O R I G I NA L A RT I C L E doi:10.1111/evo.12541
Cold adaptation shapes the robustness of metabolic networks in Drosophila melanogaster Caroline M. Williams,1,2 Miki Watanabe,3 Mario R. Guarracino,4 Maria B. Ferraro,5 Arthur S. Edison,6 Theodore J. Morgan,7 Arezue F. B. Boroujerdi,8 and Daniel A. Hahn9 1
Department of Integrative Biology, University of California, Berkeley, California 94720 2
E-mail: [email protected]
3
NMR Metabolomics Core-Mass Spectrometry Lab, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229
4
High Performance Computing and Networking Institute, National Research Council, Via Pietro Castellino, 111 - 80131,
Naples, Italy 5
Department of Statistical Sciences, Sapienza University of Rome, 00185 Rome, Italy
6
Department of Biochemistry & Molecular Biology, University of Florida, Gainesville, Florida 32610
7
Division of Biology, Kansas State University, Manhattan, Kansas 66506
8
Molecular Science Research Center, Claflin University, Orangeburg, South Carolina 29115
9
Department of Entomology and Nematology, College of Agriculture and Life Sciences, University of Florida, Gainesville,
Florida 32611
Received May 2, 2014 Accepted September 14, 2014 When ectotherms are exposed to low temperatures, they enter a cold-induced coma (chill coma) that prevents resource acquisition, mating, oviposition, and escape from predation. There is substantial variation in time taken to recover from chill coma both within and among species, and this variation is correlated with habitat temperatures such that insects from cold environments recover more quickly. This suggests an adaptive response, but the mechanisms underlying variation in recovery times are unknown, making it difficult to decisively test adaptive hypotheses. We use replicated lines of Drosophila melanogaster selected in the laboratory for fast (hardy) or slow (susceptible) chill-coma recovery times to investigate modifications to metabolic profiles associated with cold adaptation. We measured metabolite concentrations of flies before, during, and after cold exposure using nuclear magnetic resonance (NMR) spectroscopy to test the hypotheses that hardy flies maintain metabolic homeostasis better during cold exposure and recovery, and that their metabolic networks are more robust to cold-induced perturbations. The metabolites of cold-hardy flies were less cold responsive and their metabolic networks during cold exposure were more robust, supporting our hypotheses. Metabolites involved in membrane lipid synthesis, tryptophan metabolism, oxidative stress, energy balance, and proline metabolism were altered by selection on cold tolerance. We discuss the potential significance of these alterations. KEY WORDS:
Correlation networks, energy balance, insect, NMR-based metabolomics, thermal limits, winter.
Bioenergetics, or energy flow within organisms, interacts with temperature to constrain species distributions and influence global patterns in species abundance (Clarke and Gaston 2006). Rates of energy flow through metabolic pathways are strongly dependent on temperature because decreases in temperatures slow C
1
2014 The Author(s). Evolution
rates of biochemical reactions within the cells of an organism (Logan 1982). Normal physiological function requires organisms to precisely and dynamically balance energy supply and demand, but metabolic pathways differ in susceptibility to thermodynamic effects (Clarke and Fraser 2004). This creates a considerable
C A RO L I N E M . W I L L I A M S E T A L .
