effects of oxygen on growth and size: synthesis of molecular, organismal, and evolutionary studies...

PH73CH05-Harrison ARI 7 January 2011 11:55

Effects of Oxygen on Growthand Size: Synthesis ofMolecular, Organismal, andEvolutionary Studies withDrosophila melanogasterJon F. Harrison1 and Gabriel G. Haddad2,3

1School of Life Sciences, Arizona State University, Tempe, Arizona 85287-4501;email: [email protected] of Pediatrics and Neuroscience, University of California, San Diego, La Jolla,California 92093-0735; email: [email protected] Children’s Hospital, San Diego, California 92123

Annu. Rev. Physiol. 2011. 73:95–113

First published online as a Review in Advance onOctober 5, 2010

The Annual Review of Physiology is online atphysiol.annualreviews.org

This article’s doi:10.1146/annurev-physiol-012110-142155

Copyright c© 2011 by Annual Reviews.All rights reserved

0066-4278/11/0315-0095$20.00

Keywords

hypoxia, hyperoxia, metabolism, developmental plasticity

Abstract

Drosophila melanogaster is a model genetic organism with an exceptionalhypoxia tolerance relative to mammals. Forward genetic, microarray,and P-element manipulations and selection experiments have revealedmultiple mechanisms of severe hypoxia tolerance, including RNA edit-ing, downregulation of metabolism, and prevention of protein unfold-ing. Drosophila live in microbe-rich, semiliquid food in which hypoxialikely indicates deteriorating environments. Hypoxia reduces growthand size by multiple mechanisms, influencing larval feeding rates, pro-tein synthesis, imaginal cell size, and control of molting. In moderatehypoxia, these effects appear to occur without ATP limitation and are in-stead mediated by signaling systems, including hypoxia-inducible factorand atypical guanyl cyclase sensing of oxygen, with downstream actionson behavior, anabolism, and the cell cycle. In hypoxia, flies developsmaller sizes, but size does not evolve, whereas in hyperoxia, flies evolvelarger sizes without exhibiting developmental size plasticity, suggestingdifferential evolutionary responses to natural versus novel directions ofoxygen change.

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aPO2: atmosphericpartial pressure ofoxygen

INTRODUCTION

Animals vary tremendously in size across andwithin species, which strongly affects their in-dividual physiology and ultimately influencesecological processes and patterns (1, 2). Surpris-ingly, we still understand little about the mech-anisms that cause an animal to grow to a certainsize and even less about the genetic basis thatproduces evolutionary divergence in size. Thisreview explores the evidence for the hypothe-sis that signaling pathways related to internaloxygen concentrations are key elements regu-lating growth and size in animals. We focus onthe vinegar or fruit fly, Drosophila melanogaster, amodel organism particularly well suited to theseinvestigations because a synthesis of molecu-lar, organismal, and evolutionary approaches isfeasible.

Here we address oxygen effects on growthand size via direct effects, developmental plas-ticity, and evolutionary responses. Environ-mental effects on growth and size may operatedirectly, as, for example, when very low temper-ature or atmospheric partial pressure of oxygen(aPO2) directly limits ATP production, therebyreducing rates of feeding, digestion, absorption,and protein synthesis. Additionally, environ-mental effects on growth and size may also re-sult from changes in gene expression that lead todevelopmental plasticity (3). Finally, in popula-tions, environmental changes may affect growthand size via evolutionary changes. These stud-ies described in this review have all attemptedto control for the other major environmentalfactors likely to affect growth and size (temper-ature, food availability, and quality), and thuswe describe primarily the effects of oxygen onmaximal potential growth and size.

The role of oxygen in the control of growthand size of D. melanogaster has been investi-gated in different contexts. First, there has beenconsiderable interest in D. melanogaster as amodel system for investigating the mechanismsby which organisms are able to recover from ex-treme hypoxia or anoxia (4), that is, as a biomed-ical model for the study of possible noveltreatments for ischemia. Because many gene

pathways are conserved in flies and humans,gene therapies based on processes discoveredin flies may have wide utility, as studies of frag-ile X syndrome have clearly shown (5). Hy-poxia suppression of somatic growth is com-monly observed in mammals, including rodents(6), humans at altitude (7), humans with con-genital heart disease with right-to-left shunt(8), and infants with chronic lung disease (9).Also, whereas hypoxia generally suppresses so-matic growth in animals, hypoxia stimulates thegrowth of the respiratory system, and therehas been considerable interest in the evolu-tionarily conserved mechanisms that controlgrowth of respiratory structures (capillaries invertebrates, tracheae in insects) in response tooxygen. Similarly, the evolutionary conserva-tion of the major signaling pathways control-ling cell cycle and organismal growth has in-spired examination of Drosophila to elucidatethe mechanisms linking hypoxia and growthsuppression (10). Second, the response ofD. melanogaster to developmental or multigen-erational exposure to moderate depression orelevation of aPO2 has been examined experi-mentally as a model system relevant to the ques-tion of whether aPO2 affects animal body sizeacross environmental gradients and through theEarth’s evolutionary history. Correlative datasuggest that environmental aPO2 affects bodysize in aquatic systems (11, 12), and effects ofoxygen on growth or size may mediate the well-documented inverse correlation between tem-perature and body size in aquatic invertebrates(13, 14). Historical changes in atmosphericoxygen level are thought to have had majororganizational change on metabolism and evo-lution (15). aPO2 levels have changed dramati-cally through the Phanerozoic, and changes inaPO2 may be responsible for major evolution-ary changes in animal body size (16).

AN OVERVIEW OF THEREGULATION OF GROWTHAND SIZE

Organismal growth requires the coordinatedactions of multiple serial processes, including

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ILP: insulin-likepeptide

AKT: a serine/threonine proteinkinase; also known asprotein kinase B

FOXO: forkheadtranscription factor

TOR: target ofrapamycin

HIF: hypoxia-inducible factor

nutrient ingestion, digestion, absorption intoblood, absorption into cells, and anabolic as-similation of nutrients that produce cell growthand cycling. At the organismal level, coordi-nation of growth processes is partly accom-plished by behavior- and growth-regulatingsignals secreted by the neuroendocrine systemsuch as insulin. In recent years, research hasdemonstrated that insulin-like peptides (ILPs)[of which there are seven currently identified inDrosophila (17)] are circulating regulators thatare important for mediating growth and devel-opment rate in insects (18). At target tissues,ILPs bind to receptor tyrosine kinases that arestructurally very similar in Drosophila and hu-mans (19). Binding of ILPs to the receptor ac-tivates a catalytic subunit that phosphorylatesthe insulin receptor substrate protein (chico inDrosophila), which in turn activates a series of ki-nases that stimulate growth and development.

