柑橘大实蝇滞育型与非滞育型蛹的代谢谱比较

doi:10.3864/j.issn.0578-1752.2019.06.006

摘要/Abstract

摘要：

【目的】明确柑橘大实蝇（Bactrocera minax）滞育型（D）与非滞育型（ND）蛹各自在不同时间点之间的代谢谱差异,以及相同时间点上D型与ND型蛹之间的代谢谱差异。此外,发掘在不同对比组合中具有显著差异的代谢物种类。【方法】在化蛹后1 d内,注射20E溶液解除滞育,获取ND型蛹;注射10%乙醇溶剂获取对照D型蛹。在注射后1、15和30 d收集两种类型的蛹,利用核磁共振（NMR）技术对样品进行检测。运用Chenomx软件将NMR谱图信号与数据库逐一比对分析,确定代谢物种类和对应的浓度。再使用偏最小二乘判别分析法（PLS-DA）进行数据分析,明确D1对D15对D30、ND1对ND15对ND30、D1对ND1、D15对ND15、D30对ND30共5个对比组合之间的代谢谱差异。最后结合变量投影重要性分析（VIP）和单因素方差分析或t检验,发掘在不同对比组合中具有显著浓度差异的代谢物种类。【结果】在所有样品中,共获得50种代谢物及其浓度。其中,氨基酸及其衍生物20种、有机酸11种、糖类4种、核酸组分5种,其他代谢物10种。PLS-DA分析表明,D型蛹在3个时间点之间代谢谱差异显著;ND型蛹在注射后15 d和30 d之间代谢谱无显著差异,但两者均与注射后1 d之间差异显著;在3个不同时间点上,D型与ND型蛹之间代谢谱均差异显著。VIP分析和单因素方差分析或t检验结果表明,共有21种代谢物浓度至少在一个对比组合中呈现显著差异,其中,D1对D15对D30组合中有10种;ND1对ND15对ND30组合中有12种;D1对ND1组合中有6种;D15对ND15组合中有12种;D30对ND30组合中有8种。柠檬酸、葡萄糖、麦芽糖、脯氨酸、海藻糖、谷氨酸、天冬酰胺和N-乙酰谷氨酸出现显著差异的次数最多。【结论】出现显著差异次数最多的代谢物中,柠檬酸、葡萄糖、麦芽糖、脯氨酸、海藻糖、谷氨酸和天冬酰胺能直接或间接进入三羧酸循环,说明柑橘大实蝇滞育期间能量代谢和物质转化产生了显著的变化。此外,海藻糖、脯氨酸、N-乙酰谷氨酸和肌醇的显著差异有助于调控柑橘大实蝇滞育期间的耐寒性。研究结果可为后续解析重要代谢物及相关生理途径在柑橘大实蝇滞育中的作用机理提供依据。

关键词: 柑橘大实蝇, 滞育, 代谢组, 核磁共振, 20-羟基蜕皮酮（20E）

Abstract:

【Objective】The objective of this study is to determine the metabolic profile differences of diapause-destined (D) and non-diapause-destined (ND) pupae of Bactrocera minax at different time points, and between D and ND pupae at the same time point, and to identify metabolites with significant difference in different comparisons.【Method】Within 1 d after pupation, 20E solution was injected to release diapause and acquire ND pupae; 10% ethanol was injected to acquire D pupae. Both types of pupae were collected at 1, 15, and 30 d after injection, and the samples were detected by nuclear magnetic resonance (NMR). All the spectra were analyzed with the Chenomx Compound Library one by one to determine the metabolite species and the corresponding concentration. Then, the partial least squares discriminant analysis (PLS-DA) was conducted to determine the metabolomic variations in 5 comparisons, D1 vs D15 vs D30, ND1 vs ND15 vs ND30, D1 vs ND1, D15 vs ND15, and D30 vs ND30. Lastly, the variable importance in projection (VIP) and one-way ANOVA/t test were performed to find out the metabolites with significant concentration difference in different contrast combinations.【Result】In all samples, a total of 50 metabolites and their concentrations were obtained, including 20 kinds of amino acids and their derivatives, 11 kinds of organic acids, 4 kinds of sugars, 5 kinds of nucleic acid components, and 10 kinds of other metabolites. PLS-DA analysis indicated that the metabolic profile of D pupae was significantly different among the three time points. The metabolic profile of ND pupae was not significantly different between 15 d and 30 d after injection, but both of them were significantly different with that of 1 d after injection. At three different time points, the metabolic profiles of D pupae were all significantly different from those of ND pupae. VIP analysis and one-way ANOVA/t test collectively indicated that the concentrations of 21 metabolites were significantly different in at least one comparison. Of these 21 metabolites, 10, 12, 6, 12, and 8 species were found in D1 vs D15 vs D30, ND1 vs ND15 vs ND30, D1 vs ND1, D15 vs ND15, and D30 vs ND30 comparisons, respectively. Citrate, glucose, maltose, proline, trehalose, glutamate, asparagine, and N-acetylglutamate showed significantly different level in most comparisons.【Conclusion】Among the metabolites with the most significant differences, citrate, glucose, maltose, proline, trehalose, glutamate, and asparagine can directly or indirectly enter the tricarboxylic acid (TCA) cycle, indicating that the energy metabolism and substance transformation have significant changes during B. minax diapause. In addition, the significant differences of trehalose, proline, N-acetylglutamate, and inositol contribute to the regulation of freezing tolerance during B. minax diapause. The results can provide a basis for further investigation of mechanisms of important metabolites and relevant physiological pathways during B. minax diapause.

