Sensory trap leads to reliable communication without a shift in nonsexual responses to the model cue.

The sensory trap model of signal evolution suggests that males manipulate females into mating using traits that mimic cues used in a nonsexual context. Despite much empirical support for sensory traps, little is known about how females evolve in response to these deceptive signals. Female sea lamprey (Petromyzon marinus) evolved to discriminate a male sex pheromone from the larval odor it mimics and orient only toward males during mate search. Larvae and males release the attractant 3-keto petromyzonol sulfate (3kPZS), but spawning females avoid larval odor using the pheromone antagonist, petromyzonol sulfate (PZS), which larvae but not males, release at higher rates than 3kPZS. We tested the hypothesis that migratory females also discriminate between larval odor and the male pheromone and orient only to larval odor during anadromous migration, when they navigate within spawning streams using larval odor before they begin mate search. In-stream behavioral assays revealed that, unlike spawning females, migratory females do not discriminate between mixtures of 3kPZS and PZS applied at ratios typical of larval versus male odorants. Our results indicate females discriminate between the sexual and nonsexual sources of 3kPZS during but not outside of mating and show sensory traps can lead to reliable sexual communication without females shifting their responses in the original context.

Male lake char release taurocholic acid as part of a mating pheromone.

The evolutionary origins of sexual preferences for chemical signals remain poorly understood, due, in part, to scant information on the molecules involved. In the current study, we identified a male pheromone in lake char (Salvelinus namaycush) to evaluate the hypothesis that it exploits a non-sexual preference for juvenile odour. In anadromous char species, the odour of stream-resident juveniles guides migratory adults into spawning streams. Lake char are also attracted to juvenile odour but have lost the anadromous phenotype and spawn on nearshore reefs, where juvenile odour does not persist long enough to act as a cue for spawning site selection by adults. Previous behavioural data raised the possibility that males release a pheromone that includes components of juvenile odour. Using metabolomics, we found that the most abundant molecule released by males was also released by juveniles but not females. Tandem mass spectrometry and nuclear magnetic resonance were used to identify the molecule as taurocholic acid (TCA), which was previously implicated as a component of juvenile odour. Additional chemical analyses revealed that males release TCA at high rates via their urine during the spawning season. Finally, picomolar concentrations of TCA attracted pre-spawning and spawning females but not males. Taken together, our results indicate that male lake char release TCA as a mating pheromone and support the hypothesis that the pheromone is a partial match of juvenile odour.

Chemical communication and its role in sexual selection across Animalia.

Sexual selection has been studied as a major evolutionary driver of animal diversity for roughly 50 years. Much evidence indicates that competition for mates favors elaborate signaling traits. However, this evidence comes primarily from a few taxa, leaving sexual selection as a salient evolutionary force across Animalia largely untested. Here, we reviewed the evidence for sexual selection on communication across all animal phyla, classes, and orders with emphasis on chemoreception, the only sense shared across lifeforms. An exhaustive literature review documented evidence for sexual selection on chemosensory traits in 10 of 34 animal phyla and indications of sexual selection on chemosensory traits in an additional 13 phyla. Potential targets of sexual selection include structures and processes involved in production, delivery, and detection of chemical signals. Our review suggests sexual selection plays a widespread role in the evolution of communication and highlights the need for research that better reflects animal diversity.

Behavioral evidence of olfactory imprinting during embryonic and larval stages in lake sturgeon.