challenge for ectotherms, whose body temperatures fluctuate with environmental temperatures, to maintain energetic and metabolic homeostasis under fluctuating thermal conditions. Species distributions can thus be driven not only directly by exposure to lethal temperatures, but also indirectly through repeated or long-term exposures to sublethal temperatures that lead to energetic failures (Fly and Hilbish 2013). Energetic failures result from an inability to supply or use sufficient energy to perform physiological functions necessary to support life. In marine organisms, energetic failures at temperatures outside the thermal window for growth and reproduction are driven by inability to supply oxygen and thus maintain aerobic scope for normal physiological function (oxygen- and capacity limitation of thermal tolerance, P¨ortner 2001). Oxygen delivery limitations result in an increasing reliance on anaerobic metabolism and the development of low cellular energy levels (Gibbs free energy of adenosine triphosphate [ATP] hydrolysis, Zielinski and P¨ortner 1996). However, terrestrial insects have airfilled tracheal tubes that supply oxygen directly to tissues without reliance on a circulatory system, and there is currently little support for oxygen and capacity limitation of thermal tolerance in these taxa (Klok et al. 2004; Stevens et al. 2010; MacMillan et al. 2012a; McCue and De Los Santos 2013). The “weak link” in the energy supply and demand chain that explains variation in performance at extreme temperatures thus remains to be identified in terrestrial insects (and other terrestrial ectotherms). In insects, low temperatures induce a reversible loss of neuromuscular coordination (chill coma) during which mating and foraging is precluded and predators cannot be avoided. Recovery time from chill coma is a commonly used metric of cold hardiness. Because chill-coma recovery time is usually assessed after exposure to mild low temperatures that do not cause cold shock (i.e., direct chilling injury that develops rapidly as a result of protein denaturation, membrane phase transitions, or cell death; Koˇst´al et al. 2011a), variation in recovery time likely results from variation in either the ability to maintain homeostatic processes in the cold or the rate of recovery of those processes upon rewarming. Ionic regulation is a critical homeostatic process compromised in the cold. While an insect is in chill coma, membrane equilibrium potentials are progressively depolarized as extracellular [K+ ] increases due to a loss of ionic regulation (MacMillan and Sinclair 2011a; Andersen et al. 2013; Findsen et al. 2013). In the fall field cricket, this progressive loss of ionic homeostasis is accompanied by the development of chilling injury, and, upon rewarming, osmotic and ionic equilibria are gradually restored (MacMillan and Sinclair 2011b). Recovery (the resumption of coordinated movement) coincides with the restoration of osmotic and ion concentration gradients, and the speed of recovery of ion homeostasis correlates positively with cold hardiness (Findsen et al. 2013).
2
EVOLUTION 2014
Summary of the response to cold exposure and selection for cold tolerance. (A) Proportion of 20 flies still in chill coma as a function of time in replicate lines of cold-hardy (black) and Figure 1.
cold-susceptible (gray) Drosophila melanogaster at the time of collection of the samples for the metabolomics experiment. Letters indicate lines that are significantly different according to an accelerated failure time model with a log-logistic error distribution, and the “+” indicates one censored value—a single fly from one of the susceptible lines did not wake up within 60 min. Hardy lines are statistically indistinguishable from each other, and have faster recovery than either of the susceptible lines. (B) Experimental design of the metabolomics experiments. (C) Numbers within each circle indicate the corresponding number of metabolites displaying a significant main effect of cold exposure, selection regime, or an interaction between the two. The number outside all circles indicates metabolites that did not respond to selection or cold exposure.
M E TA B O L I C H O M E O S TA S I S I N C O L D - A DA P T E D D RO S O P H I L A
A
B Higher in hardy
Scores on PC1
*
Before
0.3
Scores on PC2
Hardy
Susceptible
Before
2
Fold change (susceptible/hardy)
3
4
During
After
0.2
-0.3
ADP B.Ala Treh Kynn Lys Mal HK Met.sulf Asp Asn Dimeth NAD KynA Sucr Tyr PChol Gln Glu Ser Tau His Pro Fum AMP Fruc Gly Malt Arg Prop Gluc Form Leu Val Ace Ile Thr Ala Lac Suc
1
After
Tau Glu Val Gluc Pro Arg Leu Ile Ala Gln Ace Sucr AMP Prop Mal Lac Gly NAD Ser Tyr Suc Form Fruc Thr Fum B.Ala HK Asp Malt Asn His Lys Kynn ADP KynA Treh PChol Met.sulf Dimeth
* * * * 0
During
-0.1
*
−2
Susceptible
C
*
*
*
Hardy
Loadings
* * * * * * * * ** * * *
Loadings
3−HK Asp Tyr Sucr Asn ADP Treh Tau Gluc Val Ile NAD Mal Gln Arg AMP Ala Ace Glu Malt Gly Prop Leu Fum Form KynA Kynn Ser B.Ala Lac Pro Lys Suc Fruc His Met.sulf Thr PChol Dimeth
Higher in susceptible
Figure 2. Changes in metabolite content of Drosophila melanogaster resulting from selection for fast or slow recovery from chill coma (hardy and susceptible, respectively). (A) Fold changes of metabolites from hardy (black) or susceptible (gray) flies, pooled across all time
points. Asterisks indicate significant differences between groups (false discovery rate < 0.05; Table 1). (B and C) Principal component scores (B = PC1, C = PC2), and associated loadings of each compound onto those components, for flies sampled before cold exposure, at the end of a 3-h cold exposure (during), or after 5-min recovery (after).