Control of growth by the insulin receptorpathway appears to operate via multiple mech-anisms (17, 20). Activation of the insulin recep-tor leads to activation of AKT/protein kinase B(a serine/threonine kinase), which alters cellsize, at least partly due to effects on protein syn-thesis via regulation of S6K (p70 ribosomal S6kinase) (20). A second pathway focuses on cellcycle control. Low levels of ILP/AKT signal-ing result in translocation of the transcriptionfactor FOXO (forkhead transcription factor) tothe nucleus, which reduces cell proliferationand number (21). Finally, activation of AKTleads to phosphorylation and inactivation of thetuberous sclerosis complex, turning on its targetof rapamycin (TOR). Activated TOR increasescell growth (size) by activating protein synthe-sis initiation via phosphorylation of elongationfactors, by activating translation (22), and bypromoting amino acid uptake to the cell (23).

In invertebrates such as Drosophila, devel-opment is discontinuous, with growth inter-spersed with molting periods during which theold cuticle is shed and a new larger exoskele-ton is formed. Adult size thus depends on theproduct of the growth rate within each devel-opmental stage and the duration of that stage,summed over all stages. The complex cascade

of neuroendocrine events that control molt-ing is triggered when insects grow to a criti-cal weight (defined experimentally as the massbelow which an insect will not survive pupa-tion), apparently sensed by growth of the pro-thoracic gland to a threshold size (24). Duringthe third larval instar, after attainment of thecritical weight, juvenile hormone levels beginto decline due to decreased secretion and in-creased hemolymph levels of juvenile hormoneesterase. The fall in juvenile hormone triggersthe secretion of ecdysone from the prothoracicgland. Ecdysone causes the transition from thefeeding stage to the wandering stage and thenpupation and metamorphosis.

OXYGEN SIGNALING PATHWAYSRELATED TO GROWTHAND SIZE

ATP

Oxygen directly affects cytochrome oxidase andmitochondrial ATP production throughout themetabolic process (25). In addition to direct ef-fects of severe hypoxia on ATP levels, AMP ac-cumulation activates AMP kinases, which havemultiple cellular effects related to the con-trol of energy metabolism and growth (26). Inisolated mitochondria, oxygen affinity is high(Km < 1 kPa), suggesting that hypoxia directlylimits mitochondrial oxygen consumption onlyat extremely low tissue PO2 (27). These stud-ies, combined with those that demonstrate thatwhole-body metabolic rates of flies decline sig-nificantly only at less than approximately 3 kPa(see below), suggest that these direct ATP-mediated effects are most important in this verylow aPO2 range.

Hypoxia-Inducible Factor

The components of the hypoxia-inducible fac-tor (HIF) pathway operate in D. melanogaster ina manner similar to that of HIF1 in vertebrates,although with less redundancy (28, 29). As invertebrates, HIF is a heterodimer in Drosophila.The constitutive portion of HIF (HIF1β in

www.annualreviews.org • Oxygen-Regulated Growth and Size 97

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FGFR: fibroblastgrowth factor receptor

FGF: fibroblastgrowth factor

vertebrates) is a product of the gene tango inDrosophila, and the oxygen-sensitive subunit(HIF1α in vertebrates) is termed sima (28).Both tango and sima are transcribed throughoutDrosophila development. However, sima andHIF protein accumulate only in hypoxic con-ditions, with very low levels observed in nor-moxic or anoxic conditions in most tissues (30).Oxygen regulation of HIF occurs due to theuse of oxygen as a substrate by prolyl hydroxy-lase (product of fatiga), which hydroxylates simaprotein, leading to the degradation of sima pro-tein via the ubiquitin ligase pathway. Whenoxygen levels are low, HIF accumulates andtranslocates into the nucleus, where it binds tohypoxia response elements, up- or downregu-lating many genes (28).

A key aspect of oxygen regulation of growthin animals is that growth is stimulated by hy-poxia in some tissues (e.g., respiratory tissues)while inhibited in others (most somatic tissues).In insects, HIF stimulation of the growth ofterminal tracheal cells is critical for the tra-cheal system to match oxygen delivery to needduring development and to compensate for hy-poxia (31). Terminal tracheal cells express HIFstrongly at 5 kPa aPO2, whereas somatic tissuesrequire a lower aPO2 to strongly activate HIFtissues (30). HIF induction in terminal trachealcells leads to expression of fibroblast growthfactor receptor (FGFR; encoded by a geneknown as breathless in Drosophila), perhaps dueto stabilization of the growth factor receptors,as has been shown in tumor cells (32). Secretionof fibroblast growth factor (FGF; encoded bybranchless in Drosophila) drives tracheal branch-ing and extension into underserved tissues (33).FGFR overexpression in tracheal cells enhancestracheal system growth in response to FGF.

HIF is involved in the reduction of cellulargrowth of somatic tissues in D. melanogasterby at least two mechanisms. First, HIF blocksILP-TOR-S6K stimulation of protein syn-thesis, reducing growth and cell size at thewhole-body level, specifically in eye, fat, andgut tissue (35). Second, study of mutants lack-ing or overexpressing fatiga has demonstratedthat stimulation of cellular growth by cyclin-

dependent protein kinase operates via HIFsignaling: Increased HIF signaling reduces cellsize (36–38). Stabilized HIF can also suppresstissue growth and cell size by causing expressionof the genes scylla and charybdis, which inhibitgrowth by downregulating S6K activation ofprotein synthesis (35). HIF may also indirectlyreduce protein synthesis by inhibiting mito-chondrial ATP production through pyruvatedehydrogenase kinase activation, although thishas not yet been shown in insects (34).

Cyclic GMP

Insects can respond extremely rapidly tochanges in oxygen, demonstrating changes inbehavior, spiracular opening, and convectiveventilation within seconds (39, 40). The centralnervous system modulates spiracular openingand convective ventilation via efferent outputin response to oxygen (41), although thishas not yet been studied in D. melanogaster.Recent evidence suggests that atypical guanylcyclases may mediate at least some of theserapid neuronal responses to oxygen (42). Theseatypical guanyl cyclases are heme-containingheterodimeric enzymes that are activated byhypoxia, but not by nitric oxide as are theconventional guanyl cyclases (43). Oxygensensitivity may relate to direct effects of oxygenbinding to the heme subgroup (44). Theseatypical guanyl cyclases are located in the cen-tral and peripheral neurons, including externalsensilla, supporting roles for these enzymes inboth internal and external oxygen sensing (45).Conventional, nitric oxide–sensitive guanyl cy-clases may also play a role in hypoxic responsesbecause the manipulation of nitric oxide levelsby genetic and pharmacological methodscauses changes in behavioral escape responsesthat appear to mimic responses to aPO2 (39).

