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.

Sea lamprey enlightens the origin of the coupling of retinoic acid signaling to vertebrate hindbrain segmentation.

Retinoic acid (RA) is involved in antero-posterior patterning of the chordate body axis and, in jawed vertebrates, has been shown to play a major role at multiple levels of the gene regulatory network (GRN) regulating hindbrain segmentation. Knowing when and how RA became coupled to the core hindbrain GRN is important for understanding how ancient signaling pathways and patterning genes can evolve and generate diversity. Hence, we investigated the link between RA signaling and hindbrain segmentation in the sea lamprey Petromyzon marinus, an important jawless vertebrate model providing clues to decipher ancestral vertebrate features. Combining genomics, gene expression, and functional analyses of major components involved in RA synthesis (Aldh1as) and degradation (Cyp26s), we demonstrate that RA signaling is coupled to hindbrain segmentation in lamprey. Thus, the link between RA signaling and hindbrain segmentation is a pan vertebrate feature of the hindbrain and likely evolved at the base of vertebrates.

Protocol for monitoring and analyzing pheromone-mediated behavioral response of sea lamprey in a natural system.

Olfactory-mediated behaviors in fish are often examined in artificial microcosms that enable well-controlled treatments but fail to replicate environmental and social contexts. However, observing these behaviors in nature poses challenges. Here, we describe a protocol for recording sea lamprey (Petromyzon marinus) behaviors in a natural system. We describe steps for administering and verifying accurate odorant concentrations, surveying sea lamprey abundance, and tracking sea lamprey movements. We also detail procedures to analyze treatment effects on pheromone-mediated spawning in a high-density population. For complete details on the use and execution of this protocol, please refer to Scott et al.1.

TLR5M and TLR5S Synergistically Sense Flagellin in Early Endosome in Lamprey Petromyzon marinus, Switched by the N-Glycosylation Site N239.

In mammals, TLR5 functions as a homodimer to recognize bacterial flagellin on the cytomembrane. The current investigations reveal the existence of two types of TLR5, a membrane-bound PmTLR5M, and a soluble variant PmTLR5S, in lamprey (Petromyzon marinus). Although both PmTLR5M and PmTLR5S can bind flagellin, only PmTLR5M is capable of eliciting a proinflammatory response, whereas PmTLR5S can detect the flagellin and facilitate the role of PmTLR5M in early endosomes. The trafficking chaperone UNC93B1 enhances the ligand-induced signaling via PmTLR5M or the combination of PmTLR5M and PmTLR5S. PmTLR5M recruits MyD88 as an adaptor. Furthermore, chimeric receptor studies demonstrate the indispensability of the intradomain of PmTLR5M in effective activation of the proinflammatory pathway upon flagellin stimulation, and the combination of PmTLR5S with a singular intradomain in both homodimer and heterodimer ectodomain arrangements can very significantly augment the immune response. Furthermore, the flagellin binding sites between PmTLR5M and PmTLR5S are conserved, which are essential for ligand binding and signal transduction. Moreover, investigations on N-linked glycosylation modifications reveal that the N239 site in PmTLR5M and PmTLR5S plays a switch role in both flagellin binding and immune responses. In addition, PmTLR5M exhibits the high-mannose-type and complex-type N-glycosylation modifications; however, PmTLR5S shows exclusive complex-type N-glycosylation modification. The key N239 site demonstrates complex-type N-glycosylation modification. The findings address the function and mechanism of TLR5 in ligand recognition, subcellular localization, and signaling pathway in lowest vertebrate and immune system transition species, highlight the regulatory role of N-glycosylation modification in TLRs, and augment immune evolutionary research on the TLR signaling pathway.

Cytotoxicity of the field lampricide 3-trifluoromethyl-4-nitrophenol (TFM) in tadpole cell lines from North American frogs.

