A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (<i>Petromyzon marinus</i>)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...

Michigan State University

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, 7alpha, 12alpha, 24-trihydroxy-5alpha-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 approximately 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$ 1.4 trillion of damage annually.

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

We developed an assay for measuring 7α,12α,24-trihydroxy-5a-cholan-3-one-24-sulfate (3kPZS), a mating pheromone released by male sea lampreys (Petromyzon marinus), at low picomolar concentrations in natural waters to assess the presence of invasive populations. 3kPZS was extracted from streamwater at a rate of recovery up to 90% using a single cation-exchange and reversed-phase mixed-mode cartridge, along with [(2)H(5)]3kPZS as an internal standard, and quantified using ultrahigh performance liquid chromatography-tandem mass spectrometry. The limit of detection was below 0.1 ng L(-1) (210 fM), which was the lowest concentration tested. Intra- and interday coefficients of variation were between 0.3-11.6% and 4.8-9.8%, respectively, at 1 ng 3kPZS L(-1) and 5 ng 3kPZS L(-1). This assay was validated by repeat measurements of water samples from a stream spiked with synthesized 3kPZS to reach 4.74 ng L(-1) or 0.24 ng L(-1). We further verified the utility of this assay to detect spawning populations of lampreys; in the seven tributaries to the Laurentian Great Lakes sampled, 3kPZS concentrations were found to range between 0.15 and 2.85 ng L(-1) during the spawning season in known sea lamprey infested segments and were not detectable in uninfested segments. The 3kPZS assay may be useful for the integrated management of sea lamprey, an invasive species in the Great Lakes where pheromone-based control and assessment techniques are desired.

Quantification of a male sea lamprey pheromone in tributaries of Laurentian Great Lakes by liquid chromatography-tandem mass spectrometry.

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.

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

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.

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

Secondary sexual characters in animals are exaggerated ornaments or weapons for intrasexual competition. Unexpectedly, we found that a male secondary sexual character in sea lamprey (Petromyzon marinus) is a thermogenic adipose tissue that instantly increases its heat production during sexual encounters. This secondary sexual character, developed in front of the anterior dorsal fin of mature males, is a swollen dorsal ridge known as the 'rope' tissue. It contains nerve bundles, multivacuolar adipocytes and interstitial cells packed with small lipid droplets and mitochondria with dense and highly organized cristae. The fatty acid composition of the rope tissue is rich in unsaturated fatty acids. The cytochrome c oxidase activity is high but the ATP concentration is very low in the mitochondria of the rope tissue compared with those of the gill and muscle tissues. The rope tissue temperature immediately rose up to 0.3Â°C when the male encountered a conspecific. Mature males generated more heat in the rope and muscle tissues when presented with a mature female than when presented with a male (paired t-test, P<0.05). On average, the rope generated 0.027Â±0.013 W cm(-3) more heat than the muscle in 10 min. Transcriptome analyses revealed that genes involved in fat cell differentiation are upregulated whereas those involved in oxidative-phosphorylation-coupled ATP synthesis are downregulated in the rope tissue compared with the gill and muscle tissues. Sexually mature male sea lamprey possess the only known thermogenic secondary sexual character that shows differential heat generation toward individual conspecifics.

A thermogenic secondary sexual character in male sea lamprey.

