A Multi-Component Pheromone in the Urine of Dominant Male Tilapia (Oreochromis mossambicus) Reduces Aggression in Rivals.

Males often use scent to communicate their dominance, and to mediate aggressive and breeding behaviors. In teleost fish, however, the chemical composition of male pheromones is poorly understood. Male Mozambique tilapia, Oreochromis mossambicus, use urine that signals social status and primes females to spawn. The urinary sex pheromone directed at females consists of 5ß-pregnane-3α,17α,20ß-triol 3-glucuronate and its 20α-epimer. The concentration of these is positively correlated with male social rank. This study tested whether dominant male urine reduces aggression in receiver males, and whether the pregnanetriol 3-glucuronates also reduce male-male aggression. Males were allowed to fight their mirror image when exposed to either: i) water control or a chemical stimulus; ii) dominant male urine (DMU); iii) C18-solid phase (C18-SPE) DMU eluate; iv) C18-SPE DMU eluate plus filtrate; v) the two pregnanetriol 3-glucuronates (P3Gs); or vi) P3Gs plus DMU filtrate. Control males mounted an increasingly aggressive fight against their image over time. However, DMU significantly reduced this aggressive response. The two urinary P3Gs did not replicate the effect of whole DMU. Neither did the C18-SPE DMU eluate, containing the P3Gs, alone, nor the C18-SPE DMU filtrate to which the two P3Gs were added. Only exposure to reconstituted DMU (C18-SPE eluate plus filtrate) restored the aggression-reducing effect of whole DMU. Olfactory activity was present in the eluate and the polar filtrate in electro-olfactogram studies. We conclude that P3Gs alone have no reducing effect on aggression and that the urinary signal driving off male competition is likely to be a multi-component pheromone, with components present in both the polar and non-polar urine fractions.

Social odors conveying dominance and reproductive information induce rapid physiological and neuromolecular changes in a cichlid fish.

BACKGROUND: Social plasticity is a pervasive feature of animal behavior. Animals adjust the expression of their social behavior to the daily changes in social life and to transitions between life-history stages, and this ability has an impact in their Darwinian fitness. This behavioral plasticity may be achieved either by rewiring or by biochemically switching nodes of the neural network underlying social behavior in response to perceived social information. Independent of the proximate mechanisms, at the neuromolecular level social plasticity relies on the regulation of gene expression, such that different neurogenomic states emerge in response to different social stimuli and the switches between states are orchestrated by signaling pathways that interface the social environment and the genotype. Here, we test this hypothesis by characterizing the changes in the brain profile of gene expression in response to social odors in the Mozambique Tilapia, Oreochromis mossambicus. This species has a rich repertoire of social behaviors during which both visual and chemical information are conveyed to conspecifics. Specifically, dominant males increase their urination frequency during agonist encounters and during courtship to convey chemical information reflecting their dominance status. RESULTS: We recorded electro-olfactograms to test the extent to which the olfactory epithelium can discriminate between olfactory information from dominant and subordinate males as well as from pre- and post-spawning females. We then performed a genome-scale gene expression analysis of the olfactory bulb and the olfactory cortex homolog in order to identify the neuromolecular systems involved in processing these social stimuli. CONCLUSIONS: Our results show that different olfactory stimuli from conspecifics' have a major impact in the brain transcriptome, with different chemical social cues eliciting specific patterns of gene expression in the brain. These results confirm the role of rapid changes in gene expression in the brain as a genomic mechanism underlying behavioral plasticity and reinforce the idea of an extensive transcriptional plasticity of cichlid genomes, especially in response to rapid changes in their social environment.

Identity of a tilapia pheromone released by dominant males that primes females for reproduction.

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,20ß-dihydroxypregn-4-en-3-one (17,20ß-P) [6]. Here we isolate and identify a male Mozambique tilapia urinary sex pheromone as two epimeric (20α- and 20ß-) 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 17,20ß-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].

Olfactory sensitivity to amino acids in the blackspot sea bream (Pagellus bogaraveo): a comparison between olfactory receptor recording techniques in seawater.

