Metabolic effects of CO2 anaesthesia in Drosophila melanogaster

Immobilization of insects is necessary for various experimental purposes, and CO₂ exposure remains the most popular anaesthetic method in entomological research. A number of negative side effects of CO₂ anaesthesia have been reported, but CO₂ probably brings about metabolic modifications that are poorly known. In this work, we used GC/MS-based metabolic fingerprinting to assess the effect of CO₂ anaesthesia in Drosophila melanogaster adults. We analysed metabolic variation of flies submitted to acute CO₂ exposure and assessed the temporal metabolic changes during short- and long-term recovery. We found that D. melanogaster metabotypes were significantly affected by the anaesthetic treatment. Metabolic changes caused by acute CO₂ exposure were still manifested after 14 h of recovery. However, we found no evidence of metabolic alterations when a long recovery period was allowed (more than 24 h). This study points to some metabolic pathways altered during CO₂ anaesthesia (e.g. energetic metabolism). Evidence of short-term metabolic changes indicates that CO₂ anaesthesia should be used with utmost caution in physiological studies when a short recovery is allowed. In spite of this, CO₂ treatment seems to be an acceptable anaesthetic method provided that a long recovery period is allowed (more than 24 h).     

Dopamine Dynamics and Signaling in Drosophila: An Overview of Genes,Drugs and Behavioral Paradigms

Changes in dopamine (DA) signaling have been implicated in a number of human neurologic and psychiatric disorders. Similarly, defects in DA signaling in the fruit fly, Drosophila melanogaster, have also been associated with several behavioral defects. As most genes involved in DA synthesis, transport, secretion, and signaling are conserved between species, Drosophila is a powerful genetic model organism to study the regulation of DA signaling in vivo. In this review, we will provide an overview of the genes and drugs that regulate DA biology in Drosophila. Furthermore, we will discuss the behavioral paradigms that are regulated by DA signaling in flies. By analyzing the genes and neuronal circuits that govern such behaviors using sophisticated genetic, pharmacologic, electrophysiologic, and imaging approaches in Drosophila, we will likely gain a better understanding about how this neuromodulator regulates motor tasks and cognition in humans.     

Quantitative Trait Loci Mapping of the Mouse Plasma Proteome (pQTL)

A current challenge in the era of genome-wide studies is to determine the responsible genes and mechanisms underlying newly identified loci. Screening of the plasma proteome by high-throughput mass spectrometry (MALDI-TOF MS) is considered a promising approach for identification of metabolic and disease processes. Therefore, plasma proteome screening might be particularly useful for identifying responsible genes when combined with analysis of variation in the genome. Here, we describe a proteomic quantitative trait locus (pQTL) study of plasma proteome screens in an F₂ intercross of 455 mice mapped with 177 genetic markers across the genome. A total of 69 of 176 peptides revealed significant LOD scores (≥5.35) demonstrating strong genetic regulation of distinct components of the plasma proteome. Analyses were confirmed by mechanistic studies and MALDI-TOF/TOF, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the two strongest pQTLs: A pQTL for mass-to-charge ratio (m/z) 3494 (LOD 24.9, D11Mit151) was identified as the N-terminal 35 amino acids of hemoglobin subunit A (Hba) and caused by genetic variation in Hba. Another pQTL for m/z 8713 (LOD 36.4; D1Mit111) was caused by variation in apolipoprotein A2 (Apoa2) and cosegregated with HDL cholesterol. Taken together, we show that genome-wide plasma proteome profiling in combination with genome-wide genetic screening aids in the identification of causal genetic variants affecting abundance of plasma proteins.     

Molecular and quantitative trait variation within and among small fragmented populations of the endangered plant species Psilopeganum sinense

Background and Aims

Natural selection and genetic drift are important evolutionary forces in determining genetic and phenotypic differentiation in plant populations. The extent to which these two distinct evolutionary forces affect locally adaptive quantitative traits has been well studied in common plant and animal species. However, we know less about how quantitative traits respond to selection pressures and drift in endangered species that have small population sizes and fragmented distributions. To address this question, this study assessed the relative strengths of selection and genetic drift in shaping population differentiation of phenotypic traits in Psilopeganum sinense, a naturally rare and recently endangered plant species.

