A method for quantitative determination of ice nucleating agents in insect hemolymph.

The presence of nucleating agents in the hemolymph appears to be a characteristic feature of insects tolerant to freezing (10). Such nucleators have been found in the hemolymph of insects of several phylogenetic orders, i.e., Coleoptera (4, 8, 9, 1 l), Diptera (7), and Hymenoptera (5). The physiological function of these nucleators is suggested to be to induce a protective extracellular freezing at relatively high subzero temperatures, preventing injurious freezing in intracellular compartments (10). Several studies indicate that the concentration of nucleators varies seasonally and that they may be absent from the hemolymph during summer (4, 9, 11). Unfortunately, there is no method available which allows a quantitative determination of the level of nucleating agents in the hemolymph of insects. The purpose of the present study has been to develop a method for determining the concentration of nucleating agents in insect hemolymph. The method is developed from experiments with nucleators from hemolymph of freeze-tolerant Eleodes blanchardi beetles. The practical applicability of the method is demonstrated by measurements of nucleator concentrations in the hemolymph of the gall fly larvae, Eurosta solidaginis, acclimated to different temperatures.