The current study investigated the olfactory sensitivity of the blackspot sea bream to amino acids, odorants associated with food detection in fish, and compared the efficacy of two different experimental methods: multi-unit recording from the olfactory nerve and the electro-olfactogram (EOG). Twenty essential amino acids plus L-DOPA evoked clear, concentration-dependent olfactory responses using both methods, with estimated thresholds of 10(-8.5)-10(-6.2) M (nerve recording) and 10(-7.5)-10(-4.8) M (EOG). The most potent amino acids were L-cysteine, L-methionine (both sulphur-containing), L-alanine, L-leucine (both neutral), L-glutamine (amide-containing) and L-serine (hydroxyl-containing). The least potent were L-proline (secondary α-amino group), the aromatic amino acids and glycine (simplest). Although the rank order of olfactory potency was similar for the two methods used, and the calculated thresholds given by the two methods were positively correlated, the sensitivity of the EOG was consistently lower than multi-unit recording by approximately one order of magnitude, presumably due to the electrical shunting effect of seawater. As in freshwater, the EOG could be a valid method for comparing olfactory potency of different odorants in stenohaline marine fish; however, for absolute 'biological' thresholds, a more invasive recording technique, such as multi-unit recording from the olfactory nerve, should be used.

Adaptation to reduced salinity affects the olfactory sensitivity of Senegalese sole (Solea senegalensis Kaup 1858) to Ca2+ and Na+ but not amino acids.

The Senegalese sole is a marine flatfish, which often penetrates into estuarine waters to feed. It cannot, however, survive in full freshwater. The current study investigated the effect of adaptation to low salinity (10 per thousand) on olfactory responses to changes in environmental [Ca(2+)] and [Na(+)] and amino acids by the electro-encephalogram (EEG) recorded from the olfactory bulb. The sole showed olfactory responses to increases in environmental [Na(+)] and decreases in environmental [Ca(2+)]; sensitivity to Na(+) was greater at 10 per thousand whereas sensitivity to Ca(2+) was greater at 35 per thousand. Decreased environmental [Na(+)] increased sensitivity to changes in [Ca(2+)] whereas increased environmental [Ca(2+)] decreased bulbar responses to changes in [Na(+)]. Sensitivity to amino acids was unaffected by external salinity. However, the absence of external Na(+) strongly decreased bulbar responses to amino acids in fish adapted to 35 per thousand seawater but not in those at 10 per thousand. The absence of external Ca(2+) had no such effect at either salinity. This suggests that odorant-receptor binding and/or olfactory transduction is reliant on external Na(+) (but not Ca(2+)) at higher salinities but the olfactory system is able to adapt to lower environmental [Na(+)]. Taken together, these results suggest that reductions of external salinity modulate olfactory sensitivity to environmental Ca(2+) and Na(+) but not amino acids. However, at low salinities, olfactory sensitivity to amino acids is maintained by decreasing reliance on external Na(+).

Identification, release and olfactory detection of bile salts in the intestinal fluid of the Senegalese sole (Solea senegalensis).

Olfactory sensitivity to bile salts is wide-spread in teleosts; however, which bile salts are released in sufficient quantities to be detected is unclear. The current study identified bile salts in the intestinal and bile fluids of Solea senegalensis by mass spectrometry-liquid chromatography and assessed their olfactory potency by the electro-olfactogram. The main bile salts identified in the bile were taurocholic acid (342 mM) and taurolithocholic acid (271 mM) plus a third, unidentified, bile salt of 532.3 Da. These three were also present in the intestinal fluid (taurocholic acid, 4.13 mM; taurolithocholic acid, 0.4 mM). In sole-conditioned water, only taurocholic acid (0.31 microM) was released in sufficient quantities to be measured (release rate: 24 nmol kg(-1) min(-1)). Sole had high olfactory sensitivity to taurocholic acid but not to taurolithocholic acid. Furthermore, olfactory sensitivity was higher in the upper (right) olfactory epithelium than the lower (left). These two bile acids contribute about 40% of the olfactory potency of intestinal fluid and account for the difference in potency at the two epithelia. Taurocholic acid (but not taurolithocholic acid), and possibly other types of bile acid not tested, could be used as chemical signals and the upper olfactory epithelium is specialised for their detection.

