| Honeybees are warm-changing animals,and its body temperature changes with the change of the temperature of the outside environment.In order to adapt to the threat of low temperature environment,the bee population usually adopts the strategy of behavior changes combined with physiological and biochemical substances in the body to avoid frostbite,which we call honeybee cold tolerance.The cold tolerance of different honeybee species is different.In pratice,it was found that A.c.cerana show better cold hardiness than A.m.ligustica,but the mechanisms are not clear.In this study,difference in cold hardiness of A.c.cerana and A.m.ligustica were compared,and the mechanisms of cold resistance were discussed.It is divided into the following three tests:Exp.1 Study on seasonal variation of cold tolerance of A.m.ligustica.The honeybees were collected from the early winter,the mid winter,the late winter,and 40 of each group were collected at each period.The super-cooling point,glycogen,glycerol,small molecular sugar alcohol and the m RNA levels of cold resistant genes(Vg,AFP,L(2)efl)were determined.The results are as follows:(1)the difference of supercooling points in different stages of wintering period is significant(P<0.05).The change of the super-cooling point of A.m.ligustica coincide with the change trend of the local ambient temperature,showing an obvious seasonal variation trend.(2)the changes of the cold tolerance index were as follows:the total protein content of the body of A.m.ligustica rose first and then decreased from the early winter to the late winter(P<0.05).The content of glycogen in A.m.ligustica increased rapidly to peak value in early winter,which was significantly higher than that in overwintering period(P<0.05).The content of glycerol in the wintering period was significantly different from that in the early and late stages(P<0.05).The contents of three kinds of small molecular sugars in haemolymph of A.m.ligustica showed a trend of first rise and then decrease after overwintering.(3)the expression of AFP,L(2)efl and Vg gene in A.m.ligustica during the whole overwintering period: the m RNA expression of AFP gene of A.m.ligustica kept at a low level in the middle of the winter(P<0.05).The expression of L(2)efl gene showed a trend of increasing and then decreasing(P<0.05).The expression level of the same Vg gene also showed significant changes at 6 time points(P<0.05).Exp.2 Comparison of different cold resistance between A.c.cerana and A.m.ligustica under natural honeybee colony.The supercooling point(SCP)and freezing point(FP)were measured immediately.The water,fat,glycogen,protein of content,trehalase activities,related genes expression level were determined by sampling of honeybee during wintering period,and the colony temperature and attendance ability in early spring,five colonies repeats of A.c.cerana and A.m.ligustica respectively.The results are as follows:(1)in early spring,there was no significant difference in temperature between A.c.cerana and A.m.ligustica,and the attendance time of A.c.cerana was earlier than that of A.m.ligustica,and the ability to collect pollen was stronger than that of A.m.ligustica(P<0.05).(2)Compared with A.m.ligustica,the super-cooling point was significantly higher than A.c.cerana(P<0.05).The freezing point showed no obvious difference(P>0.05).(3)The free-water content of A.c.cerana was significantly lower than A.m.ligustica(P<0.05),by contrast the bound-water content of A.c.cerana was obviously higher than A.m.ligustica(P<0.05).There was no obvious difference in the fat content between A.cerana cerana and A.m.ligustica(P>0.05).There were no significant differences in the protein content of two bee species(P>0.05);glycogen content of A.c.cerana had significantly increased than A.mellifera ligustica in mid-winter(P<0.05).The mannitol,sorbic alcohol content of A.c.cerana was twice as that of A.m.ligustica in the hemolymph(P<0.05).The content of trehalose in A.c.cerana was obviously higher than A.m.ligustica(P < 0.05).The trehalase activity of A.c.cerana significantly reduced compared with A.m.ligustica in the body and hemolymph(P<0.05).(4)Compared with A.c.cerana,the relative expression levels of Tre-2 and UGP had significantly increased in A.m.ligustica(P<0.05),but Tre-1 was obviously lower(P<0.05).There was no obvious difference in the relative expression levels of TPS between A.c.cerana and A.m.ligustica(P>0.05).Exp.3 Study on antioxidant capacity and expression of cold tolerance related genes of A.c.cerana and A.m.ligustica in out-of-colony under low temperature stress.A.c.cerana and A.m.ligustica in overwintering period was used as control group.The change of freezing stiffness was recorded at 4 ? at low temperature and the activities of three antioxidantenzymes were determined under 16 ? low temperature and stored 15 min,30min,45 min,1h and 1.5h.Five genes encoding cold-tolerance related proteins were selected and their expression level of m RNA were detected.The results are as follows:(1)the freezing stiffness rate of A.c.cerana was significantly lower than that of A.m.ligustica under 4 ? low temperature stress(P<0.05).(2)The activity of POD reached the peak after 30 minutes of cryopreservation,and it was significantly higher than the control group(P<0.05).but it had no significant effect on the activity of SOD in A.m.ligustica(P>0.05).The activity of three antioxidant enzyme increased significantly with the prolongation of low temperature stress time(P<0.05).(3)The expression level of VG,TPS and SOD gene in both A.c.cerana and A.m.ligustica were significantly up-regulated(P<0.05).CAT expression level was inhibited during cryopreservation(P<0.05).The difference was that the expression level of L(2)efl was significantly down-regulated under short-term cold stress,and was always in a low expression of the treatment group level in A.m.ligustica(P<0.05).The study showed that the cold tolerance of A.m.ligustica showed a significant seasonal variation.The variation of cold resistance was related to the main biochemical substances in the body.The cold tolerance of A.c.cerana was higher than that of A.m.ligustica,and the difference was significant.The cold-resistance of honeybees was attributed to the antioxidant capacity and the expression of cold-resistance-related genes,which constituted a cold-resistance response mechanism against cold temperatures. |
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