The solute carrier SLC25A17 sustains peroxisomal redox homeostasis in diverse mammalian cell lines.

Despite the crucial role of peroxisomes in cellular redox maintenance, little is known about how these organelles transport redox metabolites across their membrane. In this study, we sought to assess potential associations between the cellular redox landscape and the human peroxisomal solute carrier SLC25A17, also known as PMP34. This carrier has been reported to function as a counter-exchanger of adenine-containing cofactors such as coenzyme A (CoA), dephospho-CoA, flavin adenine dinucleotide, nicotinamide adenine dinucleotide (NAD+), adenosine 3',5'-diphosphate, flavin mononucleotide, and adenosine monophosphate. We found that inactivation of SLC25A17 resulted in a shift toward a more reductive state in the glutathione redox couple (GSSG/GSH) across HEK-293 cells, HeLa cells, and SV40-transformed mouse embryonic fibroblasts, with variable impact on the NADPH levels and the NAD+/NADH redox couple. This phenotype could be rescued by the expression of Candida boidinii Pmp47, a putative SLC25A17 orthologue reported to be essential for the metabolism of medium-chain fatty acids in yeast peroxisomes. In addition, we provide evidence that the alterations in the redox state are not caused by changes in peroxisomal antioxidant enzyme expression, catalase activity, H2O2 membrane permeability, or mitochondrial fitness. Furthermore, treating control and ΔSLC25A17 cells with dehydroepiandrosterone, a commonly used glucose-6-phosphate dehydrogenase inhibitor affecting NADPH regeneration, revealed a kinetic disconnection between the peroxisomal and cytosolic glutathione pools. Additionally, these experiments underscored the impact of SLC25A17 loss on peroxisomal NADPH metabolism. The relevance of these findings is discussed in the context of the still ambiguous substrate specificity of SLC25A17 and the recent observation that the mammalian peroxisomal membrane is readily permeable to both GSH and GSSG.

Functional Analysis of GSTK1 in Peroxisomal Redox Homeostasis in HEK-293 Cells.

Peroxisomes serve as important centers for cellular redox metabolism and communication. However, fundamental gaps remain in our understanding of how the peroxisomal redox equilibrium is maintained. In particular, very little is known about the function of the nonenzymatic antioxidant glutathione in the peroxisome interior and how the glutathione antioxidant system balances with peroxisomal protein thiols. So far, only one human peroxisomal glutathione-consuming enzyme has been identified: glutathione S-transferase 1 kappa (GSTK1). To study the role of this enzyme in peroxisomal glutathione regulation and function, a GSTK1-deficient HEK-293 cell line was generated and fluorescent redox sensors were used to monitor the intraperoxisomal GSSG/GSH and NAD+/NADH redox couples and NADPH levels. We provide evidence that ablation of GSTK1 does not change the basal intraperoxisomal redox state but significantly extends the recovery period of the peroxisomal glutathione redox sensor po-roGFP2 upon treatment of the cells with thiol-specific oxidants. Given that this delay (i) can be rescued by reintroduction of GSTK1, but not its S16A active site mutant, and (ii) is not observed with a glutaredoxin-tagged version of po-roGFP2, our findings demonstrate that GSTK1 contains GSH-dependent disulfide bond oxidoreductase activity.

Enhanced Levels of Peroxisome-Derived H2O2 Do Not Induce Pexophagy but Impair Autophagic Flux in HEK-293 and HeLa Cells.

Peroxisomes are functionally specialized organelles that harbor multiple hydrogen peroxide (H2O2)-producing and -degrading enzymes. Given that this oxidant functions as a major redox signaling agent, peroxisomes have the intrinsic ability to mediate and modulate H2O2-driven processes, including autophagy. However, it remains unclear whether changes in peroxisomal H2O2 (po-H2O2) emission impact the autophagic process and to which extent peroxisomes with a disturbed H2O2 metabolism are selectively eliminated through a process called "pexophagy". To address these issues, we generated and validated HEK-293 and HeLa pexophagy reporter cell lines in which the production of po-H2O2 can be modulated. We demonstrate that (i) po-H2O2 can oxidatively modify multiple selective autophagy receptors and core autophagy proteins, (ii) neither modest nor robust levels of po-H2O2 emission act as a prime determinant of pexophagy, and (iii) high levels of po-H2O2 impair autophagic flux by oxidative inhibition of enzymes involved in LC3II formation. Unexpectedly, our analyses also revealed that the autophagy receptor optineurin can be recruited to peroxisomes, thereby triggering pexophagy. In summary, these findings lend support to the idea that, during cellular and organismal aging, peroxisomes with enhanced H2O2 release can escape pexophagy and downregulate autophagic activity, thereby perpetuating the accumulation of damaged and toxic cellular debris.

