Chemicals released by predation increase the growth rate of yellow perch, Perca flavescens.

Water-soluble factors associated with walleye Sander vitreus predation on either yellow perch Perca flavescens or fathead minnows Pimephales promelas markedly increased the growth rate of P. flavescens. The findings suggest that P. flavescens possess an inducible growth-promoting mechanism regulated by water-born chemicals. It may be possible to increase the growth rate of farm-raised P. flavescens by manipulating this system.

Production and fate of the sea lamprey migratory pheromone.

Biochemical studies demonstrate that three steroids postulated to function as the sea lamprey migratory pheromone are released in sufficient quantities, and possess adequate stability and binding characteristics, to function as a multi-component pheromone in natural river waters. Mass spectrometric (MS) analyses of the holding water of recently fed larval lamprey demonstrated that each of these compounds is released at rates of 5-25 ng larva(-1) h(-1), adequate to produce picomolar (biologically relevant) concentrations in river waters. Petromyzonamine disulfate (PSDS) was released at about twice the rate of the other two components, petromyzonamine disulfate (PADS) and petromyzonol sulfate (PS). Unfed larvae also released all three steroids but only at about two-thirds the rate of fed larvae and in a different ratio. However, a behavioral test of fed and unfed larval holding waters suggested this change in pheromone ratio does not diminish pheromonal signal function in the winter when larvae are not feeding. A study of steroid degradation found that PADS and PSDS had half-lives of about 3 days, similar to values previously described for PS and sufficiently slow for the entire pheromone to persist in river mouths. Finally, both MS and electro-olfactogram recording found that contrary to previous suggestions, natural levels of natural organic matter found in streams do not bind to these steroids in ways that diminish their natural biological potency. In conclusion, it appears highly likely that a mixture of PADS, PSDS and PS is present at biologically relevant concentrations and ratios in many Great Lakes streams where it functions as a pheromonal attractant.

Sexually mature male goldfish release large quantities of androstenedione into the water where it functions as a pheromone.

Previous studies have demonstrated that ovulatory female goldfish release a variety of sex steroids into the water where they function as a pheromonal blend dominated by C21 steroids that stimulates male hormone release, sperm production and behavior. This study investigated whether male goldfish might also release sex steroids with pheromonal activity. It found that spermiated male goldfish release substantial quantities of androstenedione (AD; about 50 ng/h) together with smaller (10-20 ng/h) quantities of several other related C19 steroids but only very small quantities (<5 ng/h) of C21 steroids. Further, when sexually aroused by females and/or their pheromones, males released even greater quantities of AD (up to 1 microg/h) while C21 steroid release rate changed little. This created a ratio of C19 to C21 steroids of about 50:1 that was dramatically different from that emitted by females (1:7). The male olfactory system was also found to be extremely sensitive to AD, detecting it to near picomolar concentrations. Together with previous studies that have shown water-borne AD to increase male aggressive behavior while suppressing responsiveness to female pheromones, this study establishes AD as a male pheromone in the goldfish. Because ovulating females also release AD but in the presence of C21 steroids, recognition of the male-derived steroid pheromone is presumably mixture dependent.

Evidence that 4-pregnen-17,20beta,21-triol-3-one functions as a maturation-inducing hormone and pheromonal precursor in the percid fish, Gymnocephalus cernuus.

Behavioral, biochemical, and electrophysiological studies suggest that the trihydroxylated progestin steroid, 4-pregnen-17,20beta,21-triol-3-one (20beta-S) stimulates oocyte maturation and pheromone release in the Eurasian ruffe, a freshwater percid fish. Behavioral observations found that female ruffe undergoing oocyte maturation (OM) release a pheromonal cue that stimulates swimming activity and social interactions among conspecific males. Neither vitellogenic nor ovulated females released the cue. Pheromone production was directly associated with elevated plasma levels of 20beta-S in maturing female ruffe which in vitro incubation suggested to be a possible maturation-inducing hormone (MIH) in this species along with 4-pregnen-17,20beta-diol-3-one (17,20betaP). However, neither of these steroids appear to be the pheromone because electrophysiological and behavioral studies found them to lack olfactory (EOG) and behavioral activity. Instead, studies of the odor of steroid-injected fish suggest the pheromone is a metabolite of 20beta-S. In particular, inter-peritoneal injection of 20beta-S (but not 17,20betaP) consistently induced release of a urinary cue with strong behavioral activity. The pheromone may be a highly polar and novel metabolite because it could not be extracted using octadecylsilane resin (C18) which has proven effective for other teleost hormonal pheromones.

