The red-eared slider (Trachemys scripta elegans) is a uniquely impressive vertebrate facultative anaerobe, capable of 18 weeks without oxygen at 3 degrees C. Metabolic rate depression (similar to 85 %) is the core feature of anaerobiosis and is characterized by the suppression of costly processes like protein synthesis/decay and the cell cycle. The elucidation of microRNA (miRNA) action in support of animal extreme stress adaptation is increasing, but is currently lacking in T.s. elegans anoxia tolerance. Here, we use small RNA sequencing and subsequent bioinformatic analyses to identify differentially expressed miRNA and predicted target pathways in 20 h anoxic red skeletal muscle of red-eared slider turtles. Of the 52 mapped miRNA species, we identify two that were upregulated (miR2114-5p, let-7f-5p) and two that were downregulated (miR-1260b, miR-5100) under 20 h anoxic conditions (| FC| > 1.5; p < 0.05). KEGG and GO analysis predict miRNA contribute to the aerobic to anaerobic respiration shift and outline miRNA-mediated inhibition of numerous gene sets in (1) protein turnover, (2) RNA turnover and (3) the cell cycle. Conversely, alleviated miRNA interference in branched amino acid biosynthesis, arachidonic acid and linolenic acid metabolism suggest a role in atrophy resistance of skeletal muscles.

Hibernation is a low-energy consumption survival strategy adopted by organisms to cope with adverse environments such as low temperatures and food scarcity. This study used the Pelophylax nigromaculatus as the model organism, and for the first time comprehensively analyzed the proteomic and phosphoproteomic regulatory networks in its kidneys, liver, and lungs through DIA quantitative proteomics technology, revealing specialized organ protection mechanisms under the hypometabolic state of hibernation. The research found that the regulation of these proteins relates to glycolysis and glycogenolysis, enhanced intracellular substance transport, membrane repair and energy buffering, rapid degradation of glycation end products, mitigation of inflammatory responses, and maintenance of signal transduction and cellular morphology. Using the glycogenolysis inhibitor CP-91149 to block glycogen breakdown, the study investigated frog hibernation status when glycogenolysis was inhibited to a certain extent, with results showing these frogs exhibited higher mortality during hibernation. This research not only provides substantial reliable data for elucidating the molecular mechanisms of amphibian hibernation but also establishes a foundational framework for understanding the molecular regulation of animal low-energy metabolism.

Since stress can be transmitted to congeners via social metabolites, it is paramount to understand how the social context of abiotic stress influences aquatic organisms' responses to global changes. Here, we integrated the transcriptomic and phenotypic responses of zebrafish embryos to a UV damage/repair assay following scenarios of heat stress, its social context and their combination. Heat stress preceding UV exposure had a hormetic effect through the cellular stress response and DNA repair, rescuing and/or protecting embryos from UV damage. However, experiencing heat stress within a social context negated this molecular hormetic effect and lowered larval fitness. We discuss the molecular basis of interindividual chemical transmission within animal groups as another layer of complexity to organisms' responses to environmental stressors.

Margarine is a popular high-calorie component of the Western diet and was shown to be associated with the development of metabolic syndrome. Intermittent fasting (IF) is an effective approach to improve health and prevent metabolic disorders. This study aimed to investigate the effects of margarine consumption, both ad libitum and in combination with IF regimens, using young C57BL/6J mice of both sexes. Female mice fed margarine ad libitum as a supplement to the standard diet showed significant body mass gain, reduced food intake, lower blood paraoxonase activity, and higher lipid peroxide (LOOH) levels, along with higher activities of antioxidant enzymes in the liver. Margarine-fed males showed higher food intake and had lower blood triacylglycerol levels, higher LOOH levels in adipose tissue, and lower LOOH levels in the liver than their control counterparts. When a margarine-supplemented diet was provided to mice with an IF regimen, males gained body mass faster and experienced severe metabolic changes, including elevated fasting blood glucose levels, higher total leukocyte count, triacylglycerol accumulation, and reduced glycogen levels in the liver compared to their margarine ad libitum counterparts. Females treated with margarine + IF showed a partial improvement in metabolic status and a decrease in proinflammatory markers compared to the group receiving margarine ad libitum. Hence, responses to the diets were sex-specific. Females that consumed margarine ad libitum had higher metabolic sensitivity than males. Meanwhile, IF provided some protective effects in females but worsened metabolic outcomes in males when combined with a high-fat margarine diet.

