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Hawkmoths use nectar sugar to reduce oxidative damage from flight

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Science  17 Feb 2017:
Vol. 355, Issue 6326, pp. 733-735
DOI: 10.1126/science.aah4634

Sugar rush

Flying requires high levels of energy production, which causes muscular oxidative damage. Food-derived antioxidants can protect against such damage; however, nectar is devoid of these compounds. Levin et al. found that nectar-feeding hawkmoths fed high concentrations of sugar had lower levels of damage than unfed moths. Sugar-fed moths generated antioxidant compounds by shunting glucose through a pentose phosphate pathway. This mechanism may have allowed for the evolution of energy-intensive flying nectarivores.

Science, this issue p. 733

Abstract

Nectar-feeding animals have among the highest recorded metabolic rates. High aerobic performance is linked to oxidative damage in muscles. Antioxidants in nectar are scarce to nonexistent. We propose that nectarivores use nectar sugar to mitigate the oxidative damage caused by the muscular demands of flight. We found that sugar-fed moths had lower oxidative damage to their flight muscle membranes than unfed moths. Using respirometry coupled with δ13C analyses, we showed that moths generate antioxidant potential by shunting nectar glucose to the pentose phosphate pathway (PPP), resulting in a reduction in oxidative damage to the flight muscles. We suggest that nectar feeding, the use of PPP, and intense exercise are causally linked and have allowed the evolution of powerful fliers that feed on nectar.

Floral nectar is the primary nutrient source for many taxa, including birds, insects, and mammals. Nectar is energy rich, consisting predominantly of water and carbohydrates (1) with little to no antioxidant components (2). Foraging for nectar often involves metabolically expensive flight or hovering. Hovering flight is the most energetically expensive form of locomotion known (3), with metabolic rates reaching 170 times higher than at rest (4). To fuel their flight muscles, nectarivores oxidize energy-rich molecules, often a combination of fuel types. Nectar-feeding bats and hummingbirds incorporate nectar carbohydrates into the pool of metabolized substrate shortly after a nectar meal and use both carbohydrates and lipids on their regular diurnal activity. In contrast, lipids are the main fuel type on migratory flights (57). Nectarivorous insects oxidize a wider range of fuel types for flight [glucose, glycogen, trehalose, proline, phosphoarginine, and lipids (3, 8)]. Hawkmoths are known to initiate flight using carbohydrates and shift to lipid oxidation shortly after (9), whereas fed moths tend to burn a mixture of carbohydrates and fat during long bouts of flight (10).

Intense exercise, as in the extremely high aerobic performance of flight muscles in hovering and flying animals, produces reactive oxygen species (ROS) that can cause oxidative damage to the contracting myocytes (11, 12). We tested the effect of sugar feeding on oxidative damage in Manduca sexta (Sphingidae), a hawkmoth. Adults feed exclusively on nectar and hover when feeding. Sugar-fed moths flew about 70% farther in 180 min on flight mills than unfed moths (mean ± SD, fed, 5.8 ± 2.7 km, n = 9; unfed, 3.4 ± 1.1 km, n = 9, t = –2.4, P = 0.0334), suggesting that they should have higher levels of oxidative damage. As expected, fed moths did have higher oxidative damage to muscle protein (8.162 ± 3.2 nmol of protein-carbonyls/mg of protein for fed moths and 5.512 ± 2.7 for unfed moths; F1,27 = 5.5576; P = 0.0259; Fig. 1A). Increased protein damage with flight is expected, as damage to the flight muscle accumulates until the damaged proteins are replaced or recycled. Although damage to muscle protein might affect the function of the muscle, it is nonlethal, and in a short-lived insect, like adult M. sexta, the accumulation of protein damage may outweigh the high cost of repair. By contrast, when ROS attack cell membrane fatty acids, a chain reaction of lipid peroxidation ensues in which one lipid peroxidation event can initiate hundreds of radical reactions (13). This leads to a severe reduction in membrane functions and damage to the cellular lipid bilayers, which can result in cell death. In addition to damage to membrane fatty acids, lipid peroxidation chain reactions also lead to the production of lipid radicals that, in turn, can attack other lipids, proteins, and nucleic acids (14). In contrast to muscle protein, sugar-fed moths had lower oxidative damage to their muscle cell membranes (0.035 ± 0.01 μg of malondialdehyde/mg of protein for fed moths and 0.081 ± 0.08 for unfed moths; F1,25 = 13.154; P = 0.0013; Fig. 1B), indicating that antioxidant activity reduces the peroxidation of membranes in the fed moths.