Many migratory fishes are thought to navigate to natal streams using olfactory cues learned during early life stages. However, direct evidence for early-life olfactory imprinting is largely limited to Pacific salmon, and other species suspected to imprint show life history traits and reproductive strategies that raise uncertainty about the generality of the salmonid-based conceptual model of olfactory imprinting in fishes. Here, we studied early-life olfactory imprinting in lake sturgeon (Acipenser fulvescens), which have a life cycle notably different from Pacific salmon, but are nonetheless hypothesized to home via similar mechanisms. We tested one critical prediction of the hypothesis that early-life olfactory imprinting guides natal homing in lake sturgeon: that exposure to odorants during early-life stages results in increased activity when exposed to those odorants later in life. Lake sturgeon were exposed to artificial odorants (phenethyl alcohol and morpholine) during specific developmental windows and durations (limited to the egg, free-embryo, exogenous feeding larvae and juvenile stages), and later tested as juveniles for behavioral responses to the odorants that were demonstrative of olfactory memory. Experiments revealed that lake sturgeon reared in stream water mixed with artificial odorants for as little as 7 days responded to the odorants in behavioral assays over 50 days after the initial exposure, specifically implicating the free-embryo and larval stages as critical imprinting periods. Our study provides evidence for olfactory imprinting in a non-salmonid fish species, and supports further consideration of conservation tactics such as stream-side rearing facilities that are designed to encourage olfactory imprinting to targeted streams during early life stages. Continued research on lake sturgeon can contribute to a model of olfactory imprinting that is more generalizable across diverse fish species and will inform conservation actions for one of the world's most imperiled fish taxonomic groups.

Determination of thyroid hormones in lake sturgeon (Acipenser fulvescens) tissues at different developmental stages using a sensitive liquid chromatography-mass spectrometry method.

Thyroid hormones (TH) are known to play an important role in the growth and development of vertebrates. In fish species, TH regulates the larval-juvenile metamorphosis, and is crucial for development during early life stages. Monitoring the variations in TH levels at different life stages can provide insights into the regulation of metamorphosis and fish development. In this study, we developed an extremely sensitive method for the quantification of thyroxine (T4), triiodothyronine (T3), and reverse-triiodothyronine (rT3), in lake sturgeon (Acipenser fulvescens) tissues from eggs, free embryos, larvae, and juveniles. The target compounds were extracted by an enzymatic digestion method, followed by protein precipitation. Further cleanup was performed by liquid-liquid extraction (LLE) and solid phase extraction (SPE) using SampliQ OPT cartridges. The liquid-chromatography tandem mass spectrometry (LC-MS/MS) method used to quantify TH compounds showed remarkably high sensitivity with the limit of detection (LOD) and the limit of quantification (LOQ) ranging from < 1 pg/mL to 10 pg/mL and linearity in the range of 10-50,000 pg/mL. This method was validated for tissue samples across several early developmental stages and was checked for intra- and inter-day accuracy (78.3-111.2 %) and precision (0.1-4.9 %), matrix effect (75.4-134.1 %), and recovery (41.2-69.0 %). The method was successfully applied for the quantification and comparison of T4, T3 and rT3 in hatchery raised lake sturgeon samples collected at unique time points (i.e., days post fertilization dpf) including fertilized eggs (11 dpf), free embryos (14 dpf), larvae (22 dpf), juveniles (40 dpf) and older juveniles (74 dpf). With modifications, this method could be applied to other species important for agriculture or conservation.

Spatiotemporal segregation by migratory phenotype indicates potential for assortative mating in lake sturgeon.

Migratory diversity can promote population differentiation if sympatric phenotypes become temporally, spatially, or behaviorally segregated during breeding. In this study, the potential for spatiotemporal segregation was tested among three migratory phenotypes of lake sturgeon (Acipenser fulvescens) that spawn in the St. Clair River of North America's Laurentian Great Lakes but differ in how often they migrate into the river and in which direction they move after spawning. Acoustic telemetry over 9 years monitored use of two major spawning sites by lake sturgeon that moved north to overwinter in Lake Huron or south to overwinter in Lake St. Clair. Lake St. Clair migrants were further distinguished by whether they migrated into the St. Clair River each year (annual migrants) or intermittently (intermittent migrants). Social network analyses indicated lake sturgeon generally co-occurred with individuals of the same migratory phenotype more often than with different migratory phenotypes. A direct test for differences in space use revealed one site was almost exclusively visited by Lake St. Clair migrants whereas the other site was visited by Lake Huron migrants, intermittent Lake St. Clair migrants, and, to a lesser extent, annual Lake St. Clair migrants. Analysis of arrival and departure dates indicated opportunity for co-occurrence at the site visited by all phenotypes but showed Lake Huron migrants arrived approximately 2 weeks before Lake St. Clair migrants. Taken together, our results indicated partial spatiotemporal segregation of migratory phenotypes that may generate assortative mating and promote population differentiation.

