不同赖氨酸水平对熊蜂幼年工蜂发育及代谢影响

doi:10.13304/j.nykjdb.2022.0013

摘要/Abstract

摘要：

为了解熊蜂饲养和授粉中对氨基酸的营养需求，以18种氨基酸与糖水混合物为试验材料，设置4组不同赖氨酸水平（总氨基酸含量占比分别为0%、2%、4%、6%），研究其对幼年地熊蜂工蜂生长发育及生理代谢的影响。对1日龄工蜂饲喂20 d，记录工蜂死亡数量，测定工蜂胸部肌肉蛋白含量，采用LC-MS代谢组学方法分析工蜂血淋巴中代谢物差异。结果表明，饲粮中赖氨酸为6%时，工蜂胸部肌肉蛋白含量最高，死亡数量最少。代谢组学分析表明，饲粮缺乏赖氨酸组与6%赖氨酸组血淋巴代谢物明显分离，筛选共鉴定出28种差异代谢物，包括氨基酸、嘌呤等。差异代谢物富集分析发现，差异极显著的通路有组氨酸代谢（P<0.01），差异显著的通路有丙氨酸、天冬氨酸和谷氨酸代谢、精氨酸生物合成、嘌呤代谢、泛酸和辅酶A生物合成（P<0.05）。综合考虑上述指标，6%赖氨酸处理组的地熊蜂胸肌蛋白含量和寿命更优，但此水平下工蜂血淋巴代谢物组胺、腺苷、腺嘌呤呈下调趋势，因此地熊蜂日粮中赖氨酸比例为4%~6%更适合幼年工蜂的生长发育与生存。揭示了幼年地熊蜂工蜂对赖氨酸需求与胸部肌肉蛋白含量及寿命之间的关系，分析了不同水平赖氨酸对其代谢影响，为熊蜂规模化饲养和授粉应用中的营养补充提供理论依据。

关键词: 地熊蜂, 赖氨酸, 胸部蛋白含量, 存活率, 代谢物分析

Abstract:

In order to understand the nutritional requirement of amino acid in bumblebee feeding and pollination， this paper set 4 lysine levels （0%， 2%， 4%， 6% of total amino alids） to study the its effects on the growth， development and physiological metabolism of young bumblebee workers taking the mixture of 18 kinds of amino acids and syrup as experimental materials. The one-day-old bumblebees were fed for 20 d， and the death number of workers was recorded. The chest muscle protein content of workers was determined， and the metabolites in their hemolymph were analyzed by LC-MS metabonomics method. The results showed that when the dietary lysine level was 6%， the chest muscle protein content was the highest and the number of worker bees death rate was the lowest. Metabonomic analysis showed that hemolymph metabolites of 6% lysine group were significantly separated from those of dietary lysine deficiency group， and 28 differential metabolites were identified including amino acids， purines and so on. The results of differential metabolite enrichment analysis showed that the most significant pathway was histidine metabolism （P<0.01）， and the significant pathways were alanine， aspartic acid and glutamate metabolism， arginine biosynthesis， purine metabolism， pantothenic acid and coenzyme A biosynthesis （P<0.05）. Considering the above indicators， although the breast muscle protein content and life span of 6% lysine group were better， the hemolymph metabolites histamine， adenosine and adenine showed a downward trend at this level. 4% to 6% of the lysine level in the diet was more suitable for the growth and development of young worker bees. This study revealed the relationship between lysine requirement and breast muscle protein content and longevity of young bumblebee workers， and analyzed the effects of different levels of lysine on metabolism of bumblebee workers， which provided theoretical basis for nutritional supplement in large-scale feeding and pollination applications.

Key words: bumblebee, lysine, chest protein content, lifespan, metabolite analysis

中图分类号:

S894

郝冬梅, 樊莹, 徐进, 郭岳琴, 李继莲, 姚军. 不同赖氨酸水平对熊蜂幼年工蜂发育及代谢影响[J]. 中国农业科技导报, 2022, 24(10): 44-52.

