Thomas J. S. Merritt

Flux control and excess capacity in the enzymes of glycolysis and their relationship to flight metabolism in Drosophila melanogaster.

An important question in evolutionary and physiological genetics is how the control of flux-base phenotypes is distributed across the enzymes in a pathway. This control is often related to enzyme-specific levels of activity that are reported to be in excess of that required for demand. In glycolysis, metabolic control is frequently considered vested in classical regulatory enzymes, each strongly displaced from equilibrium. Yet the contribution of individual steps to control is unclear. To assess enzyme-specific control in the glycolytic pathway, we used P-element excision-derived mutagenesis in Drosophila melanogaster to generate full and partial knockouts of seven metabolic genes and to measure tethered flight performance. For most enzymes, we find that reduction to half of the normal activity has no measurable impact on wing beat frequency. The enzymes catalyzing near-equilibrium reactions, phosphoglucose isomerase, phosphoglucomutase, and triosephosphate isomerase fail to show any decline in flight performance even when activity levels are reduced to 17% or less. At reduced activities, the classic regulatory enzymes, hexokinase and glycogen phosphorylase, show significant drops in flight performance and are nearer to saturation. Our results show that flight performance is canalized or robust to the activity variation found in natural populations. Furthermore, enzymes catalyzing near-equilibrium reactions show strong genetic dominance down to low levels of activity. This implies considerable excess enzyme capacity for these enzymes.

PATTERNS OF GENE DUPLICATION IN LEPIDOPTERAN PHEROMONE BINDING PROTEINS

Abstract. We have isolated and characterized cDNAs representing two distinct pheromone binding proteins (PBPs) from the gypsy moth, Lymantria dispar. We use the L. dispar protein sequences, along with other published lepidopteran PBPs, to investigate the evolutionary relationships among genes within the PBP multigene family. Our analyses suggest that the presence of two distinct PBPs in genera representing separate moth superfamilies is the result of relatively recent, independent, gene duplication events rather than a single, ancient, duplication. We discuss this result with respect to the biochemical diversification of moth PBPs.

Paraquat administration in Drosophila for use in metabolic studies of oxidative stress

Paraquat (PQ) is widely used in the laboratory to induce in vivo oxidative stress, particularly in the fruit fly, Drosophila melanogaster. PQ administration to the fly traditionally involves feeding in a 1% sucrose solution; however, a diet high in sucrose can itself be stressful. We examined a novel method of PQ administration: incorporation into the flys standard cornmeal-sucrose-yeast diet. This method successfully delivers PQ to the fly at concentrations similar to those of the traditional method but with fewer possibly confounding complications.

Characterization of the microbial acid mine drainage microbial community using culturing and direct sequencing techniques

We characterized the bacterial community from an AMD tailings pond using both classical culturing and modern direct sequencing techniques and compared the two methods. Acid mine drainage (AMD) is produced by the environmental and microbial oxidation of minerals dissolved from mining waste. Surprisingly, we know little about the microbial communities associated with AMD, despite the fundamental ecological roles of these organisms and large-scale economic impact of these waste sites. AMD microbial communities have classically been characterized by laboratory culturing-based techniques and more recently by direct sequencing of marker gene sequences, primarily the 16S rRNA gene. In our comparison of the techniques, we find that their results are complementary, overall indicating very similar community structure with similar dominant species, but with each method identifying some species that were missed by the other. We were able to culture the majority of species that our direct sequencing results indicated were present, primarily species within the Acidithiobacillus and Acidiphilium genera, although estimates of relative species abundance were only obtained from direct sequencing. Interestingly, our culture-based methods recovered four species that had been overlooked from our sequencing results because of the rarity of the marker gene sequences, likely members of the rare biosphere. Further, direct sequencing indicated that a single genus, completely missed in our culture-based study, Legionella, was a dominant member of the microbial community. Our results suggest that while either method does a reasonable job of identifying the dominant members of the AMD microbial community, together the methods combine to give a more complete picture of the true diversity of this environment.

A novel ion pairing LC/MS metabolomics protocol for study of a variety of biologically relevant polar metabolites

We report a method of ion-pairing liquid chromatography coupled to mass spectrometry (IP-LC-MS) that we have developed for the sensitive detection and quantification of a variety of biologically relevant polar molecules. We use the ion-pairing agent diamyl ammonium to improve chromatographic resolution of polar compounds, such as nucleotide cofactors, sugar phosphates, and organic acids, that are generally poorly retained by conventional reverse phase chromatographic methods. This method showed good linearity (average R value of 0.996) and reproducibility (generally RSD values <10%). We demonstrate the utility of this method by investigating the metabolomic signature of three distinct biological systems: the metabolic response to lack of superoxide dismutase activity and to paraquat induced oxidative stress, and the metabolic profiles of four different Drosophila species.