11-ketotestosterone stimulates putative sex pheromone production in the male peacock blenny, Salaria pavo (Risso 1810).

Male peacock blennies (Salaria pavo) release odors from their anal glands and, possibly, from their gonads that attract reproductive females. The current study investigated the effects of 11-ketotestosterone (KT) on development of the anal glands, testes, and accessory testicular organs as well as the subsequent olfactory potency of their secretions. After 3 wk of KT treatment (5 mg/kg in silastic implants), clusters of cells secreting neutral mucins differentiated in the anal gland of all treated males, whereas this occurred in only one control male. Secretions by anal glands from KT-treated males elicited greater olfactory responses, as assessed by electro-olfactography, than those from controls. Treatment with KT stimulated testicular gland growth and sialomucin secretion but had no clear effect on the germinal region of the testis; KT also stimulated enlargement of, and fluid secretion in, the blind pouches (paired evaginations of the spermatic ducts). Secretions by the testes and fluid in blind pouches from KT-treated males elicited greater olfactory responses than those from controls. In conclusion, KT stimulates development of the anal glands and testicular accessory organs and promotes odorant secretion, the putative multicomponent male peacock blenny pheromone.

A sterol-like odorant in the urine of Mozambique tilapia males likely signals social dominance to females.

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.

Behavioral and olfactory responses of female Salaria pavo (Pisces: Blenniidae) to a putative multi-component male pheromone.

The peacock blenny, Salaria pavo (Risso 1810), typically breeds in rocky shores of the Mediterranean and adjacent Atlantic coast. Males defend a territory around a hole or cavity wherein females deposit eggs that the male guards until hatching. A pair of exocrine glands on the anal fin (anal glands) of males produces a putative pheromone involved in attraction of reproductively competent females to the nest. We used behavioral assays to assess species-specific attraction of reproductively competent females to putative male pheromones, including the anal gland pheromone. Additionally, chromatographic fractions of anal glands and male-conditioned water were tested for olfactory potency in females by electro-olfactogram analysis (EOG). In a flow-through tank or fluviarium, reproductive females were attracted to male-conditioned water and to the anal gland macerate from conspecifics but not to those of a closely related heterospecific. In addition, attraction of reproductive females to conspecific anal gland macerate occurred only during their initial upstream movement in the fluviarium; this was an ephemeral response when compared with the response to male-conditioned water that attracted females throughout the entire period of observation (5 min). Reproductive females also were attracted during the entire period of observation to water-conditioned by conspecific males whose anal glands had been removed. However, the attraction was more variable than that to water conditioned by intact males. Moreover, females were not attracted to male (without anal glands) odor during their initial upstream movement in the fluviarium. Finally, non-reproductive females were not attracted to the conspecific anal gland macerate. The EOG responses of females to molecular weight fractions and solid-phase extraction and high-performance liquid chromatography fractions of anal gland macerates and male-conditioned water (with and without anal glands) suggest that the anal glands release hydrophilic odorants that consist mainly of molecules smaller than 500 Da. Furthermore, males released potent odorants that do not originate from the anal glands. We hypothesize that females respond to a multi-component male pheromone to find mates. The putative anal gland pheromone is possibly comprised of hydrophilic odorants, whereas the other component(s), presumably of gonadal origin, may be less water-soluble.

Male urine signals social rank in the Mozambique tilapia (Oreochromis mossambicus).

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. RESULTS: Males released urine in pulses of short duration (about one second) and markedly increased urination frequency during aggressive behaviour, but did not release urine whilst submissive. In the stable hierarchy, subordinate males stored less urine than males of higher social rank; the olfactory potency of the urine was positively correlated with the rank of the male donor. CONCLUSION: Dominant males store urine and use it as a vehicle for odorants actively released during aggressive disputes. The olfactory potency of the urine is positively correlated with the social status of the male. We suggest that males actively advertise their dominant status through urinary odorants which may act as a 'dominance' pheromone to modulate aggression in rivals, thereby contributing to social stability within the lek.