Dongmei HAO, Ying FAN, Jin XU, Yueqin GUO, Jilian LI, Jun YAO. Effects of Different Concentrations of Lysine on Development and Metabolism of Young Bumblebee Workers （Bombus terrestris）[J]. Journal of Agricultural Science and Technology, 2022, 24(10): 44-52.

图/表 7

参考文献 42

1	彭文君,安建东,梁诗魁.熊蜂生物学特性及授粉机理[J].中国养蜂, 1999, 50(6): 27-28.
2	赵亚周,安建东,周志勇,等.意大利蜜蜂和小峰熊蜂在温室桃园的传粉行为及其影响因素[J]. 昆虫学报, 2011, 54(1): 89-96.
	ZHAO Y Z, AN J D, ZHOU Z Y, et al.. Pollination behavior of Apis mellifera ligustica and Bombus hypocrita (Hymenoptera, Apidae) and the influencing factors in peach greenhouse [J]. Acta Entomol. Sin., 2011, 54(1): 89-96.
3	AYASSE M, JARAU S. Chemical ecology of bumble bees [J]. Ann. Rev. Entomol., 2014, 59(1): 299-319.
4	徐希莲,王欢.国内熊蜂授粉产业化现状与发展建议[J].黑龙江畜牧兽医, 2018(16): 199-202.
5	TASEI J, AUPINEL P. Nutritive value of 15 single pollens and pollen mixes tested on larvae produced by bumblebee workers (Bombus terrestris, Hymenoptera: Apidae) [J]. Apidologie, 2008, 39(4): 397-409.
6	GENISSEL A, AUPINEL P, BRESSAC C, et al.. Influence of pollen origin on performance of Bombus terrestris micro-colonies [J]. Entomol. Exp. Appl., 2002, 104(2): 329-336.
7	WU G. Functional amino acids in growth, reproduction, and health [J]. Adv. Nutr., 2010, 1(1): 31-37.
8	BOISEN S, HVELPLUND T, WEISBJERG M R. Ideal amino acid profiles as a basis for feed protein evaluation [J]. Livest. Prod. Sci., 2000, 64(2): 239-251.
9	LEE K P. The interactive effects of protein quality and macronutrient imbalance on nutrient balancing in an insect herbivore [J]. J. Exp. Biol., 2007, 210(18): 3236-3244.
10	HENDRIKSMA H P, PACHOW C D, NIEH J C. Effects of essential amino acid supplementation to promote honey bee gland and muscle development in cages and colonies [J/OL]. J. Insect Physiol., 2019, 117: 103906 [2022-05-06]. .
11	HENDRIKSMA H P, OXMAN K L, SHAFIR S. Amino acid and carbohydrate tradeoffs by honey bee nectar foragers and their implications for plant-pollinator interactions [J]. J. Insect Physiol., 2014, 69: 56-64.
12	NICOLSON S W, HUMAN H. Chemical composition of the 'low quality’ pollen of sunflower (Helianthus annuus, Asteraceae) [J]. Apidologie, 2013, 44(2): 144-152.
13	PERNAL S F, CURRIE R W. Pollen quality of fresh and 1-year-old single pollen diets for worker honey bees (Apis mellifera L.) [J]. Apidologie, 2000, 31(3): 387-409.
14	GROOT A P D. Amino acid requirements for growth of the honeybee (Apis mellifica L.) [J]. Experientia, 1952, 8(5): 192-194.
15	LEONHARDT S D, BLüTHGEN N. The same, but different: pollen foraging in honeybee and bumblebee colonies [J]. Apidologie, 2012, 43(4): 449-464.
