Drosophila O-GlcNAcase Mutants Reveal an Expanded Glycoproteome and Novel Growth and Longevity Phenotypes.

The reversible posttranslational O-GlcNAc modification of serine or threonine residues of intracellular proteins is involved in many cellular events from signaling cascades to epigenetic and transcriptional regulation. O-GlcNAcylation is a conserved nutrient-dependent process involving two enzymes, with O-GlcNAc transferase (OGT) adding O-GlcNAc and with O-GlcNAcase (OGA) removing it in a manner that's protein- and context-dependent. O-GlcNAcylation is essential for epigenetic regulation of gene expression through its action on Polycomb and Trithorax and COMPASS complexes. However, the important role of O-GlcNAc in adult life and health span has been largely unexplored, mainly due the lack of available model systems. Cataloging the O-GlcNAc proteome has proven useful in understanding the biology of this modification in vivo. In this study, we leveraged a recently developed oga knockout fly mutant to identify the O-GlcNAcylated proteins in adult Drosophilamelanogaster. The adult O-GlcNAc proteome revealed many proteins related to cell and organismal growth, development, differentiation, and epigenetics. We identified many O-GlcNAcylated proteins that play a role in increased growth and decreased longevity, including HCF, SIN3A, LOLA, KISMET, ATX2, SHOT, and FOXO. Interestingly, oga mutant flies are larger and have a shorter life span compared to wild type flies, suggesting increased O-GlcNAc results in increased growth. Our results suggest that O-GlcNAc alters the function of many proteins related to transcription, epigenetic modification and signaling pathways that regulate growth rate and longevity. Therefore, our findings highlight the importance of O-GlcNAc in growth and life span in adult Drosophila.

Multiple roles forlaccase2 in butterfly wing pigmentation, scale development, and cuticle tanning.

Lepidopteran wing scales play important roles in a number of functions including color patterning and thermoregulation. Despite the importance of wing scales, however, we still have a limited understanding of the genetic mechanisms that underlie scale patterning, development, and coloration. Here, we explore the function of the phenoloxidase-encoding gene laccase2 in wing and scale development in the nymphalid butterfly Vanessa cardui. Somatic deletion mosaics of laccase2 generated by CRISPR/Cas9 genome editing presented several distinct mutant phenotypes. Consistent with the work in other nonlepidopteran insect groups, we observed reductions in melanin pigmentation and defects in cuticle formation. We were also surprised, however, to see distinct effects on scale development including complete loss of wing scales. This study highlights laccase2 as a gene that plays multiple roles in wing and scale development and provides new insight into the evolution of lepidopteran wing coloration.

From phenoloxidase to fecundity: food availability does not influence the costs of oxidative challenge in a wing-dimorphic cricket.

Stressed animals often struggle to maintain optimal investment into a number of fitness-related traits, which can result in some traits being more adversely affected than others. Variation in stress-related costs may also depend on the environment-costs can be facultative and only occur when resources are limited, or they may be obligate and occur regardless of resource availability. Dynamics of oxidative stress may be important in life-history evolution given their role in a range of biological processes-from reproduction to immunity to locomotion. Thus, we examined how resource (food) availability influences the costs of oxidative challenge to fitness-related traits spanning several levels of biological organization. We manipulated food availability and oxidative status in females of the wing-dimorphic sand field cricket (Gryllus firmus) during early adulthood. We then determined investment into several traits: reproduction (ovary mass), soma (body mass and flight musculature), and immune function (total phenoloxidase activity). Oxidative challenge (paraquat exposure) obligated costs to somatic tissue and a parameter of immune function regardless of food availability, but it did not affect reproduction. We show that the costs of oxidative challenge are trait-specific, but we did not detect a facultative (food-dependent) cost of oxidative challenge to any trait measured. Although the dynamics of oxidative stress are complex, our study is an important step toward a more complete understanding of the roles that resource availability and redox systems play in mediating life histories.

Salvador-Warts-Hippo pathway regulates sensory organ development via caspase-dependent nonapoptotic signaling.

