东方蜜蜂幼虫封盖信息素含量及生物合成通路

doi:10.3864/j.issn.0578-1752.2021.11.017

摘要/Abstract

摘要：

【目的】在蜜蜂中,甲基棕榈酸酯（MP）、甲基油酸酯（MO）、甲基亚油酸酯（ML）和甲基亚麻酸酯（MLN）是重要的封盖信息素成分,它们触发成年工蜂对幼虫的封盖行为。本研究旨在比较封盖信息素化学成分在不同封盖时期的东方蜜蜂（Apis cernana）工蜂与雄蜂幼虫体内的含量,并分析其在幼虫体内的生物合成通路,进一步探索蜜蜂幼虫与成年工蜂之间的信息素交流机制。【方法】以中华蜜蜂（Apis cernana cernana）为实验材料,分别取未封盖、正在封盖和已封盖的工蜂与雄蜂幼虫,利用GC/MS分析技术,比较4种封盖信息素成分在不同封盖时期的工蜂与雄蜂幼虫体内的含量;同时利用RNA-seq技术对不同封盖时期的工蜂与雄蜂幼虫进行转录组测序,分析其基因表达差异,并根据差异表达基因KEGG富集分析推测封盖信息素的生物合成通路。【结果】在工蜂幼虫中,4种封盖信息素成分在正在封盖和已封盖幼虫体内的含量均显著高于未封盖幼虫,其中MP和MO的含量均随幼虫日龄增长而显著增加,而ML和MLN的含量在正在封盖和已封盖幼虫体内差异不显著;在雄蜂幼虫中,4种信息素成分含量均随日龄增长而增加,且已封盖幼虫的信息素含量显著高于未封盖和正在封盖的幼虫。对工蜂与雄蜂3个封盖时期幼虫的基因表达量进行组间比较分析,分别从3个比较组中获得4 299和3 926个差异表达基因,并且在差异表达基因KEGG注释分析中分别获得152和130个KEGG通路。根据差异表达基因KEGG富集结果,推测出东方蜜蜂工蜂与雄蜂幼虫可能利用乙酰辅酶A合成MP、MO、ML和MLN的生物合成通路以及11个调控候选基因,并发现该生物合成通路与西方蜜蜂相同。【结论】东方蜜蜂工蜂幼虫与雄蜂幼虫在被封蜡盖的关键阶段增加了MP、MO、ML和MLN的释放量,进一步验证了它们是与蜜蜂封盖行为相关的信息素,并且推测这些信息素可能是在相关基因的调控下由乙酰辅酶A从头合成,而东方蜜蜂幼虫与西方蜜蜂幼虫可能利用相同的生物合成通路进行信息素的生物合成。

关键词: 东方蜜蜂, 封盖信息素, 含量, 转录组, 生物合成通路

Abstract:

【Objective】 In honeybees, methyl palmitate (MP), methyl oleate (MO), methyl linoleate (ML) and methyl linolenate (MLN) are important capping pheromone components, which trigger the capping behavior of adult workers. The objective of this study is to compare the contents of these four pheromone components in the larvae of workers and drones of Apis cernana at different capping stages, analyze their biosynthetic pathways, and to further explore the mechanism of pheromone communication between larvae and adult workers. 【Method】Using A. c. cernana as the experimental material, the larvae of workers and drones of prior to be capped, in the process of being capped and had been capped were collected for comparing the contents of these four pheromone components by using GC/MS. Simultaneously, RNA-seq was used for gene expression analysis, and the biosynthetic pathways were speculated based on KEGG enrichment of differential expressed genes. 【Result】In worker larvae, the contents of the four capping pheromone components were significantly higher at the capping and capped stage than those of the prior to be capped larvae, and the contents of MP and MO significantly increased with aging of the larvae, while the contents of ML and MLN were not significantly different between the capping and capped stage. Whereas in drone larvae, the contents of the four pheromone components were higher overall and increased with aging, and the content at capped stage was significantly higher than that at prior to be capped and capping. RNA-seq results showed that there were 4 299 and 3 926 differential expressed genes among the larvae groups of three stages of workers and drones, respectively. In addition, 152 and 130 KEGG pathways were obtained from the KEGG annotation analysis of the differential expressed genes, respectively. Furthermore, the possible de novo biosynthetic pathways were proposed for MP, MO, ML and MLN from acetyl-CoA, regulating under 11 related candidate genes, and these biosynthesis pathways were found to be similar to those of Apis mellifera. 【Conclusion】The release contents of MP, MO, ML and MLN were increased during the critical stage of capping in worker and drone larvae of A. cernana, which further verified that these four pheromones were related to capping behavior of honeybees, and it was speculated that they were possibly de novo biosynthesized from acetyl-CoA under the control of related genes. A. cernana larvae and A. mellifera larvae may use the same biosynthesis pathway for pheromone biosynthesis.