The common field lampricide, 3-trifluoromethyl-4-nitrophenol (TFM), is used to treat streams and creeks infested with highly invasive and destructive sea lamprey (Petromyzon marinus) in the tributaries of the Great Lakes. Unfortunately, amphibian deaths have been reported following stream treatments with TFM. Larval amphibians (tadpoles) are more susceptible to TFM toxicity than adult amphibians. The aim of this study was to test the toxicity of TFM in eight new tadpole cell lines from the green frog (Lithobates clamitans), wood frog (Lithobates sylvaticus), and American toad (Anaxyrus americanus). A cell viability bioassay using two fluorescent dyes, Alamar Blue and CFDA-AM, was performed following 24-h and 72-h exposures to a range of TFM concentrations. In general, TFM exposure reduced Alamar Blue fluorescence more rapidly than CFDA-AM fluorescence in tadpole cells, suggesting that Alamar Blue is perhaps a better diagnostic indicator of cell health for acute TFM cytotoxicity. At present, the in vivo 96-h LC50s of TFM are only available for L. clamitans and they correlated well with the in vitro EC50 values for the green frog tadpole cell lines in this study. The eight tadpole cell lines with different relative sensitivities to TFM cytotoxicity could prove to be useful tools in assessing next-generation lampricides in high-throughput bioassays to ensure safety in frogs before their sea lamprey-targeted application in the field.

Differences in the transcriptome response in the gills of sea lamprey acutely exposed to 3-trifluoromethyl-4-nitrophenol (TFM), niclosamide or a TFM:niclosamide mixture.

Sea lamprey (Petromyzon marinus) control in the Laurentian Great Lakes of North America makes use of two pesticides: 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide, which are often co-applied. Sea lamprey appear to be vulnerable to these agents resulting from a lack of detoxification responses with evidence suggesting that lampricide mixtures produce a synergistic effect. However, there is a lack of information pertaining to the physiological responses of sea lamprey to niclosamide and TFM:niclosamide mixtures. Here, we characterized the transcriptomic responses of the sea lamprey to TFM, niclosamide, and a TFM:niclosamide (1.5 %) mixture in the gill. Along with a control, larval sea lamprey were exposed to each treatment for 6 h, after which gill tissues were extracted for measuring whole-transcriptome responses using RNA sequencing. Differential gene expression patterns were summarized, which included identifying the broad roles of genes and common expression patterns among the treatments. While niclosamide treatment resulted in no differentially expressed genes, TFM- and mixture-treated fish had several differentially expressed genes that were associated with the cell cycle, DNA damage, metabolism, immune function, and detoxification. However, there was no common differential expression among treatments. For the first time, we characterized the transcriptomic response of sea lamprey to niclosamide and a TFM:niclosamide mixture and identified that these agents impact mRNA transcript abundance of genes associated with the cell cycle and cellular death, and immune function, which are likely mediated through mitochondrial dysregulation. These results may help to inform the production of more targeted and effective lampricides in sea lamprey control efforts.

A lamprey neural cell type atlas illuminates the origins of the vertebrate brain.

The vertebrate brain emerged more than ~500 million years ago in common evolutionary ancestors. To systematically trace its cellular and molecular origins, we established a spatially resolved cell type atlas of the entire brain of the sea lamprey-a jawless species whose phylogenetic position affords the reconstruction of ancestral vertebrate traits-based on extensive single-cell RNA-seq and in situ sequencing data. Comparisons of this atlas to neural data from the mouse and other jawed vertebrates unveiled various shared features that enabled the reconstruction of cell types, tissue structures and gene expression programs of the ancestral vertebrate brain. However, our analyses also revealed key tissues and cell types that arose later in evolution. For example, the ancestral brain was probably devoid of cerebellar cell types and oligodendrocytes (myelinating cells); our data suggest that the latter emerged from astrocyte-like evolutionary precursors in the jawed vertebrate lineage. Altogether, our work illuminates the cellular and molecular architecture of the ancestral vertebrate brain and provides a foundation for exploring its diversification during evolution.

Reduced steroid activation of elephant shark GR and MR after inserting four amino acids from the DNA-binding domain of lamprey corticoid receptor-1.

