Racial and Ethnic Comparisons in Satisfaction with Services Provided by the Special Supplemental Nutrition Program for Women, Infants, and Children in California.

Understanding satisfaction of nutrition education and other services provided in the Special Supplemental Nutrition Program for Women, Infants and Children (WIC) is needed to ensure the program is responsive to the needs of diverse populations. This study examined the variation of WIC participants' perceptions and satisfaction with WIC nutrition education and services by race, ethnicity, and language preference. Phone surveys were conducted in 2019 with California WIC families with children aged 1−4 years. While most participants (86%) preferred one-on-one nutrition education, online/mobile apps were also favored (69%). The majority (89%) found nutrition education equally important to receiving the WIC food package. Racial/ethnic groups differed in which WIC service they primarily valued as 20% of non-Hispanic White people rated the food package as more important than nutrition education compared to 5% of Spanish- and 6% of English-speaking Hispanic people, respectively. More Spanish (91%) and English-speaking Hispanic people (87%) than non-Hispanic white (79%) or Black people (74%) changed a behavior because of something they learned at WIC (p < 0.001). Spanish-speaking Hispanic people (90%) had the highest satisfaction with WIC nutrition education. Preferential differences among participants suggest that providing flexible options may improve program satisfaction and emphasizes the need for future studies to examine WIC services by race and ethnicity.

Complex interaction network revealed by mutation of human telomerase 'insertion in fingers' and essential N-terminal domains and the telomere protein TPP1.

In the majority of human cancer cells, cellular immortalization depends on the maintenance of telomere length by telomerase. An essential step required for telomerase function is its recruitment to telomeres, which is regulated by the interaction of the telomere protein, TPP1, with the telomerase essential N-terminal (TEN) domain of the human telomerase reverse transcriptase, hTERT. We previously reported that the hTERT 'insertion in fingers domain' (IFD) recruits telomerase to telomeres in a TPP1-dependent manner. Here, we use hTERT truncations and the IFD domain containing mutations in conserved residues or premature aging disease-associated mutations to map the interactions between the IFD and TPP1. We find that the hTERT-IFD domain can interact with TPP1. However, deletion of the IFD motif in hTERT lacking the N-terminus and the C-terminal extension does not abolish interaction with TPP1, suggesting the IFD is not essential for hTERT interaction with TPP1. Several conserved residues in the central IFD-TRAP region that we reported regulate telomerase recruitment to telomeres, and cell immortalization compromise interaction of the hTERT-IFD domain with TPP1 when mutated. Using a similar approach, we find that the IFD domain interacts with the TEN domain but is not essential for intramolecular hTERT interactions with the TEN domain. IFD-TEN interactions are not disrupted by multiple amino acid changes in the IFD or TEN, thus highlighting a complex regulation of IFD-TEN interactions as suggested by recent cryo-EM structures of human telomerase.

PCNA, a focus on replication stress and the alternative lengthening of telomeres pathway.

The maintenance of telomeres, which are specialized stretches of DNA found at the ends of linear chromosomes, is a crucial step for the immortalization of cancer cells. Approximately 10-15 % of cancer cells use a homologous recombination-based mechanism known as the Alternative Lengthening of Telomeres (ALT) pathway to maintain their telomeres. Telomeres in general pose a challenge to DNA replication owing to their repetitive nature and potential for forming secondary structures. Telomeres in ALT+ cells especially are subject to elevated levels of replication stress compared to telomeres that are maintained by the enzyme telomerase, in part due to the incorporation of telomeric variant repeats at ALT+ telomeres, their on average longer lengths, and their modified chromatin states. Many DNA metabolic strategies exist to counter replication stress and to protect stalled replication forks. The role of proliferating cell nuclear antigen (PCNA) as a platform for recruiting protein partners that participate in several of these DNA replication and repair pathways has been well-documented. We propose that many of these pathways may be active at ALT+ telomeres, either to facilitate DNA replication, to manage replication stress, or during telomere extension. Here, we summarize recent evidence detailing the role of PCNA in pathways including DNA secondary structure resolution, DNA damage bypass, replication fork restart, and DNA damage synthesis. We propose that an examination of PCNA and its post-translational modifications (PTMs) may offer a unique lens by which we might gain insight into the DNA metabolic landscape that is distinctively present at ALT+ telomeres.