16	MOERMAN R, VANDERPLANCK M, ROGER N, et al.. Growth rate of bumblebee larvae is related to pollen amino acids [J]. J. Econ. Entomol., 2016, 109(1): 25-30.
17	LE COUTEUR D G, SOLON-BIET S, COGGER V C, et al.. The impact of low-protein high-carbohydrate diets on aging and lifespan [J]. Cell. Mol. Life Sci., 2015, 73(6): 1237-1252.
18	KONASHI S, TAKAHASHI K, AKIBA Y. Effects of dietary essential amino acid deficiencies on immunological variables in broiler chickens [J]. Brit. J. Nutr., 2000, 83(4): 449-456.
19	王帅,王红芳,刘振国,等.赖氨酸在蜜蜂中的营养功能及其研究进展[J].蜜蜂杂志, 2016, 36(10): 9-11.
	WANG S, WANG H F, LIU Z G, et al.. Nutritionai function of lysine in bee and its research progress [J]. J. Bee, 2016, 36(10): 9-11.
20	陈盼盼,闵育娜,王哲鹏,等.日粮赖氨酸水平对1~21日龄肉鸡生长性能和血清生化指标的影响[J].中国家禽, 2017, 39(9): 29-34.
	CHEN PP, MIN YN, WANG ZP, et al.. Effects of dietary lysine level on growth performance and serum biochemical parameters of broilers at the age of 1 to 21 days [J]. Chin. Poultry, 2017, 39(9): 29-34.
21	CHEN C, SANDER J E, DALE N M. The effect of dietary lysine deficiency on the immune response to Newcastle disease vaccination in chickens [J]. Avian Dis., 2003, 47(4): 1346-1351.
22	LIAO S F, WANG T, REGMI N. Lysine nutrition in swine and the related monogastric animals: muscle protein biosynthesis and beyond [J]. Springerplus, 2015, 4: 147.
23	代伟伟,麦康森,徐玮,等.饲料中赖氨酸和精氨酸含量对大菱鲆幼鱼生长、体成分和肌肉氨基酸含量的影响[J].水产学报, 2015, 39(6): 876-887.
	DAI WW, MAI KS, XU W, et al.. Effects of lysine-arginine interaction on growth performance, body composition, and muscle amino acid levels of juvenile turbot (Scophthalmus maximus L. ) [J]. J. Fish. Chin., 2015, 39: 876-887.
24	BIDNER B S, ELLIS M, WITTE D P, et al.. Influence of dietary lysine level, pre-slaughter fasting, and rendement napole genotype on fresh pork quality [J]. Meat Sci., 2004, 68(1): 53-60.
25	ALTAYE S Z, PIRK C W W, CREWE R M, et al.. Convergence of carbohydrate-biased intake targets in caged worker honeybees fed different protein sources [J]. J. Exp. Biol., 2010, 213(19): 3311-3318.
26	GROOT A P D. Protein and amino acid requirements of the honeybee (Apis mellifica L.) [J]. Physiol. Comp. Oecol., 1953, 3: 197-285.
27	王帅,王红芳,胥保华.意大利蜜蜂工蜂幼虫饲粮的适宜赖氨酸水平[J].动物营养学报, 2017, 29(11): 4236-4244.
	WANG S, WANG HF, XU BH. Aietary appropriate lysine level for Apis mellifera ligustica worker bee larvae [J]. Chin. J. Anim. Nutr., 2017, 29: 4236-4244.
28	WANG L, MEEUS I, ROMBOUTS C, et al.. Metabolomics-based biomarker discovery for bee health monitoring: a proof of concept study concerning nutritional stress in Bombus terrestris [J/OL]. Sci. Rep., 2019, 9(1): 11423 [2022-05-06]. .
