petromyzonol-sulfate and petromyzonamine-disulfate

The sea lamprey, Petromyzon marinus, is a harmful invader of the Laurentian Great Lakes. The odor emitted by larval lampreys resident to streams attracts migrating adults to high quality spawning habitats. Three components of the larval pheromone have been identified and tested in laboratory settings: petromyzonol sulfate, petromyzosterol disulfate, and petromyzonamine disulfate. Here, we report the first field test of six mixtures of synthetic versions of these pheromone components, and we compare lamprey responses to these with those elicited by the complete larval odor in a natural stream. Exposure to larval odor both increased upstream movement and attracted migrants into the portion of a channel containing the odor. No tested combination of synthetic pheromone components proved similarly attractive. These findings suggest the existence of unknown additional components of the pheromone that await discovery and are likely necessary if the pheromone is to be useful in management of this pest. Further, we hypothesize that the complete pheromone mixture is necessary to attract migrants into spawning habitat at the conclusion of the migration, whereas a partial pheromone may be effective at the transition from lake to stream when natural factors both dilute and alter the ratio of components from that actually emitted by sea lamprey larvae.

Topics: Animals; Behavior, Animal; Cholestanes; Cholic Acids; Larva; Odorants; Petromyzon; Pheromones; Pyrrolidinones; Rivers

The methodology of using fish pheromones, or chemical signatures, as a tool to monitor or manage species of fish is rapidly gaining popularity. Unequivocal detection and accurate quantitation of extremely low concentrations of these chemicals in natural waters is paramount to using this technique as a management tool. Various species of lamprey are known to produce a mixture of three important migratory pheromones; petromyzonol sulfate (PS), petromyzonamine disulfate (PADS), and petromyzosterol disulfate (PSDS), but presently there are no established robust methods for quantitation of all three pheromones. In this study, we report a new, highly sensitive and selective method for the rapid identification and quantitation of these pheromones in river water samples. The procedure is based on pre-concentration, followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. The method is fast, with unambiguous pheromone determination. Practical quantitation limits of 0.25 ng/l were achieved for PS and PADS and 2.5 ng/l for PSDS in river water, using a 200-fold pre-concentration, However, lower quantitation limits can be achieved with greater pre-concentration. The methodology can be modified easily to include other chemicals of interest. Furthermore, the pre-concentration step can be applied easily in the field, circumventing potential stability issues of these chemicals.

Topics: Animal Migration; Animals; Cholestanes; Cholic Acids; Chromatography, Liquid; Lampreys; Pheromones; Pyrrolidinones; Reproducibility of Results; Rivers; Tandem Mass Spectrometry; Time Factors; Water

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.

Topics: Animals; Bile Acids and Salts; Cholestanes; Cholic Acids; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Least-Squares Analysis; Limit of Detection; Male; Petromyzon; Pyrrolidinones; Reproducibility of Results; Rivers; Solid Phase Extraction; Tandem Mass Spectrometry

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.

Topics: Animal Migration; Animals; Cholestanes; Cholic Acids; Half-Life; Larva; Mass Spectrometry; Midwestern United States; Petromyzon; Pheromones; Pyrrolidinones; Rivers

Migratory adult sea lampreys locate spawning streams by using a pheromone released by stream-resident conspecific larvae. It was recently reported that this pheromone is comprised of a mixture of three sulfated steroids: petromyzonamine disulfate (PADS), petromyzosterol disulfate (PSDS), and petromyzonol sulfate (PS). This manuscript reports in-depth details of pheromone isolation and provides new information on the olfactory potency of PADS and PSDS and the behavioral activity of synthesized PADS. Isolation was accomplished using bioassay-guided fractionation which included liquid chromatography-mass spectrometry, electro-olfactogram recording (EOG), and behavioral assays. Both highly purified and synthesized PADS stimulated the olfactory system of adult lamprey and were attractive at concentrations of 10(-13) M. PSDS also had olfactory activity at 10(-13) M. Cross-adaptation studies with EOG recording demonstrated that PADS, PSDS, and PS are detected by independent olfactory receptor sites. Finally, the mixture of all three components was as attractive as larval water to adult sea lampreys in laboratory mazes. It is believed that these steroids are the principal components of the pheromone.

Topics: Animal Migration; Animals; Cholestanes; Cholic Acids; Larva; Petromyzon; Pheromones; Pyrrolidinones; Reproduction; Smell; Water

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.

Topics: Animal Migration; Animals; Cholestanes; Cholic Acids; Larva; Molecular Structure; Odorants; Petromyzon; Pheromones; Pyrrolidinones; Steroids