Identification of Peroxisome-Derived Hydrogen Peroxide-Sensitive Target Proteins Using a YAP1C-Based Genetic Probe.

As the reversible oxidation of protein cysteine thiols is an important mechanism in signal transduction, it is essential to have access to experimental approaches that allow for spatiotemporal indexing of the cellular sulfenome in response to local changes in H2O2 levels. Here, we provide a step-by-step guide for enriching and identifying the sulfenome of mammalian cells at the subcellular level in response to peroxisome-derived H2O2 by the combined use of (i) a previously developed cell line in which peroxisomal H2O2 production can be induced in a time- and dose-dependent manner; (ii) YAP1C, a genetically encoded yeast AP-1-like transcription factor-based probe that specifically reacts with S-sulfenylated cysteines and traps them through mixed disulfide bonds; and (iii) mass spectrometry. Given that this approach includes differential labeling of reduced and reversibly oxidized cysteine residues, it can also provide additional information on the positions of the modified cysteines. Gaining more in-depth insight into the complex nature of how alterations in peroxisomal H2O2 metabolism modulate the cellular sulfenome is key to our understanding of how these organelles act as redox signaling hubs in health and disease.

Assessment of the Peroxisomal Redox State in Living Cells Using NADPH- and NAD+/NADH-Specific Fluorescent Protein Sensors.

The pyridine nucleotides NAD(H) and NADP(H) are key molecules in cellular metabolism, and measuring their levels and oxidation states with spatiotemporal precision is of great value in biomedical research. Traditional methods to assess the redox state of these metabolites are intrusive and prohibit live-cell quantifications. This obstacle was surpassed by the development of genetically encoded fluorescent biosensors enabling dynamic measurements with subcellular resolution in living cells. Here, we provide step-by-step protocols to monitor the intraperoxisomal NADPH levels and NAD+/NADH redox state in cellulo by using targeted variants of iNAP1 and SoNar, respectively.

Interrogating the Roles of Mutation-Selection Balance, Heterozygote Advantage, and Linked Selection in Maintaining Recessive Lethal Variation in Natural Populations.

For nearly a century, evolutionary biologists have observed chromosomes that cause lethality when made homozygous persisting at surprisingly high frequencies (>25%) in natural populations of many species. The evolutionary forces responsible for the maintenance of such detrimental mutations have been heavily debated-are some lethal mutations under balancing selection? We suggest that mutation-selection balance alone cannot explain lethal variation in nature and the possibility that other forces play a role. We review the potential that linked selection in particular may drive maintenance of lethal alleles through associative overdominance or linkage to beneficial mutations or by reducing effective population size. Over the past five decades, investigation into this mystery has tapered. During this time, key scientific advances have provided the ability to collect more accurate data and analyze them in new ways, making the underlying genetic bases and evolutionary forces of lethal alleles timely for study once more.

Apoptotic and histopathological defects enhanced by titanium dioxide nanoparticles in male mice after short-term exposure.

Titanium dioxide nanoparticles (TiO2NPs) are commercially utilized in diverse fields. Therefore, the current study investigated the apoptotic and histopathological defects that were caused in male mice following intraperitoneal (i.p) injection of TiO2NPs for 28 days. Doses: 2.5, 5.0, 10.0 and 20.0 mg/kg body weight were applied (10 mice for each group). Results revealed that, lactate dehydrogenase (LDH) activity was significantly increased in homogenates of liver, spleen, kidney, lung, heart, and muscles of treated animals, respect to their controls. Also, significant alterations in acid and alkaline phosphatase (ACP and ALP) activities were reported. The dose 5.0 mg/kg exhibited a significant decline in cell viability of blood samples (74.9 %) (P 0.05 = 0.0177), followed by 2.5 mg/kg (80.8 %), and finally the 10.0 mg/kg (81.8 %) with respect to control (96.3 %). Additionally, significant increases of expressed proteins of caspases-3 and-7 were noticed in cells of the treated animals. Ultrastructural investigations in sections of liver, kidney, lung and spleen of the treated animals showed significant defects, especially in the nucleus, mitochondria and rough endoplasmic reticulum (RER), compared to normal patterns of the control. Also, significant induction of nanoparticle (NPs)-phagolysosomes was visualized in sections of the treated animals. The present findings might provide evidence for the risk pattern of TiO2NPs in mammals after short-term exposure. So, TiO2NPs-based commercial products have now increased in the markets, and it is prudent to investigate their mammalian toxicology.