Key words: Apis cernana, capping pheromone, content, transcriptome, biosynthetic pathway

秦秋红,何旭江,江武军,王子龙,曾志将. 东方蜜蜂幼虫封盖信息素含量及生物合成通路[J]. 中国农业科学, 2021, 54(11): 2464-2475.

QIN QiuHong,HE XuJiang,JIANG WuJun,WANG ZiLong,ZENG ZhiJiang. The Capping Pheromone Contents and Putative Biosynthetic Pathways in Larvae of Honeybees Apis cernana[J]. Scientia Agricultura Sinica, 2021, 54(11): 2464-2475.

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参考文献 47

[1]	FREE J B, WINDER M E. Brood recognition by honey bee (Apis mellifera) workers. Animal Behavior, 1983,31:539-545. doi: 10.1016/S0003-3472(83)80077-3
[2]	HAYDAK M H. Honey bee nutrition. Annual Review of Entomology, 1970,15:143-156. doi: 10.1146/annurev.en.15.010170.001043
[3]	HUANG Z Y, OTIS G W. Inspection and feeding of larvae by worker honey bees (Hymenoptera: Apidae): Effect of starvation and food quantity. Journal of Insect Behavior, 1991,4(3):305-317. doi: 10.1007/BF01048280
[4]	HUANG Z Y, OTIS G W. Nonrandom visitation of brood cells by worker honey bees (Hymenoptera: Apidae). Journal of Insect Behavior, 1991,4(2):177-184. doi: 10.1007/BF01054610
[5]	LE CONTE Y, ARNOLD G, TROUILLER J, MASSON C, CHAPPE B, OURISSON G. Attraction of the parasitic mite Varroa to the drone larvae of honey bees by simple aliphatic esters. Science, 1989,245(4918):638-639. doi: 10.1126/science.245.4918.638
[6]	SLESSOR K N, WINSTON M L, LE CONTE Y. Pheromone communication in the honeybee (Apis mellifera L.). Journal of Chemical Ecology, 2005,31(11):2731-2745. doi: 10.1007/s10886-005-7623-9
[7]	LE CONTE Y. The recognition of larvae by worker honeybees. Naturwissenschaften, 1994,81:462-465. doi: 10.1007/BF01136651
[8]	HE X J, ZHANG X C, JIANG W J, BARRON A B, ZHANG J H, ZENG Z J. Starving honey bee (Apis mellifera) larvae signal pheromone ally to worker bees. Scientific Reports, 2016,6:22359. doi: 10.1038/srep22359
[9]	何旭江, 江武军, 颜伟玉, 曾志将. 蜜蜂蜂王与雄蜂幼虫饥饿信息素鉴定及其生物合成通路. 中国农业科学, 2016,49(23):4646-4655.
	HE X J, JIANG W J, YAN W Y, ZENG Z J. Identification and biosynthetic pathway of a hunger pheromone in honeybee queen and drone larvae. Scientia Agricultura Sinica, 2016,49(23):4646-4655. (in Chinese)
[10]	曾志将. 蜜蜂生物学. 北京: 中国农业出版社, 2007.
	ZENG Z J. Bee Biology. Beijing: China Agriculture Press, 2007. (in Chinese)
[11]	LE CONTE Y, ARNOLD G, TROUILLER J, MASSON C, CHAPPE B. Identification of a brood pheromone in honeybees. Naturwissenschaften, 1990,77:334-336. doi: 10.1007/BF01138390
[12]	QIN Q H, HE X J, BARRON A B, GUO L, JIANG W J, ZENG Z J. The capping pheromones and putative biosynthetic pathways in worker and drone larvae of honey bees Apis mellifera. Apidologie, 2019,50(6):793-803. doi: 10.1007/s13592-019-00686-9
[13]	TROUILLER J, ARNOLD G, CHAPPE B, LE CONTE Y, MASSON C. Semiochemical basis of infestation of honey bee brood by Varroa jacobsoni. Journal of Chemical Ecology, 1992,18(11):2041-2053. doi: 10.1007/BF00981926
[14]	曾志将. 养蜂学. 3 版. 北京: 中国农业出版社, 2017.
	ZENG Z J. Apiculture. 3rd ed. Beijing: China Agriculture Press, 2017. (in Chinese)
[15]	陈盛禄. 中国蜜蜂学. 北京: 中国农业出版社, 2001.
	CHEN S L. The Apicultural Science in China. Beijing: China Agriculture Press, 2001. (in Chinese)
[16]	QIN Q H, HE X J, TIAN L Q, ZHANG S W, ZENG Z J. Comparison of learning and memory of Apis cerana and Apis mellifera. Journal of Comparative Physiology A, 2012,198(10):777-786. doi: 10.1007/s00359-012-0747-9
[17]	颜伟玉, LE CONTE Y, BESLAY D, 曾志将. 中华蜜蜂幼虫信息素鉴定. 中国农业科学, 2009,42(6):2250-2254.
