Extensive DNA methylome rearrangement during early lamprey embryogenesis.

DNA methylation (5mC) is a repressive gene regulatory mark widespread in vertebrate genomes, yet the developmental dynamics in which 5mC patterns are established vary across species. While mammals undergo two rounds of global 5mC erasure, teleosts, for example, exhibit localized maternal-to-paternal 5mC remodeling. Here, we studied 5mC dynamics during the embryonic development of sea lamprey, a jawless vertebrate which occupies a critical phylogenetic position as the sister group of the jawed vertebrates. We employed 5mC quantification in lamprey embryos and tissues, and discovered large-scale maternal-to-paternal epigenome remodeling that affects ~30% of the embryonic genome and is predominantly associated with partially methylated domains. We further demonstrate that sequences eliminated during programmed genome rearrangement (PGR), are hypermethylated in sperm prior to the onset of PGR. Our study thus unveils important insights into the evolutionary origins of vertebrate 5mC reprogramming, and how this process might participate in diverse developmental strategies.

Sensory trap leads to reliable communication without a shift in nonsexual responses to the model cue.

The sensory trap model of signal evolution suggests that males manipulate females into mating using traits that mimic cues used in a nonsexual context. Despite much empirical support for sensory traps, little is known about how females evolve in response to these deceptive signals. Female sea lamprey (Petromyzon marinus) evolved to discriminate a male sex pheromone from the larval odor it mimics and orient only toward males during mate search. Larvae and males release the attractant 3-keto petromyzonol sulfate (3kPZS), but spawning females avoid larval odor using the pheromone antagonist, petromyzonol sulfate (PZS), which larvae but not males, release at higher rates than 3kPZS. We tested the hypothesis that migratory females also discriminate between larval odor and the male pheromone and orient only to larval odor during anadromous migration, when they navigate within spawning streams using larval odor before they begin mate search. In-stream behavioral assays revealed that, unlike spawning females, migratory females do not discriminate between mixtures of 3kPZS and PZS applied at ratios typical of larval versus male odorants. Our results indicate females discriminate between the sexual and nonsexual sources of 3kPZS during but not outside of mating and show sensory traps can lead to reliable sexual communication without females shifting their responses in the original context.

Evolution of a chordate-specific mechanism for myoblast fusion.

Vertebrate myoblast fusion allows for multinucleated muscle fibers to compound the size and strength of mononucleated cells, but the evolution of this important process is unknown. We investigated the evolutionary origins and function of membrane-coalescing agents Myomaker and Myomixer in various groups of chordates. Here, we report that Myomaker likely arose through gene duplication in the last common ancestor of tunicates and vertebrates, while Myomixer appears to have evolved de novo in early vertebrates. Functional tests revealed a complex evolutionary history of myoblast fusion. A prevertebrate phase of muscle multinucleation driven by Myomaker was followed by the later emergence of Myomixer that enables the highly efficient fusion system of vertebrates. Evolutionary comparisons between vertebrate and nonvertebrate Myomaker revealed key structural and mechanistic insights into myoblast fusion. Thus, our findings suggest an evolutionary model of chordate fusogens and illustrate how new genes shape the emergence of novel morphogenetic traits and mechanisms.

Diel Patterns of Pheromone Release by Male Sea Lamprey.

Costs to producing sexual signals can create selective pressures on males to invest signaling effort in particular contexts. When the benefits of signaling vary consistently across time, males can optimize signal investment to specific temporal contexts using biological rhythms. Sea lamprey, Petromyzon marinus, have a semelparous life history, are primarily nocturnal, and rely on pheromone communication for reproduction; however, whether male investment in pheromone transport and release matches increases in spawning activity remains unknown. By measuring (1) 3keto-petromyzonol sulfate (3kPZS, a main pheromone component) and its biosynthetic precursor PZS in holding water and tissue samples at six points over the course of 24 hours and (2) 3kPZS release over the course of several days, we demonstrate that 3kPZS release exhibits a consistent diel pattern across several days with elevated pheromone release just prior to sunset and at night. Trends in hepatic concentrations and circulatory transport of PZS and 3kPZS were relatively consistent with patterns of 3kPZS release and suggest the possibility of direct upregulation in pheromone transport and release rather than observed release patterns being solely a byproduct of increased behavioral activity. Our results suggest males evolved a signaling strategy that synchronizes elevated pheromone release with nocturnal increases in sea lamprey behavior. This may be imperative to ensure that male signaling effort is not wasted in a species having a single, reproductive event.

Bile acid production is life-stage and sex-dependent and affected by primer pheromones in the sea lamprey.

