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.     

Synthesis and olfactory activity of unnatural, sulfated 5β-bile acid derivatives in the sea lamprey (Petromyzon marinus)

A variety of unnatural bile acid derivatives (9a-9f) was synthesized and used to examine the specificity with which the sea lamprey (Petromyzon marinus) olfactory system detects these compounds. These compounds are analogs of petromyzonol sulfate (PS, 1), a component of the sea lamprey migratory pheromone. Both the stereochemical configuration at C5 (i.e., 5α vs. 5β) and the extent and sites of oxygenation (hydroxylation or ketonization) of the bile acid derived steroid skeleton were evaluated by screening the compounds for olfactory activity using electro-olfactogram recording. 5β-Petromyzonol sulfate (9a) elicited a considerable olfactory response at sub-nanomolar concentration. In addition, less oxygenated systems (i.e., 9b-9e) elicited olfactory responses, albeit with less potency. The sea lamprey sex pheromone mimic 9f (5β-3-ketopetromyzonol sulfate) was also examined and found to produce a much lower olfactory response. Mixture studies conducted with 9a and PS (1) suggest that stimulation is occurring via similar modes of activation, demonstrating a relative lack of specificity for recognition of the allo-configuration (i.e., 5α) in sea lamprey olfaction. This attribute could facilitate design of pheromone analogs to control this invasive species.     

Discrimination of bile acids by the rainbow trout olfactory system: evidence as potential pheromone

Electro-olfactogram recording was used to determine whether the olfactory epithelium of adult rainbow trout is specifically sensitive to bile acids, some of which have been hypothesized to function as pheromones. Of 38 bile acids that had been pre-screened for olfactory activity, 6 were selected. The rainbow trout-specific bile acids, taurocholic acid (TCA), and taurolithocholic acid 3-sulfate (TLS) were the most potent compounds tested. TLS had a distinctive dose-response curve. Cross-adaptation experiments demonstrated that sensitivity to bile acids is attributable to at least 3 independent classes of olfactory receptor sites. Our data suggest that bile acids are discriminated by olfaction in rainbow trout, supporting the possibility that these compounds function as pheromones.     

Mixtures of Two Bile Alcohol Sulfates Function as a Proximity Pheromone in Sea Lamprey

Unique mixtures of pheromone components are commonly identified in insects, and have been shown to increase attractiveness towards conspecifics when reconstructed at the natural ratio released by the signaler. In previous field studies of pheromones that attract female sea lamprey (Petromyzon marinus, L.), putative components of the male-released mating pheromone included the newly described bile alcohol 3,12-diketo-4,6-petromyzonene-24-sulfate (DkPES) and the well characterized 3-keto petromyzonol sulfate (3kPZS). Here, we show chemical evidence that unequivocally confirms the elucidated structure of DkPES, electrophysiological evidence that each component is independently detected by the olfactory epithelium, and behavioral evidence that mature female sea lamprey prefer artificial nests activated with a mixture that reconstructs the male-released component ratio of 30:1 (3kPZS:DkPES, molar:molar). In addition, we characterize search behavior (sinuosity of swim paths) of females approaching ratio treatment sources. These results suggest unique pheromone ratios may underlie reproductive isolating mechanisms in vertebrates, as well as provide utility in pheromone-integrated control of invasive sea lamprey in the Great Lakes.     

Electrophysiological evidence for detection and discrimination of pheromonal bile acids by the olfactory epithelium of female sea lampreys (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>)

