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- Chemical diplomacy in male tilapia: urinary signal increases sex hormone and decreases aggressionPublication . Saraiva, João; Keller-Costa, Tina; Hubbard, Peter; Rato, Ana; Canario, AdelinoAndrogens, namely 11-ketotestosterone (11KT), have a central role in male fish reproductive physiology and are thought to be involved in both aggression and social signalling. Aggressive encounters occur frequently in social species, and fights may cause energy depletion, injury and loss of social status. Signalling for social dominance and fighting ability in an agonistic context can minimize these costs. Here, we test the hypothesis of a 'chemical diplomacy' mechanism through urinary signals that avoids aggression and evokes an androgen response in receiver males of Mozambique tilapia (Oreochromis mossambicus). We show a decoupling between aggression and the androgen response; males fighting their mirror image experience an unresolved interaction and a severe drop in urinary 11KT. However, if concurrently exposed to dominant male urine, aggression drops but urinary 11KT levels remain high. Furthermore, 11KT increases in males exposed to dominant male urine in the absence of a visual stimulus. The use of a urinary signal to lower aggression may be an adaptive mechanism to resolve disputes and avoid the costs of fighting. As dominance is linked to nest building and mating with females, the 11KT response of subordinate males suggests chemical eavesdropping, possibly in preparation for parasitic fertilizations.
- Synthetic versus natural receptors: supramolecular control of chemical sensing in fishPublication . Silva, José P. da; Choudhury, Rajib; Porel, Mintu; Pischel, Uwe; Jockusch, Steffen; Hubbard, Peter; Ramamurthy, Vaidhyanathan; Canario, Adelino V. M.The encapsulation of odorants by the synthetic receptor cucurbit[7]uril (CB[7]) reduces the response of olfactory receptors in Mozambique tilapia (Oreochromis mossambicus) in vivo. For example, the olfactory receptor response to the odorant adamantan-1-amine, as measured by electro-olfactography, was suppressed by 92% in the presence of CB[7]. A reduction in olfactory response of 88% was observed for pentane-1,5-diamine (cadaverine), an odorant associated with carrion avoidance in some fish. The results reveal how the association constants and the concentrations of natural and synthetic receptors play a determinant role and show that synthetic receptors can be used to remove bioactive molecules from fish olfaction.
- A multi-component pheromone in the urine of dominant male tilapia (Oreochromis mossambicus) reduces aggression in rivalsPublication . Keller-Costa, Tina; Saraiva, João L.; Hubbard, Peter; Barata, Eduardo Nuno; Canário, Adelino V. M.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 5 beta-pregnane-3 alpha,17 alpha,20 beta-triol 3-glucuronate and its 20 alpha-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.
- Olfactory sensitivity of the marine flatfish Solea senegalensis to conspecific body fluidsPublication . Fatsini, Elvira; Carazo, Ignacio; Chauvigne, Francois; Manchado, Manuel; Cerda, Joan; Hubbard, Peter; Duncan, Neil J.Chemical communication is better understood in freshwater fish than marine fish. The Senegalese sole (Solea senegalensis) is a marine flatfish wherein one of the problems in aquaculture is the poor reproductive performance of hatchery-bred males. Is chemical communication involved in the reproduction of this species? Urine, intestinal fluid and mucus samples were taken from adult fish (wild-caught and hatchery-bred) over the spawning season (March-May), and assessed for olfactory potency using the electro-olfactogram (EOG). The effect of stimulation of the olfactory system with adult female urine on circulating luteinizing hormone (LH) levels was also tested in males. Intestinal fluid and urine were potent olfactory stimuli for both juvenile and adult conspecifics, evoking large-amplitude, concentration-dependent EOG responses, with thresholds of detection at approximately 1:10(6). However, the amplitude of the response to urine depended on the sex and state of maturity of both the donor and the receiver. Most olfactory activity could be extracted by C18 solid-phase cartridges. Urine from mature females evoked a slight, but significant, increase in circulating LH levels in mature males 30 min after exposure. Furthermore, the olfactory potency of urine differed between wild-caught and hatchery-bred fish; however, contrary to expectations, urine from wild-caught females was less potent than that from hatchery-bred females. Taken together, these results strongly suggest that faeces-and urine-released odorants are involved in reproduction in the Senegalese sole, and establish a basis for further investigation into pheromonal communication in marine teleosts.
- Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturationPublication . Churcher, Allison; Pujolar, Jose M.; Milan, Massimo; Hubbard, Peter; Martins, Rute S. T.; L. Saraiva, João; Huertas, Mar; Bargelloni, Luca; Patarnello, T.; Marino, Ilaria A. M.; Zane, Lorenzo; Canario, Adelino V. M.Background: The vertebrate brain plays a critical role in the regulation of sexual maturation and reproduction by integrating environmental information with developmental and endocrine status. The European eel Anguilla anguilla is an important species in which to better understand the neuroendocrine factors that control reproduction because it is an endangered species, has a complex life cycle that includes two extreme long distance migrations with both freshwater and seawater stages and because it occupies a key position within the teleost phylogeny. At present, mature eels have never been caught in the wild and little is known about most aspects of reproduction in A. anguilla. The goal of this study was to identify genes that may be involved in sexual maturation in experimentally matured eels. For this, we used microarrays to compare the gene expression profiles of sexually mature to immature males. Results: Using a false discovery rate of 0.05, a total of 1,497 differentially expressed genes were identified. Of this set, 991 were expressed at higher levels in brains (forebrain and midbrain) of mature males while 506 were expressed at lower levels relative to brains of immature males. The set of up-regulated genes includes genes involved in neuroendocrine processes, cell-cell signaling, neurogenesis and development. Interestingly, while genes involved in immune system function were down-regulated in the brains of mature males, changes in the expression levels of several receptors and channels were observed suggesting that some rewiring is occurring in the brain at sexual maturity. Conclusions: This study shows that the brains of eels undergo major changes at the molecular level at sexual maturity that may include re-organization at the cellular level. Here, we have defined a set of genes that help to understand the molecular mechanisms controlling reproduction in eels. Some of these genes have previously described functions while many others have roles that have yet to be characterized in a reproductive context. Since most of the genes examined here have orthologs in other vertebrates, the results of this study will contribute to the body of knowledge concerning reproduction in vertebrates as well as to an improved understanding of eel biology.
- Olfactory sensitivity to bile fluid and bile salts in the European eel (Anguilla anguilla), goldfish (Carassius auratus) and Mozambique tilapia (Oreochromis mossambicus) suggests a 'broad range' sensitivity not confined to those produced by conspecifics alonePublication . Huertas, Mar; Hagey, L.; Hofmann, A. F.; Cerda, J.; Canario, Adelino V. M.; Hubbard, PeterTeleosts have high olfactory sensitivity to bile salts. To assess whether this phenomenon is involved in intra-specific chemical communication alone, or is part of a more ‘broad range’ sensitivity to bile salts produced by heterospecifics, we investigated possible differences in the odour of bile between the sexes and among different species – the eel (Anguilla anguilla), goldfish (Carassius auratus) and Mozambique tilapia (Oreochromis mossambicus) – using the electro-olfactogram (EOG). We also identified the main bile constituents by liquid chromatography and mass spectrometry. There were marked differences in olfactory response of the eel to thin-layer chromatography fractions of bile from both sexes, and mature and immature conspecifics. Smaller differences were seen in the potency of fractions of bile from male and female goldfish and tilapia. Eels, goldfish and tilapia demonstrated similar olfactory sensitivity to bile from a range of different species, with no apparent correlation between the olfactory potency of bile and a phylogenetic closeness and/or similarity of diet of the donor to the receiver. The three species were able to detect odorants in thin-layer chromatography fractions of heterospecific bile even in the absence of activity in conspecific bile. Eels, goldfish and tilapia responded to both sulphated C27 bile salts (5-scymnol-sulphate and 5-cyprinol sulphate) and to taurine-conjugated C24 bile salts (taurochenodeoxycholic acid, taurolithocholic acid and taurocholic acid), irrespective of whether these bile salts were present in conspecific bile. Together, these results suggest that teleosts have a broad-range olfactory sensitivity to bile salts, with potential roles in both intra-specific chemical communication and in inter-specific interactions.
- Evidence that 1-methyl-l-tryptophan is a food-related odorant for the Senegalese sole (Solea senegalensis)Publication . Velez, Zélia; Hubbard, Peter; Hardege, Jörg D.; Welham, K.; Barata, E. N.; Canario, Adelino V. M.Nocturnal animals often rely heavily on olfactory cues to locate their food. This is especially true for fish whose prey live buried in the substrate. The aim of the current study was to identify non-polar odorants released by the ragworm (Hediste diversicolor) that are detected by the olfactory system of the Senegalese sole (Solea senegalensis) and therefore may be food-related cues. Ragworm-conditioned water was passed through a C18 solid-phase extraction cartridge (to remove polar compounds) and the eluate fractionated by reverse phase high-performance liquid chromatography (HPLC). The fractions were then tested for olfactory activity in the sole by the electro-olfactogram (EOG). Most olfactory activity was found in the first two fractions to elute from the HPLC column (0–4 min). Liquid chromatography-mass spectrometry (LC/MS) of both fractions revealed a base peak with a molecular mass of 219.4 Da and a fragment of 205.3 Da. These data are consistent with a methylated form of tryptophan; synthetic 1-methyl-l-tryptophan had a similar HPLC retention time (2.8 min) and similar LC/MS spectra. Furthermore, sole showed olfactory sensitivity to 1-methyl-l-tryptophan with the lower (left) epithelium being more sensitive than the upper (right). Cross-adaptation (using EOG recording) suggested that the olfactory receptors responding to 1-methyl-l-tryptophan are different in the lower epithelium from the upper. These results suggest that ragworms release 1-methyl-l-tryptophan, or similar tryptophan derivative, and that sole may use this chemical cue to locate and/or identify one of their main prey species. This may help the formation of artificial feeds that are more attractive to sole.