How might these fast-acting neuronal path-ways mediate effects of oxygen on growth?First, neurons expressing these atypical guanylcyclase pathways may play key roles in facili-tating molt cycles. Blocking synaptic transmis-sion from neurons expressing atypical guanylcyclases prevented adult eclosion and initiation


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of the first larval ecdysis, suggesting that sens-ing of hypoxia plays a key role in the survivalof molt cycles (46). Synaptic transmission fromthese neurons appears critical for the head ex-pansion associated with adult eclosion and forthe tracheal gas filling that must occur at eachmolt (46). These data also suggest that tissuesbecome to some degree hypoxic during the timeperiod when insects switch from the old tra-cheal system to the new tracheal system duringeach molt. Such oxygen sensors may be involvedin pathways that determine the duration of de-velopmental stages, perhaps contributing to thesensing of critical weight. In support of this hy-pothesis, the safety margin for oxygen deliverybecomes minimal at the end of the instar, anda higher aPO2 level extends the duration of ju-venile instars, at least in some insects (47, 48).

Neuronally mediated behavioral responsesto oxygen may be important for juvenilegrowth as well. The high microbial contentand semiliquid nature of the natural (rot-ting fruit) or lab (agar-based media) food ofDrosophila larvae suggest that these larvaeencounter hypoxia during foraging, whichoften involves tunneling. Inhibition of nitricoxide synthesis (39) or inhibition of transmis-sion from atypical guanyl cyclase–expressingneurons (46) inhibits the capacity of larvaeto escape from hypoxic areas. Larvae withinhibited atypical guanyl cyclase neurons oftendrown in the media in normoxic atmospheres,further suggesting the critical role of oxygensensing in the normal foraging of Drosophilalarvae (46). Manipulation of protein kinase Gand nitric oxide synthesis pathways also affectsthe response of the cell cycle to anoxia (39),suggesting that these pathways also help con-trol anabolism and cellular growth, perhaps viaeffects on circulating hormones such as insulin.

EFFECTS OF HYPOXIA ONMETABOLIC RATE

As do most insects, D. melanogaster exhibitsrelatively large safety margins for oxygendelivery when these are assessed by examiningthe response of metabolic rate or behavior to

short-term alterations in aPO2. For restingadult Drosophila, metabolic rates are unaffectedby lowering aPO2 from 21 to 3 kPa andthen fall linearly below 3 kPa, with tenfoldreductions in metabolic rate at 0.1 kPa (49).When critical aPO2 values (the PO2 values thatlimit metabolic rate or function) are assessedwith continuously dropping aPO2 rather thanstep changes, such critical values for carbondioxide emission and locomotory behavior areeven lower—approximately 1 kPa for bothadults and third-instar larvae—indicating anexceptional capacity to function at reducedaPO2 (50). The mechanisms for this remark-able capacity over such a wide range of aPO2

are unknown. On the basis of studies withother insects, responses to hypoxia in adultslikely include spiracle opening, fluid removalfrom the tracheoles, and ventilation driven bymechanisms such as proboscis pumping (40,51). Larvae lack valves in their spiracles andobvious mechanisms to promote ventilation,suggesting that they experience substantialvariation in internal PO2 as aPO2 drops.

SEVERE HYPOXIA ANDANOXIA TOLERANCE

Anoxia is an extreme example of oxygen’s limit-ing effects on function. Nonetheless, most an-imals, including D. melanogaster, run the occa-sional risk of experiencing anoxia (e.g., duringdrowning for flies), and as is discussed below,flies have an extraordinary capacity to tolerateanoxia, at least compared with mammals. Aninteresting question is to what extent responsesto anoxia represent simply the extreme end ofa generally linear set of responses to hypoxiaor an alternative set of responses activated onlyunder extreme stress.

Unlike mammals, most insects can surviveand recover from hours to days of completeanoxia. Most D. melanogaster survive 4 h ofcomplete anoxia without any evidence of cel-lular damage (52). The behavioral response ofD. melanogaster to anoxia depends on its de-velopmental stage. Larval flies continue to ex-hibit escape locomotion for at least 20 min in


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RNA editing

Compatiblesolutes/HSPs

Functional anoxia Change in ion/osmotic balance

Proteinunfolding

Active iontransport

Low ATPproduction

ATPases, kinases

Oxygen

Autonomouscellular oxygen

sensing (e.g., HIF)

Neuronal oxygensensing (e.g.,

cGMP neurons)

Behavior (spiracleopening, escape,

decreased feeding)

Somatic tissuegrowth

Tracheal cellgrowth

Figure 1Pathways of potential physiological problems (black lines) from functional anoxia and demonstrated responses(dashed red lines) of Drosophila. Functional anoxia reduces mitochondrial ATP production, which directlylimits cellular ATPases and kinases. This results in the inhibition of organismal and subcellular behavior, ofanabolic reactions such as protein synthesis, and of the ATPases of ion pumps that counterbalance diffusion-driven fluxes of ions and water across the cell membrane. Unbalanced ion and water fluxes across membraneslead to cellular conditions that promote protein unfolding, which can result in irreversible cellular injury andapoptosis. At various levels of hypoxia, oxygen signaling pathways are coupled to transcriptional andneuronal responses that ameliorate or prevent cellular damage. Some responses are directed at theprevention of alteration of ionic balance (RNA editing of ion channels) and the prevention of proteinunfolding [heat shock protein (HSP) upregulation, trehalose accumulation]. Other responses act to increaseoxygen delivery (enhancement of tracheal growth, stimulation of escape behavior) or to reduce oxygen need(reduction of feeding behavior and somatic cell growth). Arrows indicate positive, causal relationships, andTs indicate suppressing effects. HIF denotes hypoxia-inducible factor.

complete anoxia (50). In contrast, adults stopmoving and collapse into immobility within1 min (52, 53) because anoxia eliminates theelectrical responses of fly muscle (52). The re-covery time of locomotory control and electro-physiological function increases as the durationof anoxia increases (52), and the extension ofanoxia to 12 h kills all flies (49, 52).

