基于LC-MS技术研究氟氯苯氰菊酯对西方蜜蜂工蜂幼虫代谢的影响

doi:10.3864/j.issn.0578-1752.2021.12.018

摘要/Abstract

摘要：

【目的】氟氯苯氰菊酯（flumethrin）属于第二代拟除虫菊酯类杀虫、杀螨剂,应用于蜂群除螨。由于杀螨剂具有一定的毒性,在杀死螨虫的同时也会威胁到蜜蜂的健康。本研究采用液相色谱-质谱联用技术（LC-MS）检测氟氯苯氰菊酯处理后的西方蜜蜂工蜂幼虫代谢物,筛选差异代谢物,并分析涉及到的代谢通路,研究氟氯苯氰菊酯对蜜蜂的毒理作用,为蜜蜂饲养提供参考。【方法】控制蜂王在空的工蜂巢脾上产卵12 h,并将产卵区域分为4组,从第5天开始分别给各组小幼虫饲喂含氟氯苯氰菊酯的糖水（0、0.5、5、50 mg·kg^-1）,剂量从第5天至第8天逐日递增（1.5、2、2.5、3 μL）,第9天采集幼虫的淋巴液。运用代谢组学中的LC-MS技术分析蜜蜂幼虫的代谢物,结合主成分分析（PCA）、偏最小二乘法-判别分析（PLS-DA）,筛选差异显著的代谢物,并对氟氯苯氰菊酯处理组和对照组间共同差异代谢物进行代谢通路分析。【结果】0.5 mg·kg^-1组与对照组相比,共有差异代谢物190种,共鉴定87种;5 mg·kg^-1组与对照组相比,共有差异代谢物275种,共鉴定97种;50 mg·kg^-1组与对照组相比,共有差异代谢物275种,共鉴定131种。从鉴定的差异代谢物中筛选到29种氟氯苯氰菊酯处理组共同差异代谢物,其中16种代谢物上调,12种代谢物下调,1种代谢物在0.5和50 mg·kg^-1组中下调,在5 mg·kg^-1组中上调。这些差异代谢物包括核糖、嘌呤及其衍生物、脂肪酸及其偶联物等。经代谢通路富集分析,发现差异显著（P＜0.05）的代谢通路包括氨基糖和核苷酸糖代谢、药物代谢-其他酶类、α-亚麻酸代谢等途径。【结论】LC-MS技术能够有效地分析氟氯苯氰菊酯影响下蜜蜂幼虫代谢物的变化,氟氯苯氰菊酯会引起蜜蜂幼虫体内尿苷二磷酸-N-乙酰葡糖胺、硫唑嘌呤、愈伤酸、9-羟基壬酸、氢过氧化亚油酸等物质含量异常,这些差异代谢物的变化证实了氟氯苯氰菊酯引起蜜蜂体内各种物质代谢紊乱,对这些代谢过程分析可进一步阐释蜜蜂代谢有毒化合物的机制,并为杀螨剂及其他有毒化合物对蜜蜂的胁迫提供理论依据。

关键词: 西方蜜蜂, LC-MS, 代谢组学, 氟氯苯氰菊酯, 工蜂幼虫

Abstract:

