Fat body cells and calcium phosphate spherules induce ice nucleation in the freeze-tolerant larvae of the gall fly Eurosta solidaginis (Diptera, Tephritidae)

During the autumn, the third-instar larvae of the gall fly Eurosta solidaginis acquire freeze tolerance and their crystallization temperatures increase into the -8 to -10 °C range. Despite conflicting reports, efficient endogenous ice nucleators have not been identified in this freeze-tolerant insect. We found large crystalloid spheres within the Malpighian tubules of overwintering larvae. Energy-dispersive X-ray microanalysis and infrared spectroscopy indicated that the spherules were a hydrate of tribasic calcium phosphate. To test for ice-nucleating activity, we placed the calcium phosphate spherules in 10 µl of Schneider's insect medium and cooled them in a refrigerated bath. The addition of spherules increased the crystallization temperature of Schneider's medium by approximately 8 C, from -18.4±0.8 °C to -10.1±0.9 °C (mean ± s.e.m., N=20). Ice-nucleating activity (-10.9±0.9 °C) was also demonstrated in fat body cells suspended in 10 µl of Schneider's medium. Both calcium phosphate spherules and fat body cells have ice-nucleating activity sufficiently high to explain whole-body crystallization temperatures. Furthermore, other crystalloid deposits, commonly found in diapausing or overwintering insects, also exhibited significant ice-nucleating activity. These endogenous crystalloid deposits represent a new class of heterogeneous ice nucleators that potentially regulate supercooling and promote freeze tolerance in E. solidaginis and possibly in other overwintering insects.