Key words: Bactrocera minax, diapause, metabolome, nuclear magnetic resonance (NMR), 20-hydroxyecdysone (20E)

王佳,王攀,樊欢,刘映红. 柑橘大实蝇滞育型与非滞育型蛹的代谢谱比较[J]. 中国农业科学, 2019, 52(6): 1021-1031.

WANG Jia,WANG Pan,FAN Huan,LIU YingHong. Comparison of Metabolic Profile Between Diapause-Destined and Non-Diapause-Destined Pupae of Bactrocera minax[J]. Scientia Agricultura Sinica, 2019, 52(6): 1021-1031.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.06.006

https://www.chinaagrisci.com/CN/Y2019/V52/I6/1021

图/表 5

图1

图2

图3

图4

图5

参考文献 36

[1]	DORJI C, CLARKE A R , DREW R A I, FLETCHER B S, LODAY P, MAHAT K, RAGHU S, ROMIG M C . Seasonal phenology of Bactrocera minax(Diptera: Tephritidae) in western Bhutan. Bulletin of Entomological Research, 2006,96(5):531-538.
[2]	DREW R A I, DORJI C, ROMIG M C, LODAY P . Attractiveness of various combinations of colors and shapes to females and males of Bactrocera minax(Diptera: Tephritidae) in a commercial mandarin grove in Bhutan. Journal of Economic Entomology, 2006,99(5):1651-1656. doi: 10.1603/0022-0493-99.5.1651 pmid: 17066795
[3]	王小蕾, 张润杰 . 桔大实蝇生物学、生态学及其防治研究概述. 环境昆虫学报, 2009,31(1):73-79. doi: 10.3969/j.issn.1674-0858.2009.01.012
	WANG X L, ZHANG R J . Review on biology, ecology and control of Bactrocera (Tetradacus) minax Enderlein. Journal of Environmental Entomology, 2009,31(1):73-79. (in Chinese) doi: 10.3969/j.issn.1674-0858.2009.01.012
[4]	DENLINGER D L . Regulation of diapause. Annual Review of Entomology, 2002,47:93-122. doi: 10.1146/annurev.ento.47.091201.145137
[5]	HAHN D A, DENLINGER D L . Meeting the energetic demands of insect diapause: Nutrient storage and utilization. Journal of Insect Physiology, 2007,53(8):760-773. doi: 10.1016/j.jinsphys.2007.03.018 pmid: 17532002
[6]	RAGLAND G J, FULLER J, FEDER J L, HAHN D A . Biphasic metabolic rate trajectory of pupal diapause termination and post-diapause development in a tephritid fly. Journal of Insect Physiology, 2009,55(4):344-350. doi: 10.1016/j.jinsphys.2008.12.013 pmid: 19200436
[7]	WANG J, FAN H, XIONG K C, LIU Y H . Transcriptomic and metabolomic profiles of Chinese citrus fly, Bactrocera minax(Diptera: Tephritidae), along with pupal development provide insight into diapause program. PLoS ONE, 2017,12(7):e0181033.
[8]	DONG Y C, DESNEUX N, LEI C L, NIU C Y . Transcriptome characterization analysis of Bactrocera minax and new insights into its pupal diapause development with gene expression a nalysis. International Journal of Biological Sciences, 2014,10(9):1051-1063. doi: 10.7150/ijbs.9438 pmid: 25285037
[9]	MICHAUD M R, DENLINGER D L . Shifts in the carbohydrate, polyol, and amino acid pools during rapid cold-hardening and diapause-associated cold-hardening in flesh flies (Sarcophaga crassipalpis): a metabolomic comparison. Journal of Comparative Physiology B-Biochemical Systemic and Environmental Physiology, 2007,177(7):753-763. doi: 10.1007/s00360-007-0172-5 pmid: 17576567
[10]	ZHANG Q, LU Y X, XU W H . Proteomic and metabolomic profiles of larval hemolymph associated with diapause in the cotton bollworm, Helicoverpa armigera. BMC Genomics, 2013,14:751. doi: 10.1186/1471-2164-14-751 pmid: 24180224
