Evolution of the endothelin pathway drove neural crest cell diversification.

Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.

Muscle fatty acid profiles of sea lamprey (Petromyzon marinus L.) indicate the use of fast metabolized energy during ontogenesis.

A comprehensive characterization of muscle's FA composition of sea lamprey ammocoetes and adults was performed to test the hypothesis that larvae, and early spawning migrants have a similar FA profile prior to metamorphosis and to spawning migration. Subsequently, the role played by FA signature in these two highly demanding stages of life cycle was inferred. The results confirm that muscle represents an important fat reservoir, and the FA trophic markers revealed the importance of bacteria as sources of iso and anteiso FA and the strong trophic representation of benthic phytoplankton (diatoms) to larvae muscle FA profile. In early spawning migrants, the significance of marine food web to FA muscle profile is highlighted by the presence of FA signatures characteristics of herbivorous calanoid copepods. Although both life cycle phases studied do not share the same muscle FA signature, there is a part of the profile that is common, which is characterized by FA used in ß-oxidation, such as C18:1ω9 but also by medium chain FA and PUFA which points that PUFA are spared as fuel to ß-oxidation process and probably used to the development of tissues membranes (ammocoetes) and gonadal development and eicosanoid production among others (early spawning migrants). Further studies on FA profile are necessary to elucidate the FA role either during different life stages (ontogeny) or in the distinct habitats frequented (freshwater versus marine) by this diadromous species.

Morphological and functional aspects of the epidermis of the sea lamprey Petromyzon marinus throughout development.

The development of the epidermis of sea lamprey Petromyzon marinus along the whole life cycle was studied using conventional staining techniques and lectin histochemistry. The epidermis undergoes variations in morphology and thickness throughout development. The simple cuboidal epithelium found in the epidermis of prolarvae becomes stratified cubic in the adult by increasing the number of cell layers. The cuticle thickness undergoes a steady increase during the larval period. There are changes in the glycoconjugate composition of the three main cell types of the P. marinus epidermis, mucous, granular and skein cells, which are more pronounced after metamorphosis. The Alcian blue-periodic acid Schiff (AB-PAS) histochemical method shows the presence of both acidic and neutral glycoconjugates in the mucous cells, indicating their secretory function. Moreover, lectin analysis reveals a mucous secretion containing glycoconjugates such as sulphated glycosaminoglycans (N-acetylglucosamine and N-acetylgalactosamine) and N-glycoproteins rich in mannose. Although granular cells are AB-PAS negative, they exhibit a similar glycoconjugate composition to the mucous cells. Moreover, granular cells show sialic acid positivity in larvae but this monosaccharide residue is not detected after metamorphosis. The skein cells, a unique cell of lampreys, are negative to AB-PAS staining but they mostly contain l-fucose and sialic acid residues, which also disappear after metamorphosis. The function of the granular and skein cells is still unknown but the role of their glycoconjugate composition is discussed. In addition, a different cellular origin is suggested for these two types of cells.

Convergent evolution of hemoglobin switching in jawed and jawless vertebrates.

BACKGROUND: During development, humans and other jawed vertebrates (Gnathostomata) express distinct hemoglobin genes, resulting in different hemoglobin tetramers. Embryonic and fetal hemoglobin have higher oxygen affinities than the adult hemoglobin, sustaining the oxygen demand of the developing organism. Little is known about the expression of hemoglobins during development of jawless vertebrates (Agnatha). RESULTS: We identified three hemoglobin switches in the life cycle of the sea lamprey. Three hemoglobin genes are specifically expressed in the embryo, four genes in the filter feeding larva (ammocoete), and nine genes correspond to the adult hemoglobin chains. During the development from the parasitic to the reproductive adult, the composition of hemoglobin changes again, with a massive increase of chain aHb1. A single hemoglobin chain is expressed constitutively in all stages. We further showed the differential expression of other globin genes: Myoglobin 1 is most highly expressed in the reproductive adult, myoglobin 2 expression peaks in the larva. Globin X1 is restricted to the embryo; globin X2 was only found in the reproductive adult. Cytoglobin is expressed at low levels throughout the life cycle. CONCLUSION: Because the hemoglobins of jawed and jawless vertebrates evolved independently from a common globin ancestor, hemoglobin switching must also have evolved convergently in these taxa. Notably, the ontogeny of sea lamprey hemoglobins essentially recapitulates their phylogeny, with the embryonic hemoglobins emerging first, followed by the evolution of larval and adult hemoglobins.