Atlantic sea lamprey contains two corticoid receptors (CRs), CR1 and CR2, that have identical amino acid sequences, except for a four amino acid insert (Thr-Arg-Gln-Gly) in the CR1 DNA-binding domain (DBD). Steroids are stronger transcriptional activators of CR2 than of CR1 suggesting that the insert reduces the transcriptional response of lamprey CR1 to steroids. The DBD in elephant shark mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), which are descended from a CR, lack these four amino acids, suggesting that a CR2 is their common ancestor. To determine if, similar to lamprey CR1, the presence of this insert in elephant shark MR and GR decreases transcriptional activation by corticosteroids, we inserted these four CR1-specific residues into the DBD of elephant shark MR and GR. Compared to steroid activation of wild-type elephant shark MR and GR, cortisol, corticosterone, aldosterone, 11-deoxycorticosterone and 11-deoxycortisol had lower transcriptional activation of these mutant MR and GR receptors, indicating that the absence of this four-residue segment in the DBD in wild-type elephant shark MR and GR increases transcriptional activation by corticosteroids.

Upstream migrant sea lamprey (Petromyzon marinus) show signs of increasing oxidative stress but maintain aerobic capacity with age.

Following the parasitic juvenile phase of their life cycle, sea lamprey (Petromyzon marinus) mature into a reproductive but rapidly aging and deteriorating adult, and typically die shortly after spawning in May or June. However, pre-spawning upstream migrant sea lamprey can be maintained for several months beyond their natural lifespan when held in cold water (∼4-8 °C) under laboratory conditions. We exploited this feature to investigate the interactions between senescence, oxidative stress, and metabolic function in this phylogenetically ancient fish. We investigated how life history traits and mitochondria condition, as indicated by markers of oxidative stress (catalase activity, lipid peroxidation) and aerobic capacity (citrate synthase activity), changed in adult sea lamprey from June to December after capture during their upstream spawning migration. Body mass but not liver mass declined with age, resulting in an increase in hepatosomatic index. Both effects were most pronounced in males, which also tended to have larger livers than females. Lamprey experienced greater oxidative stress with age, as reflected by increasing activity of the antioxidant enzyme catalase and increasing levels of lipid peroxidation in liver mitochondrial isolates over time. Surprisingly, the activity of citrate synthase also increased with age in both sexes. These observations implicate mitochondrial dysfunction and oxidative stress in the senescence of sea lamprey. Due to their unique evolutionary position and the technical advantage of easily delaying the onset of senescence in lampreys using cold water, these animals could represent an evolutionary unique and tractable model to investigate senescence in vertebrates.

Regulation of the swimming kinematics of lampreys Petromyzon marinus across changes in viscosity.

The bodies of most swimming fishes are very flexible and deform as result of both external fluid dynamic forces and internal musculoskeletal forces. If fluid forces change, the body motion will also change unless the fish senses the change and alters its muscle activity to compensate. Lampreys and other fishes have mechanosensory cells in their spinal cords that allow them to sense how their body is bending. We hypothesized that lampreys (Petromyzon marinus) actively regulate body curvature to maintain a fairly constant swimming waveform even as swimming speed and fluid dynamic forces change. To test this hypothesis, we measured the steady swimming kinematics of lampreys swimming in normal water, and water in which the viscosity was increased by 10 or 20 times by adding methylcellulose. Increasing the viscosity over this range increases the drag coefficient, potentially increasing fluid forces up to 40%. Previous computational results suggested that if lampreys did not compensate for these forces, the swimming speed would drop by about 52%, the amplitude would drop by 39%, and posterior body curvature would increase by about 31%, while tail beat frequency would remain the same. Five juvenile sea lampreys were filmed swimming through still water, and midlines were digitized using standard techniques. Although swimming speed dropped by 44% from 1× to 10× viscosity, amplitude only decreased by 4%, and curvature increased by 7%, a much smaller change than the amount we estimated if there was no compensation. To examine the waveform overall, we performed a complex orthogonal decomposition and found that the first mode of the swimming waveform (the primary swimming pattern) did not change substantially, even at 20× viscosity. Thus, it appears that lampreys are compensating, at least partially, for the changes in viscosity, which in turn suggests that sensory feedback is involved in regulating the body waveform.

