Global level of methylation in the sea lamprey (jawless vertebrate) genome is intermediate between invertebrate and jawed vertebrate genomes.

In eukaryotes, cytosine methylation is a primary heritable epigenetic modification of the genome that regulates many cellular processes. In invertebrate, methylated cytosine generally located on specific genomic elements (e.g., gene bodies and silenced repetitive elements) to show a "mosaic" pattern. While in jawed vertebrate (teleost and tetrapod), highly methylated cytosine located genome-wide but only absence at regulatory regions (e.g., promoter and enhancer). Many studies imply that the evolution of DNA methylation reprogramming may have helped the transition from invertebrates to jawed vertebrates, but the detail remains largely elusive. In this study, we used the whole-genome bisulfite-sequencing technology to investigate the genome-wide methylation in three tissues (heart, muscle, and sperm) from the sea lamprey, an extant agnathan (jawless) vertebrate. Strikingly, we found that the methylation level of the sea lamprey is very similar to that in sea urchin (a deuterostome) and sea squirt (a chordate) invertebrates. In sum, the global pattern in sea lamprey is intermediate methylation level (around 30%), that is higher than methylation level in the genomes of pre-bilaterians and protostomes (1%-10%), but lower than methylation level appeared in jawed vertebrates (around 70%, teleost and tetrapod). We anticipate that, in addition to genetic dynamics such as genome duplications, epigenetic dynamics such as global methylation reprograming was also orchestrated toward the emergence and evolution of vertebrates.

Distribution of Brain-Derived Neurotrophic Factor in the Brain of the Small-Spotted Catshark Scyliorhinus canicula, and Evolution of Neurotrophins in Basal Vertebrates.

Neurotrophins (NTFs) are structurally related neurotrophic factors essential for differentiation, survival, neurite outgrowth, and the plasticity of neurons. Abnormalities associated with neurotrophin-signaling (NTF-signaling) were associated with neuropathies, neurodegenerative disorders, and age-associated cognitive decline. Among the neurotrophins, brain-derived neurotrophic factor (BDNF) has the highest expression and is expressed in mammals by specific cells throughout the brain, with particularly high expression in the hippocampus and cerebral cortex. Whole genome sequencing efforts showed that NTF signaling evolved before the evolution of Vertebrates; thus, the shared ancestor of Protostomes, Cyclostomes, and Deuterostomes must have possessed a single ortholog of neurotrophins. After the first round of whole genome duplication that occurred in the last common ancestor of Vertebrates, the presence of two neurotrophins in Agnatha was hypothesized, while the monophyletic group of cartilaginous fishes, or Chondrichthyans, was situated immediately after the second whole genome duplication round that occurred in the last common ancestor of Gnathostomes. Chondrichthyans represent the outgroup of all other living jawed vertebrates (Gnathostomes) and the sister group of Osteichthyans (comprehensive of Actinopterygians and Sarcopterygians). We were able to first identify the second neurotrophin in Agnatha. Secondly, we expanded our analysis to include the Chondrichthyans, with their strategic phylogenetic position as the most basal extant Gnathostome taxon. Results from the phylogenetic analysis confirmed the presence of four neurotrophins in the Chondrichthyans, namely the orthologs of the four mammalian neurotrophins BDNF, NGF, NT-3, and NT-4. We then proceeded to study the expression of BDNF in the adult brain of the Chondrichthyan Scyliorhinus canicula. Our results showed that BDNF is highly expressed in the S. canicula brain and that its expression is highest in the Telencephalon, while the Mesencephalic and Diencephalic areas showed expression of BDNF in isolated and well-defined cell groups. NGF was expressed at much lower levels that could be detected by PCR but not by in situ hybridization. Our results warrant further investigations in Chondrichthyans to characterize the putative ancestral function of neurotrophins in Vertebrates.

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.

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.

Variation in the Transcriptome Response and Detoxification Gene Diversity Drives Pesticide Tolerance in Fishes.

