Fatty acid metabolization and insulin regulation prevent liver injury from lipid accumulation in Himalayan marmots.

Fat storage and weight gain are dominant traits for hibernating mammals. However, excessive fat accumulation may cause liver damage. Here, we explore the lipid accumulation and metabolic processes of the Himalayan marmot (Marmota himalayana), a hibernating rodent species. We find that the unsaturated fatty acid (UFA) content in food was consistent with a large increase in the body mass of Himalayan marmots. Metagenomic analysis shows that Firmicutes Bacterium CAG:110 plays a synergistic role by synthesizing UFAs, which is demonstrated by fecal transplantation experiments, indicating that the gut microbiome promotes fat storage in Himalayan marmots for hibernation. Microscopic examination results indicate that the risk of fatty liver appears at maximum weight; however, liver function is not affected. Upregulations of UFA catabolism and insulin-like growth factor binding protein genes provide an entry point for avoiding liver injury.

Emergent social structure is typically not associated with survival in a facultatively social mammal.

For social animals, group social structure has important consequences for disease and information spread. While prior studies showed individual connectedness within a group has fitness consequences, less is known about the fitness consequences of group social structure for the individuals who comprise the group. Using a long-term dataset on a wild population of facultatively social yellow-bellied marmots (Marmota flaviventer), we showed social structure had largely no relationship with survival, suggesting consequences of individual social phenotypes may not scale to the group social phenotype. An observed relationship for winter survival suggests a potentially contrasting direction of selection between the group and previous research on the individual level; less social individuals, but individuals in more social groups experience greater winter survival. This work provides valuable insights into evolutionary implications across social phenotypic scales.

A novel cardiovirus species identified in feces of wild Himalayan marmots.

Recently a growing number of novel cardioviruses have been frequently discovered, which boosts interest in the search for the genetic diversity of cardioviruses. However, wild-marmot cardioviruses have been rarely reported. Here, a novel cardiovirus (tentatively named HHMCDV) was identified in fecal samples from wild Himalayan marmots in Qinghai Tibetan Plateau, China, by viral metagenomics analysis. 3 out of 99 fecal samples from Himalayan marmots were positive for HHMCDV, with the viral loads ranging from 2.7 × 105 to 1.3 × 107 gene copies/g. The complete genomic sequence of HHMCDV was 8108 nucleotides in length, with the typical cardiovirus genome organization and motifs. Coincidentally, while the data was analyzing, one marmot cardiovirus HT7 partial sequence was available in the Genbank, showing 95.1%, 95.6% and 96.0% amino acid (aa) identity in P1, P2 and P3, respectively. However, sequence analysis revealed that HHMCDV and HT7 are more closely related to species Cardiovirus F strain with 65.7%, 61.9-65.6%, 58.9-59.7%, 71.1-71.7%, 69.1-69.4% and 71.4-72.2% aa identity in polyprotein, P1, P2, P3, 2C and 3CD proteins, respectively. Phylogenetic analysis of P1, P2, P3 and 3CD aa sequences indicated that HHMCDV and HT7 clustered tightly and formed a distinct cluster in the Cardiovirus genus. Based on these data, we propose that HHMCDV and HT7 should be two different members of a potential novel species within the genus Cardiovirus. Further studies are needed to investigate the epidemiology and potential pathogenicity of the virus in Himalayan marmots.

Comprehensive Evaluation of RNA and DNA Viromic Methods Based on Species Richness and Abundance Analyses Using Marmot Rectal Samples.