The fundamental roles for the Salvador-Warts-Hippo (SWH) pathway are widely characterized in growth regulation and organ size control. However, the function of SWH pathway is less known in cell fate determination. Here we uncover a novel role of the SWH signaling pathway in determination of cell fate during neural precursor (sensory organ precursor, SOP) development. Inactivation of the SWH pathway in SOP of the wing imaginal discs affects caspase-dependent bristle patterning in an apoptosis-independent process. Such nonapoptotic functions of caspases have been implicated in inflammation, proliferation, cellular remodeling, and cell fate determination. Our data indicate an effect on the Wingless (Wg)/Wnt pathway. Previously, caspases were proposed to cleave and activate a negative regulator of Wg/Wnt signaling, Shaggy (Sgg)/GSK3ß. Surprisingly, we found that a noncleavable form of Sgg encoded from the endogenous locus after CRISPR-Cas9 modification supported almost normal bristle patterning, indicating that Sgg might not be the main target of the caspase-dependent nonapoptotic process. Collectively, our results outline a new function of SWH signaling that crosstalks to caspase-dependent nonapoptotic signaling and Wg/Wnt signaling in neural precursor development, which might be implicated in neuronal pathogenesis.

Reciprocal action of Casein Kinase Iε on core planar polarity proteins regulates clustering and asymmetric localisation.

The conserved core planar polarity pathway is essential for coordinating polarised cell behaviours and the formation of polarised structures such as cilia and hairs. Core planar polarity proteins localise asymmetrically to opposite cell ends and form intercellular complexes that link the polarity of neighbouring cells. This asymmetric segregation is regulated by phosphorylation through poorly understood mechanisms. We show that loss of phosphorylation of the core protein Strabismus in the Drosophila pupal wing increases its stability and promotes its clustering at intercellular junctions, and that Prickle negatively regulates Strabismus phosphorylation. Additionally, loss of phosphorylation of Dishevelled - which normally localises to opposite cell edges to Strabismus - reduces its stability at junctions. Moreover, both phosphorylation events are independently mediated by Casein Kinase Iε. We conclude that Casein Kinase Iε phosphorylation acts as a switch, promoting Strabismus mobility and Dishevelled immobility, thus enhancing sorting of these proteins to opposite cell edges.

The putative chitin deacetylases Serpentine and Vermiform have non-redundant functions during Drosophila wing development.

The chitin modifying deacetylases (CDA) CDA1 and CDA2 have been reported to play partially redundant roles during insect cuticle formation and molting and tracheal morphogenesis in various insect species. In order to distinguish possible functional differences between these two enzymes, we analyzed their function during wing development in the fruit fly Drosophila melanogaster. In tissue-specific RNA interference experiments, we demonstrate that DmCDA1 (Serpentine, Serp) and DmCDA2 (Vermiform, Verm) have distinct functions during Drosophila adult wing cuticle differentiation. Chitosan staining revealed that Serp is the major enzyme responsible for chitin deacetylation during wing cuticle formation, while Verm does not seem to be needed for this process. Indeed, it is questionable whether Verm is a chitin deacetylase at all. Atomic force microscopy suggested that Serp and Verm have distinct roles in establishing the shape of nanoscale bumps at the wing surface. Moreover, our data indicate that Verm but not Serp is required for the laminar arrangement of chitin. Both enzymes participate in the establishment of the cuticular inward barrier against penetration of xenobiotics. Taken together, correct differentiation of the wing cuticle involves both Serp and Verm in parallel in largely non-overlapping functions.

BmCHSA-2b, a Lepidoptera specific alternative splicing variant of epidermal chitin synthase, is required for pupal wing development in Bombyx mori.

Insect chitin synthase A (CHSA) is an epidermis-specific enzyme that plays an essential role in insect development. In this study, the function and regulation of CHSA-2b, an alternative splicing variant of Bombxy mori CHSA that is discovered only in Lepidopteran insects, were investigated. Analysis of mRNA level showed that BmCHSA-2b was responsive to 20-hydroxyecdysone (20E) in pupal wing unlike BmCHSA-2a, which shares almost the identical sequence as BmCHSA-2b except the first 31 amino acids, suggesting that the expression of these two alternative splicing variants is driven by different promoters of CHSA gene. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed that BmCHSA-2b was up-regulated in the wing of mid-pupa unlike BmCHSA-2a, which was up-regulated in epidermis and wing disc at the beginning and end of pupal stage. Further analysis reveals that the up-regulations of BmCHSA-2a and BmCHSA-2b in pupal wing were consistent with the increase of chitin content and wing area at the same stages, respectively. Furthermore, the higher transcription level of BmCHSA-2b in the mid-pupal wing of male than that in female was consistent with the chitin content of pupal wing between genders. Injection of double-stranded RNAs of BmCHSA-2b resulted in the decrease in the area and chitin content of the wing, and irregular and crimpled vein. All these results together suggest that B. mori evolves an extra promoter in CHSA gene to activate BmCHSA-2b expression in the wing of mid-pupal stage in response to 20E, and BmCHSA-2b is required for the wing development in the mid-pupa of B. mori.

Identification and functional analysis of chitinase 7 gene in white-backed planthopper, Sogatella furcifera.

