Biology subjects

Hubert, D. L.

Publications and source records attributed to Hubert, D. L..

6 recordsLinked to original sources

bioRxiv2026

Cold, dark, and hungry: Dynamic transcriptional regulation across eight months of brumation

Long-term winter dormancy in ectotherms (brumation) defines the annual cycle of many temperate-zone reptiles, yet the transcriptional regulation that supports survival across months of cold and aphagy remains poorly understood. We generated time-resolved transcriptomic profiles of liver and testis from male red-sided garter snakes (Thamnophis sirtalis parietalis) at five timepoints spanning the eight-month brumation cycle: pre-brumation, early, mid-, and late brumation, and post-arousal under continued aphagy. Time-course negative-binomial regression (maSigPro) followed by gene-set enrichment analysis identified 3,715 transcripts in liver and 5,828 in testis with significant temporal expression structure organized into five overarching temporal patterns: sustained downregulation, downregulation with post-arousal recovery, sustained upregulation, brumation-specific upregulation and cyclic modulation. Liver showed coordinated upregulation of fatty acid mobilization enzymes (ATGL, FOXO1, PPAR, CPT1A) and gluconeogenic regulators (CREBBP, PCK1) coincident with sustained low temperatures. Additionally, low temperature transcriptional activity was suggestive of a shift toward hepatic lipid mobilization and alanine-supported gluconeogenesis. Testis showed sustained suppression of meiosis, reproduction, and DNA-metabolism gene sets that did not fully recover at arousal consistent with this species dissociated reproductive pattern. Both tissues showed coordinated upregulation of stress-response pathways involving heat-shock proteins, HIF1 and a glutathione-based antioxidant defense. Interestingly, three vitellogenin transcripts and 17{beta}-hydroxysteroid dehydrogenases associated with estradiol-favoring steroid metabolism were upregulated in male liver during late brumation, which is not expected during natural physiology in adult males. Together these data support a framework in which temperature- and starvation-associated transcriptional programs contribute to survival of one of the longest, coldest brumations documented in a squamate. Summary statementA time-resolved transcriptomic analysis of liver and testis spanning eight months of winter brumation in Thamnophis sirtalis parietalis reveals gene expression patterns consistent with a temperature-associated shift toward hepatic lipid mobilization, sustained reproductive suppression, and vitellogenin response in males.

physiology↗

bioRxiv2026

Signal, noise, and sampling: How pool size and replication shape metabolomic inference

Metabolomics provides a direct readout of physiological state and is increasingly used in evolutionary and systems biology. In small organisms such as Drosophila melanogaster, metabolomic analyses typically require pooling individuals to obtain sufficient material, yet pool sizes vary widely across studies with little justification. How pooling and biological replication influence metabolome characterization and the detection of biological signal remains poorly understood. Here, we evaluate the effects of pool size and biological replication on metabolomic profiles and signal detection using two complementary experimental designs. In the first, we assess how pooling (5, 50, or 100 individuals) influences metabolomic structure and reproducibility in inbred and outbred populations. In the second, we test how pool size interacts with systematic variation in replicate number to affect detection of diet-associated metabolite changes under a high-sugar perturbation. Pool size strongly influenced metabolomic profiles, with samples pooled at five individuals consistently differing from larger pools, while profiles from 50 and 100 individuals were more similar. Larger pools improved reproducibility in a dataset-dependent manner. In the dietary experiment, smaller pool sizes substantially reduced sensitivity, leading to loss of true diet-associated metabolites without increasing false discoveries. Replicate downsampling further revealed that both pool size and biological replication jointly determine signal retention, with smaller pools accelerating the loss of detectable metabolites under reduced replication. Across all analyses, the ability to detect metabolite signals was strongly dependent on effect size and variability. Metabolites with larger and more stable effect estimates were consistently retained, whereas those with smaller or more variable effects were rapidly lost under reduced sampling. Linear mixed-effects modeling confirmed that detection probability is governed by a balance between biological signal strength and measurement variability, with pool size and replication jointly modulating this relationship. More broadly, our results demonstrate that metabolomic inference is governed by the interplay of signal, noise, and sampling design, with pool size and replication jointly shaping the detectability, stability, and interpretation of biological signals.

molecular biology↗

bioRxiv2025

Trade-offs between surviving and thriving: A careful balance of physiological limitations and reproductive effort under thermal stress