New solid-state membrane and coated wire potentiometric sensors for the determination of Zn(II) ions based on nanoparticles.

The first, novel solid-state membrane sensor for Zn(II) determination is developed based on ZnS nanoparticles. ZnS nanoparticles are synthesized by chemical co-precipitation and investigated via X-ray diffraction, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and impedance study. X-ray diffraction shows that the prepared ZnS nanoparticles have an average domain size of 5.72 nm, which is very close to the particle size obtained from TEM observations (6.30 nm). The ZnS nanoparticles are pressed into disks and examined as electroactive solid-state membrane. Solid-state membrane and coated wire sensors are fabricated. They display linear responses over concentration ranges of 1.0 × 10-5 to 1.0 × 10-1 mol L-1 Zn2+ ions with cationic slopes of 28.9±0.2 and 25.9±0.2 mV decade-1 for the solid-state membrane and coated wire sensors, respectively. The lower limits of detection are 2.86 × 10-6 and 4.60 × 10-6 mol L-1 Zn2+ ions for the solid-state membrane and coated wire sensors, respectively. The response time for the two sensors is instantaneous (1 s), and the useful lifetimes for the solid-state membrane and coated wire sensors are long (10 and 6 months, respectively). The solid-state membrane sensor is utilized for the quantification of Zn(II) ions in brass alloys and pharmaceutical preparations.

Gene flow biases population genetic inference of recombination rate.

Accurate estimates of the rate of recombination are key to understanding a host of evolutionary processes as well as the evolution of the recombination rate itself. Model-based population genetic methods that infer recombination rates from patterns of linkage disequilibrium in the genome have become a popular method to estimate rates of recombination. However, these linkage disequilibrium-based methods make a variety of simplifying assumptions about the populations of interest that are often not met in natural populations. One such assumption is the absence of gene flow from other populations. Here, we use forward-time population genetic simulations of isolation-with-migration scenarios to explore how gene flow affects the accuracy of linkage disequilibrium-based estimators of recombination rate. We find that moderate levels of gene flow can result in either the overestimation or underestimation of recombination rates by up to 20-50% depending on the timing of divergence. We also find that these biases can affect the detection of interpopulation differences in recombination rate, causing both false positives and false negatives depending on the scenario. We discuss future possibilities for mitigating these biases and recommend that investigators exercise caution and confirm that their study populations meet assumptions before deploying these methods.

Peroxisome-Derived Hydrogen Peroxide Modulates the Sulfenylation Profiles of Key Redox Signaling Proteins in Flp-In T-REx 293 Cells.

The involvement of peroxisomes in cellular hydrogen peroxide (H2O2) metabolism has been a central theme since their first biochemical characterization by Christian de Duve in 1965. While the role of H2O2 substantially changed from an exclusively toxic molecule to a signaling messenger, the regulatory role of peroxisomes in these signaling events is still largely underappreciated. This is mainly because the number of known protein targets of peroxisome-derived H2O2 is rather limited and testing of specific targets is predominantly based on knowledge previously gathered in related fields of research. To gain a broader and more systematic insight into the role of peroxisomes in redox signaling, new approaches are urgently needed. In this study, we have combined a previously developed Flp-In T-REx 293 cell system in which peroxisomal H2O2 production can be modulated with a yeast AP-1-like-based sulfenome mining strategy to inventory protein thiol targets of peroxisome-derived H2O2 in different subcellular compartments. By using this approach, we identified more than 400 targets of peroxisome-derived H2O2 in peroxisomes, the cytosol, and mitochondria. We also observed that the sulfenylation kinetics profiles of key targets belonging to different protein families (e.g., peroxiredoxins, annexins, and tubulins) can vary considerably. In addition, we obtained compelling but indirect evidence that peroxisome-derived H2O2 may oxidize at least some of its targets (e.g., transcription factors) through a redox relay mechanism. In conclusion, given that sulfenic acids function as key intermediates in H2O2 signaling, the findings presented in this study provide valuable insight into how peroxisomes may be integrated into the cellular H2O2 signaling network.

Autophagy Blockage Reduces the Incidence of Pancreatic Ductal Adenocarcinoma in the Context of Mutant Trp53.