Complete recovery of ionic and osmotic homeostasis following cold exposure is an energetically expensive process that can take several hours (MacMillan et al. 2012b). Although it is clear that the maintenance and recovery of ionic homeostasis plays a proximate role in modulating cold hardiness, the mechanisms underlying evolutionary variation in ion homeostasis are still unknown. Energy supply limitations may compromise the function of ion-motive pumps that regulate ionic and osmotic homeostasis, but to date there are few indications that ATP supply is compromised in insects at low temperatures. Aerobic metabolism and ATP supply is maintained down to the freezing point of the body tissues ( –10°C) in freeze-tolerant pupae of the gall-fly, Eurosta solidaginus (Storey and Storey 1981), and the firebug, Pyrrhocoris apterus, maintains its energy state
right until death from cold injury (Koˇst´al et al. 2004). Some studies document an ATP decline in insects at low temperatures (Pullin et al. 1990; Dollo et al. 2010), but this decline develops slowly and may represent secondary effects rather than the cause of energetic failure. In other cases, ATP accumulates during prolonged or lethal cold exposures (Coulson et al. 1992; Macmillan et al. 2012a), suggesting that the ability to use, rather than synthesize, ATP may be reduced in the cold. Low temperatures may inhibit energy-demanding processes directly through thermal effects on ion-motive pumps or metabolic enzymes, or indirectly via modulation of the lipid membrane environment of cells and organelles (Koˇst´al et al. 2011a; Macmillan and Sinclair 2011a; Findsen et al. 2013). Alterations to lipid membrane fluidity will affect rates of proton leak and thus oxidative
EVOLUTION 2014
3
C A RO L I N E M . W I L L I A M S E T A L .
stress, and performance of membrane-bound proteins such as ionmotive pumps and metabolic enzymes. Whether the effects of temperature are direct or indirect, alterations to energy homeostasis will occur and these alterations should be associated with distinct metabolomic fingerprints. Metabolomics provides a snapshot of a portion of the metabolome at a point in time, and thus does not provide information on flux through underlying pathways. However, correlations among metabolites across biological samples at steady state can be used to infer the relative importance of various enzymatic reactions, and the underlying regulatory structure of the system as a whole (Weckwerth and Morgenthal 2005). The metabolic network of an organism is the complete set of biochemical reactions occurring within its cells, including all metabolic pathways and regulatory interactions among them. Interdependencies arise between metabolites that interact through the metabolic network as a result of fluctuations induced by environmental (e.g., temperature) or intrinsic variability (e.g., enzyme concentration; Steuer 2006). Reaction interdependencies generate patterns of correlations among metabolites that provide a fingerprint of the underlying physiological state of the organism (Camacho et al. 2005; Morgenthal et al. 2006). Therefore, if cold exposure disturbs the physiological state of an organism, we expect alterations to the structure of correlation-based metabolic networks. The degree of disruption to these correlation networks may be associated with underlying genetic variation in chill-coma recovery time. Although no studies have investigated the maintenance of metabolic networks during cold exposure, there is evidence for loss of metabolic homeostasis in insects at low temperatures (Overgaard et al. 2007; Colinet et al. 2012). This manifests in increases in free amino acid concentrations that may result from cold-induced protein breakdown (Lalouette et al. 2007; Koˇst´al et al. 2011b; Colinet et al. 2012) or an imbalance between glycolysis and the tricarboxylic acid (TCA) cycle (Michaud and Denlinger 2007; Koˇst´al et al. 2011b). Cold exposure also increases sugars (Overgaard et al. 2007; Colinet et al. 2012; Teets et al. 2012) and sugar alcohols such as glycerol and sorbitol (Michaud and Denlinger 2007). It is clear that both acclimation and adaptation can modulate the degree of cold-induced disruption to homeostatic processes. Acclimation to low temperatures improves both ionic (Findsen et al. 2013) and metabolic homeostasis in the cold (Overgaard et al. 2007; Colinet et al. 2012). At the whole organism level, chill-coma recovery time is evolutionarily labile—latitudinal variation within and among species is consistent with local adaptation (David et al. 2003; Sinclair et al. 2012), and cold hardiness traits respond to artificial selection in the laboratory, including chill-coma recovery time (Mori and Kimura 2008; Goto et al. 2010). However, we still do not know whether genetically based
4
EVOLUTION 2014
variation in the ability to maintain metabolic processes in the cold underpins variation in susceptibility to cold stress. A strong evolutionary perspective on this question has potential to advance our understanding of the mechanisms of low temperature tolerance in insects. Here, we test the hypothesis that fly lines selected for coldhardiness will maintain metabolic homeostasis to a higher degree than cold-susceptible flies during cold exposure and recovery. Specifically, we predict that the metabolic networks of cold-hardy flies will be more resistant to cold stress, maintaining their structure better during cold exposure than the metabolic networks of cold-susceptible flies, and that the network structure of cold-hardy flies will also be more tolerant of cold stress, reestablishing their structure more quickly during recovery from cold stress. We use replicate experimental evolution lines of Drosophila melanogaster selected in the laboratory for fast or slow recovery from chill coma. These lines originate from a natural population in Raleigh, North Carolina, and have stable and genetically based divergence in chill-coma recovery. We use nuclear magnetic resonance (NMR) spectroscopy to profile the water-soluble metabolome, demonstrating that selection for rapid chill-coma recovery results in improved metabolic homeostasis and more robust metabolic networks.