Exposure to very low aPO2 (below 2 kPa)results in a strong depression in ATP produc-tion, which inhibits all cellular ATPases, withconsequent effects on most aspects of cellularfunction (Figure 1). Unlike most of the anoxia-tolerant vertebrates that have been studied,evidence suggests that most adult insects do notrely significantly on anaerobic ATP productionto survive anoxia (54, 55). In the insects studiedto date, heat flux drops to less than 5% of

normoxic rates during anoxia, with ATP levelsfalling dramatically (55). Although anaerobicenergy production during anoxia has not yetbeen quantified for adult D. melanogaster, thefact that there is an only moderate elevationin the respiratory exchange ratio during deephypoxia and a minimal oxygen debt after anoxicexposure (49, 56) suggests that anoxia is notaccompanied by substantial accumulation ofanaerobic end products. Thus, the survival ofadults during anoxia likely depends substan-tially on the suppression of ATP synthesiswith the concurrent minimization of damagingeffects of low-energy states such as ionicand osmotic imbalance. However, becauseD. melanogaster larvae exhibit escape locomo-tion for many minutes under complete anoxia(50), and they express lactate dehydrogenase


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Hsp: heat shockprotein

genes (57), anaerobic ATP production via lac-tate synthesis is likely a significant componentof at least the initial responses of larvae tosevere anoxia.

In addition to suppressing locomotion, adecrease in available ATP below 3 kPa aPO2

induces a dose-dependent negative effect onthe cell cycle of D. melanogaster embryos. Em-bryos carrying a kinesin–green fluorescent pro-tein (GFP) fusion, which permits in vivo vi-sualization of the cell cycle, showed that mildhypoxia (7 kPa aPO2) has no apparent effecton cycle length. By contrast, severe hypoxia(3 kPa aPO2) or anoxia arrests the cell cycle ei-ther at metaphase or just before S phase. Devel-opmental arrest during anoxia is associated withcondensation of chromosomes along the wallof a swollen nucleus (58). Anoxia causes rapidrelocation of motor, centrosome, and kineto-chore proteins and the inhibition of chromo-some segregation. Lack of ATP appears tomediate these structural changes in the mi-totic spindle apparatus in complete anoxia, asblockers of oxidative phosphorylation mimicthe anoxic phenotypes (59). In addition, un-der conditions of anoxia, manipulation of G1/S(e.g., dE2F1, RBF2) and G2/M (e.g., cyclinA, cyclin B, dWee1) proteins affected develop-ment, suggesting that stabilized cyclin A andE2F1 also mediate hypoxia-induced arrest (59).

The response of animals to hypoxia differsdepending on whether they experience constantor intermittent hypoxia (60–64). Intermittenthypoxia is of considerable biomedical interest,as it occurs in several pathological and diseaseconditions, including asthma and obstructivesleep apnea (65). Flies that experience inter-mittent hypoxia exhibit oxygen reperfusiondamage, with decreased metabolic rates andloss of spiracular control (56). However, fliesthat experience intermittent hypoxia (1 kPaaPO2 and 21 kPa aPO2, cycled every 20 minfor 2.5 h) show many fewer significantly alteredgenes (12 upregulated and 4 downregulatedgenes) compared with flies that experiencecontinuous 1 kPa aPO2 (94 upregulated and 70downregulated genes) (65). Also, different genefamilies tend to be regulated in constant versus

intermittent hypoxia. Gene families overrep-resented in constant hypoxia included thoseinvolved in the metabolism of chitin, lipid, andcarboxylic acid; the immune response; and theresponse to protein unfolding. Indeed, the heatshock protein family was the most upregulatedgroup in the flies exposed to constant hypoxia.In contrast, during intermittent hypoxia,biological processes involved primarily inneurotransmitter transport and multidrugresistance proteins (52, 53) were upregulated.

Genetic screens in Drosophila have iden-tified X-linked, autosomal-dominant, andautosomal-recessive mutants that influence tol-erance to constant or intermittent severehypoxia/anoxia (66–68). Electrophysiologicalstudies have demonstrated that polysynaptictransmission in the central nervous system isabnormally long in mutants that exhibit longrecovery times from anoxia (66, 67). Crossesmade among survivors of 12 days of exposure to1.5 kPa aPO2 had almost full survival (an ∼97%survival rate compared with controls, which hada 6% survival rate), strongly supporting the hy-pothesis that variation in the capacity to sur-vive severe hypoxia/anoxia has a genetic basisin Drosophila. P-element manipulations of genedosage have identified a variety of genes criticalfor the capacity of flies to survive anoxia.

Lack of ATP during anoxia inhibitsmembrane-bound ATPases such as theNa+/K+-ATPase, leading to changes in cyto-plasmic ionic and osmotic conditions, proteinunfolding, and irreversible cellular damage(Figure 1). During exposure to continuoushypoxia, fruit flies exhibit strong upregulationof the chaperone heat shock proteins Hsp70and Hsp23. Doubling Hsp expression usingP-element strain flies approximately doubledsurvival rates after 7 days at 1.5 kPa aPO2

relative to controls (65), whereas deletion ofthe Hsp70 P-elements eliminated the increasein survival rates. Fly lines that had no or halfcopies of Hsp70 or Hsp70Bb, -Bbb, and -Bchad markedly reduced survival. Overexpressionof Hsp70 in heart and hemocytes utilizing theUAS-Gal4 system increased the survival ofadult flies remarkably. Thus, chaperoning of


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tps1: trehalose-6-phosphate synthase

dADAR: Drosophilaadenosine deaminase

ROS: reactive oxygenspecies

unfolded proteins via induction of heat shockproteins is a major mechanism of anoxia/severehypoxia tolerance in fruit flies.

A second mechanism linked with survival ofanoxia via prevention of protein unfolding inDrosophila is trehalose accumulation (69). Tre-halose plays a protective role in a variety ofstresses, including heat, freezing and thawing,dehydration, hyperosmotic shock, and oxidantinjury (69, 70). Overexpression of trehalose-6-phosphate synthase (tps1), which synthesizestrehalose, increased levels of trehalose and in-creased tolerance to anoxia (71). In vitro exper-iments showed that trehalose reduces proteinaggregation caused by anoxia. The homozygoustps1 mutant (with a P-element insertion into thethird intron of the gene) dies at an early lar-val stage, and excision of the P-element rescuesthe phenotype. Thus, trehalose contributes toanoxia tolerance in flies by reducing protein ag-gregation. Excitingly, human HEK-293 cellstransfected with the Drosophila tps1 producedtrehalose, exhibited reduced protein damage,and were more resistant to low-oxygen stressrelative to control cells, demonstrating that tre-halose can also protect human cells from hy-poxic injury (71).