The GLP-1 Receptor Agonist Liraglutide Increases Myocardial Glucose Oxidation Rates via Indirect Mechanisms and Mitigates Experimental Diabetic Cardiomyopathy.

BACKGROUND: Type 2 diabetes (T2D) increases risk for cardiovascular disease. Of interest, liraglutide, a therapy for T2D that activates the glucagon-like peptide-1 receptor to augment insulin secretion, reduces cardiovascular-related death in people with T2D, though it remains unknown how liraglutide produces these actions. Notably, the glucagon-like peptide-1 receptor is not expressed in ventricular cardiac myocytes, making it likely that ventricular myocardium-independent actions are involved. We hypothesized that augmented insulin secretion may explain how liraglutide indirectly mediates cardioprotection, which thereby increases myocardial glucose oxidation. METHODS: C57BL/6J male mice were fed either a low-fat diet (lean) or were subjected to experimental T2D and treated with either saline or liraglutide 3× over a 24-hour period. Mice were subsequently euthanized and had their hearts perfused in the working mode to assess energy metabolism. A separate cohort of mice with T2D were treated with either vehicle control or liraglutide for 2 weeks for the assessment of cardiac function via ultrasound echocardiography. RESULTS: Treatment of lean mice with liraglutide increased myocardial glucose oxidation without affecting glycolysis. Conversely, direct treatment of the isolated working heart with liraglutide had no effect on glucose oxidation. These findings were recapitulated in mice with T2D and associated with increased circulating insulin levels. Furthermore, liraglutide treatment alleviated diastolic dysfunction in mice with T2D, which was associated with enhanced pyruvate dehydrogenase activity, the rate-limiting enzyme of glucose oxidation. CONCLUSIONS: Our data demonstrate that liraglutide augments myocardial glucose oxidation via indirect mechanisms, which may contribute to how liraglutide improves cardiovascular outcomes in people with T2D.

An investigation into the impact of deleting one copy of the glutaredoxin-2 gene on diet-induced weight gain and the bioenergetics of muscle mitochondria in female mice fed a high fat diet.

Our group recently documented that male mice containing a deletion for one copy of the glutaredoxin-2 (Grx2) gene were completely protected from developing diet-induced obesity (DIO). Objectives: Here, we conducted a similar investigation but with female littermates. Results: In comparison to our recent publication using male mice, exposure of WT and GRX2+/- female mice to a HFD from 3-to-10 weeks of age did not induce any changes in body mass, circulating blood glucose, food intake, hepatic glycogen levels, or abdominal fat pad mass. Examination of the bioenergetics of muscle mitochondria revealed no changes in the rate of superoxide ( O 2 ∙ - )/hydrogen peroxide (H2O2) or O2 consumption under different states of respiration or alterations in lipid peroxidation adduct levels regardless of mouse strain or diet. Additionally, we measured the bioenergetics of mitochondria isolated from liver tissue and found that partial loss of GRX2 augmented respiration but did not alter ROS production. Discussion: Overall, our findings demonstrate there are sex differences in the protection of female GRX2+/- mice from DIO, fat accretion, intrahepatic lipid accumulation, and the bioenergetics of mitochondria from muscle and liver tissue.

C57BL/6J mice upregulate catalase to maintain the hydrogen peroxide buffering capacity of liver mitochondria.

