Abstract：To study the effects of exogenous sorbitol and trehalose on the cold resistance of Apis mellifera ligustica, the contents of sorbitol, trehalose and other related biochemical substances in honeybees, the changes of antioxidant enzyme activity and the expression of cold-resistant genes were measured to explore the possible physiological and biochemical mechanisms in the cold-resistant process of honeybees. From the A.m. ligustica sister colony, 900 young bees which were just out of the house were randomly selected and divided into 6 groups with 3 replicates per each group and 50 bees per replicate. The bees were fed sucrose solution, trehalose solution and sorbitol solution,respectively. All the 6 groups were fed at normal temperature (30 ℃) for 12 days, after feeding for 12 days, three of the groups were cold treated (4 ℃ cold training for 2 h). After treatment, supercooling point, freezing point were detected for comparing the difference of cold resistance; the contents of free moisture, glycogen, fat, the activities of sorbitol dehydrogenase (SDH) and trehalase (THL), and the mRNA relative expession levels of trehalose monophosphate synthase (TPS) and SDH were determined. Another 900 young bees were bred according to the above groups for the determination of small molecular sugar alcohols contents in hemolymph. Results showed as follows: 1) compared with the control group, supercooling point in sorbitol group and trehalose group reduced by 0.65 (P<0.05) and 0.41 ℃(P<0.05). 2) Compared with the control group, under normal temperature and cold treatment conditions, the content of free moisture in sorbitol group and trehalose group had no significant change (P>0.05). 3) Compared with the control group, under normal temperature and cold treatment conditions, the body fat content in sorbitol group and trehalose group had no significant change (P>0.05), but the glycogen content significantly increased (P<0.05). 4) Compared with the control group, under normal temperature and cold treatment conditions, glucose content in hemolymph in sorbitol group and trehalose group increased significantly (P<0.05); under normal temperature condition, the content of fructose in hemolymph in sorbitol group significantly increased (P<0.05), under cold treatment condition, the content of fructose in hemolymph in trehalose group decreased significantly (P<0.05); under normal temperature condition, the sorbitol content in hemolymph in sorbitol group increased significantly (P<0.05), while under cold treatment condition, the results showed no significant difference (P>0.05). 5) Compared with the control group, under normal temperature and cold treatment conditions, the SDH and THL activities in sorbitol group and trehalose group significantly increased (P<0.05). 6) Compared with the control group, under normal temperature and cold treatment conditions, the mRNA relative expression level of SDH in sorbitol group significantly up-regulated (P<0.05). Under normal temperature condition, the mRNA relative expression level of TPS in trehalose group significantly decreased (P<0.05). To sum up, the exogenous addition of sorbitol and trehalose in the diets of bees raised in the room significantly reduce the supercooling point and freezing point of bees, thus improving the cold tolerance. Sorbitol and trehalose improve the cold tolerance of bees by regulating the content of small molecule sugar alcohol in hemolymph and the expression of genes related to sorbitol and trehalose metabolic pathways.
褚亚平.氟铃脲对斜纹夜蛾海藻糖酶活性及基因表达的影响[D].硕士学位论文.泰安:山东农业大学,2013. ZHU Y P.Effects of fluoroflurea on trehalase activity and gene expression in C.terrai[D].Master's Thesis.Tai'an:Shandong Agricultural University,2013.(in Chinese)
[7]
李正杨.高温对抗性和敏感小菜蛾海藻糖及其酶活性的影响[D].硕士学位论文.福州:福建农林大学,2013. LI Z Y.Effects of high temperature on the activity of trehalose and its enzyme in resistant and sensitive Plutella xylosa[D].Master's Thesis.Fuzhou:Fujian Agriculture and Forestry University,2013.(in Chinese)
[11]
王洪亮,仵均祥,王丙丽.麦红吸浆虫滞育期间海藻糖酶和山梨醇脱氢酶活性的变化[J].西北农林科技大学学报(自然科学版),2006,34(8):139-142. WANG H L,WU J X,WANG B L.Changes of trehalase and sorbitol dehydrogenase activities during diapause in wheat red plasmids[J].Journal of Northwest A&F University (Natural Science),2006,34(8):139-142.(in Chinese)
[14]
DANKS H V.Life history and biology of Einfeldia synchrona (Diptera:Chironomidae)[J].The Canadian Entomologist,1971,103(11):1597-1606.
