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- Behavioral and olfactory responses of female Salaria pavo (Pisces : Blenniidae) to a putative multi-component male pheromonePublication . Serrano, Rui M.; Barata, E. N.; Birkett, Michael A.; Hubbard, Peter; Guerreiro, Patrícia; Canario, Adelino V. M.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 speciesspecific 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-conditio ned 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.
- Olfactory sensitivity to conspecific bile fluid and skin mucus in the European eel Anguilla anguilla (L.)Publication . Huertas, Mar; Hubbard, Peter; Canario, Adelino V. M.; Cerda, J.The present study assessed the olfactory potency of conspecific bile fluid and skin mucus in the European eel Anguilla anguilla by the electro-olfactogram. Immature males showed high olfactory sensitivity to conspecific bile, giving large amplitude responses in a concentrationdependent manner with estimated thresholds of detection of <1:107 (n = 6). Mucus also proved to contain highly potent odorants with thresholds of detection of c. 1:106 (n = 6). Crude solidphase extraction of bile fluid (C-18 and C-2/ENV+ cartridges) showed that the majority of olfactory activity in bile fluid was contained in the eluate of C-18 cartridges (n = 6). There were quantitative differences, however, between the sexes; female bile fluid had a higher proportion of activity in this fraction. Similar solid-phase extraction of mucus showed that it contains a higher proportion of odorants in the C-18 filtrate than bile fluid. Mucus from mature eels, however, had a higher proportion of olfactory activity in the eluate than immature fish (n = 6). Cross-adaptation experiments suggest that there are qualitative differences in the odorants contained in bile and mucus depending on both the sex and state of sexual maturation of the donor (n = 6). These results are consistent with a role for chemical communication in the reproduction of the European eel and suggest that both bile and mucus are potential sources of the odorants involved.
- Olfactory discrimination of female reproductive status by male tilapia (Oreochromis mossambicus)Publication . Miranda, A.; Almeida, O. G.; Hubbard, Peter; Barata, E. N.; Canario, Adelino V. M.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.
- 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.
- 11-ketotestosterone stimulates putative sex pheromone production in the male peacock blenny, Salaria pavo (Risso 1810)Publication . Serrano, Rui M.; Lopes, Orlando; Hubbard, Peter; Araújo, J.; Canario, Adelino V. M.; Barata, E. N.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.
- Tilapia male urinary pheromone stimulates female reproductive axisPublication . Huertas, Mar; Almeida, O. G.; Canario, Adelino V. M.; Hubbard, PeterMozambique tilapia males congregate in leks where they establish dominance hierarchies and attract females to spawn in sandy pits. Dominant males store more urine than subordinates and the pattern of urination and the high sensitivity of females to male urine suggest chemical signalling via the urine. Here we show that pre-ovulated and post-spawn females when exposed to dominant male urine increased significantly, in less than 1 h, the release rate of the maturation-inducing steroid 17,20bdihydroxypregn- 4-en-3-one which is maintained elevated for at least 6 h. This indicates a pheromonal role for male urine in the synchronisation of spawning. Furthermore, we show that the lack of affinity of 17,20bP to sex steroid binding globulin explains, at least partly, its rapid release and lack of detection in the blood. Thus tilapia urine involvement in several communication processes confirms that cichlids have evolved a sophisticated chemical signalling system together with their complex visual, acoustic and behavioural displays.
- Bile acids as putative social signals in Mozambique tilapia (Oreochromis mossambicus)Publication . Ashouri, Samyar; Da Silva, José Paulo; Canario, Adelino; Hubbard, PeterChemical cues provide potential mates with information about reproductive status and resource-holding potential. In the Mozambique tilapia (Oreochromis mossambicus), males can distinguish female reproductive status through chemical cues, and accessibility of males to females depends on their position in the hierarchy, determined in part by chemical cues. Here, we hypothesized that tilapia faecal cues are attractive to conspecifics once released into the water. C18 solid-phase extracts of faeces from dominant males and pre-ovulatory females evoked stronger olfactory epithelium electrical responses (EOG) than, respectively, subordinate males and postspawning females. Mass spectrometry of the reverse-phase C18 high-performance liquid chromatography fractions of these extracts with highest EOG, identified by amino acids and bile acids. Faeces from pre-ovulatory females contain significantly higher concentrations of cholic acid (CA) and taurocholic acid (TCH) than both post-spawning females and males. A pool of amino acids had no effect on aggression or attraction in males. However, males were attracted to the scent of pre-ovulatory female faeces, as well as CA and TCH, when applied separately. This attraction was accompanied by increased digging behaviour compared to the odour of postspawning females. CA and TCH exert their action through separate receptor mechanisms. These findings are consistent with a role for faeces - and bile acids therein - in chemical communication in this species, acting as an attractant for males to reproductive females.
- Production, release and olfactory detection of sex steroids by the tench (Tinca tinca L.)Publication . Pinillos, M. L.; Guijarro, A. I.; Delgado, M. J.; Hubbard, Peter; Canario, Adelino V. M.; Scott, A. P.The present study is concerned with pheromone communication in tench (Tinca tinca L.), establishing firstly whethermales have a high olfactory sensitivity to some typical teleost sex steroids and prostaglandins; and secondly whether males and females might be able to synthesise and release some of these steroids into the water. The C21 steroid, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)was found to give large electro-olfactogram responses with an estimated threshold of detection of 10−12 M. The male tench were equally sensitive to glucuronidated 17,20β-P (10−11.6 M) but 100 times less sensitive to sulphated 17,20β-P (11−9.7 M). Preliminary data from cross-adaptation studies suggest that both the free and conjugated forms are detected by the same olfactory receptor(s). Male tench also had high olfactory sensitivity to prostaglandin F2α (PGF2α) and 15-keto PGF2α (11−11.5 and 10−11.4 M). They were relatively insensitive, however, to testosterone (T), androstenedione (AD), 11-ketotestosterone (11-KT), 17β-oestradiol (E2), 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) and 17,20α-dihydroxy-4-pregnen-3- one (17,20α-P). Radioimmunoassays were used to measure the steroids in plasma and water and all samples were processed for themeasurement of free, sulphated and glucuronidated fractions. In females, free 17,20β-P, 17,20α-P, free and glucuronidated T, and AD in plasma showed the largest increases in response to injection with mammalian gonadotropin-releasing hormone analogue (GnRHa) or Ovaprim (a mixture of GnRHa and a dopamine inhibitor). Free 17,20β-P was released into the water at the greatest rate. Plasma concentrations of the two conjugated forms of 17,20β-P were also elevated 18 h after the administration of GnRHa, but not by as much as the free steroid. In males, AD and 11-KT showed the greatest increase in response to GnRHa and were moreover released into the water at a higher rate in the treated group than in the control. The data support a possible pheromonal role for free and glucuronidated 17,20β-P.
- A sterol-like odorant in the urine of mozambique tilapia males likely signals social dominance to femalesPublication . Barata, E. N.; Fine, J. M.; Hubbard, Peter; Almeida, O. G.; Frade, P.; Sorensen, P. W.; Canario, Adelino V. M.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.