Estimating Metabolic Rates for Small Terrestrial Arthropods Using Stop-Flow Gas Respirometry and Overwintering Megachile rotundata

Gas respirometry is widely used as a proxy of metabolic rate and provides valuable insights into an organism's energetic demands and performance. Compared to traditional flow-through respirometry, stop-flow respirometry is a versatile and effective method for quantifying low metabolic rates. Stop-flow respirometry involves placing an organism in a sealed chamber for a set period of time, then measuring the total CO2 produced and/or O2 consumed, which is then used to calculate the rate of gas exchange. When both CO2 and O2 are measured, researchers can calculate the respiratory exchange ratio (RER), the ratio of CO2 to O2, which indicates the primary energy substrate used by an organism (i.e., lipids, carbohydrates, or proteins). Stop-flow respirometry also allows higher sampling throughput than flow-through respirometry, which often relies on multiplexer setups to process multiple samples at a time. Here, we demonstrate a protocol to measure gas exchange using overwintering solitary bees (Megachile rotundata) in their prepupal stage as the representative example due to their low metabolic rates. We describe the general setup and the timing protocol used to optimize sampling throughout. Results for overwintering M. rotundata show the RER to be approximately 0.7, indicating that lipids are their primary metabolic fuel during overwintering. This simple setup enables the quantification of organismal metabolic rates, even during dormancy. Finally, this method may also be applied to active individuals, if careful consideration is taken for locomotor activity and other physiological states that contribute to overall energy use.