Drosophila Melanogaster Locomotion in Cold Thin Air Michael E
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364 The Journal of Experimental Biology 209, 364-371 Published by The Company of Biologists 2006 doi:10.1242/jeb.01999 Drosophila melanogaster locomotion in cold thin air Michael E. Dillon* and Melanie R. Frazier Department of Biology, Box 351800, University of Washington, Seattle, WA 98195-1800, USA *Author for correspondence (e-mail: [email protected]) Accepted 16 November 2005 Summary The alpine environment is likely to challenge insect performance were more pronounced at colder locomotion because of low mean temperatures and temperatures. Reduced flight performance in high altitude reduced barometric pressure. In this study, we measured conditions was primarily driven by an increased the direct and interactive effects of these factors on reluctance for flies to initiate flight rather than outright walking and flight performance of wild-caught Drosophila failure to fly. Such reluctance to fly in high altitude melanogaster Meigen. We found that decreased conditions may in part explain the prevalence of aptery temperature and decreased air pressure both reduced and brachyptery in high altitude insects. The observed walking speed and flight performance. Flies walked more interactive effects of temperature and air pressure on slowly at 18°C and in the lowest air pressure treatment flight performance confirm the importance of (34·kPa). This treatment, equivalent in air pressure to the simultaneously manipulating both of these factors when top of Mount Everest, was the only air pressure that studying the impact of altitudinal conditions on insect significantly reduced fly walking speed. Therefore, physiology and behavior. walking performance in the wild is likely limited by temperature, but not oxygen availability. In contrast to walking performance, low but ecologically realistic air Key words: Drosophila melanogaster, high altitude, mountain, flight, pressures dramatically reduced overall flight walking speed, temperature, physiology, interaction, air density, air performance. The effects of reduced air pressure on flight pressure, oxygen. Introduction whether these factors, independently and in concert, Altitudinal gradients are characterized by rapid changes in compromise insect locomotory performance. the physical environment. Mean air temperature drops by Decreased temperatures at high altitude affect insect 6°C·km–1, such that the temperature change in climbing from performance because physiological reaction rates are strongly sea level to 4000·m is roughly equivalent to the change determined by temperature (Huey and Kingsolver, 1989). Low experienced in traveling 4500·km in latitude (Dillon et al., in temperatures reduce metabolic rates and change the dynamics press). Accompanying this rapid temperature change, at of muscle contraction (Josephson, 1981), impairing muscle 4000·m the partial pressure of oxygen (PO2) falls to half its physiology (Scaraffia and De Burgos, 2000; Hosler et·al., sea level value, and air density decreases by 40%. These 2000) and therefore locomotory performance of fruit flies. physical changes should, both individually and in concert, Walking speed of D. melanogaster is slower in colder compromise the physiology of ectothermic insects (Mani, temperatures (Gibert et al., 2001; Crill et al., 1996). Similarly, 1968; Sømme, 1989, 1995). Despite these challenges, in tethered flies, less power is produced for flight at low however, many ectothermic insects maintain robust temperatures (Curtsinger and Laurie-Ahlberg, 1981; Lehmann, populations on mountains (e.g. Mani, 1968; Brehm et al., 1999). 2003; Romero-Alcaraz and Avila, 2000). Fruit flies in the Reduced PO2 may compromise fruit fly locomotion by genus Drosophila (family Drosophilidae) are found from less reducing metabolic rate. The dramatic altitudinal reduction than 100·m to over 3000·m in the Californian Sierra Nevada in PO2 may challenge tissue oxygen delivery because (Dobzhansky, 1948) and above 5000·m in the Indian insects depend on diffusion (at least through the terminal Himalayas (Khare et al., 2002). To maintain robust tracheoles) for which PO2 is the driving force (Denny, 1993). populations on high mountains, these small ectothermic However, oxygen delivery may not limit insects at high insects must locate food and mates, and evade predators, altitude because insects are generally very resistant to despite the combined physiological challenges of low low oxygen levels (Krishnan et al., 1997; Hoback and temperature, low PO2 and low air density. Here we ask Stanley, 2001; but see below for potential interactions THE JOURNAL OF EXPERIMENTAL BIOLOGY Drosophila locomotion at high altitude 365 between PO2 and temperature and their effects on oxygen female flies to fresh food vials every 24·h and allowed the flies delivery). to mature for 3–4 days before testing their locomotory Reduced air density should make it difficult to fly at high performance (we only tested virgin flies). We chose this age altitude because aerodynamic forces produced by wing because wing-beat frequency and