Cell lineage-specific mitochondrial resilience during mammalian organogenesis.

Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments.

Niche-Selective Inhibition of Pathogenic Th17 Cells by Targeting Metabolic Redundancy.

Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

Atlas of Subcellular RNA Localization Revealed by APEX-Seq.

We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

Mitochondrial Protein Synthesis Adapts to Influx of Nuclear-Encoded Protein.

Mitochondrial ribosomes translate membrane integral core subunits of the oxidative phosphorylation system encoded by mtDNA. These translation products associate with nuclear-encoded, imported proteins to form enzyme complexes that produce ATP. Here, we show that human mitochondrial ribosomes display translational plasticity to cope with the supply of imported nuclear-encoded subunits. Ribosomes expressing mitochondrial-encoded COX1 mRNA selectively engage with cytochrome c oxidase assembly factors in the inner membrane. Assembly defects of the cytochrome c oxidase arrest mitochondrial translation in a ribosome nascent chain complex with a partially membrane-inserted COX1 translation product. This complex represents a primed state of the translation product that can be retrieved for assembly. These findings establish a mammalian translational plasticity pathway in mitochondria that enables adaptation of mitochondrial protein synthesis to the influx of nuclear-encoded subunits.

MOF Acetyl Transferase Regulates Transcription and Respiration in Mitochondria.

A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism.

Transforming growth factor-ß-regulated mTOR activity preserves cellular metabolism to maintain long-term T cell responses in chronic infection.

Antigen-specific CD8+ T cells in chronic viral infections and tumors functionally deteriorate, a process known as exhaustion. Exhausted T cells are sustained by precursors of exhausted (Tpex) cells that self-renew while continuously generating exhausted effector (Tex) cells. However, it remains unknown how Tpex cells maintain their functionality. Here, we demonstrate that Tpex cells sustained mitochondrial fitness, including high spare respiratory capacity, while Tex cells deteriorated metabolically over time. Tpex cells showed early suppression of mTOR kinase signaling but retained the ability to activate this pathway in response to antigen receptor signals. Early transient mTOR inhibition improved long-term T cell responses and checkpoint inhibition. Transforming growth factor-ß repressed mTOR signaling in exhausted T cells and was a critical determinant of Tpex cell metabolism and function. Overall, we demonstrate that the preservation of cellular metabolism allows Tpex cells to retain long-term functionality to sustain T cell responses during chronic infection.

A Metabolism-Based Quorum Sensing Mechanism Contributes to Termination of Inflammatory Responses.

Recruitment of immune cells with antimicrobial activities is essential to fight local infections but has the potential to trigger immunopathology. Whether the immune system has the ability to sense inflammation intensity and self-adjust accordingly to limit tissue damage remains to be fully established. During local infection with an intracellular pathogen, we have shown that nitric oxide (NO) produced by recruited monocyte-derived cells was essential to limit inflammation and cell recruitment. Mechanistically, we have provided evidence that NO dampened monocyte-derived cell cytokine and chemokine production by inhibiting cellular respiration and reducing cellular ATP:ADP ratio. Such metabolic control operated at the tissue level but only when a sufficient number of NO-producing cells reached the site of infection. Thus, NO production and activity act as a quorum sensing mechanism to help terminate the inflammatory response.

NRF2 is a spatiotemporal metabolic hub essential for the polyfunctionality of Th2 cells.

Upon encountering allergens, CD4+ T cells differentiate into IL-4-producing Th2 cells in lymph nodes, which later transform into polyfunctional Th2 cells producing IL-5 and IL-13 in inflamed tissues. However, the precise mechanism underlying their polyfunctionality remains elusive. In this study, we elucidate the pivotal role of NRF2 in polyfunctional Th2 cells in murine models of allergic asthma and in human Th2 cells. We found that an increase in reactive oxygen species (ROS) in immune cells infiltrating the lungs is necessary for the development of eosinophilic asthma and polyfunctional Th2 cells in vivo. Deletion of the ROS sensor NRF2 specifically in T cells, but not in dendritic cells, significantly abolished eosinophilia and polyfunctional Th2 cells in the airway. Mechanistically, NRF2 intrinsic to T cells is essential for inducing optimal oxidative phosphorylation and glycolysis capacity, thereby driving Th2 cell polyfunctionality independently of IL-33, partially by inducing PPARγ. Treatment with an NRF2 inhibitor leads to a substantial decrease in polyfunctional Th2 cells and subsequent eosinophilia in mice and a reduction in the production of Th2 cytokines from peripheral blood mononuclear cells in asthmatic patients. These findings highlight the critical role of Nrf2 as a spatial and temporal metabolic hub that is essential for polyfunctional Th2 cells, suggesting potential therapeutic implications for allergic diseases.

