Effect of PirAB Toxin from Xenorhabdus nematophila on the Activities of Related Enzymes in Helicoverpa armigera
GUO Xiao-Xiao1, YANG Qing2, LIU Shu-Qin1, WANG Qin-Ying1, NANGONG Zi-Yan1*
1 College of Plant Protection, Hebei Agricultural University, Baoding 071000, China; 2 College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China
Abstract:Entomopathogenic nematode symbiotic bacteria and insecticidal toxins have been a research hotspot in the field of pest control. PirAB (photorhabdus insect related toxin AB) toxin is a special binary toxin from symbiotic bacteria of entomopathogenic nematodes with high insecticidal activity, and the insecticidal characteristics of the toxin and its effect on the enzyme activity of host insects were carried out in present study. Firstly, the pirAB gene of Xenorhabdus nematophila HB310 was expressed in prokaryotic expression system, and the biological activity of the recombinant PirAB protein was determinated on the 4th instar larvae of Helicoverpa armigera by intracavity injection method, and the influence of PirAB against related enzymes (acetylcholinesterase, carboxylesterase, polyphenol oxidase, tyrosinase and peroxidase) was further detected. The results showed that recombinant PirAB protein demonstrated a high intravenous injection activity (LD50=2.003 μg/larva). After injection of PirAB toxin with LD50 dose, there was no significant difference in acetylcholinesterase activity between the treatment and the control group; Carboxylesterase activity showed a dramatic change of activation, inhibition and re-activation, and tended to be stable after 36 h treatment; Polyphenol oxidase activity also showed a trend of first activation and then inhibition, and the change of activity tended to be stable after 24 h, and was significantly lower than that of the control group (P<0.05). Tyrosinase activity was basically inhibited after toxin treatment, and there was a significant change between 12 and 36 h which decreased sharply and increased again, and the tyrosinase activity was the same as that of the control; The overall change trend of peroxidase activity was not obvious and continued to be lower than that of the control. This study provides basic information for revealing the immune mechanism of host insects against PirAB toxin.
郭笑笑, 杨晴, 刘淑琴, 王勤英, 南宫自艳. 嗜线虫致病杆菌PirAB毒素对棉铃虫相关酶活性的影响[J]. 农业生物技术学报, 2019, 27(7): 1266-1274.
GUO Xiao-Xiao, YANG Qing, LIU Shu-Qin, WANG Qin-Ying, NANGONG Zi-Yan. Effect of PirAB Toxin from Xenorhabdus nematophila on the Activities of Related Enzymes in Helicoverpa armigera. 农业生物技术学报, 2019, 27(7): 1266-1274.
[1] 陈清西, 宋康康. 2006. 酪氨酸酶的研究进展[J]. 厦门大学学报(自然版), 45(5): 731-737. (Chen Q X, Song K K, 2006. Research progress in tyrosinase[J]. Journal of Xiamen University (Natural Science), 45(5): 731-737.) [2] 蒋晴, 苏宏华, 杨益众. 2013. 昆虫对Bt毒素的抗性与中肠蛋白酶、解毒酶及保护酶系活性的关系[J]. 环境昆虫学报, 35(1): 95-101. (Jiang Q, Su H H, Yang Y Z.2013. Relationship between midgut proteinases, detoxification enzymes, protective enzymes in insect and the resistance to Bt[J]. Journal of Environmental Entomology, 35(1): 95-101.) [3] 刘丹, 严善春, 曹传旺, 等. 2012. 多杀菌素对黄褐天幕毛虫解毒酶及保护酶的影响[J]. 林业科学, 48(4): 67-74. (Liu D, Yan S C, Cao C W, et al.2012. Effect of spinosad on detoxifying and protective enzymes of Malacosoma neustria testaces[J]. Scientia Silvae Sinicae, 48(4): 67-74.) [4] 李星月. 2015. 昆虫病原线虫新种Heterorhabditis beicherriana的分类鉴定及其生物防治的应用探讨与机理研究[D]. 