Fig. 1 Oxidative damage and GSH/GSSG ratio in flight muscles in sugar-fed and starved moths.

(A) Levels of protein oxidative damage. (B)Levels of lipid oxidative damage. (C) Ratios of reduced to oxidized glutathione (GSH/GSSG) (mean ± SD).

Glutathione in its reduced form (GSH) is a key antioxidant in protecting cell membranes by reducing oxidative damage. GSH donates electrons to lipid peroxide, which stops the peroxidation chain reaction (15, 16). We found significantly higher ratios of GSH/GSSG in fed moths (1.9765 ± 0.5 μM glutathione disulfide/mg of protein for fed moths and 1.557 ± 0.5 for unfed moths; F1,29 = 5.146; P = 0.0309; Fig. 1C), indicating that fed moths are better able to recycle oxidized glutathione (GSSG) to its reduced active form (GSH) to control oxidative damage to flight muscle membranes.

GSSG is recycled to GSH by glutathione reductase using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor. The main source of NADPH is the pentose phosphate pathway (PPP). The PPP is an essential and conserved metabolic pathway in bacteria, plants, and animals and is involved in the synthesis of nucleic acid precursors (ribose-5-phosphate) and reducing power in the form of NADPH, two processes vital for the maintenance of cell function (17). The PPP is divided into oxidative (ox-PPP) and nonoxidative (nonox-PPP) branches. Ox-PPP is an irreversible pathway that produces ribulose-5-phosphate, NADPH, and CO2 (from carbon atom 1 of glucose). Ribulose-5-phosphate is a major source of metabolic precursors for biosynthetic processes such as nucleic acids, whereas NADPH functions as reducing power for anabolic process such as fatty acid synthesis, cholesterol synthesis, and the maintenance of a pool of reduced glutathione. Both NADPH and glutathione are important antioxidants used to reduce cellular oxidative damage. The nonox-PPP branch is a reversible pathway that interconverts pentose phosphate and other sugar phosphates. It contributes to the synthesis of ribose-5-phosphate and redirects excess pentose phosphate toward glycolysis (17) and the production of the insect blood sugar trehalose (18), which is important in enantiostasis (8).

When carbohydrates are the sole source of metabolic fuel, for every molecule of O2 consumed, one molecule of CO2 is produced. This yields a respiratory quotient (RQ = VCO2/VO2) of 1.0. The published RQs of many nectarivores are greater than 1.0. Over the past 100 years, high RQ values have been recorded in a variety of animals, including nectar-feeding bats [RQ values up to 1.7 (19)], sunbirds, and their convergent New World hummingbirds [1.3 and 1.4, respectively (20)]. We found that the peak RQ of sugar-fed M. sexta ranged between 1.11 and 1.74 (n = 10, mean ± SD = 1.38 ± 0.19). Such high values have been previously interpreted as “lipid synthesis,” but a mechanism by which lipid synthesis causes a high RQ has never been adequately explained (2123). Declining body mass is characteristic of many adult Lepidoptera, even when fed as adults (24), which suggests that lipid synthesis is not significant in these insects. To the best of our knowledge, under aerobic conditions, these high RQ values can only be explained by the use of the PPP and the release of CO2 from glucose carbon atom 1 (C1) in this metabolic pathway. We suggest that the reduction potential of the PPP can also be used to build endogenous antioxidant potential.