Chitinase is used to degrade chitin in insect cuticles and the peritrophic matrix. In this study, the full-length cDNA sequence of a Cht gene (SfCht7) was identified and characterized from the white-black planthopper, Sogatella furcifera. The SfCht7 cDNA was 3148bp, contained an open reading frame of 2877bp and encoded 958 amino acids with a predicted molecular weight of 107.9kDa. Homology analysis indicated that SfCht7 has typical chitinase features include a chitin-binding domain, two catalytic domains and a signal peptide region. Phylogenetic analysis suggested that SfCht7 belonged to the group III chitinases. Quantitative real-time PCR analyses showed that SfCht7 was highly expressed before molting. After injecting SfCht7 double-stranded RNA in the nymph stage, insects exhibited phenotypes of difficulty in molting and wing development. A lethal phenotype was that nymph bodies exuviated from the head but the old cuticle did not detach completely from the body. Another lethal phenotype was that elongated distal wing pads of fifth-instar nymphs with junctions between the thorax and abdomen in the treatment group that were thinner than in the control group, giving a "wasp-waisted" appearance. In another phenotype that was not lethal, nymphs exuviated and old cuticles detached completely from the body, but the wings of adults did not stretch normally.

APC/CFzr/Cdh1-Dependent Regulation of Planar Cell Polarity Establishment via Nek2 Kinase Acting on Dishevelled.

The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase, well known for its role in cell-cycle progression. However, it has been linked to additional functions, mainly in neuronal contexts, when using the co-activator Cdh1/Fzr. Here, our data indicate a post-mitotic requirement for the APC/CFzr/Cdh1 in epithelial cell patterning and planar cell polarity (PCP) in Drosophila. PCP signaling is critical for development by establishing cellular asymmetries and orientation within the plane of an epithelium, via differential localization of distinct complexes of core PCP factors. Loss of APC/C function leads to reduced levels of Dishevelled (Dsh), a core PCP factor. The effect of APC/C on Dsh is mediated by Nek2 kinase, which can phosphorylate Dsh and is a direct APC/CFzr/Cdh1 substrate. We have thus uncovered a pathway of regulation whereby APC/CFzr/Cdh1 negatively regulates Nek2, which negatively regulates Dsh, to ensure its proper stoichiometric requirement and localization during PCP establishment.

Curly Encodes Dual Oxidase, Which Acts with Heme Peroxidase Curly Su to Shape the Adult Drosophila Wing.

Curly, described almost a century ago, is one of the most frequently used markers in Drosophila genetics. Despite this the molecular identity of Curly has remained obscure. Here we show that Curly mutations arise in the gene dual oxidase (duox), which encodes a reactive oxygen species (ROS) generating NADPH oxidase. Using Curly mutations and RNA interference (RNAi), we demonstrate that Duox autonomously stabilizes the wing on the last day of pupal development. Through genetic suppression studies, we identify a novel heme peroxidase, Curly Su (Cysu) that acts with Duox to form the wing. Ultrastructural analysis suggests that Duox and Cysu are required in the wing to bond and adhere the dorsal and ventral cuticle surfaces during its maturation. In Drosophila, Duox is best known for its role in the killing of pathogens by generating bactericidal ROS. Our work adds to a growing number of studies suggesting that Duox's primary function is more structural, helping to form extracellular and cuticle structures in conjunction with peroxidases.

Localization of Hippo signalling complexes and Warts activation in vivo.

Hippo signalling controls organ growth and cell fate by regulating the activity of the kinase Warts. Multiple Hippo pathway components localize to apical junctions in epithelial cells, but the spatial and functional relationships among components have not been clarified, nor is it known where Warts activation occurs. We report here that Hippo pathway components in Drosophila wing imaginal discs are organized into distinct junctional complexes, including separate distributions for Salvador, Expanded, Warts and Hippo. These complexes are reorganized on Hippo pathway activation, when Warts shifts from associating with its inhibitor Jub to its activator Expanded, and Hippo concentrates at Salvador sites. We identify mechanisms promoting Warts relocalization, and using a phospho-specific antisera and genetic manipulations, identify where Warts activation occurs: at apical junctions where Expanded, Salvador, Hippo and Warts overlap. Our observations define spatial relationships among Hippo signalling components and establish the functional importance of their localization to Warts activation.

Scaling the Drosophila Wing: TOR-Dependent Target Gene Access by the Hippo Pathway Transducer Yorkie.