博士学位论文, 中国农业大学, 导师: 刘奇志, pp. 1-75. (Li X Y.2015. Classification and identification of Heterorhabditis beicherriana, a new species of entomopathogenic nematode, and application and mechanism of biological control[D]. Thesis for Ph.D., China Agricultural University, Suppervisor: Liu Q Z, pp. 1-75.) [5] 任娜娜, 谢苗, 尤燕春, 等. 2014. 羧酸酯酶及其介导昆虫抗药性的研究进展[J]. 福建农林大学学报(自然版), 43(4): 337-344. (Ren N N, Xie M, You Y C, et al.2014. An overview on the study of insect carboxylesterases (COEs) and the COE-mediated resistance to insecticides[J]. Journal of Fujian Agriculture & Forestry University, 43(4): 337-344.) [6] 孙建宇, 柳春林, 邱礼鸿. 2012. 荧光发光杆菌TT01品系pirA2B2基因的克隆表达与杀虫活性[J]. 微生物学报, 52(4): 532-537. (Sun J Y, Liu C L, Qiu LH.2012. Cloning, expression and insecticidal activity of the pirA2B2 gene from Photorhabdus luminescens TT01[J]. Acta Microbiologica Sinica, 52(4): 532-537.) [7] 杨君, 王勤英, 宋萍, 等. 2008. 嗜线虫致病杆菌血腔毒素Tp40对大蜡螟幼虫体内酶活性和中肠组织的影响[J]. 昆虫学报, 51(6): 601-608. (Yang J, Wang Q Y, Song P, et al.2008. Influence of the haemocoel toxin (Tp40) from Xenorhabdus nematophila on enzyme activities and midgut tissues in Galleria mellonella (Lepidoptera: Pyralidae) larvae[J]. Acta Entomologica Sinica, 51(6): 601-608.) [8] 鄢杰明, 钟华, 严俊鑫, 等. 2012. 多杀菌素对舞毒蛾幼虫解毒酶活性的影响[J]. 林业科学, 48(9): 82-87. (Yan J M, Zhong H, Yan J X, et al.2012. Effect of spinosad to detoxifying enzymes activity in Lymantria dispar larva[J]. Scientia Silvae Sinicae, 48(9): 82-87.) [9] 苑胜垒, 管京敏, 杨兵, 等. 2016. 昆虫免疫蛋白多酚氧化酶的研究进展[J]. 生命科学, (1): 70-76. (Yuan S L, Guan J M, Yang B, et al. 2016. Recent achievements on the insect important innate immunity protein prophenoloxidase[J]. Chinese Bulletin of Life Sciences, (1): 70-76.) [10] 赵爱平, 展恩玲, 孙聪, 等. 2017. Cry1Ac毒素对小菜蛾幼虫中肠蛋白酶和羧酸酯酶活性的影响[J]. 植物保护学报, 44(5): 713-720. (Zhao A P, Zhan E L, Sun C, et al.2017. Effects of Cry1Ac toxin on proteases and carboxylesterase activities in the larvael midgut of Plutella xylostella[J]. Journal of Plant Protection, 44(5): 713-720.) [11] Ahantarig A, Chantawat N, Waterfield N R, et al.2009. PirAB toxin from Photorhabdus asymbiotica as a larvicide against dengue vectors[J]. Applied and Environmental Microbiology, 75(13): 4627-4629. [12] Alonso V, Nasrolahi S, Dillman A R.2018. Host-specific activation of entomopathogenic nematode infective juveniles[J]. Insects, 9(2): 59-69. [13] Blackburn M B, Farrar R R, Novak N G, et al.2006. Remarkable susceptibility of the diamondback moth (Plutella xylostella) to ingestion of Pir toxins from Photorhabdus luminescens[J]. Entomologia Experimentalis Applicata, 121(1): 31-37. [14] Bowen D, Rocheleau T A, Blackburn M, et al.1998. Insecticidal toxins from the bacterium Photorhabdus luminescens[J]. Pest Management Science, 280(5372): 2129-2132. [15] Casida J E, Durkin K A.2013. Neuroactive insecticides: Targets, selectivity, resistance, and secondary effects[J]. Annual Review of Entomology, 58(1): 99-117. [16] Crosland R D, Fitch R W, Hines H B.2005. Characterization of beta-leptinotarsin-h and the effects of calcium flux antagonists on its activity[J]. Toxicon, 45(7): 829-841. [17] Cruz-Martínez H, Ruiz-Vega J, Matadamas-Ortíz P T, et al.2017. Formulation of entomopathogenic nematodes for crop pest control: A review[J]. Plant Protection Science, 53(1): 1-10. [18] Duchaud E, Rusniok C, Frangeul L, et al.2003. The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens[J]. Nature Biotechnology, 21(11): 1307-1313. [19] Ellman GL, Courtney KD, Jr AV, et al.1961. A new and rapid colorimetric determination of acetylcholine esterase activity[J]. Biochemical Pharmacology, 7(2): 88-90, IN1, 91-95. [20] Jiang X C, Jiang X Y, Liu S.2018. Molecular characterization and expression analysis of two