Transcriptomic impacts and potential routes of detoxification in a lampricide-tolerant teleost exposed to TFM and niclosamide.

Sea lamprey (Petromyzon marinus) control in the Laurentian Great Lakes of North America often relies on the application of 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide mixtures to kill larval sea lamprey. Selectivity of TFM against lampreys appears to be due to differential detoxification ability in these jawless fishes compared to bony fishes, particularly teleosts. However, the proximate mechanisms of tolerance to the TFM and niclosamide mixture and the mechanisms of niclosamide toxicity on its own are poorly understood, especially among non-target fishes. Here, we used RNA sequencing to identify specific mRNA transcripts and functional processes that responded to niclosamide or a TFM:niclosamide mixture in bluegill (Lepomis macrochirus). Bluegill were exposed to niclosamide or TFM:niclosamide mixture, along with a time-matched control group, and gill and liver tissues were sampled at 6, 12, and 24 h. We summarized the whole-transcriptome patterns through gene ontology (GO) term enrichment and through differential expression of detoxification genes. The niclosamide treatment resulted in an upregulation of several transcripts associated with detoxification (cyp, ugt, sult, gst), which may help explain the relatively high detoxification capacity in bluegill. Conversely, the TFM:niclosamide mixture resulted in an enrichment of processes related to arrested cell cycle and growth, and cell death alongside a diverse detoxification gene response. Detoxification of both lampricides likely involves the use of phase I and II biotransformation genes. Our findings strongly suggest that the unusually high tolerance of bluegill to lampricides is due to these animals having an inherently high capacity and flexible detoxification response to such compounds.

The GH/IGF axis in the sea lamprey during metamorphosis and seawater acclimation.

How the growth hormone (GH)/insulin-like growth factor (IGF) system affects osmoregulation in basal vertebrates remains unknown. We examined changes in the expression of components of the GH/IGF axis and gill ion transporters during metamorphosis and following seawater (SW) exposure of sea lamprey. During metamorphosis, increases in gill nka and nkcc1 and salinity tolerance were accompanied by increases in pituitary gh, liver igf1, gill ghr and igf1, but not liver ghr. SW exposure of fully metamorphosed sea lamprey resulted in slight increases in plasma chloride concentrations after SW exposure, indicating a high level of SW tolerance, but no major changes in mRNA levels of gill ion transporters or components of the GH/IGF axis. Our results indicate that metamorphosis is a critical point in the lifecycle of sea lamprey for stimulation of the GH/IGF axis and is temporally associated with and likely promotes metamorphosis and SW tolerance.

An improved germline genome assembly for the sea lamprey Petromyzon marinus illuminates the evolution of germline-specific chromosomes.

Programmed DNA loss is a gene silencing mechanism that is employed by several vertebrate and nonvertebrate lineages, including all living jawless vertebrates and songbirds. Reconstructing the evolution of somatically eliminated (germline-specific) sequences in these species has proven challenging due to a high content of repeats and gene duplications in eliminated sequences and a corresponding lack of highly accurate and contiguous assemblies for these regions. Here, we present an improved assembly of the sea lamprey (Petromyzon marinus) genome that was generated using recently standardized methods that increase the contiguity and accuracy of vertebrate genome assemblies. This assembly resolves highly contiguous, somatically retained chromosomes and at least one germline-specific chromosome, permitting new analyses that reconstruct the timing, mode, and repercussions of recruitment of genes to the germline-specific fraction. These analyses reveal major roles of interchromosomal segmental duplication, intrachromosomal duplication, and positive selection for germline functions in the long-term evolution of germline-specific chromosomes.

N-terminal domain influences steroid activation of the Atlantic sea lamprey corticoid receptor.