【Objective】Flumethrin belongs to the second generation pyrethroid insecticides and acaricides, which is used for the control of honeybee mites. Because of the toxicity of acaricides, it can not only kill mites, but also threat the health of honeybees. The different metabolites of Apis mellifera worker larvae treated with different concentrations of flumethrin were tested using liquid chromatography-mass spectrometry (LC-MS) technology and the metabolic pathways involved of different metabolites were analyzed, so as to explore the toxicological effect of flumethrin on honeybees and provide references for scientific using in beekeeping.【Method】The queen was controlled to lay eggs on an empty worker frame for 12 h, and the spawning area was divided into four groups. From the 5th day, the small larvae of each group were fed with sugar water containing different concentrations of flumethrin (0, 0.5, 5, 50 mg·kg^-1), the dose was increased daily from day 5 to day 8 (1.5, 2, 2.5, 3 μL), and lymph fluid from larvae was collected on day 9. The metabolites of A. mellifera larvae were analyzed by LC-MS and the metabolites with significant difference were screened by principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), while the metabolic pathways of common differential metabolites in flumethrin treatment groups were analyzed.【Result】Compared with the control group, a total of 190 different metabolites were found and 87 types were identified in 0.5 mg·kg^-1 group, and a total of 275 different metabolites were identified and 97 types were identified in 5 mg·kg^-1 group, while there were a total of 275 different metabolites and a total of 131 species were identified in 50 mg·kg^-1 group. Meanwhile, 29 common differential metabolites in treatment groups were screened, of which 16 metabolites were up-regulated, 12 metabolites were down-regulated, and 1 metabolite was down-regulated in 0.5 and 50 mg·kg^-1 groups while it was up-regulated in 5 mg·kg^-1 group. These differential metabolites include ribose, purine and its derivatives, fatty acids with their conjugates. After enrichment analysis of metabolic pathways, significant differences (P＜0.05) were found in the metabolic pathways, which include amino sugar and nucleotide sugar metabolism, drug metabolism-other enzymes, α-linolenic acid metabolism and other pathways.【Conclusion】The LC-MS technology can effectively analyze the changes of metabolites in the honeybee larvae treated with flumethrin, and flumethrin can cause the contents of UDP-N-acetylglucosamine, azathioprine, traumatic acid, 9-oxononanoic acid and 13(s)-HPODE abnormal in honeybee larvae. The changes of these different metabolites confirm that flumethrin causes various substance metabolism disorders in honeybees. The analysis of these metabolic processes can further explain the mechanism of honeybees metabolizing toxic compounds, and provide a theoretical basis for the stress of acaricides and other toxic compounds on honeybees.

Key words: Apis mellifera, LC-MS, metabolomics, flumethrin, worker larva

虞龙涛,杨何妍,苏宇晨,颜伟玉,吴小波. 基于LC-MS技术研究氟氯苯氰菊酯对西方蜜蜂工蜂幼虫代谢的影响[J]. 中国农业科学, 2021, 54(12): 2689-2698.

YU LongTao,YANG HeYan,SU YuChen,YAN WeiYu,WU XiaoBo. The Effect of Flumethrin on Metabolism of Worker Larvae of Apis mellifera with LC-MS Technique[J]. Scientia Agricultura Sinica, 2021, 54(12): 2689-2698.

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图/表 7

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表1

表2

对照组与氟氯苯氰菊酯处理组之间差异代谢物"