Electro-olfactograms were used to determine sensitivity and specificity of olfactory organs of female sea lampreys (Petromyzon marinus) to four bile acids: 3-keto petromyzonol sulfate and 3-keto allocholic acid from spermiating males and petromyzonol sulfate and allocholic acid from larvae. Spermiating male bile acids are thought to function as a mating pheromone and larval bile acids as a migratory pheromone. The response threshold was 10^–12 mol l^–1 for 3-keto petromyzonol sulfate and 10^–10 mol l^–1 for the other bile acids. At concentrations above 10^–9 mol l^–1, the sulfated bile acids showed almost identical potency, as did the non-sulfated bile acids. The two sulfated bile acids were more potent than the two non-sulfated ones. In addition, 3-keto petromyzonol sulfate and water conditioned with spermiating males induced similar concentration-response curves and response thresholds. Cross-adaptation experiments demonstrated that the sulfated and non-sulfated bile acids represent different odors to the olfactory epithelium of females. Further exploration revealed that 3-keto petromyzonol sulfate represents a different odor than petromyzonol sulfate, while 3-keto allocholic acid and allocholic acid represent the same odor. Results indicate that male-specific bile acids are potent and specific stimulants to the female olfactory organ, supporting the previous hypothesis that these bile acids function as a pheromone.Abbreviations 3kACA 3-keto allocholic acid - 3kPZS 3-keto petromyzonol sulfate - ACA allocholic acid - ANOVA analysis of variance - ELISA enzyme-linked immunosorbent assay - EOG electro-olfactogram - PIR percent initial response - PZS petromyzonol sulfate - SMW spermiating male washings     

Multiplex quantification of lamprey specific bile acid derivatives in environmental water using UHPLC-MS/MS

Larval and adult sea lampreys (Petromyzon marinus) release bile salts and acids into the surrounding aquatic environment. Some of these bile salts and acids, such as petromyzonol sulfate (PZS), 3-keto petromyzonol sulfate (3k PZS), petromyzonamine disulfate (PADS), petromyzosterol disulfate (PSDS), and 3-keto allocholic acid (3k ACA), may function as pheromones. To examine the release and distribution patterns of these metabolites, which this study has termed bile acid derivatives, we developed a novel UHPLC-MS/MS method that was characterized by simple sample preparation, baseline separation, and short analysis time for all studied compounds. These five analytes were separated in 7 min using a reversed-phase C18 column containing 1.7 μm particles and a gradient elution at pH 8.9. Once separated, the analytes were subjected to electrospray ionization-mass spectrometry (negative ion mode) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) using the multiple reaction monitoring (MRM) mode. Deuterated 3k PZS ([(2)H(5)]3k PZS) was added as the internal standard (IS) to the sample prior to solid phase extraction (SPE). Among the three types of SPE sorbent tested, mixed-mode cation-exchange and reversed-phase sorbent for bases (MAX) and acids (MCX), and reversed-phase C18 sorbent (Sep-pak), the best recoveries (84.1-99.7%) were obtained with MCX cartridges. The calibration curves of all five analytes were linear between 0.15 and 1200 ng/mL, with R(2)≥0.9997. This method had a precision of relative standard deviation (RSD) ≤9.9% and an accuracy of deviation (DEV) ≥92.5%. The developed method was successfully used to quantify bile acid derivatives found in streams where lampreys spawn (SD<1.4) and water conditioned with male sea lampreys (SD<4.8). Utilizing this method provides a routine analysis of lamprey bile acid derivatives and may prove useful for sea lamprey population estimates in future studies and applications.     

High correlation between microvillous olfactory receptor cell abundance and sensitivity to pheromones in olfactory nerve-sectioned goldfish

1. To determine whether microvillous olfactory receptor cells mediate responses to pheromonal cues, the olfactory nerves of mature male goldfish were axotomized and both the olfactory and behavioral sensitivity of these animals to olfactory stimuli investigated after which the histological condition of their olfactory epithelia was determined. 2. Behavioral responsiveness to food odor returned within 2 weeks but responsiveness to sexually-active females (pheromones) took 4–10 weeks to return. 3. Electro-olfactogram recordings from the olfactory epithelium of axotomized fish found that olfactory responsiveness to amino acids and pheromones changed little during the first week subsequent to axotomy. However, olfactory sensitivity decreased rapidly during the second week. During the course of the third week, electro-olfactogram sensitivity to amino acids remained while exposure to pheromones evoked no recordable electro-olfactogram. During week 4, sensitivity to amino acids increased further, and weak sensitivity to some pheromones became evident. Further recovery of electro-olfactogram sensitivity to all odorants was slow and erratic over the next 6 months, particularly to the pheromones. 4. Histological examination of the olfactory epithelia of axotomized fish demonstrated that while ciliated receptor cells were present within 2 weeks, microvillous receptor cells took approximately 4 weeks to regenerate. 5. Together these data suggest that microvillous receptor cells mediate responsiveness to pheromones in this species. Accepted: 22 August 1996     

