Water loss, not overheating, limits the activity period of an endothermic Sonoran Desert bee

Animals In June 2020, we studied a mating aggregation of C. caesalpiniae males in rural Scottsdale, Arizona (GPS coordinates: 33.727, -111.799), focusing on respirometric measurements (Johnson et al., 2022), critical maximum temperatures, and field body temperatures. In May 2021, C. caesalpiniae males and females emerged at the same site. We measured crop volumes and sugar content, water loss rate, microclimate selection, and field body temperatures. In 2022, we measured critical water contents for C. pallida males and females collected in the flood plains of the Tonto National Forest (GPS coordinates: 33.552, -111.566), and for C. caesalpiniae females collected in Cottonwood, Arizona (GPS coordinates: 34.726, -112.017). We took all mass measures using a Mettler Toledo XPE56 XPE micro-analytical balance (accurate to 0.000001 g) unless otherwise specified. Crop volume and sugar content We measured the crop volume and sugar content of C. caesalpiniae males flying across 24 to 45 °C air temperatures in 2021. After netting a bee, we squeezed the abdomen caudally to cranially until the proboscis extended to extrude crop contents. We collected crop contents with a 10 µL glass microcapillary tube, using multiple tubes if necessary. We measured the length of the microcapillary tube using digital calipers (accurate to 0.01 mm) to estimate volume. We transferred the contents of the microcapillary tube(s) to a BRIX refractometer (V·RESOURCING, model VLT032) to measure sugar content. Mark-recapture To provide measures of how long males persist at an aggregation site within a day and across days, we conducted a mark-recapture study. On May 11th, 2021, from 6:30-7:30 AM, we marked ~200 large morph C. caesalpiniae males on the thorax with a dot of blue acrylic paint. On May 12th, 13th, 17th, 18th, and 20th, we netted bees through the morning (6:30 – 11:30 AM), counting the total number of marked and unmarked males. We released all captured males. Microclimate selection To record changes in microclimate selection through the activity period in 2021, we marked four, one-by-one-meter patches with two in shade and two in direct sunlight. Every 30 minutes, from sunrise to midday, we recorded 20-second videos of each patch, and measured ground and air temperature with a BAT-12 thermometer and copper-constantan thermocouple. We analyzed videos by manually counting the number of large males on the ground or in flight. We did not count small morph males or females; both were easily identifiable by their dark black abdomens (Fig. 1A). We coded behavior based on the initial observed location of the individual. We summed number of bees in each location for each patch to determine the relative number of bees in sun or shade and flying versus on the ground. Metabolic water production for flying Centris caesalpiniae males We calculated metabolic water production from previously published carbon dioxide emission rates (Johnson et al., 2022). We estimated metabolic water production (MWP) assuming carbohydrate metabolism. Although the respiratory quotient of flying C. caesalpiniae has not been measured, bees have been reported to utilize carbohydrates (Bertsch, 1984; Gäde and Auerswald, 1999; Suarez et al., 2005). We estimated metabolic water production in mg H2O·g-1·h-1 in Equation 1: MWP = [VCO2 · (22.4 L·mol-1)-1] ·18000 mg·mol-1 (1) Critical water content (CWC) In 2022, we recognized the need for CWC measures, but were unable to capture C. caesalpiniae males. We measured CWC for C. pallida males and females and C. caesalpiniae nesting females. For both species, we netted the first bees to begin flying and those with little to no wing wear or hair loss on the thorax. Bees were transported from the field sites (45 – 70 minutes) to the laboratory in pre-weighed 10 mL tubes with ventilation holes. Bees remained still in the tubes and did not fly, defecate, or regurgitate. We weighed the bees and placed them in a sealed plastic container containing silica gel, at a relative humidity < 1% at 30 ˚C. We recorded masses every hour, and then immediately after death of the bee. We dried all individuals in an oven at 50 °C until masses were constant (dry mass, md). Critical water content was calculated as: CWC = [(mf - md) · mi-1] ·100% (2) where mf is the final wet mass and mi is the initial wet mass. Field validation of water loss rates Because wind and solar radiation conditions differ for bees in the field versus in the respirometer, and because water loss rates during flight were only measured for a few minutes in the respirometer, we performed a field-validation of water loss rates for large C. caesalpiniae males in 2021. Before sunrise, we caught ten large morph males and individually marked them with acrylic paint on the thorax or abdomen. We put five in a cage in the sun and five in a shaded location. The 30 cm3 cages were constructed with aluminum 6.4 mm mesh, allowing bees to fly or walk. Bees in the sun were usually flying, while bees in the shade tended to remain still. Every 30 minutes, we measured air temperature, ground temperature, and total body mass of each bee using a portable field balance (Fisher SLF103, accurate to 0.001 g). Water loss rates were calculated as the change in mass per unit time. Critical thermal maxima (CTmax) To ensure the ecological relevance of our CTmax measures, we defined CTmax as the temperature at which bees lost control of flight. We caught large and small morph C. caesalpiniae males in the field in 2020 and transported them to the first author’s kitchen in individual tubes within a dark, insulated bag. Before the experiment, we allowed bees to adjust to room temp (26-30 °C) for one hour. We placed one individual in a 500 mL glass chamber coated with fluon to prevent landing. We turned on a 200-Watt lightbulb in an insulated box, and gently stimulated the bee to fly if they were not already flying. The chamber was heated at a rate of about 0.5 °C per min (Fig. S1). We measured chamber temperature using a copper-constantan thermocouple thermometer (Type T, Gauge/diameter [MJ1] ) connected to a Pico Technology USB TC-08 Thermocouple Data Logger (Tyler, TX, USA). We defined flight failure as occurring when the bees could not sustain flight; usually they continued to buzz around the bottom of the chamber. Within five seconds of flight failure, we transferred the bee to a Styrofoam board and measured head, thorax, and abdomen temperature as described in Johnson et al., 2022.