Neuropeptide Y family receptors Y1 and Y2 from sea lamprey, Petromyzon marinus.

The vertebrate gene family for neuropeptide Y (NPY) receptors expanded by duplication of the chromosome carrying the ancestral Y1-Y2-Y5 gene triplet. After loss of some duplicates, the ancestral jawed vertebrate had seven receptor subtypes forming the Y1 (including Y1, Y4, Y6, Y8), Y2 (including Y2, Y7) and Y5 (only Y5) subfamilies. Lampreys are considered to have experienced the same chromosome duplications as gnathostomes and should also be expected to have multiple receptor genes. However, previously only a Y4-like and a Y5 receptor have been cloned and characterized. Here we report the cloning and characterization of two additional receptors from the sea lamprey Petromyzon marinus. Sequence phylogeny alone could not with certainty assign their identity, but based on synteny comparisons of P. marinus and the Arctic lamprey, Lethenteron camtschaticum, with jawed vertebrates, the two receptors most likely are Y1 and Y2. Both receptors were expressed in human HEK293 cells and inositol phosphate assays were performed to determine the response to the three native lamprey peptides NPY, PYY and PMY. The three peptides have similar potencies in the nanomolar range for Y1. No obvious response to the three peptides was detected for Y2. Synteny analysis supports identification of the previously cloned receptor as Y4. No additional NPY receptor genes could be identified in the presently available lamprey genome assemblies. Thus, four NPY-family receptors have been identified in lampreys, orthologs of the same subtypes as in humans (Y1, Y2, Y4 and Y5), whereas many other vertebrate lineages have retained additional ancestral subtypes.

Evidence that progestins play an important role in spermiation and pheromone production in male sea lamprey (Petromyzon marinus).

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 1week 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 0.53pmol/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.

Iso-petromyroxols: novel dihydroxylated tetrahydrofuran enantiomers from sea lamprey (Petromyzon marinus).

An enantiomeric pair of new fatty acid-derived hydroxylated tetrahydrofurans, here named iso-petromyroxols, were isolated from sea lamprey larvae-conditioned water. The relative configuration of iso-petromyroxol was elucidated with 1D and 2D NMR spectroscopic analyses. The ratio of enantiomers (er) in the natural sample was measured by chiral-HPLC-MS/MS to be ca. 3:1 of (-)- to (+)-antipodes.

Changes in the gut microbiome of the sea lamprey during metamorphosis.

Vertebrate metamorphosis is often marked by dramatic morphological and physiological changes of the alimentary tract, along with major shifts in diet following development from larva to adult. Little is known about how these developmental changes impact the gut microbiome of the host organism. The metamorphosis of the sea lamprey (Petromyzon marinus) from a sedentary filter-feeding larva to a free-swimming sanguivorous parasite is characterized by major physiological and morphological changes to all organ systems. The transformation of the alimentary canal includes closure of the larval esophagus and the physical isolation of the pharynx from the remainder of the gut, which results in a nonfeeding period that can last up to 8 months. To determine how the gut microbiome is affected by metamorphosis, the microbial communities of feeding and nonfeeding larval and parasitic sea lamprey were surveyed using both culture-dependent and -independent methods. Our results show that the gut of the filter-feeding larva contains a greater diversity of bacteria than that of the blood-feeding parasite, with the parasite gut being dominated by Aeromonas and, to a lesser extent, Citrobacter and Shewanella. Phylogenetic analysis of the culturable Aeromonas from both the larval and parasitic gut revealed that at least five distinct species were represented. Phenotypic characterization of these isolates revealed that over half were capable of sheep red blood cell hemolysis, but all were capable of trout red blood cell hemolysis. This suggests that the enrichment of Aeromonas that accompanies metamorphosis is likely related to the sanguivorous lifestyle of the parasitic sea lamprey.

Field evaluation of larval odor and mixtures of synthetic pheromone components for attracting migrating sea lampreys in rivers.

