First successful artificial insemination of the endangered Louisiana pinesnake, Pituophis ruthveni.

The Louisiana pinesnake (Pituophis ruthveni) is considered one of the rarest snakes in North America and was federally listed under the Endangered Species Act in 2018. Captive breeding and reintroduction of zoo-bred hatchlings has been successful, however, limited founders in the captive population and the inability to bring new, wild genes into the captive colony presents a major concern for the conservation of this species. The use of artificial insemination (AI) was first applied to snakes in the 1980s but further development of the technique has since received little attention. Our goal was to develop a method of AI for use in breeding Louisiana pinesnakes to facilitate gene flow from wild to captive populations. We inseminated two captive Louisiana pinesnakes with semen collected from one donor male, novel to both females. Timing of AI occurred following the emergence of females from brumation, and when large, distinct follicles were detected using digital palpation. Females were inseminated four and five times over a period of 14 and 19 days, respectively, using fresh and 2-day refrigerator stored semen. One female laid seven eggs, which resulted in four fertile eggs and two viable hatchlings, while the second female produced three fertile of seven eggs laid but no viable hatchlings. Genetic analyses confirmed the donor male was the sire of hatchlings. This is the first successful AI of an endangered snake species and provides a framework for the use and optimization of assisted reproductive technologies for use in conservation breeding programs.

Effect of MHC and inbreeding on disassortative reproduction: A data revisit, extension and inclusion of fertilization in sand lizards.

The harmful effects of close inbreeding have been recognized for centuries and, with the rise of Mendelian genetics, was realized to be an effect of homozygosis. This historical background led to great interest in ways to quantify inbreeding, its depression effects on the phenotype and flow-on effects on mate choice and other aspects of behavioral ecology. The mechanisms and cues used to avoid inbreeding are varied and include major histocompatibility complex (MHC) molecules and the peptides they transport as predictors of the degree of genetic relatedness. Here, we revisit and complement data from a Swedish population of sand lizards (Lacerta agilis) showing signs of inbreeding depression to assess the effects of genetic relatedness on pair formation in the wild. Parental pairs were less similar at the MHC than expected under random mating but mated at random with respect to microsatellite relatedness. MHC clustered in groups of RFLP bands but no partner preference was observed with respect to partner MHC cluster genotype. Male MHC band patterns were unrelated to their fertilization success in clutches selected for analysis on the basis of showing mixed paternity. Thus, our data suggest that MHC plays a role in pre-copulatory, but not post-copulatory partner association, suggesting that MHC is not the driver of fertilization bias and gamete recognition in sand lizards.

Limited effects of inbreeding on breeding coloration.

Animal color signals may function as indicators of fighting ability when males compete for access to females. This allows opponents to settle aggressive interactions before they escalate into physical combat and injury. Thus, there may be strong directional selection on these traits, toward enhanced signal quality. This renders sexually selected traits particularly susceptible to inbreeding depression, due to relatively low ratios of additive genetic variance to dominance variance. We measured the effects of inbreeding on an intrasexually selected color signal (the badge) in a population of Swedish sand lizards (Lacerta agilis) using the Rhh software based on 17 to 21 microsatellites. Males of this sexually dichromatic species use the badge during aggressive interactions to display, and assess, fighting ability. We found negative effects of homozygosity on badge size, saturation, and brightness. However, no such effects were observed on color hue. Pairwise correlations between badge size, hue, and saturation were all statistically significant. Thus, the sand lizard "badge" is a multicomponent signal with variation explained by covariation in badge size, saturation, and color hue. Body mass corrected for skeletal size (body condition) positively predicted badge size and saturation, encouraging future research on the extent that sexual signals may convey information on multigene targets (i.e. "genic capture").

Sprint training interacts with body mass to affect hepatic insulin-like growth factor expression in female green anoles (Anolis carolinensis).