Amphibians, notably Xenopus laevis, exhibit remarkable dehydration tolerance, yet the tissue-specific proteomic adaptations remain poorly understood. Here, we used data-independent acquisition-based proteomics to analyze molecular responses in five organs and tissues (heart, kidney, liver, lung, skeletal muscle) of X. laevis during graded dehydration (15 %, 30 %) and rehydration. We identified 844 differentially expressed proteins (DEPs) in heart, 334 in kidney, 1057 in liver, 560 in lung, and 374 in muscle, respectively. DEPs in heart, liver, and kidney were significantly enriched in energy metabolism pathways, highlighting metabolic remodeling in response to dehydration and rehydration stresses. Progressively down-regulated proteins in heart during dehydration were enriched in NAD/NADH and ATP metabolic processes as well as glycolysis, aligning with metabolic rate depression to conserve energy and reduce oxidative stress. Conversely, lung and skeletal muscle prioritized cytoskeletal integrity (actin-myosin reorganization) over metabolic adjustments. Heart tissue exhibited activation of p38-MAPK signaling and up-regulation of MAPKAPK2, which is important in implementing the response to dehydration. Tissue-specific antioxidant responses showed that kidney and muscle catalase were up-regulated during 15 % dehydration, whereas lung delayed induction until rehydration to mitigate ischemia-reperfusion damage. Chaperone dynamics varied, with HSP27 up-regulated in heart and lung during dehydration and HSP60 sustained in liver, which contribute to maintaining the structural integrity of mitochondrial proteins. Moreover, X. laevis up-regulates proteins involved in oxygen transport, blood circulation and blood coagulation in order to counteract dehydration-induced hemoconcentration and hypovolemia. Five conserved DEPs shared in all examined tissues displayed dynamic expression, including Na+/K+-ATPase, plectin, annexin, electron transfer flavoprotein, and aconitate hydratase, indicating systemic adjustments in ion homeostasis, cytoskeletal stability, and mitochondrial metabolism. Overall, these findings highlight tissue-specific and conserved responses to dehydration stress, elucidate the importance of inhibiting metabolic pathways and eliciting protective mechanisms, and provide valuable insights for future studies exploring animal adaptation to stressful environments.

Northern Crayfish, Faxonius virilis, displays various strategies that allow them to survive extended periods of oxygen deprivation. However, certain epigenetic adaptations that these crayfish use have not been studied in detail, and the role of specific mechanisms used such as histone modifications remain unknown. Epigenetic studies offer a new perspective on how crayfish can regulate gene expression to redirect energy to essential functions needed for survival. This study investigates the regulation of histone modifications of proteins including acetylation and deacetylation in F. virilis in response to 20-h anoxia exposure. These histone modifications were studied via analysis of writer, reader, and eraser proteins such as lysine acetyltransferases (KATs), bromodomain proteins (BRDs), histone deacetylases (HDAC), and sirtuin proteins (SIRTs). Significant upregulation was seen in one histone protein and one lysine acetyltransferase: H3K14Ac and KAT2A. These proteins are known to be regulated by BRD2; a protein that specifically reads and targets H3K14Ac. In response to anoxia, a larger number of histone deacetylases and sirtuin proteins were upregulated in comparison to lysine acetyltransferases suggesting a focus on suppression of gene expression. The histone deacetylases and sirtuin proteins with significant upregulation were HDAC2, HDAC3, SIRT2, SIRT3, and SIRT6. These proteins have also all been implicated in DNA damage regulation which further suggests that crayfish focus limited energy on ensuring cell survival. This study provides an understanding of how histone acetylation and deacetylation are regulated in crayfish as a component of metabolic rate suppression under anoxia.