Multiple excitatory receptor types on individual olfactory neurons: implications for coding of mixtures in the spiny lobster

The aim of our paper was to investigate whether single olfactory receptor neurons (ORNs) of the spiny lobster Panulirus argus functionally express more than one type of receptor, examine the consequences of this on coding of mixtures, and compare principles of odorant mixture coding by spiny lobsters with that by the channel catfish, which has been studied extensively using the same experimental and analytical procedures (Caprio et al. 1989; Kang and Caprio 1991). We examined responses of individual taurine-sensitive ORNs to binary mixtures of excitatory compounds, either competitive agonists (taurine, β-alanine, hypotaurine) or non-competitive agonists (taurine, l-glutamate, ammonium chloride, adenosine-5′-monophosphate). Responses to mixtures were compared to two indices: mixture discrimination index (MDI) and independent component index (ICI). Binary mixtures of competitive agonists had MDI values close to 1.0, as expected for competitors. Mixtures of non-competitive agonists had ICI values averaging 0.83, indicating the effects of the components are not independent. We conclude that individual olfactory cells of spiny lobsters can express more than one type of receptor mediating excitation, one of which typically has a much higher density or affinity, and that spiny lobster and catfish olfactory cells encode mixtures of two excitatory agonists using similar rules. Accepted: 20 December 1996     

Morphological and electrophysiological examination of olfactory sensory neurons during the early developmental prolarval stage of the sea lamprey Petromyzon marinus L

This study examined olfactory sensory neuron morphology and physiological responsiveness in newly hatched sea lamprey, Petromyzon marinus L. These prolarvae hatch shortly after neural tube formation, and stay within nests for approximately 18 days, before moving downstream to silty areas where they burrow, feed and pass to the larval stage. To explore the possibility that the olfactory system is functioning during this prolarval stage, morphological and physiological development of olfactory sensory neurons was examined. The nasal cavity contained an olfactory epithelium with ciliated olfactory sensory neurons. Axons formed aggregates in the basal portion of the olfactory epithelium and spanned the narrow distance between the olfactory epithelium and the brain. The presence of asymmetric synapses with agranular vesicles within fibers in the brain, adjacent to the olfactory epithelium suggests that there was synaptic connectivity between olfactory sensory axons and the brain. Neural recordings from the surface of the olfactory epithelium showed responses following the application of L-arginine, taurocholic acid, petromyzonol sulfate (a lamprey migratory pheromone), and water conditioned by conspecifics. These results suggest that lampreys may respond to olfactory sensory input during the prolarval stage.     

HPLC and ELISA analyses of larval bile acids from Pacific and western brook lampreys

Comparative studies were performed on two native lamprey species, Pacific lamprey (Lampetra tridentata) and western brook lamprey (Lampetra richardsoni) from the Pacific coast along with sea lamprey (Petromyzon marinus) from the Great Lakes, to investigate their bile acid production and release. HPLC and ELISA analyses of the gall bladders and liver extract revealed that the major bile acid compound from Pacific and western brook larval lampreys was petromyzonol sulfate (PZS), previously identified as a migratory pheromone in larval sea lamprey. An ELISA for PZS has been developed in a working range of 20 pg-10 ng per well. The tissue concentrations of PZS in gall bladder were 127.40, 145.86, and 276.96 micro g/g body mass in sea lamprey, Pacific lamprey, and western brook lamprey, respectively. Releasing rates for PZS in the three species were measured using ELISA to find that western brook and sea lamprey released PZS 20 times higher than Pacific lamprey did. Further studies are required to determine whether PZS is a chemical cue in Pacific and western brook lampreys.     

