Michael J. Siefkes

Male Sea Lampreys, Petromyzon marinus L., Excrete a Sex Pheromone from Gill Epithelia

Abstract During the period when they are producing sperm, male sea lampreys (Petromyzon marinus L.) release a sex pheromone 7α, 12α, 24-trihydroxy-5α-cholan-3-one-24-sulfate (3 keto-petromyzonol sulfate, 3ketoPZS) that induces search and preference behaviors in ovulating females. In this study, we conducted a series of experiments to demonstrate that release of this pheromone into water takes place exclusively through the gills. In a behavioral maze, water conditioned with the anterior region of spermiating males induced an increase of search and preference behaviors in ovulating females. Similar behavior was not elicited by water conditioned by the posterior region. The anterior region washings and whole-body washings from spermiating males also elicited large and virtually identical electro-olfactogram responses from female sea lampreys, while the posterior washings produced negligible responses. Further, mass spectrometry and immunoassay confirmed that virtually all the 3ketoPZS released into water was through the gills. Immunocytochemistry revealed some gill epithelial cells and hepatocytes from spermiating males contained dense immunoreactive 3ketoPZS, but not those from prespermiating males. These results demonstrate that 3ketoPZS is released through the gill epithelia and suggest that this pheromone or its precursor may be produced in the liver.

Sex Pheromone Communication in the Sea Lamprey: Implications for Integrated Management

Studies in the past two decades have demonstrated that sexually mature sea lampreys (Petromyzon marinus) release sex pheromones that attract conspecific individuals of the opposite sex. Recently, electrophysiological, behavioral, and biochemical studies have shown that male sea lampreys, during spermiation (spermatogenesis), release 7α,12α,24-trihydroxy-3-one-5α-cholan-24-sulfate, or 3 keto-petromyzonol sulfate, which induces searching behavior in ovulatory females. This behavior appears to lead the females to nests occupied by males. The biosynthesis and release of this pheromone are likely to be regulated by the endocrine system and by environmental factors such as water temperature. Synthetic copies of this pheromone, if available, could be used for attraction and annihilation of mature female lampreys, or for disruption of mating. It may also be possible to develop procedures that promote pheromone biosynthesis in sterile males, which when stocked into spawning grounds, may make them highly effective in competing for mates with resident males. To develop protocols for implementing these potential strategies in integrated sea lamprey management, it is critical to fully understand pheromone biosynthesis and release in male sea lampreys, and to determine the role of sex pheromones in male reproductive fitness.

Capture of Ovulating Female Sea Lampreys in Traps Baited with Spermiating Male Sea Lampreys

Abstract This study was conducted as an initial step in the development of a trapping technique for sexually mature female sea lampreys Petromyzon marinus. Recent research has demonstrated that spermiating male sea lampreys release a sex pheromone that attracts ovulating females. This discovery prompted us to hypothesize that traps baited with spermiating males would capture more ovulating females than empty traps or traps baited with nonspermiating males. We found that traps baited with spermiating males captured nearly 74% of the ovulating females released, whereas empty traps and traps baited with nonspermiating males did not capture any ovulating females. We conclude that pheromone-baited traps may complement current sea lamprey management through direct removal of ripe females from spawning grounds.

Mating Pheromone Reception and Induced Behavior in Ovulating Female Sea Lampreys

Abstract This study was conducted to determine how ovulating female sea lampreys Petromyzon marinus respond to water conditioned with spermiating males (spermiating male washings) and how trap efficiency can be improved through their use. The capture rate of ovulating female sea lampreys was observed in traps baited with continuous or pulsed spermiating male washings. The behavior of ovulating females around baited traps was quantified. Within 2 h, traps baited with continuous spermiating male washings captured 52% of ovulating females (n = 27) and traps baited with pulsed washings captured 28% (n = 25) of ovulating females. Unbaited traps did not capture ovulating females. The behavior of females near traps baited with pulsed spermiating male washings was characterized by significantly more downstream and side-stream movements than that of females near traps with continuous washings. We occluded the olfactory organ of ovulating females and tested whether they were attracted to spermiating male washings i...

Characterization of a Novel Bile Alcohol Sulfate Released by Sexually Mature Male Sea Lamprey (Petromyzon marinus)

A sulphate-conjugated bile alcohol, 3,12-diketo-4,6-petromyzonene-24-sulfate (DKPES), was identified using bioassay-guided fractionation from water conditioned with sexually mature male sea lamprey (Petromyzon marinus). The structure and relative stereochemistry of DKPES was established using spectroscopic data. The electro-olfactogram (EOG) response threshold of DKPES was 10−7 Molar (M) and that of 3-keto petromyzonol sulfate (3 KPZS; a known component of the male sea lamprey sex pheromone) was 10−10 M. Behavioural studies indicated that DKPES can be detected at low concentrations by attracting sexually mature females to nests when combined with 3 KPZS. Nests baited with a mixture of DKPES and 3 KPZS (ratio 1∶29.8) attracted equal numbers of sexually mature females compared to an adjacent nest baited with 3 KPZS alone. When DKPES and 3 KPZS mixtures were applied at ratios of 2∶29.8 and 10∶29.8, the proportion of sexually mature females that entered baited nests increased to 73% and 70%, respectively. None of the sexually mature females released were attracted to nests baited with DKPES alone. These results indicated that DKPES is a component of the sex pheromone released by sexually mature male sea lamprey, and is the second biologically active compound identified from this pheromone. DKPES represents the first example that a minor component of a vertebrate pheromone can be combined with a major component to elicit critical sexual behaviors. DKPES holds considerable promise for increasing the effectiveness of pheromone-baited trapping as a means of sea lamprey control in the Laurentian Great Lakes.