Evidence that male sea lamprey increase pheromone release after perceiving a competitor.

Sexual signals evolve via selective pressures arising from male-male competition and female choice, including those originating from unintended receivers that detect the signal. For example, males can acquire information from other males signaling to females and alter their own signal. Relative to visual and acoustic signals, less is known about how such communication networks influence chemical signaling among animals. In sea lamprey (Petromyzon marinus), the chemical communication system is essential for reproduction, offering a useful system to study a pheromone communication network that includes signalers and both intended and unintended receivers. Male sea lamprey aggregate on spawning grounds, where individuals build nests and signal to females using sex pheromones. We examined how exposure to a major component of the male pheromone, 3keto-petromyzonol sulfate (3kPZS), influenced male pheromone signaling, and whether females had a preference for males that altered their signal. Exposure to 3kPZS, at a concentration of 5×10-10 mol l-1, simulated the presence of other male(s) and led to increased 3kPZS release rates within 10 min, followed by a return to baseline levels within 30 min. Exposure also led to increases in hepatic synthesis and circulatory transport of pheromone components. In behavioral assays, females preferred the odor of males that had been exposed to 3kPZS; therefore, males likely benefit from upregulating 3kPZS release after detecting competition for mates. Here, we define how a specific pheromone component influences chemical signaling during intrasexual competition, and show a rare example of how changes in chemical signaling strategies resulting from male competition may influence mate choice.

Behavioural responses of female lake trout Salvelinus namaycush to male chemical stimuli and prostaglandin F2α.

Male olfactory cues may guide aggregation on spawning reefs, mate evaluation and synchronized gamete release in lake trout Salvelinus namaycush, but a lack of information on the source and identity of the cues precludes direct tests of their function. Using a two-channel flume, we found ovulated female lake trout increased time spent in the channel treated with spermiated male-conditioned water, urine and bile but not synthesized prostaglandin F2α . We suggest future efforts to characterize male olfactory cues focus on urine and postulate that bile acids contribute to its behavioural activity.

A pheromone antagonist liberates female sea lamprey from a sensory trap to enable reliable communication.

The evolution of male signals and female preferences remains a central question in the study of animal communication. The sensory trap model suggests males evolve signals that mimic cues used in nonsexual contexts and thus manipulate female behavior to generate mating opportunities. Much evidence supports the sensory trap model, but how females glean reliable information from both mimetic signals and their model cues remains unknown. We discovered a mechanism whereby a manipulative male signal guides reliable communication in sea lamprey (Petromyzon marinus). Migratory sea lamprey follow a larval cue into spawning streams; once sexually mature, males release a pheromone that mimics the larval cue and attracts females. Females conceivably benefit from the mimetic pheromone during mate search but must discriminate against the model cue to avoid orienting toward larvae in nearby nursery habitats. We tested the hypothesis that spawning females respond to petromyzonol sulfate (PZS) as a behavioral antagonist to avoid attraction to the larval cue while tracking the male pheromone despite each containing attractive 3-keto petromyzonol sulfate (3kPZS). We found 1) PZS inhibited electrophysiological responses to 3kPZS and abated preferences for 3kPZS when mixed at the same or greater concentrations, 2) larvae released more PZS than 3kPZS whereas males released more 3kPZS than PZS, and 3) mixtures of 3kPZS and PZS applied at ratios measured in larval and male odorants resulted in the discrimination observed between the natural odors. Our study elucidates how communication systems that arise via deception can facilitate reliable communication.

Intra- and Interspecific Variation in Production of Bile Acids That Act as Sex Pheromones in Lampreys.