Identification of plant volatiles activating single receptor neurons in the pine weevil (Hylobius abietis)

Naturally produced plant volatiles, eliciting responses of single olfactory receptor neurons in the pine weevil, have been identified by gas chromatography linked with mass spectrometry. The receptor neurons (n = 72) were classified in 30 types, according to the compound which elicited the strongest response in each neuron, 20 of which compounds were identified. Most potent for 14 types of neurons (n = 50) were monoterpenes, including bicyclic (e.g. α-pinene, camphor and myrtenal) for 8 types (n = 32), monocyclic (limonene, carvone, α-terpinene) for 3 types (n = 12) and acyclic (e.g. β-myrcene and linalool) for 3 types (n = 6). Other compounds eliciting strongest responses of a neuron were five sesquiterpenes, including α-copaene and a farnesene-isomer, and an anethole type which has no biosynthetic relationship with terpenes. Within one type, receptor neurons with quite selective responses to the most potent compound as well as neurons with additional responses to several, structurally similar compounds were found, indicating that the neurons may have the same functional types of membrane receptors, but different sensitivities. Response spectra of neurons within the bicyclic-, mono-cyclic and acyclic types showed more overlapping than across the neuron types. Minimal overlapping response spectra was found between monoterpene and sesquiterpene neurons. The results suggest that this structure-activity relationship is significant for encoding plant odour information in the pipe weevil. Accepted: 6 January 1997     

Mixture of new sulfated steroids functions as a migratory pheromone in the sea lamprey

The sea lamprey is an ancient, parasitic fish that invaded the Great Lakes a century ago, where it triggered the collapse of many fisheries. Like many fishes, this species relies on chemical cues to mediate key aspects of its life, including migration and reproduction. Here we report the discovery of a multicomponent steroidal pheromone that is released by stream-dwelling larval lamprey and guides adults to spawning streams. We isolated three compounds with pheromonal activity (in submilligram quantities from 8,000 l of larval holding water) and deduced their structures. The most important compound contains an unprecedented 1-(3-aminopropyl)pyrrolidin-2-one subunit and is related to squalamine, an antibiotic produced by sharks. We verified its structure by chemical synthesis; it attracts adult lamprey at very low (subpicomolar) concentrations. The second component is another new sulfated steroid and the third is petromyzonol sulfate, a known lamprey-specific bile acid derivative. This mixture is the first migratory pheromone identified in a vertebrate and is being investigated for use in lamprey control.     

Lake char (<Emphasis Type="Italic">Salvelinus namaycush</Emphasis>) olfactory neurons are highly sensitive and specific to bile acids

Bile acids have been implicated as chemical signals in spawning behaviour of lake char (Salvelinus namaycush). In this study, we investigated olfactory responses of lake char to bile acids by using the electro-olfactogram recording. Lake char detected 9 out of 38 bile acids tested at thresholds 0.02–0.5 nM. The most stimulatory included chenodeoxycholic acid, cholic acid, taurochenodeoxycholic acid, taurocholic acid, and taurolithocholic acid 3α-sulphate. Structure–activity analysis indicated that substituents in the side chain or hydroxyl sulphation were determinant elements for the recognition of individual bile acid receptors, while the position and orientation of hydroxyls or the type of amidation were important for effective stimulation. Three distinct types of concentration–response relationships were found, representing free, taurine- or glycine-amidated, and 3α-sulphated bile acids. Cross-adaptation and binary mixture experiments revealed the presence of multiple olfactory receptors for bile acids. Lake char were also capable of detecting petromyzonol sulphate at 1 nM, possibly via its own receptors. Our study further showed that the olfactory responses to bile acids were independent of those of known odorants including amino acids, prostaglandins and gonadal steroids. We conclude that lake char possess multiple olfactory receptors capable of discriminating bile acids produced and released by conspecifics.     

