Environmental salinity modulates olfactory sensitivity in the euryhaline European seabass, Dicentrarchus labrax, acclimated to seawater and brackish water.

The olfactory epithelium of fish is - of necessity - in intimate contact with the surrounding water. In euryhaline fish, movement from seawater to freshwater (and vice versa) exposes the epithelium to massive changes in salinity and ionic concentrations. How does the olfactory system function in the face of such changes? The current study compared olfactory sensitivity in seawater- (35‰) and brackish water-adapted seabass (5‰) using extracellular multi-unit recording from the olfactory nerve. Seawater-adapted bass had higher olfactory sensitivity to amino acid odorants when delivered in seawater than in freshwater. Conversely, brackish water-adapted bass had largely similar sensitivities to the same odorants when delivered in seawater or freshwater, although sensitivity was still slightly higher in seawater. The olfactory system of seawater-adapted bass was sensitive to decreases in external [Ca2+], whereas brackish water-adapted bass responded to increases in [Ca2+]; both seawater- and brackish water-adapted bass responded to increases in external [Na+] but the sensitivity was markedly higher in brackish water-adapted bass. In seawater-adapted bass, olfactory sensitivity to l-alanine depended on external Ca2+ ions, but not Na+; brackish water-adapted bass did respond to l-alanine in the absence of Ca2+, albeit with lower sensitivity, whereas sensitivity was unaffected by removal of Na+ ions. A possible adaptation of the olfactory epithelium was the higher number of mucous cells in brackish water-adapted bass. The olfactory system of seabass is able to adapt to low salinities, but this is not immediate; further studies are needed to identify the processes involved.

Minimizing the time to evaluate pheromone-mediated reduction of aggressive behavior in Mozambique tilapia (Oreochromis mossambicus).

Some cichlid fishes release urine-containing chemical cues that lower aggression in their opponents. Bioassays to identify the aggression-modulating pheromone include assessing the effect of urine fractions on the behavior towards a mirror image or in interactions with another male. However, many of these methods can be time-consuming and require many fish. The objective of the present study was to assess the behavior of male Mozambique tilapia (Oreochromis mossambicus) towards male urine using two methods with the intent of simplifying the bioassays: aggression towards a mirror image (mirror assay) and real opponents in which the urogenital papilla was tied using surgical silk to prevent urination. The results confirm the aggression-reducing effect of dominant male urine in both experimental approaches. Ten minutes of biting or 15 min of tail-beating behaviors in the mirror assay, or 5 min of opercular expansion or 15 min of lateral display in interactions with real opponents were necessary to detect a statistically significant reduction in aggressive behavior towards dominant male urine. We also found that males with subordinate status had lower latency to initiate aggressive behaviors towards the mirror than dominants in the same condition, even though fish had been isolated for 1 week. However, no such differences in latency were found in the real opponent assay. We conclude that 5 min of opercular expansion behavior in real opponent fights or 10 min of biting behavior in the mirror assay are the shortest times necessary to test aggressive behavior in urine fractions in bioassay-guided identification of pheromones.

Anatomy of the olfactory system and potential role for chemical communication in the sound-producing Lusitanian toadfish, Halobatrachus didactylus.

The 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 Gαolf-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.

What do oysters smell? Electrophysiological evidence that the bivalve osphradium is a chemosensory organ in the oyster, Magallana gigas.

The sensing of chemical cues is essential for several aspects of bivalve biology, such as the detection of food and pheromones. However, little is known about chemical communication systems in bivalves or the possible role of the osphradium as a chemosensory organ. To address this, we adapted an electrophysiological technique extensively used in vertebrates-the electro-olfactogram-to record from the osphradium in the Pacific oyster, Magallana gigas. This technique was validated using amino acids as stimulants. The osphradium proved to be sensitive to most proteinogenic L-amino acids tested, evoking tonic, negative, concentration-dependent 'electro-osphradiogram' (EOsG) voltage responses, with thresholds of detection in the range of 10- 6 to 10- 5 M. Conversely, it was insensitive to L-arginine and L-glutamic acid. The current study supports the hypothesis that the osphradium is, indeed, a chemosensory organ. The 'electro-osphradiogram' may prove to be a powerful tool in the isolation and characterization of pheromones and other important chemical cues in bivalve biology.

GABA receptors in the olfactory epithelium of the gilthead seabream (Sparus aurata).

