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

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

Diel Patterns of Pheromone Release by Male Sea Lamprey

Synopsis 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

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

Extensive DNA methylome rearrangement during early lamprey embryogenesis

Abstract 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