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 ...

U.S. Geological Survey

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.

There is an abundance of experimental studies and reviews that describe odorant-mediated behaviors of fish in laboratory microcosms, but research in natural field conditions has received considerably less attention. Fish pheromone studies in laboratory settings can be highly productive and allow for controlled experimental designs; however, laboratory tanks and flumes often cannot replicate all the physical, physiological and social contexts associated with natural environments. Field experiments can be a critical step in affirming and enhancing understanding of laboratory discoveries and often implicate the ecological significance of pheromones employed by fishes. When findings from laboratory experiments have been further tested in field environments, often different and sometimes contradictory conclusions are found. Examples include studies of sea lamprey (Petromyzon marinus) mating pheromones and fish alarm substances. Here, we review field research conducted on fish pheromones and alarm substances, highlighting the following topics: (1) contradictory results obtained in laboratory and field experiments, (2) how environmental context and physiological status influences behavior, (3) challenges and constraints of aquatic field research and (4) innovative techniques and experimental designs that advance understanding of fish chemical ecology through field research.

Understanding behavioral responses of fish to pheromones in natural freshwater environments.

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.

Delta-V (ΔV), the magnitude of the velocity change experienced by a vehicle during a crash, is widely used as a predictor of injury risk. The National Automotive Sampling System/Crashworthiness Data System (NASS-CDS) uses the WinSMASH computer code to reconstruct ΔV based on postcrash vehicle deformation. WinSMASH, a direct descendant of CRASH3, first uses vehicle damage to estimate absorbed energy and then applies momentum conservation to estimate ΔV. This study aims to determine the accuracy of WinSMASH ΔV reconstructions for NHTSA side crash tests.

Accuracy of a damage-based reconstruction method in NHTSA side crash tests.

Vertebrate 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.

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

Reproductive functions can be modulated by both stimulatory and inhibitory primer pheromones released by conspecifics. Many stimulatory primer pheromones have been documented, but relatively few inhibitory primer pheromones have been reported in vertebrates. The sea lamprey male sex pheromone system presents an advantageous model to explore the stimulatory and inhibitory primer pheromone functions in vertebrates since several pheromone components have been identified. We hypothesized that a candidate sex pheromone component, 7α, 12α-dihydroxy-5α-cholan-3-one-24-oic acid (3 keto-allocholic acid or 3kACA), exerts priming effects through the hypothalamic-pituitary-gonadal (HPG) axis. To test this hypothesis, we measured the peptide concentrations and gene expressions of lamprey gonadotropin releasing hormones (lGnRH) and the HPG output in immature male sea lamprey exposed to waterborne 3kACA. Exposure to waterborne 3kACA altered neuronal activation markers such as jun and jun N-terminal kinase (JNK), and lGnRH mRNA levels in the brain. Waterborne 3kACA also increased lGnRH-III, but not lGnRH-I or -II, in the forebrain. In the plasma, 3kACA exposure decreased all three lGnRH peptide concentrations after 1h exposure. After 2h exposure, 3kACA increased lGnRH-I and -III, but decreased lGnRH-II peptide concentrations in the plasma. Plasma lGnRH peptide concentrations showed differential phasic patterns. Group housing condition appeared to increase the averaged plasma lGnRH levels in male sea lamprey compared to isolated males. Interestingly, 15α-hydroxyprogesterone (15α-P) concentrations decreased after prolonged 3kACA exposure (at least 24h). To our knowledge, this is the only known synthetic vertebrate pheromone component that inhibits steroidogenesis in males.

An anti-steroidogenic inhibitory primer pheromone in male sea lamprey (Petromyzon marinus).

Synchronization of male and female locomotor rhythmicity can play a vital role in ensuring reproductive success. Several physiological and environmental factors alter these locomotor rhythms. As sea lamprey, Petromyzon marinus, progress through their life cycle, their locomotor activity rhythm changes multiple times. The goal of this study was to elucidate the activity patterns of adult female sea lamprey during the sexual maturation process and discern the interactions of these patterns with exposure to male pheromones. During these stages, preovulated and ovulated adult females are exposed to sex pheromone compounds, which are released by spermiated males and attract ovulated females to the nest for spawning. The locomotor behavior of adult females was monitored in a natural stream with a passive integrated tag responder system as they matured, and they were exposed to a sex pheromone treatment (spermiated male washings) or a control (prespermiated male washings). Results showed that, dependent on the hour of day, male sex pheromone compounds reduce total activity (p < 0.05) and cause increases in activity during several daytime hours in preovulated and ovulated females. These results are one of the first examples of how sex pheromones modulate a locomotor rhythm in a vertebrate, and they suggest that the interaction between maturity stage and sex pheromone exposure contributes to the differential locomotor rhythms found in adult female sea lamprey. This phenomenon may contribute to the reproductive synchrony of mature adults, thus increasing reproductive success in this species.

Effects of sex pheromones and sexual maturation on locomotor activity in female sea lamprey (Petromyzon marinus).

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.

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.