29	BORSUK G, PTASZYŃSKA A A, OLSZEWSKI K, et al.. A new method for quick and easy hemolymph collection from Apidae adults [J/OL]. PLoS One, 2017, 12(1): e170487 [2022-05-06]. .
30	CANFIELD L M, CHYTIL F. Effect of low lysine diet on rat protein metabolism [J]. J. Nutr., 1978, 108(8): 1343-1347.
31	KOIE M, TANAKA M, HAYASE K, et al.. Effects of adding dietary lysine to a low gluten diet on the brain protein synthesis rate in aged rats [J]. Biosci. Biotechnol. Biochem., 2000, 64(7): 1466-1471.
32	曾佩玲,张常明,王修启,等.日粮不同赖氨酸水平对生长猪养分表观消化率、血清氨基酸含量和生化指标的影响[J].华北农学报, 2009, 24(S2): 116-120.
	ZENG P L, ZHANG C M, WANG X Q, et al.. Effects of different dietary lysine levels on apparent nutrient digestibility and serum amino acid concent ration and serum biochemical indexes in growing pig [J]. Acta Agric. Boreali-Sin., 2009, 24(S2): 116-120.
33	夏济平.尿刊酸的免疫抑制作用研究进展[J].国外医学(皮肤性病学分册), 1998, 24(4): 213-216.
34	胡孟,武书庚,王晶,等.组氨酸的生理功能及在动物生产中的应用[J].中国饲料, 2018(7): 38-45.
	HU M, WU S, WANG J, et al.. Physiological function and application of histidine in animal production [J]. Chin. Feed, 2018 (7): 38-45.
35	康培,张亚,刘双,等.大肠杆菌生产L-组氨酸研究进展[J].食品与发酵工业, 2016, 42(11): 249-254.
	KANG P, ZHANG Y, LIU S, et al.. Research on L-histidine production by Escherichia coli [J]. Food Ferment. Ind., 2016, 42(11): 249-254.
36	ROSENFELDT F L, KORCHAZHKINA O V, RICHARDS S M, et al.. Aspartate improves recovery of the recently infarcted rat heart after cardioplegic arrest [J]. Eur. J. Cardio-thoracic., 1998, 14(2): 185-190.
37	RUSSELL R R, TAEGTMEYER H. Changes in citric acid cycle flux and anaplerosis antedate the functional decline in isolated rat hearts utilizing acetoacetate [J]. J. Clin. Invest., 1991, 87(2): 384-390.
38	陈家顺,苏文璇,符晨星,等.α-酮戊二酸的生理功能及其在动物生产中的应用[J].动物营养学报, 2018, 30(10): 3818-3827.
	CHEN J, SU W E, FU C, et al.. Physiological function of alpha-ketoglutarate and its application in animal production [J]. Chin. J. Anim. Nutr., 2018, 30(10): 3818-3827.
39	张再明,杨琳,侯水生.动物营养中赖氨酸与精氨酸互作关系的研究进展[J].饲料广角, 2010(21): 26-28.
40	刘劲,代方银,朱勇,等.家蚕突变体的嘌呤代谢[J].蚕学通讯, 2005, 25(3): 9-14.
	LIU J, DAI F Y, ZHU Y, et al.. Purine metabolism in silkworm mutants [J]. Newsl. Sericul. Sci., 2005, 25(3): 9-14.
41	BECKER B F. Towards the physiological function of uric acid [J]. Free Radical Biol. Med., 1993, 14(6): 615-631.
42	栾贻宏,路宁,王基伟,等.辅酶A的生化功能和应用[J].中国生化药物杂志, 2003, 24(3): 159-161.
	LUAN Y H, LU N, WANG J W, et al. Biochemical functionand applicationof coenzyme A [J]. Chin. J. Biochem. Phar., 2003, 24(3): 159-161.