Pheromones are important sexual signals in most animals, but research into their evolution is largely biased toward insects. Lampreys are a jawless fish with a relatively well-understood pheromone communication system, and they offer a useful opportunity to study pheromone evolution in a vertebrate. Once sexually mature, male sea lamprey (Petromyzon marinus) and likely other lampreys produce and release bile acids that act as sex pheromones. Spawning males do not feed and therefore produce bile acids primarily for sexual communication, whereas larvae produce the same bile acids but for digestion, offering an opportunity to compare the evolution of bile acids produced for sexual versus nonsexual functions. We profiled eight pheromone-related bile acids in livers from larvae and males and determined the effect of life stage on intra- and interspecific variation in bile acid production. Our results indicate less variation among males than larvae within P. marinus but more variation among species for males than larvae. We postulate that bile acid production in males is shaped by directional or stabilizing selection that reduces variance within P. marinus and directional or disruptive selection that promotes diversification across species. Although our results offer support for the role of sexual selection in the evolution of lamprey pheromones, they do not eliminate possible roles of other aspects of lamprey ecology.

Petromylidenes A⁻C: 2-Alkylidene Bile Salt Derivatives Isolated from Sea Lamprey (Petromyzon marinus).

Three novel bile acid derivatives, petromylidenes A⁻C (1⁻3), featuring uncommon alkylidene adductive scaffolds, were isolated from water conditioned with sexually mature male sea lampreys (Petromyzon marinus). Their structures were elucidated by mass spectrometry and NMR spectroscopy, and by comparison to spectral data of related structures. The identification of compounds 1⁻3, further illustrates the structural diversity of the 5α bile salt family. Compounds 1⁻3 exhibited notable biological properties as well, including high olfactory potencies in adult sea lampreys and strong behavioral attraction of ovulated female sea lampreys. Electro-olfactogram recordings indicated that the limit of detection for 1 was 10-9 M, 2 was 10-11 M, and 3 was less than 10-13 M. These results suggested 1⁻3 were likely male pheromones, which guide reproductive behaviors in the sea lamprey.

High-performance liquid chromatography quantification of enantiomers of a Dihydroxylated tetrahydrofuran natural product.

Both enantiomers of petromyroxol are putative pheromones in sea lamprey (Petromyzon marinus). Here, we describe the separation and quantification of the petromyroxol enantiomers using high-performance liquid chromatography tandem mass spectrometry. The separation was tested on a wide range of chiral columns with normal phases, and effects of the chromatographic parameters such as mobile phase and temperature on the separation were optimized. The AD-H column showed the best separation of enantiomers with n-hexane and ethanol as the mobile phase. The enantiomers were detected by multiple reaction monitoring with a positive atmospheric-pressure chemical ionization on triple quadrupole mass spectrometer. Validation revealed that the method was specific, accurate, and precise. The validated method was applied to measure the amount of petromyroxol enantiomers in water conditioned with sea lamprey larvae, the source of the putative pheromone. This method will be applied in quantifying the natural scalemic petromyroxol mixture, enabling further investigations of a rare non-racemic enantiomeric pheromone mixture in a vertebrate species.

Discovery and characterization of natural products that act as pheromones in fish.

Covering: up to 2018 Fish use a diverse collection of molecules to communicate with conspecifics. Since Karlson and Lüscher termed these molecules 'pheromones', chemists and biologists have joined efforts to characterize their structures and functions. In particular, the understanding of insect pheromones developed at a rapid pace, set, in part, by the use of bioassay-guided fractionation and natural product chemistry. Research on vertebrate pheromones, however, has progressed more slowly. Initially, biologists characterized fish pheromones by screening commercially available compounds suspected to act as pheromones based upon their physiological function. Such biology-driven screening has proven a productive approach to studying pheromones in fish. However, the many functions of fish pheromones and diverse metabolites that fish release make predicting pheromone identity difficult and necessitate approaches led by chemistry. Indeed, the few cases in which pheromone identification was led by natural product chemistry indicated novel or otherwise unpredicted compounds act as pheromones. Here, we provide a brief review of the approaches to identifying pheromones, placing particular emphasis on the promise of using natural product chemistry together with assays of biological activity. Several case studies illustrate bioassay-guided fractionation as an approach to pheromone identification in fish and the unexpected diversity of pheromone structures discovered by natural product chemistry. With recent advances in natural product chemistry, bioassay-guided fractionation is likely to unveil an even broader collection of pheromone structures and enable research that spans across disciplines.

