•Determination of oxosteroids using quaternary aminooxy derivatization and UPLC-MS/MS.•Method validation on sea lamprey brain and plasma according to FDA guidelines.•Application to real samples reveals biological changes on neurosteroid levels.This article describes the development and validation of a sensitive and robust method for the determination of neurosteroids in sea lamprey, an ancestral animal in vertebrate evolution. Chemical derivatization was used to enhance the detection of neurosteroids containing ketone function by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Iminooxy derivatives of 12 oxosteroids and three internal standards were monitored by positive electrospray tandem mass spectrometry using the neutral loss of sulfate. Limit of quantification, extraction recovery and matrix effect were first evaluated and SPE using C18 sorbent was selected after comparison with liquid/liquid extraction and protein precipitation. Matrix effect ranged from 89.6% to 113.1% in plasma and from 79.8% to 100.0% in the brain. Recovery values ranged from 80.0% to 103.8% in plasma and from 86.3% to 107.9% in the brain. Chromatographic separation was achieved by reverse phase chromatography (2.1 mm×100 mm, 1.7 µm particle size, C18) with a binary gradient between methanol and 0.1% formic acid in water. Limit of quantification ranged from 5 to 10 pg/mL and was up to 80 times lower than those for non-derivatized steroids. Accuracy and precision parameters were determined and inter- and intra-day at three concentrations: 50, 500 and 5000 pg/mL. This method was applied to analyze real samples. progesterone (P), pregnenolone (P5), 7-hydroxy-pregnenolpne (7P5), 17-hydroxy-pregnenolpne (17P5)dehydroepiandrosterone (DHEA), androstenedienone (A4), testosterone (T), dihydrotestosterone (DHT), allopregnanolone (THP), 11-hydroxy-androstenedienone (11A4) and 11-Deoxycortisol (S) were measured in sea lamprey brain and plasma matrixes.

(+)- and (−)-Petromyroxol [(+)-1 and (−)-1, respectively], two novel tetrahydrofuran (THF)-diol fatty acid enantiomers, were isolated from water conditioned with larval sea lamprey. We herein describe their isolation and subsequent resolution using chiral chromatography. The absolute configuration of each enantiomer was determined by a combination of Mosher ester analysis and comparison with related natural and synthetic products. Electro-olfactogram (EOG) assays indicated that (+)-petromyroxol (1) possesses potent olfactory activity for sea lamprey.

This is the first study to use electrochemistry to generate a nitro reduction metabolite as a standard for a liquid chromatography–mass spectrometry-based quantitative assay. This approach is further used to quantify 3-trifluoromethyl-4-nitrophenol (TFM) reductive metabolism. TFM is a widely used pesticide for the population control of sea lamprey (Petromyzon marinus), an invasive species of the Laurentian Great Lakes. Three animal models, sea lamprey, lake sturgeon (Acipenser fulvescens), and rainbow trout (Oncorhynchus mykiss), were selected to evaluate TFM reductive metabolism because they have been known to show differential susceptibilities to TFM toxicity. Amino-TFM (aTFM; 3-trifluoromethyl-4-aminophenol) was the only reductive metabolite identified through liquid chromatography–high-resolution mass spectrometry screening of liver extracts incubated with TFM and was targeted for electrochemical synthesis. After synthesis and purification, aTFM was used to develop a quantitative assay of the reductive metabolism of TFM through liquid chromatography and tandem mass spectrometry. The concentrations of aTFM were measured from TFM-treated cellular fractions, including cytosolic, nuclear, membrane, and mitochondrial protein extracts. Sea lamprey extracts produced the highest concentrations (500 ng/mL) of aTFM. In addition, sea lamprey and sturgeon cytosolic extracts showed concentrations of aTFM substantially higher than thoose of rainbow trout. However, other fractions of lake sturgeon extracts tend to show aTFM concentrations similar to those of rainbow trout but not with sea lamprey. These data suggest that the level of reductive metabolism of TFM may be associated with the sensitivities of the animals to this particular pesticide.