- Muscular hypertrophy of urinary bladders in dominant tilapia facilitates the control of aggression through urinary signalsPublication . Keller-Costa, Tina; Lopes, O. S.; Almeida, O. G.; Hubbard, Peter; Iacovella, A.; Lima, M.; Barata, E. N.; Canario, Adelino V. M.The urination pattern of the Mozambique tilapia (Oreochromis mossambicus) depends on social context, and the olfactory potency of urine released depends on social rank (males) and reproductive status (females). This strongly suggests that urine mediates chemical communication in this species. The current study tested, firstly, whether urine production rate depends on sex or social status and, secondly, whether differences in urination pattern and volume of urine stored are associated with variation in the morphology of the urinary bladder. Finally, the effect of urination during aggressive male–male interactions was assessed. Urine production in catheterized fish depended neither on sex nor social status (males). Nevertheless, males had larger kidneys than females. Dominant males had heavier urinary bladders than subordinate males or females, mainly due to enlarged muscle fibres, thicker urothelium and a thicker smooth muscle layer. In male pairs wherein urination was prevented by temporary constriction of the genital papillae, social interaction escalated to aggression (mouth-to-mouth fighting) more rapidly and frequently than control pairs. This was accompanied by elevated plasma testosterone and 11-ketotestosterone levels. In control encounters, the male that initiated the aggressive behaviour was usually the winner of the subsequent fight; this did not happen when the males could not urinate. These results suggest that the larger, more muscular bladder of dominant males is an adaptation, facilitating higher urination frequency, post-renal modulation and storage of larger urine volumes for longer. It is likely that urinary pheromones modulate aggression in male–male encounters by providing information on the social rank and/or motivation of the emitter; males are unlikely to invest in costly highly aggressive fights if they judge their opponent to be more dominant. Thus, a morphological explanation for the differing urination patterns of dominant and subordinant males, and females, has been provided, and a possible function for this behaviour in male–male interactions is suggested.
- Olfactory transduction pathways in the Senegalese sole Solea senegalensisPublication . Velez, Zélia; Hubbard, Peter; Barata, E. N.; Canario, Adelino V. M.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 Lphenylalanine (food-related odorants) in the lower epithelium, and for taurocholic acid (conspecificderived 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.
- Chemical communication in tilapia: a comparison of Oreochromis mossambicus with O. niloticusPublication . Hubbard, Peter; Mota, Vasco; Keller-Costa, Tina; Silva, José P. da; Canario, Adelino V. M.In allopatric speciation species differentiation generally results from different selective pressures in different environments, and identifying the traits responsible helps to understand the isolation mechanism(s) involved. Male Mozambique tilapia (Oreochromis mossambicus) use urine to signal dominance; furthermore, 5-pregnane-3,17,20-triol-3-glucuronide (and its -epimer, 5-pregnane-3,17,20-triol-3-glucuronide), in their urine is a potent pheromone, the concentration of which is correlated with social status. The Nile tilapia (O. niloticus) is a close relative; species divergence probably resulted from geographical separation around 6 million years ago. This raises the question of whether the two species use similar urinary chemical cues during reproduction. The olfactory potency of urine, and crude extracts, from either species was assessed by the electro-olfactogram and the presence of the steroid glucuronides in urine from the Nile tilapia by liquid-chromatography/mass spectrometry. Both species showed similar olfactory sensitivity to urine and respective extracts from either species, and similar sensitivity to the steroid glucuronides. 5-pregnan-3,17,20-triol-3-glucuronide was present at high concentrations (approaching 0.5 mM) in urine from Nile tilapia, with 5-pregnan-3,17,20-triol-3-glucuronide present at lower concentrations, similar to the Mozambique tilapia. Both species also had similar olfactory sensitivity to estradiol-3-glucuronide, a putative urinary cue from females. Together, these results support the idea that reproductive chemical cues have not been subjected to differing selective pressure. Whether these chemical cues have the same physiological and behavioural roles in O. niloticus as O. mossambicus remains to be investigated.