Locomotor performance is a key predictor of fitness in many animal species. As such, locomotion integrates the output of a number of morphological, physiological, and molecular levels of organization, yet relatively little is known regarding the major molecular pathways that bolster locomotor performance. One potentially relevant pathway is the insulin and insulin-like signaling (IIS) network, a significant regulator of physiological processes such as reproduction, growth, and metabolism. Two primary hormones of this network, insulin-like growth factor 1 (IGF1) and insulin-like growth factor 2 (IGF2) are important mediators of these processes and, consequently, of life-history strategies. We sprint-trained green anole (Anolis carolinensis) females to test the responsiveness of IGF1 and IGF2 hepatic gene expression to exercise training. We also tested how sprint training would affect glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 2 (EEF2). The former is a crucial enzyme for glycolytic function in a cell, and the latter is necessary for protein synthesis. Resistance exercise forces animals to increase investment of resources towards skeletal muscle growth. Because IGF1 and IGF2 are important hormones for growth, and GAPDH and EEF2 are crucial for proper cellular function, we hypothesized that these four genes would be affected by sprint training. We found that sprint training affects IGF and EEF2 expression, such that larger sprint-trained lizards express hepatic IGF1, IGF2, and EEF2 to a lesser extent than similarly sized untrained lizards. These results demonstrate that the IIS, and pathways connected to it, can react in a size-dependent manner and are implicated in the exercise response in reptiles.

Reduced Mitochondrial Respiration in Hybrid Asexual Lizards.

AbstractThe scarcity of asexual reproduction in vertebrates alludes to an inherent cost. Several groups of asexual vertebrates exhibit lower endurance capacity (a trait predominantly sourced by mitochondrial respiration) compared with congeneric sexual species. Here we measure endurance capacity in five species of Aspidoscelis lizards and examine mitochondrial respiration between sexual and asexual species using mitochondrial respirometry. Our results show reduced endurance capacity, reduced mitochondrial respiration, and reduced phenotypic variability in asexual species compared with parental sexual species, along with a positive relationship between endurance capacity and mitochondrial respiration. Results of lower endurance capacity and lower mitochondrial respiration in asexual Aspidoscelis are consistent with hypotheses involving mitonuclear incompatibility.

Postnatal expression of IGF2 is the norm in amniote vertebrates.

The insulin and insulin-like signalling (IIS) network plays an important role in mediating several life-history traits, including growth, reproduction and senescence. Although insulin-like growth factors (IGFs) 1 and 2 are both key hormones in the vertebrate IIS network, research on IGF2 in juveniles and adults has been largely neglected because early biomedical research on rodents found negligible IGF2 postnatal expression. Here, we challenge this assumption and ask to what degree IGF2 is expressed during postnatal life across amniotes by quantifying the relative gene expression of IGF1 and IGF2 using publicly available RNAseq data for 82 amniote species and quantitative polymerase chain reaction on liver cDNA at embryonic, juvenile and adult stages for two lizard, bird and mouse species. We found that (i) IGF2 is expressed postnatally across amniote species and life stages-often at a higher relative expression than IGF1, contradicting rodent models; (ii) the lack of rodent postnatal IGF2 expression is due to phylogenetic placement, not inbreeding or artificial selection; and (iii) adult IGF2 expression is sex-biased in some species. Our results demonstrate that IGF2 expression is typical for amniotes throughout life, suggesting that a comprehensive understanding of the mechanisms mediating variation in life-history traits will require studies that measure both IGFs.

Evolutionary physiology at 30+: Has the promise been fulfilled?: Advances in Evolutionary Physiology: Advances in Evolutionary Physiology.

Three decades ago, interactions between evolutionary biology and physiology gave rise to evolutionary physiology. This caused comparative physiologists to improve their research methods by incorporating evolutionary thinking. Simultaneously, evolutionary biologists began focusing more on physiological mechanisms that may help to explain constraints on and trade-offs during microevolutionary processes, as well as macroevolutionary patterns in physiological diversity. Here we argue that evolutionary physiology has yet to reach its full potential, and propose new avenues that may lead to unexpected advances. Viewing physiological adaptations in wild animals as potential solutions to human diseases offers enormous possibilities for biomedicine. New evidence of epigenetic modifications as mechanisms of phenotypic plasticity that regulate physiological traits may also arise in coming years, which may also represent an overlooked enhancer of adaptation via natural selection to explain physiological evolution. Synergistic interactions at these intersections and other areas will lead to a novel understanding of organismal biology.

Draft genomes for one Microcystis-resistant and one Microcystis-sensitive strain of the water flea, Daphnia pulicaria.