Viral metagenomics is the most powerful tool to profile viromic composition for a given sample. Different viromic methods, including amplification-free ones, have been developed, but choosing them for different purposes requires comprehensive benchmarks. Here, we assessed the performance of four routinely used methods, i.e., multiple displacement amplification (MDA), direct metagenomic sequencing (MTG), sequence-independent single-primer amplification (SIA), and metatranscriptomic sequencing (MTT), using marmot rectal samples as the templates spiked with five known viruses of different genome types. The obtained clean data were differently contaminated by host and bacterial genomes, resulting in MDA having the most, with ~72.1%, but MTT had only ~7.5% data, useful for follow-up viromic analysis. MDA showed a broader spectrum with higher efficiency to profile the DNA virome, and MTT captured almost all RNA viruses with extraordinary sensitivity; hence, they are advisable in richness-based viromic studies. MTG was weak in capturing single-stranded DNA viruses, and SIA could detect both RNA and DNA viruses but with high randomness. Due to biases to certain types of viruses, the four methods caused different alterations to species abundance compared to the initial virus composition. SIA and MDA introduced greater stochastic errors to relative abundances of species, genus, and family taxa, whereas the two amplification-free methods were more tolerant toward such errors and thus are recommendable in abundance-based analyses. In addition, genus taxon is a compromising analytic level that ensures technically supported and biologically and/or ecologically meaningful viromic conclusions. IMPORTANCE Viral metagenomics can be roughly divided into species richness-based studies and species abundance-based analyses. Viromic methods with different principles have been developed, but rational selection of these techniques according to different purposes requires comprehensive understanding of their properties. By assessing the four most widely used methods using template samples, we found that multiple displacement amplification (MDA) and metatranscriptomic sequencing (MTT) are advisable for species richness-based viromic studies, as they show excellent efficiency to detect DNA and RNA viruses. Meanwhile, metagenomic sequencing (MTG) and MTT are more compatible with stochastic errors of methods introduced into relative abundance of viromic taxa and hence are rational choices in species abundance-based analyses. This study also highlights that MTG needs to tackle host genome contamination and ameliorate the capacity to detect single-stranded DNA viruses in the future, and the MTT method requires an improvement in bacterial rRNA depletion prior to library preparation.

Anaplasma phagocytophilum in Marmota himalayana.

BACKGROUND: Human granulocytic anaplasmosis is a tick-borne zoonotic disease caused by Anaplasma phagocytophilum. Coinfections with A. phagocytophilum and other tick-borne pathogens are reported frequently, whereas the relationship between A. phagocytophilum and flea-borne Yersnia pestis is rarely concerned. RESULTS: A. phagocytophilum and Yersnia pestis were discovered within a Marmota himalayana found dead in the environment, as determined by 16S ribosomal rRNA sequencing. Comparative genomic analyses of marmot-derived A. phagocytophilum isolate demonstrated its similarities and a geographic isolation from other global strains. The 16S rRNA gene and GroEL amino acid sequence identity rates between marmot-derived A. phagocytophilum (JAHLEX000000000) and reference strain HZ (CP000235.1) are 99.73% (1490/1494) and 99.82% (549/550), respectively. 16S rRNA and groESL gene screenings show that A. phagocytophilum is widely distributed in marmots; the bacterium was more common in marmots found dead (24.59%, 15/61) than in captured marmots (19.21%, 29/151). We found a higher Y. pestis isolation rate in dead marmots harboring A. phagocytophilum than in those without it (2 = 4.047, p < 0.05). Marmot-derived A. phagocytophilum was able to live in L929 cells and BALB/c mice but did not propagate well. CONCLUSIONS: In this study, A. phagocytophilum was identified for the first time in Marmota himalayana, a predominant Yersinia pestis host. Our results provide initial evidence for M. himalayana being a reservoir for A. phagocytophilum; moreover, we found with the presence of A. phagocytophilum, marmots may be more vulnerable to plague. Humans are at risk for co-infection with both pathogens by exposure to such marmots.

Hibernation slows epigenetic ageing in yellow-bellied marmots.

Species that hibernate generally live longer than would be expected based solely on their body size. Hibernation is characterized by long periods of metabolic suppression (torpor) interspersed by short periods of increased metabolism (arousal). The torpor-arousal cycles occur multiple times during hibernation, and it has been suggested that processes controlling the transition between torpor and arousal states cause ageing suppression. Metabolic rate is also a known correlate of longevity; we thus proposed the 'hibernation-ageing hypothesis' whereby ageing is suspended during hibernation. We tested this hypothesis in a well-studied population of yellow-bellied marmots (Marmota flaviventer), which spend 7-8 months per year hibernating. We used two approaches to estimate epigenetic age: the epigenetic clock and the epigenetic pacemaker. Variation in epigenetic age of 149 samples collected throughout the life of 73 females was modelled using generalized additive mixed models (GAMM), where season (cyclic cubic spline) and chronological age (cubic spline) were fixed effects. As expected, the GAMM using epigenetic ages calculated from the epigenetic pacemaker was better able to detect nonlinear patterns in epigenetic ageing over time. We observed a logarithmic curve of epigenetic age with time, where the epigenetic age increased at a higher rate until females reached sexual maturity (two years old). With respect to circannual patterns, the epigenetic age increased during the active season and essentially stalled during the hibernation period. Taken together, our results are consistent with the hibernation-ageing hypothesis and may explain the enhanced longevity in hibernators.