赖氨酸水平Lysine level	0%	2%	4%
2%	0.053	—	—
4%	0.084	0.802	—
6%	<0.001	0.009	0.005

赖氨酸水平Lysine level	0%	2%	4%
2%	0.053	—	—
4%	0.084	0.802	—
6%	<0.001	0.009	0.005

代谢物 Metabolite	保留时间 Retention time/min	VIP	差异倍数Fold change	P值P-value	趋势 Trend
黄嘌呤 Xanthine	6.23	1.17	10.21	1.40e-12	上升Up
S-腺苷高半胱氨酸 S-adenosylhomocysteine	9.37	1.17	7.37	3.39e-12	上升Up
哌啶酸Pipecolinic acid	10.31	1.17	28.34	1.33e-10	上升Up
L-高胱氨酸 L-homocystine	10.40	1.17	7.75	2.85e-10	上升Up
N-乙酰组胺 N-acetylhistamine	6.63	1.17	0.38	2.10e-10	下降Down
胞嘧啶 Cytosine	6.46	1.16	0.18	8.13e-10	下降Down
N8-乙酰基亚精胺 N8-acetylspermidine	9.50	1.16	0.08	6.18e-10	下降Down
腺苷Adenosine	5.56	1.16	0.37	1.62e-09	下降Down
戊二酸 Glutaric acid	2.46	1.16	5.79	2.29e-09	上升Up
腺嘌呤 Adenine	5.23	1.16	0.42	2.97e-09	下降Down
咪唑乳酸 Imidazolelactic acid	8.49	1.16	0.10	7.77e-09	下降Down
3-磷酸甘油 Glycerol 3-phosphate	9.38	1.15	0.32	1.27e-08	下降Down
组胺 Histamine	6.68	1.15	0.24	3.35e-08	下降Down
泛醇 Panthenol	2.20	1.15	0.26	4.57e-08	下降Down
L-酵母氨酸 L-saccharopine	10.75	1.15	18.11	6.08e-08	上升Up
L-缬氨酸 L-valine	1.92	1.14	0.36	8.50e-08	下降Down
N-乙酰-L-赖氨酸 N-acetyl-L-lysine	8.65	1.14	2.38	1.65e-07	上升Up
α-酮戊二酸 α-oxoglutaric acid	8.18	1.13	2.18	4.94e-07	上升Up
5-氨基乙酰丙酸 5-aminolevulinic acid	8.36	1.13	0.13	4.41e-07	下降Down
L-天冬氨酸 L-aspartic acid	9.88	1.13	2.09	6.20e-07	上升Up
5-脱氧-5-甲硫腺苷 5’-s-methyl-5’-thioadenosine	2.91	1.12	0.40	1.54e-06	下降Down
左旋多巴 L-dopa	8.62	1.12	2.09	1.89e-06	上升Up
油酸酰胺 Oleamide	1.28	1.11	0.26	2.20e-06	下降Down
尿刊酸 Urocanic acid	2.07	1.11	0.33	4.57e-06	下降Down
天冬酰胺 Asparagine	9.33	1.10	0.41	6.26e-06	下降Down
己二酸 Adipic acid	1.86	1.10	0.29	8.24e-06	下降Down
托品碱 Tropine	8.77	1.09	0.41	1.14e-05	下降Down
7-甲基鸟嘌呤 7-methylguanine	6.09	1.07	0.33	3.56e-05	下降Down

