ABSTRACT
Megachile rotundata, the alfalfa leafcutting bee, is a solitary, cavity-nesting bee. M. rotundata develop from eggs laid inside brood cells constructed from leaf pieces and placed in series in an existing cavity. Due to the cavity nesting behavior of M. rotundata, developing bees may experience hypoxic conditions. The brood cell itself and the position of cell inside the cavity may impact the rates of oxygen diffusion creating hypoxic conditions for developing animals. We hypothesized that bees would be adapted to living in hypoxia and predicted that they would be highly tolerant of hypoxic conditions. To test the hypothesis, we measured critical PO2 (Pcrit) in pupal M. rotundata of varying ages. Defined as the atmospheric O2 level below which metabolic rate cannot be sustained, Pcrit is a measure of an animal's respiratory capacity. Using flow through respirometry, we measured CO2 emission rates of developing bees exposed to 21, 10, 6, 5, 4, 3, 2, 1, and 0 kPa PO2 and statistically determined Pcrit. Mean Pcrit was 4 kPa PO2 and ranged from 0 to 10 kPa PO2, similar to those of other insects. Pcrit was positively correlated with age, indicating that as pupae aged, they were less tolerant of hypoxia. To determine if there were developmental changes in tracheal structure that accounted for the increase in Pcrit, we used synchrotron X-ray imaging and measured the diameter of several tracheae in the head and abdomen of developing bees. Analyses of tracheal diameters showed that tracheae increased in size as animals aged, but the magnitude of the increase varied depending on which trachea was measured. Tracheal diameters increased as pupae molted and decreased as they neared adult emergence, possibly accounting for the decrease in hypoxia tolerance. Little is known about respiratory structures during metamorphosis in bees, and this study provides the first description of tracheal system structure and function in developing M. rotundata. Studies such as this are important for understanding how basic physiological parameters change throughout development and will help to maintain healthy pollinator populations.