Pheromonal bile salts are important for sea lampreys (Petromyzon marinus Linnaeus) to complete their life cycle. The synthesis and release of a releaser/primer pheromone 3-keto petromyzonol sulfate (3kPZS) by spermiating males have been well characterized. 3kPZS evokes sexual behaviors in ovulatory females, induces immediate 3kPZS release in spermiating males, and elicits neuroendocrine responses in prespawning adults. Another primer pheromone released by spermiating males, 3-keto allocholic acid (3kACA), antagonizes the neuroendocrine effects of 3kPZS in prespermiating males. However, the effects of 3kACA and 3kPZS on pheromone production in prespawning adults is unclear. To understand the foundation of pheromone production, we examined sea lamprey bile salt levels at different life stages. To investigate the priming effects of 3kACA and 3kPZS, we exposed prespawning adults with vehicle or synthetic 3kACA or 3kPZS. We hypothesized that endogenous bile salt levels were life-stage and sex-dependent, and differentially affected by 3kACA and 3kPZS in prespawning adults. Using ultra-performance liquid chromatography tandem mass spectrometry, we found that sea lampreys contained distinct mixtures of bile salts in the liver and plasma at different life stages. Males usually contained higher amounts of bile salts than females. Petromyzonamine disulfate was the most abundant C27 bile salt and petromyzonol sulfate was the most abundant C24 bile salt. Waterborne 3kACA and 3kPZS exerted differential effects on bile salt production in the liver and gill, their circulation and clearance in the plasma, and their release into water. We conclude that bile salt levels are life-stage and sex-dependent and differentially affected by primer pheromones.

Sex-dependent pheromonal effects on steroid hormone levels in sea lampreys (Petromyzon marinus).

Sea lampreys (Petromyzon marinus) are basal vertebrates that exhibit reproductive control via a hypothalamic-pituitary-gonadal axis. The function and evolution of the hypothalamic and pituitary peptide hormones are well studied in this species, whereas the functions of classical sex steroid hormones have not been well established due to their low or non-detectable plasma levels. Sea lamprey pheromone 3-keto petromyzonol sulfate (3kPZS) has been shown to increase while 3-keto allocholic acid (3kACA) decreases plasma 15α-hydroxyprogesterone (15αP) levels in prespermiating males (PSM) but not in preovulatory females (POF). However, spermiating male washings that contain both 3kPZS and 3kACA facilitate spawning in both sexes. Therefore, we wondered if the effects of pheromones on POF were elicited by classical steroid hormones such as progesterone, androstenedione, testosterone and estradiol. We hypothesized that waterborne 3kACA and 3kPZS differentially alter steroid hormone levels in prespawning sea lampreys. We determined the sex differences and pheromonal effects on steroid hormone levels in prespawning sea lampreys using sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods. Some PSM became spermiating (SM) at the time of sample collection, and those data were treated as a separate group. We found that males contained more androstenedione and progesterone in the gonad and more estradiol and testosterone in the plasma than POF, whereas POF contained more gonadal testosterone than males (no detectable gonadal testosterone). In POF, 3kPZS decreased gonadal androstenedione but increased gonadal progesterone and plasma estradiol, whereas 3kACA only increased gonadal progesterone levels. Exposure to 3kPZS for 4 h increased plasma 15αP in POF and SM, and gonadal 15αP in POF. Interestingly, 3kACA or 3kPZS depleted gonadal or plasma 15αP in PSM at various time points. On the other hand, both pheromones had no significant effect on androstenedione, progesterone or estradiol levels in males. Plasma testosterone levels did not change after pheromone exposure in both sexes. We conclude that sea lamprey pheromones 3kACA and 3kPZS induced differential steroidal responses in POF, PSM and SM.

Evidence that male sea lamprey increase pheromone release after perceiving a competitor.

Sexual signals evolve via selective pressures arising from male-male competition and female choice, including those originating from unintended receivers that detect the signal. For example, males can acquire information from other males signaling to females and alter their own signal. Relative to visual and acoustic signals, less is known about how such communication networks influence chemical signaling among animals. In sea lamprey (Petromyzon marinus), the chemical communication system is essential for reproduction, offering a useful system to study a pheromone communication network that includes signalers and both intended and unintended receivers. Male sea lamprey aggregate on spawning grounds, where individuals build nests and signal to females using sex pheromones. We examined how exposure to a major component of the male pheromone, 3keto-petromyzonol sulfate (3kPZS), influenced male pheromone signaling, and whether females had a preference for males that altered their signal. Exposure to 3kPZS, at a concentration of 5×10-10 mol l-1, simulated the presence of other male(s) and led to increased 3kPZS release rates within 10 min, followed by a return to baseline levels within 30 min. Exposure also led to increases in hepatic synthesis and circulatory transport of pheromone components. In behavioral assays, females preferred the odor of males that had been exposed to 3kPZS; therefore, males likely benefit from upregulating 3kPZS release after detecting competition for mates. Here, we define how a specific pheromone component influences chemical signaling during intrasexual competition, and show a rare example of how changes in chemical signaling strategies resulting from male competition may influence mate choice.