We determined whole-fish total mercury (Hg) concentrations of 40 male and 40 female adult sea lampreys (Petromyzon marinus) captured in the Cheboygan River, a tributary to Lake Huron, during May 2011. In addition, bioenergetics modeling was used to explore the effects of sex-related differences in activity and resting (standard) metabolic rate (SMR) on mercury accumulation. The grand mean for Hg concentrations was 519 ng/g (standard error of the mean=46 ng/g). On average, males were 16% higher in Hg concentration than females. Bioenergetics modeling results indicated that 14% higher activity and SMR in males would account for this observed sex difference in Hg concentrations. We concluded that the higher Hg concentration in males was most likely due to higher rate of energy expenditure in males, stemming from greater activity and SMR. Our findings have implications for estimating the effects of sea lamprey populations on mercury cycling within ecosystems, as well as for the proposed opening of sea lamprey fisheries. Eventually, our results may prove useful in improving control of sea lamprey, a pest responsible for substantial damage to fisheries in lakes where it is not native.

Mercury accumulation in sea lamprey (Petromyzon marinus) from Lake Huron.

In higher vertebrates, in response to stress, the hypothalamus produces corticotropin-releasing hormone (CRH), which stimulates cells in the anterior pituitary to produce adrenocorticotropic hormone (ACTH), which in turn stimulates production of either cortisol (F) or corticosterone (B) by the adrenal tissues. In lampreys, however, neither of these steroids is present. Instead, it has been proposed that the stress steroid is actually 17,21-dihydroxypregn-4-ene-3,20-dione (11-deoxycortisol; S). However, there have been no studies yet to determine its mechanism of regulation or site of production. Here we demonstrate that (1) intraperitoneal injections of lamprey-CRH increase plasma S in a dose dependent manner, (2) intraperitoneal injections of four lamprey-specific ACTH peptides at 100μg/kg, did not induce changes in plasma S concentrations in either males or females; (3) two lamprey-specific gonadotropin-releasing hormones (GnRH I and III) and arginine-vasotocin (AVT), all at single doses, stimulated S production as well as, or to an even greater extent than CRH; (4) sea lamprey mesonephric kidneys, in vitro, converted tritiated 17α-hydroxyprogesterone (17α-P) into a steroid that had the same chromatographic properties (on HPLC and TLC) as S; (5) kidney tissues released significantly more immunoassayable S into the incubation medium than gill, liver or gonad tissues. One interpretation of these results is that the corticosteroid production of the sea lamprey, one of the oldest extant vertebrates, is regulated through multiple pathways rather than the classical HPI-axis. However, the responsiveness of this steroid to the GnRH peptides means that a reproductive rather than a stress role for this steroid cannot yet be ruled out.

Regulation of a putative corticosteroid, 17,21-dihydroxypregn-4-ene,3,20-one, in sea lamprey, Petromyzon marinus.

Abstract Objective Planar crash severity is most commonly defined by delta-V (ΔV), which is the change in a vehicle's velocity vector during a crash. All ΔV estimates contained in the United States' National Automotive Sampling System-Crashworthiness Data System (NASS-CDS) are generated using a damage-based program called WinSMASH. WinSMASH ΔV accuracy in side crashes has not previously been validated against real-world crash data. This study will investigate the accuracy of WinSMASH ΔV estimates in real-world side crashes. Methods This study uses biaxial ΔV data from event data recorders (EDRs) to assess the accuracy of side crash ΔV estimates in NASS-CDS crash cases. Single-event side crashes were identified in the NASS-CDS for which a) WinSMASH ΔV had been coded and b) biaxial EDR data was available. For these crashes, the WinSMASH-estimated resultant ΔV was compared with the EDR-recorded resultant ΔV to assess the accuracy of the former. EDR ΔV values were adjusted for an assumed average restitution value of 10 %, based on values reported in the literature. Principal Direction of Force (PDOF) is the orientation of the net crash impulse relative to the vehicle and is a key parameter in WinSMASH reconstructions. NASS-CDS PDOF estimates were compared to the PDOF computed from the EDR data to assess their accuracy as well. Results WinSMASH systematically overestimates EDR ΔV by about 12.9 % for cars struck by cars, and by about 2.4 % for cars struck by LTVs. ΔV error was significantly different (at 95% confidence) for crashes to different areas of the vehicle side. The mean discrepancy in NASS-CDS PDOF was -0.9° with a standard deviation of 12.6°. Discussion WinSMASH systematically overestimates ΔV at common velocity by about 13 % for cars struck by cars and about 2 % for cars struck by LTVs. Since WinSMASH's assumption of zero restitution is accounted for in this analysis, this suggests that WinSMASH stiffness parameters represent LTV impacts better than car impacts in side crashes. ΔV error differs by impacted area, suggesting that side impact stiffness parameters used in damage-based ΔV reconstruction must closely represent the crash mode being reconstructed for best results. NASS-CDS estimates of PDOF do not appear to exhibit any systematic discrepancy. The amount of random PDOF discrepancy observed here is consistent with prior studies.

Nicholas S. Johnson

Hammond Bay Biological Station,
Great Lakes Science Center,
Hammond Bay Biological Station, Great Lakes Science Center

U.S. Geological Survey

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

Nicholas S. Johnson

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U.S. Geological Survey

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

Hammond Bay Biological Station,
Great Lakes Science Center,
Hammond Bay Biological Station, Great Lakes Science Center