Chemical cues and pheromones guide decisions in organisms throughout the animal kingdom. The neurobiology, function, and evolution of olfaction are particularly well described in insects, and resulting concepts have driven novel approaches to pest control. However, aside from several exceptions, the olfactory biology of vertebrates remains poorly understood. One exception is the sea lamprey (Petromyzon marinus), which relies heavily upon olfaction during reproduction. Here, we provide a broad review of the chemical cues and pheromones used by the sea lamprey during reproduction, including overviews of the sea lamprey olfactory system, chemical cues and pheromones, and potential applications to population management. The critical role of olfaction in mediating the sea lamprey life cycle is evident by a well-developed olfactory system. Sea lamprey use chemical cues and pheromones to identify productive spawning habitat, coordinate spawning behaviors, and avoid risk. Manipulation of olfactory biology offers opportunities for management of populations in the Laurentian Great Lakes, where the sea lamprey is a destructive invader. We suggest that the sea lamprey is a broadly useful organism with which to study vertebrate olfaction because of its simple but well-developed olfactory organ, the dominant role of olfaction in guiding behaviors during reproduction, and the direct implications for vertebrate pest management.

•A new UHPLC–MS/MS is developed to assay bile acids in feces.•The liquid–liquid extraction is applied for sample treatment.•The assay is applicable for studying bile acids release by fish function as chemical cues.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 7.5 mM ammonium acetate and 0.1% formic acid) as mobile phase at a flow rate of 0.25 mL/min for 12 min. 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 1.00–5,000 ng/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 36.2 ng/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 38.2 ng/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.

•Peptides profile and target peptides identification in complex biological samples.•Lamprey GnRH peptides characterization by high resolution Q-TOF MS.•Endogenous GnRH peptides quantification by a fully validated LC–MS/MS method in brain tissues and plasma.•Reported firstly for simultaneous quantification of three lGnRHs in sea lamprey brain tissues and plasma.Gonadotropin-releasing hormones (GnRH) are neuropeptide hormones that regulate reproduction in vertebrates. Twenty-five unique chordate GnRH isoforms have been identified, all with very similar molecular architecture. Identification and quantification of endogenous GnRH in biological samples is extremely challenging due to the high levels of sequence similarity among these GnRH peptides and complexity of the biological matrices laden with large numbers of other peptides and protein degradation fragments, and due to low levels of GnRH abundance. In this study, three lamprey GnRH (lGnRH-I, -II, and -III) were extracted from sea lamprey brain tissue and plasma samples by solid-phase extraction (SPE) and identified by a high resolution Q-TOF mass spectrometry (MS). A rapid quantitation method was developed and validated to determine the concentrations of these three lGnRHs by using a UPLC coupled tandem MS in positive ESI multiple reaction monitoring (MRM) mode. Luteinizing hormone-release hormone (LHRH, one of the mammalian GnRHs) was used as the internal standard. The developed quantitation method was fully validated for its recovery, matrix effect, linearity, repeatability, precision and accuracy, and storage stability. This method exhibited excellent linearity in a broad concentration range for all three lGnRHs (R2 > 0.99) and limits of detection (LOD; as low as 0.007 ng/mL). Brain and plasma samples from a total of 280 sea lampreys were analyzed with the developed method to investigate the biological relevance of the lGnRH levels. The concentrations of these three lGnRHs were detected at levels of pictogram per microgram brain tissue and milliliter of plasma. The obtained analytical performance parameters and collected data from real biological samples have proven that this is a robust, sensitive, and fully validated LC–MS/MS method to simultaneously quantify three neuropeptide hormones in complex biological matrices.

Sulfated bile salts function as chemical cues that coordinate reproduction in sea lamprey, Petromyzon marinus. 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3kPZS) is the most abundant known bile salt released by sexually mature male sea lampreys and attracts ovulated females. However, previous studies showed that the male-produced pheromone consists of unidentified components in addition to 3kPZS. Here, analysis of water conditioned with mature male sea lampreys indicated the presence of 4 oxidized, unsaturated compounds with molecular weights of 466 Da, 468 Da, and 2 of 470 Da. These compounds were not detectable in water conditioned with immature male sea lampreys. By using mass spectrometry, 4 A-ring unsaturated sulfated bile salts were tentatively identified from male washings as 2 4-ene, a 1-ene, and a 1,4-diene analogs. These were synthesized to determine if they attracted ovulated female sea lampreys to spawning nests in natural streams. One of the novel synthetic bile salts, 3 keto-1-ene PZS, attracted ovulated females to the point of application at a concentration of 10−12 M. This study reveals the structural diversity of bile salts in sea lamprey, some of which have been demonstrated to be pheromonal cues.