化合物名称 Compound name	50 mg·kg^-1-Control				5 mg·kg^-1-Control				0.5 mg·kg^-1-Control
化合物名称 Compound name	VIP	FC	P	趋势 Trend	VIP	FC	P	趋势 Trend	VIP	FC	P	趋势 Trend
尿苷二磷酸-N-乙酰葡糖胺 UDP-N-acetylglucosamine	2.1451	3.7838	0.0175	↑	1.0349	2.0512	0.0494	↑	1.4664	2.4830	0.0277	↑
硫唑嘌呤Azathioprine	3.5178	22.9131	0.0038	↑	2.2240	6.6142	0.0216	↑	2.8184	12.1241	0.0135	↑
反玉米素Trans-zeatin	3.4573	27.3706	0.0012	↑	2.1436	10.783	0.0109	↑	3.5919	29.5094	0	↑
焦炭酸二乙酯Diethylpyrocarbonate	1.8239	2.6369	0.0126	↑	1.3799	2.1616	0.0312	↑	1.8167	2.6558	0.0272	↑
阿斯巴甜 Methyl alpha-aspartylphenylalaninate	2.4076	6.6251	0.0210	↑	1.5393	3.6759	0.0056	↑	2.5201	7.6114	0.0272	↑
草完隆Noruron	2.5040	5.7411	0.0237	↑	1.8949	5.3422	0.0312	↑	2.4082	5.7124	0.0272	↑
四聚乙醛Ethanal tetramer	3.1358	10.8657	0.0022	↑	1.6834	4.3428	0.0168	↑	1.9323	4.0922	0.0240	↑
扎莫特罗Xamoterol	1.7021	2.5619	0.0303	↑	1.6402	2.9414	0.0271	↑	2.3289	4.8191	0.0272	↑
二甲弗林Dimetofrine	2.0830	5.3298	0.0337	↑	1.9187	6.1752	0.0216	↑	2.3776	6.4440	0.0434	↑
细辛脑Asarone	3.5178	22.9131	0.0038	↑	2.2168	7.7327	0.0301	↑	2.4104	5.5813	0.0399	↑
异丙酚Propofol	1.9412	3.3387	0.0169	↑	1.7439	3.4319	0.0121	↑	2.2298	4.1785	0.0135	↑
芹黄素Apigetrin	2.7393	7.0597	0	↑	2.1954	5.8356	0.008	↑	2.2192	5.1848	0.0070	↑
4,4'-二硝基联苄4,4'-dinitrobibenzyl	3.5924	36.459	0.0024	↑	1.7819	5.2543	0.0073	↑	3.1705	15.6981	0.0019	↑
早熟素Precocene ii	2.8581	11.2521	0.0012	↑	1.0312	2.0035	0.0439	↑	2.1354	3.8515	0.0428	↑
贝那普利拉Benazeprilat	1.3429	2.0242	0.0438	↑	1.8099	6.4223	0.0188	↑	2.6353	10.2252	0.0135	↑
氟扎可特Azacortid	2.6960	12.7132	0.0305	↑	2.4081	15.5769	0.0188	↑	3.5251	35.6758	0.0048	↑
甲苯Toluene	1.5881	0.4467	0.0226	↓	1.4841	0.2876	0.0301	↓	1.9802	0.2721	0.0235	↓
愈伤酸Traumatic acid	2.4552	0.1909	0.0021	↓	1.5634	0.2683	0.0121	↓	2.0103	0.2575	0.0079	↓
9-羟基壬酸9-oxononanoic acid	2.9634	0.0980	0.0006	↓	1.3122	0.2638	0.0139	↓	2.7705	0.1129	0.0015	↓
普瑞巴林Pregabalin	1.6876	0.4322	0.0028	↓	1.2720	0.4581	0.0178	↓	1.0768	0.5848	0.0389	↓
阿伐那非Avanafil	1.9936	0.3237	0.0253	↓	1.1258	0.4234	0.0347	↓	1.7236	0.3553	0.0235	↓
1-壬酸1-nonanoic acid	2.6990	0.1060	0.0104	↓	2.0052	0.1232	0.0238	↓	2.5335	0.1151	0.0260	↓
匹伐加宾Pivagabine	2.8956	0.1266	0.0057	↓	2.0864	0.1468	0.0560	↓	2.1990	0.1935	0.0135	↓
美噻吨Metixene	2.6297	0.1776	0.0012	↓	1.1710	0.3815	0.0271	↓	1.4653	0.3801	0.0277	↓
樟脑(+/-)-camphor	2.2568	0.2106	0.0169	↓	1.4478	0.2932	0.0247	↓	2.2074	0.2177	0.0216	↓
扎莫特罗Xamoterol [usan:ban:inn]	1.8083	0.2538	0.0229	↓	2.1189	0.1402	0.0056	↓	1.8550	0.2617	0.0235	↓
二甲福林Dimetofrine	2.5825	0.1265	0.0139	↓	2.1321	0.1535	0.0238	↓	2.6898	0.1118	0.0199	↓
苯乙酮Acetophenone	1.7796	0.3617	0.0213	↓	1.8288	0.2989	0.0139	↓	1.5460	0.4091	0.0387	↓
氢过氧化亚油酸13(s)-HPODE	1.1855	0.5253	0.0414	↓	1.6963	4.7824	0.0331	↑	1.5777	0.4183	0.0235	↓

表2

表3

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化合物名称Compound name	Pathway ID	通路Pathway
尿苷二磷酸-N-乙酰葡糖胺 UDP-N-acetylglucosamine	Map00520	氨基糖与核糖代谢 Amino sugar and nucleotide sugar metabolism
硫唑嘌呤 Azathioprine	Map00983	药物代谢-其他酶类代谢 Drug metabolism-other enzymes
反玉米素 Trans-zeatin	Map01100	其他代谢途径 Metabolic pathways
甲苯 Toluene	Map01100	其他代谢途径 Metabolic pathways
愈伤酸 Traumatic acid	Map00592	ɑ-亚麻酸代谢 alpha-Linolenic acid metabolism
9-羟基壬酸 9-oxononanoic acid	Map00592	ɑ-亚麻酸代谢 alpha-Linolenic acid metabolism
氢过氧化亚油酸 13(s)-HPODE	Map01100	其他代谢途径 Metabolic pathways