Daphnia species are well-suited for studying local adaptation and evolutionary responses to stress(ors) including those caused by algal blooms. Algal blooms, characterized by an overgrowth (bloom) of cyanobacteria, are detrimental to the health of aquatic and terrestrial members of freshwater ecosystems. Some strains of Daphnia pulicaria have demonstrated resistance to toxic algae and the ability to mitigate toxic algal blooms. Understanding the genetic mechanism associated with this toxin resistance requires adequate genomic resources. Using whole-genome sequence data mapped to the Daphnia pulex reference genome (PA42), we present reference-guided draft assemblies from one tolerant and one sensitive strain of D. pulicaria, Wintergreen-6 (WI-6), and Bassett-411 (BA-411), respectively. Assessment of the draft assemblies reveal low contamination levels, and high levels (95%) of genic content. Reference scaffolds had coverage breadths of 98.9-99.4%, and average depths of 33X and 29X for BA-411 and WI-6, respectively. Within, we discuss caveats and suggestions for improving these draft assemblies. These genomic resources are presented with a goal of contributing to the resources necessary to understand the genetic mechanisms and associations of toxic prey resistance observed in this species.

A chromosome-level genome assembly for the eastern fence lizard (Sceloporus undulatus), a reptile model for physiological and evolutionary ecology.

BACKGROUND: High-quality genomic resources facilitate investigations into behavioral ecology, morphological and physiological adaptations, and the evolution of genomic architecture. Lizards in the genus Sceloporus have a long history as important ecological, evolutionary, and physiological models, making them a valuable target for the development of genomic resources. FINDINGS: We present a high-quality chromosome-level reference genome assembly, SceUnd1.0 (using 10X Genomics Chromium, HiC, and Pacific Biosciences data), and tissue/developmental stage transcriptomes for the eastern fence lizard, Sceloporus undulatus. We performed synteny analysis with other snake and lizard assemblies to identify broad patterns of chromosome evolution including the fusion of micro- and macrochromosomes. We also used this new assembly to provide improved reference-based genome assemblies for 34 additional Sceloporus species. Finally, we used RNAseq and whole-genome resequencing data to compare 3 assemblies, each representing an increased level of cost and effort: Supernova Assembly with data from 10X Genomics Chromium, HiRise Assembly that added data from HiC, and PBJelly Assembly that added data from Pacific Biosciences sequencing. We found that the Supernova Assembly contained the full genome and was a suitable reference for RNAseq and single-nucleotide polymorphism calling, but the chromosome-level scaffolds provided by the addition of HiC data allowed synteny and whole-genome association mapping analyses. The subsequent addition of PacBio data doubled the contig N50 but provided negligible gains in scaffold length. CONCLUSIONS: These new genomic resources provide valuable tools for advanced molecular analysis of an organism that has become a model in physiology and evolutionary ecology.

Expression of insulin-like growth factors depends on both mass and resource availability in female green anoles (Anolis carolinensis).

The insulin and insulin-like signaling (IIS) network is an important mediator of cellular growth and metabolism in animals, and is sensitive to environmental conditions such as temperature and resource availability. The two main hormones of the IIS network, insulin-like growth factor 1 (IGF1) and insulin-like growth factor 2 (IGF2), are present in all vertebrates, yet little is known regarding the responsiveness of IGF2 in particular to external stimuli in non-mammalian animals. We manipulated diet (low or high quantity of food: low and high diet group, respectively) in adult green anole (Anolis carolinensis) females to test the effect of energetic state on hepatic gene expression of IGF1 and IGF2. The absolute expression of IGF2 in female green anoles was 100 times higher than that of IGF1 regardless of diet treatment, and IGF1 and IGF2 expression interacted with post-treatment body mass and treatment, as did the expression of the purported housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 2 (EEF2). The low diet group showed a negative relationship between body mass and gene expression for all genes, whereas the relationships between body mass and gene expression in the high diet group were either absent (in the case of IGF1) or positive (for all other genes). After accounting for total change in mass, the low diet group expressed IGF2, GAPDH and EEF2 at higher levels compared with individuals in the high diet group of a similar change in mass. These results illustrate that expression of IGF1 and IGF2, and of the housekeeping genes is affected by energe-tic status in reptiles.

Multiple Paternity in Garter Snakes With Evolutionarily Divergent Life Histories.