Temporal constraints on the potential role of fry odors as cues of past reproductive success for spawning lake trout.

Deciding where to reproduce is a major challenge for most animals. Many select habitats based upon cues of successful reproduction by conspecifics, such as the presence of offspring from past reproductive events. For example, some fishes select spawning habitat following odors released by juveniles whose rearing habitat overlaps with spawning habitat. However, juveniles may emigrate before adults begin to search for spawning habitat; hence, the efficacy of juvenile cues could be constrained by degradation or dissipation rates. In lake trout (Salvelinus namaycush), odors deposited by the previous year's offspring have been hypothesized to guide adults to spawning reefs. However, in most extant populations, lake trout fry emigrate from spawning reefs during the spring and adults spawn during the fall. Therefore, we postulated that the role of fry odors in guiding habitat selection might be constrained by the time between fry emigration and adult spawning. Time course chemical, physiological, and behavioral assays indicated that the odors deposited by fry likely degrade or dissipate before adults select spawning habitats. Furthermore, fry feces did not attract wild lake trout to constructed spawning reefs in Lake Huron. Taken together, our results indicate fry odors are unlikely to act as cues for lake trout searching for spawning reefs in populations whose juveniles emigrate before the spawning season, and underscore the importance of environmental constraints on social cues.

High-sensitivity determination of estrogens in fish plasma using chemical derivatization upstream UHPLC-MSMS.

This article describes the development and validation of a sensitive LC-MSMS method for determination of estrogen in fish plasma. Dansyl chloride derivatization of the phenol functional group in estrogen was used to enhance the response to atmospheric pressure ionization leading to improve the sensitivity. Individual 13C internal standards were selected after comparison with deuterated standards. Liquid-liquid extraction (ethyl acetate or methyl tert-butyl ether) and protein precipitation (acetonitrile, methanol or acetone) were compared for the extraction and clean-up of estrogens from fish plasma. Ethyl acetate was selected as the best alternative with recovery ranging from 61 to 96% and matrix effect ranging from 88 to 106%. Limits of quantification ranged from 0.5 to 1pg/mL showing a gain in sensitivity of 10,000 times over electrospray ionization of underivatized estrogens. Accuracy and precision were validated over three consecutive days and the method was applied to measure estrogen in sea lamprey (Petromyzon marinus) and lake trout (Salvelinus namaycush) plasma. Estrone and estriol were detected in fish below 1ng/mL in plasma, justifying the need of a highly sensitive LC-MSMS quantification method.

Evidence for partial overlap of male olfactory cues in lampreys.

Animals rely on a mosaic of complex information to find and evaluate mates. Pheromones, often consisting of multiple components, are considered to be particularly important for species-recognition in many species. Although the evolution of species-specific pheromone blends is well described in many insects, very few vertebrate pheromones have been studied in a macro-evolutionary context. Here, we report a phylogenetic comparison of multi-component male odours that guide reproduction in lampreys. Chemical profiling of sexually mature males from eleven species of lamprey, representing six of ten genera and two of three families, indicated that the chemical profiles of sexually mature male odours are partially shared among species. Behavioural assays conducted with four species sympatric in the Laurentian Great Lakes indicated asymmetric female responses to heterospecific odours, where Petromyzon marinus were attracted to male odour collected from all species tested, but other species generally preferred only the odour of conspecifics. Electro-olfactogram recordings from P. marinus indicated that although P. marinus exhibited behavioural responses to odours from males of all species, at least some of the compounds that elicited olfactory responses were different in conspecific male odours compared with heterospecific male odours. We conclude that some of the compounds released by sexually mature males are shared among species and elicit olfactory and behavioural responses in P. marinus, and suggest that our results provide evidence for partial overlap of male olfactory cues among lampreys. Further characterization of the chemical identities of odour components is needed to confirm shared pheromones among species.