The genome of the American groundhog, Marmota monax.

We sequenced the genome of the North American groundhog, Marmota monax, also known as the woodchuck. Our sequencing strategy included a combination of short, high-quality Illumina reads plus long reads generated by both Pacific Biosciences and Oxford Nanopore instruments. Assembly of the combined data produced a genome of 2.74 Gbp in total length, with an N50 contig size of 1,094,236 bp. To annotate the genome, we mapped the genes from another M. monax genome and from the closely related Alpine marmot, Marmota marmota, onto our assembly, resulting in 20,559 annotated protein-coding genes and 28,135 transcripts. The genome assembly and annotation are available in GenBank under BioProject PRJNA587092.

Genetic and molecular features for hepadnavirus and plague infections in the Himalayan marmot.

The Himalayan marmot (Marmota himalayana), a natural host and transmitter of plague, is also susceptible to the hepadnavirus infection. To reveal the genetic basis of the hepadnavirus susceptibility and the immune response to plague, we systematically characterized the features of immune genes in Himalayan marmot with those of human and mouse. We found that the entire major histocompatibility complex region and the hepatitis B virus pathway genes of the Himalayan marmot were conserved with those of humans. A Trim (tripartite motif) gene cluster involved in immune response and antiviral activity displays dynamic evolution, which is reflected by the duplication of Trim5 and the absence of Trim22 and Trim34. Three key regions of Ntcp, which is critical for hepatitis B virus entry, had high identity among seven species of Marmota. Moreover, we observed a severe alveolar hemorrhage, inflammatory infiltrate in the infected lungs and livers from Himalayan marmots after infection of EV76, a live attenuated Yersinia pestis strain. Lots of immune genes were remarkably up-regulated, which several hub genes Il2rγ, Tra29, and Nlrp7 are placed at the center of the gene network. These findings suggest that Himalayan marmot is a potential animal model for study on the hepadnavirus and plague infection.

iMarmot: an integrative platform for comparative and functional genomics of marmots.

BACKGROUND: Marmots are large Holarctic rodents with unique biological features, making them potential animal models in various research fields. Due to the rapid accumulation of the genetic data in marmots, a highly integrative database is urgent needed. DESCRIPTION: iMarmot is freely available on the web at http://www.marmotdb.org/ and currently contains the biological information of 14 marmots, genomic sequence of 6 marmots, syntenic relationship and orthologs among 3 marmots, and expression profiles of several hibernators and plague hosts. To assist with the genomic and transcriptomic analysis, we also integrated a set of analysis and visualization tools, such as KEGG or GO enrichment analysis, PCA, Blast, Muscle, GeneWise, Lastz, and JBrowse. Particularly, one DEGs (differentially expressed genes) module has been implemented in this database to visualize the gene expression changes in hibernators and plague hosts. CONCLUSION: This database will provide comprehensive information and analysis platform for researchers interested in understanding the biological features of marmots.

Older mothers produce more successful daughters.

Annual reproductive success and senescence patterns vary substantially among individuals in the wild. However, it is still seldom considered that senescence may not only affect an individual but also affect age-specific reproductive success in its offspring, generating transgenerational reproductive senescence. We used long-term data from wild yellow-bellied marmots (Marmota flaviventer) living in two different elevational environments to quantify age-specific reproductive success of daughters born to mothers differing in age. Contrary to prediction, daughters born to older mothers had greater annual reproductive success on average than daughters born to younger mothers, and this translated into greater lifetime reproductive success. However, in the favorable lower elevation environment, daughters born to older mothers also had greater age-specific decreases in annual reproductive success. In the harsher higher elevation environment on the other hand, daughters born to older mothers tended to die before reaching ages at which such senescent decreases could be observed. Our study highlights the importance of incorporating environment-specific transgenerational parent age effects on adult offspring age-specific life-history traits to fully understand the substantial variation observed in senescence patterns in wild populations.

