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- Possible disruption of pheromonal communication by humic acid in the goldfish, Carassius auratusPublication . Hubbard, Peter; Barata, E. N.; Canario, Adelino V. M.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 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P), its sulphated conjugate (17,20β-P-SO4) and prostaglandin F2α (PGF2α), all at 10−11 to 10−8 M, in the absence and presence of humic acids (1–1000 mg l−1). At nearly all concentrations of humic acid tested, there was a significant attenuation of the amplitude of the initial (phasic) response to 17,20β-P compared to 17,20β-P alone. At higher concentrations of humic acid, the EOG response to 17,20β-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 17,20β-P per se. Furthermore, simultaneous recording of electro-encephalograms from the olfactory bulb demonstrated that the nervous activity evoked by the same concentration of 17,20β-P was less intense in the presence of humic acid than its absence. PGF2α is non-steroidal and much more soluble in water. In contrast to 17,20β-P, only the higher concentrations of humic acid (100 and 1000 mg l−1) significantly diminished the EOG amplitude. 17,20β-P-SO4 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 17,20β-P-SO4 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 17,20β-P and 17,20β-P-SO4 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 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.
- Sexually mature European eels (Anguilla anguilla L.) stimulate gonadal development of neighbouring males: possible involvement of chemical communicationPublication . Huertas, Mar; Scott, A. P.; Hubbard, Peter; Canario, Adelino V. M.; Cerda, J.This study was aimed to investigate whether sexual maturation of immature male eels could be stimulated indirectly by placing them in contact with either male (Minj) or female (Finj) eels in which sexual maturation had been stimulated directly by weekly injections of human chorionic gonadotropin (hCG) or salmon pituitary extract (SPE), respectively. Untreated males were placed either in the same tank or in a separate tank that was linked to the injected Wsh via a recirculation system. The hormonal treatments stimulated spermatogenesis and spermiation in Minj, and ovulation in Finj as well as an increase of the ocular (Io) and gonadosomatic (GSI) indices in both sexes. Plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) increased in Minj and T and 17 -estradiol (E2) in Finj. A small peak of plasma 17,20 -dihydroxypregn-4-en-3-one (17,20 P) occurred during ovulation, while the plasma levels of 17 -hydroxypregn-4- ene-3,20-dione (17P) were undetectable in both males and females. The water conditioned by Minj and Finj induced signiWcant, though relatively minor, increases in Io and GSI in uninjected males. In addition, uninjected Wsh showed small changes in plasma T and 11-KT levels, apparently related to the timing of spermiation and ovulation of Minj and Finj, respectively, as well as an activation of spermatogenesis (but not spermiation). Injected Wsh released free and conjugated T, 11-KT and E2 into the water, although immature eels were unable to smell (by electro-olfactogram) any of these steroids or prostaglandin F2 . However, immature males were highly sensitive to water extracts conditioned by spermiating Minj and pre-ovulatory and ovulated Finj. These preliminary results suggest the existence of chemical communication between maturing eels and immature males that stimulates gonad development, although the putative pheromone(s) involved has/have not yet been identiWed.
- Chemical communication in the Genus Anguilla: a minireviewPublication . Hubbard, Peter; Canario, Adelino V. M.; Huertas, MarEels of the Genus Anguilla are important fish in both scientific and economic terms. Unfortunately, current stocks of the European eel in particular (Anguilla anguilla L.) are becoming increasingly endangered. Chemical communication plays important roles in several key aspects of fish biology. Due to its complex life-cycle, the eel offers a unique opportunity to study various aspects of olfactory biology and chemical communication almost in isolation, feeding (during freshwater ‘yellow’ stage), adaptation to seawater, migration and, finally, reproduction. The aim of this brief review is to summarise what is known about chemical communication in the eel (during reproduction in particular) and, more importantly, stimulate further research. To date, eels have been shown to have high olfactory sensitivity to substances released by conspecifics. Both the bile fluid and skin mucus have been implicated as the routes of release of these odorants but other routes, such as the urine, cannot be excluded. Furthermore, the nature of these odorants depends on both the sex and reproductive status of the donor; exposure to water conditioned by mature con-specifics causes a stimulation of sexual maturation in immature eels. These results are consistent with a role for chemical communication in eel reproduction. The European eel is, therefore, proposed as a model species for studies on olfaction and chemical communication in fish because of its ancestral status, its high olfactory sensitivity and its physiological plasticity. However, more research is necessary; specifically, identification of the key odorants involved, their behavioural and physiological effects and how the olfactory system may change at various stages of its life-cycle.
- Differential detection of conspecific-derived odorants by the two olfactory epithelia of the Senegalese sole (Solea senegalensis)Publication . Velez, Zélia; Hubbard, Peter; Barata, E. N.; Canario, Adelino V. M.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 C24 and C27 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.
- 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.
- 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.
- Identify of a tilapia pheromone released by dominant males that primes females for reproductionPublication . Keller-Costa, Tina; Hubbard, Peter; Paetz, Christian; Nakamura, Yoko; Silva, José P. da; Rato, Ana; Barata, E. N.; Schneider, Bernd; Canario, Adelino V. M.Knowledge of the chemical identity and role of urinary pheromones in fish is scarce, yet necessary to understand the integration of multiple senses in adaptive responses and the evolution of chemical communication. In nature, Mozambique tilapia (Oreochromis mossambicus) males form hierarchies and females mate preferentially with dominant territorial males which they visit in aggregations or leks.
- 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.
- Anatomy of the olfactory system and potential role for chemical communication in the sound‐producing lusitanian toadfish, halobatrachus didactylusPublication . Modesto, Teresa; Gregório, Beatriz Neves; Marcelino, Gonçalo; Marquet, Nathalie; Costa, Rita; Guerreiro da Costa Guerreiro, Pedro Miguel; Velez, Zélia; Hubbard, PeterThe current study investigated the structure and function of the olfactory system of the Lusitanian toadfish, Halobatrachus didactylus, using histology and electrophysiology (electro-olfactogram [EOG]), respectively. The olfactory system consists of a digitated anterior peduncle, of unknown function, containing the inhalant nostril. This then leads to a U-shaped olfactory chamber with the olfactory epithelium-identified by G(alpha olf)-immunoreactivity-on the ventral surface. A large lacrimal sac is connected to this tube and is likely involved in generating water movement through the olfactory chamber (this species is largely sedentary). The exhalent nostril lies by the eye and is preceded by a bicuspid valve to ensure one-way flow of water. As do other teleosts, H. didactylus had olfactory sensitivity to amino acids and bile acids. Large-amplitude EOG responses were evoked by fluid from the anterior and posterior testicular accessory glands, and bile and intestinal fluids. Anterior gland and intestinal fluids from reproductive males were significantly more potent than those from non-reproductive males. Male urine and skin mucus proved to be the least potent body fluids tested. These results suggest that chemical communication-as well as acoustic communication-may be important in the reproduction of this species and that this may be mediated by the accessory glands and intestinal fluid.