Lampreys are jawless fish that evolved about 550 million years ago at the base of the vertebrate line. Modern lampreys contain a corticoid receptor (CR), the common ancestor of the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), which first appear in cartilaginous fish, such as sharks. Until recently, 344 amino acids at the amino terminus of adult lamprey CR were not present in the lamprey CR sequence in GenBank. A search of the recently sequenced lamprey germline genome identified two CR sequences, CR1 and CR2, containing the 344 previously un-identified amino acids. CR1 also contains a novel four amino acid insertion in the DNA-binding domain (DBD). We studied corticosteroid and progesterone activation of CR1 and CR2 and found their strongest response was to 11-deoxycorticosterone and 11-deoxycortisol, the two circulating corticosteroids in lamprey. Based on steroid specificity, both CRs are close to elephant shark MR and distant from elephant shark GR. HEK293 cells that were transfected with full-length CR1 or CR2 and the MMTV promoter have about 3-fold higher steroid-mediated activation compared to HEK293 cells transfected with these CRs and the TAT3 promoter. Deletion of the amino-terminal domain (NTD) of lamprey CR1 and CR2 to form truncated CRs decreased transcriptional activation by about 70% in HEK293 cells that were transfected with MMTV, but increased transcription by about 6-fold in cells transfected with TAT3. This indicated that the promoter has an important effect on NTD regulation of transcriptional activation of the CR by steroids. Our results also indicate that the entire lamprey CR sequence is needed for an accurate determination of steroid-mediated transcription.

Hair cell morphological patterns and polarity organization in the sea lamprey vestibular cristae.

The inner ear of the sea lamprey was examined by scanning electron microscopy, antibody labeling with tubulin, Myo7a, Spectrin, and Phalloidin stain to elucidate the canal cristae organization and the morphology and polarity of the hair cells. We characterized the hair cell stereocilia bundles and their morphological polarity with respect to the kinocilia. We identified three types of hair cells. In Type 1 hair cells, the kinocilia were slightly longer than the tallest stereocilia. This type was located along the medial bank of the crista and their polarity, based on kinocilia location, was uniformly pointed ampullipetally. Type 2 hair cells that had kinocilia that were much longer than the stereocilia, were most abundant in the central region of the crista. This type of hair cell displayed variable polarity. Type 3 hair cells had extremely long kinocilia (~40-50 µm long) and with extremely short stereocilia. They were mostly located in the lateral zone crista and displayed ampullipetal polarity. Myo7a and tubulin antibodies revealed that hair cells and vestibular afferents are distributed across the canal cristae in the lamprey, covering the area of cruciate eminence; a feature that is absent in more derived vertebrates. Spectrin shows hair cells of varying polarities in the central zone. In this zone, some cells followed the main polarity vector (lateral) like those in medial and lateral zones, whereas other cells displayed polarities that carried up to 40° from the main polarity vector.

Organization of the corticotropin-releasing hormone and corticotropin-releasing hormone-binding protein systems in the central nervous system of the sea lamprey Petromyzon marinus.

The expression of the corticotropin-releasing hormone (PmCRH) and the CRH-binding protein (PmCRHBP) mRNAs was studied by in situ hybridization in the brain of prolarvae, larvae, and adults of the sea lamprey Petromyzon marinus. We also generated an antibody against the PmCRH mature peptide to study the distribution of PmCRH-immunoreactive cells and fibers. PmCRH immunohistochemistry was combined with antityrosine hydroxylase immunohistochemistry, PmCRHBP in situ hybridization, or neurobiotin transport from the spinal cord. The most numerous PmCRH-expressing cells were observed in the magnocellular preoptic nucleus-paraventricular nucleus and in the superior and medial rhombencephalic reticular formation. PmCRH expression was more extended in adults than in larvae, and some cell populations were mainly (olfactory bulb) or only (striatum, ventral hypothalamus, prethalamus) observed in adults. The preopto-paraventricular fibers form conspicuous tracts coursing toward the neurohypophysis, but many immunoreactive fibers were also observed coursing in many other brain regions. Brain descending fibers in the spinal cord mainly come from cells located in the isthmus and in the medial rhombencephalic reticular nucleus. The distribution of PmCRHBP-expressing neurons was different from that of PmCRH cells, with cells mainly present in the septum, striatum, preoptic region, tuberal hypothalamus, pretectum, pineal complex, isthmus, reticular formation, and spinal cord. Again, expression in adults was more extended than in larvae. PmCRH- and PmCRHBP-expressing cells are different, excluding colocalization of these substances in the same neuron. Present findings reveal a complex CRH/CRHBP system in the brain of the oldest extant vertebrate group, the agnathans, which shows similarities but important divergences with that of mammals.