power output of flies remains flapping increase linearly with air density (Dudley, 2000). constant from 2 to 8 days of age (Curtsinger and Laurie- Despite this theoretical limitation, many insects fly on high Ahlberg, 1981). To assess performance, we first starved flies mountains (Mani, 1968). Furthermore, in the laboratory, orchid for 16–20·h in individual 1.5·ml Eppendorf tubes containing bees fly in pure heliox (21% O2, balance helium), despite the fresh agar for water; moderate starvation increases average 64% reduction in air density (approximately equivalent to the flight speed (David, 1978) and duration (Tammero and top of Mount Everest; Dudley, 1995). To do so, orchid bees Dickinson, 2002), and preliminary experiments demonstrated increase mechanical power output by 40–50%, suggesting that that starvation motivated flight. Then, individual flies were flight in low air densities, though possible, may be randomly assigned to a single temperature/air pressure energetically expensive. treatment to test either walking or flight performance. Some research exists on the independent effects of Experiments were performed in a walk-in environmental temperature, PO2, and air density on insect locomotory chamber; humidity was kept near 100%, and temperature was performance. However, high altitude ecosystems are uniquely monitored using a calibrated thermocouple thermometer characterized by the combination of these factors, yet no study (Physitemp Bat–12, Bailey Instruments Inc., Saddlebrook, NJ, has investigated the interactions among these factors and their USA). Given the relationship between air density, pressure and potential synergistic effects on insect locomotion. Recent work temperature, we could not simultaneously keep both air documenting large interactive effects of temperature and pressure and air density constant at different temperatures. We oxygen on insect development (Frazier et al., 2001; Woods and chose to keep air density constant because of its direct link to Hill, 2004) strongly suggests that such interactions should be production of flight forces; consequently, at a given air density, considered in studies of insect physiology. air pressure increases slightly with temperature. Here we measure the direct and interactive effects of temperature and barometric pressure on walking and flight Walking performance performance of wild-caught Drosophila melanogaster. We use We modified the technique of Gilchrist et al. (1997) to assess a standard technique to measure walking speed (Crill et al., walking speed of flies at three temperatures and four air 1996; Gilchrist, 1996) and introduce a novel method to densities: 18, 25, 30°C (18.09±0.38°C, 25.34±0.18°C and measure whole animal flight ability and flight motivation. We 29.61±0.33°C, means ± s.d., respectively); 33%, 50%, 66%, find strong negative effects of reduced temperature and and 100% sea-level air pressure (34.27±0.74·kPa, reduced air pressure on flight ability and walking speed. Low 51.19±0.68·kPa, 67.06±1.04·kPa and 100.97±1.75·kPa, means temperatures and low air pressures interact to reduce flight ± s.d., respectively). We placed 10 flies individually inside performance more than predicted by the additive effects of 10·ml (6·ml volume after we had cut them down) plastic these variables. Our results suggest that future studies on high graduated burets that were connected in series to a vacuum altitude physiology incorporate the suite of physical factors pump. After adjusting air pressure inside the burets, we that characterize high altitude ecosystems. allowed the flies to acclimate for 5·min. We then knocked each fly to the bottom of its buret and timed to the nearest 0.01·s how long it took to walk 12·cm vertically. This was repeated Materials and methods twice more for each fly and the average was used to calculate Experimental animals walking speed. If a fly jumped, flew or walked in a spiral, the In July 2001, we collected female D. melanogaster Meigen time was discarded and the fly was knocked to the bottom of from a pear orchard near Wenatchee, WA, USA (47°34ЈN, the buret and retimed. 120°36ЈW). We combined the 38 resulting isofemale lines into a single colony, which served as the source of our experimental Flight performance flies. To maintain the colony, we collected eggs approximately We assessed flight performance at four temperatures and at every 10 days and reared them in controlled conditions (50 four air densities: 18, 25, 30, 32°C (18.05±0.23, 25.22±0.51, eggs/vial; ~23°C and ~16·h:8·h L:D; diet of cornmeal, 30.11±0.28, 31.91±0.28, mean ± s.d., respectively); 33%, 50%, molasses, yeast, agar, tegosept) before transferring newly 66% and 100% sea level (33.28±1.81·kPa, 50.87±1.34·kPa, emerged adult flies into the colony. Colony food bottles 67.46±1.22·kPa and 101.82±1.6·kPa, means ± s.d., (150·ml) were replaced every 3–5 days, ensuring that all respectively). The flight chambers were 250·ml glass milk colony flies developed in controlled conditions. The colony bottles inverted over rubber stoppers to form an air-tight seal was maintained at 1000–1500 flies for 3–6 generations prior to (Fig.·1).