Methods

Population differentiation at five quantitative traits (Q_ST) obtained from a common garden experiment was compared with differentiation at putatively neutral microsatellite markers (F_ST) in seven populations of P. sinense. Q_ST estimates were derived using a Bayesian hierarchical variance component method.

Key Results

Trait-specific Q_ST values were equal to or lower than F_ST. Neutral genetic diversity was not correlated with quantitative genetic variation within the populations of P. sinense.

Conclusions

Despite the prevalent empirical evidence for Q_ST > F_ST, the results instead suggest a definitive role of stabilizing selection and drift leading to phenotypic differentiation among small populations. Three traits exhibited a significantly lower Q_ST relative to F_ST, suggesting that populations of P. sinense might have experienced stabilizing selection for the same optimal phenotypes despite large geographical distances between populations and habitat fragmentation. For the other two traits, Q_ST estimates were of the same magnitude as F_ST, indicating that divergence in these traits could have been achieved by genetic drift alone. The lack of correlation between molecular marker and quantitative genetic variation suggests that sophisticated considerations are required for the inference of conservation measures of P. sinense from neutral genetic markers.     

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.     

Effect of semolina-jaggery diet on survival and development of Drosophila melanogaster

Drosophila melanogaster is an ideal model organism for developmental studies. This study tests the potential of semolina-jaggery (SJ) diet as a new formulation for bulk rearing of flies. Semolina and jaggery are organic products obtained from wheat endosperm and cane sugar, respectively. Semolina is a rich source of carbohydrates and protein. Jaggery has a high content of dietary sugars. Moreover, preparation of semolina jaggery diet is cost-effective and easy. Thus, the current study aimed to compare survival and developmental parameters of flies fed the SJ diet to flies fed the standard cornmeal-sugar-yeast (CSY) diet. SJ diet enhanced survival of flies without affecting fecundity; male flies showed increased resistance to starvation. A higher number of flies emerged at F₂ and F₃ generation when fed the SJ diet than when fed the control CSY diet. SJ diet did not increase fly body weight and lipid percentage. Therefore, SJ diet can be used for bulk rearing of healthy flies at par with the standard cornmeal-sugar-yeast diet.     

Thermoregulatory responses in seasonally acclimatized captive Southern White-faced Scops-owls

Seasonal thermoregulatory responses that are associated with cold tolerance have been reported for many species that inhabit regions where winters are severe (e.g. Holarctic), but relatively few studies have focused on species from regions where the climate is more unpredictable (e.g. Southern Africa). In this study, metabolic rate (VO₂) and body temperature (T_b) was measured during summer and winter in captive Southern White-faced Scops-owl (Ptilopsis granti), to test for thermoregulatory responses representing energy conservation in winter. During winter the Southern White-faced Scops-owls increased resting metabolic rate (RMR) by 45% to regulate a set point T_b—a result similar to what had been shown in small passerines from the Holarctic region. Increased RMR and increased conductance at cold T_a's are suggestive of improved cold tolerance. Basal metabolic rate (BMR) was 0.60 mL O₂ g⁻¹ h⁻¹ and showed no seasonal flexibility. Thus, contrary to expectation, the Southern White-faced Scops-owls showed seasonal thermoregulatory responses that are unlikely to represent energy conservation which was expected for a medium-sized bird inhabiting unpredictable climates in Southern Africa.     

Estimating Population-Level Coancestry Coefficients by an Admixture F Model

In this article, we develop an admixture F model (AFM) for the estimation of population-level coancestry coefficients from neutral molecular markers. In contrast to the previously published F model, the AFM enables disentangling small population size and lack of migration as causes of genetic differentiation behind a given level of F_ST. We develop a Bayesian estimation scheme for fitting the AFM to multiallelic data acquired from a number of local populations. We demonstrate the performance of the AFM, using simulated data sets and real data on ninespine sticklebacks (Pungitius pungitius) and common shrews (Sorex araneus). The results show that the parameterization of the AFM conveys more information about the evolutionary history than a simple summary parameter such as F_ST. The methods are implemented in the R package RAFM.     