Pesticides are critical for invasive species management but often have negative effects on nontarget native biota. Tolerance to pesticides should have an evolutionary basis, but this is poorly understood. Invasive sea lamprey (Petromyzon marinus) populations in North America have been controlled with a pesticide lethal to them at lower concentrations than native fishes. We addressed how interspecific variation in gene expression and detoxification gene diversity confer differential pesticide sensitivity in two fish species. We exposed sea lamprey and bluegill (Lepomis macrochirus), a tolerant native species, to 3-trifluoromethyl-4-nitrophenol (TFM), a pesticide commonly used in sea lamprey control. We then used whole-transcriptome sequencing of gill and liver to characterize the cellular response in both species. Comparatively, bluegill exhibited a larger number of detoxification genes expressed and a larger number of responsive transcripts overall, which likely contributes to greater tolerance to TFM. Understanding the genetic and physiological basis for pesticide tolerance is crucial for managing invasive species.

A noninvasive eDNA tool for detecting sea lamprey larvae in river sediments: Analytical validation and field testing in a low-abundance ecosystem.

Anthropogenic activities are increasingly threatening aquatic biodiversity, especially anadromous species. Monitoring and conservation measures are thus required to protect, maintain and restore imperilled populations. While many species can be surveyed using traditional capture and visual census techniques, species that use riverine habitats in a less conspicuous manner, such as sea lamprey Petromyzon marinus, can be more challenging to monitor. Sea lamprey larvae (ammocoetes) can spend several years in freshwater burrowed within soft sediments, inhibiting their detection and assessment. Here, we present a qPCR assay based on the detection of environmental DNA (eDNA) to identify the presence of ammocoetes burrowed in the sediment. We present an extensively validated method that ensured both species-specificity of the assay as well as the capacity to detect ammocoetes when abundances are low. Experiments on burrowing activity suggested that most of the DNA released into the sediment occurs during burrowing. Overall, we demonstrate this new molecular-based tool is an efficient and effective complement to traditional monitoring activities targeting larval stages of sea lampreys.

Engineering a Minimal Leucine-rich Repeat IgG-binding Module.

Sea lamprey immunization can yield leucine-rich repeat (LRR) protein binders analogous to globular antibodies developed from mammals. A novel minimal LRR was discovered through lamprey immunization with human immunoglobulin G Fc domain (IgG Fc). Initial attempts to solubly express this LRR protein, VLRB.IgGFc, in Escherichia coli proved challenging, so it was analyzed using the cell-free method ribosome display. In ribosome display, VLRB.IgGFc was found to bind specifically to the Fc domain of IgG, with little observed cross-reactivity to IgA or IgM. The minimal repeat protein architecture of VLRB.IgGFc may facilitate modular LRR extensions to incorporate additional or augmented functionality within a continuous, structurally defined scaffold. We exploited this modularity to design a chimera of a well-characterized, soluble LRR repebody and the initially insoluble VLRB.IgGFc to produce soluble Repe-VLRB.IgGFc. The minimal IgG Fc-binding module, Repe-VLRB.IgGFc, and future-engineered variants thereof should be useful additions to the biotechnological toolbox for detecting, purifying, or targeting IgGs. More generally, this two-step approach of minimal LRR binder discovery via sea lamprey immunization followed by modular augmentation of functionality may be of general utility in protein engineering.

Disturbances to energy metabolism in juvenile lake sturgeon (Acipenser fulvescens) following exposure to niclosamide.