Exposure to high PCO2/low pH seawater induces behavioural alterations in fish; a possible explanation for this is a reversal of Cl-/HCO3- currents through GABAA receptors (the GABAA receptor theory). However, the main evidence for this is that gabazine, a GABAA receptor antagonist, reverses these effects when applied to the water, assuming that exposure to systems other than the CNS would be without effect. Here, we show the expression of both metabotropic and ionotropic GABA receptors, and the presence of GABAA receptor protein, in the olfactory epithelium of gilthead seabream. Furthermore, exposure of the olfactory epithelium to muscimol (a specific GABAA receptor agonist) increases or decreases the apparent olfactory sensitivity to some odorants. Thus, although the exact function of GABAA receptors in the olfactory epithelium is not yet clear, this may complicate the interpretation of studies wherein water-borne gabazine is used to reverse the effects of high CO2 levels on olfactory-driven behaviour in fish.

Independent effects of seawater pH and high PCO2 on olfactory sensitivity in fish: possible role of carbonic anhydrase.

Ocean acidification may alter olfactory-driven behaviour in fish by direct effects on the peripheral olfactory system; olfactory sensitivity is reduced in CO2-acidified seawater. The current study tested whether this is due to elevated PCO2  or the consequent reduction in seawater pH and, if the former, the possible involvement of carbonic anhydrase, the enzyme responsible for the hydration of CO2 and production of carbonic acid. Olfactory sensitivity to amino acids was assessed by extracellular multi-unit recording from the olfactory nerve of the gilthead seabream (Sparus aurata L.) in normal seawater (pH ∼8.2), and after acute exposure to acidified seawater (pH ∼7.7) but normal PCO2  (∼340 µatm) or to high PCO2  seawater (∼1400 µatm) at normal pH (∼8.2). Reduced pH in the absence of elevated PCO2  caused a reduction in olfactory sensitivity to l-serine, l-leucine, l-arginine and l-glutamine, but not l-glutamic acid. Increased PCO2  in the absence of changes in pH caused reduced olfactory sensitivity to l-serine, l-leucine and l-arginine, including increases in their threshold of detection, but had no effect on sensitivity to l-glutamine and l-glutamic acid. Inclusion of 1 mmol l-1 acetazolamide (a membrane-permeant inhibitor of carbonic anhydrase) in the seawater reversed the inhibition of olfactory sensitivity to l-serine caused by high PCO2 Ocean acidification may reduce olfactory sensitivity by reductions in seawater pH and intracellular pH (of olfactory receptor neurones); the former by reducing odorant-receptor affinity, and the latter by reducing the efficiency of olfactory transduction. The physiological role of carbonic anhydrase in the olfactory receptor neurones remains to be explored.

Acidification can directly affect olfaction in marine organisms.

In the past decade, many studies have investigated the effects of low pH/high CO2 as a proxy for ocean acidification on olfactory-mediated behaviours of marine organisms. The effects of ocean acidification on the behaviour of fish vary from very large to none at all, and most of the maladaptive behaviours observed have been attributed to changes in acid-base regulation, leading to changes in ion distribution over neural membranes, and consequently affecting the functioning of gamma-aminobutyric acid-mediated (GABAergic) neurotransmission. Here, we highlight a possible additional mechanism by which ocean acidification might directly affect olfaction in marine fish and invertebrates. We propose that a decrease in pH can directly affect the protonation, and thereby, 3D conformation and charge distribution of odorants and/or their receptors in the olfactory organs of aquatic animals. This can sometimes enhance signalling, but most of the time the affinity of odorants for their receptors is reduced in high CO2/low pH; therefore, the activity of olfactory receptor neurons decreases as measured using electrophysiology. The reduced signal reception would translate into reduced activation of the olfactory bulb neurons, which are responsible for processing olfactory information in the brain. Over longer exposures of days to weeks, changes in gene expression in the olfactory receptors and olfactory bulb neurons cause these neurons to become less active, exacerbating the problem. A change in olfactory system functioning leads to inappropriate behavioural responses to odorants. We discuss gaps in the literature and suggest some changes to experimental design in order to improve our understanding of the underlying mechanisms and their effects on the associated behaviours to resolve some current controversy in the field regarding the extent of the effects of ocean acidification on marine fish.

Olfactory sensitivity of the marine flatfish Solea senegalensis to conspecific body fluids.

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 1:106 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.

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.