As ectotherms highly sensitive to environmental temperature fluctuations, skinks (a small lizard) are increasingly vulnerable to population instability due to global heatwaves. A clade model analysis of four Chinese skink species (Plestiodon capito, Plestiodon chinensis, Sphenomorphus indicus, and Scincella modesta) revealed positive selection acting on the ND6 gene in Sp. indicus. This species exhibits codon alterations in ND6, shifts its expression pathway and potentially decouples ND6 from high-temperature stress response mechanisms. To validate these findings, transcriptomic profiling was conducted to assess mitochondrial protein-coding gene (PCG) expression patterns under thermal stress. Using RT-qPCR, liver mitochondrial PCG transcript levels were compared between high-temperature (34 °C) and control (25 °C) groups in skink populations from distinct latitudes. Low-latitude species (P. chinensis and Sc. modesta) exhibited metabolic downregulation, characterized by a significant suppression of mitochondrial gene expression. Specifically, P. chinensis showed the downregulation of six mitochondrial genes (COII, COIII, ATP6, ND2, ND4, ND6) while upregulating one (ND1). By contrast, Sc. modesta showed the downregulation of nine genes (COI, COII, COIII, ATP8, ND1, ND3, ND4, ND4L, CYTB) and upregulated two (ND5, ND6). By contrast, high-latitude species exhibited divergent patterns: P. capito downregulated four genes (COI, COII, COIII, ND4L) and upregulated four others (ND1, ND2, ND3, ND4), whereas Sp. indicus downregulated six genes (COI, COII, ND2, ND3, ND4, ND4L) and upregulated one (ND5). These regulatory disparities suggest that low-latitude skinks have a greater capacity for metabolic depression to cope with chronic stress, whereas their high-latitude counterparts exhibit different adaptations. The findings provide valuable insights into assessing the adaptive potential of species in warming environments, particularly for ectotherms with limited thermoregulatory capacities.

The African clawed frog, Xenopus laevis, is able to survive prolonged arid conditions during seasonal droughts. During these conditions, X. laevis enters aestivation whereby its metabolic rate is suppressed, urea and ammonia levels increase, and its physiological functions slow. Various molecular mechanisms are employed by X. laevis to mitigate the deleterious effects of severe dehydration and hypometabolism, including pro-survival cellular processes that protect cells and tissues from damage and atrophy. While previous research has focused on antioxidant proteins' role in preventing oxidative stress, information on the role of anti-apoptotic signaling in X. laevis is lacking. As such, we investigated the role of nuclear factor-kappa B (NF-κB) signaling and its downstream target genes in liver and skeletal muscle tissue of X. laevis. The transcription factor, NF-κB, and its downstream target genes work to inhibit apoptotic machinery and promote cell survival. Herein, we found that NF-κB signaling activation in liver tissue leads to the selective upregulation of downstream anti-apoptotic proteins. In contrast, this upregulation occurs independently of NF-κB signaling in skeletal muscle tissue. Overall, our results serve to expand our knowledge of the anti-apoptotic mechanisms underlying the natural dehydration-tolerance of X. laevis, including its likely use in mitigating tissue atrophy during aestivation.

