensitive to amino acids with thresholds of detection from 10(-8.1) M (L-leucine) to 10(-6.1) M (L-phenylalanine). However, a range of other known fish odorants (steroids, bile acids, and prostaglandins) failed to evoke significant responses. Given the high olfactory sensitivity to intestinal fluid and the low urine release rates of marine compared with freshwater fish, we suggest that chemical communication is likely to be mediated via compounds present in the intestinal fluid rather than urine. Furthermore, the types of chemicals involved are likely to be different from those of freshwater fish. Their exact chemical identity and biological roles remain to be established.

Olfactory sensitivity to catecholamines and their metabolites in the goldfish.

The current study assessed the olfactory sensitivity of the goldfish (Carassius auratus L.) to the catecholamines, their immediate precursors and metabolites by use of the electro-olfactogram (EOG). The olfactory system of the goldfish was found to be sensitive to both adrenaline and dopamine with thresholds of detection of 10(-7.8) and 10(-7.9) M respectively, but less so to noradrenaline (threshold of detection 10(-6.3) M). The 3-O-methoxy metabolites (metadrenaline, normetadrenaline and 3-O-methoxytyramine) evoked larger amplitude EOGs than the non-metabolized form with lower thresholds of detection. However, the olfactory system was less sensitive to the amino acid precursors L-tyrosine and L-DOPA, and markedly less so to the alpha-deaminated metabolites (3,4-dihydroxyphenyl glycol, 3,4-dihydroxy mandelic acid and dihydroxyphenyacetic acid). Sensitivity to metabolites, both alpha-deaminated and 3-O-methoxylated, was similar to the alpha-deaminated forms. Cross-adaptation studies suggested that, while there is some degree of commonality of the receptor mechanisms with L-tyrosine and L-serine, a proportion of the response to the catecholamines is due to distinct receptor subtypes. Similarly, the 3-O-methoxy metabolites also had (a) separate receptor mechanism(s), although, again, there was overlap with the adrenaline/dopamine receptor site(s). Presence of the alpha-adrenoreceptor antagonist prazosin or the peripheral DA(2) dopamine receptor antagonist domperidone caused partial attenuation of the EOG responses to adrenaline and dopamine, but had much less effect on the responses to their 3-O-methoxy metabolites. The beta-adrenoreceptor antagonist sotalol had no such effect. This suggests that the olfactory catecholamine receptors are structurally and functionally distinct from systemic adreno- and dopamine receptors. The current study raises the possibility that release of catecholamines or their 3-O-methoxy metabolites to the water may play a role in chemical communication.

Possible disruption of pheromonal communication by humic acid in the goldfish, Carassius auratus.

Humic acids are large, complex, organic molecules which are ubiquitous components of aquatic environments as products of degradation of plant material. In aqueous solution they form microvesicles. As many teleost pheromones are steroidal in nature, we hypothesised that they would preferentially dissolve in the organic, hydrophobic core of these vesicles instead of in water and therefore be unavailable for detection. This would have obvious and profound effects on many aspects of fish biology. To test this hypothesis we recorded electro-olfactogram (EOG) response of the goldfish (Carassius auratus) olfactory epithelium to the pheromones 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (1720 beta-P), its sulphated conjugate (1720 beta-P-SO(4)) and prostaglandin F(2alpha) (PGF(2alpha)), all at 10(-11) to 10(-8) M, in the absence and presence of humic acids (1-1000 m x gl(-1)). At nearly all concentrations of humic acid tested, there was a significant attenuation of the amplitude of the initial (phasic) response to 1720 beta-P compared to 1720 beta-P alone. At higher concentrations of humic acid, the EOG response to 1720 beta-P was often completely obliterated, suggesting that the concentration of the pheromone available to the olfactory epithelium was below the threshold of detection. Exposure of the olfactory epithelium to humic acid did not cause any short-term loss of sensitivity to 1720 beta-P per se. Furthermore, simultaneous recording of electro-encephalograms from the olfactory bulb demonstrated that the nervous activity evoked by the same concentration of 1720 beta-P was less intense in the presence of humic acid than its absence. PGF(2alpha) is non-steroidal and much more soluble in water. In contrast to 1720 beta-P, only the higher concentrations of humic acid (100 and 1000 mg x l(-1)) significantly diminished the EOG amplitude. 1720 beta-P-SO(4) is detected via a distinct olfactory mechanism to the free form. Given that the sulphate group increases the water solubility, we predicted that the effect of humic acid would be reduced. However, the effect of humic acid on EOG amplitude in response to 1720 beta-P-SO(4) was similar to that of the free form. We suggest that the steroid portion of the molecule adsorbs onto the surface of the humic acid microvesicles and is still effectively unavailable for olfactory detection. In conclusion, humic acid may significantly reduce the concentration of 1720 beta-P and 1720 beta-P-SO(4) available for detection by Carassius auratus in natural environments. Furthermore, as many teleost pheromones are steroid derivatives, this phenomenon may be applicable to chemical communication systems in teleosts in general.