[15]
常志光,李志勇,高寿增,等.卡尼鄂拉蜂越冬期体内水分、蛋白质、脂肪的变化与抗寒性的关系研究[J].蜜蜂杂志,2008,28(1):6-7. CHANG Z G,LI Z Y,GAO S Z,et al.Study on the relationship between the changes of water,protein and fat and cold resistance of the Kaneorrah bee during its overwintering period[J].Journal of Bee,2008,28(1):6-7.(in Chinese)
[19]
罗立平,王小艺,唐艳龙,等.外源海藻糖对花绒寄甲成虫存活和耐寒性的影响[J].昆虫学报,2019,62(5):586-593. LUO L P,WANG X Y,TANG Y L,et al.Effects of exogenous trehalose on survival and cold tolerance of adult Phyllostachys grandiflori[J].Acta Entomologica Sinica,2019,62(5):586-593.(in Chinese)
[22]
LEHMANN P,LYYTINEN A,SINISALO T,et al.Population dependent effects of photoperiod on diapause related physiological traits in an invasive beetle (Leptinotarsa decemlineata)[J].Journal of Insect Physiology,2012,58(8):1146-1158.
[1]
ELBEIN A D,MITCHELL M.Levels of glycogen and trehalose in Mycobacterium smegmatis and the purification and properties of the glycogen synthetase[J].Journal of Bacteriology,1973,113(2):863-873.
[5]
刘晓健,崔淼,李大琪,等.飞蝗几丁质合成酶2基因的表达特性、功能及调控[J].中国农业科学,2014,47(7):1330-1340. LIU X J,CUI M,LI D Q,et al.Expression characteristics,function and regulation of chitinase 2 gene in migratory locust[J].Chinese Agricultural Science,2014,47(7):1330-1340.(in Chinese)
[9]
CHILDERS C C,ACHOR D S.Thrips feeding and oviposition injuries to economic plants,subsequent damage and host responses to infestation[M]//PARKER B L,SKINNER M,LEWIS T.Thrips biology and management.Boston:Springer,1995:31-51.
[13]
李志勇,薛运波,王志,等.东西方蜜蜂越冬期过冷却点的测试及过冷却点与越冬性能的关系[J].蜜蜂杂志,2006,26(11):12-13. LI Z Y,XUE Y B,WANG Z,et al.The test of overcooling point and the relationship between overcooling point and overwintering performance of eastern and Western honeybees[J].Journal of Honeybees,2006,26(11):12-13.(in Chinese)
[17]
韩召军,王荫长,尤子平.陆生昆虫的抗寒性机制[J].昆虫知识,1989(1):39-42. HAN Z J,WANG Y C,YOU Z P.Mechanism of cold resistance of terrestrial insects[J].Insect Knowledge,1989(1):39-42.(in Chinese)
[4]
聂凌鸿,宁正祥.海藻糖的生物保护作用[J].生命的化学,2001,21(3):206-209. NIE L H,NING Z X.Biological protection of trehalose[J].Chemistry of Life,2001,21(3):206-209.(in Chinese)
[8]
明照岳.高温胁迫对二点委夜蛾海藻糖代谢的影响与其卵黄蛋白特性的研究[D].硕士学位论文.泰安:山东农业大学,2014. MING Z Y.Effects of high temperature stress on trehalose metabolism and its yolk protein characteristics in C.japonicus[D].Master's Thesis.Tai'an:Shandong Agricultural University,2014.(in Chinese)
[12]
杜晓云.山梨醇脱氢酶抑制剂的筛选、合成与活性研究[D].硕士学位论文.大连:大连理工大学,2018. DU X Y.Screening,synthesis and activity of sorbitol dehydrogenase inhibitors[D].Master's Thesis.Dalian:Dalian University of Technology,2018.(in Chinese)
[16]
李浩.沙葱萤叶甲耐寒性及其机理的研究[D].硕士学位论文.呼和浩特:内蒙古农业大学,2014. LI H.Study on cold tolerance and mechanism of Firefly armour[D].Master's Thesis.Hohhot:Inner Mongolia Agricultural University,2014.(in Chinese)
[20]
BEMANI M,IZADI H,MAHDIAN K,et al.Study on the physiology of diapause,cold hardiness and supercooling point of overwintering pupae of the pistachio fruit hull borer,Arimania comaroffi[J].Journal of Insect Physiology,2012,58(7):897-902.