Chlorpyrifos (CPF) is an organophosphate insecticide that is extensively utilized globally due to its effectiveness against over 200 pest species. CPF exhibits its toxicity primarily through the inhibition of the acetylcholinesterase (AChE) enzyme, while mitochondrial damage and dysfunction have also been observed. The present study quantified the transcript levels of mitochondria protein-coding genes (mtPCGs) using quantitative real-time polymerase chain reaction (RT-qPCR) in samples of larvae of three dragonfly species (A. parthenope, E. elegans, and G. confluens) under different levels of CPF stress. By exposing larvae from uncontaminated populations to 0.05 μg/L CPF for 24 h, the transcript levels of seven mtPCGs in A. parthenope were significantly increased (p < 0.05) by 1.89 ± 0.42-fold for COI, 4.30 ± 0.24-fold for COIII, 5.94 ± 0.17-fold for ND1, 4.69 ± 0.56-fold for ND2, 3.44 ± 0.48-fold for ND4, 2.19 ± 0.53-fold for ND4L, and 5.05 ± 0.36-fold for Cytb, respectively. In E. elegans, the transcript levels of ND1, ND2, and ND4 increased by 1.23 ± 0.15, 1.48 ± 0.31, and 1.98 ± 0.25-fold, respectively (p < 0.05). In G. confluens, the transcript levels of COI, COIII, and ND4 increased by 1.56 ± 0.13, 1.50 ± 0.26, and 3.74 ± 0.40-fold, respectively (p < 0.01). It was demonstrated that the transcript levels of different mtPCGs showed significant up-regulation in the three different dragonfly larvae under CPF stress in the absence of mortality. ND4 was significantly increased in all three species, indicating that it is an important target gene. The present study underscores the response of mitochondrial gene expression in larvae of three different species in response to CPF pollutants, indicating that pesticide influences can potentially alter mitochondrial gene expression and potentially act as a method for assessing aquatic ecosystem health.

Background: Freeze tolerance is an uncommon but highly effective strategy that allows certain vertebrates to survive prolonged exposure to subzero temperatures in a frozen, ischemic state. While past studies have characterized the metabolic and biochemical adaptations involved, including cryoprotectant accumulation and metabolic rate suppression, the contribution of post-transcriptional gene regulation by microRNAs (miRNAs) remains largely unexplored. This study investigated freeze-responsive miRNAs in cardiac tissue of the gray tree frog, Dryophytes versicolor, to better understand the molecular mechanisms that support ischemic survival and tissue preservation. Methods: Adult frogs were subjected to controlled freezing at -2.5 °C, and cardiac tissue was collected from frozen and control animals. Total RNA was extracted and analyzed via small RNA sequencing to identify differentially expressed miRNAs, followed by target gene prediction and KEGG pathway enrichment analysis. Results: A total of 3 miRNAs were differentially expressed during freezing, with significant upregulation of miR-93-5p and let-7b-5p and downregulation of miR-4485-3p. Predicted targets of upregulated miRNAs included genes involved in immune signaling pathways (e.g., cytokine-cytokine receptor interaction), steroid hormone biosynthesis, and neuroactive ligand-receptor interaction, suggesting suppression of energetically costly signaling processes. Downregulation of miRNAs targeting cell cycle, insulin signaling, and WNT pathways indicates possible selective preservation of cytoprotective and repair functions. Conclusion: Overall, these results suggest that D. versicolor employs miRNA-mediated regulatory networks to support metabolic suppression, maintain essential signaling, and prevent damage during prolonged cardiac arrest. This work expands our understanding of freeze tolerance at the molecular level and may offer insights into biomedical strategies for cryopreservation and ischemia-reperfusion injury.

As one of the four primary evolutionary groups within myriapods, centipedes (Chilopoda) comprise approximately 3150 valid species. Recent molecular studies have begun to elucidate the phylogeny and time to divergence in Chilopoda; yet, identifying scutigeromorphs at the species level remains a notoriously challenging task. In this study, we obtained seven new complete mitogenomes of Thereuopoda clunifera (Wood, 1862) to investigate the phylogeny and divergence times of Chilopoda. Both maximum likelihood (ML) and Bayesian inference (BI) analyses recovered the relationship of (Scutigeromorpha + (Scolopendromorpha + (Lithobiomorpha + Geophilomorpha))). For Scutigeromorpha, seven newly sequenced mitogenomes of T. clunifera were divided into four distinct clades. Divergence time estimates suggest that the basal split of Chilopoda occurred during the Middle Ordovician period, with the origins of Scolopendromorpha, Lithobiomorpha, and Geophilomorpha dating to the Devonian period. Factors such as warm climates, coevolution between predator and prey, and the rifting of the Hainan Island may have driven the diversification of Scutigeromorpha. Based on genetic distance, the delimitation of molecular species, phylogenetic relationships, and divergence time analyses, we identified three cryptic species that existed within T. clunifera. This exceptionally high degree of hidden diversity can be ascribed to the morphological stasis that has occurred since the Paleozoic era and taxonomic impediment.