We coupled real-time respirometry with real-time δ13C analysis to determine fuel oxidation strategies (25) of fed and unfed moths at rest and during activity. Moths (n = 12) were fed nectar containing either 13C1- or 13C2-labeled glucose (1.0 mg/ml), then placed in a metabolic chamber at rest. It is more energetically efficient to oxidize ingested sugar directly than to first convert it to lipids, as has been shown for nectarivorous bats and hummingbirds (5). δ13C in the breath of the fed moth started to rise immediately after feeding, reaching a steady-state RQ within 30 to 60 min. When moths were disturbed from rest by shaking the chamber, they activated their flight muscles in a preflight warm-up (4), causing an immediate drop in RQ (Fig. 2). These changes in RQ might also reflect different metabolisms in different organs. For example, when moths are at rest and fed, the fat body and digestive system are active. By contrast, when moths are flying, flight muscles are the primary active organ. During postfeeding rest, more C1 is exhaled as CO2 than for the other five glucose carbons (C2-6). This would occur if C1 were selectively released as CO2 in the PPP (decarboxylation of 6P-gluconate, fig. S1). This additional CO2 results in an RQ >> 1. When the muscles were activated in the preflight warm-up (black arrows in Fig. 2), the δ13C1 decreased (Fig. 2A), suggesting that the 13C1 in the exhaled CO2 is in equal proportion with the other five glucose carbons released as CO2 in muscle mitochondria (Fig. 2A). When the moth is back at rest (red arrows in Fig. 2), RQ increases again as a result of a reduction in the use of glycolysis and the tricarboxylic acid cycle and an increase in the PPP activity by the moth. With 13C2 (second glucose carbon labeled), RQ dropped after muscle activity because of an increase in muscle metabolism as described above, but δ13C2 increased (Fig. 2B). The δ13C2 increase reflects the decrease in the proportion of nonlabeled C1 exhaled as CO2 from PPP activity and an increase in all of the other five carbons (including 13C2) released in the mitochondria. Together, these results support our hypothesis that the “extra” CO2 that causes an RQ >>1 originates from glucose carbon atom 1, as only C1 from glucose leaves through the PPP as CO2 (fig. S1).

Fig. 2 Change in RQ and δ13C over time.

(A) Change in RQ and δ13C for C1-labeled glucose-fed moth. (B) Change in RQ and δ13C for C2-labeled glucose-fed moth. Black arrows indicate when the moth was disturbed and muscle activity started. Red arrows indicate when movement and muscle activity stop.

It has been argued that endogenous antioxidant defense is costly for animals to produce (26). This has led to the suggestion that migrating birds must consume exogenous antioxidants (e.g., anthocyanins and carotenoids) in their food, such as berries, to meet this need (26, 27). However, it is known that insectivorous songbirds also consume sugary fruits and flower nectar during stops along the migratory route (28). Long-distance (>1000 km) migrating Lepidoptera, such as the monarch butterfly, as well as migrating hummingbirds, refuel only with carbohydrates (29). During these refueling stops, shunting nectar glucose through the PPP may provide these animals with the endogenous antioxidant potential needed during the intense exercise of migration. The energetic cost of antioxidant generation by the PPP is low, as adenosine 5′-triphosphate is not required for any of its enzymatic reactions. The two branches of the PPP also make it adaptable to the organism’s needs; when in need of immediate energy, three ribulose-5P molecules arising from the oxidative branch of the PPP can be redirected into glycolysis by converting them into two molecules of fructose-6P and one molecule of glyceraldehyde-3P, and still produce antioxidant potential in the form of NADPH (18). We propose that in flying animals, consuming carbohydrate-rich diets and the use of the PPP during rest are causally linked with the ability to reduce oxidative damage caused by bouts of intense exercise. We suggest that this causal linkage has enabled the evolution of physiological traits that can sustain nectarivores’ metabolically demanding modes of locomotion such as hovering and long-distance migration while feeding only on nectar.

Supplementary Materials

www.sciencemag.org/content/355/6326/733/suppl/DC1

Materials and Methods

Fig. S1

References (3035)

References and Notes

Acknowledgments: This study was supported by NSF grant IOS-1053318 to G.D. We thank H. Costa for maintaining the moth colony and R. Tzach and L. Braulke for fruitful discussion. The authors declare no conflicts of interest.
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