Here, we demonstrate that the upregulation of catalase is required to compensate for the loss of nicotinamide nucleotide transhydrogenase (NNT) to maintain hydrogen peroxide (H2O2) steady-state levels in C57BL/6J liver mitochondria. Our investigations using the closely related mouse strains C57BL/6NJ (6NJ; +NNT) and C57BL/6J (6J; -NNT) revealed that NNT is required for the provision of NADPH and that the upregulation of isocitrate dehydrogenase-2 (IDH2) activity is not enough to compensate for the absence of NNT, which is consistent with previous observations. Intriguingly, despite the absence of NNT, 6J mitochondria had rates of H2O2 production (58.56 ±â€¯3.79 pmol mg-1 min-1) that were similar to samples collected from 6NJ mice (72.75 ±â€¯14.26 pmol mg-1 min-1) when pyruvate served as the substrate. However, 6NJ mitochondria energized with succinate produced significantly less H2O2 (59.95 ±â€¯2.13 pmol mg-1 min-1) when compared to samples from 6J mice (116.39 ±â€¯20.74 pmol mg-1 min-1), an effect that was attributed to the presence of NNT. Further investigations into the H2O2 eliminating capacities of these mitochondria led to the novel observation that 6J mitochondria compensate for the loss of NNT by upregulating catalase. Indeed, 6NJ and 6J mitochondria energized with pyruvate or succinate displayed similar rates for H2O2 elimination, quenching ~84% and ~86% of the H2O2, respectively, in the surrounding medium within 30 s. However, inclusion of palmitoyl-CoA, an NNT inhibitor, significantly limited H2O2 degradation by 6NJ mitochondria only (~55% of H2O2 eliminated in 30 s). Liver mitochondria from 6J mice treated with palmitoyl-CoA still cleared ~80% of the H2O2 from the surrounding environment. Inhibition of catalase with triazole compromised the capacity of 6J mitochondria to maintain H2O2 steady-state levels. By contrast, disabling NADPH-dependent antioxidant systems had a limited effect on the H2O2 clearing capacity of 6J mitochondria. Liver mitochondria collected from 6NJ mice, on the other hand, were more reliant on the GSH and TRX systems to clear exogenously added H2O2. However, catalase still played an integral in eliminating H2O2 in 6NJ liver mitochondria. Immunoblot analyses demonstrated that catalase protein levels were ~7.7-fold higher in 6J mitochondria. Collectively, our findings demonstrate for the first time that 6J liver mitochondria compensate for the loss of NNT by increasing catalase levels for the maintenance of H2O2 steady-state levels. In general, our observations reveal that catalase is an integral arm of the antioxidant response in liver mitochondria.

Lactate dehydrogenase supports lactate oxidation in mitochondria isolated from different mouse tissues.

Research over the past seventy years has established that mitochondrial-l-lactate dehydrogenase (m-L-LDH) is vital for mitochondrial bioenergetics. However, in recent report, Fulghum et al. concluded that lactate is a poor fuel for mitochondrial respiration [1]. In the present study, we have followed up on these findings and conducted an independent investigation to determine if lactate can support mitochondrial bioenergetics. We demonstrate herein that lactate can fuel the bioenergetics of heart, muscle, and liver mitochondria. Lactate was just as effective as pyruvate at stimulating mitochondrial coupling efficiency. Inclusion of LDH (sodium oxamate or GSK 2837808A) and pyruvate dehydrogenase (PDH; CPI-613) inhibitors abolished respiration in mitochondria energized with lactate. Lactate also fueled mitochondrial ROS generation and was just as effective as pyruvate at stimulating H2O2 production. Additionally, lactate-induced ROS production was inhibited by both LDH and PDH inhibitors. Enzyme activity measurements conducted on permeabilized mitochondria revealed that LDH is localized in mitochondria. In aggregate, we can conclude that mitochondrial LDH fuels bioenergetics in several tissues by oxidizing lactate.

Sex-dependent Differences in the Bioenergetics of Liver and Muscle Mitochondria from Mice Containing a Deletion for glutaredoxin-2.

Our group recently published a study demonstrating that deleting the gene encoding the matrix thiol oxidoreductase, glutaredoxin-2 (GRX2), alters the bioenergetics of mitochondria isolated from male C57BL/6N mice. Here, we conducted a similar study, examining H2O2 production and respiration in mitochondria isolated from female mice heterozygous (GRX2+/-) or homozygous (GRX2-/-) for glutaredoxin-2. First, we observed that deleting the Grx2 gene does not alter the rate of H2O2 production in liver and muscle mitochondria oxidizing pyruvate, α-ketoglutarate, or succinate. Examination of the rates of H2O2 release from liver mitochondria isolated from male and female mice revealed that (1) sex has an impact on the rate of ROS production by liver and muscle mitochondria and (2) loss of GRX2 only altered ROS release in mitochondria collected from male mice. Assessment of the bioenergetics of these mitochondria revealed that loss of GRX2 increased proton leak-dependent and phosphorylating respiration in liver mitochondria isolated from female mice but did not alter rates of respiration in liver mitochondria from male mice. Furthermore, we found that deleting the Grx2 gene did not alter rates of respiration in muscle mitochondria collected from female mice. This contrasts with male mice where loss of GRX2 substantially augmented proton leaks and ADP-stimulated respiration. Our findings indicate that some fundamental sexual dimorphisms exist between GRX2-deficient male and female rodents.