[11]	KOŠTÁL V, ŠTĚTINA T, POUPARDIN R, KORBELOVÁ J, BRUCE A W . Conceptual framework of the eco-physiological phases of insect diapause development justified by transcriptomic profiling. Proceedings of the National Academy of Sciences of the United States of America, 2017,114(32):8532-8537. doi: 10.1073/pnas.1707281114 pmid: 28720705
[12]	TU X, WANG J, HAO K, WHITMAN D W, FAN Y, CAO G, ZHANG Z . Transcriptomic and proteomic analysis of pre-diapause and non-diapause eggs of migratory locust, Locusta migratoria L.(Orthoptera: Acridoidea). Scientific Reports, 2015,5:11402. doi: 10.1038/srep11402 pmid: 4650673
[13]	ZHANG Q, LU Y X, XU W H . Integrated proteomic and metabolomic analysis of larval brain associated with diapause induction and preparation in the cotton bollworm, Helicoverpa armigera. Journal of Proteome Research, 2012,11(2):1042-1053. doi: 10.1021/pr200796a pmid: 22149145
[14]	WISHART D S . Quantitative metabolomics using NMR. Trends in Analytical Chemistry, 2008,27(3):228-237. doi: 10.1016/j.trac.2007.12.001
[15]	夏建飞, 梁琼麟, 胡坪, 王义明, 罗国安 . 代谢组学研究策略与方法的新进展. 分析化学, 2009,37(1):136-143. doi: 10.3321/j.issn:0253-3820.2009.01.029
	XIA J F, LIANG Q L, HU P, WANG Y M, LUO G A . Recent trends in strategies and methodologies for metabonomics. Chinese Journal of Analytical Chemistry, 2009,37(1):136-143. (in Chinese) doi: 10.3321/j.issn:0253-3820.2009.01.029
[16]	WANG J, ZHOU H Y, ZHAO Z M, LIU Y H . Effects of juvenile hormone analogue and ecdysteroid on adult eclosion of the fruit fly Bactrocera minax(Diptera: Tephritidae). Journal of Economic Entomology, 2014,107(4):1519-1525. doi: 10.1603/EC13539 pmid: 25195444
[17]	CHEN Z, DONG Y, WANG Y, ANDONGMA A A, RASHID M A, KRUTMUANG P, NIU C . Pupal diapause termination in Bactrocera minax: an insight on 20-hydroxyecdysone induced phenotypic and genotypic expressions. Scientific Reports, 2016,6:27440. doi: 10.1038/srep27440 pmid: 27273028
[18]	WANG J, XIONG K C, LIU Y H . De novo transcriptome analysis of Chinese citrus fly, Bactrocera minax( Diptera: Tephritidae), by high-throughput Illumina sequencing. PLoS ONE, 2016,11(6):e0157656.
[19]	MEVIK B H, WEHRENS R . The pls package: Principal component and partial least squares regression in R. Journal of Statistical Software, 2007,18(2):1-23. doi: 10.1360/jos180001
[20]	BERG J M, TYMOCZKO J L, STRYER L . Biochemistry. 5th ed. New York: W.H. Freeman & Co Ltd, 2002.
[21]	LU Y X, ZHANG Q, XU W H . Global metabolomic analyses of the hemolymph and brain during the initiation, maintenance, and termination of pupal diapause in the cotton bollworm, Helicoverpa armigera. PLoS ONE, 2014,9(6):e99948. doi: 10.1371/journal.pone.0099948 pmid: 4057385
[22]	XU W H, LU Y X, DENLINGER D L . Cross-talk between the fat body and brain regulates insect developmental arrest. Proceedings of the National Academy of Sciences of the United States of America, 2012,109(36):14687-14692. doi: 10.1073/pnas.1212879109 pmid: 22912402
[23]	刘遥, 张礼生, 陈红印, 黄凤霞, 蒋莎, 任小云 . 苹果酸脱氢酶与异柠檬酸脱氢酶在滞育七星瓢虫中的差异表达. 中国生物防治学报, 2014,30(5):593-599. doi: 10.3969/j.issn.2095-039X.2014.05.004
	LIU Y, ZHANG L S, CHEN H Y, HUANG F X, JIANG S, REN X Y . Differential expression of malate dehydrogenase and isocitrate dehydrogenase in diapaused ladybird, Coccinella septempunctata L. Chinese Journal of Biological Control, 2014,30(5):593-599. (in Chinese) doi: 10.3969/j.issn.2095-039X.2014.05.004