Chemical cues and pheromones in the sea lamprey (Petromyzon marinus).

Chemical cues and pheromones guide decisions in organisms throughout the animal kingdom. The neurobiology, function, and evolution of olfaction are particularly well described in insects, and resulting concepts have driven novel approaches to pest control. However, aside from several exceptions, the olfactory biology of vertebrates remains poorly understood. One exception is the sea lamprey (Petromyzon marinus), which relies heavily upon olfaction during reproduction. Here, we provide a broad review of the chemical cues and pheromones used by the sea lamprey during reproduction, including overviews of the sea lamprey olfactory system, chemical cues and pheromones, and potential applications to population management. The critical role of olfaction in mediating the sea lamprey life cycle is evident by a well-developed olfactory system. Sea lamprey use chemical cues and pheromones to identify productive spawning habitat, coordinate spawning behaviors, and avoid risk. Manipulation of olfactory biology offers opportunities for management of populations in the Laurentian Great Lakes, where the sea lamprey is a destructive invader. We suggest that the sea lamprey is a broadly useful organism with which to study vertebrate olfaction because of its simple but well-developed olfactory organ, the dominant role of olfaction in guiding behaviors during reproduction, and the direct implications for vertebrate pest management.

Quantification of 15 bile acids in lake charr feces by ultra-high performance liquid chromatography-tandem mass spectrometry.

Many fishes are hypothesized to use bile acids (BAs) as chemical cues, yet quantification of BAs in biological samples and the required methods remain limited. Here, we present an UHPLC-MS/MS method for simultaneous, sensitive, and rapid quantification of 15 BAs, including free, taurine, and glycine conjugated BAs, and application of the method to fecal samples from lake charr (Salvelinus namaycush). The analytes were separated on a C18 column with acetonitrile-water (containing 7.5mM ammonium acetate and 0.1% formic acid) as mobile phase at a flow rate of 0.25mL/min for 12min. BAs were monitored with a negative electrospray triple quadrupole mass spectrometer (Xevo TQ-S™). Calibration curves of 15 BAs were linear over the concentration range of 1.00-5,000ng/mL. Validation revealed that the method was specific, accurate, and precise. The method was applied to quantitative analysis of feces extract of fry lake charr and the food they were eating. The concentrations of analytes CA, TCDCA, TCA, and CDCA were 242.3, 81.2, 60.7, and 36.2ng/mg, respectively. However, other taurine conjugated BAs, TUDCA, TDCA, and THDCA, were not detected in feces of lake charr. Interestingly, TCA and TCDCA were detected at high concentrations in food pellets, at 71.9 and 38.2ng/mg, respectively. Application of the method to feces samples from lake charr supported a role of BAs as chemical cues, and will enhance further investigation of BAs as chemical cues in other fish species.

Bile salts as semiochemicals in fish.

Bile salts are potent olfactory stimuli in fishes; however the biological functions driving such sensitivity remain poorly understood. We provide an integrative review of bile salts as semiochemicals in fish. First, we present characteristics of bile salt structure, metabolism, and function that are particularly relevant to chemical communication. Bile salts display a systematic pattern of structural variation across taxa, are efficiently synthesized, and are stable in the environment. Bile salts are released into the water via the intestine, urinary tract, or gills, and are highly water soluble. Second, we consider the potential role of bile salts as semiochemicals in the contexts of detecting nearby fish, foraging, assessing risk, migrating, and spawning. Lastly, we suggest future studies on bile salts as semiochemicals further characterize release into the environment, behavioral responses by receivers, and directly test the biological contexts underlying olfactory sensitivity.