PRUITT, Nancy L.; SHAPIRO, Craig: Evidence for a cryoprotective protein in freeze tolerant larvae of the goldenrod gall fly, Eurosta solidaginis

Third instar larvae of the goldenrod gall fly, Eurosta solidaginis, overwinter in a ball gall on the stem of the goldenrod, Solidago spp., where they are exposed to a wide range of environmental extremes. Populations of gall fly larvae from central New York (USA) undergo a transition from freeze-susceptible to freeze-tolerant in early October. This transition is accompanied by the production of several sugars, including trehalose, glycerol, and sorbitol. We found that freeze tolerant larvae (collected between November and March) also produce a soluble, heat stable, cryoprotective protein (CRP) capable of preserving up to 80 percent of the activity of freeze-labile rabbit muscle lactate dehydrogenase against freeze-thaw denaturation. The CRP is more than 10 times more effective than sugars and about 4 times more effective than bovine serum albumin. Preliminary studies indicate that freeze susceptible larvae (collected in August and early September) may also produce the CRP, but at significantly lower levels than freeze-tolerant larvae. Partial purification of the CRP from freeze tolerant larvae using DEAE ion exchange chromatography resulted in an active fraction significantly enriched with a 29 kDa protein compared with crude preparations. This molecular mass compares well with that of cryoprotective proteins reported from ice nucleating bacteria (29 kDa) and Arabidopsis (15 and 26 kDa), but is not similar to heat shock proteins previously reported from gall fly larvae (71 and 94 kDa). This indicates that the CRP may be a formerly unknown protein in Eurosta.