Les caract&egrave;res sexuels secondaires chez l&#39;animal sont des ornements exag&eacute;r&eacute;es ou armes de comp&eacute;tition intrasexuelle. Contre toute attente, nous avons constat&eacute; qu&#39;un caract&egrave;re sexuel secondaire masculin dans la grande lamproie marine (Petromyzon marinus) est un tissu adipeux thermog&eacute;nique qui instantan&eacute;ment augmente sa production de chaleur au cours de rencontres sexuelles. Ce caract&egrave;re sexuel secondaire, mis au point en face de la nageoire dorsale ant&eacute;rieure des m&acirc;les matures, est une cr&ecirc;te dorsale gonfl&eacute;e, connue comme le tissu &#39;corde&#39;. Il contient des faisceaux nerveux, multivacuolar adipocytes et cellules interstitielles remplis de gouttelettes lipidiques petit et mitochondries &agrave; cr&ecirc;tes denses et tr&egrave;s bien organis&eacute;s. La composition en acides gras du tissu corde est riche en acides gras insatur&eacute;s. L&#39;activit&eacute; cytochrome c oxydase est &eacute;lev&eacute;e, mais la concentration d&#39;ATP est tr&egrave;s faible dans les mitochondries du tissu corde compar&eacute;es &agrave; celles des tissus gill et muscle. La temp&eacute;rature des tissus corde a imm&eacute;diatement augment&eacute; jusqu&#39;&agrave; 0,3 &deg; C lorsque le m&acirc;le rencontre un cong&eacute;n&egrave;re. Les m&acirc;les matures a g&eacute;n&eacute;r&eacute; plus de chaleur dans les tissus des corde et muscle lorsqu&#39;une femelle adulte que quand pr&eacute;sent&eacute; avec un m&acirc;le (jumel&eacute; t-test, P &lt; 0,05). En moyenne, la corde g&eacute;n&eacute;r&eacute; 0.027&plusmn;0.013 W cm(-3) que plus de chaleur que le muscle en 10 min. du Transcriptome analyses ont r&eacute;v&eacute;l&eacute; que les g&egrave;nes impliqu&eacute;s dans la diff&eacute;renciation des cellules de graisse sont surexprim&eacute;s ATTENDU que ceux impliqu&eacute;s dans la phosphorylation oxydative-coupl&eacute; la synth&egrave;se d&#39;ATP sont r&eacute;prim&eacute;s dans le tissu de corde compar&eacute; avec les tissus gill et muscle. Lamproie de mer m&acirc;le sexuellement mature poss&egrave;dent le seul connu thermog&eacute;nique caract&egrave;re sexuel secondaire qui montre la g&eacute;n&eacute;ration de chaleur diff&eacute;rentiels envers leurs cong&eacute;n&egrave;res individuels.

Un caract&egrave;re sexuel secondaire thermog&egrave;ne dans le m&acirc;le de la lamproie.

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.

Characterization of a novel bile alcohol sulfate released by sexually mature male sea lamprey (Petromyzon marinus).

In vertebrates, bile salts are primarily synthesized in the liver and secreted into the intestine where they aid in absorption of dietary fats. Small amounts of bile salts that are not reabsorbed into enterohepatic circulation are excreted with waste. In sexually mature male sea lamprey (Petromyzon marinus L.) a bile salt is released in large amounts across gill epithelia into water where it functions as a pheromone. We postulate that the release of this pheromone is associated with a dramatic increase in its biosynthesis and transport to the gills upon sexual maturation.

Biosynthesis and release of pheromonal bile salts in mature male sea lamprey.

Mechanisms for orienting toward and locating an odor source are sought in both biology and engineering. Chemical ecology studies have demonstrated that adult female sea lamprey show rheotaxis in response to a male pheromone with dichotomous outcomes: sexually mature females locate the source of the pheromone whereas immature females swim by the source and continue moving upstream. Here we introduce a simple switching mechanism modeled after odor-conditioned rheotaxis for the sea lamprey as they search for the source of a pheromone in a one-dimensional riverine environment. In this strategy, the females move upstream only if they detect that the pheromone concentration is higher than a threshold value and drifts down (by turning off control action to save energy) otherwise. In addition, we propose various uncertainty models such as measurement noise, actuator disturbance, and a probabilistic model of a concentration field in turbulent flow. Based on the proposed model with uncertainties, a convergence analysis showed that with this simplistic switching mechanism, the lamprey converges to the source location on average in spite of all such uncertainties. Furthermore, a slightly modified model and its extensive simulation results explain the behaviors of immature female lamprey near the source location.

Odor-conditioned rheotaxis of the sea lamprey: modeling, analysis and validation.

A new hexahydrophenanthrene sulfate was identified from water conditioned with sea lamprey larvae ( Petromyzon marinus ) and named petromyzonin. Its structure was unequivocally elucidated on the basis of spectroscopic analyses including comparison with spectra of known compounds. The absolute configuration was determined by electronic circular dichroism. Petromyzonin may function as a chemical signal, as it elicited responses in electro-olfactogram recording with a dynamic concentration-response relationship and a detection threshold of 10(-11) M.

Cory O. Brant

Department of Fisheries & Wildlife

Michigan State University

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Cory O. Brant

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Fisheries & Wildlife

Michigan State University

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Department of Fisheries & Wildlife