Since the 1960s, invasive sea lamprey (Petromyzon marinus) populations in the Laurentian Great Lakes have been controlled by applying two chemicals, 3-trifluoromethyl-4-nitrophenol (TFM) and 2',5-dichloro-4'-nitrosalicylanilide (niclosamide, aka. Bayluscide®), to streams infested with larval sea lamprey. These "lampricide" applications primarily rely on TFM, and are often combined with 1-2% niclosamide, which increases treatment effectiveness. Niclosamide is also used alone to treat lentic habitats and in rivers with high discharge. However, little is known about niclosamide's possible adverse physiological effects on non-target organisms. Of particular concern is the lake sturgeon (Acipenser fulvescens), which is threatened throughout the Great Lakes basin where its habitat often overlaps with larval lamprey. Because niclosamide is believed to impair ATP production by uncoupling oxidative phosphorylation, we determined how it altered metabolic processes and acid-base balance in young-of-the-year (YOY) lake sturgeon exposed to their 9-h LC50 of niclosamide (0.11 mg L-1) for 9 h. Exposure to niclosamide led to decreased brain ATP and glucose reserves, and increased lactate, with no effect on brain glycogen. In contrast, substantial (60%) reductions in glycogen were observed in liver, suggesting that hepatic glycogen reserves were mobilized to meet the brain's glucose requirements when ATP supply was impaired during niclosamide exposure. Disturbances in carcass included reduced phosphocreatine (65-70%), 2- and 4-fold increases in pyruvate and lactate, and a slight metabolic acidosis, characterized by a 0.1 unit decrease in intracellular pH (pHi). Each of these disturbances were corrected within 24 h following depuration in clean (niclosamide-free) water. We conclude that if lake sturgeon survive exposure to niclosamide, they are able to rapidly replenish their energy stores (glycogen, ATP, phosphocreatine) and correct any corresponding metabolic disturbances within 24 h.

Reversible disruptions to energy supply and acid-base balance in larval sea lamprey exposed to the pesticide: Niclosamide (2',5-dichloro-4'-nitrosalicylanilide).

Since the 1960s, chemical control of larval sea lamprey has been achieved using the pesticides 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide (Bayluscide®). Much more potent, niclosamide is often used as an adjuvant for TFM, and on its own to treat lentic habitats, rivers with high discharge and currents, and for population surveys. Yet, little is known about its mode of action or physiological effects on sea lamprey. Like TFM, niclosamide is thought to impair mitochondrial ATP production by uncoupling oxidative phosphorylation. We therefore tested the hypothesis that niclosamide would result in metabolic perturbations and disturbances to acid-base balance in larval lamprey due to their need to balance ATP supply with ATP demands. When larval sea lamprey were exposed to the nominal 9-h niclosamide LC50 (0.11 mg L-1) over 9 h, it resulted in significant decreases in brain, phosphocreatine (35 %) and glycogen (50 %), accompanied by a 5-fold increase in lactate. In carcass, there were 25-30 % decreases in glycogen, corresponding increases in pyruvate and lactate, and a pronounced 0.5 unit decrease in intracellular pH. Calculation of the NAD+/NADH ratio in the carcass indicated that neither oxygen delivery nor the flux of reducing equivalents through the mitochondrial electron transport chain were impaired by niclosamide, supporting the hypothesis that niclosamide interferes with mitochondrial ATP production by uncoupling oxidative phosphorylation. Thus, greater reliance on glycogen, characterized by higher rates of glycolysis, temporarily mitigates the corresponding shortfall in ATP supply caused by niclosamide. Notably, all lamprey that survived niclosamide exposure readily restored ATP, phosphocreatine, glycogen and acid-base balance after recovery in niclosamide-free water. This resilience suggests that sea lamprey that survive or escape niclosamide treatment could compromise sea lamprey control efforts by subsequently completing their larval stage and developing into parasitic juvenile sea lamprey that could ultimately threaten Great Lake's fisheries populations.

Corrigendum: Gut Microbiota Associated With Different Sea Lamprey (Petromyzon marinus) Life Stages.

[This corrects the article DOI: 10.3389/fmicb.2021.706683.].

Gut Microbiota Associated With Different Sea Lamprey (Petromyzon marinus) Life Stages.