Diet of the brown bear in Himalaya: Combining classical and molecular genetic techniques.

The ecological requirements of brown bears are poorly known in the Himalaya region, which complicates conservation efforts. We documented the diet of the Himalayan brown bear (Ursus arctos isabellinus) by combining classical scat analysis and a newly developed molecular genetic technique (the trnL approach), in Deosai National Park, Pakistan. Brown bears consumed over 50 plant species, invertebrates, ungulates, and several rodents. Eight plant families; Poaceae, Polygonaceae, Cyperaceae, Apiaceae, Asteraceae, Caryophyllaceae, Lamiaceae, and Rubiaceae were commonly eaten with graminoids comprising the bulk of the diet. Golden marmots comprised the major mammalian biomass in the park, and were also the main meat source for bears. Animal matter, making 36% of dietary content, contributed half of the digestible energy, due to its higher nutritious value. We did not find a significant temporal pattern in diet, perhaps because the availability of the major diet (graminoids) did not change over the foraging period. Male brown bears were more carnivorous than females, probably because of their larger size, which requires higher energy and also makes them more efficient in capturing marmots. Frequencies of three plant species were also significantly higher in male brown bears; Bistorta affinis, Carex diluta, and Carex sp. Diet of the brown bear differed significantly between the park and surrounding valleys. In valleys, diet consisted predominantly of graminoids and crops, whereas the park provided more nutritious and diverse foodThe estimated digestible energy available to brown bears in Deosai was the lowest documented among brown bear populations, due to the lack of fruits and a relatively lower meat content. The low nutritious diet and high cost of metabolism in a high-altitude environment, probably explains the very low reproductive potential of this population.

The Genome Sequence of the Eastern Woodchuck (Marmota monax) - A Preclinical Animal Model for Chronic Hepatitis B.

The Eastern woodchuck (Marmota monax) has been extensively used in research of chronic hepatitis B and liver cancer because its infection with the woodchuck hepatitis virus closely resembles a human hepatitis B virus infection. Development of novel immunotherapeutic approaches requires genetic information on immune pathway genes in this animal model. The woodchuck genome was assembled with a combination of high-coverage whole-genome shotgun sequencing of Illumina paired-end, mate-pair libraries and fosmid pool sequencing. The result is a 2.63 Gigabase (Gb) assembly with a contig N50 of 74.5 kilobases (kb), scaffold N50 of 892 kb, and genome completeness of 99.2%. RNA sequencing (RNA-seq) from seven different tissues aided in the annotation of 30,873 protein-coding genes, which in turn encode 41,826 unique protein products. More than 90% of the genes have been functionally annotated, with 82% of them containing open reading frames. This genome sequence and its annotation will enable further research in chronic hepatitis B and hepatocellular carcinoma and contribute to the understanding of immunological responses in the woodchuck.

Ice-Age Climate Adaptations Trap the Alpine Marmot in a State of Low Genetic Diversity.

Some species responded successfully to prehistoric changes in climate [1, 2], while others failed to adapt and became extinct [3]. The factors that determine successful climate adaptation remain poorly understood. We constructed a reference genome and studied physiological adaptations in the Alpine marmot (Marmota marmota), a large ground-dwelling squirrel exquisitely adapted to the "ice-age" climate of the Pleistocene steppe [4, 5]. Since the disappearance of this habitat, the rodent persists in large numbers in the high-altitude Alpine meadow [6, 7]. Genome and metabolome showed evidence of adaptation consistent with cold climate, affecting white adipose tissue. Conversely, however, we found that the Alpine marmot has levels of genetic variation that are among the lowest for mammals, such that deleterious mutations are less effectively purged. Our data rule out typical explanations for low diversity, such as high levels of consanguineous mating, or a very recent bottleneck. Instead, ancient demographic reconstruction revealed that genetic diversity was lost during the climate shifts of the Pleistocene and has not recovered, despite the current high population size. We attribute this slow recovery to the marmot's adaptive life history. The case of the Alpine marmot reveals a complicated relationship between climatic changes, genetic diversity, and conservation status. It shows that species of extremely low genetic diversity can be very successful and persist over thousands of years, but also that climate-adapted life history can trap a species in a persistent state of low genetic diversity.