Organ growth is controlled by patterning signals that operate locally (e.g., Wingless/Ints [Wnts], Bone Morphogenetic Proteins [BMPs], and Hedgehogs [Hhs]) and scaled by nutrient-dependent signals that act systemically (e.g., Insulin-like peptides [ILPs] transduced by the Target of Rapamycin [TOR] pathway). How cells integrate these distinct inputs to generate organs of the appropriate size and shape is largely unknown. The transcriptional coactivator Yorkie (Yki, a YES-Associated Protein, or YAP) acts downstream of patterning morphogens and other tissue-intrinsic signals to promote organ growth. Yki activity is regulated primarily by the Warts/Hippo (Wts/Hpo) tumour suppressor pathway, which impedes nuclear access of Yki by a cytoplasmic tethering mechanism. Here, we show that the TOR pathway regulates Yki by a separate and novel mechanism in the Drosophila wing. Instead of controlling Yki nuclear access, TOR signaling governs Yki action after it reaches the nucleus by allowing it to gain access to its target genes. When TOR activity is inhibited, Yki accumulates in the nucleus but is sequestered from its normal growth-promoting target genes--a phenomenon we term "nuclear seclusion." Hence, we posit that in addition to its well-known role in stimulating cellular metabolism in response to nutrients, TOR also promotes wing growth by liberating Yki from nuclear seclusion, a parallel pathway that we propose contributes to the scaling of wing size with nutrient availability.

Deubiquitinase USP47/UBP64E Regulates ß-Catenin Ubiquitination and Degradation and Plays a Positive Role in Wnt Signaling.

Wnt signaling plays important roles in development and tumorigenesis. A central question about the Wnt pathway is the regulation of ß-catenin. Phosphorylation of ß-catenin by CK1α and GSK3 promotes ß-catenin binding to ß-TrCP, leading to ß-catenin degradation through the proteasome. The phosphorylation and ubiquitination of ß-catenin have been well characterized; however, it is unknown whether and how a deubiquitinase is involved. In this study, by screening RNA interference (RNAi) libraries, we identified USP47 as a deubiquitinase that prevents ß-catenin ubiquitination. Inactivation of USP47 by RNAi increased ß-catenin ubiquitination, attenuated Wnt signaling, and repressed cancer cell growth. Furthermore, USP47 deubiquitinates itself, whereas ß-TrCP promotes USP47 ubiquitination through interaction with an atypical motif in USP47. Finally, in vivo studies in the Drosophila wing suggest that UBP64E, the USP47 counterpart in Drosophila, is required for Armadillo stabilization and plays a positive role in regulating Wnt target gene expression.

An Opposite Pattern to the Conventional Thermal Hypothesis: Temperature-Dependent Variation in Coloration of Adults of Saccharosydne procerus (Homoptera: Delphacidae).

Melanism is a common polymorphism in many insect species that also influences immune function. According to the thermal melanin hypothesis, ectothermic individuals from cooler environments have darker cuticles and higher polyphenol oxidase (PO) levels, which represent a better immunocompetence. In this study, the links among environmental temperature, melanism, and PO activity of Saccharosydne procerus (Matsumura) were examined. Most S. procerus have a black spot on their forewings at high temperatures in the field and in the laboratory. In PO activity assay, a positive association between PO level and temperature was found. Our results showed that a diversification of melanism occurred under different temperatures and that melanism in S. procerus presented an opposite pattern to the one proposed by the thermal hypothesis.

Loss of Drosophila pseudouridine synthase triggers apoptosis-induced proliferation and promotes cell-nonautonomous EMT.

Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called 'undead cells', amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted. Collectively, these data point out that cell-cell communication and long-range signaling can take a relevant role in the response to pseudouridine synthase decline. Considering that all the affected pathways are highly conserved throughout evolution, it is plausible that the response to pseudouridine synthase depletion has been widely preserved. On this account, our results can add new light on the still unexplained tumor predisposition that characterizes X-linked dyskeratosis, the human disease caused by reduced pseudouridine synthase activity.

Two insulin receptors determine alternative wing morphs in planthoppers.