Sea lamprey (SL; Petromyzon marinus), one of the oldest living vertebrates, have a complex metamorphic life cycle. Following hatching, SL transition into a microphagous, sediment burrowing larval stage, and after 2-10+ years, the larvae undergo a dramatic metamorphosis, transforming into parasitic juveniles that feed on blood and bodily fluids of fishes; adult lamprey cease feeding, spawn, and die. Since gut microbiota are critical for the overall health of all animals, we examined the microbiota associated with SLs in each life history stage. We show that there were significant differences in the gut bacterial communities associated with the larval, parasitic juvenile, and adult life stages. The transition from larval to the parasitic juvenile stage was marked with a significant shift in bacterial community structure and reduction in alpha diversity. The most abundant SL-associated phyla were Proteobacteria, Fusobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria, and Firmicutes, with their relative abundances varying among the stages. Moreover, while larval SL were enriched with unclassified Fusobacteriaceae, unclassified Verrucomicrobiales and Cetobacterium, members of the genera with fastidious nutritional requirements, such as Streptococcus, Haemophilus, Cutibacterium, Veillonella, and Massilia, were three to four orders of magnitude greater in juveniles than in larvae. In contrast, adult SLs were enriched with Aeromonas, Iodobacter, Shewanella, and Flavobacterium. Collectively, our findings show that bacterial communities in the SL gut are dramatically different among its life stages. Understanding how these communities change over time within and among SL life stages may shed more light on the role that these gut microbes play in host growth and fitness.

Incursions of sea lamprey, Petromyzon marinus, and striped bass, Morone saxatilis, in Labrador waters: Episodic events or evidence of a northward range expansion?

We describe observations of sea lamprey (Petromyzon marinus) and striped bass (Morone saxatilis) incursions into Labrador, Canada. While P. marinus have been periodically observed in similar latitudes, their numbers have conspicuously increased in estuarine environments in 2020. In contrast, M. saxatilis were not observed from Labrador until 2017 but appear to be declining after the initial surge in abundance that peaked in 2018. It remains unclear whether spawning populations of either species exist. Given the potential to negatively affect species of commercial and cultural importance through predation, follow-up surveys are warranted.

Detection and Neutralization of SARS-CoV-2 Using Non-conventional Variable Lymphocyte Receptor Antibodies of the Evolutionarily Distant Sea Lamprey.

SARS-CoV-2 is a newly emerged betacoronavirus and the causative agent for the COVID-19 pandemic. Antibodies recognizing the viral spike protein are instrumental in natural and vaccine-induced immune responses to the pathogen and in clinical diagnostic and therapeutic applications. Unlike conventional immunoglobulins, the variable lymphocyte receptor antibodies of jawless vertebrates are structurally distinct, indicating that they may recognize different epitopes. Here we report the isolation of monoclonal variable lymphocyte receptor antibodies from immunized sea lamprey larvae that recognize the spike protein of SARS-CoV-2 but not of other coronaviruses. We further demonstrate that these monoclonal variable lymphocyte receptor antibodies can efficiently neutralize the virus and form the basis of a rapid, single step SARS-CoV-2 detection system. This study provides evidence for monoclonal variable lymphocyte receptor antibodies as unique biomedical research and potential clinical diagnostic reagents targeting SARS-CoV-2.

Contrasting physiological responses between invasive sea lamprey and non-target bluegill in response to acute lampricide exposure.

Control of invasive sea lamprey (Petromyzon marinus) in the Laurentian Great Lakes of North America uses lampricides, which consist of 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide. Lampricides are thought to inhibit aerobic energy synthesis, with TFM having a relatively greater selective action against lampreys. While the toxicity and physiological effects of TFM are known, the impacts associated with exposure to niclosamide and TFM:niclosamide mixtures are poorly characterized in fishes. Therefore, focusing on energy metabolism, we quantified the physiological responses of larval sea lamprey and bluegill (Lepomis macrochirus), a non-target, native species. Exposures consisted of each lampricide alone (TFM at the species-specific 24 h LC10; niclosamide at 1.5% of the mixture's TFM concentration) or a mixture of the two (larval sea lamprey at TFM 24 h LC10 + 1.5% niclosamide; bluegill at sea lamprey's TFM 24 h LC99.9 + 1.5% niclosamide) for 24 h. Tissues (brain, skeletal muscle, and liver) were sampled at 6, 12, and 24 h of exposure and assayed for concentrations of ATP, phosphocreatine, glycogen, lactate, and glucose and tissue lampricide levels. In larval sea lamprey, TFM had little effect on brain and skeletal muscle, but niclosamide resulted in a depletion of high energy substrates in both tissues. Mixture-exposed lamprey showed depletion of high energy substrates, accumulation of lactate, and high mortality rates. Bluegill were largely unaffected by toxicant exposures. However, bluegill liver showed lower glycogen and lactate under all three toxicant exposures suggesting increased metabolic turnover. Bluegill also had lower concentrations of TFM and niclosamide in their tissues when compared to lamprey. Our results indicate that lampricide toxicity in sea lamprey larvae is mediated through a depletion of high energy substrates because of impaired aerobic ATP synthesis. We also confirmed that non-target bluegill showed high tolerance to lampricide exposure, an effect potentially mediated through a high detoxification capacity relative to lampreys.