Materials and Methods FLY STOCKS AND EXPERIMENTAL DESIGN
Flies from two base populations, each collected at the same locale in Raleigh, North Carolina, the same populations that gave rise to the D. melanogaster genetic reference panel (Mackay et al. 2012). These two base populations were selected for 31 generations for fast (hardy) or slow (susceptible) recovery from chill coma at Kansas State University, and thereafter maintained at 25°C (Supporting Information). These two base populations gave rise to two independent lines each of hardy and susceptible flies that were used in all metabolomics experiments. Flies for this study were reared at University of Florida at 25°C on a 12:12 L:D cycle in 235-mL bottles on molasses-cornmeal-yeast medium under controlled density, achieved by allowing 10 females (accompanied by five males) to oviposit for 48 h. This resulted in uncrowded conditions and relatively synchronous emergence. On day 12 following the beginning of oviposition, emerged flies were cleared and discarded. Twenty-four hours later emerged flies were transferred to holding bottles for 24 h to ensure mating, and groups of 20 females were sorted under light CO2 anesthesia (48 h before use in experiments (yielding 5to 8-day-old mated females). Eight replicate pools of 10 flies from each line were frozen in liquid nitrogen at one of three time points: (1) before cold exposure, (2) at the end of a 3-h exposure at 0°C, and (3) after
M E TA B O L I C H O M E O S TA S I S I N C O L D - A DA P T E D D RO S O P H I L A
Table 1. The effect of cold exposure and selection for cold tolerance of levels of metabolites in Drosophila melanogaster, measured using 1 H NMR spectroscopy.
Cold exposure
Selection regime
Exposure × selection
Metabolite
Abbreviations.
F
Q
F
Q
F
Q
3-Hydroxykynurenine O-Phosphocholine L-Glutamate L-Tyrosine Taurine Sucrose L-Aspartate Nicotinamide adenine dinucleotide Adenosine diphosphate Malate L-Histidine D-Glucose L-Alanine L-Proline Dimethylamine Methionine sulfoxide Acetate Threonine Glycine L-Valine Lactate Trehalose L-Arginine Adenosine monophosphate L-Glutamine Succinate β-Alanine Kynurenine Fumarate L-Lysine L-Isoleucine Propionate Maltose L-Serine L-Asparagine L-Leucine Fructose Kynurenate Formate
3-HK PChol Glu Tyr Tau Sucr Asp NAD ADP Mal His Gluc Ala Pro Dimeth Met.sulf Ace Thr Gly Val Lac Treh Arg AMP Gln Suc B.Ala Kynn Fum Lys Ile Prop Malt Ser Asn Leu Fruc KynA Form
4.03 2.27 27.62 1.85 0.99 5.83 19.92 1.3 14.04 13.09 2.64 2.43 3.83 8.95 3.99 1.47 8.54 3.52 2.43 0.47 6.76 6.86 2.5 0.49 1.33 6.09 4.02 6.39 0.88 2.35 0.18 3.36 0.34 0.27 2.16 0.31 0.03 0.51 1.36
0.061 0.185
Cold adaptation shapes the robustness of metabolic networks in Drosophila melanogaster Caroline M. Williams,1,2 Miki Watanabe,3 Mario R. Guarracino,4 Maria B. Ferraro,5 Arthur S. Edison,6 Theodore J. Morgan,7 Arezue F. B. Boroujerdi,8 and Daniel A. Hahn9 1
Department of Integrative Biology, University of California, Berkeley, California 94720 2
E-mail: [email protected]
3