[24]	JAIN N K, ROY I . Effect of trehalose on protein structure. Protein Science, 2009,18(1):24-36.
[25]	THOMPSON S N . Trehalose-The insect ‘blood’ sugar//SIMPSON S J. Advances in Insect Physiology. Elsevier Ltd., 2003,31:205-285.
[26]	唐斌, 张露, 熊旭萍, 汪慧娟, 王世贵 . 海藻糖代谢及其调控昆虫几丁质合成研究进展. 中国农业科学, 2018,51(4):697-707. doi: 10.3864/j.issn.0578-1752.2018.04.009
	TANG B, ZHANG L, XIONG X P, WANG H J, WANG S G . Advances in trehalose metabolism and its regulation of insect chitin synthesis. Scientia Agricultura Sinica, 2018,51(4):697-707. (in Chinese) doi: 10.3864/j.issn.0578-1752.2018.04.009
[27]	GUO Q, HAO Y J, LI Y, ZHANG Y J, REN S, SI F L, CHEN B . Gene cloning, characterization and expression and enzymatic activities related to trehalose metabolism during diapause of the onion maggot Delia antiqua(Diptera: Anthomyiidae). Gene, 2015,565(1):106-115. doi: 10.1016/j.gene.2015.03.070 pmid: 25841989
[28]	KOŠTÁL V, ŠIMEK P . Dynamics of cold-hardiness, supercooling and cryoprotectants in diapausing and non-diapausing pupae of the cabbage root fly, Delia radicum L. Journal of Insect Physiology, 1995,41(7):627-634. doi: 10.1016/0022-1910(94)00124-Y
[29]	ROZSYPAL J , KOŠTÁL V, ZAHRADNÍČKOVÁ H, ŠIMEK P . Overwintering strategy and mechanisms of cold tolerance in the codling moth ( Cydia pomonella). PLoS ONE, 2013,8(4):e61745. doi: 10.1371/journal.pone.0061745 pmid: 3629207
[30]	KOŠTÁL V, ŠIMEK P, ZAHRADNÍČKOVÁ H, CIMLOVA J, STETINA T . Conversion of the chill susceptible fruit fly larva (Drosophila melanogaster) to a freeze tolerant organism. Proceedings of the National Academy of Sciences of the United States of America, 2012,109(9):3270-3274.
[31]	KOŠTÁL V, ZAHRADNÍČKOVÁ H, ŠIMEK P . Hyperprolinemic larvae of the drosophilid fly, Chymomyza costata, survive cryopreservation in liquid nitrogen. Proceedings of the National Academy of Sciences of the United States of America, 2011,108(32):13041-13046.
[32]	BEMANI M, IZADI H, MANDIAN K, KHANI A, SAMIH M A . Study on the physiology of diapause, cold hardiness and supercooling point of overwintering pupae of the pistachio fruit hull borer, Arimania comaroffi. Journal of Insect Physiology, 2012,58(7):897-902. doi: 10.1016/j.jinsphys.2012.04.003 pmid: 22542495
[33]	WATANABE M, TANAKA K . Seasonal change of the thermal response in relation to myo-inositol metabolism in adults of Aulacophora nigripennis(Coleoptera Chrysomelidae). Journal of Insect Physiology, 1999,45(2):167-172.
[34]	LI J, LI G . Transamination of 3-hydroxykynurenine to produce xanthurenic acid: a major branch pathway of tryptophan metabolism in the mosquito, Aedes aegypti, during larval development. Insect Biochemistry and Molecular Biology, 1997,27(10):859-867. doi: 10.1016/S0965-1748(97)00068-4 pmid: 9474782
[35]	MACKENZIE S M, HOWELLS A J, COX G B, EWART G D . Sub-cellular localisation of the White/Scarlet ABC transporter to pigment granule membranes within the compound eye of Drosophila melanogaster. Genetica, 2000,108(3):239-252. doi: 10.1023/A:1004115718597 pmid: 11294610
[36]	CALDOVIC L, TUCHMAN M . N-acetylglutamate and its changing role through evolution. The Biochemical Journal, 2003,372(2):279-290.