Heritability and quantitative genetic divergence of serotiny,a fire-persistence plant trait

Background and Aims

Although it is well known that fire acts as a selective pressure shaping plant phenotypes, there are no quantitative estimates of the heritability of any trait related to plant persistence under recurrent fires, such as serotiny. In this study, the heritability of serotiny in Pinus halepensis is calculated, and an evaluation is made as to whether fire has left a selection signature on the level of serotiny among populations by comparing the genetic divergence of serotiny with the expected divergence of neutral molecular markers (Q_ST–F_ST comparison).

Methods

A common garden of P. halepensis was used, located in inland Spain and composed of 145 open-pollinated families from 29 provenances covering the entire natural range of P. halepensis in the Iberian Peninsula and Balearic Islands. Narrow-sense heritability (h²) and quantitative genetic differentiation among populations for serotiny (Q_ST) were estimated by means of an ‘animal model’ fitted by Bayesian inference. In order to determine whether genetic differentiation for serotiny is the result of differential natural selection, Q_ST estimates for serotiny were compared with F_ST estimates obtained from allozyme data. Finally, a test was made of whether levels of serotiny in the different provenances were related to different fire regimes, using summer rainfall as a proxy for fire regime in each provenance.

Key Results

Serotiny showed a significant narrow-sense heritability (h²) of 0·20 (credible interval 0·09–0·40). Quantitative genetic differentiation among provenances for serotiny (Q_ST = 0·44) was significantly higher than expected under a neutral process (F_ST = 0·12), suggesting adaptive differentiation. A significant negative relationship was found between the serotiny level of trees in the common garden and summer rainfall of their provenance sites.

Conclusions

Serotiny is a heritable trait in P. halepensis, and selection acts on it, giving rise to contrasting serotiny levels among populations depending on the fire regime, and supporting the role of fire in generating genetic divergence for adaptive traits.     

In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB1 receptor antagonist for the management of obesity

Cannabinoid CB₁ receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB₁ receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB₁ receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB₁ receptors in both binding (K_i = 0.7 nM) and functional assays (K_i = 0.2 nM). The compound has low affinity (K_i = 7600 nM) for human CB₂ receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10 mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB₁ receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB₁ receptor competitive antagonist that may further our understanding of the endocannabinoid system.     

Genetic consequences of a century of protection: serial founder events and survival of the little spotted kiwi (Apteryx owenii)

We present the outcome of a century of post-bottleneck isolation of a long-lived species, the little spotted kiwi (Apteryx owenii, LSK) and demonstrate that profound genetic consequences can result from protecting few individuals in isolation. LSK were saved from extinction by translocation of five birds from South Island, New Zealand to Kapiti Island 100 years ago. The Kapiti population now numbers some 1200 birds and provides founders for new populations. We used 15 microsatellite loci to compare genetic variation among Kapiti LSK and the populations of Red Mercury, Tiritiri Matangi and Long Islands that were founded with birds from Kapiti. Two LSK native to D''Urville Island were also placed on Long Island. We found extremely low genetic variation and signatures of acute and recent genetic bottleneck effects in all four populations, indicating that LSK have survived multiple genetic bottlenecks. The Long Island population appears to have arisen from a single mating pair from Kapiti, suggesting there is no genetic contribution from D''Urville birds among extant LSK. The N_e/N_C ratio of Kapiti Island LSK (0.03) is exceptionally low for terrestrial vertebrates and suggests that genetic diversity might still be eroding in this population, despite its large census size.     