Polyamines as olfactory stimuli in the goldfish Carassius auratus.

Electrophysiological responses of goldfish olfactory receptor neurons (ORNs) and goldfish behavioral responses to polyamines were investigated in vivo. Electro-olfactogram (EOG) recordings indicated that polyamines (putrescine, cadaverine and spermine) are potent olfactory stimuli for goldfish with estimated electrophysiological thresholds of 10(-8)-10(-7) mol l(-1), similar to that for L-arginine, the most stimulatory amino acid. Although thresholds were similar, the magnitude of the EOG responses to intermediate (10(-5)-10(-4) mol l(-1)) and high (10(-3) mol l(-1)) concentrations of polyamines dwarfed the responses to amino acids and related single amine containing compounds (amylamine and butylamine). The EOG responses to 0.1 mmol l(-1) putrescine, cadaverine and spermine were, respectively, 4.2x, 4.3x and 10.3x the response of the standard, 0.1 mmol l(-1) L-arginine. Electrophysiological cross-adaptation experiments indicated that polyamine receptor sites are independent from those to L-amino acids (alanine, arginine, glutamate, lysine, methionine and ornithine), bile salts (sodium taurocholate and taurolithocholate), the single amine containing compounds (amylamine and butylamine) and ATP. Further, the cross-adaptation experiments revealed the existence of independent receptor sites for the different polyamines tested. Pharmacological experiments suggested that polyamine odorant transduction does not primarily involve the cyclic AMP and IP(3) second messenger pathways. Behavioral assays indicated that polyamines are attractants that elicit feeding behavior similar to that elicited by L-amino acids.

The three steroidal components of the goldfish preovulatory pheromone signal evoke different behaviors in males.

The goldfish sex pheromone system is the best understood among the teleost fishes. Pheromones in this species are unspecialized hormonal products, which are released in ratios that vary with reproductive status. This study examined behavioral responses of male goldfish to three steroidal components of the female preovulatory pheromone: 17,20beta-dihydroxy-4-pregnen-3-one (1720betaP); 17,20beta-dihydroxy-4-pregnen-3-one-20-sulfate (1720betaP-S); and androstenedione (AD). Males were observed during exposure to nanomolar concentrations of each steroid over a 2-h period. We observed chasing, nudging (courtship behaviors) and pushing (an aggressive behavior). Each steroid elicited a different set of behaviors. 1720betaP, which is released by ovulatory females, elicited a low level of chasing and nudging that persisted throughout the experiment. Exposure to 1720betaP-S, which is released primarily by ovulatory females, triggered a large increase in nudging and chasing that lasted for only 5 min. In contrast, AD, which is released by females early in the ovulatory cycle and by mature males, elicited increases in aggressive behavior. 1720betaP and 1720betaP-S both caused increases in GtH-II release while AD did not. These results demonstrate that goldfish can discriminate components found in the female pheromone blend, suggesting that goldfish, and likely other fish species, may employ blends of hormonal products as pheromones.

Functional identification of a goldfish odorant receptor.

The vertebrate olfactory system utilizes odorant receptors to receive and discriminate thousands of different chemical stimuli. An understanding of how these receptors encode information about an odorant's molecular structure requires a characterization of their ligand specificities. We employed an expression cloning strategy to identify a goldfish odorant receptor that is activated by amino acids-potent odorants for fish. Structure-activity analysis indicates that the receptor is preferentially tuned to recognize basic amino acids. The receptor is a member of a multigene family of G protein-coupled receptors, sharing sequence similarities with the calcium sensing, metabotropic glutamate, and V2R class of vomeronasal receptors. The ligand tuning properties of the goldfish amino acid odorant receptor provide information for unraveling the molecular mechanisms underlying olfactory coding.

