Peter C. Hubbard

Non-invasive measurement of steroids in fish-holding water: important considerations when applying the procedure to behaviour studies

Fish behaviourists are increasingly turning to non-invasive measurement of steroid hormones in holding water, as opposed to blood plasma. When some of us met at a workshop in Faro, Portugal, in September, 2007, we realised that there were still many issues concerning the application of this procedure that needed resolution, including: Why do we measure release rates rather than just concentrations of steroids in the water? How does one interpret steroid release rates when dealing with fish of different sizes? What are the merits of measuring conjugated as well as free steroids in water? In the ‘static’ sampling procedure, where fish are placed in a separate container for a short period of time, does this affect steroid release—and, if so, how can it be minimised? After exposing a fish to a behavioural stimulus, when is the optimal time to sample? What is the minimum amount of validation when applying the procedure to a new species? The purpose of this review is to attempt to answer these questions and, in doing so, to emphasize that application of the non-invasive procedure requires more planning and validation than conventional plasma sampling. However, we consider that the rewards justify the extra effort.

Olfactory discrimination of female reproductive status by male tilapia(Oreochromis mossambicus)

SUMMARY 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.

A Sterol-Like Odorant in the Urine of Mozambique Tilapia Males Likely Signals Social Dominance to Females

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.

Possible disruption of pheromonal communication by humic acid in the goldfish, Carassius auratus.

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 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (1720 beta-P), its sulphated conjugate (1720 beta-P-SO(4)) and prostaglandin F(2alpha) (PGF(2alpha)), all at 10(-11) to 10(-8) M, in the absence and presence of humic acids (1-1000 m x gl(-1)). At nearly all concentrations of humic acid tested, there was a significant attenuation of the amplitude of the initial (phasic) response to 1720 beta-P compared to 1720 beta-P alone. At higher concentrations of humic acid, the EOG response to 1720 beta-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 1720 beta-P per se. Furthermore, simultaneous recording of electro-encephalograms from the olfactory bulb demonstrated that the nervous activity evoked by the same concentration of 1720 beta-P was less intense in the presence of humic acid than its absence. PGF(2alpha) is non-steroidal and much more soluble in water. In contrast to 1720 beta-P, only the higher concentrations of humic acid (100 and 1000 mg x l(-1)) significantly diminished the EOG amplitude. 1720 beta-P-SO(4) 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 1720 beta-P-SO(4) 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 1720 beta-P and 1720 beta-P-SO(4) 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.

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.

Identity of a Tilapia Pheromone Released by Dominant Males that Primes Females for Reproduction

Knowledge of the chemical identity and role of urinary pheromones in fish is scarce, yet it is necessary in order to understand the integration of multiple senses in adaptive responses and the evolution of chemical communication [1]. In nature, Mozambique tilapia (Oreochromis mossambicus) males form hierarchies, and females mate preferentially with dominant territorial males, which they visit in aggregations or leks [2]. Dominant males have thicker urinary bladder muscular walls than subordinates or females and store large volumes of urine, which they release at increased frequency in the presence of subordinate males or preovulatory, but not postspawned, females [3-5]. Females exposed to dominant-male urine augment their release of the oocyte maturation-inducing steroid 17α,20β-dihydroxypregn-4-en-3-one (17,20β-P) [6]. Here we isolate and identify a male Mozambique tilapia urinary sex pheromone as two epimeric (20α- and 20β-) pregnanetriol 3-glucuronates. We show that both males and females have high olfactory sensitivity to the two steroids, which cross-adapt upon stimulation. Females exposed to both steroids show a rapid, 10-fold increase in production of 17,20β-P. Thus, the identified urinary steroids prime the female endocrine system to accelerate oocyte maturation and possibly promote spawning synchrony. Tilapia are globally important as a food source but are also invasive species, with devastating impact on local freshwater ecosystems [7, 8]. Identifying the chemical cues that mediate reproduction may lead to the development of tools for population control [9-11].