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.

We present a comparison of two sensitive methods, HPLC with fluorescence detector (HPLC/FLD) and UPLC with electrospray tandem mass spectrometry (UPLC/MS/MS), for the determination of indoleamine neurotransmitters (NTs) and their metabolites in sea lamprey plasma samples. Liquid–liquid extraction (LLE) and solid-phase extraction (SPE) were also tested for recovery and matrix effect. The recoveries of SPE determined by HPLC/FLD and UPLC/MS/MS ranged from 75 to 123% and 78 to 105%, respectively, while the recoveries of LLE ranged from 45 to 73% and 48 to 75%, respectively. SPE combined with HPLC/FLD and UPLC/MS/MS to determine the target analytes in plasma samples were validated of the sensitivity, reproducibility, accuracy and precision. Both methods exhibited excellent linearity in the range of 0.2–50 ng mL−1 for all analytes. The limits of detection (LOD) varied from 0.04 ng mL−1 to 0.13 ng mL−1 for HPLC/FLD method and 0.003 ng mL−1 to 0.02 ng mL−1 for UPLC/MS/MS method. The inter-day accuracy ranged from 82.5 to 127.0% for HPLC/FLD and 93.0 to 113.0% for UPLC/MS/MS. The inter-day precision ranged from 9.9 to 32.3% for HPLC/FLD and 5.4 to 13.2% for UPLC/MS/MS. These results demonstrated that the values obtained by both methods were within the satisfactory range and the UPLC/MS/MS method provided more accurate and precise measurements than HPLC/FLD method. The comparison is of great importance to determine the available detectors, considering the complexity and expensiveness versus quality parameters. These two methods were applied to the analysis of four important indoleamine neurotransmitter analytes (5-hydroxytryptamine, 5-hydroxyindole-3-acetic acid, tryptamine and melatonin) in sea lamprey plasma samples.Graphical abstractSensitivity comparison of UPLC–MS/MS and HPLC/FLD method for the determination of melatonin.Highlights► Compared two sensitive analytical methods: HPLC/FLD and UPLC/MS/MS. ► Reported firstly on the measurements of neurotransmitters in the sea lamprey by two methods. ► Studied recovery and matrix effects of SPE and LLE. ► Determined four indoleamine neurotransmitters. ► Analyzed plasma samples from sea lampreys.

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 1 mM 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.Highlights► Developed and validated a rapid LC–MS/MS method. ► Determined three monoamine neurotransmitters and melatonin in brain tissues. ► Studied recovery and matrix effects of different sample pretreatment methods. ► Analyzed brain tissues from a total of 360 sea lampreys. ► Reported firstly on the measurements of neurotransmitters and melatonin in the sea lamprey.

Bile salt synthesis is a specialized liver function in vertebrates. Bile salts play diverse roles in digestion and signaling, and their homeostasis is maintained by controlling input (biosynthesis) and intestinal conservation. Patients with biliary atresia (i.e., obliteration of the biliary tree) suffer liver fibrosis and cirrhosis. In contrast, sea lamprey thrives despite developmental biliary atresia. We discovered that the sea lamprey adapts to biliary atresia through a unique mechanism of de novo synthesis and secretion of bile salts in intestine after developmental biliary atresia, in addition to known mechanisms, such as the reduction of bile salt synthesis in liver. During and after developmental biliary atresia, expression of cyp7a1 in intestine increased by more than 100-fold (P < 0.001), whereas in liver it decreased by the same magnitude (P < 0.001). Concurrently, bile salt pools changed in similar patterns and magnitudes in these two organs and the composition shifted from C24 bile alcohol sulfates to taurine-conjugated C24 bile acids. In addition, both in vivo and ex vivo experiments showed that aductular sea lamprey secreted taurocholic acid into its intestinal lumen. Our results indicate that the sea lamprey, a jawless vertebrate, may be in an evolutionarily transitional state where bile salt synthesis occurs in both liver and intestine. Understanding the molecular basis of these mechanisms may shed light on the evolution of bile salt synthesis and possible therapy for infant biliary atresia.

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 [2H5]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.

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 ([2H5]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 R2 ≥ 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.Highlights► Simultaneous measurement of bile acid derivatives in environmental water and animal conditioned samples. ► Using of MCX cartridge in sample process resulted in excellent recoveries. ► UHPLC/MS/MS accomplished high-resolution separation and provided lower LODs. ► The developed method is an appealing methodology for routine analysis.