Ultrastructure of the olfactory epithelium in intact, axotomized, and bulbectomized goldfish, Carassius auratus.

The ultrastructure of the olfactory epithelium in intact, axotomized, and bulbectomized goldfish was studied by scanning and transmission electron microscopy. A total of 58 adult goldfish of various survival times were examined to determine whether the different types of surgery--either olfactory nerve transection or bulbectomy--yielded differences in the extent or time course of cellular degeneration and renewal. Control animals were also examined in detail to elucidate previous controversial findings concerning the types of olfactory receptor neurons present in goldfish. We found that the intact olfactory epithelium of unoperated control goldfish contains the previously observed ciliated and microvillous receptor neurons, and the crypt cell, a cell type not yet seen in the goldfish but recently reported in other species of teleosts. Following either olfactory nerve transection or bulbectomy, the olfactory receptor neurons showed similar signs of degeneration and subsequent cell death, but, surprisingly, the thickness of the olfactory epithelium did not change significantly with either treatment. The time course of receptor cell renewal was different in axotomized and bulbectomized goldfish. In axotomized goldfish, the amount of receptor cells decreased continuously until 8-13 days after surgery, followed by rapid cell renewal. For bulbectomized goldfish, cell replacement began almost immediately after surgery, with degeneration and cell renewal occurring simultaneously. Six weeks after bulbectomy, cell death and cell proliferation reached a "steady state," and the epithelia did not further improve.

Discrimination of pheromonal cues in fish: emerging parallels with insects.

Many fish species employ hormonal products as sex pheromones, and these cues are often mixtures that are released with a temporal pattern. This behavior is strikingly similar to that of insects, as moths use precise blends of odorants as sex pheromones and are skillful at tracking them in spite of changes in odor intensity associated with aerial dispersal. New studies in both groups of animals suggest many parallels in the functional anatomy of olfactory pathways and the organization of information-coding circuits.

Sex pheromones and amino acids evoke distinctly different spatial patterns of electrical activity in the goldfish olfactory bulb.

Until now, electrophysiological studies of the vertebrate olfactory bulb have tested only 'generalist' cues. These studies suggest that odorants are discriminated by a broadly distributed spatial map. In this study, we tested for the first time in a vertebrate the hypothesis that 'specialist' cues (pheromones) are discriminated by a more restricted component of the olfactory bulb. Our model is the male goldfish, Carassius auratus, for which five sex pheromones with both behavioral and physiological activity have now been identified. Electrical activity (electroencephalography: EEG) was recorded over a 12-point grid from the surface of the olfactory bulb, while fish were exposed to one of ten stimuli including: five sex pheromones, two amino acids, two bile steroids and a control. Evoked activity was evaluated by time series analysis. Power ratios were calculated by dividing the power of the dominant frequency in the power spectrum before stimulation by the power of the dominant frequency during stimulation. Next, the average magnitudes of odorant responses were compared using analysis of variance (ANOVA). The spatial patterning of these responses was also described using cluster analysis, which grouped odorants based on the similarity of their spatial patterns of activity. Although all odorants elicited EEG responses with similar dominant frequencies, odorant-specific differences were evident in the size and distribution of these responses. Sex pheromones and bile steroids elicited relatively small responses that were spatially restricted in distinctive manners, although some overlap was evident. In contrast, amino acids consistently produced large responses at all positions. These results are consistent with the hypothesis that vertebrate pheromones are discriminated by a distinctive subcomponent of the vertebrate olfactory system comprised of a relatively small number of olfactory neurons.

Pheromones as tools for olfactory research.Introduction.

