Anne M. Scott

Quantification of monoamine neurotransmitters and melatonin in sea lamprey brain tissues by high performance liquid chromatography-electrospray ionization tandem mass spectrometry

A rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of three monoamine neurotransmitters (NTs) and melatonin in sea lamprey brain tissues. Separation was performed on a reversed-phase column with mobile phases of 1mM perfluoroheptanoic acid (PFHA) water solution/acetonitrile and mass spectra were acquired in positive electrospray ionization multiple reaction monitoring (MRM) mode. Solid-phase extraction (SPE) was employed to purify and extract the target compounds from the tissue samples. The matrix effects as well as the influence of two extraction methods, protein precipitation (PPT) and SPE, on matrix effects were examined for the first time on the quantification of NTs from brain tissue extracts. The matrix effects with SPE (2.4 to -14.9%) were about 30% lower on average than those with the ACN PPT method (-29 to -38%). The recoveries of three types of SPE cartridges were tested and Bond-Elut C18 cartridge was selected to process the samples because of its good extraction efficiencies (71.3-95.3%) and low matrix effects (-6.6 to -14.9%) for all four analytes. This method exhibited excellent linearity for all analytes with regression coefficients higher than 0.99. The limits of detection were between 0.03 ng/mL (melatonin) and 0.14 ng/mL (norepinephrine). The precisions, expressed as coefficients of variation (CV), ranged from 4.8 to 14.1% for intra-day analyses and from 6.1 to 16.2% for inter-day analyses. Brain tissues from 360 sea lampreys were analyzed by the developed method and the concentrations for four analytes were found to be at the level of nanogram per gram of brain tissues. To our knowledge, this is the first report on the quantification of NTs and melatonin in the sea lamprey using the LC-MS/MS method.

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

BackgroundIn 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.ResultsWe show an 8000-fold increase in transcription of cyp7a1, a three-fold increase in transcription of cyp27a1, and a six-fold increase in transcription of cyp8b1 in the liver of mature male sea lamprey over immature male adults. LC–MS/MS data on tissue-specific distribution and release rates of bile salts from mature males show a high concentration of petromyzonol sulfate (PZS) in the liver and gills of mature males. 3-keto petromyzonol sulfate (3kPZS, known as a male sex pheromone) is the primary compound released from gills, suggesting a conversion of PZS to 3kPZS in the gill epithelium. The PZS to 3kPZS conversion is supported by greater expression of hsd3b7 in gill epithelium. High expression of sult2b1 and sult2a1 in gill epithelia of mature males, and tissue-specific expression of bile salt transporters such as bsep, slc10a1, and slc10a2, suggest additional sulfation and transport of bile salts that are dependent upon maturation state.ConclusionsThis report presents a rare example where specific genes associated with biosynthesis and release of a sexual pheromone are dramatically upregulated upon sexual maturation in a vertebrate. We provide a well characterized example of a complex mechanism of bile salt biosynthesis and excretion that has likely evolved for an additional function of bile salts as a mating pheromone.

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.

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.

Quantification of Oxidized and Unsaturated Bile Alcohols in Sea Lamprey Tissues by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

A sensitive and reliable method was developed and validated for the determination of unsaturated bile alcohols in sea lamprey tissues using liquid-liquid extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The liver, kidney, and intestine samples were extracted with acetonitrile and defatted by n-hexane. Gradient UHPLC separation was performed using an Acquity BEH C18 column with a mobile phase of water and methanol containing 20 mM triethylamine. Multiple reaction monitoring modes of precursor-product ion transitions for each analyte was used. This method displayed good linearity, with correlation coefficients greater than 0.99, and was validated. Precision and accuracy (RSD %) were in the range of 0.31%–5.28%, while mean recoveries were between 84.3%–96.3%. With this technique, sea lamprey tissue samples were analyzed for unsaturated bile alcohol analytes. This method is practical and particularly suitable for widespread putative pheromone residue analysis.

Pheromone 3kPZS evokes context-dependent serotonin sexual dimorphism in the brain of the sea lamprey (Petromyzon marinus).