Deep sequencing of the olfactory epithelium reveals specific chemosensory receptors are expressed at sexual maturity in the European eel Anguilla anguilla.

Vertebrate genomes encode a diversity of G protein-coupled receptor (GPCR) that belong to large gene families and are used by olfactory systems to detect chemical cues found in the environment. It is not clear however, if individual receptors from these large gene families have evolved roles that are specific to certain life stages. Here, we used deep sequencing to identify differentially expressed receptor transcripts in the olfactory epithelia (OE) of freshwater, seawater and sexually mature male eels (Anguilla anguilla). This species is particularly intriguing because of its complex life cycle, extreme long-distance migrations and early-branching position within the teleost phylogeny. In the A. anguillaOE, we identified full-length transcripts for 13, 112, 6 and 38 trace amine-associated receptors, odorant receptors (OR) and type I and type II vomeronasal receptors (V1R and V2R). Most of these receptors were expressed at similar levels at different life stages and a subset of OR and V2R-like transcripts was more abundant in sexually mature males suggesting that ORs and V2R-like genes are important for reproduction. We also identified a set of GPCR signal transduction genes that were differentially expressed indicating that eels make use of different GPCR signal transduction genes at different life stages. The finding that a diversity of chemosensory receptors is expressed in the olfactory epithelium and that a subset is differentially expressed suggests that most receptors belonging to large chemosensory gene families have functions that are important at multiple life stages, while a subset has evolved specific functions at different life stages.

Chemical communication in cichlids: A mini-review.

The family Cichlidae is well-known for pair-formation, parental care, territoriality, elaborate courtship and social organization. Do cichlids use chemical communication to mediate any of these behaviours? Early studies suggest that parent cichlids can discriminate between conspecific and heterospecific wrigglers (but not eggs) using olfactory cues. Some species are able to discriminate between their own brood and other conspecific broods based on olfaction. The young recognise conspecific adults (although not necessarily their parents) through the odorants they release. In both scenarios, protection of the young from predation is the likely selective force. Some male cichlids use urinary pheromones during courtship and spawning to attract females and induce ovulation. Females--in their turn--may base their mate-choice in part on assessment of those self-same pheromones. The same pheromonal system may be involved in establishing and maintaining the social hierarchies in lek-breeding cichlids. Individual recognition is also mediated by chemical communication. Finally, there is ample behavioural evidence that cichlids--like ostariophysan fish--release alarm cues that alert conspecifics to predation danger. Although the effects of these cues may be similar (e.g., increased shelter use, tighter schooling), they are different substances which remain to be identified. Cichlids, then, use chemical communication associated with many different behaviours. However, given the diversity of cichlids, little is known about the mechanisms of chemical communication or the chemical identity of the cues involved. The aim of this mini-review is to persuade those working with cichlids to consider the involvement of chemical communication, and those working in chemical communication to consider using cichlids.

Lack of evidence for a role of olfaction on first maturation in farmed sea bass Dicentrarchus labrax.

Chemical communication is widespread in the animal kingdom and olfaction constitutes a powerful channel for social and environmental cues. In fish, olfactory stimuli are known to influence physiological processes, including reproduction. Here we investigate the effects of olfaction on puberty in European sea bass Dicentrarchus labrax males. Intact sea bass coming to first maturity (puberty) are able to smell conspecific odours. However, induced anosmia during most of the spermatogenesis period had no effect on the sex ratio, gonad maturation state or gonado-somatic index at the time of reproduction. Furthermore anosmia decreased mRNA expression of brain KISS2 and pituitary LHb and FSHb, but not brain GnRH1 and GnRH3. Thus, although anosmia seems to modify gene expression of key reproduction related genetic factors, it seems to be insufficient to stop or delay growth or gonadal development and maturation.

Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturation.

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 steroid glucuronates in Mozambique tilapia suggests two distinct and specific receptors for pheromone detection.