Many animal species exhibit multiple paternity, defined as multiple males genetically contributing to a single female reproductive event, such as a clutch or litter. Although this phenomenon is well documented across a broad range of taxa, the underlying causes and consequences remain poorly understood. For example, it is unclear how multiple paternity correlates with life-history strategies. Furthermore, males and females may differ in mating strategies and these patterns may shift with ecological context and life-history variation. Here, we take advantage of natural life-history variation in garter snakes (Thamnophis elegans) to address these questions in a robust field setting where populations have diverged along a slow-to-fast life-history continuum. We determine both female (observed) and male (using molecular markers) reproductive success in replicate populations of 2 life-history strategies. We find that despite dramatic differences in annual female reproductive output: 1) females of both life-history ecotypes average 1.5 sires per litter and equivalent proportions of multiply-sired litters, whereas 2) males from the slow-living ecotype experience greater reproductive skew and greater variance in reproductive success relative to males from the fast-living ecotype males despite having equivalent average reproductive success. Together, these results indicate strong intrasexual competition among males, particularly in the fast-paced life-history ecotype. We discuss these results in the context of competing hypotheses for multiple paternity related to population density, resource variability, and life-history strategy.

Tails of reproduction: Regeneration leads to increased reproductive investment.

Trade-offs between life-history traits are due to limited resources or constraints in the regulation of genetic and physiological networks. Tail autotomy, with subsequent regeneration, is a common anti-predation mechanism in lizards and is predicted to trade-off with life-history traits, such as reproduction. We utilize the brown anole lizard with its unusual reproductive pattern of single-egg clutches every 7-10 days to test for a trade-off in reproductive investment over 8 weeks of tail regeneration on a limited diet. In contrast to predictions, we found that investing in tissue regeneration had a positive effect on reproduction in terms of egg size (11.7% relative to controls) and hatchling size (11.5% relative to controls), and no effect on egg number or survival, with the increase in reproduction starting at peak regeneration. We discuss mechanistic hypotheses that the process of regeneration may cause increased energetic efficiency or utilized shared physiological pathways with reproductive investment.

Addressing the Unique Qualities of Upper-Level Biology Course-based Undergraduate Research Experiences through the Integration of Skill-Building.

Early exposure to course-based undergraduate research experiences (CUREs) in introductory biology courses can promote positive student outcomes such as increased confidence, critical thinking, and views of applicability in lower-level courses, but it is unknown if these same impacts are achieved by upper-level courses. Upper-level courses differ from introductory courses in several ways, and one difference that could impact these positive student outcomes is the importance of balancing structure with independence in upper-level CUREs where students typically have more autonomy and greater complexity in their research projects. Here we compare and discuss two formats of upper-level biology CUREs (Guided and Autonomous) that vary along a continuum between structure and independence. We share our experiences teaching an upper-level CURE in two different formats and contrast those formats through student reported perceptions of confidence, professional applicability, and CURE format. Results indicate that the Guided Format (i.e., a more even balance between structure and independence) led to more positive impacts on student outcomes than the Autonomous Format (less structure and increased independence). We review the benefits and drawbacks of each approach while considering the unique elements of upper-level courses relative to lower-level courses. We conclude with a discussion of how implementing structured skill-building can assist instructors in adapting CUREs to their courses.

The untapped potential of reptile biodiversity for understanding how and why animals age.

1. The field of comparative aging biology has greatly expanded in the past 20 years. Longitudinal studies of populations of reptiles with a range of maximum lifespans have accumulated and been analyzed for evidence of mortality senescence and reproductive decline. While not as well represented in studies of amniote senescence, reptiles have been the subjects of many recent demographic and mechanistic studies of the biology of aging. 2. We review recent literature on reptile demographic senescence, mechanisms of senescence, and identify unanswered questions. Given the ecophysiological and demographic diversity of reptiles, what is the expected range of reptile senescence rates? Are known mechanisms of aging in reptiles consistent with canonical hallmarks of aging in model systems? What are the knowledge gaps in our understanding of reptile aging? 3. We find ample evidence of increasing mortality with advancing age in many reptiles. Testudines stand out as slower aging than other orders, but data on crocodilians and tuatara are sparse. Sex-specific analyses are generally not available. Studies of female reproduction suggest that reptiles are less likely to have reproductive decline with advancing age than mammals. 4. Reptiles share many physiological and molecular pathways of aging with mammals, birds, and laboratory model organisms. Adaptations related to stress physiology coupled with reptilian ectothermy suggest novel comparisons and contrasts that can be made with canonical aging phenotypes in mammals. These include stem cell and regeneration biology, homeostatic mechanisms, IIS/TOR signaling, and DNA repair. 5. To overcome challenges to the study of reptile aging, we recommend extending and expanding long-term monitoring of reptile populations, developing reptile cell lines to aid cellular biology, conducting more comparative studies of reptile morphology and physiology sampled along relevant life-history axes, and sequencing more reptile genomes for comparative genomics. Given the diversity of reptile life histories and adaptations, achieving these directives will likely greatly benefit all aging biology.