	YAN W Y, LE CONTE Y, BESLAY D, ZENG Z J. Identification of brood pheromone in Chinese honeybee [Apis cerana cerana (Hymenoptera: Apidae)]. Scientia Agricultura Sinica, 2009,42(6):2250-2254. (in Chinese)
[18]	张含, 曾志将, 颜伟玉, 吴小波, 郑云林. 信息素中三种酯类对中华蜜蜂工蜂发育和采集行为的影响. 昆虫学报, 2010,53(1):55-60.
	ZHANG H, ZENG Z J, YAN W Y, WU X B, ZHENG Y L. Effects of three aliphatic esters of brood pheromone on development and foraging behavior of Apis cerana cerana workers. Acta Entomologica Sinica, 2010,53(1):55-60. (in Chinese)
[19]	TREFZ P, KISCHKEL S, HEIN D, JAMES E S, SCHUBERTA J K, MIEKISCH W. Needle trap micro-extraction for VOC analysis: Effects of packing materials and desorption parameters. Journal of Chromatography A, 2012,1219:29-38. doi: 10.1016/j.chroma.2011.10.077
[20]	余爱丽, 赵晋锋, 成锴, 王振华, 张鹏, 刘鑫, 田岗, 赵太存, 王玉文. 谷子萌发吸水期关键代谢途径的筛选与分析. 中国农业科学, 2020,53(15):3005-3019.
	YU A L, ZHAO J F, CHENG K, WANG Z H, ZHANG P, LIU X, TIAN G, ZHAO T C, WANG Y W. Screening and analysis of key metabolic pathways in foxtail millet during different water uptake phases of germination. Scientia Agricultura Sinica, 2020,53(15):3005-3019. (in Chinese)
[21]	LOVE M I, HUBER W, ANDERS S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 2014,15:550. doi: 10.1186/s13059-014-0550-8
[22]	XIE C, MAO X, HUANG J, DING Y, WU J M, DONG S, KONG L, GAO G, LI C Y, WEI L P. KOBAS 2.0: A web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Research, 2010,39:W316-W322. doi: 10.1093/nar/gkr483
[23]	LIU W, SAINT D A. A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Analytical Biochemistry, 2002,302(1):52-59. doi: 10.1006/abio.2001.5530
[24]	DVINGE H, BERTONE P. HTqPCR: High-throughput analysis and visualization of quantitative real-time PCR data in R. Bioinformatics, 2009,25(24):3325-3326. doi: 10.1093/bioinformatics/btp578
[25]	ROELOFS W, BJOSTAD L. Biosynthesis of lepidopteran pheromones. Bioorganic Chemistry, 1984,12(4):279-298. doi: 10.1016/0045-2068(84)90011-7
[26]	ANDO T, HASE T, ARIMA R, UCHIYAMA M. Biosynthetic pathway of bombykol, the sex pheromone of the female silkworm moth. Agricultural and Biological Chemistry, 1998,52(2):473-478.
[27]	TANG J D, CHARLTON R E, JURENKA R A, WOLF W A, PHELAN P L, SRENG L, ROELOFS W L. Regulation of pheromone biosynthesis by a brain hormone in two moth species. Proceedings of the National Academy of Sciences of the United States of America, 1989,86(6):1806-1810.
[28]	LE CONTE Y, ELLIS M, RITTER W. Varroa mites and honey bee health: Can Varroa explain part of the colony losses? Apidologie, 2010,41(3):353-363. doi: 10.1051/apido/2010017
[29]	曾云峰, 曾志将, 颜伟玉, 吴小波. 幼虫信息素中三种酯类对中华蜜蜂和意大利蜜蜂工蜂哺育和封盖行为以及蜂王发育影响. 昆虫学报, 2010,53(2):154-159.
	ZENG Y F, ZENG Z J, YAN W Y, WU X B. Effects of three aliphatic esters of brood pheromone on worker feeding and capping behavior and queen development of Apis cerana cerana and A. mellifera ligustica. Acta Entomologica Sinica, 2010,53(2):154-159. (in Chinese)
[30]	TILLMAN J A, SEYBOLD S J, JURENKA R A, BLOMQUIST G J. Insect pheromones—An overview of biosynthesis and endocrine regulation. Insect Biochemistry and Molecular Biology, 1999,29(6):481-514. doi: 10.1016/S0965-1748(99)00016-8
[31]	MCGEE R, SPECTOR A A. Fatty acid biosynthesis in Erlich cells. The mechanism of short term control by exogenous free fatty acids. The Journal of Biological Chemistry, 1975,250(14):5419-5425. doi: 10.1016/S0021-9258(19)41198-8
[32]	CRIPPS C, BLOMQUIST G J, DE RENOBALES M. De novo biosynthesis of linoleic acid in insects. Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1986,876(3):572-580.