Coordinating mitochondrial translation with assembly of the OXPHOS complexes.

The mitochondrial oxidative phosphorylation (OXPHOS) system produces the majority of energy required by cells. Given the mitochondrion's endosymbiotic origin, the OXPHOS machinery is still under dual genetic control where most OXPHOS subunits are encoded by the nuclear DNA and imported into mitochondria, while a small subset is encoded on the mitochondrion's own genome, the mitochondrial DNA (mtDNA). The nuclear and mtDNA encoded subunits must be expressed and assembled in a highly orchestrated fashion to form a functional OXPHOS system and meanwhile prevent the generation of any harmful assembly intermediates. While several mechanisms have evolved in eukaryotes to achieve such a coordinated expression, this review will focus on how the translation of mtDNA encoded OXPHOS subunits is tailored to OXPHOS assembly.

SPICE-Met: profiling and imaging energy metabolism at the single-cell level using a fluorescent reporter mouse.

The regulation of cellular energy metabolism is central to most physiological and pathophysiological processes. However, most current methods have limited ability to functionally probe metabolic pathways in individual cells. Here, we describe SPICE-Met (Single-cell Profiling and Imaging of Cell Energy Metabolism), a method for profiling energy metabolism in single cells using flow cytometry or imaging. We generated a transgenic mouse expressing PercevalHR, a fluorescent reporter for cellular ATP:ADP ratio. Modulation of PercevalHR fluorescence with metabolic inhibitors was used to infer the dependence of energy metabolism on oxidative phosphorylation and glycolysis in defined cell populations identified by flow cytometry. We applied SPICE-Met to analyze T-cell memory development during vaccination. Finally, we used SPICE-Met in combination with real-time imaging to dissect the heterogeneity and plasticity of energy metabolism in single macrophages ex vivo and identify three distinct metabolic patterns. Functional probing of energy metabolism with single-cell resolution should greatly facilitate the study of immunometabolism at a steady state, during disease pathogenesis or in response to therapy.

BCL7A-containing SWI/SNF/BAF complexes modulate mitochondrial bioenergetics during neural progenitor differentiation.

Mammalian SWI/SNF/BAF chromatin remodeling complexes influence cell lineage determination. While the contribution of these complexes to neural progenitor cell (NPC) proliferation and differentiation has been reported, little is known about the transcriptional profiles that determine neurogenesis or gliogenesis. Here, we report that BCL7A is a modulator of the SWI/SNF/BAF complex that stimulates the genome-wide occupancy of the ATPase subunit BRG1. We demonstrate that BCL7A is dispensable for SWI/SNF/BAF complex integrity, whereas it is essential to regulate Notch/Wnt pathway signaling and mitochondrial bioenergetics in differentiating NPCs. Pharmacological stimulation of Wnt signaling restores mitochondrial respiration and attenuates the defective neurogenic patterns observed in NPCs lacking BCL7A. Consistently, treatment with an enhancer of mitochondrial biogenesis, pioglitazone, partially restores mitochondrial respiration and stimulates neuronal differentiation of BCL7A-deficient NPCs. Using conditional BCL7A knockout mice, we reveal that BCL7A expression in NPCs and postmitotic neurons is required for neuronal plasticity and supports behavioral and cognitive performance. Together, our findings define the specific contribution of BCL7A-containing SWI/SNF/BAF complexes to mitochondria-driven NPC commitment, thereby providing a better understanding of the cell-intrinsic transcriptional processes that connect metabolism, neuronal morphogenesis, and cognitive flexibility.

E3 ubiquitin ligase Hul6 modulates iron-dependent metabolism by regulating Php4 stability.