NMR Metabolomics Core-Mass Spectrometry Lab, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229
4
High Performance Computing and Networking Institute, National Research Council, Via Pietro Castellino, 111 - 80131,
Naples, Italy 5
Department of Statistical Sciences, Sapienza University of Rome, 00185 Rome, Italy
6
Department of Biochemistry & Molecular Biology, University of Florida, Gainesville, Florida 32610
7
Division of Biology, Kansas State University, Manhattan, Kansas 66506
8
Molecular Science Research Center, Claflin University, Orangeburg, South Carolina 29115
9
Department of Entomology and Nematology, College of Agriculture and Life Sciences, University of Florida, Gainesville,
Florida 32611
Received May 2, 2014 Accepted September 14, 2014 When ectotherms are exposed to low temperatures, they enter a cold-induced coma (chill coma) that prevents resource acquisition, mating, oviposition, and escape from predation. There is substantial variation in time taken to recover from chill coma both within and among species, and this variation is correlated with habitat temperatures such that insects from cold environments recover more quickly. This suggests an adaptive response, but the mechanisms underlying variation in recovery times are unknown, making it difficult to decisively test adaptive hypotheses. We use replicated lines of Drosophila melanogaster selected in the laboratory for fast (hardy) or slow (susceptible) chill-coma recovery times to investigate modifications to metabolic profiles associated with cold adaptation. We measured metabolite concentrations of flies before, during, and after cold exposure using nuclear magnetic resonance (NMR) spectroscopy to test the hypotheses that hardy flies maintain metabolic homeostasis better during cold exposure and recovery, and that their metabolic networks are more robust to cold-induced perturbations. The metabolites of cold-hardy flies were less cold responsive and their metabolic networks during cold exposure were more robust, supporting our hypotheses. Metabolites involved in membrane lipid synthesis, tryptophan metabolism, oxidative stress, energy balance, and proline metabolism were altered by selection on cold tolerance. We discuss the potential significance of these alterations. KEY WORDS:
Correlation networks, energy balance, insect, NMR-based metabolomics, thermal limits, winter.
Bioenergetics, or energy flow within organisms, interacts with temperature to constrain species distributions and influence global patterns in species abundance (Clarke and Gaston 2006). Rates of energy flow through metabolic pathways are strongly dependent on temperature because decreases in temperatures slow C
1
2014 The Author(s). Evolution
rates of biochemical reactions within the cells of an organism (Logan 1982). Normal physiological function requires organisms to precisely and dynamically balance energy supply and demand, but metabolic pathways differ in susceptibility to thermodynamic effects (Clarke and Fraser 2004). This creates a considerable
C A RO L I N E M . W I L L I A M S E T A L .