We have long been fascinated by the unique ability of odors to stir our emotions and to evoke long-forgotten memories, but certain odors play a much more fundamental role in that they vastly improve an organism's chances for reproductive success and survival. These odorants are called pheromones, a term commonly applied to semiochemicals that are released by one member of a species and evoke a specific reaction or reactions from members of the same species. Pheromones are known for both the specificity and the potency of their actions, which can be behavioral and/or neuroendocrinological. Pheromones can stimulate individuals to aggregate, to disperse, or to react defensively in the presence of a predator, but they are probably best known for bringing the sexes together. Some pheromones have also been found to trigger a dramatic release of pituitary hormones in several vertebrate species. Although first identified in insects, more recent studies show that sex pheromones influence the lives of a wide range of organisms, from microbes to man. The hormonally-derived sex pheromones in teleost fish, and the airborne pheromones of moths are two systems that illustrate how scientists have used these specialized chemical signals as important tools to investigate the morphology, physiology and biochemistry of olfactory-receptor systems, the mechanisms of odor-information processing in the brain, and the diverse range of behaviors and endocrinological changes associated with pheromonal communication. While our focus is on these two animal models, other examples, including mammalian pheromone systems, are also discussed.

Biological responsiveness to pheromones provides fundamental and unique insight into olfactory function.

When exposed to the odor of conspecifics, most organisms exhibit an adaptive behavioral response, particularly if the individuals are sexually mature. Evidence increasingly suggests that behavioral responsiveness to these odors, which are termed 'pheromones', reflects neuroethological mechanisms associated with olfactory function. Reproductive pheromones, which are the best understood, are commonly used by both invertebrates and vertebrates. In both instances they are generally comprised of mixtures of compounds and behavioral responsiveness to them is largely instinctual, sexually-dimorphic, and attributable to a specialized component(s) of the olfactory system. While pheromonal responsiveness in some systems (e.g. moths) appears highly stereotypic and symptomatic of a relatively simple 'labeled line', behavioral responsiveness of other animals (e.g. rodents) can be modified by experience, suggesting a more complex underlying central mechanism. In any case, our understanding of these fascinating systems is progressing only because of an active dialogue between behavioral and neurological investigations. This review briefly examines how behavioral studies have provided fundamental insight into the neuroethology of olfactory function by drawing comparisons between some of the better understood sex pheromone systems which have been described in heliothine moths, the goldfish, and the pig. Many similarities between invertebrate and vertebrate pheromone systems are noted.

Effect of dominance status on sex hormone levels in laboratory and wild-spawning male trout.

We investigated the relationship between male social status and hormone levels in salmonids spawning under laboratory and field conditions. In small groups of rainbow trout (Onchorhynchus mykiss) spawning in the laboratory, dominant males had higher plasma levels of testosterone (T) and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) compared with subordinates. Steroid levels increased in subordinate males that became dominant after dominant males were experimentally removed; higher steroid levels in dominant males appears to be a result rather than a cause of their social status. In free-ranging brown trout (Salmo trutta) sampled in the field, we found higher levels of 11-ketotestosterone (11KT) but not T in dominant males. No significant differences in levels of either androgen were found between dominant and subordinate male brook trout (Salvelinus fontinalis) sampled at the same field location. Furthermore, in marked contrast with the laboratory fish, there were no significant differences in plasma 17,20 beta-P between dominant and subordinate males in either species of fish in the wild. The different findings in the laboratory and field may indicate species differences in behavioral endocrinology among brook, brown, and rainbow trout. Alternatively, the greater differential in hormonal profile of dominant and subordinate males in the laboratory may reflect the relative uniformity of the laboratory environment; this simple environment may allow competitively superior males to more completely dominate less competitive tank-mates and to exclude them from female sexual cues. In any case, these results suggest that the relationship between steroid hormones and spawning behavior in male salmonids is likely more complex than suggested by experiments conducted solely on laboratory-held rainbow trout.

Sulfated 17,20 beta-dihydroxy-4-pregnen-3-one functions as a potent and specific olfactory stimulant with pheromonal actions in the goldfish.