Gene expression of the IGF hormones and IGF binding proteins across time and tissues in a model reptile.

The insulin and insulin-like signaling (IIS) network regulates cellular processes including pre- and postnatal growth, cellular development, wound healing, reproduction, and longevity. Despite their importance in the physiology of vertebrates, the study of the specific functions of the top regulators of the IIS network, insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs), has been mostly limited to a few model organisms. To expand our understanding of this network, we performed quantitative gene expression of IGF hormones in liver and qualitative expression of IGFBPs across tissues and developmental stages in a model reptile, the brown anole lizard (Anolis sagrei). We found that lizards express IGF2 across all life stages (preoviposition embryos to adulthood) and at a higher level than IGF1, which is opposite to patterns seen in laboratory rodents but similar to those seen in humans and other vertebrate models. IGFBP expression was ubiquitous across tissues (brain, gonad, heart, liver, skeletal muscle, tail, and regenerating tail) in adults, apart from IGFBP5, which was variable. These findings provide an essential foundation for further developing the anole lizard as a physiological and biomedical reptile model, as well as expanding our understanding of the function of the IIS network across species.

Characterization of two MHC II genes (DOB, DRB) in white-tailed deer (Odocoileus virginianus).

BACKGROUND: The major histocompatibility complex (MHC) is responsible for detecting and addressing foreign pathogens inside the body. While the general structure of MHC genes is relatively well conserved among mammalian species, it is notably different among ruminants due to a chromosomal inversion that splits MHC type II genes into two subregions (IIa, IIb). Recombination rates are reportedly high between these subregions, and a lack of linkage has been documented in domestic ruminants. However, no study has yet examined the degree of linkage between these subregions in a wild ruminant. The white-tailed deer (Odocoileus virginianus), a popular ruminant of the Cervidae family, is habitually plagued by pathogens in its natural environment (e.g. Haemonchus contortus, Elaeophora). Due to the association between MHC haplotypes and disease susceptibility, a deeper understanding of MHC polymorphism and linkage between MHC genes can further aid in this species' successful management. We sequenced MHC-DRB exon 2 (IIa) and MHC-DOB exon 2 (IIb) on the MiSeq platform from an enclosed white-tailed deer population located in Alabama. RESULTS: We identified 12 new MHC-DRB alleles, and resampled 7 alleles, which along with other published alleles brings the total number of documented alleles in white-tailed deer to 30 for MHC-DRB exon 2. The first examination of MHC-DOB in white-tailed deer found significantly less polymorphism (11 alleles), as was expected of a non-classical MHC gene. While MHC-DRB was found to be under positive, diversifying selection, MHC-DOB was found to be under purifying selection for white-tailed deer. We found no significant linkage disequilibrium between MHC-DRB and MHC-DOB, suggesting that these loci are unlikely to be closely linked. CONCLUSIONS: Overall, this study identified 12 new MHC-DRB exon 2 alleles and characterized a new, non-classical, MHC II gene (MHC-DOB) for white-tailed deer. We also found a lack of significant linkage between these two loci, which supports previous findings of a chromosomal inversion within the MHC type II gene region in ruminants, and suggests that white-tailed deer may have a recombination hotspot between these MHC regions similar to that found for Bos taurus.

The Promises and the Challenges of Integrating Multi-Omics and Systems Biology in Comparative Stress Biology.

Comparative stress biology is inherently a systems biology approach with the goal of integrating the molecular, cellular, and physiological responses with fitness outcomes. In this way, the systems biology approach is expected to provide a holistic understanding of how different stressors result in different fitness outcomes, and how different individuals (or populations or species) respond to stressors differently. In this perceptive article, I focus on the use of multiple types of -omics data in stress biology. Targeting students and those researchers who are considering integrating -omics approaches in their comparative stress biology studies, I discuss the promise of the integration of these measures for furthering our holistic understanding of how organisms respond to different stressors. I also discuss the logistical and conceptual challenges encountered when working with -omics data and the current hurdles to fully utilize these data in studies of stress biology in non-model organisms.

Mitochondria as central characters in a complex narrative: Linking genomics, energetics, pace-of-life, and aging in natural populations of garter snakes.