Sea lamprey nests promote the diversity of benthic macroinvertebrate assemblages.

The habitat heterogeneity hypothesis states that increased habitat heterogeneity promotes species diversity through increased availability of ecological niches. We aimed at describing the local-scale (i.e. nest and adjacent substrate) effects of nests of the sea lamprey (Petromyzon marinus L.) as ecosystem engineer on macroinvertebrate assemblages. We hypothesized that increased streambed physical heterogeneity caused by sea lamprey spawning would modify invertebrate assemblages and specific biologic traits and promote reach-scale diversity. We sampled thirty lamprey nests of the Nive River, a river of the south western France with a length of 79.3 km and tributary of the Adour River, in three zones: the unmodified riverbed (upstream) and zones corresponding to the nest: the area excavated (pit) and the downstream accumulation of pebbles and cobbles (mound). The increased habitat heterogeneity created by lamprey was accompanied by biological heterogeneity with a reduced density of invertebrates (3777 ± 1332 individuals per m2 in upstream, 2649 ± 1386 individuals per m2 in pit and 3833 ± 1052 individuals per m2 in mound) and number of taxa (23.5 ± 3.9 taxa for upstream, 18.6 ± 3.9 taxa in pit and 21.2 ± 4.5 taxa for mound) in the pit compared to other zones. However the overall taxa diversity in nest increased with 82 ± 14 taxa compared to the 69 ± 8 taxa estimated in upstream zone. Diversity indices were consistent with the previous results indicating a loss of α diversity in pit but a higher ß diversity between a pit and a mound than between two upstream zones, especially considering Morisita index accounting for taxa abundance. Trait analysis showed high functional diversity within zones with a reduced proportion of collectors, scrapers, shredders, litter/mud preference and small invertebrates in mound, while the proportion of "slabs, blocks, stones and pebbles" preference and largest invertebrates increased. Pit presented the opposite trend, while upstream had globally intermediate trait proportions. Our results highlight important effects on species and functional diversity due to habitat heterogeneity created by a nest-building species, what can ultimately influence food webs and nutrient processes in river ecosystems.

Plasma metabolomic profiles reveal sex- and maturation-dependent metabolic strategies in sea lamprey (Petromyzon marinus).

INTRODUCTION: Adult sea lamprey (Petromyzon marinus) cease feeding and migrate to spawning streams where males build nests, undergo final sexual maturation, and subsequently produce and release large quantities of bile acid pheromones that attract mature females. These animals are predicted to rearrange their metabolic pathways drastically to support their reproductive strategies, presenting advantageous opportunities to examine how sex and the maturation processes affect metabolism. OBJECTIVES: The objective is to investigate the metabolic differences between sexes and maturation states in sea lamprey that support changes in physiological functions. METHODS: We compared plasma metabolomes of spawning and prespawning sea lamprey in both sexes using both non-targeted and targeted metabolomics approaches using UPLC/MS-MS with electrospray ionization in both positive and negative modes. The data were processed using Progenesis QI, Compound Discoverer and XCMS softwares for alignment, peak picking, and deconvolution of the peaks. Principle component analyses (PCA) and partial least squares discriminant analyses (PLS-DA) were performed using SIMCA and Metaboanalyst softwares to identify discriminating features, followed by fragmentation matching with extensive database search and pathway mapping. RESULTS: The pheromonal bile acid biosynthesis was upregulated significantly in males compared to females. Spermiating males further upregulated bile acid biosynthesis by altering amino acid metabolisms, upregulating cofactors and nucleotide metabolisms, but downregulating carbohydrate and energy metabolisms. CONCLUSION: Plasma metabolomes are sex- and maturation-dependent and reflect the special metabolic demands at each life stage and reproductive strategy.