Macroautophagy (hereafter referred to as autophagy) is a homeostatic process that preserves cellular integrity. In mice, autophagy regulates pancreatic ductal adenocarcinoma (PDAC) development in a manner dependent on the status of the tumor suppressor gene Trp53. Studies published so far have investigated the impact of autophagy blockage in tumors arising from Trp53-hemizygous or -homozygous tissue. In contrast, in human PDACs the tumor suppressor gene TP53 is mutated rather than allelically lost, and TP53 mutants retain pathobiological functions that differ from complete allelic loss. In order to better represent the patient situation, we have investigated PDAC development in a well-characterized genetically engineered mouse model (GEMM) of PDAC with mutant Trp53 (Trp53 R172H ) and deletion of the essential autophagy gene Atg7. Autophagy blockage reduced PDAC incidence but had no impact on survival time in the subset of animals that formed a tumor. In the absence of Atg7, non-tumor-bearing mice reached a similar age as animals with malignant disease. However, the architecture of autophagy-deficient, tumor-free pancreata was effaced, normal acinar tissue was largely replaced with low-grade pancreatic intraepithelial neoplasias (PanINs) and insulin expressing islet ß-cells were reduced. Our data add further complexity to the interplay between Atg7 inhibition and Trp53 status in tumorigenesis.

Acute systemic knockdown of Atg7 is lethal and causes pancreatic destruction in shRNA transgenic mice.

The notion that macroautophagy/autophagy is a potentially attractive therapeutic target for a variety of diseases, including cancer, largely stems from pre-clinical mouse studies. Most of these examine the effects of irreversible and organ confined autophagy deletion using site specific Cre-loxP recombination of the essential autophagy regulating genes Atg7 or Atg5. Model systems with the ability to impair autophagy systemically and reversibly at all disease stages would allow a more realistic approach to evaluate the consequences of authophagy inhibition as a therapeutic concept and its potential side effects. Here, we present shRNA transgenic mice that via doxycycline (DOX) regulable expression of a highly efficient miR30-E-based shRNA enabled knockdown of Atg7 simultaneously in the majority of organs, with the brain and spleen being noteable exceptions. Induced animals deteriorated rapidly and experienced profound destruction of the exocrine pancreas, severe hypoglycemia and depletion of hepatic glycogen storages. Cessation of DOX application restored apparent health, glucose homeostasis and pancreatic integrity. In a similar Atg5 knockdown model we neither observed loss of pancreatic integrity nor diminished survival after DOX treatment, but identified histological changes consistent with steatohepatitis and hepatic fibrosis in the recovery period after termination of DOX. Regulable Atg7-shRNA mice are valuable tools that will enable further studies on the role of autophagy impairment at various disease stages and thereby help to evaluate the consequences of acute autophagy inhibition as a therapeutic concept.Abbreviations: ACTB: actin, beta; AMY: amylase complex; ATG4B: autophagy related 4B, cysteine peptidase; ATG5: autophagy related 5; ATG7: autophagy related 7; Cag: CMV early enhancer/chicken ACTB promoter; Col1a1: collagen, type I, alpha 1; Cre: cre recombinase; DOX: doxycycline; GCG: glucagon; GFP: green fluorescent protein; INS: insulin; LC3: microtubule-associated protein 1 light chain 3; miR30-E: optimized microRNA backbone; NAFLD: non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis; PNLIP: pancreatic lipase; rtTA: reverse tetracycline transactivator protein; SQSTM1/p62: sequestome 1; TRE: tetracycline responsive element.

Intraspecific Genetic Variation for Behavioral Isolation Loci in Drosophila.

Behavioral isolation is considered to be the primary mode of species isolation, and the lack of identification of individual genes for behavioral isolation has hindered our ability to address fundamental questions about the process of speciation. One of the major questions that remains about behavioral isolation is whether the genetic basis of isolation between species also varies within a species. Indeed, the extent to which genes for isolation may vary across a population is rarely explored. Here, we bypass the problem of individual gene identification by addressing this question using a quantitative genetic comparison. Using strains from eight different populations of Drosophila simulans, we genetically mapped the genomic regions contributing to behavioral isolation from their closely related sibling species, Drosophila mauritiana. We found extensive variation in the size of contribution of different genomic regions to behavioral isolation among the different strains, in the location of regions contributing to isolation, and in the ability to redetect loci when retesting the same strain.

COVID-19 Pandemic and Maternal Perspectives.