Petromyzonol sulfate and its derivatives: the chemoattractants of the sea lamprey

Petromyzonol sulfate (PZS) and 3 keto-PZS are bile alocohol derivatives that serve as chemoattractants during the life cycle of sea lamprey (Petromyzon marinus). The sulfonate moiety is crucial perhaps conferring the required solubility for the pheromone that is released into the streams and for the specificity to bind to its receptor. During the life cycle of lamprey, larvae produce copious amounts of 5 alpha-cholan-PZS, and trace amounts of allocholic acid (ACA), which attracts adults to the same breeding ground. Later the spermeating males produce 3keto-PZS, and trace amounts of 3-keto-ACA, which attracts the ovulating females, signaling both its reproductive status and its nesting location for successful reproduction. In both stages, a mixture of components serves as pheromone plume, similar to insects. The receptors for the migratory and the reproductive pheromones need to be molecularly cloned and characterized in order to understand the molecular biology of olfaction in the sea lamprey.     

Leaf phenology, seasonal changes in leaf quality and herbivory pattern of Sanguisorba tenuifolia at different altitudes

Leaf demography, seasonal changes in leaf quality and leaf-beetle herbivory of a herbaceous perennial plant, Sanguisorba tenuifolia, were compared between low- and high-elevation sites. Leaf nitrogen concentration was higher and leaf mass per area (LMA) was lower at the higher site than at the lower one. At the lower site, with a long growth period, plants produced many leaf cohorts and leaves emerged throughout the growing season. At the higher site, with a short growth period, however, leaf emergence was concentrated early in the growing season. The improvement of leaf quality and acceleration of leaf emergence at higher altitude are seen as adaptations to a short growing season. Results of a feeding trial suggested that leaf quality for the leaf-beetle Galerucella grisescens was higher at the higher site, but plants at the higher site showed less damage. Oviposition of G. grisescens was seasonal and unimodal at both altitudes, but the period of oviposition was shorter and its density lower at the higher site. The low temperature and short growth period at the higher site appear to reduce the activity of the leaf-beetles, resulting in a decrease in damage by herbivory, despite better leaf quality. Received: 11 December 1997 / Accepted: 24 July 1998     

Seed dispersal in Cakile edentula var. lacustris : decoupling the fitness effects of density and distance from the home site

A factorial design of three densities of siblings at three local distances from seed parents was employed to distinguish effects of density from effects of dispersal distance on lifespan and fruit production of Cakile edentula var. lacustris, a plant with heteromorphic seeds. The segmented fruits produce two seed types: proximal and distal, with distal seeds having greater mass and greater dispersibility. Effects of longer distances (0.5 km and 30 km) on lifespan and fruit production were investigated using plants at low density. The prediction was tested that the greater seed mass of distal seeds increases fitness when seeds are dispersed into sites of unknown quality away from the home site or when seeds are dispersed to low density. High density caused earlier mortality and lower probability of reproduction. Distance from the maternal plant did not influence lifespan or reproduction at distances of 15 m or less, but lifespan was longer 0.5 km from the home site. No interaction was detected between the effects of density and distance on either lifespan or total fitness. Environmental conditions that influence fitness did not vary as a function of dispersal distance in this system, and favorable conditions at the home site did not persist between generations. Therefore, selection on dispersion patterns in natural conditions is likely to be through effects of density rather than dispersal distance. Proximal seeds had greater reproduction than distal seeds at the home site, and distal seeds had greater reproduction at the more distant sites (but not the most distant site), as expected, but these performance differences could not be attributed to differences in mass between the two seed types. Reduced seed mass was favored at the most distant site, but larger seed mass was favored most strongly at low density. Seeds that are dispersed to low density are larger, suggesting that although kin selection may limit the effectiveness of individual selection to increase seed mass under conditions of sibling competition, density-dependent individual selection on seed mass, rather than distant-dependent selection, also contributes to the observed associations among seed type, seed mass and dispersal ability. Received: 21 October 1996 / Accepted: 4 December 1996     

