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.

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.

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.

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.

Quantifying the Surface Strain Field Induced by Active Sources with Distributed Acoustic Sensing: Theory and Practice.

Quantitative dynamic strain measurements of the ground would be useful for engineering scale problems such as monitoring for natural hazards, soil-structure interaction studies, and non-invasive site investigation using full waveform inversion (FWI). Distributed acoustic sensing (DAS), a promising technology for these purposes, needs to be better understood in terms of its directional sensitivity, spatial position, and amplitude for application to engineering-scale problems. This study investigates whether the physical measurements made using DAS are consistent with the theoretical transfer function, reception patterns, and experimental measurements of ground strain made by geophones. Results show that DAS and geophone measurements are consistent in both phase and amplitude for broadband (10 s of Hz), high amplitude (10 s of microstrain), and complex wavefields originating from different positions around the array when: (1) the DAS channels and geophone locations are properly aligned, (2) the DAS cable provides good deformation coupling to the internal optical fiber, (3) the cable is coupled to the ground through direct burial and compaction, and (4) laser frequency drift is mitigated in the DAS measurements. The transfer function of DAS arrays is presented considering the gauge length, pulse shape, and cable design. The theoretical relationship between DAS-measured and pointwise strain for vertical and horizontal active sources is introduced using 3D elastic finite-difference simulations. The implications of using DAS strain measurements are discussed including directionality and magnitude differences between the actual and DAS-measured strain fields. Estimating measurement quality based on the wavelength-to-gauge length ratio for field data is demonstrated. A method for spatially aligning the DAS channels with the geophone locations at tolerances less than the spatial resolution of a DAS system is proposed.

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.

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.

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.

Physiology, endocrinology and chemical communication in aggressive behaviour of fishes.

Fishes show remarkably diverse aggressive behaviour. Aggression is expressed to secure resources; adjusting aggression levels according to context is key to avoid negative consequences for fitness and survival. Nonetheless, despite its importance, the physiological basis of aggression in fishes is still poorly understood. Several reports suggest hormonal modulation of aggression, particularly by androgens, but contradictory studies have been published. Studies exploring the role of chemical communication in aggressive behaviour are also scant, and the pheromones involved remain to be unequivocally characterized. This is surprising as chemical communication is the most ancient form of information exchange and plays a variety of other roles in fishes. Furthermore, the study of chemical communication and aggression is relevant at the evolutionary, ecological and economic levels. A few pioneering studies support the hypothesis that aggressive behaviour, at least in some teleosts, is modulated by "dominance pheromones" that reflect the social status of the sender, but there is little information on the identity of the compounds involved. This review aims to provide a global view of aggressive behaviour in fishes and its underlying physiological mechanisms including the involvement of chemical communication, and discusses the potential use of dominance pheromones to improve fish welfare. Methodological considerations and future research directions are also outlined.

Extracellular Multi-Unit Recording from the Olfactory Nerve of Teleosts.

Recent studies have shown that ocean acidification affects olfactory-driven behavior in fish. This may be due in part to a reduction in olfactory sensitivity in high PCO2/low pH water. To assess the effects of ocean acidification, or olfactory sensitivity in marine fish in general, we propose that extracellular multi-unit recording from the olfactory nerve is the method of choice. Although invasive, it is sensitive, robust, reproducible and independent of external salinity (unlike the electro-olfactogram [EOG], for example). Furthermore, it records a primary sensory input into the CNS, prior to any central processing. We show that this method can show a reduction in olfactory sensitivity that is both temporary and odorant-dependent, using a range of amino acids to construct concentration-response curves and calculate the thresholds of 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.

Chemicals released by male sea cucumber mediate aggregation and spawning behaviours.

The importance of chemical communication in reproduction has been demonstrated in many marine broadcast spawners. However, little is known about the use of chemical communication by echinoderms, the nature of the compounds involved and their mechanism(s) of action. Here, the hypothesis that the sea cucumber Holothuria arguinensis uses chemical communication for aggregation and spawning was tested. Water conditioned by males, but not females, attracted both males and females; gonad homogenates and coelomic fluid had no effect on attraction. Male spawning water, but not female spawning water, stimulated males and females to release their gametes; the spermatozoa alone did not induce spawning. H. arguinensis male spawning water also induced spawning in the phylogenetically related H. mammata. This indicates that males release pheromones together with their gametes that induce spawning in conspecifics and possibly sympatric species. Finally, the male pheromone seems to be a mixture with at least one labile compound (biological activity is lost after four hours at ambient temperature) possibly including phosphatidylcholines. The identification of pheromones in sea cucumbers offers a new ecological perspective and may have practical applications for their aquaculture.

Chemical diplomacy in male tilapia: urinary signal increases sex hormone and decreases aggression.

Androgens, 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.

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.

'Smelling' the cerebrospinal fluid: olfactory signaling molecules are expressed in and mediate chemosensory signaling from the choroid plexus.

The olfactory-type signaling machinery has been known to be involved not only in odorant detection but also in other tissues with unsuspected sensory roles. As a barrier, the choroid plexus (CP) is an active participant in the monitoring of the cerebrospinal fluid (CSF), promptly responding to alterations in its composition. We hypothesized that olfactory signaling could be active in CP, contributing to the surveillance of the CSF composition. We determined the mRNA and protein expression of the major components of the olfactory transduction pathway in the rat CP, including odorant receptors, the olfactory G-protein (Gαolf), adenylate cyclase 3 and cyclic nucleotide-gated channel 2. The functionality of the transduction pathway and the intracellular mechanisms involved were analyzed by DC field potential recording electrophysiological analysis, in an ex vivo CP-brain setup, using polyamines as stimuli and blockers of the downstream signaling pathways. Concentration-dependent responses were obtained for the polyamines studied (cadaverine, putrescine, spermine and spermidine), all known to be present in the CSF. Transfection of a CP epithelial cell line with siRNA against Gαolf effectively knocked down protein expression and reduced the CP cells' response to spermine. Thus, the key components of the olfactory chemosensory apparatus are present and are functional in murine CP, and polyamines seem to trigger both the cAMP and the phospholipase C-inositol 1,4,5-trisphosphate pathways. Olfactory-like chemosensory signaling may be an essential component of the CP chemical surveillance apparatus to detect alterations in the CSF composition, and to elicit responses to modulate and maintain brain homeostasis.

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.

Transcriptomic profiling of male European eel (Anguilla anguilla) livers at sexual maturity.

The European eel Anguilla anguilla has a complex life cycle that includes freshwater, seawater and morphologically distinct stages as well as two extreme long distance migrations. Eels do not feed as they migrate across the Atlantic to the Sargasso Sea but nevertheless reach sexual maturity before spawning. It is not yet clear how existing energy stores are used to reach the appropriate developmental state for reproduction. Since the liver is involved in energy metabolism, protein biosynthesis and endocrine regulation it is expected to play a key role in the regulation of reproductive development. We therefore used microarrays to identify genes that may be involved in this process. Using this approach, we identified 231 genes that were expressed at higher and 111 genes that were expressed at lower levels in sexually mature compared with immature males. The up-regulated set includes genes involved in lipid metabolism, fatty acid synthesis and transport, mitochondrial function, steroid transport and bile acid metabolism. Several genes with putative enzyme functions were also expressed at higher levels at sexual maturity while genes involved in immune system processes and protein biosynthesis tended to be down-regulated at this stage. By using a high-throughput approach, we have identified a subset of genes that may be linked with the mobilization of energy stores for sexual maturation and migration. These results contribute to an improved understanding of eel reproductive biology and provide insight into the role of the liver in other teleosts with a long distance spawning migrations.