A second important family of genes involvedin anoxia tolerance are those related to RNAediting. Adenosine deaminase acting on RNAworks through RNA editing and alters proteinstructure and function. The gene hypnos-2encodes a Drosophila pre-mRNA adenosinedeaminase (dADAR) and is expressed almostexclusively in the adult central nervous sys-tem. Disruption of the dADAR gene resultsin unedited sodium, calcium, and chloridechannels (72–82); a prolonged recovery fromanoxic stupor; a vulnerability to heat shockand increased oxygen demands; and prematureneuronal degeneration in older flies (83). Thesedata demonstrate that, through the editing ofion channels, dADAR is essential for adap-tation to altered environmental stresses suchas oxygen deprivation (78). ADAR also playsa regulatory role in reactive oxygen species(ROS) metabolism (84–86), which can also beimportant in tolerating intermittent hypoxia.

DEVELOPMENTAL EFFECTS OFMODERATE HYPOXIA ANDHYPEROXIA ON GROWTHAND SIZE OF DROSOPHILA

Wild-type flies can develop and reproduce in at-mospheres containing as low as 7 kPa aPO2 (87,88). Over a range of 21 to 7 kPa aPO2, body sizedecreases linearly with decreasing aPO2, to ap-proximately 60% of the mass of normoxic flies(87) (Figure 2). The magnitude of the effectof hypoxia on size increases with temperature,probably because metabolic processes increasemore dramatically with temperature than doesoxygen diffusion rate (89). Hypoxia decreasesgrowth rate and extends development time (89).Hypoxic suppression of size is associated with adecrease in both cell number and size in the flywing (87).

These strong effects of moderate hypoxia onbody size and survival occur well above the crit-ical aPO2 values for metabolic rate (49), sug-gesting that such effects occur due to the activa-tion of genetic programs that reduce body sizein response to stress. Hypoxia during the egg,larval, or pupal stage can reduce adult size—pupal hypoxia is more specifically associatedwith changes in wing cell size—suggesting thatmultiple mechanisms for hypoxia effects on sizeare likely (90).

Although locomotory behavior andmetabolic rate exhibit large safety margins,persisting down to 1 kPa, other behaviors maybe more sensitive to hypoxia and may explaineffects of moderate hypoxia on growth. Larvaetested at 10 kPa aPO2 show significantly re-duced feeding rates compared with those testedat 21 kPa aPO2; the percentage decrease infeeding rate is similar to the 15–20% decreaseobserved in body mass for flies reared at thisoxygen level (91). In addition to reducingfeeding rates, hypoxia (5 kPa aPO2) causesadult flies to reduce consumption of foodscontaining protein (yeast) relative to foods con-taining carbohydrate (sucrose) (92), suggestingthat hypoxia modulates appetite in specificways rather than simply suppressing feeding.Exposure to hypoxia can also cause larvae to


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Individual fly

Seven-generation population

Eleven-generation large sizepopulation

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Figure 2Effects of atmospheric oxygen level on mean adult mass of male Drosophila melanogaster with five differentprotocols. Open symbols denote single-generation exposure; filled symbols, multiple-generation exposure.Blue circles denote animals reared individually from egg to adult (developmental plasticity) (97); purpletriangles, populations of flies reared for 7 generations at the indicated oxygen level (97); orange circles,populations of flies selected for large size at the indicated oxygen level for 11 generations (100); red circles,populations of flies selected for hypoxia tolerance over many generations (96); cyan squares, populations offlies selected for hyperoxia tolerance over many generations (99).

cease feeding and to attempt to escape from thelocal area (39). Hypoxia’s differential effects oncarbohydrate and protein consumption in flies(92), and its link with signaling cascades thatsuppress protein synthesis and the cell cycle,suggest that there are signaling pathways thatcause hypoxia to suppress appetite and proteinintake, as has been shown for other stressfulenvironmental stimuli (93). Such signaling mayoccur via atypical guanyl cyclase–expressingneurons and/or via hormonal pathways suchas ILP. These effects of moderate hypoxia onfeeding may be key factors causing develop-mentally mediated reductions in body size. Akey unanswered question is whether hypoxiaaffects the critical weight or the hormonalsignaling processes associated with molting.

The developmental responses to hypoxiainclude processes that increase oxygen deliv-ery as well as suppress oxygen demand. First,rapid, neuronally mediated responses, such asincreased spiracular opening, enhance oxygen

delivery (40). HIF expression in tracheal cellsincreases tracheal diameters and stimulates tra-cheole proliferation and tortuosity (33, 94). Theobserved effect of hypoxia on growth and size,and the lack of rearing aPO2 effect on maximaltracheal conductance, suggests that responsesof the tracheal system are insufficient to pre-vent drops in internal aPO2 associated with de-creased aPO2 (50).

An even greater developmental plasticity ofbody size in response to hypoxia occurs whenfly populations are selected for oxygen toler-ance by rearing over multiple generations atprogressively lower oxygen levels (95). An out-bred population was created by crossing 27 iso-genic lines that differed in oxygen tolerance anddevelopment time. Oxygen level was loweredfrom 8 kPa aPO2 to 4 kPa aPO2, in steps of1 kPa, with three to five generations at each oxy-gen level. This selection protocol produced flypopulations able to live in normally lethal levelsof hypoxia, with a variety of genetic changes (see


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below). When reared at 4 kPa aPO2, fly bodymass decreased by 40% (96), with changes inboth wing cell number and size (95). Eggs fromthese populations that were reared in normoxiawere the same size as eggs of flies from controlpopulations reared continuously in normoxia,demonstrating that the reduced body size in hy-poxia is due to a developmental response ratherthan an evolutionary response (95, 96).

