Analysis Of The Cold Hardness And The Thermal Hysteresis Activity Of The Hemolymph In Anatolica Polita Borealis

Insects experience a seasonal low-temperature,which tend to injury, their ability to adapt to the environment and tolerance that determine the size of their population and distribution range. The weather in Xinjiang desert region is a kind of typical dry land weather. The temperature fluctuations are greatly during the year and extreamly in a day. Anatolica polita boreali successfully survive and reproduce in desert after long time evolution. Studies show that insect antifreeze proteins (AFPs) play a very important role in body fluids to reduce the supercooling points and reduce the ice crystals which may lead to fatal injury. Is this high cold resistance of Anatolica polita boreali related to the seasonal AFPs expression? And which factors impact insect AFPs seasonal expression? It is necessary to study the cold resistance of desert region insects.The hemolymph of Anatolica polita boreali adults from March to September were collected in a small tube placed in a bigger tube by centrifugation and primary purified at 70℃water bath. The thermal hysteresis activity (THA) of the hemolymph samples were determined by osmometer. The seasonal expression of the antifreeze protein was analyzed with Tricine SDS-PAG Eelectrophoresis, Western blotting and ELISA. The cryoprotective activity of the natural antifreeze protein was identified by the survival rate of bacteria protected at low temperature by AFP, The effect of the hemolymph on changing the ice crystal shape was observed by Nanolitre Osmometer connected to a microscope. The influence of cold acclimation on the expression of AFP and the expression with larval development was preliminaryly studied.The results showed that the AFP expression and the THA of Anatolica polita boreali adults has obvious seasonal variation from March to September. The THA of hemolymph was 2.2℃in March, 0.83℃in July and 2.1℃in September. The THA of the A. polita hemolymph in spring and autumn were significantly higher than that in summer (P<0.001). The adult AFP in March and April showed molecular weights of 10KD and 12KD, respectively, by Western bloting, and was not detected in other months due to the lower AFPs content. ELISA analysis showed that the AFP content in March and September were significantly higher than that in summer (P <0.001). The protective effect of the crude purified hemolymph on bacteria at low temperature was higher in March than that in September, and was low in July. The shape of a single ice crystal was changed to spindle and needle by the hemolymph in March, which is consistent with the other measurements of the AFP activities. Accordingly, the shape of the single ice crystal was modified as hexagona by hemolymph in September and almost no ice modification in July. Cold acclimation at 4℃for 5 days or 13 days enhanced the AFP content in the adult beetles, but appeared to some fluctuations during the cold acclimation. The AFPs of the larvae increased with the larval development, and increased significantly from the third instar to the seventh instar.In order to explore the cold tolerance and the possible mechanisms of the cold resistance of the beetle, the low-temperature exposure of the beetles from different months at - 10℃were tested. The results showed that the beetles in spring and autumn were extremely cold resistance, and less cold resistance in summer. The results suggest that A. polita borealis obtains high cold tolerance by keeping low SCP via increasing the ratio of bound water to free water and the amount of antifreeze protein and glycerol.