Woodchuck VEGF (wVEGF) characteristics: Model for angiogenesis and human hepatocellular carcinoma directed therapies.

Vascular endothelial growth factor (VEGF) stimulates angiogenesis. Human hepatocellular carcinoma (HCC) is a VEGF-driven tumor often associated with chronic hepatitis B or C virus infection. The woodchuck is a well-characterized model of hepatitis B virus related HCC and a valuable tool for translational studies of novel VEGF targeted agents. We cloned the cDNA encoding woodchuck VEGF (wVEGF), transiently expressed it in COS cells and functionally characterized the recombinant protein. The open reading frame of wVEGF contained 645 nucleotides encoding a protein of 214 amino acids. Two protein bands (17 and 25 kDa) were detected in conditioned media of wVEGF expressing COS-1 cells and a single band of 25 kDa was identified in cell lysates. Addition of recombinant wVEGF to COS cells enhanced cell proliferation and stimulated VEGFR2, Akt, ERK1/2, and FAK phosphorylation. Sunitinib, a tyrosine kinase inhibitor, inhibited wVEGF- induced VEGFR2 phosphorylation in a dose-dependent manner. Finally, development of HCC in woodchucks was accompanied by increased laminin and PECAM1 expressing vessels, VEGFR2 expression, increased ligation of VEGF to VEGFR2, and a decrease in collagen IV-positive blood vessels. Our results suggest that woodchuck model can be used further to study angiogenesis and the effect of VEGF directed therapies in human HCC.

Features of Interspecific Contacts and Hybridization of Ground Squirrels (Marmotinae, Sciuridae, Rodentia) in Mongolia.

Using molecular genetic methods, we investigated the secondary contact zones of two pairs of species of ground squirrels of Mongolia. In common colonies of marmots M. sibirica and M. baibacina, we revealed a high frequency of occurrence of hybrid individuals that were viable and fertile. A hybridization between S. alaschanicus and S. pallidicauda was sporadic in nature. The hypothesis about an extensive hybrid zone in these species of ground squirrels was not confirmed. An occupation of suboptimal biotopes by individuals of different species was the main factor contributing to hybridization in both case. The complex social behavior in marmots affected on the localization and diffusion of the hybridization process.

Extrinsic and intrinsic constraints interact to drive extra-pair paternities in the Alpine marmot.

To reproduce, animals have to form pairs and large variations in the degree of mate switching are observed. Extrinsic and intrinsic factors can constrain individual's mate switching. Among intrinsic factors, genes involved in pair-bonding, such as Avpr-1a, receive increasing attention. The length of microsatellites present in the regulatory region of Avpr-1a determines the neural densities and distributions of the vasopressin receptors known to impact pair-bonding behaviours. For the first time, we investigated whether and how the genetic makeup at Avpr-1a, an intrinsic factor, and the social context, an extrinsic factor, experienced by wild Alpine marmot (Marmota marmota) females affect the proportion of extra-pair young. This proportion was positively correlated with the length of their Avpr-1a regulatory region but only when the social constraints were relaxed, that is when mature male subordinates were present. When ignoring the interactive effect between the length of their Avpr-1a regulatory region and the social constraints, the genetic makeup at Avpr-1a was not associated with the proportion of extra-pair young. Under natural conditions, the genetic regulation of pair-bonding could be hidden by extrinsic factors constraining mate choice.

Chinese woodchucks with different susceptibility to WHV infection differ in their genetic background exemplified by cytochrome B and MHC-DRB molecules.