Cichlids offer an exciting opportunity to understand vertebrate speciation; chemical communication could be one of the drivers of African cichlid radiation. Chemical signals mediate key aspects in the lives of vertebrates and often are species specific. Dominant male Mozambique tilapia [Oreochromis mossambicus (Peters 1852)] release a sex pheromone, 5ß-pregnan-3α,17α,20ß-triol 3-glucuronate and its 20α-epimer, via their urine. The objective of this study was to assess the sensitivity, specificity and versatility of the olfactory system of O. mossambicus to other steroids and their conjugates using the electro-olfactogram. Oreochromis mossambicus was sensitive to several 3-glucuronidated steroids, but did not respond to prostaglandins, unconjugated steroids or 17- or 20-conjugated steroids. Stimulation of the olfactory epithelium with increasing concentrations (1 pmol l(-1) to 10 µmol l(-1)) of 5ß-pregnan-3α,17α,20ß-triol 3-glucuronate, 5ß-pregnan-3α,17α,20α-triol 3-glucuronate, 3α,17α-dihydroxy-5ß-pregnan-20-one 3-glucuronate, etiocholanolone 3α-glucuronate and 17ß-estradiol 3-glucuronate produced characteristic sigmoidal concentration-response curves. However, tilapia were most sensitive to 17ß-estradiol-3-glucuronate, which also had the lowest apparent EC50 and maximal response amplitude. Cross-adaptation and binary mixture experiments suggested that 5ß,3α-reduced pregnan- and androstan-3-glucuronates share (a) common olfactory receptor(s), whereas 17ß-estradiol 3-glucuronate is detected via (a) distinct olfactory receptor(s). In conclusion, the Mozambique tilapia has evolved high olfactory sensitivity and specificity to 3-glucuronidated steroids through two distinct olfactory receptor types; one detecting a male sex pheromone and a second detecting 17ß-estradiol 3-glucuronate, a putative female-derived signal. However, O. mossambicus differs markedly in its olfactory perception from the more recently derived East African cichlid Astatotilapia burtoni, suggesting that chemical communication could, indeed, be involved in speciation.

Tilapia male urinary pheromone stimulates female reproductive axis.

Mozambique 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 1h, the release rate of the maturation-inducing steroid 17,20ß-dihydroxypregn-4-en-3-one which is maintained elevated for at least 6h. This indicates a pheromonal role for male urine in the synchronisation of spawning. Furthermore, we show that the lack of affinity of 17,20ßP 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.

Chemical communication in tilapia: a comparison of Oreochromis mossambicus with O. niloticus.

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, 5ß-pregnane-3α,17,20ß-triol-3α-glucuronide (and its α-epimer, 5ß-pregnane-3α,17,20α-triol-3α-glucuronide), in their urine is a potent pheromone, the concentration of which is correlated with social status. The Nile tilapia (Oreochromisniloticus) 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. 5ß-Pregnan-3α,17α,20ß-triol-3α-glucuronide was present at high concentrations (approaching 0.5mM) in urine from Nile tilapia, with 5ß-pregnan-3α,17α,20α-triol-3α-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.

Bile acids as putative social signals in Mozambique tilapia (Oreochromis mossambicus).

Chemical 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 post-spawning 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 post-spawning 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.

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.

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.

Short- and Medium-Term Exposure to Ocean Acidification Reduces Olfactory Sensitivity in Gilthead Seabream.

The effects of ocean acidification on fish are only partially understood. Studies on olfaction are mostly limited to behavioral alterations of coral reef fish; studies on temperate species and/or with economic importance are scarce. The current study evaluated the effects of short- and medium-term exposure to ocean acidification on the olfactory system of gilthead seabream (Sparus aurata), and attempted to explain observed differences in sensitivity by changes in the protonation state of amino acid odorants. Short-term exposure to elevated PCO2 decreased olfactory sensitivity to some odorants, such as L-serine, L-leucine, L-arginine, L-glutamate, and conspecific intestinal fluid, but not to others, such as L-glutamine and conspecific bile fluid. Seabream were unable to compensate for high PCO2 levels in the medium term; after 4 weeks exposure to high PCO2, the olfactory sensitivity remained lower in elevated PCO2 water. The decrease in olfactory sensitivity in high PCO2 water could be partly attributed to changes in the protonation state of the odorants and/or their receptor(s); we illustrate how protonation due to reduced pH causes changes in the charge distribution of odorant molecules, an essential component for ligand-receptor interaction. However, there are other mechanisms involved. At a histological level, the olfactory epithelium contained higher densities of mucus cells in fish kept in high CO2 water, and a shift in pH of the mucus they produced to more neutral. These differences suggest a physiological response of the olfactory epithelium to lower pH and/or high CO2 levels, but an inability to fully counteract the effects of acidification on olfactory sensitivity. Therefore, the current study provides evidence for a direct, medium term, global effect of ocean acidification on olfactory sensitivity in fish, and possibly other marine organisms, and suggests a partial explanatory mechanism.