Insect eggs must obtain oxygen across the eggshell to support embryonic development. Because eggs are small, obtaining enough oxygen would seem trivial. Recent work, however, has shown that eggs of a moth, Manduca sexta, are oxygen limited at high but realistic temperatures (32-37 degrees C) and that P(O2) drops steeply across the eggshell. Here we use theoretical and experimental approaches to partition the total resistance to oxygen flux among several steps in the oxygen cascade from environment to embryo. Standard mass-transfer analysis suggests that boundary layers of air around eggs, and around substrates to which they are attached, offer negligible resistance. Likewise, a mathematical model, parameterized using published and newly obtained morphological data, predicts that air-filled parts of the chorion also do not resist oxygen flux. This prediction was confirmed by experiments that measured rates of carbon dioxide emission from batches of eggs subjected simultaneously to hypoxia and inert gas substitution: depression of metabolic rate by hypoxia was not rescued when the diffusion coefficient of oxygen in air was doubled by substituting helium for nitrogen. The model did predict, however, that a set of subchoral layers (a crystalline chorionic layer, a wax layer and the vitelline membrane) could account for most or all of the total resistance to oxygen flux. Support for this prediction was obtained from two sequential experiments. First, eggs extracted with chloroform:methanol had highly elevated rates of water loss, suggesting that indeed eggs of M. sexta are waterproofed by wax. Second, rates of water loss and carbon dioxide emission from batches of eggs, measured from laying to hatching, changed in parallel over development. These data suggest that a single layer, likely a wax layer or a combination of wax and other subchoral layers, provides the main resistance to water efflux and oxygen influx.