The sea lamprey, Petromyzon marinus, is a harmful invader of the Laurentian Great Lakes. The odor emitted by larval lampreys resident to streams attracts migrating adults to high quality spawning habitats. Three components of the larval pheromone have been identified and tested in laboratory settings: petromyzonol sulfate, petromyzosterol disulfate, and petromyzonamine disulfate. Here, we report the first field test of six mixtures of synthetic versions of these pheromone components, and we compare lamprey responses to these with those elicited by the complete larval odor in a natural stream. Exposure to larval odor both increased upstream movement and attracted migrants into the portion of a channel containing the odor. No tested combination of synthetic pheromone components proved similarly attractive. These findings suggest the existence of unknown additional components of the pheromone that await discovery and are likely necessary if the pheromone is to be useful in management of this pest. Further, we hypothesize that the complete pheromone mixture is necessary to attract migrants into spawning habitat at the conclusion of the migration, whereas a partial pheromone may be effective at the transition from lake to stream when natural factors both dilute and alter the ratio of components from that actually emitted by sea lamprey larvae.

Sea lamprey (Petromyzon marinus) contain four developmentally regulated serum thyroid hormone distributor proteins.

Thyroid hormones (THs) are very lipophilic molecules which require a distribution network for efficient transport in serum. Despite observations that THs function in a wide variety of processes, including aspects of fish development (i.e., flat fish metamorphosis and smoltification), the proteins responsible for TH distribution in fish serum remain poorly studied. We chose to investigate the serum TH distributor proteins (THDPs) in lampreys. As one of only two extant agnathans, data on lamprey THDPs may offer new insights into the evolution of the vertebrate TH distribution network and serum proteins in general. Moreover, lampreys appear to contradict the vertebrate model of an increase in TH concentrations initiating and driving vertebrate metamorphosis. We show for the first time that sea lamprey serum contains at least four THDPs and that their presence in serum is temporally regulated throughout the life cycle. The albumin, glycoprotein AS is the dominant THDP present in the sera of larval and metamorphosing sea lamprey. In stage seven of metamorphosis, three additional THDPs appear, including the albumin, glycoprotein SDS-1; the glycolipoprotein CB-III; and an unidentified low molecular weight protein temporarily named Spot-5. The sera of parasitic and upstream migrant sea lampreys lack AS; their serum THDPs are SDS-1, CB-III, and Spot-5. Our data indicate that despite the change in type and number of THDPs, the overall total TH binding capacity of sea lamprey serum remains fairly stable until stage 7 of metamorphosis when a only modest decrease in total binding capacity is observed. Collectively these data indicate that the decline in serum TH concentrations observed during lamprey metamorphosis is not a consequence of a reduction in the distribution and storage capacity of the serum.

Functional re-organization of the gills of metamorphosing sea lamprey (Petromyzon marinus): preparation for a blood diet and the freshwater to seawater transition.

Sea lamprey (Petromyzon marinus) begin life as filter-feeding larvae (ammocoetes) before undergoing a complex metamorphosis into parasitic juveniles, which migrate to the sea where they feed on the blood of large-bodied fishes. The greater protein intake during this phase results in marked increases in the production of nitrogenous wastes (N-waste), which are excreted primarily via the gills. However, it is unknown how gill structure and function change during metamorphosis and how it is related to modes of ammonia excretion, nor do we have a good understanding of how the sea lamprey's transition from fresh water (FW) to sea water (SW) affects patterns and mechanisms of N-waste excretion in relation to ionoregulation. Using immunohistochemistry, we related changes in the gill structure of larval, metamorphosing, and juvenile sea lampreys to their patterns of ammonia excretion (Jamm) and urea excretion (Jurea) in FW, and following FW to artificial seawater (ASW) transfer. Rates of Jamm and Jurea were low in larval sea lamprey and increased in feeding juvenile, parasitic sea lamprey. In freshwater-dwelling ammocoetes, immunohistochemical analysis revealed that Rhesus glycoprotein C-like protein (Rhcg-like) was diffusely distributed on the lamellar epithelium, but following metamorphosis, Rhcg-like protein was restricted to SW mitochondrion-rich cells (MRCs; ionocytes) between the gill lamellae. Notably, these interlamellar Rhcg-like proteins co-localized with Na+/K+-ATPase (NKA), which increased in expression and activity by almost tenfold during metamorphosis. The distribution of V-type H+-ATPase (V-ATPase) on the lamellae decreased following metamorphosis, indicating it may have a more important role in acid-base regulation and Na+ uptake in FW, compared to SW. We conclude that the re-organization of the sea lamprey gill during metamorphosis not only plays a critical role in allowing them to cope with greater salinity following the FW-SW transition, but that it simultaneously reflects fundamental changes in methods used to excrete ammonia.