challenge for ectotherms, whose body temperatures fluctuate with environmental temperatures, to maintain energetic and metabolic homeostasis under fluctuating thermal conditions. Species distributions can thus be driven not only directly by exposure to lethal temperatures, but also indirectly through repeated or long-term exposures to sublethal temperatures that lead to energetic failures (Fly and Hilbish 2013). Energetic failures result from an inability to supply or use sufficient energy to perform physiological functions necessary to support life. In marine organisms, energetic failures at temperatures outside the thermal window for growth and reproduction are driven by inability to supply oxygen and thus maintain aerobic scope for normal physiological function (oxygen- and capacity limitation of thermal tolerance, P¨ortner 2001). Oxygen delivery limitations result in an increasing reliance on anaerobic metabolism and the development of low cellular energy levels (Gibbs free energy of adenosine triphosphate [ATP] hydrolysis, Zielinski and P¨ortner 1996). However, terrestrial insects have airfilled tracheal tubes that supply oxygen directly to tissues without reliance on a circulatory system, and there is currently little support for oxygen and capacity limitation of thermal tolerance in these taxa (Klok et al. 2004; Stevens et al. 2010; MacMillan et al. 2012a; McCue and De Los Santos 2013). The “weak link” in the energy supply and demand chain that explains variation in performance at extreme temperatures thus remains to be identified in terrestrial insects (and other terrestrial ectotherms). In insects, low temperatures induce a reversible loss of neuromuscular coordination (chill coma) during which mating and foraging is precluded and predators cannot be avoided. Recovery time from chill coma is a commonly used metric of cold hardiness. Because chill-coma recovery time is usually assessed after exposure to mild low temperatures that do not cause cold shock (i.e., direct chilling injury that develops rapidly as a result of protein denaturation, membrane phase transitions, or cell death; Koˇst´al et al. 2011a), variation in recovery time likely results from variation in either the ability to maintain homeostatic processes in the cold or the rate of recovery of those processes upon rewarming. Ionic regulation is a critical homeostatic process compromised in the cold. While an insect is in chill coma, membrane equilibrium potentials are progressively depolarized as extracellular [K+ ] increases due to a loss of ionic regulation (MacMillan and Sinclair 2011a; Andersen et al. 2013; Findsen et al. 2013). In the fall field cricket, this progressive loss of ionic homeostasis is accompanied by the development of chilling injury, and, upon rewarming, osmotic and ionic equilibria are gradually restored (MacMillan and Sinclair 2011b). Recovery (the resumption of coordinated movement) coincides with the restoration of osmotic and ion concentration gradients, and the speed of recovery of ion homeostasis correlates positively with cold hardiness (Findsen et al. 2013).
2
EVOLUTION 2014
Summary of the response to cold exposure and selection for cold tolerance. (A) Proportion of 20 flies still in chill coma as a function of time in replicate lines of cold-hardy (black) and Figure 1.
cold-susceptible (gray) Drosophila melanogaster at the time of collection of the samples for the metabolomics experiment. Letters indicate lines that are significantly different according to an accelerated failure time model with a log-logistic error distribution, and the “+” indicates one censored value—a single fly from one of the susceptible lines did not wake up within 60 min. Hardy lines are statistically indistinguishable from each other, and have faster recovery than either of the susceptible lines. (B) Experimental design of the metabolomics experiments. (C) Numbers within each circle indicate the corresponding number of metabolites displaying a significant main effect of cold exposure, selection regime, or an interaction between the two. The number outside all circles indicates metabolites that did not respond to selection or cold exposure.
M E TA B O L I C H O M E O S TA S I S I N C O L D - A DA P T E D D RO S O P H I L A
A
B Higher in hardy
Scores on PC1
*
Before
0.3
Scores on PC2
Hardy
Susceptible
Before
2
Fold change (susceptible/hardy)
3
4
During
After
0.2
-0.3
ADP B.Ala Treh Kynn Lys Mal HK Met.sulf Asp Asn Dimeth NAD KynA Sucr Tyr PChol Gln Glu Ser Tau His Pro Fum AMP Fruc Gly Malt Arg Prop Gluc Form Leu Val Ace Ile Thr Ala Lac Suc
1
After
Tau Glu Val Gluc Pro Arg Leu Ile Ala Gln Ace Sucr AMP Prop Mal Lac Gly NAD Ser Tyr Suc Form Fruc Thr Fum B.Ala HK Asp Malt Asn His Lys Kynn ADP KynA Treh PChol Met.sulf Dimeth
* * * * 0
During
-0.1
*
−2
Susceptible
C
*
*
*
Hardy
Loadings
* * * * * * * * ** * * *
Loadings
3−HK Asp Tyr Sucr Asn ADP Treh Tau Gluc Val Ile NAD Mal Gln Arg AMP Ala Ace Glu Malt Gly Prop Leu Fum Form KynA Kynn Ser B.Ala Lac Pro Lys Suc Fruc His Met.sulf Thr PChol Dimeth
Higher in susceptible
Figure 2. Changes in metabolite content of Drosophila melanogaster resulting from selection for fast or slow recovery from chill coma (hardy and susceptible, respectively). (A) Fold changes of metabolites from hardy (black) or susceptible (gray) flies, pooled across all time
points. Asterisks indicate significant differences between groups (false discovery rate < 0.05; Table 1). (B and C) Principal component scores (B = PC1, C = PC2), and associated loadings of each compound onto those components, for flies sampled before cold exposure, at the end of a 3-h cold exposure (during), or after 5-min recovery (after).
