Strong tolerance of freezing is an important strategy for insects living in extremely cold regions. They produce highly effective cryoprotectant systems consisting of ice-nucleating proteins and polyols, which enables tolerable freezing of the body fluid. Therefore, the measurement of the concentrations of polyols and the activity of ice nucleators in the haemolymph is an essential tool for describing tolerance to ice formation in insects occurring in particularly cold places. This study evaluates three parameters: insect body supercooling point (SCP), haemolymph glycerol content and the profile of haemolymph ice nucleating activity that characterize the strategies of cold adaptation and cold hardiness in two previously unstudied beetles, Chrysolina graminis graminis L. and Galerucella nymphaea L., inhabiting Yakutia (Russian Far East, latitude 62°N). The high SCP values, ice nucleating activity and survival of the chrysomelids after freezing indicate that both species are tolerant of freezing. According to the profiles of ice-nucleating activity, the haemolymph from C. graminis graminis is characterized by a higher nucleating potential than that from G. nymphaea. The glycerol level is also higher in C. graminis graminis. The results indicate that both species develop tolerance to low temperatures, but the cold hardiness potential of C. graminis graminis is greater than that of G. nymphaea. This was revealed by the survival test, in which beetles were frozen to a temperature of -22°C for 30 min; 86% of C. graminis graminis and 72% of G. nymphaea survived the test. Thus, the freeze-tolerance of these beetles seems to be based on the production of an integrated cryoprotectant system, the quality of which apparently influences the range of their cold resistance., Natalia G. Li., and Obsahuje bibliografii
Four case studies are used to examine the relationships of water, ice nucleators and desiccation in the cold survival of invertebrates and the viability of frozen plant material: the freeze intolerant Antarctic springtail Cryptopygus antarcticus (Willem) (Collembola, Isotomidae), the freeze tolerant larvae of the fly Heleomyza borealis Boh. (Diptera: Heleomyzidae), the freeze intolerant Arctic springtail Onychiurus arcticus (Tullberg) (Collembola, Onychiuridae) and meristems of the currant Ribes ciliatum Humb. & Bonpl.(Grossulariaceae) from Mexico. Prevention of ice nucleation, lowering the water content by removal of osmotically active (freezable) water are critical features of the different cold survival strategies of the three species of invertebrates. In C. antarcticus, which desiccates rapidly by losing water via the cuticle to the atmosphere, the number of ice nucleators (and their activity) increases with lowered ambient temperature. During prolonged cold exposure ice nucleators are masked, but re-activated rapidly by water uptake in this species. Larval H. borealis do not readily desiccate and conserve their body water, 20-25% of it being bound (osmotically inactive). Experiments showed that a high proportion (c. 80%) of slowly cooled larvae survived exposure to -60°C. By comparison O. arcticus is able to sustain up to 40% loss of its body water and desiccation lowers its supercooling point to promote over winter survival. Dehydration leading to partial vitrification of currant (R. ciliatum) meristems improves their viability after cryopreservation in liquid nitrogen. From this comparison of four biological systems, it is concluded that the role of water and its activity at sub-zero temperatures are fundamental to the survival of freezing conditions by all the species studied. Although similar features exist in the four systems, no common basic mechanism was found.