Effects of Hg(II) Exposure on MAPK Phosphorylation and Antioxidant System in D. melanogaster

The heavy metal mercury is a known toxin, but while the mechanisms involved in mercury toxicity have been well demonstrated in vertebrates, little is known about toxicological effects of this metal in invertebrates. Here, we present the results of our study investigating the effects associated with exposure of fruit fly Drosophila melanogaster to inorganic mercury (HgCl2). We quantify survival and locomotor performance as well as a variety of biochemical parameters including antioxidant status, MAPK phosphorylation and gene expression following mercury treatment. Our results demonstrate that exposure to Hg(II) through diet induced mortality and affected locomotor performance as evaluated by negative geotaxis, in D. melanogaster. We also saw a significant impact on the antioxidant system including an inhibition of acetylcholinesterase (Ache), glutathione S‐transferase (GST) and superoxide dismutase (SOD) activities. We found no significant alteration in the levels of mRNA of antioxidant enzymes or NRF‐2 transcriptional factor, but did detect a significant up regulation of the HSP83 gene. Mercury exposure also induced the phosphorylation of JNK and ERK, without altering p38MAPK and the concentration of these kinases. In parallel, Hg(II) induced PARP cleavage in a 89 kDa fragment, suggesting the triggering of apoptotic cell death in response to the treatment. Taken together, this data clarifies and extends our understanding of the molecular mechanisms mediating Hg(II) toxicity in an invertebrate model.

Cold acclimation wholly reorganizes the Drosophila melanogaster transcriptome and metabolome

Cold tolerance is a key determinant of insect distribution and abundance, and thermal acclimation can strongly influence organismal stress tolerance phenotypes, particularly in small ectotherms like Drosophila. However, there is limited understanding of the molecular and biochemical mechanisms that confer such impressive plasticity. Here, we use high-throughput mRNA sequencing (RNA-seq) and liquid chromatography – mass spectrometry (LC-MS) to compare the transcriptomes and metabolomes of D. melanogaster acclimated as adults to warm (rearing) (21.5 °C) or cold conditions (6 °C). Cold acclimation improved cold tolerance and led to extensive biological reorganization: almost one third of the transcriptome and nearly half of the metabolome were differentially regulated. There was overlap in the metabolic pathways identified via transcriptomics and metabolomics, with proline and glutathione metabolism being the most strongly-supported metabolic pathways associated with increased cold tolerance. We discuss several new targets in the study of insect cold tolerance (e.g. dopamine signaling and Na+-driven transport), but many previously identified candidate genes and pathways (e.g. heat shock proteins, Ca2+ signaling, and ROS detoxification) were also identified in the present study, and our results are thus consistent with and extend the current understanding of the mechanisms of insect chilling tolerance.

Quantifying Interactions Within the NADP(H) Enzyme Network in Drosophila melanogaster

In this report, we use synthetic, activity-variant alleles in Drosophila melanogaster to quantify interactions across the enzyme network that reduces nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. We examine the effects of large-scale variation in isocitrate dehydrogenase (IDH) or glucose-6-phosphate dehydrogenase (G6PD) activity in a single genetic background and of smaller-scale variation in IDH, G6PD, and malic enzyme across 10 different genetic backgrounds. We find significant interactions among all three enzymes in adults; changes in the activity of any one source of a reduced cofactor generally result in changes in the other two, although the magnitude and directionality of change differs depending on the gene and the genetic background. Observed interactions are presumably through cellular mechanisms that maintain a homeostatic balance of NADPH/NADP, and the magnitude of change in response to modification of one source of reduced cofactor likely reflects the relative contribution of that enzyme to the cofactor pool. Our results suggest that malic enzyme makes the largest single contribution to the NADPH pool, consistent with the results from earlier experiments in larval D. melanogaster using naturally occurring alleles. The interactions between all three enzymes indicate functional interdependence and underscore the importance of examining enzymes as components of a network.

Metabolic regulation of Drosophila apoptosis through inhibitory phosphorylation of Dronc.

Apoptosis ensures tissue homeostasis in response to developmental cues or cellular damage. Recently reported genome‐wide RNAi screens have suggested that several metabolic regulators can modulate caspase activation in Drosophila. Here, we establish a previously unrecognized link between metabolism and Drosophila apoptosis by showing that cellular NADPH levels modulate the initiator caspase Dronc through its phosphorylation at S130. Depletion of NADPH removed this inhibitory phosphorylation, resulting in the activation of Dronc and subsequent cell death. Conversely, upregulation of NADPH prevented Dronc‐mediated apoptosis upon DIAP1 RNAi or cycloheximide treatment. Furthermore, this CaMKII‐mediated phosphorylation of Dronc hindered Dronc activation, but not its catalytic activity. Blockade of NADPH production aggravated the death‐inducing activity of Dronc in specific neurons, but not in the photoreceptor cells of the eyes of transgenic flies; similarly, non‐phosphorylatable Dronc was more potent than wild type in triggering specific neuronal apoptosis. Our observations reveal a novel regulatory circuitry in Drosophila apoptosis, and, as NADPH levels are elevated in cancer cells, also provide a genetic model to understand aberrations in cancer cell apoptosis resulting from metabolic alterations.

Triglyceride Pools, Flight and Activity Variation at the Gpdh Locus in Drosophila melanogaster

We have created a set of P-element excision-derived Gpdh alleles that generate a range of GPDH activity phenotypes ranging from zero to full activity. By placing these synthetic alleles in isogenic backgrounds, we characterize the effects of minor and major activity variation on two different aspects of Gpdh function: the standing triglyceride pool and glycerol-3-phosphate shuttle-assisted flight. We observe small but statistically significant reductions in triglyceride content for adult Gpdh genotypes possessing 33–80% reductions from normal activity. These small differences scale to a notable proportion of the observed genetic variation in triglyceride content in natural populations. Using a tethered fly assay to assess flight metabolism, we observed that genotypes with 100 and 66% activity exhibited no significant difference in wing-beat frequency (WBF), while activity reductions from 60 to 10% showed statistically significant reductions of ∼7% in WBF. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection in the triglyceride pool.