This study demonstrates that in addition to using the maturational steroid hormone 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) as a potent sex pheromone, the goldfish uses its sulfated metabolite 17,20 beta-dihydroxy-4-pregnen-3-one 20-sulfate (17,20 beta-P-20S). As measured by electro-olfactogram recording (EOG), the goldfish olfactory epithelium is extremely sensitive to both free and sulfated 17,20 beta-P (detection thresholds of approximately 10(-12) and 10(-11) M) but not to glucuronidated or phosphated 17,20 beta-P. Furthermore, both structure-activity and cross-adaptation studies using EOG indicated that 17,20 beta-P-20S and 17,20 beta-P are detected by different olfactory receptor sites, suggesting that these cues function as a distinguishable mixture. Finally, although the pheromonal activity of 17,20 beta-P-20S appears to be slightly less than that of 17,20 beta-P, it too stimulates gonadotropin release and sperm production in male goldfish. The precise function of 17,20 beta-P-20S remains somewhat enigmatic, however, because it does not appear to enhance the actions of 17,20 beta-P and is released by a different mechanism. In any case, our findings clearly establish that a fish olfactory system can detect particular conjugated steroidal compounds in a highly specific manner and that naturally released conjugated steroids can function as components of a pheromonal mixture.

The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae.

Larval sea lamprey inhabit freshwater streams and migrate to oceans or lakes to feed after a radical metamorphosis; subsequently, mature adults return to streams to spawn. Previous observations suggested that lamprey utilize the odor of conspecific larvae to select streams for spawning. Here we report biochemical and electrophysiological evidence that this odor is comprised of two unique bile acids released by larvae. High performance liquid chromatography and mass spectrometry demonstrated that larval sea lamprey produce and release two unique bile acids, allocholic acid (ACA) and petromyzonol sulfate (PS). Electro-olfactogram (EOG) recording also demonstrated that the olfactory system of migratory adult sea lamprey is acutely and specifically sensitive to ACA and PS; detection thresholds for these compounds were approximately 10(-12) M. ACA and PS were the most potent of 38 bile acids tested and cross-adaptation experiments suggested that adult sea lamprey have specific olfactory receptor sites associated with independent signal transduction pathways for these bile acids. These receptor sites specifically recognize the key substituents of ACA and PS such as a 5 alpha-hydrogen, three axial hydroxyls, and a C-24 sulfate ester or carboxyl. In conclusion, the unique lamprey bile acids, ACA and PS, are potent and specific stimulants of the adult olfactory system, strongly supporting the hypothesis that these unique bile acids function as migratory pheromones in lamprey.

Time course of release of pheromonally active gonadal steroids and their conjugates by ovulatory goldfish.

Previous studies have demonstrated that ovulatory goldfish synthesize and release a variety of steroids into the water, where some of them function as sex pheromones. Among the steroids which have been measured are free androstenedione, testosterone, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (17,20 beta,21-P), 17 alpha-hydroxy-4-pregnene-3,20-dione (17-P), 11-deoxycortisol (17,21-P), and 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P), glucuronidated testosterone, 17,20 beta-P, and 17,20 beta,21-P, and sulfated 17,21-P, 17,20 beta-P, and 17,20 beta,21-P. This study reports the changes in the amounts of these steroids released into the water, at 3-hr intervals, by 12 female goldfish injected with human chorionic gonadotrophin (HCG). Eight fish ovulated. The amounts of steroids released into the water were significantly higher in the fish which ovulated than in those which did not. The peak rate of release (ng per hour) of free androstenedione (91), glucuronidated testosterone (143), and sulfated 17,21-P (33) occurred at ca. 4 hr postinjection; free 17-P (163) and free 17,21-P (217) at ca. 6 hr.; free 17,20 beta-P (58) and free 17,20 beta,21-P (58) at ca. 7.5 hr; and glucuronidated 17,20 beta-P (96), glucuronidated 17,20 beta,21-P (264), sulfated 17,20 beta-P (64) and sulfated 17,20 beta,21-P (153) at ca. 9 hr. Amounts of all of the steroids were at their lowest between 12 and 15 hr and, in the ovulated fish, had risen significantly again between 18 and 21 hr. The amounts of glucuronidated testosterone, 17,20 beta-P and 17,20 beta,21-P, and of sulfated 17,20 beta-P and 17,20 beta,21-P, in water exceeded those of free steroid. Elevated levels of free and glucuronidated steroids were also found in the blood plasma of HCG-injected fish. Considerably more androstenedione than testosterone was released into the water. Assessment of the "pheromonal effectiveness" of each steroid indicated that the free and sulfated forms of 17,20 beta-P and 17,20 beta,21-P, and androstenedione were the most important pheromonal steroids released by female goldfish.