Evidence for functional asymmetry in the olfactory system of the senegalese sole ( solea senegalensis )

The two olfactory epithelia of flatfish of the family Soleidae are essentially in contact with two distinct environments; the upper (right) side samples open water while the lower (left) side samples interstitial water. This study assessed whether there are differences in the responsiveness of the two epithelia by use of the electro‐olfactogram in the Senegalese sole (Solea senegalensis). The upper epithelium was significantly more responsive to the basic amino acids (l‐lysine and l‐arginine), glycine, and l‐threonine than the lower epithelium. The lower epithelium was significantly more responsive to aromatic amino acids (l‐tryptophan, l‐tyrosine, l‐DOPA, and l‐phenylalanine), l‐leucine, and l‐asparagine than the upper. Both epithelia had similar responsiveness to the sulphur‐containing amino acids (l‐cysteine and l‐methionine), l‐alanine, l‐serine, and l‐glutamine. Neither side was responsive to the acidic amino acids (l‐aspartate and l‐glutamate) or the D‐isomers of any amino acid tested. The upper olfactory organ was much more responsive to conspecific‐derived stimuli (bile and intestinal fluid) than the lower organ. We suggest that these differences in responsiveness may be related to different functional roles of the upper and lower epithelia in feeding and chemical communication.

Effect of acute copper sulfate exposure on olfactory responses to amino acids and pheromones in goldfish (Carassius auratus).

Exposure of olfactory epithelium to environmentally relevant concentrations of copper disrupts olfaction in fish. To examine the dynamics of recovery at both functional and morphological levels after acute copper exposure, unilateral exposure of goldfish olfactory epithelia to 100 microM CuSO(4) (10 min) was followed by electro-olfactogram (EOG) recording and scanning electron microscopy. Sensitivity to amino acids (l-arginine and l-serine), generally considered food-related odorants, recovered most rapidly (three days), followed by that to catecholamines (3-O-methoxytyramine), bile acids (taurolithocholic acid) and the steroid pheromone, 17,20beta-dihydroxy-4-pregnen-3-one 20-sulfate, which took 28 days to reach full recovery. Sensitivity to the postovulatory pheromone prostaglandin F(2alpha) had not fully recovered even at 28 days. These changes in sensitivity were correlated with changes in the recovery of ciliated and microvillous receptor cell types. Microvillous cells appeared largely unaffected by CuSO(4) treatment. Cilia in ciliated receptor neurones, however, appeared damaged one day post-treatment and were virtually absent after three days but had begun to recover after 14 days. Together, these results support the hypothesis that microvillous receptor neurones detect amino acids whereas ciliated receptor neurones were not functional and are responsible for detection of social stimuli (bile acids and pheromones). Furthermore, differences in sensitivity to copper may be due to different transduction pathways in the different cell types.

Olfactory sensitivity of the gilthead seabream (Sparus auratus L) to conspecific body fluids.

The potential for intraspecific chemical communication in the gilthead seabream (a marine perciform) was investigated by assessing the olfactory sensitivity to conspecific body-fluids (water occupied by conspecifics, intestinal fluid, urine, semen, egg fluid) by multiunit electrophysiological recording from the olfactory nerve. The olfactory system was responsive to water previously occupied by conspecifics, and the active compound(s) could be extracted by solid-phase extraction. The olfactory system was extremely sensitive to body fluids of sexually mature conspecifics: thresholds of detection were 1:107.4 (intestinal fluid), 1:106.1 (gametes), and 1:104.2 (urine). The olfactory system was also sensitive to amino acids with thresholds of detection from 10−8.1 M (l-leucine) to 10−6.1 M (l-phenylalanine). However, a range of other known fish odorants (steroids, bile acids, and prostaglandins) failed to evoke significant responses. Given the high olfactory sensitivity to intestinal fluid and the low urine release rates of marine compared with freshwater fish, we suggest that chemical communication is likely to be mediated via compounds present in the intestinal fluid rather than urine. Furthermore, the types of chemicals involved are likely to be different from those of freshwater fish. Their exact chemical identity and biological roles remain to be established.

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.