MicroRNAs (miRNAs), a critical class of short non-coding RNAs, regulate metabolic processes associated with mammalian torpor (e.g., Mus musculus), though their precise functional mechanisms remain incompletely characterized. Here, we employ RNA-seq to profile miRNA expression in white adipose tissue (WAT) of active versus torpid C57BL/6 mice. Among 863 detected miRNAs, 12 showed significant differential expression during torpor. In silico prediction of miRNA targets revealed these miRNAs preferentially target cancer-related pathways, indicating their potential role in suppressing cell proliferation during metabolic depression. Intriguingly, steroid biosynthesis genes escaped miRNA-mediated inhibition, suggesting active endocrine modulation by WAT during torpor. Machine learning helped to identify biomarkers for torpor in the mice, specifically a minimum of three miRNAs were sufficient to distinguish adipose samples from control versus torpid conditions. Taken together, this study demonstrates the role of miRNAs as transcriptional regulators of cell signalling pathways within WAT during mouse torpor.

Animals inhabiting altitudinal gradients exhibit a variety of adaptations to environmental variations. However, to date, changes in metabolomic profiles with altitude have been poorly characterized. Here, we used target and non-target metabolomic analyses of liver to investigate the environmental adaptations of Asiatic toads (Bufo gargarizans) along an altitudinal gradient (50 m, 1200 m, 2300 m, and 3400 m above sea level). Non-targeted metabolomics analysis identified 775 metabolites, and k-means clustering analysis showed that up-regulated metabolites along the altitudinal gradient were significantly enriched in the thiamine and sphingolipid metabolism pathways. Down-regulated metabolites were mainly enriched in alanine, aspartate and glutamate metabolism and glycolysis/glycogenesis. Weighted gene co-expression network analysis showed that metabolites positively correlated with altitude were mainly related to sphingolipid metabolism and glycerophospholipid metabolism, whereas those negatively correlated were involved in glycolysis/gluconeogenesis and glycerolipid metabolism. Moreover, a total of 52 metabolites were identified by the targeted metabolomics analysis. K-means clustering analysis showed that down-regulated metabolites along the altitudinal gradient were mainly enriched in pentose phosphate pathway and glycolysis/gluconeogenesis. In addition, toads from different altitudes exhibited significant variation in the activities of key metabolic enzymes, including phosphofructokinase, lactate dehydrogenase, and α-ketoglutarate dehydrogenase. In conclusion, the metabolic profiles of Asiatic toads along an altitudinal gradient differed significantly. These findings enhance our understanding of the physiological adaptations of toads to different environments along an altitudinal gradient.

The wood frog (Rana sylvatica) possesses remarkable adaptation mechanisms that ensures it survival in extreme environmental conditions, including enduring whole body freezing. The present study investigates the epigenetic mechanisms, specifically histone lysine acetylation and deacetylation, which are critical for regulating gene expression and conserving energy, underlying the wood frog’s ability to endure whole-body freezing. We investigated the expression patterns of lysine acetyltransferases (KATs) and lysine deacetylases (HDACs) in wood frog kidney over the freeze-thaw cycle. Our results reveal a significant downregulation of KATs in kidneys of frozen frogs, with specific KATs showing considerable reductions. This suggests that histone acetylation may play a vital role in suppressing gene expression and conserving energy during freezing. Furthermore, histone acetylation marks, including H2AK5ac, H2BK5ac, H3K9ac, H3K23ac, H3K27ac, and H3K56ac, showed repression under frozen and thawed conditions, indicating a role in silencing specific genes. HDACs exhibited dynamic regulation, with HDAC3 and HDAC11 showing significant repression in frozen frog kidneys, while HDAC5, p-HDAC4, and p-HDAC8 were downregulated during the recovery phase, suggesting their involvement in the thawing process. This research provides crucial insights into the epigenetic control of freeze tolerance in wood frog kidneys and offers a foundation for further exploration of epigenetic modifications that mediate the wood frog’s remarkable adaptations for freezing survival.

