063 Functions of insect antifreeze proteins and glycolipids

Thermal hysteresis (TH), indicative of antifreeze proteins (AFPs) or antifreeze glycolipids (AFGLs), has been identified in numerous species of insects. The AFPs or AFGLs responsible for the TH in most of these have not been described, and, given the diversity of insects, there are numerous new TH-producing substances yet to be discovered. While it is assumed that AFPs and AFGLs function to prevent freezing, details of their function(s) in vivo are sparse. Freeze avoiding larvae of the beetle Dendroides canadensis produce a family of over 30 AFPs (DAFPs) that are structurally similar to those of other beetles such as Tenebrio molitor. There is tissue specific expression with certain DAFPs present in hemolymph, mid- and hind-gut, primary urine, and epidermis, suggesting variation in function specific to the site. Some DAFPs exhibit self-enhancement whereby activity is increased by certain other DAFPs, as well as by endogenous glycerol (∼0.5M in hemolymph), a thaumatin-like protein, or AFGL. Hemolymph and epidermal DAFPs inhibit inoculative freezing across the cuticle when larvae are in contact with ice. Hemolymph and gut DAFPs inhibit ice nucleator proteins and bacteria, thereby promoting supercooling. Likewise, DAFPs in the primary urine inhibit ice nucleating crystals present in insect urine. Similar AFPs in freeze avoiding larvae of the beetle Cucujus clavipes assist (along with high glycerol and cryoprotective dehydration) in the extreme supercooling ability of the Alaskan subspecies whereby when in a mid-winter deep supercooling state they do not freeze even at −150 °C, but vitrify at ∼−60 to −70 °C. While low hemolymph TH is present in several freeze tolerant insects, AFPs have not been sequenced from these sources. However, a xylomannan-AFGL with TH activity similar to insect AFPs was purified from freeze tolerant Alaskan beetles, Upis ceramboides and a few other freeze tolerant insects, plants, and frogs. As AFPs and the AFGL inhibit recrystallization, this is likely to be a function in freeze tolerant species. However, most of the AFGL is present on cell membranes where it could also inhibit inoculation of the cytoplasm by extracellular ice. There may be additional functions of AFPs unrelated to antifreeze. Several insects have low TH in summer, and D. canadensis larvae synthesize certain DAFPs, but not AFGL, through the summer. TH was first identified by Ramsay in his classic studies of the water reabsorption ability of the cryptonephridial rectal complex of Tenebrio molitor. TH was present in hemolymph, but was especially high in the perirectal space and Ramsay speculated that TH-proteins were involved in water reabsorption. Later we demonstrated that T. molitor held at low relative humidity increase hemolymph TH, and any acclimation that increased hemolymph TH (low temperature, short photoperiod or low humidity) greatly extended their ability to survive 15% RH at 22 °C. DAFP and/or AFGLs may be involved (perhaps by membrane stabilization) in the higher upper lethal temperatures exhibited by D. canadensis in winter (∼41 °C) relative to summer (∼35 °C), even though lower lethal temperatures are −20 to −25 °C in winter but only ∼4 °C in summer. Source of funding: National Science Foundation IOS0618342 and 1025929. Conflict of interest: None declared. duman.1@nd.edu