Wing polyphenism is an evolutionarily successful feature found in a wide range of insects. Long-winged morphs can fly, which allows them to escape adverse habitats and track changing resources, whereas short-winged morphs are flightless, but usually possess higher fecundity than the winged morphs. Studies on aphids, crickets and planthoppers have revealed that alternative wing morphs develop in response to various environmental cues, and that the response to these cues may be mediated by developmental hormones, although research in this area has yielded equivocal and conflicting results about exactly which hormones are involved. As it stands, the molecular mechanism underlying wing morph determination in insects has remained elusive. Here we show that two insulin receptors in the migratory brown planthopper Nilaparvata lugens, InR1 and InR2, have opposing roles in controlling long wing versus short wing development by regulating the activity of the forkhead transcription factor Foxo. InR1, acting via the phosphatidylinositol-3-OH kinase (PI(3)K)-protein kinase B (Akt) signalling cascade, leads to the long-winged morph if active and the short-winged morph if inactive. InR2, by contrast, functions as a negative regulator of the InR1-PI(3)K-Akt pathway: suppression of InR2 results in development of the long-winged morph. The brain-secreted ligand Ilp3 triggers development of long-winged morphs. Our findings provide the first evidence of a molecular basis for the regulation of wing polyphenism in insects, and they are also the first demonstration--to our knowledge--of binary control over alternative developmental outcomes, and thus deepen our understanding of the development and evolution of phenotypic plasticity.

Nucleoside diphosphate kinases (NDPKs) in animal development.

In textbooks of biochemistry, nucleoside diphosphate conversion to a triphosphate by nucleoside diphosphate 'kinases' (NDPKs, also named NME or NM23 proteins) merits a few lines of text. Yet this essential metabolic function, mediated by a multimeric phosphotransferase protein, has effects that lie beyond a simple housekeeping role. NDPKs attracted more attention when NM23-H1 was identified as the first metastasis suppressor gene. In this review, we examine these NDPK enzymes from a developmental perspective because of the tractable phenotypes found in simple animal models that point to common themes. The data suggest that NDPK enzymes control the availability of surface receptors to regulate cell-sensing cues during cell migration. NDPKs regulate different forms of membrane enclosure that engulf dying cells during development. We suggest that NDPK enzymes have been essential for the regulated uptake of objects such as bacteria or micronutrients, and this evolutionarily conserved endocytic function contributes to their activity towards the regulation of metastasis.

The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of "upstream" Planar Cell Polarity proteins.

The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.

Negative regulation of MAP kinase signaling in Drosophila by Ptp61F/PTP1B.

PTP1B is an important negative regulator of insulin and other signaling pathways in mammals. However, the role of PTP1B in the regulation of RAS-MAPK signaling remains open to deliberation, due to conflicting evidence from different experimental systems. The Drosophila orthologue of mammalian PTP1B, PTP61F, has until recently remained largely uncharacterized. To establish the potential role of PTP61F in the regulation of signaling pathways in Drosophila and particularly to help resolve its fundamental function in RAS-MAPK signaling, we generated a new allele of Ptp61F as well as employed both RNA interference and overexpression alleles. Our results validate recent data showing that the activity of insulin and Abl kinase signaling is increased in Ptp61F mutants and RNA interference lines. Importantly, we establish negative regulation of the RAS/MAPK pathway by Ptp61F activity in whole animals. Of particular interest, our results document the modulation of hyperactive MAP kinase activity by Ptp61F alleles, showing that the phosphatase intervenes to directly or indirectly regulate MAP kinase itself.

Purification and characterization of 6-phosphogluconate dehydrogenase from the wing-polymorphic cricket, Gryllus firmus, and assessment of causes of morph-differences in enzyme activity.

Considerable information exists on the physiological correlates of life history adaptation, while molecular data on this topic are rapidly accumulating. However, much less is known about the enzymological basis of life history adaptation in outbred populations. In the present study, we compared developmental profiles of fat body specific activity, kinetic constants of homogeneously purified and unpurified enzyme, and fat body enzyme concentration of the pentose-shunt enzyme, 6-phosphogluconate dehydrogenase (6PGDH, E.C.1.1.1.44) between the dispersing [long-winged, LW(f)] and flightless [short-winged, SW] genotypes of the cricket Gryllus firmus. Neither kcat nor the Michaelis constant for 6-phosphogluconate differed between 6PGDH from LW(f) versus SW morphs for either homogeneously purified or unpurified enzyme. Purified enzyme from the LW(f) morph exhibited reduced KM for NADP(+), but this was not observed for multiple KM(NADP+) estimates for unpurified enzyme. A polyclonal antibody was generated against 6PGDH which was used to develop a chemiluminescence assay to quantify 6PGDH concentration in fat body homogenates. Elevated enzyme concentration accounted for all of the elevated 6PGDH specific activity in the LW(f) morph during the juvenile and adult stages. Finally, activity of another pentose-shunt enzyme, glucose-6-phosphate dehydrogenase, strongly covaried with 6PGDH activity suggesting that variation in 6PGDH activity gives rise to variation in pentose shunt flux. This is one of the first life-history studies and one of the few studies of intraspecific enzyme adaptation to identify the relative importance of evolutionary change in enzyme concentration vs. kinetic constants to adaptive variation in enzyme activity in an outbred population.