Differential detection of conspecific-derived odorants by the two olfactory epithelia of the Senegalese sole (Solea senegalensis).

The two olfactory epithelia of members of the family Soleidae sample two distinct water sources; the upper (right) side is in contact with the open water column whilst the lower (left) side is in contact with interstitial water. To evaluate whether there are differences in the sensitivities, and therefore functional roles, of the two epithelia the olfactory activity of conspecific-derived odorants was assessed in both using the electro-olfactogram (EOG). The upper nostril was significantly more sensitive to conspecific bile fluid, intestinal fluid and mucus than the lower nostril. Crude fractionation of these samples (solid-phase extraction with C18 and C2/ENV+ cartridges) revealed that olfactory activity in each body fluid was likely due to a mixture of compounds. In each case, the upper olfactory epithelium was significantly more sensitive than the lower. Similarly, olfactory sensitivity to a range of C(24) and C(27) bile acids was greater in the upper epithelium. These results suggest that intra-specific chemical communication is mediated mainly, if not entirely, by the upper olfactory epithelium. The odorants involved, and their functional roles, remain to be established.

Evidence for functional asymmetry in the olfactory system of the Senegalese sole (Solea senegalensis).

The two olfactory epithelia of flatfish of the family Soleidae are essentially in contact with two distinct environments; the upper (right) side samples open water while the lower (left) side samples interstitial water. This study assessed whether there are differences in the responsiveness of the two epithelia by use of the electro-olfactogram in the Senegalese sole (Solea senegalensis). The upper epithelium was significantly more responsive to the basic amino acids (L-lysine and L-arginine), glycine, and L-threonine than the lower epithelium. The lower epithelium was significantly more responsive to aromatic amino acids (L-tryptophan, L-tyrosine, L-DOPA, and L-phenylalanine), L-leucine, and L-asparagine than the upper. Both epithelia had similar responsiveness to the sulphur-containing amino acids (L-cysteine and L-methionine), L-alanine, L-serine, and L-glutamine. Neither side was responsive to the acidic amino acids (L-aspartate and L-glutamate) or the D-isomers of any amino acid tested. The upper olfactory organ was much more responsive to conspecific-derived stimuli (bile and intestinal fluid) than the lower organ. We suggest that these differences in responsiveness may be related to different functional roles of the upper and lower epithelia in feeding and chemical communication.

Fine structure of antennal sensilla basiconica and their detection of plant volatiles in the eucalyptus woodborer, Phoracantha semipunctata Fabricius (Coleoptera: Cerambycidae).

The ultrastructure and distribution pattern of two types of basiconic sensilla (I and II) on the antennal flagellum of both sexes of Phoracantha semipunctata (Coleoptera: Cerambycidae) was investigated by scanning and transmission electron microscope. Both types are thin-walled multiporous sensilla and occur mostly along the anterior border of the Fl1-Fl6 flagellomeres, while on the distal flagellomeres (Fl7-Fl9) they are more evenly distributed on both surfaces. Clusters of sensilla basiconica II are found on the distal half of the anterior border of the Fl1-Fl6 flagellomeres. Sensilla basiconica I have one bipolar sensory cell with a branched distal dendrite, whereas the sensilla basiconica II contain two bipolar sensory cells with branched distal dendrites. No sexual dimorphism was found in the fine structure and distribution pattern of both types of sensilla basiconica. Responses from single sensory cells to host and non-host plant odors were examined, using gas chromatography linked with electrophysiological recordings. Most cells associated with each sensillum type were narrowly tuned, each specialized for the detection of one or two chemically related compounds. No clear functional distinction between the two morphological types of sensilla was found, although the few cells that responded specifically to non-host volatiles were associated with sensilla basiconica II.