A pheromone antagonist liberates female sea lamprey from a sensory trap to enable reliable communication.

The evolution of male signals and female preferences remains a central question in the study of animal communication. The sensory trap model suggests males evolve signals that mimic cues used in nonsexual contexts and thus manipulate female behavior to generate mating opportunities. Much evidence supports the sensory trap model, but how females glean reliable information from both mimetic signals and their model cues remains unknown. We discovered a mechanism whereby a manipulative male signal guides reliable communication in sea lamprey (Petromyzon marinus). Migratory sea lamprey follow a larval cue into spawning streams; once sexually mature, males release a pheromone that mimics the larval cue and attracts females. Females conceivably benefit from the mimetic pheromone during mate search but must discriminate against the model cue to avoid orienting toward larvae in nearby nursery habitats. We tested the hypothesis that spawning females respond to petromyzonol sulfate (PZS) as a behavioral antagonist to avoid attraction to the larval cue while tracking the male pheromone despite each containing attractive 3-keto petromyzonol sulfate (3kPZS). We found 1) PZS inhibited electrophysiological responses to 3kPZS and abated preferences for 3kPZS when mixed at the same or greater concentrations, 2) larvae released more PZS than 3kPZS whereas males released more 3kPZS than PZS, and 3) mixtures of 3kPZS and PZS applied at ratios measured in larval and male odorants resulted in the discrimination observed between the natural odors. Our study elucidates how communication systems that arise via deception can facilitate reliable communication.

Waterborne pheromones modulate gonadotropin-inhibitory hormone levels in sea lamprey (Petromyzon marinus).

The relationships between pheromone stimuli and neuropeptides are not well established in vertebrates due to the limited number of unequivocally identified pheromone molecules. The sea lamprey (Petromyzon marinus) is an advantageous vertebrate model to study the effects of pheromone exposure on neuropeptides since many pheromone molecules and neuropeptides have been identified in this species. Sexually mature male sea lamprey release pheromones 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3 keto-petromyzonol sulfate, 3kPZS) and 7α, 12α-dihydroxy-5α-cholan-3-one-24-oic acid (3-keto allocholic acid, 3kACA) that differentially regulate gonadotropin-releasing hormone (lGnRH) and steroid levels in sexually immature sea lamprey. However, the effects of these pheromones on gonadotropin-inhibitory hormones (GnIHs), hypothalamic neuropeptides that regulate lGnRH release, are still elusive. In this report, we sought to examine the effects of waterborne pheromones on lamprey GnIH-related neuropeptide levels in sexually immature sea lamprey. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analyses revealed sex differences in GnIH-related neuropeptide levels in the brain and plasma of immature sea lamprey. Exposure to 3kPZS and 3kACA exerted differential effects on GnIH-related neuropeptide levels in both sexes, but the effects were more prominent in female brains. We conclude that sea lamprey pheromones regulate GnIH-related neuropeptide levels in a sexually dimorphic manner.

Petromylidenes A⁻C: 2-Alkylidene Bile Salt Derivatives Isolated from Sea Lamprey (Petromyzon marinus).

Three novel bile acid derivatives, petromylidenes A⁻C (1⁻3), featuring uncommon alkylidene adductive scaffolds, were isolated from water conditioned with sexually mature male sea lampreys (Petromyzon marinus). Their structures were elucidated by mass spectrometry and NMR spectroscopy, and by comparison to spectral data of related structures. The identification of compounds 1⁻3, further illustrates the structural diversity of the 5α bile salt family. Compounds 1⁻3 exhibited notable biological properties as well, including high olfactory potencies in adult sea lampreys and strong behavioral attraction of ovulated female sea lampreys. Electro-olfactogram recordings indicated that the limit of detection for 1 was 10-9 M, 2 was 10-11 M, and 3 was less than 10-13 M. These results suggested 1⁻3 were likely male pheromones, which guide reproductive behaviors in the sea lamprey.

High-performance liquid chromatography quantification of enantiomers of a Dihydroxylated tetrahydrofuran natural product.

Both enantiomers of petromyroxol are putative pheromones in sea lamprey (Petromyzon marinus). Here, we describe the separation and quantification of the petromyroxol enantiomers using high-performance liquid chromatography tandem mass spectrometry. The separation was tested on a wide range of chiral columns with normal phases, and effects of the chromatographic parameters such as mobile phase and temperature on the separation were optimized. The AD-H column showed the best separation of enantiomers with n-hexane and ethanol as the mobile phase. The enantiomers were detected by multiple reaction monitoring with a positive atmospheric-pressure chemical ionization on triple quadrupole mass spectrometer. Validation revealed that the method was specific, accurate, and precise. The validated method was applied to measure the amount of petromyroxol enantiomers in water conditioned with sea lamprey larvae, the source of the putative pheromone. This method will be applied in quantifying the natural scalemic petromyroxol mixture, enabling further investigations of a rare non-racemic enantiomeric pheromone mixture in a vertebrate species.