Mature male sea lampreys (Petromyzon marinus) release a sex pheromone, 3-keto-petromyzonol sulfate (3kPZS), that induces sexually dimorphic behavioral responses in conspecifics. However, the neural mechanism of such responses is mostly unknown. We examined the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) and the expression of 5-HT1A receptors in the forebrain and brainstem of sea lamprey exposed to the vehicle (0.91 ppm methanol) or 10(-10) M 3kPZS for 2 h using high performance liquid chromatography-electrospray ionization tandem mass spectrometry, immunohistochemistry and real-time quantitative polymerase chain reaction. Exposure to 3kPZS for 2 h increased 5-HT concentration in the forebrain of adult females, whereas 5-HT was not detected in the forebrain of adult males. On the contrary, 3kPZS exposure decreased 5-HT concentration in the brainstem of adult females and had no effect in adult males. Pheromone exposure evoked context-dependent sexual dimorphism in brain 5-HT1A receptor immunoreactivity, but had no effect on 5-HT1A mRNA concentrations in the brain with 2 h exposure time. It appears that in sea lamprey pheromone 3kPZS affects the 5-HT system in the brain in a context-dependent, sexually dimorphic manner.

Fatty-acid derivative acts as a sea lamprey migratory pheromone

Significance Anadromous fishes are those that migrate from the ocean into freshwater to reproduce and need to orient toward a suitable spawning stream or risk leaving no offspring. These migrants are known to use olfactory cues to guide stream selection, but the cues’ identities are not known. Incidentally, it is well-known that stream-dwelling larval sea lampreys emit a pheromone that guides conspecific adults ascending freshwater streams. We identified this pheromone as a fatty-acid derivative that attracts migratory adults into the baited channel in a natural spawning stream. These results illustrate an olfactory mechanism whereby lampreys reliably identify and orient toward a proven spawning ground over a large spatial scale and implicate a potential strategy for sea lamprey control. Olfactory cues provide critical information for spatial orientation of fish, especially in the context of anadromous migrations. Born in freshwater, juveniles of anadromous fish descend to the ocean where they grow into adults before migrating back into freshwater to spawn. The reproductive migrants, therefore, are under selective pressures to locate streams optimal for offspring survival. Many anadromous fish use olfactory cues to orient toward suitable streams. However, no behaviorally active compounds have been identified as migratory cues. Extensive studies have shown that the migratory adult sea lampreys (Petromyzon marinus), a jawless fish, track a pheromone emitted by their stream-dwelling larvae, and, consequently, enter streams with abundant larvae. We fractionated extracts of larval sea lamprey washings with guidance from a bioassay that measures in-stream migratory behaviors of adults and identified four dihydroxylated tetrahydrofuran fatty acids, of which (+)-(2S,3S,5R)-tetrahydro-3-hydroxy-5-[(1R)-1-hydroxyhexyl]-2-furanoctanoic acid was shown as a migratory pheromone. The chemical structure was elucidated by spectroscopies and confirmed by chemical synthesis and X-ray crystallography. The four fatty acids were isomer-specific and enantiomer-specific in their olfactory and behavioral activities. A synthetic copy of the identified pheromone was a potent stimulant of the adult olfactory epithelium, and, at 5 × 10−13 M, replicated the extracts of larval washings in biasing adults into a tributary stream. Our results reveal a pheromone that bridges two distinct life stages and guides orientation over a large space that spans two different habitats. The identified molecule may be useful for control of the sea lamprey.

Automated Inference of Chemical Discriminants of Biological Activity

Ligand-based virtual screening has become a standard technique for the efficient discovery of bioactive small molecules. Following assays to determine the activity of compounds selected by virtual screening, or other approaches in which dozens to thousands of molecules have been tested, machine learning techniques make it straightforward to discover the patterns of chemical groups that correlate with the desired biological activity. Defining the chemical features that generate activity can be used to guide the selection of molecules for subsequent rounds of screening and assaying, as well as help design new, more active molecules for organic synthesis.The quantitative structure-activity relationship machine learning protocols we describe here, using decision trees, random forests, and sequential feature selection, take as input the chemical structure of a single, known active small molecule (e.g., an inhibitor, agonist, or substrate) for comparison with the structure of each tested molecule. Knowledge of the atomic structure of the protein target and its interactions with the active compound are not required. These protocols can be modified and applied to any data set that consists of a series of measured structural, chemical, or other features for each tested molecule, along with the experimentally measured value of the response variable you would like to predict or optimize for your project, for instance, inhibitory activity in a biological assay or ΔGbinding. To illustrate the use of different machine learning algorithms, we step through the analysis of a dataset of inhibitor candidates from virtual screening that were tested recently for their ability to inhibit GPCR-mediated signaling in a vertebrate.

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.

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.