Rapid genetic adaptation to recently colonized environments is driven by genes underlying life history traits.

BACKGROUND: Uncovering the mechanisms underlying rapid genetic adaptation can provide insight into adaptive evolution and shed light on conservation, invasive species control, and natural resource management. However, it can be difficult to experimentally explore rapid adaptation due to the challenges associated with propagating and maintaining species in captive environments for long periods of time. By contrast, many introduced species have experienced strong selection when colonizing environments that differ substantially from their native range and thus provide a "natural experiment" for studying rapid genetic adaptation. One such example occurred when sea lamprey (Petromyzon marinus), native to the northern Atlantic, naturally migrated into Lake Champlain and expanded their range into the Great Lakes via man-made shipping canals. RESULTS: Utilizing 368,886 genome-wide single nucleotide polymorphisms (SNPs), we calculated genome-wide levels of genetic diversity (i.e., heterozygosity and π) for sea lamprey collected from native (Connecticut River), native but recently colonized (Lake Champlain), and invasive (Lake Michigan) populations, assessed genetic differentiation between all populations, and identified candidate genes that responded to selection imposed by the novel environments. We observed a 14 and 24% reduction in genetic diversity in Lake Michigan and Lake Champlain populations, respectively, compared to individuals from the Connecticut River, suggesting that sea lamprey populations underwent a genetic bottleneck during colonization. Additionally, we identified 121 and 43 outlier genes in comparisons between Lake Michigan and Connecticut River and between Lake Champlain and Connecticut River, respectively. Six outlier genes that contained synonymous SNPs in their coding regions and two genes that contained nonsynonymous SNPs may underlie the rapid evolution of growth (i.e., GHR), reproduction (i.e., PGR, TTC25, STARD10), and bioenergetics (i.e., OXCT1, PYGL, DIN4, SLC25A15). CONCLUSIONS: By identifying the genomic basis of rapid adaptation to novel environments, we demonstrate that populations of invasive species can be a useful study system for understanding adaptive evolution. Furthermore, the reduction in genome-wide levels of genetic diversity associated with colonization coupled with the identification of outlier genes underlying key life history traits known to have changed in invasive sea lamprey populations (e.g., growth, reproduction) illustrate the utility in applying genomic approaches for the successful management of introduced species.

Positive Effects of NPY1 Receptor Activation on Islet Structure Are Driven by Pancreatic Alpha- and Beta-Cell Transdifferentiation in Diabetic Mice.