What is the function of this developmen-tally induced reduction in body size in responseto hypoxia? Certainly the reduction in size al-lows flies to reproduce with a lower total en-ergy and oxygen requirement and may elevatetissue PO2 by reducing diffusion distances foroxygen. Drosophila has evolved adaptations thatallow it to feed on rotting fruit, an ephemeralresource that experiences increasing hypoxia asbacterial and fungal growth progresses. Thus,hypoxia may be an indicator of deterioratingenvironmental conditions. Hypoxia decreasesgrowth rate and increases development time,potentially compounding problems with dete-riorating food quality in highly rotted fruit.Thus, the reduction in body size required formaturation to adult under hypoxic conditionsmay be important to survival and reproductionin ephemeral, deteriorating environments. Thecapacity to manipulate genes involved in hy-poxic signaling in D. melanogaster provides anexciting model with which to test the functionalconsequences of this developmental plasticity.

Hyperoxia (40 kPa aPO2) increases the meanmass of fruit fly populations in a single gen-eration, primarily by extending developmenttime (89). However, when flies are reared in-dividually, hyperoxia (25–40 kPa aPO2) hasno effect on body mass or development time(Figure 2), and mild hyperoxia does not in-crease feeding rates (91), suggesting that theeffect of hyperoxia on the mean mass of flypopulations is an evolutionary effect (see be-low). These data suggest that from 21 to 40 kPaaPO2, oxygen is saturating and effects of oxida-tive stress are minimal (or that the mild benefitsof hyperoxia trade off with the mild costs of ox-idative stress) (97).

MULTIGENERATIONALEFFECTS OF HYPOXIA ANDHYPEROXIA ON DROSOPHILAEvolutionary studies of oxygen effects on fruitflies have been performed with several alterna-tive protocols. One approach has been to rearpopulations of D. melanogaster for multiple gen-erations in different oxygen atmospheres, ob-serving the changes in mean size, and then totest for evolution by returning the populationsto 21 kPa aPO2 for at least two generations toeliminate developmental and parental effects ofoxygen on size (97). With this type of labora-tory natural selection (98), which we term oxy-gen selection, the question is whether aPO2 se-lects for different body sizes within the ecologyof the laboratory rearing environment. A sec-ond approach has been to expose fly popula-tions to aPO2 that decreases or increases slowlyover multiple generations to oxygen levels be-low those at which D. melanogaster can normallysurvive and reproduce (95, 96, 99). This ap-proach, which we term hypoxia or hyperoxiatolerance selection, selects for the ability to sur-vive and reproduce in very low or high aPO2

and provides a way to determine whether thereis a genetically based capacity for increased hy-poxic/hyperoxic tolerance. A third approach hasbeen to select for large body size with trun-cation selection on populations in hyperoxic,normoxic, and hypoxic atmospheres (100). Thisapproach allows tests for interactions betweenaPO2 and selection for large size, which are par-ticularly useful for determining whether aPO2

can limit body size in insects.Studies using both oxygen selection and oxy-

gen tolerance selection approaches have foundthat all changes in body size in response to hy-poxic rearing can be attributed to developmen-tal effects. In the oxygen selection study, bodysize dropped by 15% within one generation ofrearing at 10 kPa aPO2, remained at that sizeover seven generations in hypoxia, and then re-verted to normal size within two generations ofreturn to normoxic rearing (97). Male flies weresignificantly smaller than control, normoxia-reared flies in the first generation after return


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to normoxia, hinting that parental effects mayhave mediated some effects of hypoxia on size.Similarly, although flies were 40% smaller af-ter a number of generations that was sufficientfor flies to reproduce at 4 kPa aPO2 in theoxygen tolerance selection protocols, such fliesreturned to the size of normoxic control flieswhen returned to normoxia for a single gener-ation (95, 96).

Multigenerational exposure to hypoxic con-ditions produces flies with many phenotypicand biochemical differences, some of which aregenetically mediated. Rearing under hypoxicconditions strongly reduces survival, providinga strong selective force, with only 70% of eggssurviving to adulthood at 10 kPa aPO2 (89) anda survival rate of less than 10% at 6 kPa (95).The oxygen tolerance selection regime stronglyimproves survival in hypoxia and the speed atwhich flies recover from anoxia (95). This im-proved hypoxic tolerance is at least partially ge-netically based, as flies derived from these linesstill demonstrated the capacity to develop toadults at the normally lethal 5 kPa aPO2 level,even after eight generations of rearing in nor-moxia (95). The oxygen tolerance selection pro-tocol produced flies with triose phosphate iso-merase genes that differed in multiple regions(cis-regulatory, coding, noncoding, and down-stream regions) compared with normoxic con-trols, definitively demonstrating evolution ofthese lines (96).

One possible strategy for coping with re-duced oxygen availability would be a reductionin oxygen consumption rates, potentially re-lated to lower growth rates or higher efficienciesin oxygen utilization. However, neither larvaenor adults reared for six to seven generations at10 kPa aPO2 differed from normoxic controlsin gas exchange rates in either normoxic or hy-poxic conditions, suggesting that oxygen selec-tion at this moderate level of hypoxia does notstrongly affect metabolism or maximal trachealconductance (50). The oxygen tolerance selec-tion approach demonstrated that adult flies thatsurvived and thrived at 4 kPa aPO2 had highermetabolic rates at 3 kPa aPO2, but not at 21 kPaaPO2, relative to control flies, suggesting that

part of the increased oxygen tolerance of theseflies is due to an improved oxygen delivery sys-tem (95). This increased oxygen tolerance maybe partially due to evolution of a more exten-sive tracheal system in hypoxia. Larvae rearedfor six to seven generations at 10 kPa aPO2 andthen returned to normoxia for two generationshave larger-diameter tracheae than do controlflies, demonstrating compensatory evolution oftracheal diameters (94).

To begin to assess mechanisms of the multi-generational responses of D. melanogaster forextreme hypoxia tolerance, whole-genome mi-croarray analyses were performed to comparegene expression in flies evolved and living in4 kPa aPO2 with gene expression in control,normoxia-reared flies, using third-instar larvae(wandering stage) and adults (3–5 days, malesand females) (96). More than 2,700 genes weredifferentially expressed during the larval stage,compared with 138 genes in adults, suggest-ing that hypoxia is a greater strain for larvae.This may be because of the energy demands forgrowth and the reduced capacity of the trachealsystem in the larval stage [adults have a muchmore extensive tracheal system and can supple-ment diffusion with convection (51, 101)]. In-terestingly, many of the upregulated genes inthe hypoxia-tolerant flies were related to im-mune defense (96), suggesting that inflamma-tory processes or interactions with microbes areimportant components of surviving hypoxia. Aclear pattern was downregulation of metabolicgenes in larvae, particularly genes relating tocarbohydrate catabolism and the tricarboxylicacid (TCA) cycle and genes encoding compo-nents of the respiratory chain complexes (96).These data strongly suggest that these highlyhypoxia-tolerant larvae have reduced oxygenconsumption rates compared with controls, al-though this remains to be tested directly. Thelarvae may be less able than adults to remodeltheir tracheal system to compensate for hy-poxia, given their lower relative tracheal con-tent and likely higher reliance on diffusion forgas exchange.