BACKGROUND: Chinese woodchucks (M. himalayana) were recently found to be susceptible to woodchuck hepatitis virus (WHV) infection. In this study, we aimed to determine the susceptibility to WHV infection of M. himalayana from different areas and their association with the animal genetic background exemplified by cytochrome B and MHC-DRB molecules. METHODS: Animals from four different areas in Qinghai province were inoculated with WHV59 strains. The virological markers including WHV surface antigen (WHsAg), WHV core antibody (WHcAb), and WHV DNA in serum were measured by ELISA and Real-time PCR, respectively. The sequences of cytochrome B gene and MHC-DRB molecules were obtained and sorted with Clustalx software. The nucleotide variation sites were identified using MEGA5 software. RESULTS: The animals from four different areas had different susceptibility to WHV infection. Animals from TR and TD areas had a high level of long-lasting viremia, while those from GD and WL areas had a low level of transient viremia after WHV inoculation. All of the animals belong to the same subspecies M. himalayana robusta identified by cytochrome B gene sequences. Based on their nucleotide variation pattern, 8 alleles of cytochrome B gene were identified, and 7 MHC-DRB alleles were identified. Allele A of cytochrome B and Allele Mamo-DRB1*02 of MHC-DRB was found to be frequent in animals from TR and TD areas, while Allele H of cytochrome B and Allele Mamo-DRB1*07 of MHC-DRB was predominant in animals from GD and WL areas. CONCLUSION: Chinese woodchucks from different areas differed in their susceptibility to WHV infection, though they belong to the same subspecies M. himalayana robusta. The genetic background exemplified by cytochrome B and MHC-DRB differed in Chinese woodchucks with different susceptibility to WHV infection.

Genetic basis of between-individual and within-individual variance of docility.

Between-individual variation in phenotypes within a population is the basis of evolution. However, evolutionary and behavioural ecologists have mainly focused on estimating between-individual variance in mean trait and neglected variation in within-individual variance, or predictability of a trait. In fact, an important assumption of mixed-effects models used to estimate between-individual variance in mean traits is that within-individual residual variance (predictability) is identical across individuals. Individual heterogeneity in the predictability of behaviours is a potentially important effect but rarely estimated and accounted for. We used 11 389 measures of docility behaviour from 1576 yellow-bellied marmots (Marmota flaviventris) to estimate between-individual variation in both mean docility and its predictability. We then implemented a double hierarchical animal model to decompose the variances of both mean trait and predictability into their environmental and genetic components. We found that individuals differed both in their docility and in their predictability of docility with a negative phenotypic covariance. We also found significant genetic variance for both mean docility and its predictability but no genetic covariance between the two. This analysis is one of the first to estimate the genetic basis of both mean trait and within-individual variance in a wild population. Our results indicate that equal within-individual variance should not be assumed. We demonstrate the evolutionary importance of the variation in the predictability of docility and illustrate potential bias in models ignoring variation in predictability. We conclude that the variability in the predictability of a trait should not be ignored, and present a coherent approach for its quantification.

Molecular cloning, characterization and expression analysis of Tim-3 and Galectin-9 in the woodchuck model.

In recent years, a critical role for T cell immunoglobulin mucin domain 3 (Tim-3) and its ligand Galectin-9 (Gal-9) has emerged in infectious disease, autoimmunity and cancer. Manipulating this immune checkpoint may have immunotherapeutic potential and could represent an alternative approach for improving immune responses to viral infections and cancer. The woodchuck (Marmot monax) infected by woodchuck hepatitis virus (WHV) represents an informative animal model to study HBV infection and HCC. In the current study, the cDNA sequences of woodchuck Tim-3 and Gal-9 were cloned, sequenced and characterized. The extracellular domain of Tim-3 cDNA sequence consisted of 576bp coding sequence (CDS) that encoded 192 amino acids. The 1076bp full-length Gal-9 cDNA sequence consisted of 1059bp coding sequence (CDS) that encoded 352 amino acids with a molecular weight of 39.7kDa. The phylogenetic tree analysis revealed that the woodchuck Tim-3 and Gal-9 had the closest genetic relationship with Ictidomys tridecemlineatus. The result of quantification PCR analysis showed that ubiquitous expression of Gal-9 but not Tim-3 in different tissues of naive woodchucks. Elevated liver Gal-9 expression was observed in woodchucks with chronic WHV infection. Moreover, a polyclonal antibody against the extracellular domain of woodchuck Tim-3 were generated and identified by flow cytometry. Our results serve as a foundation for further insight into the role of Tim-3/Galectin-9 signaling pathway in viral hepatitis and HCC in the woodchuck model.