Enzymatically stable and specific neuropeptide Y1 receptor (NPYR1) agonists, such as sea lamprey PYY(1-36) (SL-PYY(1-36)), are believed to improve glucose regulation in diabetes by targeting pancreatic islets. In this study, streptozotocin (STZ) diabetic transgenic GluCreERT2 ;ROSA26-eYFP and Ins1Cre/+;Rosa26-eYFP mouse models have been used to study effects of sustained NPYR1 activation on islet cell composition and alpha- and beta-cell lineage transitioning. STZ induced a particularly severe form of diabetes in Ins1Cre/+;Rosa26-eYFP mice, but twice-daily administration (25 nmol/kg) of SL-PYY(1-36) for 11 days consistently improved metabolic status. Blood glucose was decreased (p < 0.05 - p < 0.001) and both fasted plasma and pancreatic insulin significantly increased by SL-PYY(1-36). In both GluCreERT2 ;ROSA26-eYFP and Ins1Cre/+; Rosa26-eYFP mice, STZ provoked characteristic losses (p < 0.05 - p < 0.001) of islet numbers, beta-cell and pancreatic islet areas together with increases in area and central islet location of alpha-cells. With exception of alpha-cell area, these morphological changes were fully, or partially, returned to non-diabetic control levels by SL-PYY(1-36). Interestingly, STZ apparently triggered decreased (p < 0.001) alpha- to beta-cell transition in GluCreERT2 ;ROSA26-eYFP mice, together with increased loss of beta-cell identity in Ins1Cre/+;Rosa26-eYFP mice, but both effects were significantly (p < 0.001) reversed by SL-PYY(1-36). SL-PYY(1-36) also apparently reduced (p < 0.05) beta- to alpha-cell conversion in Ins1Cre/+;Rosa26-eYFP mice and glucagon expressing alpha-cells in GluCreERT2 ;ROSA26-eYFP mice. These data indicate that islet benefits of prolonged NPY1R activation, and especially restoration of beta-cell mass, are observed irrespective of diabetes status, being linked to cell lineage alterations including transdifferentiation of alpha- to beta-cells.

Generation and characterisation of C-terminally stabilised PYY molecules with potential in vivo NPYR2 activity.

BACKGROUND: Activation of neuropeptide Y2 receptors (NPYR2) by the N-terminally truncated, dipeptidyl peptidase-4 (DPP-4) generated, Peptide YY (PYY) metabolite, namely PYY(3-36), results in satiating actions. However, PYY(3-36) is also subject to C-terminal enzymatic cleavage, which annuls anorectic effects. METHODS: Substitution of l-Arg35 with d-Arg35 in the DPP-4 stable sea lamprey PYY(1-36) peptide imparts full C-terminal stability. In the current study, we have taken this molecule and introduced DPP-4 susceptibility by Iso3 substitution. RESULTS: As expected, [Iso3]sea lamprey PYY(1-36) and [Iso3](d-Arg35)sea lamprey PYY(1-36) were N-terminally degraded to respective PYY(3-36) metabolites in plasma. Only [Iso3](d-Arg35)sea lamprey PYY(1-36) was C-terminally stable. Both peptides possessed similar insulinostatic and anti-apoptotic biological actions to native PYY(1-36) in beta-cells. Unlike native PYY(1-36) and [Iso3](d-Arg35)sea lamprey PYY(1-36), [Iso3]sea lamprey PYY(1-36) displayed some proliferative actions in Npyr1 knockout beta-cells. In addition, [Iso3]sea lamprey PYY(1-36) induced more rapid NPYR2-dependent appetite suppressive effects in mice than its C-terminally stable counterpart. Twice daily administration of either peptide to high fat fed (HFF) mice resulted in significant body weight reduction and improvements in circulating triglyceride levels. [Iso3]sea lamprey PYY(1-36) treatment also prevented elevations in glucagon. Both peptides, and especially [Iso3]sea lamprey PYY(1-36), improved glucose tolerance. The treatment interventions also partially reversed the deleterious effects of sustained high fat feeding on pancreatic islet morphology. CONCLUSION: The present study confirms that sustained NPYR2 receptor activation by [Iso3](d-Arg35)sea lamprey induced significant weight lowering actions. However, identifiable benefits of this peptide over [Iso3]sea lamprey PYY(1-36), which was not protected against C-terminal degradation, were not pronounced.

Investigation on Endocrine Disruption of the Larval Lampricide 3-Trifluoromethyl-4-Nitrophenol: Short-Term Reproduction Assay with Fathead Minnow (Pimephales promelas).