How is the coordinated suppression ofmetabolic genes accomplished? At least for the


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TCA cycle enzymes, the transcriptional sup-pression factor encoded by hairy appears to be akey factor (96). Hairy is upregulated in hypoxia-tolerant flies, downregulated TCA genes havebinding sites for hairy in their cis-regulatory re-gions, and chromatin immunoprecipitation as-says revealed that hairy binds to these regulatoryregions during hypoxia. Finally, mutants lack-ing hairy function did not exhibit downregula-tion of the TCA cycle in hypoxia. The signalingpathways linking oxygen to hairy (possibly HIF)remain unknown.

The multigenerational effects of hyperoxiaon flies have been less examined, but the effectson growth and size appear to differ markedlyfrom the responses to hypoxia. Flies exposedto 40 kPa aPO2 for seven generations becamesignificantly larger and retained most of thislarger size when returned for more than twogenerations to normoxia, indicating evolutionof body size (Figure 2) (97). The mechanismsthat drive this size increase are unclear and arelikely complex. Parental effects are likely in-volved because body sizes increased stronglywhen flies were returned from 40 kPa aPO2 tonormoxia (97). Male and female mass showeddifferent patterns across generations; adult malemass increased strongly in generation one andthen remained relatively constant over the nextsix generations, whereas adult female mass rosesteadily over three generations at 40 kPa aPO2.Adult and third-instar larvae from populationsreared for seven generations at 40 kPa aPO2 didnot have higher metabolic rates when tested at21 kPa aPO2 or in hypoxic conditions, suggest-ing that hyperoxia does not directly enhanceATP turnover (50). Fly populations exposed to40 kPa aPO2 for a single generation hadlonger development times (89). Conceivably,decreases in oxygen near the end of an instarare involved in the regulation of critical weightbecause the safety margin for oxygen deliveryfalls dramatically as insects approach the end ofan instar (47). Thus, hyperoxia may allow fliesto gain a larger size by permitting a longer pe-riod of feeding and growth. This scenario mayallow a weak developmental effect of hyperoxiaon body size that may operate in individuals

with genes that allow them to take advantageof increased oxygen availability. Under such ascenario, the increase in size over multiple gen-erations in hyperoxia may be driven by generalreproductive advantages for larger flies, withoxygen enabling larger size in a subset of thepopulation. Another possibility is that largersize reduces the oxidative stress imposed by hy-peroxia. Rearing at 40 kPa aPO2 causes theevolution of smaller tracheae, suggesting ei-ther that prevention of oxidative damage isan important selective factor at 40 kPa aPO2

or that the costs of the tracheal system aresignificant.

ATMOSPHERIC OXYGEN LEVELAND THE EVOLUTION OFINSECT BODY SIZE

aPO2 levels have changed dramatically duringanimal evolution, likely ranging between 12 and31 kPa aPO2 during the Phanerozoic (102).Fossil and geological evidence shows that in-creases in aPO2 level are correlated with in-creases in body sizes of animals, leading to thesuggestion that the well-known giant insects ofthe late Paleozoic were enabled by increasedoxygen availability (16, 103). The relativelysmall response of body size to strong increasesin aPO2 in developmental or evolutionary tests(Figure 2) suggests that simple stimulatory ef-fects of oxygen on growth are not the explana-tion. More likely potential mechanisms for suchoxygen limitation of insect size are spatial ormaterial costs of tracheal systems because largerinsects have a greater fraction of their body in-vested as tracheae or generally positive relation-ships between aPO2 and insect performance,survival, or fecundity (16). Large animals likelyarose from natural selection for larger size dueto competition, predation, or sexual selection.One way to test whether aPO2 influences theevolution of larger insects is to examine theeffect of aPO2 on the trajectory of body sizeunder artificial selection for large mass. Whenoutbred populations of D. melanogaster weresubjected to truncation selection (the largest15% of population were allowed to found the


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next generation), body size increased stronglywith time (100). Populations in hypoxia (10 kPaaPO2) were smaller (Figure 2), but when thesepopulations were returned to normoxia, theywere the same size (as large) as populations se-lected for large size at 21 kPa aPO2, demon-strating a strong constraint of hypoxia on size.In contrast, flies selected for larger body sizesin hyperoxia (40 kPa aPO2) were not signifi-cantly larger than flies experiencing the sameselection pressure in normoxia (Figure 2), sug-gesting that normoxic conditions are sufficientto allow considerable evolutionary increases inbody size (100).

Hyperoxia tolerance selection also increasedthe size of adult flies (Figure 2). Body weightof hyperoxia-tolerant flies (those able to live at60–90 kPa aPO2) was at least 20% greater thanthat of control flies (99). Hyperoxia-tolerantflies also had a larger wing area and total wingcell number compared with control flies. Cer-tainly the magnitude of the size increase withhyperoxia in these laboratory selection exper-iments is small relative to the five- to tenfoldincrease in maximal size observed in the latePaleozoic. However, greater effects might beobserved with more time, genetic variation, andnatural or sexual selection for large size.

Oxygen availability may affect all individ-uals (or species) equally, causing shifts in themean size without changing the shape of thesize distribution. Alternatively, oxygen may af-fect only the largest species or individuals in aspecies, possibly because oxygen gradients maybe greater in larger animals. In support of thismodel, Peck & Chappelle (104) pointed outthat the best fit between size and environmen-tal aPO2 in amphipods occurs when a measureof the maximal size is used (these investigatorsutilized the size below which 95% of the indi-viduals occur as the index of maximal size).

Do evolutionary and developmental studiesof aPO2 effects on flies support the hypothe-sis that oxygen affects primarily the largest in-dividuals? One way to assess this question isto ask whether changes in the shape of bodysize distributions occur in response to aPO2.We reanalyzed the data from Reference 97 to

examine this question. When flies were rearedindividually for a single generation at seven dif-ferent oxygen levels (10–40 kPa), the distribu-tion of body masses did not differ significantlyfrom a normal distribution (according to theKolmogorov-Smirnov and Lilliefors tests fornormality), and there were no trends in skew-ness or kurtosis with aPO2. Also, the upper 95thpercentile of body mass showed the identicalpattern with aPO2 as did the mean masses, withsmaller sizes at 10 kPa and similar sizes at allhigher aPO2 values tested. Thus, developmen-tal effects of aPO2 seem to shift the entire dis-tribution of body sizes; measures of the centraltendency are appropriate indices of oxygen ef-fects. Similar effects have been found for evo-lutionary studies; oxygen shifted the means ofdistributions, rather than specifically affectingthe upper tail of body masses (96, 100).