The garden dormouse (Eliomys quercinus) is a fat-storing mammal that undergoes annual periods of hibernation to mitigate the effects of food scarcity, low ambient temperatures, and reduced photoperiod that characterize winter. Like other hibernating species, this animal suppresses its metabolic rate by downregulating nonessential genes and processes in order to prolong available energy stores and limit waste accumulation throughout the season. MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that bind to mRNA and mediate post-transcriptional suppression, making miRNA ideal for modulating widespread changes in gene expression, including global downregulation typified by metabolic rate depression. Using next-generation sequencing, we analyzed an RNA-seq dataset to determine which miRNAs are differentially regulated during hibernation in the dormouse liver. We found that the expression of 19 miRNAs was altered during hibernation; however, only one major miRNA (miR-34a-5p) remained significantly downregulated after correcting for false discovery rate. Gene Ontology, KEGG Pathway Analysis, and DIANA-miRPath predicted that energy metabolism, nuclear-related functions such as histone binding, chromatin- and chromosomal binding, and the cell cycle are processes that may be differentially regulated during hibernation due to miRNA regulation. Taken together, our data suggest that miRNA influence appears to be strongly directed toward suppressing energy-intensive processes in the nucleus hence contributing to extend the animal's endogenous fuel reserves for the duration of hibernation.

The evolutionary relationships within the mantid subfamily Hierodulinae remain contentious, particularly concerning the morphological diversity and classification of species with leaf-like pronotum, which are broad and flattened thoracic structures. Traditionally regarded as a defining characteristic of the genus Rhombodera, specifically as a specialized pronotal trait, mitochondrial phylogenomics has cast doubt on this assumption, suggesting potential homoplasy. In this study, we generated 30 high-quality transcriptomes of Hierodulinae and established the first Mantodea-specific orthologous database (Mantodea_odb) to identify universal single-copy orthologues (USCOs) for phylogenomic inference. By integrating genomic and transcriptomic data, we reconstructed well-supported phylogenetic trees using both maximum likelihood and coalescent-based approaches, which provided the framework to assess evolutionary patterns of morphological traits. Divergence time estimation and ancestral state reconstruction suggested that the leaf-like pronotum evolved independently on multiple occasions within Hierodulinae, corresponding temporally to the late Paleogene to early Neogene (Oligocene-Miocene transition, ca. 20-23 Ma). Notably, our molecular phylogeny exhibits a strong correlation with distinct clades delineated by male genital morphology, thereby resolving longstanding taxonomic inconsistencies. These findings reveal decoupled evolutionary patterns between homoplastic pronotal traits and conserved genital morphology in Hierodulinae, showcasing how phylogenomics can discriminate between convergent and conserved traits.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) are obligate hibernators capable of reducing their metabolic rates by up to 99 % during winter. Their ability to remain dormant without food for an extended period in cold conditions has made them compelling subjects for research. Developing a clearer understanding of mechanisms surrounding the pre-transcriptional control of hibernating tissues is crucial for cryobiological applications such as organ preservation. Thus, we investigated the differential expression of 24 modified histones (MH) in the livers of torpid and euthermic free-ranging ground squirrels by immunoblotting histone-enriched extracts (p < 0.05). We identified the torpor-responsive downregulation of multiple permissive MHs (H2BK5ac, H3K18ac, H3K23ac, H3K27ac, H3K4me2, H3K4me3, H4K20me1, H4R3me2s), including total H2B and H4, while the linker histone H1.0 was the only histone species that was upregulated. The present study provides valuable insights into the involvement of histone post-translational modifications in the epigenetic landscape of deeply torpid ground squirrel livers.

