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 sol idaginis acquire freeze tolerance and their crystallization temperatur es increase into the -8 to -10 degrees C range. Despite conflicting re ports, efficient endogenous ice nucleators have not been identified in this freeze-tolerant insect. We found large crystalloid spheres withi n the Malpighian tubules of overwintering larvae. Energy-dispersive X- ray microanalysis and infrared spectroscopy indicated that the spherul es were a hydrate of tribasic calcium phosphate, To test for ice-nucle ating activity, we placed the calcium phosphate spherules in 10 mu l o f Schneider's insect medium and cooled them in a refrigerated bath. Th e addition of spherules increased the crystallization temperature of S chneider's medium by approximately 8 degrees C, from -18.4+/-0.8 degre es C to -10.1+/-0.9 degrees C (mean +/- S.E.M., N=20). Ice-nucleating activity (-10.1+/-0.9+/-0.9 degrees C) was also demonstrated in fat bo dy cells suspended in 10 mu l of Schneider's medium. Both calcium phos phate spherules and fat body cells have ice-nucleating activity suffic iently high to explain whole-body crystallization temperatures. Furthe rmore, other crystalloid deposits, commonly found in diapausing or ove rwintering insects, also exhibited significant ice-nucleating activity . These endogenous crystalloid deposits represent a new class of heter ogeneous ice nucleators that potentially regulate supercooling and pro mote freeze tolerance in E. solidaginis and possibly in other overwint ering insects.