Schizosaccharomyces pombe Php4 is the regulatory subunit of the CCAAT-binding complexes and plays an important role in the regulation of iron homeostasis and iron-dependent metabolism. Here, we show that Php4 undergoes ubiquitin-dependent degradation in the late logarithmic and stationary phases. The degradation and ubiquitination of Php4 could be attenuated by deletion of hul6, a gene encoding a putative HECT-type E3 ubiquitin ligase. The expression levels of Hul6 and Php4 are oppositely regulated during cell growth. Hul6 interacts with the C-terminal region of Php4. Two lysine residues (K217 and K274) located in the C-terminal region of Php4 are required for its polyubiquitination. Increasing the levels of Php4 by deletion of hul6 or overexpression of php4 decreased expression of Php4 target proteins involved in iron-dependent metabolic pathways such as the tricarboxylic cycle and mitochondrial oxidative phosphorylation, thus causing increased sensitivity to high-iron and reductions in succinate dehydrogenase and mitochondrial complex II activities. Hul6 is located primarily in the mitochondrial outer membrane and most likely targets cytosolic Php4 for ubiquitination and degradation. Taken together, our data suggest that Hul6 regulates iron-dependent metabolism through degradation of Php4 under normal growth conditions. Our results also suggest that Hul6 promotes iron-dependent metabolism to help the cell to adapt to a nutrient-starved growth phase.

Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome.

So-called ρ0 cells lack mitochondrial DNA and are therefore incapable of aerobic ATP synthesis. How cells adapt to survive ablation of oxidative phosphorylation remains poorly understood. Complexome profiling analysis of ρ0 cells covered 1,002 mitochondrial proteins and revealed changes in abundance and organization of numerous multiprotein complexes including previously not described assemblies. Beyond multiple subassemblies of complexes that would normally contain components encoded by mitochondrial DNA, we observed widespread reorganization of the complexome. This included distinct changes in the expression pattern of adenine nucleotide carrier isoforms, other mitochondrial transporters, and components of the protein import machinery. Remarkably, ablation of mitochondrial DNA hardly affected the complexes organizing cristae junctions indicating that the altered cristae morphology in ρ0 mitochondria predominantly resulted from the loss of complex V dimers required to impose narrow curvatures to the inner membrane. Our data provide a comprehensive resource for in-depth analysis of remodeling of the mitochondrial complexome in response to respiratory deficiency.

Blood progenitor redox homeostasis through olfaction-derived systemic GABA in hematopoietic growth control in Drosophila.

The role of reactive oxygen species (ROS) in myeloid development is well established. However, its aberrant generation alters hematopoiesis. Thus, a comprehensive understanding of events controlling ROS homeostasis forms the central focus of this study. We show that, in homeostasis, myeloid-like blood progenitor cells of the Drosophila larvae, which reside in a specialized hematopoietic organ termed the lymph gland, use TCA to generate ROS. However, excessive ROS production leads to lymph gland growth retardation. Therefore, to moderate blood progenitor ROS, Drosophila larvae rely on olfaction and its downstream systemic GABA. GABA internalization and its breakdown into succinate by progenitor cells activates pyruvate dehydrogenase kinase (PDK), which controls inhibitory phosphorylation of pyruvate dehydrogenase (PDH). PDH is the rate-limiting enzyme that connects pyruvate to the TCA cycle and to oxidative phosphorylation. Thus, GABA metabolism via PDK activation maintains TCA activity and blood progenitor ROS homeostasis, and supports normal lymph gland growth. Consequently, animals that fail to smell also fail to sustain TCA activity and ROS homeostasis, which leads to lymph gland growth retardation. Overall, this study describes the requirement of animal odor-sensing and GABA in myeloid ROS regulation and hematopoietic growth control.

Sesaminol alters phospholipid metabolism and alleviates obesity-induced NAFLD.

The prevalence of obesity-induced non-alcoholic fatty liver disease (NAFLD) and insulin resistance is increasing worldwide. We previously demonstrated that sesaminol increases thermogenesis in adipocytes, improves insulin sensitivity, and mitigates obesity in mice. In this study, we demonstrated that sesaminol increased mitochondrial activity and reduced ROS production in hepatocytes. Therefore, we delve into the metabolic action of sesaminol in obesity-induced NAFLD or metabolic dysfunction-associated liver disease (MAFLD). Here, we report that sesaminol induces OXPHOS proteins and mitochondrial function in vivo. Further, our data suggest that sesaminol administration reduces hepatic triacylglycerol accumulation and LDL-C levels. Prominently, the lipidomics analyses revealed that sesaminol administration decreased the major phospholipids such as PC, PE, PI, CL, and PS to maintain membrane lipid homeostasis in the liver upon HFD challenge. Besides, SML reduced ePC and SM molecular species and increased PA levels in the HFD-fed mice. Also, sesaminol renders anti-inflammatory properties and dampens fibrosis markers in the liver. Remarkably, SML lowers the hepatic levels of ALT and AST enzymes and alleviates NAFLD in diet-induced obese mice. The molecular docking analysis identifies peroxisome proliferator-activated receptors as potential endogenous receptors for sesaminol. Together, our study demonstrates plant lignan sesaminol as a potential small molecule that alters the molecular species of major phospholipids, including sphingomyelin and ether-linked PCs in the liver tissue, improves metabolic parameters, and alleviates obesity-induced fatty liver disease in mice.