The evolution of hormonal sex pheromones in teleost fish: poor correlation between the pattern of steroid release by goldfish and olfactory sensitivity suggests that these cues evolved as a result of chemical spying rather than signal specialization.

It is becoming increasingly evident that many teleost fish use hormones and their metabolites as sex pheromones. Although hormonal pheromone systems of several species of fish have now been characterized, that of the goldfish is the best understood. Reproductively-active female goldfish sequentially release derivatives of three steroidal hormones and two prostaglandins which have specific and potent actions on the goldfish olfactory sense and subsequently conspecific reproductive behaviour and physiology. Three goldfish hormonal pheromones are unmodified sex hormones which are also found in the blood of many other species of fish and are therefore unlikely to be species-specific. This scenario evokes two related questions. First, how did these systems evolve? Second, do hormonal pheromones represent specialized signals used for communication or rather simple metabolites which fish have evolved the ability to detect because of their intrinsic meaning ('spying')? Here we review hormonal pheromone function in fish and then describe a study of the goldfish which tests whether hormonal pheromones are specialized communicatory signals by comparing the profile of steroids released by goldfish with their olfactory responsiveness to these compounds. Little correlation between signal production and detection was found and we conclude that the goldfish hormonal pheromone system most likely exemplifies spying.

Pheromonal and reproductive function of F prostaglandins and their metabolites in teleost fish.

Although the function of prostaglandins in fish reproduction has not been well studied, it is becoming increasingly clear that prostaglandin F2 alpha or a compound closely resembling it serves three critical roles mediating reproductive activities in teleost fish. First, it appears to play a paracrine role in the ovary stimulating and/or modulating follicular rupture. Second, circulating levels of F prostaglandins rise at the time of ovulation and travel to the brain where they elicit female sexual behavior. Third, recent studies indicate that F prostaglandin is metabolized and released to the water where it functions as a sex pheromone stimulating male sexual behavior. Although these roles have been best characterized in the goldfish, ongoing studies indicate that metabolites of prostaglandin F2 alpha may commonly function as pheromones in many fish. Many questions remain about the identity(ies), origins, and species-specificity of the prostaglandin pheromone.

Hormonally Derived Sex Pheromones in Goldfish: A Model for Understanding the Evolution of Sex Pheromone Systems in Fish.

It is now well established that female goldfish release unmodified and metabolized sex hormones to the water and that some of these compounds function as potent sex pheromones detected by the male's olfactory sense. In goldfish, both olfactory pheromonal receptors and their corresponding hormonal receptors appear to be transmembrane-domain receptors coupled with G proteins. Recent studies of other teleost fish indicate that fish commonly use `hormonal-pheromones.' Taken together, these data suggest that fish pheromone systems may have evolved as a consequence of a chance expression of hormone receptor molecules on olfactory receptor cells. Isolation and identification of olfactory and hormonal receptors may be the next step in resolving this question.

Sex pheromones selectively stimulate the medial olfactory tracts of male goldfish.

The olfactory tracts of teleost fish are comprised of medial and lateral sub-tracts which previous studies suggest convey responses to pheromones and food odors respectively. This study tested this possibility by recording electrical responses from the medial and lateral tracts of male goldfish exposed to sex pheromones and food odors. Only the medial olfactory tract responded to pheromones and both tracts responded to an L-amino acid and crude food odor. These findings verify earlier studies of peripheral olfactory sensitivity to pheromones and confirm that pheromonal information is carried within the medial tracts. They also suggest that the neural processes responsible for food recognition are more complex than previously supposed.