Corticosteroid hormones are critical for controlling metabolism, hydromineral balance, and the stress response in vertebrates. Although corticosteroid hormones have been well characterized in most vertebrate groups, the identity of the earliest vertebrate corticosteroid hormone has remained elusive. Here we provide evidence that 11-deoxycortisol is the corticosteroid hormone in the lamprey, a member of the agnathans that evolved more than 500 million years ago. We used RIA, HPLC, and mass spectrometry analysis to determine that 11-deoxycortisol is the active corticosteroid present in lamprey plasma. We also characterized an 11-deoxycortisol receptor extracted from sea lamprey gill cytosol. The receptor was highly specific for 11-deoxycortisol and exhibited corticosteroid binding characteristics, including DNA binding. Furthermore, we observed that 11-deoxycortisol was regulated by the hypothalamus–pituitary axis and responded to acute stress. 11-Deoxycortisol implants reduced sex steroid concentrations and up-regulated gill Na⁺, K⁺-ATPase, an enzyme critical for ion balance. We show here that 11-deoxycortisol functioned as both a glucocorticoid and a mineralocorticoid in the lamprey. Our findings indicate that a complex and highly specific corticosteroid signaling pathway evolved at least 500 million years ago with the arrival of the earliest vertebrate.

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.

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, 7α, 12α, 24-trihydroxy-5α-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 ≈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.

The sea lamprey (Petromyzon marinus) is one of the earliest extant vertebrates for which the hypothalamic-pituitary-gonadal (HPG) axis has been shown to control and regulate reproduction in a similar fashion to gnathostome vertebrates. While the two forms of gonadotropin-releasing hormones in the sea lamprey (GnRH I and GnRH III) have been studied extensively, their in vivo effect on synthesis of 15α-hydroxytestosterone (15α-T) and 15α-hydroxyprogesterone (15α-P) have only been partially characterized. In the present study, plasma concentrations of 15α-T and 15α-P were measured in prespermiating sea lampreys that were given a single injection of either GnRH I or GnRH III in doses ranging from 5 to 100 μg/kg, or of pituitary extract (as a source of gonadotropin). Plasma was sampled at 1–6 h and 6–48 h post-injection, in separate experiments, in order to characterize the peak and duration of responses. 15α-T plasma concentrations increased slightly in response to all three treatments, but not in a dose-dependent manner, and the timing of peak concentrations varied between doses. However, 15α-P plasma concentrations showed a greater range of response (between 1 and 100 ng/ml) and were clearly correlated with the injection dose. Plasma concentrations of 15α-P also responded to far lower doses of GnRH I and GnRH III than any other steroid previously investigated in lampreys. The plasma concentrations of 15α-P peaked at 6 h after injection for all three treatments, and levels reached a mean of 53.1 ng/ml. In female lampreys that were injected twice with 50 μg/ml GnRH I or III, 15α-T concentrations did not exceed 0.5 ng/ml and 15α-P concentrations did not exceed 1 ng/ml. These results lend further support to the hypothesis that 15α-P plays an important role in the reproductive endocrinology of male lampreys.

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.Highlights► Petromyzestrosterol was isolated from the sexually mature male sea lamprey. ► The structure and configuration were determined by MS and NMR analysis. ► Petromyzestrosterol is the first steroidal tetrol estrogen from the sea lamprey. ► The sensitivity of olfactory system to Petromyzestrosterol was determined.

The Great Lakes Fishery Commission (GLFC) considers the application of sea lamprey pheromones a promising alternative-control method for its sea lamprey management program. Several components of two pheromones that regulate migration and reproduction, respectively, have been identified and synthesized. Potential utility of these pheromone compounds in lamprey management have been demonstrated in a series of field experiments. These discoveries have laid a solid foundation for development of pheromone-based management. In order to identify potential strategies that will be practical, effective, and economical, we propose a hypothesis driven approach that integrates concepts and experimental methods from several disciplines of biological science, such as neurobiology, biochemistry, and behavioral ecology to illustrate the exact function of identified compounds. In the interim, we identify the necessary steps, or issues critical to eventual implementation, to charter a pathway that leads from laboratory research to effective deployment of pheromones. Finally, we highlight a strategy that fosters collaboration among scientists across disciplines, as well as among research institutes and lamprey control agencies, to accomplish this research agenda.