Bile Salt-like Dienones Having a Novel Skeleton or a Rare Substitution Pattern Function as Chemical Cues in Adult Sea Lamprey.

Two novel sulfated bile salt-like dienones, featuring either a unique, rearranged side chain or a rare cis-11,12-diol on the steroidal C-ring, herein named petromyzene A (1) and B (2), respectively, were isolated from water conditioned with spawning male sea lamprey (Petromyzon marinus; a jawless vertebrate animal). The structures of these natural products were elucidated by mass spectrometry and NMR spectroscopy. Petromyzenes A and B exhibited high olfactory potency for adult sea lamprey and strong behavioral attraction for spawning females.

Three Novel Bile Alcohols of Mature Male Sea Lamprey (Petromyzon marinus) Act as Chemical Cues for Conspecifics.

Sea lamprey, Petromyzon marinus, rely heavily on chemical cues that mediate their life history events, such as migration and reproduction. Here, we describe petromyzone A-C (1-3), three novel bile alcohols that are highly oxidized and sulfated, isolated from water conditioned with spermiated male sea lamprey. Structures of these compounds were unequivocally established by spectroscopic analyses and by comparison with spectra of known compounds. Electro-olfactogram recordings showed that 1 at 10-11 M was stimulatory to the adult sea lamprey olfactory epithelium, while 2 and 3 were stimulatory at 10-13 M. Behavioral assays indicated that 1 is attractive, 2 is not attractive or repulsive, and 3 is repulsive to ovulated female sea lamprey. The results suggest that 1 and 2 may be putative pheromones that mediate chemical communication in sea lamprey. The identification of these three components enhances our understanding of the structures and functions of sex pheromone components in this species and may provide useful behavioral manipulation tools for the integrated management of sea lamprey, a destructive invader in the Laurentian Great Lakes.

A validated LC-MS/MS method for thyroid hormone determination in sea lamprey (Petromyzon marinus) plasma, gill, kidney and liver.

A UPLC-MS/MS method was developed to provide a reproducible, sensitive and quantitative assay to determine thyroid hormones in sea lamprey tissues and plasma. l-Thyroxine (T4) and its two triiodo-thyronine isomers have been simultaneously quantified and validated for plasma, gill, liver, and kidney matrices. Multiple sample preparation techniques were investigated to achieve optimal sample matrix digestion and clean-up. Enzymatic digestion followed by protein precipitation was selected to process the samples. The developed method exhibited excellent linearity for all analytes with regression coefficients higher than 0.99 for concentrations ranged from 10 to 50,000pg/mL. The limit of detection (LOD) was under 1pg/mL while the limit of quantification (LOQ) was estimated as 10pg/mL. This method was validated according to the FDA guidance and applied to determine thyroid hormone levels in plasma, gill and kidney of sea lamprey exposed to a sex pheromone. With appropriate implementation and further validation, this method could be applied to tissues in other species including humans.

Quantification of 15 bile acids in lake charr feces by ultra-high performance liquid chromatography-tandem mass spectrometry.

Many fishes are hypothesized to use bile acids (BAs) as chemical cues, yet quantification of BAs in biological samples and the required methods remain limited. Here, we present an UHPLC-MS/MS method for simultaneous, sensitive, and rapid quantification of 15 BAs, including free, taurine, and glycine conjugated BAs, and application of the method to fecal samples from lake charr (Salvelinus namaycush). The analytes were separated on a C18 column with acetonitrile-water (containing 7.5mM ammonium acetate and 0.1% formic acid) as mobile phase at a flow rate of 0.25mL/min for 12min. BAs were monitored with a negative electrospray triple quadrupole mass spectrometer (Xevo TQ-S™). Calibration curves of 15 BAs were linear over the concentration range of 1.00-5,000ng/mL. Validation revealed that the method was specific, accurate, and precise. The method was applied to quantitative analysis of feces extract of fry lake charr and the food they were eating. The concentrations of analytes CA, TCDCA, TCA, and CDCA were 242.3, 81.2, 60.7, and 36.2ng/mg, respectively. However, other taurine conjugated BAs, TUDCA, TDCA, and THDCA, were not detected in feces of lake charr. Interestingly, TCA and TCDCA were detected at high concentrations in food pellets, at 71.9 and 38.2ng/mg, respectively. Application of the method to feces samples from lake charr supported a role of BAs as chemical cues, and will enhance further investigation of BAs as chemical cues in other fish species.