DOI: 10.5061/dryad.xsj3tx9jn
title: The thermal performance curve for aerobic metabolism of a flying endotherm

description: Performance benefits of stable, warm muscles are believed to be important
 for the evolution of endothermy in mammals, birds, and flying insects.
 However, thermal performance curves have never been measured for a
 free-flying endotherm, as it is challenging to vary body temperatures of
 these animals, and maximal flight performance is difficult to elicit. We
 varied air temperatures and gas densities to manipulate thoracic
 temperatures of flying honey bees from 29-44°C, with low air densities
 used to increase flight metabolic rates to maximal values. Honey bees
 showed a clear thermal performance curve with an optimal temperature of
 39°C. Maximal flight metabolic rates increased by ~2% per 1°C increase in
 thoracic temperature at suboptimal thoracic temperatures, but decreased
 ~5% per 1°C increase as the bees continued to heat up. This study provides
 the first quantification of the maximal metabolic performance benefit of
 thermoregulation in an endotherm. These data directly support aerobic
 capacity models for benefits of thermoregulation in honey bees, and
 suggest that improved aerobic capacity likely contributes to the multiple
 origins of endothermic heterothermy in bees and other insects.
description: We manipulated body temperatures and assessed maximal capacities
 of bees by flying them in various air densities and temperatures (Table 1,
 2). Foragers were collected in random order from three colonies of the
 Italian honey bee, Apis mellifera ligustica, maintained on the third-story
 balcony of the Interdisciplinary Science and Technology Building 1 at
 Arizona State University in Tempe, AZ, USA. Unloaded, outgoing foragers
 were captured when leaving the colony by holding an opened plastic bag
 (~950 mL) approximately 15 centimeters from the colony entrance. After a
 single forager flew directly into the opened bag, it was sealed and the
 bee was quickly transported to a room regulated at 23±0.5°C or 35±0.5°C
 (EGC, Chagrin Falls, Ohio, USA) and its flight metabolism was assessed at
 a single air density. Substituting helium for nitrogen
 in air lowers its density, requiring bees to generate more lift in order
 to fly (19,22). This substitution will also increase heat loss rates
 because helium has a thermal conductivity about six-times higher than
 nitrogen (21,23,24). To further manipulate heat loss, we examined
 metabolic rates and body temperatures at two air temperatures, 23 and
 35°C.  Heat loss rates are proportional to thermal gradient between an
 animal’s body and ambient temperature. Thus, we predicted that flight in
 gases enriched in helium at low air temperatures would induce the greatest
 heat loss rates and therefore the coolest body temperatures, whereas heat
 loss would be lowest in nitrox mixtures at 35°C air
 temperatures. Metabolism during free flight was
 assessed in a cylindrical, transparent acrylic flight chamber (350 mL).
 After placing the bee in the chamber, it was sealed and covered with a
 dark cloth to encourage reduced activity of the bee and the chamber was
 flushed to completely replace atmospheric air and water with the desired
 gas mixture. Gas mixtures were created by using cylinders of pure O2, N2,
 and He, which were regulated at a total flow rate of 2 L·min-1 by a
 multi-channeled Sable Systems FB8 flow meter system (Las Vegas, NV, USA).
 Each flow meter was calibrated for the different gas densities using a
 soap-film bubble meter. The gases from the flow meters flowed sequentially
 through a CaSO4 and soda lime column to remove H2O and CO2, the reference
 cell of a LI-COR 6262 CO2/H2O analyzer (Lincoln, NE, USA), the
 respirometry chamber, a small column of MgSO4 to remove water produced by
 the bee, and then the sample cell of the LI-COR. Differential analog
 output from the LI-COR was digitized (Sable Systems UI-2) and recorded
 each second (Expedata, Sable Systems, Las Vegas, NV). The LI-COR was
 calibrated using 252 ppm CO2 and Ultra-Zero calibration gases at the same
 flow rate and pressure (761.5-761.8 mm Hg) as during the flight
 respirometry, and baseline recordings were taken before and after each
 measurement period. Flight was then encouraged for
 two minutes by shining a 150W dual goose-neck Fiber Optical Illuminator
 (China) over the chamber. Bees that landed were immediately encouraged to
 fly by gently tapping and inverting the chamber. Flight behavior was
 categorized based on ability, duration, and control (i.e., quality; 25).
 Flight was categorized and ranked as: 1 – no flight, 2 – flapping wings
 with brief periods of flight (&lt; 3 sec), 3 – intermittent flight
 characterized by frequent crashing (i.e., bee usually ends upside down),
 4 – intermittent flight characterized by frequent controlled landing
 (i.e., bee gently lands on its feet), or 5 – continual, stable flight.
 Expedata (Sable Systems, Las Vegas, NV) was used to find and average the
 10 seconds with the highest CO2 readings during each trial. Flight
 CO2 emission rates (mL·hr-1) were calculated by multiplying the decimal
 CO2 fraction times the STP flow rate through the flight chamber. After
 flight CO2 emission rates were measured, the bee was shaken into a plastic
 bag, which was flattened to restrict the bee’s movement. Thoracic
 temperature was then measured by inserting a Physitemp model MT29/1
 hypodermic microprobe (Clifton, New Jersey, USA; 29-gauge, time
 constant=0.025·s) through the bag and into the center of the thorax. The
 temperature data were recorded with a Pico Technology USB TC-08
 Thermocouple Data Logger (Tyler, TX, USA). Thoracic temperatures were
 measured within five seconds of cessation of flight, and the highest
 temperature reported by the thermometer was recorded. After measurement,
 the bee was weighed (±0.1 mg) using an A&D HR-120 Analytical
 Balance (Tokyo, Japan) and stored at -20°C. Data were
 analyzed using R (3.6.2; R Foundation for Statistical Computing, Vienna,
 Austria). Two-tailed significance was determined at α = 0.05. We used a
 linear mixed-effects model to test the independent and interactive effects
 of air temperature and gas density on flight metabolic rate [i.e.,
 milliwatts (mJ·sec-1)] and thoracic temperature, with hive included as a
 random effect. To determine the independent effect of gas density on
 thorax temperature, we ran a linear model for each separate air
 temperature. We also ran a similar model for the above independent
 variables, with body mass included in the model. We used an ordinal
 logistic regression model analysis to test the independent and interactive
 effects of air temperature, gas density, and thoracic temperature on
 flight quality. Models were chosen using the Akaike Information Criterion
 (AIC) and the Bayesian Information Criterion (BIC).
description: Data were analyzed using R (3.6.2; R Foundation for Statistical
 Computing, Vienna, Austria).