Balancing survival and reproduction presents a fundamental evolutionary challenge, especially in extreme and unpredictable environments. Thermoregulatory behavior in particular imposes a costly trade-off, as time spent maintaining optimal body temperature precludes other essential activities and forces individuals to balance competing selective pressures. By combining field observations, behavioral assays, and gene expression profiling of red-sided garter snakes (Thamnophis sirtalis parietalis), we describe how an ectothermic vertebrate navigates this trade-off in an extreme thermal environment through a combination of finely tuned mechanisms of behavioral and physiological plasticity. Snakes demonstrated remarkably consistent critical thermal limits, with minimal individual variation, suggesting a hard physiological constraint. Importantly, behavioral temperature thresholds for voluntary withdrawal and courtship cessation occurred within 5 {degrees}C of lethal, demonstrating that males routinely operate perilously close to lethal body temperatures in order to maximize reproductive opportunities. Context-dependent plasticity allows males to prioritize mating opportunities over thermoregulation until survival becomes an immediate threat. Molecular analyses of multiple tissues revealed rapid, tissue-specific transcriptomic responses activated within one hour of thermal challenge, well before critical limits were reached. Heat shock proteins were universally upregulated across all tissues under both heat and cold stress, suggesting anticipatory protective mechanisms that support risky behavioral decisions rather than simply responding to thermal damage. Tissue-specific patterns of gene expression reflected functional priorities with liver showing extensive metabolic flexibility, heart maintaining conservative stability, while brain and testis appeared to balance critical functions with stress responses. These findings demonstrate precise thermal reaction norms that integrate behavioral and molecular plasticity to maximize reproductive effort without compromising survival. The narrow margin between behavioral thresholds and lethal limits suggests this system may already be approaching evolutionary constraints. While phenotypic plasticity currently buffers populations against extreme thermal variability, it may paradoxically limit long-term adaptive potential as increasing environmental temperatures further narrow safety margins. Precise thermal decision-making, rapid physiological responses, and context-dependent behavioral switching suggest an evolved solution to the problem of balancing competing selective pressures.

zoology↗

bioRxiv2024

Selection for early reproduction leads to accelerated aging and extensive metabolic remodeling in Drosophila melanogaster populations

Experimental evolution studies that feature selection on life-history characters are a proven approach for studying the evolution of aging and variation in rates of senescence. Recently, the incorporation of genomic and transcriptomic approaches into this framework has led to the identification of hundreds of genes associated with different aging patterns. However, our understanding of the specific molecular mechanisms underlying these aging patterns remains limited. Here, we incorporated extensive metabolomic profiling into this framework to generate mechanistic insights into aging patterns in Drosophila melanogaster. Specifically, we characterized metabolomic change over adult lifespan in populations of D. melanogaster where selection for early reproduction has led to an accelerated aging phenotype relative to their controls. Using these data we: i) evaluated evolutionary repeatability across the metabolome; ii) assessed the value of the metabolome as a predictor of "biological age" in this system; and iii) identified specific metabolites associated with accelerated aging. Generally, our findings suggest that selection for early reproduction resulted in highly repeatable alterations to the metabolome and the metabolome itself is a reliable predictor of "biological age". Specifically, we find clusters of metabolites that are associated with the different rates of senescence observed between our accelerated aging population and their controls, adding new insights into the metabolites that may be driving the accelerated aging phenotype. SignificanceWhile experimental evolution studies featuring Drosophila melanogaster have generated significant insights into the forces that shape aging and life history patterns, more recent efforts incorporating genomic and transcriptomic data have had comparatively little success identifying the molecular mechanisms underlying these patterns. Here we work to incorporate molecular phenotyping into this general framework as a way forward. Specifically, we characterize how the metabolome changes with age in populations of D. melanogaster where hundreds of generations of selection for early reproduction have led to enrichment for an accelerated aging phenotype. By comparing to control populations, we show that the metabolome does appear to capture true signals of "biological age" and provides a new avenue for understanding the factors that underlie complex trait variation in real populations.

evolutionary biology↗

bioRxiv2023

Human Heart Failure Alters Mitochondria and Fiber 3D Structure Triggering Metabolic Shifts

This study, utilizing SBF-SEM, reveals structural alterations in mitochondria and myofibrils in human heart failure (HF). Mitochondria in HF show changes in structure, while myofibrils exhibit increased cross-sectional area and branching. Metabolomic and lipidomic analyses indicate concomitant dysregulation in key pathways. The findings underscore the need for personalized treatments considering individualized structural changes in HF.

physiology↗

bioRxiv2023

Enhanced reproductive outcomes under worst-case climate change scenarios in Red-sided Garter snakes (Thamnophis sirtalis parietalis)

As global temperatures continue to increase, understanding the impacts of warming environments is increasingly relevant. Temperature is especially relevant for ectothermic organisms which depend upon consistent and predictable annual temperature cycles for reproduction and development. However, additional research is required in this area to elucidate the potential impacts of climate change on future generations. To understand how projected increases in environmental temperatures may impact reproductive outcomes within natural populations of ectothermic vertebrates, we manipulated minimum ambient temperatures during gestation in Red-sided garter snakes (Thamnophis sirtalis parietalis). Wild snakes were collected in the Interlake region of Manitoba, Canada during their spring mating season and allowed to mate in controlled conditions. For the duration of gestation, mated females were placed into one of two ambient thermal conditions: temperatures emulating those found in the species natural habitat or temperatures with a consistent 5 {degrees}C increase to match end-of-century climate change projections. We recorded observations for each litter and all neonates resulting from controlled mating trials. We observed no difference in litter sizes or birth rates between thermal conditions. However, we observed a significant reduction in gestation length and significant increase to neonate body mass and body condition associated with increased ambient temperatures. These results suggest that increased minimum temperatures during gestation may confer reproductive benefits for the northern populations of this species even under the most extreme current modeled warming predictions. We discuss the broader implications of this effect, including possible negative ecological outcomes.

ecology↗