3-Trifluoromethyl-4-nitrophenol (TFM) has been used for more than 60 yr to control the invasive parasitic sea lamprey (Petromyzon marinus) in the Great Lakes Basin (USA/Canada). In the early 1990s, researchers reported that TFM induced vitellogenin in fish and that TFM was an agonist for the rainbow trout estrogen receptor. To support continued registration of TFM for sea lamprey control, regulatory agencies required further testing to evaluate potential endocrine disruption effects. Fathead minnow (Pimephales promelas) were exposed to TFM at measured concentrations of 0.0659, 0.181, 0.594, 1.79, and 5.11 mg active ingredient (a.i.)/L for 21 d. No-observable- and lowest-observable-effect concentrations (NOEC and LOEC, respectively) were determined to be 1.79 mg/L or greater for each endpoint. Male survival in the highest treatment group was reduced relative to the controls. Percentage of egg fertility was reduced in the highest treatment group, resulting in an estimated NOEC of 1.79 mg/L. Whereas no effect on the gonadosomatic index (GSI) was observed for males, female GSI was increased in the 5.11-mg/L treatment. Vitellogenin production was not altered relative to the controls for all TFM treatment groups. However, female testosterone was elevated in the 5.11-mg/L treatment. The results suggest that prolonged exposure to TFM at concentrations exceeding 1.79 mg/L has the potential to disrupt endocrine function. Biologically relevant effects were found at the highest exposure concentration following a 21-d exposure. However, the duration of exposure in our study is not consistent with typical treatment durations (12 h) for sea lamprey control. Environ Toxicol Chem 2020;39:1599-1607. © 2020 SETAC.

Vasotocin and the origins of the vasopressin/oxytocin receptor gene family.

We previously characterized the arginine vasotocin receptor sequences in the jawless vertebrate sea lamprey. These gene and protein sequences provide clues to the origins of the various arginine vasopressin and oxytocin receptor family members in jawed vertebrates. However, orthological relationships between the jawless and jawed receptors is unclear. The current work is a closer examination and comparison between these G protein-coupled receptor sequences of the lamprey, the early jawed vertebrate elephant shark, and the boned fish and tetrapods. Our objective was to gain more insight into the differentiation of key signaling domains, which may then aid in discerning the pattern and timing of whole genome duplication early in the vertebrate lineage. The lamprey receptors remain less differentiated than shark receptors, due in part to the single vasotocin ligand in the lamprey and the selection pressure of a second ligand, oxytocin, in the shark. However, variation in G proteins utilized among the V1A, V1B and oxytocin receptor types has also contributed to differentiation, as well as leading to a change in second-messenger signaling pathway in the V2-type receptors. Conservation of gene regulatory elements may provide additional evidence of receptor gene orthology. These molecular evolution studies can ultimately be informative in applications such as drug discovery and environmental toxicology to determine cross-species sensitivity to chemicals.

RAPTURE (RAD capture) panel facilitates analyses characterizing sea lamprey reproductive ecology and movement dynamics.

Genomic tools are lacking for invasive and native populations of sea lamprey (Petromyzon marinus). Our objective was to discover single nucleotide polymorphism (SNP) loci to conduct pedigree analyses to quantify reproductive contributions of adult sea lampreys and dispersion of sibling larval sea lampreys of different ages in Great Lakes tributaries. Additional applications of data were explored using additional geographically expansive samples. We used restriction site-associated DNA sequencing (RAD-Seq) to discover genetic variation in Duffins Creek (DC), Ontario, Canada, and the St. Clair River (SCR), Michigan, USA. We subsequently developed RAD capture baits to genotype 3,446 RAD loci that contained 11,970 SNPs. Based on RAD capture assays, estimates of variance in SNP allele frequency among five Great Lakes tributary populations (mean F ST 0.008; range 0.00-0.018) were concordant with previous microsatellite-based studies; however, outlier loci were identified that contributed substantially to spatial population genetic structure. At finer scales within streams, simulations indicated that accuracy in genetic pedigree reconstruction was high when 200 or 500 independent loci were used, even in situations of high spawner abundance (e.g., 1,000 adults). Based on empirical collections of larval sea lamprey genotypes, we found that age-1 and age-2 families of full and half-siblings were widely but nonrandomly distributed within stream reaches sampled. Using the genomic scale set of SNP loci developed in this study, biologists can rapidly genotype sea lamprey in non-native and native ranges to investigate questions pertaining to population structuring and reproductive ecology at previously unattainable scales.