[33]	MOSHITZKY P, MILOSLAVSKI I, AIZENSHTAT Z, APPLEBAUM S W. Methyl palmitate: A novel product of the Medfly (Ceratitis capitata) corpus allatum. Insect Biochemistry and Molecular Biology, 2003,33(12):1299-1306. doi: 10.1016/j.ibmb.2003.06.008
[34]	STERN N, SHENBERG E, TIETZ A. Studies on the metabolism of fatty acids in Leptospira: The biosynthesis of Δ9- and Δ11- monounsaturated acids. European Journal of Biochemistry, 1969,8(1):101-108. doi: 10.1111/ejb.1969.8.issue-1
[35]	BACHLAVA E, DEWEY R E, BURTON J W, CARDINAL A J. Mapping candidate genes for oleate biosynthesis and their association with unsaturated fatty acid seed content in soybean. Molecular Breeding, 2009,23(2):337-347. doi: 10.1007/s11032-008-9246-7
[36]	KNIPPLE D C, ROSENFIELD C L, MILLER S J, LIU W, TANG J, MA P W K, ROELOFS W L. Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Δ¹¹-desaturase of the cabbage looper moth, Trichoplusia ni. Proceedings of the National Academy of Sciences of the United States of America, 1998,95:15287-15292.
[37]	LIENARD M A, STRANDH M, HEDENSTROM E, JOHANSSON T, LÖFSTEDT C. Key biosynthetic gene subfamily recruited for pheromone production prior to the extensive radiation of Lepidoptera. BMC Evolutionary Biology, 2008,8:270. doi: 10.1186/1471-2148-8-270
[38]	DING B J, LIENARD M A, WANG H L, ZHAO C H, LÖFSTEDT C. Terminal fatty-acyl-CoA desaturase involved in sex pheromone biosynthesis in the winter moth (Operophtera brumata). Insect Biochemistry and Molecular Biology, 2011,41(9):715-722. doi: 10.1016/j.ibmb.2011.05.003
[39]	HAGSTROM A K, LIENARD M A, GROOT A T, HEDENSTROM E, LÖFSTEDT C. Semi-selective fatty acyl reductases from four heliothine moths influence the specific pheromone composition. PLoS ONE, 2012,7(5):e37230. doi: 10.1371/journal.pone.0037230
[40]	LIENARD M A, WANG H L, LASSANCE J M, LÖFSTEDT C. Sex pheromone biosynthetic pathways are conserved between moths and the butterfly Bicyclus anynana. Nature Communications, 2014,5:3957. doi: 10.1038/ncomms4957
[41]	WANG H L, BRATTSTRÖM O, BRAKEFIELD P M, FRANCKE W, LÖFSTEDT C. Identification and biosynthesis of novel male specific esters in the wings of the tropical butterfly, Bicyclus martius sanaos. Journal of Chemical Ecology, 2014,40(6):549-559. doi: 10.1007/s10886-014-0452-y
[42]	王镜岩, 朱圣庚, 徐长法. 生物化学. 3版. 北京: 高等教育出版社, 2002.
	WANG J Y, ZHU S G, XU C F. Biological Chemistry. 3rd ed. Beijing: Higher Education Press, 2002. (in Chinese)
[43]	SERRA M, PIÑA B, BUJONS J, CAMPS F. FABRIAS G. Biosynthesis of 10, 12-dienoic fatty acids by a bifunctional Δ11 desaturase in Spodoptera littoralis. Insect Biochemistry and Molecular Biology, 2006,36(8):634-641. doi: 10.1016/j.ibmb.2006.05.005
[44]	LOFSTEDT C, ELMFORS A, SJÖGREN M, WIJK E. Confirmation of sex pheromone biosynthesis from (16-D₃) palmitic acid in the turnip moth using capillary gas chromatography. Experientia, 1986,42(9):1059-1061. doi: 10.1007/BF01940732
[45]	RODRIGUEZ F, HALLAHAN D L, PICKETT J A, CAMPS F. Characterization of the Δ ¹¹-palmitoyl-CoA-desaturase from Spodoptera littoralis (Lepidoptera: Noctuidae) . Insect Biochemistry and Molecular Biology, 1992,22(2):143-148. doi: 10.1016/0965-1748(92)90152-5
[46]	FOSTER S P. Sex pheromone biosynthesis in the tortricid moth Planotortrix excessana (Walker) involves chain-shortening of palmitoleate and oleate. Archives of Insect Biochemistry and Physiology, 1998,37(2):158-167. doi: 10.1002/(ISSN)1520-6327
[47]	ROELOFS W, BJOSTAD L. Biosynthesis of lepidopteran pheromones. Bioorganic Chemistry, 1984,12(4):279-298. doi: 10.1016/0045-2068(84)90011-7