Divergent Pathways of Ammonia and Urea Production and Excretion during the Life Cycle of the Sea Lamprey.

Little is known about nitrogenous waste (N waste) handling and excretion (JN waste) during the complex life cycle of the sea lamprey (Petromyzon marinus), an extant jawless fish that undergoes a complete metamorphosis from a filter-feeding larva (ammocoete) into a parasitic juvenile that feeds on the blood of larger, jawed fishes. Here, we investigate the ammonia- and urea-handling profiles of sea lampreys before, during, and after metamorphosis. The rates of ammonia excretion (Jamm) and urea excretion (Jurea) significantly decreased after the onset of metamorphosis, with the lowest rates observed during midmetamorphosis. Near the completion of metamorphosis, rates of JN waste (JN waste=Jamm+Jurea) significantly increased as sea lampreys entered the juvenile period. Feeding juvenile lampreys had greater than 10- to 15-fold higher Jamm and fivefold higher Jurea compared to nonfed juveniles, which corresponded to higher postprandial (postfeeding) concentrations of plasma ammonia and urea. The routes of Jamm and Jurea completely diverged following metamorphosis. In larvae, Jamm was equally split between branchial (gills) and extrabranchial (skin plus renal) pathways, but following metamorphosis, >80% of ammonia was excreted via the gills in nonfeeding juvenile lampreys, and >95% of ammonia was excreted via the gills in adult sea lampreys. Urea, on the other hand, was predominantly excreted via extrabranchial routes and, to a lesser extent, the gills in larvae and in nonfeeding juveniles. In adults, however, virtually all urea was excreted via urine. Reverse transcription polymerase chain reaction and in silico analyses also indicated that a urea transporter encoded by a slc4a2-like gene is present in lampreys. The branchial expression of this transporter is modulated throughout sea lamprey life history, as it is higher in the larvae and steadily decreases until the adult stage. We conclude that the divergent pathways of Jamm and Jurea during the sea lamprey life cycle reflect changes in their habitat, lifestyle, and diet. Further, the near-complete reliance on renal routes for Jurea in adult sea lampreys is unique among fishes and may reflect the ancestral condition of how this N waste product was handled and excreted by the earliest vertebrates.

Sea Lamprey Alarm Cue Comprises Water- and Chloroform- Soluble Components.

A diversity of aquatic organisms manage predation risk by avoiding waters activated with conspecific alarm cues, a chemical mixture released from injuries. The sea lamprey (Petromyzon marinus) is a nocturnal migratory species that relies on its alarm cue to navigate around areas of predation risk when moving through river channels. Identification of the cue's chemistry would allow managers to harness this innate behavioral response to guide migrating sea lamprey to traps (invasive population in the Laurentian Great Lakes) or to fish passage devices where dams block migrations in their native range. We pursued isolation of the sea lamprey alarm cue through behaviorally guided fractionation, fractionating the alarm cue into water-soluble and chloroform-soluble fractions, each of which elicited a substantial avoidance response. Recombining the two fractions restored full reactivity, suggesting the alarm cue mixture contains components that exhibit high solubility in water (e.g., nitrogenous compounds), chloroform (e.g., lipids), or perhaps materials that dissolve readily in either solvent. We further screened 13 individual compounds or pure isolates and 6 sub-fractions from the water-soluble fraction and found one of the pure isolates, isoleucine, evoked an avoidance response on its own, but not consistently when found in other mixtures. In a third experiment, we observed no behavioral response after recombining 32 compounds isolated and identified from the water-soluble fraction. These results confirm other suggestions that the process of elucidating alarm cue constituents is challenging. However, we suggest the pursuit is worthwhile given the strong evidence for the utility of alarm cues for use in the conservation and management of fishes and other aquatic organisms.