Deletion of the Glutaredoxin-2 Gene Protects Mice from Diet-Induced Weight Gain, Which Correlates with Increased Mitochondrial Respiration and Proton Leaks in Skeletal Muscle.

Aims: The aim of this study was to determine whether deleting the gene encoding glutaredoxin-2 (GRX2) could protect mice from diet-induced weight gain. Results: Subjecting wild-type littermates to a high fat diet (HFD) induced a significant increase in overall body mass, white adipose tissue hypertrophy, lipid droplet accumulation in hepatocytes, and higher circulating insulin and triglyceride levels. In contrast, GRX2 heterozygotes (GRX2+/-) fed an HFD had a body mass, white adipose tissue weight, and hepatic and circulating lipid and insulin levels similar to littermates fed a control diet. Examination of the bioenergetics of muscle mitochondria revealed that this protective effect was associated with an increase in respiration and proton leaks. Muscle mitochondria from GRX2+/- mice had a ∼2- to 3-fold increase in state 3 (phosphorylating) respiration when pyruvate/malate or succinate served as substrates and a ∼4-fold increase when palmitoyl-carnitine was being oxidized. Proton leaks were ∼2- to 3-fold higher in GRX2+/- muscle mitochondria. Treatment of mitochondria with either guanosine diphosphate, genipin, or octanoyl-carnitine revealed that the higher rate of O2 consumption under state 4 conditions was associated with increased leaks through uncoupling protein-3 and adenine nucleotide translocase. GRX2+/- mitochondria also had better protection from oxidative distress. Innovation: For the first time, we demonstrate that deleting the Grx2 gene can protect from diet-induced weight gain and the development of obesity-related disorders. Conclusions: Deleting the Grx2 gene protects mice from diet-induced weight gain. This effect was related to an increase in muscle fuel combustion, mitochondrial respiration, proton leaks, and reactive oxygen species handling. Antioxid. Redox Signal. 31, 1272-1288.

Estimation of the hydrogen peroxide producing capacities of liver and cardiac mitochondria isolated from C57BL/6N and C57BL/6J mice.

Here, we examined the hydrogen peroxide (H2O2) producing capacities of pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), proline dehydrogenase (PRODH), glycerol-3-phosphate dehydrogenase (G3PDH), succinate dehydrogenase (SDH; complex II), and branched-chain keto acid dehydrogenase (BCKDH), in cardiac and liver mitochondria isolated from C57BL/6N (6N) and C57BL/6J (6J) mice. Various inhibitor combinations were used to suppress ROS production by complexes I, II, and III and estimate the native rates of H2O2 production for these enzymes. Overall, liver mitochondria from 6N mice produced ∼2-fold more ROS than samples enriched from 6J mice. This was attributed, in part, to the higher levels of glutathione peroxidase-1 (GPX1) and catalase (CAT) in 6J mitochondria. Intriguingly, PDH, KGDH, and SDH comprised up to ∼95% of the ROS generating capacity of permeabilized 6N liver mitochondria, with PRODH, G3PDH, and BCKDH making minor contributions. By contrast, BCKDH accounted for ∼34% of the production in permeabilized 6J mitochondria with KGDH and PRODH accounting for ∼23% and ∼19%. G3PDH produced high amounts of ROS, accounting for ∼52% and ∼39% of the total H2O2 generating capacity in 6N and 6J heart mitochondria. PRODH was also an important ROS source in 6J mitochondria, accounting for ∼43% of the total H2O2 formed. In addition, 6J cardiac mitochondria produced significantly more ROS than 6N mitochondria. Taken together, our findings demonstrate that these other generators can also serve as important sources of H2O2. Additionally, we found that mouse strain influences the rate of production from the individual sites that were studied.

Protein S-glutathionylation: The linchpin for the transmission of regulatory information on redox buffering capacity in mitochondria.