基因编号 Gene ID	基因名称 Gene name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
gene-APICC_00421	Kat	CCACGGCCTCAAACGACTC	CGGCACCATCAGAAATACCAG
gene-APICC_06057	Hadha	GGAGGAGGCTTGGAGATGG	GCACCAGGCAAGATACCTAACA
gene-APICC_00389	Mecr	TGCCTTCACCAAAATTAGCCC	CCATGCTGTCATCCAAAATCC
gene-APICC_01118	Δ11 desaturase	GCTTGTCAATTCCGCTGCTC	AACCTTCGCCAAGTGCTCCTA
gene-APICC_02088	Hacd	GCAGCTTCATCTTTTGCTCTTC	CTATAACTCCATGCTTCTGGGTG
	β-actin	TCCTGGAATCGCAGATAGAATG	GGAAGGTGGACAAAGAAGCAAG

基因编号Gene ID	基因注释Gene annotation
gene-APICC_00421	3-ketoacyl-CoA thiolase (Apis cerana cerana)
gene-APICC_06057	Trifunctional enzyme subunit alpha (Apis cerana cerana)
gene-APICC_10032	Enoyl-CoA hydratase (Apis cerana cerana)
gene-APICC_00389	PREDICTED: probable trans-2-enoyl-CoA reductase, mitochondrial (Apis cerana)
gene-APICC_05477	PREDICTED: elongation of very long chain fatty acids protein 4-like isoform X1 (Apis dorsata)
gene-APICC_02088	PREDICTED: very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase hpo-8 (Apis cerana)
gene-APICC_05451	Trans-2,3-enoyl-CoA reductase (Apis cerana cerana)
gene-APICC_01118	Acyl-CoA Delta (11) desaturase (Apis cerana cerana)
gene-APICC_08222	Acyl-CoA desaturase (Apis cerana cerana)
gene-APICC_01590	PREDICTED: acyl-CoA Delta (11) desaturase-like (Apis cerana)
gene-APICC_03413	Acyl-CoA desaturase (Apis cerana cerana)