Repeated sprint training in hypoxia induces specific skeletal muscle adaptations through S100A protein signaling.

Athletes increasingly engage in repeated sprint training consisting in repeated short all-out efforts interspersed by short recoveries. When performed in hypoxia (RSH), it may lead to greater training effects than in normoxia (RSN); however, the underlying molecular mechanisms remain unclear. This study aimed at elucidating the effects of RSH on skeletal muscle metabolic adaptations as compared to RSN. Sixteen healthy young men performed nine repeated sprint training sessions in either normoxia (FIO2 = 0.209, RSN, n = 7) or normobaric hypoxia (FIO2 = 0.136, RSH, n = 9). Before and after the training period, exercise performance was assessed by using repeated sprint ability (RSA) and Wingate tests. Vastus lateralis muscle biopsies were performed to investigate muscle metabolic adaptations using proteomics combined with western blot analysis. Similar improvements were observed in RSA and Wingate tests in both RSN and RSH groups. At the muscle level, RSN and RSH reduced oxidative phosphorylation protein content but triggered an increase in mitochondrial biogenesis proteins. Proteomics showed an increase in several S100A family proteins in the RSH group, among which S100A13 most strongly. We confirmed a significant increase in S100A13 protein by western blot in RSH, which was associated with increased Akt phosphorylation and its downstream targets regulating protein synthesis. Altogether our data indicate that RSH may activate an S100A/Akt pathway to trigger specific adaptations as compared to RSN.

The activator protein-1 complex governs a vascular degenerative transcriptional programme in smooth muscle cells to trigger aortic dissection and rupture.

BACKGROUND AND AIMS: Stanford type A aortic dissection (AD) is a degenerative aortic remodelling disease marked by an exceedingly high mortality without effective pharmacologic therapies. Smooth muscle cells (SMCs) lining tunica media adopt a range of states, and their transformation from contractile to synthetic phenotypes fundamentally triggers AD. However, the underlying pathomechanisms governing this population shift and subsequent AD, particularly at distinct disease temporal stages, remain elusive. METHODS: Ascending aortas from nine patients undergoing ascending aorta replacement and five individuals undergoing heart transplantation were subjected to single-cell RNA sequencing. The pathogenic targets governing the phenotypic switch of SMCs were identified by trajectory inference, functional scoring, single-cell regulatory network inference and clustering, regulon, and interactome analyses and confirmed using human ascending aortas, primary SMCs, and a ß-aminopropionitrile monofumarate-induced AD model. RESULTS: The transcriptional profiles of 93 397 cells revealed a dynamic temporal-specific phenotypic transition and marked elevation of the activator protein-1 (AP-1) complex, actively enabling synthetic SMC expansion. Mechanistically, tumour necrosis factor signalling enhanced AP-1 transcriptional activity by dampening mitochondrial oxidative phosphorylation (OXPHOS). Targeting this axis with the OXPHOS enhancer coenzyme Q10 or AP-1-specific inhibitor T-5224 impedes phenotypic transition and aortic degeneration while improving survival by 42.88% (58.3%-83.3% for coenzyme Q10 treatment), 150.15% (33.3%-83.3% for 2-week T-5224), and 175.38% (33.3%-91.7% for 3-week T-5224) in the ß-aminopropionitrile monofumarate-induced AD model. CONCLUSIONS: This cross-sectional compendium of cellular atlas of human ascending aortas during AD progression provides previously unappreciated insights into a transcriptional programme permitting aortic degeneration, highlighting a translational proof of concept for an anti-remodelling intervention as an attractive strategy to manage temporal-specific AD by modulating the tumour necrosis factor-OXPHOS-AP-1 axis.