•Optimization of sample preparation for endogenous thyroid hormones.•Method validation in gill, liver, plasma and kidney of sea lamprey.•T3, rT3 and T4 determined in sea lamprey, a vertebrate model for biliary atresia.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 50,000 pg/mL. The limit of detection (LOD) was under 1 pg/mL while the limit of quantification (LOQ) was estimated as 10 pg/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.

•Optimized cortisol extraction and clean-up from fish eggs matrix.•Liquid chromatography method validated for routine analysis.•Cortisol determined in fish eggs as a biomarker of stress in lake sturgeon.Quantifying cortisol concentrations in fish eggs is important to understand the effects of environmental conditions on maternal physiological condition and on egg provisioning and quality. Data are particularly relevant to studies of the ecology of threatened species such as lake sturgeon (Aciperser fulvescens) as well as assessments of larval physical and behavioral phenotypes, fish health and caviar quality in sturgeon aquaculture. This study focuses on development of bioanalytical methods for high sensitivity and robust determination of cortisol in sturgeon eggs. Sample preparation was optimized after investigating protein precipitation and liquid-liquid extraction techniques. Ethyl acetate was found to be the most efficient solvent (recovery parameter) and also provided the best sample clean up (matrix effect parameter). The method was determined to be linear for cortisol concentrations between 0.025 and 100 ng/mL. The limits of detection and quantification were 0.025 and 0.1 ng/mL respectively. Intra- and inter-day performances of the method were validated at three concentrations (0.25; 10 and 100 ng/mL). The method was applied to field-collected samples for the determination of endogenous cortisol in lake sturgeon eggs. Cortisol was detected in all egg samples and statistical analysis showed significant differences between fertilized and non-fertilized eggs.

•Differential expression of PGRMC1 and nPR in target tissues at different life stage.•Differential response of PGRMC1 and nPR to lGnRH-I and -III.•P (or 15α-P) regulates the production of male sex pheromone in sea lamprey.•Increases in nPR and PGRMC1 gene expressions correlate with the late stages of sexual maturation in male sea lamprey.•Progesterone accelerates the onset of spermiation in sea lamprey.Progestins (progestogens, C21 steroids) have been shown to regulate key physiological activities for reproduction in both sexes in all classes of vertebrates except for Agnathans. Progesterone (P) and 15α-hydroxyprogesterone (15α-P) have been detected in sea lamprey (Petromyzon marinus) plasma, but the expression patterns and functions of putative progestin receptor genes have not yet been investigated. The first objective of this study was to determine the differences in mRNA expression levels of nuclear progestin receptor (nPR) and the membrane receptor adaptor protein ‘progesterone receptor membrane component 1’ (pgrmc1) in putative target tissues in males at different life stages, with and without lamprey GnRH-I and -III treatment. The second objective was to demonstrate the function of progestins by implanting prespermiating males (PSM) with time-release pellets of P and measuring the latency to the onset of spermiation and plasma concentrations of sex pheromones and steroids. The third objective was to measure the binding affinity of P in the nuclear and membrane fractions of the target tissues. Expression levels of nPR and pgrmc1 differed between life stages and tissues, and in some cases were differentially responsive to lamprey GnRH-I and -III. Increases in nPR and pgrmc1 gene expressions were correlated to the late stages of sexual maturation in males. The highest expression levels of these genes were found in the liver and gill of spermiating males. These organs are, respectively, the site of production and release of the sex pheromone 3 keto-petromyzonol sulfate (3kPZS). The hypothesis that pheromone production may be under hormonal control was tested in vivo by implanting PSM with time-release pellets of P. Concentrations of 3kPZS in plasma after 1 week were 50-fold higher than in controls or in males that had been implanted with androstenedione, supporting the hypothesis that P is responsible for regulating the production of the sex pheromone. P treatment also accelerated the onset of spermiation. Saturation and Scatchard analyses of the target tissues showed that both nuclear and membrane fractions bound P with high affinity and low capacity (KD 0.53 pmol/g testis and 0.22 pmol/g testis, and Bmax 1.8 and 5.7 nM, respectively), similar to the characteristics of nPR and mPR in other fish. The fact that a high proportion of P was also converted in vivo to 15α-P means that it is not yet possible to determine which of these two steroids is the natural ligand in the sea lamprey.