Complete recovery of ionic and osmotic homeostasis following cold exposure is an energetically expensive process that can take several hours (MacMillan et al. 2012b). Although it is clear that the maintenance and recovery of ionic homeostasis plays a proximate role in modulating cold hardiness, the mechanisms underlying evolutionary variation in ion homeostasis are still unknown. Energy supply limitations may compromise the function of ion-motive pumps that regulate ionic and osmotic homeostasis, but to date there are few indications that ATP supply is compromised in insects at low temperatures. Aerobic metabolism and ATP supply is maintained down to the freezing point of the body tissues ( –10°C) in freeze-tolerant pupae of the gall-fly, Eurosta solidaginus (Storey and Storey 1981), and the firebug, Pyrrhocoris apterus, maintains its energy state
right until death from cold injury (Koˇst´al et al. 2004). Some studies document an ATP decline in insects at low temperatures (Pullin et al. 1990; Dollo et al. 2010), but this decline develops slowly and may represent secondary effects rather than the cause of energetic failure. In other cases, ATP accumulates during prolonged or lethal cold exposures (Coulson et al. 1992; Macmillan et al. 2012a), suggesting that the ability to use, rather than synthesize, ATP may be reduced in the cold. Low temperatures may inhibit energy-demanding processes directly through thermal effects on ion-motive pumps or metabolic enzymes, or indirectly via modulation of the lipid membrane environment of cells and organelles (Koˇst´al et al. 2011a; Macmillan and Sinclair 2011a; Findsen et al. 2013). Alterations to lipid membrane fluidity will affect rates of proton leak and thus oxidative
EVOLUTION 2014
3
C A RO L I N E M . W I L L I A M S E T A L .
stress, and performance of membrane-bound proteins such as ionmotive pumps and metabolic enzymes. Whether the effects of temperature are direct or indirect, alterations to energy homeostasis will occur and these alterations should be associated with distinct metabolomic fingerprints. Metabolomics provides a snapshot of a portion of the metabolome at a point in time, and thus does not provide information on flux through underlying pathways. However, correlations among metabolites across biological samples at steady state can be used to infer the relative importance of various enzymatic reactions, and the underlying regulatory structure of the system as a whole (Weckwerth and Morgenthal 2005). The metabolic network of an organism is the complete set of biochemical reactions occurring within its cells, including all metabolic pathways and regulatory interactions among them. Interdependencies arise between metabolites that interact through the metabolic network as a result of fluctuations induced by environmental (e.g., temperature) or intrinsic variability (e.g., enzyme concentration; Steuer 2006). Reaction interdependencies generate patterns of correlations among metabolites that provide a fingerprint of the underlying physiological state of the organism (Camacho et al. 2005; Morgenthal et al. 2006). Therefore, if cold exposure disturbs the physiological state of an organism, we expect alterations to the structure of correlation-based metabolic networks. The degree of disruption to these correlation networks may be associated with underlying genetic variation in chill-coma recovery time. Although no studies have investigated the maintenance of metabolic networks during cold exposure, there is evidence for loss of metabolic homeostasis in insects at low temperatures (Overgaard et al. 2007; Colinet et al. 2012). This manifests in increases in free amino acid concentrations that may result from cold-induced protein breakdown (Lalouette et al. 2007; Koˇst´al et al. 2011b; Colinet et al. 2012) or an imbalance between glycolysis and the tricarboxylic acid (TCA) cycle (Michaud and Denlinger 2007; Koˇst´al et al. 2011b). Cold exposure also increases sugars (Overgaard et al. 2007; Colinet et al. 2012; Teets et al. 2012) and sugar alcohols such as glycerol and sorbitol (Michaud and Denlinger 2007). It is clear that both acclimation and adaptation can modulate the degree of cold-induced disruption to homeostatic processes. Acclimation to low temperatures improves both ionic (Findsen et al. 2013) and metabolic homeostasis in the cold (Overgaard et al. 2007; Colinet et al. 2012). At the whole organism level, chill-coma recovery time is evolutionarily labile—latitudinal variation within and among species is consistent with local adaptation (David et al. 2003; Sinclair et al. 2012), and cold hardiness traits respond to artificial selection in the laboratory, including chill-coma recovery time (Mori and Kimura 2008; Goto et al. 2010). However, we still do not know whether genetically based
4
EVOLUTION 2014
variation in the ability to maintain metabolic processes in the cold underpins variation in susceptibility to cold stress. A strong evolutionary perspective on this question has potential to advance our understanding of the mechanisms of low temperature tolerance in insects. Here, we test the hypothesis that fly lines selected for coldhardiness will maintain metabolic homeostasis to a higher degree than cold-susceptible flies during cold exposure and recovery. Specifically, we predict that the metabolic networks of cold-hardy flies will be more resistant to cold stress, maintaining their structure better during cold exposure than the metabolic networks of cold-susceptible flies, and that the network structure of cold-hardy flies will also be more tolerant of cold stress, reestablishing their structure more quickly during recovery from cold stress. We use replicate experimental evolution lines of Drosophila melanogaster selected in the laboratory for fast or slow recovery from chill coma. These lines originate from a natural population in Raleigh, North Carolina, and have stable and genetically based divergence in chill-coma recovery. We use nuclear magnetic resonance (NMR) spectroscopy to profile the water-soluble metabolome, demonstrating that selection for rapid chill-coma recovery results in improved metabolic homeostasis and more robust metabolic networks.