Diel activity of newly metamorphosed juvenile sea lamprey (Petromyzon marinus).

Timing of activity, especially for juvenile anadromous fishes undertaking long migrations can be critical for survival. River-resident larval sea lamprey metamorphose into juveniles and migrate from their larval stream habitats in fall through spring, but diel timing of this migratory behavior is not well understood. Diel activity was determined for newly metamorphosed sea lamprey using day/night net sampling and passive integrated transponder (PIT) telemetry in two natural streams and PIT telemetry in an artificial stream. Downstream migration was primarily nocturnal in all studies. All but one of 372 sea lamprey were captured during night sampling in the day/night net collections and all detections (N = 56) for the in-stream PIT telemetry occurred within a few hours after sunset. Most (81% of 48) tagged lamprey moved downstream during the first night following release and moved at speeds consistent with observed water velocities. During long-term observation of behavior in the artificial stream most sea lamprey movement occurred during the night with limited occurrence of movement during daylight hours. Understanding seasonal and diel timing of downstream migration behavior may allow more effective management of sea lamprey for both conservation and control.

Control of invasive sea lampreys using the piscicides TFM and niclosamide: Toxicology, successes & future prospects.

The invasion of the Laurentian Great Lakes of North America by sea lampreys (Petromyzon marinus) in the early 20th century contributed to the depletion of commercial, recreational and culturally important fish populations, devastating the economies of communities that relied on the fishery. Sea lamprey populations were subsequently controlled using an aggressive integrated pest-management program which employed barriers and traps to prevent sea lamprey from migrating to their spawning grounds and the use of the piscicides (lampricides) 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide to eliminate larval sea lampreys from their nursery streams. Although sea lampreys have not been eradicated from the Great Lakes, populations have been suppressed to less than 10% of their peak numbers in the mid-1900s. The ongoing use of lampricides provides the foundation for sea lamprey control in the Great Lakes, one of the most successful invasive species control programs in the world. Yet, significant gaps remain in our understanding of how lampricides are taken-up and handled by sea lampreys, how lampricides exert their toxic effects, and how they adversely affect non-target invertebrate and vertebrates species. In this review we examine what has been learned about the uptake, handling and elimination, and the mode of TFM and niclosamide toxicity in lampreys and in non-target animals, particularly in the last 10 years. It is now clear that the mode of TFM toxicity is the same in non-target fishes and lampreys, in which TFM interferes with oxidative phosphorylation by the mitochondria leading to decreased ATP production. Vulnerability to TFM is related to abiotic factors such as water pH and alkalinity, which we propose changes the relative amounts of the bioavailable un-ionized form of TFM in the gill microenvironment. Niclosamide, which is also a molluscicide used to control snails in areas prone to schistosomiasis infections of humans, also likely works by uncoupling oxidative phosphorylation, but less is known about other aspects of its toxicology. The effects of TFM include reductions in energy stores, particularly glycogen and high energy phosphagens. However, non-target fishes readily recover from sub-lethal TFM exposure as demonstrated by the rapid restoration of energy stores and clearance of TFM. Although both TFM and niclosamide are non-persistent in the environment and critical for sea lamprey control, increasing public and institutional concerns about pesticides in the environment makes it imperative to explore other means of sea lamprey control. Accordingly, we also address possible "next-generation" strategies of sea lamprey control including genetic tools such as RNA interference and CRISPR-Cas9 to impair critical physiological processes (e.g. reproduction, digestion, metamorphosis) in lamprey, and the use of green chemistry to develop more environmentally benign chemical methods of sea lamprey control.

Epicardial development in lamprey supports an evolutionary origin of the vertebrate epicardium from an ancestral pronephric external glomerulus.