A male pheromone in the sea lamprey (Petromyzon marinus): an overview

We conclude that spermiating male sea lamprey release a large amount of 7α,12α,24-trihydroxy-5α-cholan-3-one 24-sulfate via their gills. We suggest that this compound is used to signal the location of their nests to ovulated females downstream. The role of the minor component, 3kACA, has yet to be determined. The selection pressure to signal over a long distance in rapid flowing water may have favored the evolution of a bile acid derivative (as opposed to a gonadal steroid) as a pheromone. Considering the size of the liver, bile acids can be produced in relatively large quantities. Interference with this pheromone system offers an attractive target for selective and environmentally benign control of the sea lamprey.

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.

Development and application of an ELISA for a sex pheromone released by the male sea lamprey (Petromyzon marinus L.)

An enzyme-linked immunosorbent assay (ELISA) has been developed for a conjugated bile acid, 7alpha,12alpha,24-trihydroxy-5alpha-cholan-3-one 24-sulfate (commonly referred to as 3-keto petromyzonol sulfate [3kPZS]), a pheromone released by reproductively mature male sea lampreys to attract sexually mature females. A polyclonal antiserum against the pheromone was raised by injecting 3-keto petromyzonol 24-hemisuccinate (3kPZ-HS) conjugated to bovine serum albumin into rabbits. The enzyme label was prepared by conjugating 3kPZ-HS to acetylcholinesterase. The standard curve had a working range of 20 pg-10 ng/well. Intra- and inter-assay variations were less than 5 and 12%, respectively. The antiserum had 100% cross-reaction with 3-keto petromyzonol and 3-keto allocholic acid but less than 0.2% cross-reaction with petromyzonol, allocholic acid, cholic acid, and taurolithocholic acid sulfate. The assay was applied to water which had been conditioned for 4h by either larvae, parasitic juveniles, ovulating females, pre-spermiating males, or spermiating males. Immunoactive material (average 200 ng/ml, which is equivalent to 500 microg animal/h) was only found in water from the reproductively mature males and diluted parallel with the standard curve. Assay of water samples collected from male lampreys in bisected aquaria also established that 99.6% of the immunoactive material emanated from the front end of the fish. This assay has applications in both physiological and ecological aspects of sea lamprey reproduction.

Pheromonal bile acid 3-ketopetromyzonol sulfate primes the neuroendocrine system in sea lamprey

BackgroundVertebrate pheromones are known to prime the endocrine system, especially the hypothalamic-pituitary-gonadal (HPG) axis. However, no known pheromone molecule has been shown to modulate directly the synthesis or release of gonadotropin releasing hormone (GnRH), the main regulator of the HPG axis. We selected sea lamprey (Petromyzon marinus) as a model system to determine whether a single pheromone component alters the output of GnRH.Sea lamprey male sex pheromones contain a main component, 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3 keto-petromyzonol sulfate or 3kPZS), which has been shown to modulate behaviors of mature females. Through a series of experiments, we tested the hypothesis that 3kPZS modulates both synthesis and release of GnRH, and subsequently, HPG output in immature sea lamprey.ResultsThe results showed that natural male pheromone mixtures induced differential steroid responses but facilitated sexual maturation in both sexes of immature animals (χ2 = 5.042, dF = 1, p < 0.05). Exposure to 3kPZS increased plasma 15α-hydroxyprogesterone (15α-P) concentrations (one-way ANOVA, p < 0.05) and brain gene expressions (genes examined: three lamprey (l) GnRH-I transcripts, lGnRH-III, Jun and Jun N-terminal kinase (JNK); one-way ANOVA, p < 0.05), but did not alter the number of GnRH neurons in the hypothalamus in immature animals. In addition, 3kPZS treatments increased lGnRH peptide concentrations in the forebrain and modulated their levels in plasma. Overall, 3kPZS modulation of HPG axis is more pronounced in immature males than in females.ConclusionsWe conclude that a single male pheromone component primes the HPG axis in immature sea lamprey in a sexually dimorphic manner.

Isolation and identification of petromyzestrosterol, a polyhydroxysteroid from sexually mature male sea lamprey (Petromyzon marinus L.)

Petromyzestrosterol (1), a novel polyhydroxylated steroid, was identified from water conditioned with sexually mature male sea lamprey (Petromyzon marinus), a jawless vertebrate animal. Along with this novel steroid, two known steroids, 7α,12α,24-trihydroxy-5α-cholan-3-one-24-sulfate (3k PZS) and 7α,12α,24-trihydroxy-5α-cholan-24-sulfate (PZS), were isolated. Structures of these compounds were unequivocally established by spectroscopic analyses and by comparison with spectra of known compounds. Electro-olfactogram recordings (EOG) showed that 1 at nanomolar concentrations was stimulatory for the olfactory epithelium of adult females. 3k PZS, known to function as a male sex pheromone, was more stimulatory than 1 for the female olfactory epithelia. The concentration-response curve of 3k PZS was exponential in shape with steep slopes between 10(-10) and 10(-6) mol L(-1). The concentration-response curve for 1 was shallower than that for 3k PZS.