acetylcholinesterase genes from the small white butterfly Pieris rapae (Lepidoptera: pieridae)[J]. Journal of Insect Science, 18(5): 1-9. [21] Hurst M R H, Beattie A, Altermann E, et al.2016. The draft genome sequence of the Yersinia entomophaga entomopathogenic type strain MH96T[J]. Toxins, 8(5): 143-162. [22] Ishaaya I, Casida J E.1974. Dietary TH 6040 alters composition and enzyme activity of housefly larval cuticle[J]. Pesticide Biochemistry & Physiology, 4(4): 484-490. [23] Karuppaiah V, Srivastava C, Padaria J C, et al.2017. Quantitative changes of the carboxylesterase associated with pyrethroid susceptibility in Spodoptera litura (lepidoptera: noctuidae)[J]. African Entomology, 25(1): 175-182. [24] Kim Y H, Lee S H.2018. Invertebrate acetylcholinesterases: Insights into their evolution and non-classical functions[J]. Journal of Asia-Pacific Entomology, 21(1): 186-195. [25] Lee C T, Chen I T, Yang Y T, et al.2015. The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin[J]. Proceedings of the National Academy of Sciences of the USA, 112(34): 10798-10803. [26] Li Y, Hu X, Zhang X, et al.2014. Photorhabdus luminescens PirAB-fusion protein exhibits both cytotoxicity and insecticidal activity[J]. Fems Microbiology Letters, 356(1): 23-31. [27] Lin S J, Hsu K C, Wang H C.2017. Structural insights into the cytotoxic mechanism of Vibrio parahaemolyticus PirAvp and PirBvp toxins[J]. Marine Drugs, 15(12): 373-385. [28] Lu D, Macchietto M, Chang D, et al.2017. Activated entomopathogenic nematode infective juveniles release lethal venom proteins[J]. PLoS Pathogens, 13(4): e1006302. [29] Lu D, Sepulveda C, Dillman A R.2017. Infective juveniles of the entomopathogenic nematode Steinernema scapterisci are preferentially activated by cricket tissue[J]. PLoS One, 12(1): e0169410. [30] Michaelb B, Robertr F, Nicoleg N, et al.2006. Remarkable susceptibility of the diamondback moth (Plutella xylostella) to ingestion of Pir toxins from Photorhabdus luminescens[J]. Entomologia Experimentalis Applicata, 121(1): 31-37. [31] Miljanich G P, Yeager R E, Hsiao T H.1988. Leptinotarsin-D, a neurotoxic protein, evokes neurotransmitter release from, and calcium flux into, isolated electric organ nerve terminals[J]. Journal of Neurobiology, 19(4): 373-386. [32] Nangong Z Y, Wang Q Y, Song P, et al.2016. Synergism between Bacillus thuringiensis and Xenorhabdus nematophila against resistant and susceptible Plutella xylostella (Lepidoptera: Plutellidae)[J]. Biocontrol Science & Technology, 26(10): 1-22. [33] Ramsden C A, Riley P A,2014. Tyrosinase: The four oxidation states of the active site and their relevance to enzymatic activation, oxidation and inactivation[J]. Bioorganic & Medicinal Chemistry, 22(8): 2388-2395. [34] van Asperen K.1962. A study of house fly esterase by means of a sensitive colorimetric method[J]. Journal of Insect Physiology, 8(4): 401-414, IN3, 415-416. [35] Wang Q Y, Nangong Z Y, Yang J, et al.2012. Toxic activity of a protein complex purified from Xenorhabdus nematophila HB310 to Plutella xylostella larvae[J]. Insect Science, 19(3): 329-336. [36] Waterfield N, Kamita S, Hammock B C R.2005. The Photorhabdus Pir toxins are similar to a developmentally regulated insect protein but show no juvenile hormone esterase activity[J]. Fems Microbiology Letters, 245(1): 47-52. [37] Yang Q, Zhang J, Li T, et al.2017. PirAB protein from Xenorhabdus nematophila HB310 exhibits a binary toxin with insecticidal activity and cytotoxicity in Galleria mellonella[J]. Journal of Invertebrate Pathology, 148: 43-50.