Monitoring mitochondrial translation by pulse SILAC.

Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation (MT) control, it is challenging to identify and quantify the mitochondrial-encoded proteins because of their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry-based proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture. Our method provides the highest protein identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 of the 13 mitochondrial-encoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of oxidative phosphorylation complexes. We found that inhibition of MT led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrial-encoded subunits. This method should be readily applicable to study MT programs in many contexts, including oxidative stress and mitochondrial disease.

IRX5 suppresses osteogenic differentiation of hBMSCs by inhibiting protein synthesis.

In our previous study, IRX5 has been revealed a significant role in adipogenesis of hBMSCs. Considering the expansion of adipose tissue in bone marrow in aged and ovariectomy-related osteoporosis, the effect of IRX5 on the osteogenesis of BMSCs still needs to be elucidated. In vivo, models of aging-induced and ovariectomy-induced osteoporotic mice, and in vitro studies of IRX5 gene gain- and loss-of-function in hBMSCs were employed. Histology, immunofluorescence, qRT-PCR, and Western blot analysis were performed to detect the functions of IRX5 in hBMSCs osteogenic differentiation. RNA-seq, transmission electron microscopy, Seahorse mito-stress assay, and Surface Sensing of Translation assay were conducted to explore the effect of mammalian/mechanistic target of rapamycin (mTOR)-mediated ribosomal translation and mitochondrial functions in the regulation of hBMSCs differentiation by IRX5. As a result, elevated IRX5 protein expression levels were observed in the bone marrow of osteoporotic mice compared to normal mice. IRX5 overexpression attenuated osteogenic processes, whereas IRX5 knockdown resulted in enhanced osteogenesis in hBMSCs. RNA-seq and enrichment analysis unveiled that IRX5 overexpression exerted inhibitory effects on ribosomal translation and mitochondrial functions. Furthermore, the application of the mTOR activator, MHY1485, effectively reversed the inhibitory impact of IRX5 on osteogenesis and mitochondrial functions in hBMSCs. In summary, our findings suggest that IRX5 restricts mTOR-mediated ribosomal translation, consequently impairing mitochondrial OxPhos, which in turn results in osteogenic dysfunction of hBMSCs.

Comparative mitogenomic analysis of subterranean and surface amphipods (Crustacea, Amphipoda) with special reference to the family Crangonyctidae.

Mitochondrial genomes play important roles in studying genome evolution, phylogenetic analyses, and species identification. Amphipods (Class Malacostraca, Order Amphipoda) are one of the most ecologically diverse crustacean groups occurring in a diverse array of aquatic and terrestrial environments globally, from freshwater streams and lakes to groundwater aquifers and the deep sea, but we have a limited understanding of how habitat influences the molecular evolution of mitochondrial energy metabolism. Subterranean amphipods likely experience different evolutionary pressures on energy management compared to surface-dwelling taxa that generally encounter higher levels of predation and energy resources and live in more variable environments. In this study, we compared the mitogenomes, including the 13 protein-coding genes involved in the oxidative phosphorylation (OXPHOS) pathway, of surface and subterranean amphipods to uncover potentially different molecular signals of energy metabolism between surface and subterranean environments in this diverse crustacean group. We compared base composition, codon usage, gene order rearrangement, conducted comparative mitogenomic and phylogenomic analyses, and examined evolutionary signals of 35 amphipod mitogenomes representing 13 families, with an emphasis on Crangonyctidae. Mitogenome size, AT content, GC-skew, gene order, uncommon start codons, location of putative control region (CR), length of rrnL and intergenic spacers differed between surface and subterranean amphipods. Among crangonyctid amphipods, the spring-dwelling Crangonyx forbesi exhibited a unique gene order, a long nad5 locus, longer rrnL and rrnS loci, and unconventional start codons. Evidence of directional selection was detected in several protein-encoding genes of the OXPHOS pathway in the mitogenomes of surface amphipods, while a signal of purifying selection was more prominent in subterranean species, which is consistent with the hypothesis that the mitogenome of surface-adapted species has evolved in response to a more energy demanding environment compared to subterranean amphipods. Overall, gene order, locations of non-coding regions, and base-substitution rates points to habitat as an important factor influencing the evolution of amphipod mitogenomes.