The epicardium is the outer layer of the vertebrate heart. Both the embryonic epicardium and its derived mesenchyme are critical to heart development, contributing to the coronary vasculature and modulating the proliferation of the ventricular myocardium. The embryonic epicardium arises from an extracardiac, originally paired progenitor tissue called the proepicardium, a proliferation of coelomic cells found at the limit between the liver and the sinus venosus. Proepicardial cells attach to and spread over the cardiac surface giving rise to the epicardium. Invertebrate hearts always lack of epicardium, and no hypothesis has been proposed about the origin of this tissue and its proepicardial progenitor in vertebrates. We herein describe the epicardial development in a representative of the most basal living lineage of vertebrates, the agnathan Petromyzon marinus (lamprey). The epicardium in lampreys develops by migration of coelomic cells clustered in a paired structure at the roof of the coelomic cavity, between the pronephros and the gut. Later on, these outgrowths differentiate into the pronephric external glomerulus (PEG), a structure composed of capillary networks, mesangial cells, and podocytes. This observation is consistent with the conclusion that the primordia of the most anterior pair of PEG in agnathans have been retained and transformed into the proepicardium in gnathostomes. Glomerular progenitor cells are highly vasculogenic and probably allowed for the vascularization of a cardiac tube primarily devoid of coronary vessels. This new hypothesis accounts for the striking epicardial expression of Wt1 and Pod1, two transcription factors essential for development of the excretory system.

Neurochemical differentiation of horizontal and amacrine cells during transformation of the sea lamprey retina.

The sea lamprey is a modern representative of the earliest vertebrates (the agnathans) in which development of the eye and retina shows unique patterns. In larval stages the retina is poorly developed, and although a small central region has developed glutamatergic vertical pathways, there is no evidence of chemical differentiation of amacrine and horizontal cells in the central or lateral larval retina [Villar-Cerviño, V., Abalo, X.M., Villar-Cheda, B., Meléndez-Ferro, M., Pérez-Costas, E., Holstein, G.R., Martinelli, G.P., Rodicio, M.C., Anadón, R., 2006. Presence of glutamate, glycine, and gamma-aminobutyric acid in the retina of the larval sea lamprey: comparative immunohistochemical study of classical neurotransmitters in larval and postmetamorphic retinas. J. Comp. Neurol. 499, 810-827.]. However, in adults all the retina was differentiated and both amacrine and horizontal cells are well developed. Present immunocytochemical results show that the horizontal and amacrine cells of the retina begin their neurochemical differentiation during metamorphosis, when they start to express GABA, glycine, serotonin and dopamine; this occurs several years after the onset of development. Immunoreactivity for GABA, glycine and serotonin was found at early metamorphic stages, while expression of the markers of catecholaminergic amacrine cells, dopamine and tyrosine hydroxylase, was found to be delayed until intermediate metamorphic stages. GABA, which is found in some amacrine and horizontal cells of adults, was first observed in amacrine cells during early stages of transformation and then in horizontal cells during middle stages. All cells immunoreactive to serotonin or tyrosine hydroxylase/dopamine were amacrine cells. Interestingly, all these markers began expression before the appearance of opsin-immunoreactive photoreceptors in the lateral retina. The pattern of chemical differentiation of amacrine and horizontal cells was compared with that of other vertebrates and their significance was discussed.

Development and organization of the descending serotonergic brainstem-spinal projections in the sea lamprey.

The organization and development of the descending spinal projections from serotonergic rhombencephalic neurons in the larval sea lamprey were investigated by double labeling, tract-tracing methods and immunocytochemistry against serotonin. The results showed that two serotonergic populations of the isthmic and vagal reticular regions present reticulospinal neurons from the beginning of the larval period. Of the three serotonergic subpopulations recognized in the isthmic reticular group [Abalo, X.M., Villar-Cheda, B., Meléndez-Ferro, M., Pérez-Costas, E., Anadón, R., Rodicio, M.C., 2007. Development of the serotonergic system in the central nervous system of the sea lamprey. J. Chem. Neuroanat. 34, 29-46], only two - the medial and ventral subpopulations - project to the spinal cord, with most of the projecting cells in the caudal part of the medial isthmic subpopulation. Occasional cells projecting to the spinal cord were observed in the ventral subpopulation. The vagal reticular serotonergic nucleus situated in the caudal rhombencephalon also presents cells with descending projections. The early development of the brainstem serotonergic projections to the spinal cord appears to be a conserved trait in all vertebrates studied. Although a serotonergic hindbrain-spinal projection system appears to have been present before the divergence of agnathans and gnathostomes, no serotonergic cells were observed in the raphe region in lamprey. Moreover, proportionally more rostral hindbrain serotonergic cells contribute to the spinal serotonergic projections in the sea lamprey than in jawed vertebrates.