Honeybees establish specific sites on the comb for their waggle dances

Successful honeybee foragers perform dances on the surface of the comb where they interact with nectar receivers and dance followers. We have recorded the sites at which dances take place in large ten-frame hives and in two-frame observation hives. We find that dancing bees are most commonly found on particular combs in large hives and in particular areas on the combs in the observation hives. Although the site where dances take place may change from day to day, dancers will keep to the same site during the foraging period in any one day. Furthermore, if an established dance site is artificially relocated in the hive during the day, dancers seek these sites out before commencing their dances. We conclude that the dance sites are labelled in some way and so promote the congregation of both dancers and dance followers at the same site. Accepted: 27 November 1996     

Evidence for a receiver bias underlying female preference for a male mating pheromone in sea lamprey

Receiver bias models suggest that a male sexual signal became exaggerated to match a pre-existing sensory, perceptual or cognitive disposition of the female. Accordingly, these models predict that females of related taxa possessing the ancestral state of signalling evolved preference for the male trait in a non-sexual context. We postulated that female preference for the male-released bile alcohol mating pheromone, 3 keto petromyzonol sulfate (3kPZS), of the sea lamprey (Petromyzon marinus) evolved as a result of a receiver bias. In particular, we propose that migratory silver lamprey (Ichthyomyzon unicuspis), a basal member of the Petromyzontidae, evolved a preference for 3kPZS released by stream-resident larvae as a means of identifying productive habitat for offspring. Larval silver lamprey released 3kPZS at rates sufficient to be detected by migratory lampreys. Females responded to 3kPZS by exhibiting upstream movement behaviours relevant in a migratory context, but did not exhibit proximate behaviours important to mate search and spawning. Male silver lamprey did not release 3kPZS at rates sufficient to be detected by females in natural high-volume stream environments. We infer that female silver lamprey cue onto 3kPZS excreted by stream-resident larvae as a mechanism to locate habitat conducive to offspring survival and that males do not signal with 3kPZS. We suggest that this female preference for a male signal in a non-sexual context represents a bias leading to the sexual signalling observed in sea lamprey.     

Physiological characterization of antennular flicking reflexes in the crayfish

The cellular substrates of antennular flicking behavior in the crayfish Procambarus clarkii were investigated. Flicking involves fast downward movements of the external filament of each biramous antennule (1st antenna), and is mediated by phasic contractions of a short muscle, the external filament depressor. Phasic contractions of the external filament depressor depend upon stereotyped impulse bursts in a single motorneuron (P1). These bursts have a characteristic impulse frequency profile that is consistent upon successive occurrences. The temporal characteristics of the impulse burst suggest that the central depolarizations generating each burst may be similar to driver potentials described for motor neurons in crustacean cardiac ganglia. Responses of the external filament to odorants have a long latency and are characterized by repetitive bursts and tonic activity in some external filament depressor fibers. Tonic activity in a slowly contracting muscle, the antennular depressor muscle, is also evoked by chemical stimulation. Flicking is consistently evoked only by mechanical or hydrodynamic stimulation of the cephalothorax, antennae and antennules. The sensitivity and short latency of the hydrodynamic antennule-generated flick reflex is consistent with the sensitivity of rapidly conducting, hydrodynamically activated mechanoreceptor neurons in both antennular filaments. I propose that antennular flicking, which has been shown to enhance the dynamic response characteristics of olfactory receptor neurons on the external antennular filament, has evolved as a response to the turbulence associated with fluid movement, within which chaotic odorant concentration fronts may be imbedded. Accepted: 23 October 1996