Inoculation triggers freezing at high subzero temperatures in a freeze-tolerant frog (<i>Rana sylvatica</i>) and insect (<i>Eurosta solidaginis</i>)

Abstract

Freezing of animals is often portrayed to begin after they supercool several degrees Celsius below the melting point of body fluids. This supposition is based on laboratory protocol that usually exposes animals only to dry air during freezing episodes, whereas, in nature, animals may be in direct contact with external ice or snow at temperatures above the supercooling point of their body fluids. This raises the possibility that ice nucleation may occur across the epithelium or cuticle, causing the freezing of body fluids. We tested this possibility in two freeze-tolerant animals, the wood frog, Rana sylvatica, and the goldenrod gall fly, Eurosta solidaginis. Frogs remained supercooled for 3 h at −1.5 to −2.0 °C when kept on an unfrozen surface; however, they began to freeze in less than 30 s after coming in direct contact with ice crystals. Seeding occurred in less than 1 min across isolated patches of frog skin held in a modified Ussing chamber. Similarly, externally moistened larvae of E. solidaginis froze at higher temperatures than dry larvae. Likewise, within galls containing a relatively high water content (65.8%), larvae froze at higher temperatures than ones found in drier galls (19.6%). Therefore, animals may freeze at high subzero temperatures, at or near the melting point of their body fluids, owing to transepithelial ice inoculation. In E. solidaginis, exposure to sufficient moisture to trigger inoculation declines as winter approaches; thus, this avenue for freezing seems limited to autumn, when plant galls have a high water content. This study further emphasizes the need to use care in extrapolating laboratory-determined supercooling points as an approximation of the response of animals in the field.