Olfactory sensitivity to changes in environmental [Ca(2+)] in the freshwater teleost Carassius auratus: an olfactory role for the Ca(2+)-sensing receptor?

Olfactory sensitivity to changes in environmental Ca(2+) has been demonstrated in two teleost species; a salmonid (Oncorhynchus nerka) and a marine/estuarine perciform (Sparus aurata). To assess whether this phenomenon is restricted to species that normally experience large fluctuations in external ion concentrations (e.g. moving from sea water to fresh water) or is present in a much wider range of species, we investigated olfactory Ca(2+) sensitivity in the goldfish (Carassius auratus), which is a stenohaline, non-migratory freshwater cyprinid. Extracellular recording from the olfactory bulb in vivo by electroencephalogram (EEG) demonstrated that the olfactory system is acutely sensitive to changes in external Ca(2+) within the range that this species is likely to encounter in the wild (0.05-3 mmol l(-1)). The olfactory system responded to increases in external calcium with increasing bulbar activity in a manner that fitted a conventional Hill plot with an apparent EC(50) of 0.9+/-0.3 mmol l(-1) (close to both ambient and plasma free [Ca(2+)]) and an apparent Hill coefficient of 1.1+/-0.3 (means +/- S.E.M., N=6). Thresholds of detection were below 50 micro mol l(-1). Some olfactory sensitivity to changes in external [Na(+)] was also recorded, but with a much higher threshold of detection (3.7 mmol l(-1)). The olfactory system of goldfish was much less sensitive to changes in [Mg(2+)] and [K(+)]. Preliminary data suggest that Ca(2+) and Mg(2+) are detected by the same mechanism, although with a much higher affinity for Ca(2+). Olfactory sensitivity to Na(+) may warn freshwater fish that they are reaching the limit of their osmotic tolerance when in an estuarine environment. Olfaction of serine, a potent odorant in fish, was not dependent on the presence of external Ca(2+) or Na(+). Finally, the teleost Ca(2+)-sensing receptor (Ca-SR) was shown to be highly expressed in a subpopulation of olfactory receptor neurones by both immunocytochemistry and in situ hybridisation. The olfactory sensitivity to Ca(2+) (and Mg(2+)) is therefore likely to be mediated by the Ca-SR. We suggest that olfactory Ca(2+) sensitivity is a widespread phenomenon in teleosts and may have an input into the physiological mechanisms regulating internal calcium homeostasis.

Olfactory sensitivity to changes in environmental [Ca(2+)] in the marine teleost Sparus aurata.

Estuarine and/or migratory teleosts may experience large and rapid changes in external [Ca(2+)]. Previous studies have largely centred on the physiological mechanisms that maintain a constant plasma [Ca(2+)] in the face of such external fluctuations, but little work has been directed to examining how these changes may originally be detected. We present evidence that the olfactory system of the gilthead seabream (Sparus aurata) is highly sensitive to reductions in environmental [Ca(2+)] and suggest a possible mechanism by which this may be mediated. Multi-unit extracellular recordings were made from the olfactory nerve of Sparus aurata while the [Ca(2+)] of artificial sea water flowing over the olfactory epithelium was varied from 10 to 0 mmol l(-)(1). Reductions in [Ca(2+)] caused a large, non-accommodating increase in the firing rate of the olfactory nerve (apparent IC(50)=1.67+/-0.26 mmol l(-)(1), apparent Hill coefficient=-1.22+/-0.14; means +/- s.e.m., N=6). This response was not due to the concomitant reduction in osmolality and was specific for Ca(2+). During continuous exposure of the olfactory epithelium to Ca(2+)-free sea water, the apparent IC(50) and Hill coefficient in response to increases in [Ca(2+)] were 0.48+/-0.14 mmol l(-)(1) and -0.76+/-0.16 (means +/- s.e.m., N=6), respectively, suggesting an adaptation of the Ca(2+)-sensing system to low-[Ca(2+)] environments. Ca(2+) is intimately involved in signal transduction in the olfactory receptor neurones, but our data support a true olfactory response, rather than a non-specific effect to lowering of external [Ca(2+)]. The absence of Ca(2+) from sea water only partially and temporarily blunted the olfactory response to the odorant l-serine; the response amplitude recovered to control levels within 20 min. This suggests that the olfactory system in general is able to adapt to low-[Ca(2+)] environments. We suggest that the Ca(2+ )sensitivity is mediated by an extracellular Ca(2+)-sensing receptor similar to the recently characterized mammalian Ca(2+)-sensing receptor.