METABOLISM OFHYPOXIA-ADAPTED FLIES

Many of the important biochemical pathwaysthat change in response to hypoxia in tissuesrelate to energy metabolism. Metabolic profilesof Drosophila exposed to acute or multigenera-tional oxygen deprivation have been examinedusing 1H NMR (nuclear magnetic resonance)spectroscopy (96, 105). Compared with controlflies, hypoxia-adapted flies produced moreATP per glucose and created fewer protons,had lower pyruvate carboxylase flux, and hadgreater usage of complex I over complex II inthe electron transport chain. Simulations sug-gested that hypoxia-adapted metabolism hasgreater ATP-per-oxygen efficiency. Further-more, experiments with isolated mitochondriashowed downregulation of complex II inhypoxia-adapted flies (D. Zhou, S. Yin & G.G.Haddad, unpublished observations).

How do these increases in ATP/oxygenefficiency arise? Because both populations offlies are in a stupor at the oxygen level used(0.5 kPa aPO2), most ATP consumption islikely for cellular maintenance and biosyntheticprocesses. Complex I produces more ATP andconsumes more protons per oxygen and per


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glucose substrate than does complex II. Also,because complex I uses mitochondrial NADHand complex II uses reducing equivalents shut-tled from the cytosol, the ratio of I:II is an indi-cator of the coupling between glycolysis and theTCA cycle. The model suggests that hypoxia-adapted flies tend to have decreased glycoly-sis and pyruvate carboxylase fluxes relative tothe amount of oxygen consumed and tend toproduce acetate rather than oxaloacetate (105).These differences in fluxes are linked to tightercoupling of glycolysis with the TCA cycle, less

anapleurotic flux (suggesting less biosynthesis),and more efficient ATP production per oxygenconsumed. Similar patterns may occur in mod-erate hypoxia because the comparatively smallsize of adapted flies indicates downregulatedanabolic activity (95). Indeed, suppression ofbiosynthesis is a common mechanism for sur-viving hypoxic conditions (106). The benefits ofthis overall strategy are reinforced by the recentdiscovery that hypoxia tolerance in Caenorhab-ditis elegans is improved when protein transla-tion is suppressed (107).

SUMMARY POINTS

1. The gas-filled tracheal respiratory system of insects allows Drosophila to restore oxygendelivery by diffusion to its tissues when reexposed to oxygen after anoxia. In contrast,once anoxia stops a vertebrate heart, oxygen delivery to the brain and to other vital tissuesis impossible. This whole-system property of insects has likely stimulated the evolutionof the multiple pathways that promote tissue survival during and after anoxia. Thesepathways mostly involve systems conserved across organisms, suggesting that they mayhave broad application in comparative biology and cure of disease.

2. Matching protein synthesis and growth to resource availability is a central principleof organismal development. During the Earth’s evolution, oxygen was a key resourceaffecting function and evolutionary innovation, and the ancient biochemical pathways foroxygen utilization and ROS disposal are integrated into virtually every function of aerobicorganisms (15). As for many resources, oxygen must be supplied sufficiently, but excess istoxic, further promoting the need for matching oxygen supply and demand. Dissectingthe biochemical, developmental, and evolutionary components to achieving this match-ing will have applications in medicine, nutrition, ecology, and evolutionary biology.

FUTURE ISSUES

Although the literature on oxygen regulation of growth and metabolism has exploded inrecent years, many key questions remain relevant to central questions of biomedical andevolutionary physiology. Here we mention only the most obvious few.1. Organisms must match oxygen transport systems with tissue oxygen needs during de-

velopment and so have evolved signaling mechanisms by which reduced oxygen levelspositively regulate respiratory growth and negatively regulate somatic growth. These dif-ferential responses occur due to differential responses to growth factor receptor signalingdownstream of HIF (31, 32). However, there is much work to be done to understand themechanisms of these pathways, their tissue specificity, their ontogeny, and their responseto environmental stress. Studies of oxygen-regulated growth pathways may broadly en-hance our understanding of developmental physiology, in addition to providing keyinsights into the mechanisms of diseases ranging from cancer to diabetes.


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2. Although the regulation of growth by oxygen at the cellular level has advanced verysignificantly in recent years, neuroendocrine pathways that operate at the whole-bodylevel have received much less attention. Effects of oxygen level on hormones and bindingproteins, as well as on behavior such as foraging, are likely to be extremely important inmodulating real-world effects of altered oxygen concentrations.

3. There are likely to be many medical applications of the information gleaned from studyof the responses of flies to oxygen. The finding that manipulation of trehalose levelsaffects hypoxia tolerance both in flies and in human cells suggests that gene therapiesinspired by fly physiology are likely.

4. In comparative and evolutionary physiology, there is great theoretical interest in thematching of oxygen delivery to metabolism across body sizes. The pathways that recipro-cally regulate respiratory and somatic system development may provide the long-elusivemechanistic answer to how this occurs. Correlative data suggest that environmental aPO2

affects body size in aquatic systems (11, 12), and the recent findings that oxygen levelscan directly affect growth and size support the hypothesis that effects of oxygen mediatethe well-documented inverse correlation between temperature and body size in aquaticinvertebrates (14). Developmental and evolutionary studies that independently manipu-late oxygen and temperature may provide experimental tests of this exciting hypothesis.Oxygen may affect animal body size through evolutionary time, and further study of thedevelopmental and evolutionary responses of animals to oxygen will provide key tests ofthis hypothesis (16).

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

J. Harrison and G. Haddad were partially supported by grants during the writing of this arti-cle (NSF EAR 0746352 and ASU sabbatical award to J.F.H.; NIH PPG PO1 HD032573 andNIH RO1 037756 to G.G.H.). We appreciate the helpful comments of Melanie Frazier, KendraGreenlee, James Waters, Jaco Klok, and our editor on the manuscript.

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effects of oxygen on growth and size: synthesis of molecular, organismal, and evolutionary studies...

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