Materials and Methods FLY STOCKS AND EXPERIMENTAL DESIGN
Flies from two base populations, each collected at the same locale in Raleigh, North Carolina, the same populations that gave rise to the D. melanogaster genetic reference panel (Mackay et al. 2012). These two base populations were selected for 31 generations for fast (hardy) or slow (susceptible) recovery from chill coma at Kansas State University, and thereafter maintained at 25°C (Supporting Information). These two base populations gave rise to two independent lines each of hardy and susceptible flies that were used in all metabolomics experiments. Flies for this study were reared at University of Florida at 25°C on a 12:12 L:D cycle in 235-mL bottles on molasses-cornmeal-yeast medium under controlled density, achieved by allowing 10 females (accompanied by five males) to oviposit for 48 h. This resulted in uncrowded conditions and relatively synchronous emergence. On day 12 following the beginning of oviposition, emerged flies were cleared and discarded. Twenty-four hours later emerged flies were transferred to holding bottles for 24 h to ensure mating, and groups of 20 females were sorted under light CO2 anesthesia (48 h before use in experiments (yielding 5to 8-day-old mated females). Eight replicate pools of 10 flies from each line were frozen in liquid nitrogen at one of three time points: (1) before cold exposure, (2) at the end of a 3-h exposure at 0°C, and (3) after
M E TA B O L I C H O M E O S TA S I S I N C O L D - A DA P T E D D RO S O P H I L A
Table 1. The effect of cold exposure and selection for cold tolerance of levels of metabolites in Drosophila melanogaster, measured using 1 H NMR spectroscopy.
Cold exposure
Selection regime
Exposure × selection
Metabolite
Abbreviations.
F
Q
F
Q
F
Q
3-Hydroxykynurenine O-Phosphocholine L-Glutamate L-Tyrosine Taurine Sucrose L-Aspartate Nicotinamide adenine dinucleotide Adenosine diphosphate Malate L-Histidine D-Glucose L-Alanine L-Proline Dimethylamine Methionine sulfoxide Acetate Threonine Glycine L-Valine Lactate Trehalose L-Arginine Adenosine monophosphate L-Glutamine Succinate β-Alanine Kynurenine Fumarate L-Lysine L-Isoleucine Propionate Maltose L-Serine L-Asparagine L-Leucine Fructose Kynurenate Formate
3-HK PChol Glu Tyr Tau Sucr Asp NAD ADP Mal His Gluc Ala Pro Dimeth Met.sulf Ace Thr Gly Val Lac Treh Arg AMP Gln Suc B.Ala Kynn Fum Lys Ile Prop Malt Ser Asn Leu Fruc KynA Form
4.03 2.27 27.62 1.85 0.99 5.83 19.92 1.3 14.04 13.09 2.64 2.43 3.83 8.95 3.99 1.47 8.54 3.52 2.43 0.47 6.76 6.86 2.5 0.49 1.33 6.09 4.02 6.39 0.88 2.35 0.18 3.36 0.34 0.27 2.16 0.31 0.03 0.51 1.36
0.061 0.185