Whether regulation of protein synthesis, autophagy, and regeneration are involved in slow/fast muscles in two disuse models (hindlimb unloading Sprague-Dawley rats ( Rattus norvegicus domestica (Berkenhout, 1769))) (HLU) and hibernating ground squirrels ( Spermophilus dauricus Brandt, 1843 (HIB)) is still unclear. Our results showed (1) fiber cross-sectional area was reduced in soleus (SOL) and extensor digitorum longus (EDL) of HLU whereas no change in HIB. The satellite cells/fiber was reduced and myonuclei/fiber was increased in SOL of HLU, while the percentage of satellite cells was significantly reduced in EDL of HIB. (2) Protein levels of phosphorylated- (P-)Akt, mTORC1, P-mTORC1, and P-S6K1 were reduced in SOL of HLU, whereas phosphorylated S6K1 was increased only in EDL of HIB. (3) Myostatin decreased in EDL of HLU but decreased in SOL of HIB. (4) Beclin1 increased in EDL of HLU and in SOL of HIB. (5) The activity of cathepsin L increased in SOL of HIB. (6) Collagen III increased in both SOL and EDL of HLU, but myogenin and collagen III increased in SOL, and collagen III was reduced in EDL of HIB. Taken together, Akt-mTORC1, Beclin1, and myogenin signaling showed muscle-specific responses in slow-twitch versus fast-twitch muscles, which may contribute to disuse atrophy in non-hibernators and anti-atrophy in hibernators.

Archaeognatha (bristletails) represent an evolutionarily significant but understudied insect group. Notably, the morphological identification method proposed by Mendes for Archaeognatha has certain limitations, which may lead to the underestimation or misidentification of some cryptic species. To address this issue, we employed an integrated strategy that combines morphological and molecular identification methods. Therefore, this study aimed to (1) identify cryptic diversity within Pedetontus silvestrii using mitogenomic data; (2) clarify phylogenetic relationships among Archaeognatha lineages; and (3) estimate divergence times for key taxonomic splits. We analyzed mitochondrial genomes from six P. silvestrii populations (Liaoning, Jilin, and Hebei Provinces) alongside 14 published Archaeognatha genomes. Key findings include the following: (1) Integrative analyses of genetic distances, phylogenetic reconstruction, bPTP-based molecular species delimitation, and divergence time estimation collectively revealed four evolutionarily distinct lineages within P. silvestrii. (2) Machilidae and Machilinae were non-monophyletic, whereas Petrobiellinae showed close affinity to Meinertellidae. (3) Archaeognatha originated ~301.19 Mya (Late Carboniferous); the Machilinae–Petrobiinae split occurred approximately 153.99 Mya (Jurassic). This study underscores the critical importance of mitogenomic analysis in elucidating cryptic biodiversity, while emphasizing the necessity of integrating morphological identification with molecular characterization for comprehensive species delineation in future taxonomic investigations.

Hibernation involves a profound metabolic rate depression (MRD) that enables certain species to survive prolonged periods of low energy availability. The thirteen-lined ground squirrel uses MRD to arrange cellular and biochemical pathways which suppress nonvital genetic and cellular pathways to conserve internal energy while preserving all essential processes. This study investigates the role of microRNAs (miRNAs) in controlling key signaling pathways and cellular processes in pancreatic tissue during hibernation. Using next-generation sequencing and broad genomic analysis, we analyzed and identified seven differentially expressed miRNAs (miR-29a-3p, miR-22-3p, miR-125-5p, miR-200a-3p, miR-328-3p, miR-21-5p, and miR-148-3p) in the pancreas of hibernating 13-lined ground squirrels (Ictidomys tridecemlineatus). Our findings reveal that these miRNAs regulate pathways involved in glucose homeostasis, including insulin secretion and metabolic regulation, contributing to the unique adaptations of hibernation. These insights advance our understanding of the molecular adaptations underlying hibernation and may have implications for therapeutic strategies targeting metabolic disorders such as diabetes.