Comparison of warfarin sensitivity between rat and bird species

Scattering coumarin derivative rodenticides in broad areas have caused primary- and secondary-poisoning incidents in non-target wild birds. In this study, we compared factors determining warfarin sensitivity between bird species and rats based on vitamin K 2,3-epoxide reductase (VKOR) kinetics, VKOR inhibition by warfarin and warfarin metabolism assays. In VKOR characterization, chickens and ostriches showed significantly lower enzymatic efficiencies than rats (one-sixth and one-third, respectively), suggesting bird species depend more on a non-VKOR vitamin K source. On the other hand, the inhibition constants (K_i) of VKOR for warfarin were significantly different between chickens and ostriches (11.3 ± 2.5 μM and 0.64 ± 0.39 μM, respectively). Interestingly, the ostrich K_i was similar to the values for rats (0.28 ± 0.09 μM). The K_i results reveal a surprising possibility that VKOR in some bird species are easily inhibited by warfarin. Warfarin metabolism assays also showed a large inter-species difference in bird species. Chickens and ostriches showed higher metabolic activity than that of rats, while mallards and owls showed only a slight ability to metabolize warfarin. In this study, we clarified the wide inter-species difference that exists among birds in xenobiotic metabolism and sensitivity to a rodenticide.     

Comparative understanding of UTS2 and UTS2R genes for their involvement in type 2 diabetes mellitus

Several reports have shown that urotensin 2 (UTS2) and its receptor (UTS2R) are involved in glucose metabolism and insulin resistance, which lead to development of type 2 diabetes mellitus (T2DM) in humans. In the present study, we annotated both bovine UTS2 and UTS2R genes and identified 5 single nucleotide polymorphisms (SNPs) for the former gene and 14 mutations for the latter gene. Four mutations were genotyped on a Wagyu x Limousin reference population, including 6 F₁ bulls, 113 F₁ dams and ~250 F₂ progeny. Among 12 phenotypes related to fat deposition and fatty acid composition, we observed that the UTS2 gene was significantly associated with the amount of skeletal saturated fatty acids, while its receptor (UTS2R) gene had significant effects on amounts of saturated and monounsaturated fatty acids, Δ⁹ desaturase activity for converting 16:0 into 16:1, muscle fat (marbling) score and Longissimus Dorsi muscle area. However, in this population, these markers were not associated with subcutaneous fat depth or percent kidney, pelvic and heart fat. We also found that mutations in the promoter regions altered the promoter activities in both genes and coding SNPs might affect the mRNA stability in the UTS2R gene. Overall, our present study provides the first evidence that both UTS2 and UTS2R genes regulate skeletal muscle fat accumulation and fatty acid metabolism, thus indicating their potential pathological functions related to obesity and T2DM in humans.     

Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

Potassium tellurite (K₂TeO₃) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.     

Ack kinase regulates CTP synthase filaments during Drosophila oogenesis

The enzyme CTP synthase (CTPS) dynamically assembles into macromolecular filaments in bacteria, yeast, Drosophila, and mammalian cells, but the role of this morphological reorganization in regulating CTPS activity is controversial. During Drosophila oogenesis, CTPS filaments are transiently apparent in ovarian germline cells during a period of intense genomic endoreplication and stockpiling of ribosomal RNA. Here, we demonstrate that CTPS filaments are catalytically active and that their assembly is regulated by the non-receptor tyrosine kinase DAck, the Drosophila homologue of mammalian Ack1 (activated cdc42-associated kinase 1), which we find also localizes to CTPS filaments. Egg chambers from flies deficient in DAck or lacking DAck catalytic activity exhibit disrupted CTPS filament architecture and morphological defects that correlate with reduced fertility. Furthermore, ovaries from these flies exhibit reduced levels of total RNA, suggesting that DAck may regulate CTP synthase activity. These findings highlight an unexpected function for DAck and provide insight into a novel pathway for the developmental control of an essential metabolic pathway governing nucleotide biosynthesis.     

Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A2-VIA (iPLA2β)-knockout mice

Calcium-independent phospholipase A₂ group VIA (iPLA₂β) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA₂β gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA₂β in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA₂β^+/+) and knockout (iPLA₂β^−/−) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA₂, cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA₂β^+/+ mice, iPLA₂β^−/− mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA₂β^−/− mice, brain levels of iPLA₂β mRNA, protein, and activity were decreased, as was the iPLA₂γ (Group VIB PLA₂) mRNA level, while levels of secretory sPLA₂-V mRNA, protein, and activity and cytosolic cPLA₂-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA₂β deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.