Aim Coronavirus (COVID-19) pandemic has affected perinatal women worldwide. Our study aimed to describe the opinions of perinatal women about COVID-19 related knowledge, attitude, and practices. Methods Pregnant and Postnatal women (n=223) were included and those who did not consent, and less than 16 weeks' gestation, were excluded. SPSS version 26 was used for descriptive statistics. Results Most of the women had good knowledge about COVID 19 regarding its nature, transmission, & symptoms. Their information sources were news (139/206=67.5%) and the internet (85/206=41%). Women understood the uncertainty around its effect on pregnancy; as it is a novel infection. A substantial number of women were concerned (130/206=63%), upset by social isolation (86/206=42%), negatively impacted by the visitor restrictions in hospital (154/206=75%), and faced COVID-19 related reduced household finances (97/206=47%). Most of them used hand washing (201/206=98%) & social distancing (191/206=93%) as preventive measures. They reported compromised contact with General Physician (GP) service as compared to the hospital service (85/206=41% Vs 31/206=15% respectively) during the pandemic. Conclusions The main challenges of the COVID-19 pandemic for perinatal women are the jeopardized GP & hospital services & psychological distress. It is imperative to incorporate telemedicine & virtual visits to tackle the burden of the COVID-19 pandemic. Perinatal women, are particularly vulnerable to the psychological impacts of the COVID-19 pandemic & societal lockdown, thus necessitating holistic interventions.

Inversions shape the divergence of Drosophila pseudoobscura and Drosophila persimilis on multiple timescales.

By shaping meiotic recombination, chromosomal inversions can influence genetic exchange between hybridizing species. Despite the recognized importance of inversions in evolutionary processes such as divergence and speciation, teasing apart the effects of inversions over time remains challenging. For example, are their effects on sequence divergence primarily generated through creating blocks of linkage disequilibrium prespeciation or through preventing gene flux after speciation? We provide a comprehensive look into the influence of inversions on gene flow throughout the evolutionary history of a classic system: Drosophila pseudoobscura and Drosophila persimilis. We use extensive whole-genome sequence data to report patterns of introgression and divergence with respect to chromosomal arrangements. Overall, we find evidence that inversions have contributed to divergence patterns between D. pseudoobscura and D. persimilis over three distinct timescales: (1) segregation of ancestral polymorphism early in the speciation process, (2) gene flow after the split of D. pseudoobscura and D. persimilis, but prior to the split of D. pseudoobscura subspecies, and (3) recent gene flow between sympatric D. pseudoobscura and D. persimilis, after the split of D. pseudoobscura subspecies. We discuss these results in terms of our understanding of evolution in this classic system and provide cautions for interpreting divergence measures in other systems.

Homage to Felsenstein 1981, or why are there so few/many species?

If there are no constraints on the process of speciation, then the number of species might be expected to match the number of available niches and this number might be indefinitely large. One possible constraint is the opportunity for allopatric divergence. In 1981, Felsenstein used a simple and elegant model to ask if there might also be genetic constraints. He showed that progress towards speciation could be described by the build-up of linkage disequilibrium among divergently selected loci and between these loci and those contributing to other forms of reproductive isolation. Therefore, speciation is opposed by recombination, because it tends to break down linkage disequilibria. Felsenstein then introduced a crucial distinction between "two-allele" models, which are subject to this effect, and "one-allele" models, which are free from the recombination constraint. These fundamentally important insights have been the foundation for both empirical and theoretical studies of speciation ever since.

PseudoBase: a genomic visualization and exploration resource for the Drosophila pseudoobscura subgroup.

Drosophila pseudoobscura is a classic model system for the study of evolutionary genetics and genomics. Given this long-standing interest, many genome sequences have accumulated for D. pseudoobscura and closely related species D. persimilis, D. miranda, and D. lowei. To facilitate the exploration of genetic variation within species and comparative genomics across species, we present PseudoBase, a database that couples extensive publicly available genomic data with simple visualization and query tools via an intuitive graphical interface, amenable for use in both research and educational settings. All genetic variation (SNPs and indels) within the database is derived from the same workflow, so variants are easily comparable across data sets. Features include an embedded JBrowse interface, ability to pull out alignments of individual genes/regions, and batch access for gene lists. Here, we introduce PseudoBase, and we demonstrate how this resource facilitates use of extensive genomic data from flies of the Drosophila pseudoobscura subgroup.

The Peroxisome-Autophagy Redox Connection: A Double-Edged Sword?