The early scaffold of axon tracts in the brain of a primitive vertebrate, the sea lamprey.

The development of the early axonal scaffold formed by early-differentiating neurons was studied in a primitive vertebrate (the sea lamprey), by immunohistochemistry against acetylated alpha-tubulin and a cell surface marker (HNK-1 antibodies), to determine the degree of conservation of this process in vertebrate evolution. The medial and dorsolateral longitudinal fascicles were the first longitudinal axonal bundles observed to develop in the neural tube, followed by the tract of the postoptic commissure and the supraoptic tract. Establishment of the first dorso-ventral tracts occurs after the appearance of the tract of the postoptic commissure and the medial longitudinal fascicle, the basal plate longitudinal axonal system. The dorsolateral longitudinal fascicle appears to be equivalent to the "descending tract" of the mesencephalic nucleus of the trigeminal nerve of mouse and birds; the possible homologies between other early scaffold tracts of the sea lamprey and those of other vertebrates are also discussed. In addition, present results suggest the presence of highly conserved brain regions that would allow for early neuronal differentiation and axonal pathfinding in vertebrates, which were probably defined before the divergence of Agnathans and Gnathostomes.

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.

Influence of body size, metabolic rate and life history stage on the uptake and excretion of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) by invasive sea lampreys (Petromyzon marinus).

Invasive sea lamprey (Petromyzon marinus) are controlled in the Great Lakes using the lampricide 3-trifluoromethyl-4-nitrophenol (TFM), which is applied to streams infested with larval lamprey. However, lamprey that survive treatments (residuals) remain a challenge because they may subsequently undergo metamorphosis into parasitic juvenile animals that migrate downstream to the Great Lakes, where they feed on important sport and commercial fishes. The goal of this study was to determine if body size and life stage could potentially influence sea lamprey tolerance to TFM by influencing patterns of TFM uptake and elimination. Because mass specific rates of oxygen consumption (M˙O2) are lower in larger compared to smaller lamprey, we predicted that TFM uptake would be negatively correlated to body size, suggesting that large larvae would be more tolerant to TFM exposure. Accordingly, TFM uptake and M˙O2 were measured in larvae ranging in size from 0.2-4.2g using radio-labelled TFM (14C-TFM) and static respirometry. Both were inversely proportional to wet mass (M), and could be described usingthe allometric power relationship: Y=aMb, in which M˙O2=1.86M0.53 and TFM Uptake=7.24M0.34. We also predicted that body size would extend to rates of TFM elimination, which was measured following the administration of 14C-TFM (via intraperitoneal injection). However, there were no differences in the half-lives of elimination of TFM (T 1/2-TFM). There were also no differences in M˙O2 or TFM uptake amongst size-matched larval, metamorphosing (stages 6-7), or post-metamorphic (juvenile) sea lamprey. However, the T1/2-TFM was significantly lower in larval than post-metamorphic lamprey (juvenile), indicating the larval lamprey cleared TFM more efficiently than juvenile lamprey. We conclude that larger larval sea lamprey are more likely to survive TFM treatments suggesting that body size might be an important variable to consider when treating streams with TFM to control these invasive species.

Olfactory sensory neurons in the sea lamprey display polymorphisms.

The sea lamprey (Petromyzon marinus) is an ancient jawless fish phyletically removed from modern (teleost) fishes. It is an excellent organism in the study of olfaction due to its accessible olfactory pathway, which is susceptible to manipulation, and its important location in the evolution of vertebrates. There are many similarities in the olfactory systems of all fishes, and they also share characteristics with the olfactory system of mammals. Teleost fishes lack the distinctive vomeronasal organ of mammals; rather all odours are processed initially by olfactory sensory neurons (OSNs) of three morphotypes within the olfactory epithelium. We sought to identify olfactory sensory neuron polymorphisms in the sea lamprey. Using retrograde tracing with dyes injected into the olfactory bulb, we identified three morphotypes which are highly similar to those found in teleosts. This study provides the first evidence of morphotypes in the sea lamprey peripheral olfactory organ, and indicates that olfactory sensory neuron polymorphism may be a trait highly conserved throughout vertebrate evolution.