Sang-Seon Yun

A synthesized pheromone induces upstream movement in female sea lamprey and summons them into traps

Female insect pheromone blends induce robust tracking responses in males and direct them into traps. In vertebrates, pheromones that induce strong and precise tracking responses in natural habitats have rarely been described. Here, we show in the sea lamprey (Petromyzon marinus), a vertebrate invader of the Laurential Great Lakes, that a synthesized component of the male mating pheromone, 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3kPZS), when released into a stream to reach concentrations of 10−14, 10−13, 10−12, 10−11, or 10−10 M, triggers robust upstream movement in ovulated females drawing ≈50% into baited traps. Experiments conducted in diverse stream segments demonstrate the level of behavioral response was not affected by habitat conditions and is effective over hundreds of meters. 3kPZS is equally effective at luring ovulated females as the whole pheromone blend released by males between 10−14 and 10−11 M. 3kPZS diverts ovulated females away from and disrupts orientation to male washings when applied at concentrations higher than washings. Indeed, a single pheromone compound is able to redirect female sea lampreys away from a natural pheromone source and lure them into traps, which should be more effective than targeting males when applied in population control. Our findings may spur the discovery of other potent and environmentally benign agents to combat biological invasion, a process accelerated by globalization, exacerbated by climate change, and costing the global economy US

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

1.4 trillion of damage annually.

11-Deoxycortisol is a corticosteroid hormone in the lamprey

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.

Pheromones of the male sea lamprey, Petromyzon marinus L.: structural studies on a new compound, 3-keto allocholic acid, and 3-keto petromyzonol sulfate.

Corticosteroid hormones are critical for controlling metabolism, hydromineral balance, and the stress response in vertebrates. Although corticosteroid hormones have been well characterized in most vertebrate groups, the identity of the earliest vertebrate corticosteroid hormone has remained elusive. Here we provide evidence that 11-deoxycortisol is the corticosteroid hormone in the lamprey, a member of the agnathans that evolved more than 500 million years ago. We used RIA, HPLC, and mass spectrometry analysis to determine that 11-deoxycortisol is the active corticosteroid present in lamprey plasma. We also characterized an 11-deoxycortisol receptor extracted from sea lamprey gill cytosol. The receptor was highly specific for 11-deoxycortisol and exhibited corticosteroid binding characteristics, including DNA binding. Furthermore, we observed that 11-deoxycortisol was regulated by the hypothalamus–pituitary axis and responded to acute stress. 11-Deoxycortisol implants reduced sex steroid concentrations and up-regulated gill Na+, K+-ATPase, an enzyme critical for ion balance. We show here that 11-deoxycortisol functioned as both a glucocorticoid and a mineralocorticoid in the lamprey. Our findings indicate that a complex and highly specific corticosteroid signaling pathway evolved at least 500 million years ago with the arrival of the earliest vertebrate.

Multiple functions of a multi-component mating pheromone in sea lamprey Petromyzon marinus

This study reports the results of chemical and chromatographic studies which establish the presence of 3-keto allocholic acid (3kACA) in water extracts from spermiating male sea lamprey, Petromyzon marinus. This is the second compound to be isolated and identified from these extracts. The first was 3-keto petromyzonol sulfate (3kPZS), which was shown to act as strong pheromonal attractant for ovulated females. Some new characterization data on 3kPZS (utilizing an only recently available synthetic preparation of the compound) is also included. The possibility that a mixture of 3kACA and 3kPZS might be a more potent pheromonal attractant than either compound alone is discussed.

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

The role of the C24 sulphate in the mating pheromone component, 7α,12α,24-trihydroxy-5α-cholan-3-one 24-sulphate (3kPZS), to specifically induce upstream movement in ovulated female sea lampreys Petromyzon marinus was investigated. 7α,12α-dihydroxy-5α-cholan-3-one 24-oic acid (3kACA), a structurally similar bile acid released by spermiated males, but lacking the C24 sulphate ester, was tested in bioassays at concentrations between 10(-11) and 10(-14) molar (M). 3kACA did not induce upstream movement in females or additional reproductive behaviours. In contrast, spermiated male washings induced upstream movement, prolonged retention on a nest and induced an array of nesting behaviours. Differential extraction and elution by solid-phase extraction resins showed that components other than 3kPZS + 3kACA are necessary to retain females on nests and induce nest cleaning behaviours. All pheromone components, including components in addition to 3kPZS + 3kACA that retain females and induce nest cleaning behaviours were released from the anterior region of the males, as had been reported for 3kPZS. It is concluded that the sea lamprey male mating pheromone has multiple functions and is composed of multiple components.

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

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.

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

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.

Identification of squalamine in the plasma membrane of white blood cells in the sea lamprey, Petromyzon marinus

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.

Reply to Thornton and Carroll: Lamprey possess a highly specific corticosteroid signaling system

It is well established that innate mechanisms play an important role in the immunity of fish. Antimicrobial peptides have been isolated and characterized from several species of teleosts. Here, we report the isolation of an antimicrobial compound from the blood of bacterially challenged sea lamprey, Petromyzon marinus. An acetic acid extract from the blood cells of challenged fish was subjected to solid-phase extraction, cation-exchange chromatography, gel-filtration chromatography, and reverse-phase high-performance liquid chromatography, with the purified fractions assayed for antimicrobial activity. Surprisingly, antimicrobial activity in these fractions originated from squalamine, an aminosterol previously identified in the dogfish shark, Squalus acanthias. Further chromatographic and mass spectrometric analyses confirmed the identity of squalamine, an antimicrobial and antiangiogenic agent, in the active fraction from the sea lamprey blood cells. Immunocytochemical analysis localized squalamine to the plasma membrane of white blood cells. Therefore, we postulate that squalamine has an important role in the innate immunity that defends the lamprey against microbial invasion. The full biochemical and immunological roles of squalamine in the white blood cell membrane remain to be investigated.