As a pacesetter for physiological processes, variation in metabolic rate can determine the shape of energetic trade-offs and thereby drive variation in life-history traits. In turn, such variation in metabolic performance and life-histories can have profound consequences for lifespan and lifetime fitness. Thus, the extent to which metabolic rate variation is due to phenotypic plasticity or fixed genetic differences among individuals or populations is likely to be shaped by natural selection. Here, we first present a generalized framework describing the central role of mitochondria in processes linking environmental, genomic, physiological, and aging variation. We then present a test of these relationships in an exemplary system: populations of garter snakes (Thamnophis elegans) exhibiting contrasting life-history strategies - fast-growing, early-reproducing, and fast-aging (FA) versus slow-growing, late-reproducing, and slow-aging (SA). Previous work has characterized divergences in mitochondrial function, reactive oxygen species processing, and whole-organism metabolic rate between these contrasting life-history ecotypes. Here, we report new data on cellular respiration and mitochondrial genomics and synthesize these results with previous work. We test hypotheses about the causes and implications of mitochondrial genome variation within this generalized framework. First, we demonstrate that snakes of the FA ecotype increase cellular metabolic rate across their lifespan, while the opposite pattern holds for SA snakes, implying that reduced energetic throughput is associated with a longer life. Second, we show that variants in mitochondrial genomes are segregating across the landscape in a manner suggesting selection on the physiological consequences of this variation in habitats varying in temperature, food availability, and rates of predation. Third, we demonstrate functional variation in whole-organism metabolic rate related to these mitochondrial genome sequence variants. With this synthesis of numerous datasets, we are able to further characterize how variation across levels of biological organization interact within this generalized framework and how this has resulted in the emergence of distinct life-history ecotypes that vary in their rates of aging and lifespan.

The utility of reptile blood transcriptomes in molecular ecology.

Reptiles and other nonmammalian vertebrates have transcriptionally active nucleated red blood cells. If blood transcriptomes can provide quantitative data to address questions relevant to molecular ecology, this could circumvent the need to euthanize animals to assay tissues. This would allow longitudinal sampling of animals' responses to treatments, as well as sampling of protected taxa. We developed and annotated blood transcriptomes from six reptile species and found on average 25,000 proteins are being transcribed in the blood, and there is a CORE group of 9,282 orthogroups that are found in at least four of six species. In comparison to liver transcriptomes from the same taxa, approximately two-thirds of the orthogroups were found in both blood and liver; and a similar percentage of ecologically relevant gene groups (insulin and insulin-like signalling, electron transport chain, oxidative stress, glucocorticoid receptors) were found transcribed in both blood and liver. As a resource, we provide a user-friendly database of gene ids identified in each blood transcriptome. Although on average 37% of reads mapped to haemoglobin, importantly, the majority of nonhaemoglobin transcripts had sufficient depth (e.g., 97% at ≥10 reads) to be included in differential gene expression analysis. Thus, we demonstrate that RNAseq blood transcriptomes from a very small blood sample (<10 µl) is a minimally invasive option in nonmammalian vertebrates for quantifying expression of a large number of ecologically relevant genes that would allow longitudinal sampling and sampling of protected populations.

Five months of voluntary wheel running downregulates skeletal muscle LINE-1 gene expression in rats.

Transposable elements (TEs) are mobile DNA and constitute approximately half of the human genome. LINE-1 (L1) is the only active autonomous TE in the mammalian genome and has been implicated in a number of diseases as well as aging. We have previously reported that skeletal muscle L1 expression is lower following acute and chronic exercise training in humans. Herein, we used a rodent model of voluntary wheel running to determine whether long-term exercise training affects markers of skeletal muscle L1 regulation. Selectively bred high-running female Wistar rats (n = 11 per group) were either given access to a running wheel (EX) or not (SED) at 5 wk of age, and these conditions were maintained until 27 wk of age. Thereafter, mixed gastrocnemius tissue was harvested and analyzed for L1 mRNA expression and DNA content along with other L1 regulation markers. We observed significantly (P < 0.05) lower L1 mRNA expression, higher L1 DNA methylation, and less L1 DNA in accessible chromatin regions in EX versus SED rats. We followed these experiments with 3-h in vitro drug treatments in L6 myotubes to mimic transient exercise-specific signaling events. The AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR; 4 mM) significantly decreased L1 mRNA expression in L6 myotubes. However, this effect was not facilitated through increased L1 DNA methylation. Collectively, these data suggest that long-term voluntary wheel running downregulates skeletal muscle L1 mRNA, and this may occur through chromatin modifications. Enhanced AMPK signaling with repetitive exercise bouts may also decrease L1 mRNA expression, although the mechanism of action remains unknown.