[21]
BEHROOZI E,IZADI H,SAMIH M A,et al.Physiological strategy in overwintering larvae of pistachio white leaf borer,Ocneria terebinthina Strg.(Lepidoptera:Lymantriidae) in Rafsanjan,Iran[J].Italian Journal of Zoology,2012,79(1):44-49.
[24]
郭海波,许永玉,鞠珍,等.中华通草蛉成虫抗寒能力季节性变化[J].生态学报,2006,26(10):3238-3244. GUO H B,XU Y Y,JU Z,et al.Seasonal variation of cold resistance of adult Lacewing beetle[J].Acta Ecologica Sinica,2006,26(10):3238-3244.(in Chinese)
[23]
马延龙,候凤,马纪.荒漠昆虫光滑鳖甲的耐寒性季节变化及其生理机制[J].昆虫学报,2009,52(4):372-379. MA Y L,HOU F,MA J.Seasonal variation of cold tolerance and its physiological mechanism in smooth turtle armour of desert insect[J].Acta Entomologica Sinica,2009,52(4):372-379. (in Chinese)
[25]
蒋智林,文礼章,李有志,等.甘薯天蛾过冷却点变化与生化成分的关系[J].昆虫知识,2009,46(2):294-297. JIANG Z L,WEN L Z,LI Y Z,et al.Relationship between the changes of supercooling point and biochemical components of the sweet potato moth[J].Insect Knowledge,2009,46(2):294-297.(in Chinese)
[26]
李冰祥,陈永林,蔡惠罗.飞蝗不同地理种群抗寒性研究(英文)[J].生态学报,2001,21(12):2023-2030. LI B X,CHEN Y L,CAI H L.Study on cold resistance of migratory locust populations in different geographical populations (English)[J].Acta Ecologica Sinica,1999,21(12):2023-2030.(in Chinese)
[27]
SU Z H,SATO Y,YAMASHITA O.Purification,cDNA cloning and Northern blot analysis of trehalase of pupal midgut of the silkworm,Bombyx mori[J].Biochimica et Biophysica Acta:Gene Structure and Expression,1993,1173(2):217-224.
[2]
WYATT G R,COOK K E,FIRKO H,et al.Juvenile hormone action on locust fat body[J].Insect Biochemistry,1987,17(7):1071-1073.
[10]
陈田飞,乐波灵.家蚕滞育机理研究概况[J].广西蚕业,2004,41(3):12-16. CHEN T F,LE B L.Study on diapause mechanism of silkworm[J].Guangxi Sericulture,2004,41(3):12-16.(in Chinese)
[18]
秦玉川,杨建才.一种便携式测定昆虫过冷却点的方法[J].应用昆虫学报,2000,37(4):236-238. QIN Y C, YANG J C.A new simple method to test insect super-cooling point[J].Chinese Bulletin of Entomology,2000,37(4):236-238. (in Chinese)
[3]
THOMPSON S N.Trehalose-the insect ‘blood’ sugar[J].Advances in Insect Physiology,2003,31:205-285.
[28]
张江伟.PHKA2基因突变致11例糖原累积症Ⅸa型的临床表型与遗传学特征分析及两种单基因病的报道[D].硕士学位论文.北京:北京协和医学院,2014. ZHANG J W.Clinical phenotypic and genetic characteristics analysis of 11 cases of glycogen accumulation syndrome TYPE ⅨA caused by PHKA2 mutation and two monogenic diseases reported[D].Master's Thesis.Beijing:Peking Union Medical College,2014.(in Chinese)
[29]
CROWE J H,CROWE L M,CHAPMAN D.Preservation of membranes in anhydrobiotic organisms:the role of trehalose[J].Science,1984,223(4637):701-703.
[30]
NⅡMI T,YAGINUMA T.Biosynthesis of NAD-sorbitol dehydrogenase is induced by acclimation at 5℃ in diapause eggs of the silkworm,Bombyx mori[J].Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1992,102(1):169-173.
[31]
NⅡMI T.YAMASHITA O.YAGINUMA T.A cold-inducible Bombyx gene encoding a protein similar to mammalian sorbitol dehydrogenase[J].European Journal of Biochemistry,1993,213(3):1125-1131.
[32]
梁中贵,张卫光,刘学辉,等.松阿扁叶蜂越冬幼虫体内抗寒物质分析[J].昆虫知识,2005,42(6):695-699. LIANG Z G,ZHANG W G,LIU X H,et al.Analysis of cold resistant substances in the overwintering larvae of Parasitophus sonata[J].Insect Knowledge,2005,42(6):695-699.(in Chinese)