代谢物 Metabolite	保留时间 Retention time/min	VIP	差异倍数Fold change	P值P-value	趋势 Trend
黄嘌呤 Xanthine	6.23	1.17	10.21	1.40e-12	上升Up
S-腺苷高半胱氨酸 S-adenosylhomocysteine	9.37	1.17	7.37	3.39e-12	上升Up
哌啶酸Pipecolinic acid	10.31	1.17	28.34	1.33e-10	上升Up
L-高胱氨酸 L-homocystine	10.40	1.17	7.75	2.85e-10	上升Up
N-乙酰组胺 N-acetylhistamine	6.63	1.17	0.38	2.10e-10	下降Down
胞嘧啶 Cytosine	6.46	1.16	0.18	8.13e-10	下降Down
N8-乙酰基亚精胺 N8-acetylspermidine	9.50	1.16	0.08	6.18e-10	下降Down
腺苷Adenosine	5.56	1.16	0.37	1.62e-09	下降Down
戊二酸 Glutaric acid	2.46	1.16	5.79	2.29e-09	上升Up
腺嘌呤 Adenine	5.23	1.16	0.42	2.97e-09	下降Down
咪唑乳酸 Imidazolelactic acid	8.49	1.16	0.10	7.77e-09	下降Down
3-磷酸甘油 Glycerol 3-phosphate	9.38	1.15	0.32	1.27e-08	下降Down
组胺 Histamine	6.68	1.15	0.24	3.35e-08	下降Down
泛醇 Panthenol	2.20	1.15	0.26	4.57e-08	下降Down
L-酵母氨酸 L-saccharopine	10.75	1.15	18.11	6.08e-08	上升Up
L-缬氨酸 L-valine	1.92	1.14	0.36	8.50e-08	下降Down
N-乙酰-L-赖氨酸 N-acetyl-L-lysine	8.65	1.14	2.38	1.65e-07	上升Up
α-酮戊二酸 α-oxoglutaric acid	8.18	1.13	2.18	4.94e-07	上升Up
5-氨基乙酰丙酸 5-aminolevulinic acid	8.36	1.13	0.13	4.41e-07	下降Down
L-天冬氨酸 L-aspartic acid	9.88	1.13	2.09	6.20e-07	上升Up
5-脱氧-5-甲硫腺苷 5’-s-methyl-5’-thioadenosine	2.91	1.12	0.40	1.54e-06	下降Down
左旋多巴 L-dopa	8.62	1.12	2.09	1.89e-06	上升Up
油酸酰胺 Oleamide	1.28	1.11	0.26	2.20e-06	下降Down
尿刊酸 Urocanic acid	2.07	1.11	0.33	4.57e-06	下降Down
天冬酰胺 Asparagine	9.33	1.10	0.41	6.26e-06	下降Down
己二酸 Adipic acid	1.86	1.10	0.29	8.24e-06	下降Down
托品碱 Tropine	8.77	1.09	0.41	1.14e-05	下降Down
7-甲基鸟嘌呤 7-methylguanine	6.09	1.07	0.33	3.56e-05	下降Down

通路名称 Map title	富集编号 Map ID	显著性P-value
组氨酸代谢Histidine metabolism	map00340	0.003
丙氨酸、天冬氨酸和谷氨酸代谢 Alanine， aspartate and glutamate metabolism	map00250	0.013
精氨酸生物合成 Arginine biosynthesis	map00220	0.026
嘌呤代谢Purine metabolism	map00230	0.029
泛酸和辅酶A生物合成 Pantothenate and CoA biosynthesis	map00770	0.046
氨酰tRNA生物合成 Aminoacyl-tRNA biosynthesis	map00970	0.056
D-谷氨酰胺与D-谷氨酰胺代谢 D-Glutamine and D-glutamate metabolism	—	0.106
半胱氨酸和蛋氨酸代谢 Cysteine and methionine metabolism	map00270	0.121
缬氨酸、亮氨酸和异亮氨酸生物合成 Valine， leucine and isoleucine biosynthesis	map00290	0.138
丁酸代谢 Butanoate metabolism	map00650	0.244
烟酸和烟酰胺代谢 Nicotinate and nicotinamide metabolism	map00760	0.244
甘油酯代谢 Glycerolipid metabolism	map00561	0.258
柠檬酸循环（TCA循环） Citrate cycle （TCA cycle）	map00020	0.312
β-丙氨酸代谢 β-alanine metabolism	map00410	0.325
赖氨酸降解 Lysine degradation	map00310	0.374
卟啉与叶绿素代谢 Porphyrin and chlorophyll metabolism	map00860	0.430
甘氨酸、丝氨酸和苏氨酸代谢 Glycine， serine and threonine metabolism	map00260	0.462
甘油磷脂代谢 Glycerophospholipid metabolism	map00564	0.492
缬氨酸、亮氨酸和异亮氨酸降解 Valine， leucine and isoleucine degradation	map00280	0.529
酪氨酸代谢 Tyrosine metabolism	map00350	0.546