Protein S-glutathionylation reactions are a ubiquitous oxidative modification required to control protein function in response to changes in redox buffering capacity. These reactions are rapid and reversible and are, for the most part, enzymatically mediated by glutaredoxins (GRX) and glutathione S-transferases (GST). Protein S-glutathionylation has been found to control a range of cell functions in response to different physiological cues. Although these reactions occur throughout the cell, mitochondrial proteins seem to be highly susceptible to reversible S-glutathionylation, a feature attributed to the unique physical properties of this organelle. Indeed, mitochondria contain a number of S-glutathionylation targets which includes proteins involved in energy metabolism, solute transport, reactive oxygen species (ROS) production, proton leaks, apoptosis, antioxidant defense, and mitochondrial fission and fusion. Moreover, it has been found that conjugation and removal of glutathione from proteins in mitochondria fulfills a number of important physiological roles and defects in these reactions can have some dire pathological consequences. Here, we provide an updated overview on mitochondrial protein S-glutathionylation reactions and their importance in cell functions and physiology.

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases.

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2●-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems and side reactions that also form O2●-/H2O2. Use of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake.

Protein S-glutathionylation is a reversible redox modification that regulates mitochondrial metabolism and reactive oxygen species (ROS) production in liver and cardiac tissue. However, whether or not it controls ROS release from skeletal muscle mitochondria has not been explored. In the present study, we examined if chemically-induced protein S-glutathionylation could alter superoxide (O2●-)/hydrogen peroxide (H2O2) release from isolated muscle mitochondria. Disulfiram, a powerful chemical S-glutathionylation catalyst, was used to S-glutathionylate mitochondrial proteins and ascertain if it can alter ROS production. It was found that O2●-/H2O2 release rates from permeabilized muscle mitochondria decreased with increasing doses of disulfiram (100-500 µM). This effect was highest in mitochondria oxidizing succinate or palmitoyl-carnitine, where a ~80-90% decrease in the rate of ROS release was observed. Similar effects were detected in intact mitochondria respiring under state 4 conditions. Incubation of disulfiram-treated mitochondria with DTT (2 mM) restored ROS release confirming that these effects were associated with protein S-glutathionylation. Disulfiram treatment also inhibited phosphorylating and proton leak-dependent respiration. Radiolabelled substrate uptake experiments demonstrated that disulfiram inhibited pyruvate import but had no effect on carnitine uptake. Immunoblot analysis of complex I revealed that it contained several protein S-glutathionylation targets including NDUSF1, a subunit required for NADH oxidation. Taken together, these results demonstrate that O2●-/H2O2 release from muscle mitochondria can be altered by protein S-glutathionylation. We attribute these changes to the protein S-glutathionylation complex I and inhibition of mitochondrial pyruvate carrier.

Physiological levels of formate activate mitochondrial superoxide/hydrogen peroxide release from mouse liver mitochondria.

Here, we found that formate, an essential one-carbon metabolite, activates superoxide (O2·-)/hydrogen peroxide (H2 O2 ) release from mitochondria. Sodium formate (30 µm) induces a significant increase in O2·-/H2 O2 production in liver mitochondria metabolizing pyruvate (50 µm). At concentrations deemed to be toxic, formate does not increase O2·-/H2 O2 production further. It was observed that the formate-mediated increase in O2·-/H2 O2 production is not associated with cytochrome c oxidase (COX) inhibition or changes in membrane potential and NAD(P)H levels. Sodium formate supplementation increases phosphorylating respiration without altering proton leaks. Finally, it was observed that the 2-oxoglutarate dehydrogenase (OGDH) inhibitors 3-methyl-2-oxovaleric acid (KMV) and CPI-613 inhibit the formate-induced increase in pyruvate-driven ROS production. The importance of these findings in one-carbon metabolism and physiology are discussed herein.

Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria.

Pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) are important sources of reactive oxygen species (ROS). In addition, it has been found that mitochondria can also serve as sinks for cellular hydrogen peroxide (H2O2). However, the ROS forming and quenching capacity of liver mitochondria has never been thoroughly examined. Here, we show that mouse liver mitochondria use catalase, glutathione (GSH), and peroxiredoxin (PRX) systems to quench ROS. Incubation of mitochondria with catalase inhibitor 3-amino-1,2,4-triazole (triazole) induced a significant increase in pyruvate or α-ketoglutarate driven O2-/H2O2 formation. 1-Choro-2,4-dinitrobenzene (CDNB), which depletes glutathione (GSH), elicited a similar effect. Auranofin (AF), a thioredoxin reductase-2 (TR2) inhibitor which disables the PRX system, did not significantly change O2-/H2O2 formation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial H2O2. In this study, the ROS forming potential of PDHC, KGDHC, Complex I, and Complex III was also profiled. Titration of mitochondria with 3-methyl-2-oxovaleric acid (KMV), a specific inhibitor for O2-/H2O2 production by KGDHC, induced a ~86% and ~84% decrease in ROS production during α-ketoglutarate and pyruvate oxidation. Titration of myxothiazol, a Complex III inhibitor, decreased O2-/H2O2 formation by ~45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating that Complex I does not contribute to ROS production during forward electron transfer from NADH. Taken together, our results indicate that KGDHC and Complex III are high capacity sites for O2-/H2O2 production in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial H2O2.

Choline and dimethylglycine produce superoxide/hydrogen peroxide from the electron transport chain in liver mitochondria.

Here, we report that choline and dimethylglycine can stimulate reactive oxygen species (ROS) production in liver mitochondria. Choline stimulated O2 ˙- /H2 O2 formation at a concentration of 5 µm. We also observed that Complex II and III inhibitors, atpenin A5 and myxothiazol, collectively induced a 95% decrease in O2 ˙- /H2 O2 production indicating both sites serve as the main sources of ROS during choline oxidation. Dimethylglycine, an intermediate of choline oxidation, was a more effective ROS generator. Rates of production were ~ 43% higher than choline-mediated O2 ˙- /H2 O2 production. The main site for dimethylglycine-mediated ROS production was via reverse electron transfer to Complex I. Our results demonstrate that metabolism of essential metabolites involved in methionine and folic acid biosynthesis can stimulate mitochondrial ROS production.

Contrasting theory with the empirical data of species recognition.

We tested hypotheses on how animals should respond to heterospecifics encountered in the environment. Hypotheses were formulated from models parameterized to emphasize four factors that are expected to influence species discrimination: mating and territorial interactions; sex differences in resource value; environments in which heterospecifics were common or rare; and the type of identity cues available for species recognition. We also considered the role of phylogeny on contemporary responses to heterospecifics. We tested the extent these factors explained variation among taxa in species discrimination using a meta-analysis of three decades of species recognition research. A surprising outcome was the absence of a general predictor of when species discrimination would most likely occur. Instead, species discrimination is dictated by the benefits and costs of responding to a conspecific or heterospecific that are governed by the specific circumstances of a given species. The phylogeny of species recognition provided another unexpected finding: the evolutionary relationships among species predicted whether courting males within species-but not females-would discriminate against heterospecifcs. This implies that species recognition has evolved quite differently in the sexes. Finally, we identify common pitfalls in experimental design that seem to have affected some studies (e.g., poor statistical power) and provide recommendations for future research.

Monogamy evolves through multiple mechanisms: evidence from V1aR in deer mice.

Genetic variation in Avpr1a, the locus encoding the arginine vasopressin receptor 1A (V1aR), has been implicated in pair-bonding behavior in voles (genus Microtus) and humans, raising the possibility that this gene may contribute commonly to mating-system variation in mammals. In voles, differential expression of V1aR in the brain is associated with male partner-preference behavior in a comparison of a monogamous (Microtus ochrogaster) and promiscuous (Microtus montanus) species. This expression difference is correlated, in turn, with a difference in length of a 5' regulatory microsatellite in Avpr1a. Here, we use a combination of comparative sequencing of coding and regulatory regions, analysis of neural expression patterns, and signaling assays to test for differences in V1aR expression and function among eight species of deer mice (genus Peromyscus). Despite well-documented variation in Peromyscus social behavior, we find no association between mating system and length variation in the microsatellite locus linked to V1aR expression in voles. Further, there are no consistent differences in V1aR expression pattern between monogamous and promiscuous species in regions of the brain known to influence mating behavior. We do find statistical evidence for positive selection on the V1aR coding sequence including several derived amino acid substitutions in a monogamous Peromyscus lineage, yet these substitutions have no measurable effect on V1aR signaling activity. Together, these results suggest that mating-system variation in rodents is mediated by multiple genetic mechanisms.