Peroxisomes harbor numerous enzymes that can produce or degrade hydrogen peroxide (H2O2). Depending on its local concentration and environment, this oxidant can function as a redox signaling molecule or cause stochastic oxidative damage. Currently, it is well-accepted that dysfunctional peroxisomes are selectively removed by the autophagy-lysosome pathway. This process, known as "pexophagy," may serve a protective role in curbing peroxisome-derived oxidative stress. Peroxisomes also have the intrinsic ability to mediate and modulate H2O2-driven processes, including (selective) autophagy. However, the molecular mechanisms underlying these phenomena are multifaceted and have only recently begun to receive the attention they deserve. This review provides a comprehensive overview of what is known about the bidirectional relationship between peroxisomal H2O2 metabolism and (selective) autophagy. After introducing the general concepts of (selective) autophagy, we critically examine the emerging roles of H2O2 as one of the key modulators of the lysosome-dependent catabolic program. In addition, we explore possible relationships among peroxisome functioning, cellular H2O2 levels, and autophagic signaling in health and disease. Finally, we highlight the most important challenges that need to be tackled to understand how alterations in peroxisomal H2O2 metabolism contribute to autophagy-related disorders.

Effect of dietary supplementation of alpha-galactosidase on the growth performance, ileal digestibility, intestinal morphology, and biochemical parameters in broiler chickens.

BACKGROUND: This study was performed to investigate the effect of Alpha-galactosidase (AlphaGal) supplementation with two energy levels on the growth performance, amino acid ileal digestibility coefficient "AID%," economic value, intestinal histology, and blood biochemical parameters of broiler chickens. Two-hundred 3-day-old broiler chicks (average body weight 74.34 g ±0.52 Ross 308) were randomly assigned to a 2 × 2 factorial arrangement consisting of two energy diets groups: in the first group, the birds were fed on a recommended energy diet (RED) while the second group was reduced 120 kcal/kg diet as a low energy diet (LED) and two levels of AlphaGal (0 or 50 mg/kg diet) for RED and LED for the 35-day feeding period. RESULTS: The interaction effects between the energy level and the AlphaGal supplementations resulted in significant decrease (P ≤ 0.05) in the body weight, body weight gain, and the relative growth rate. The feed conversion ratio was signficantly increased in LED without supplementation of AlphaGal group during the entire experimental period, this negative effect on the growth performance was corrected by AlphaGal supplementation. The AID% value was increased significantly by AlphaGal supplementation. Blood triglyceride concentrations were significantly decreased (P = 0.02) in the LED group with or without AlphaGal supplementation, while the level of high-density lipoprotein (HDL) was significantly decreased (P = 0.01) in the LED or RED groups supplemented with 50 mg RED AlphaGal. Histologically, the number of intestinal glands and goblet cells increased in both RED and LED groups supplemented with AlphaGal and their secretions were mainly neutral mucopolysaccharides and less acidic mucopolysaccharides. CONCLUSION: AlphaGal supplementation improved the growth performance of broiler chickens fed LED and the growth performance is similar to those fed RED, thereby consequently improving the economic value of these diets. AlphaGal supplementation improves intestinal histology and morphology as well.

Natural Selection Shapes Variation in Genome-wide Recombination Rate in Drosophila pseudoobscura.

While recombination is widely recognized to be a key modulator of numerous evolutionary phenomena, we have a poor understanding of how recombination rate itself varies and evolves within a species. Here, we performed a comprehensive study of recombination rate (rate of meiotic crossing over) in two natural populations of Drosophila pseudoobscura from Utah and Arizona, USA. We used an amplicon sequencing approach to obtain high-quality genotypes in approximately 8,000 individual backcrossed offspring (17 mapping populations with roughly 530 individuals each), for which we then quantified crossovers. Interestingly, variation in recombination rate within and between populations largely manifested as differences in genome-wide recombination rate rather than remodeling of the local recombination landscape. Comparing populations, we discovered individuals from the Utah population displayed on average 8% higher crossover rates than the Arizona population, a statistically significant difference. Using a QST-FST analysis, we found that this difference in crossover rate was dramatically higher than expected under neutrality, indicating that this difference may have been driven by natural selection. Finally, using a combination of short- and long-read whole-genome sequencing, we found no significant association between crossover rate and structural variation at the 200-400 kb scale. Our results demonstrate that (1) there is abundant variation in genome-wide crossover rate in natural populations, (2) at the 200-400 kb scale, recombination rate appears to vary largely genome-wide, rather than in specific intervals, and (3) interpopulation differences in recombination rate may be the result of local adaptation.