Please wait a minute...
Journal of Integrative Agriculture  2021, Vol. 20 Issue (6): 1585-1595    DOI: 10.1016/S2095-3119(20)63356-1
Special Issue: 昆虫合辑Plant Protection—Entomolgy 害虫抗药性和毒理学合辑Pest Toxicology
Plant Protection Advanced Online Publication | Current Issue | Archive | Adv Search |
Molecular characterization of the ryanodine receptor from Adoxophyes orana and its response to lethal and sublethal doses of chlorantraniliprole
SUN Li-na, LIU Yan-di, ZHANG Huai-jiang, YAN Wen-tao, YUE Qiang, QIU Gui-sheng
Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, P.R.China
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      

昆虫鱼尼丁受体(RyRs)是邻甲酰氨基苯甲酰胺类和邻苯二甲酰胺类等二酰胺类杀虫剂的靶标。目前,已经有几种二酰胺类杀虫剂在我国果园害虫防治中应用。为了加强二酰胺类杀虫剂作用机制的了解,本文克隆了危害仁果和核果类较为严重的害虫苹小卷叶蛾的RyR(AoRyR)基因cDNA全长。此外,还研究了二酰胺杀虫剂对AoRyR mRNA表达调控的影响。通过本研究获得AoRyR mRNA的开放阅读框(ORF)为15402 bp,可编码5113个氨基酸。氨基酸序列比对结果显示,AoRyR与其他昆虫和哺乳动物RyRs分别具有77-92%和45-47%的一致性。在AoRyR中发现了一个可变剪接位点为互斥外显子(a/b),其在卵和幼虫中的使用频率高于蛹和成虫。实时荧光定量qPCR显示,AoRyR mRNA在各发育阶段均有表达,特别是在卵、蛹和雄成虫中高表达。3龄幼虫经LC10、LC20和LC50剂量氯虫苯甲酰胺处理后,AoRyR mRNA表达水平明显上调。研究结果为进一步研究苹小卷叶蛾鱼尼丁受体的功能和开发具有抗虫选择性活性的新化合物提供了依据。

The insect ryanodine receptor (RyR) is a novel target of the anthranilic and phthalic insecticides, which have high activity against lepidopteran insects.  Several diamide insecticides have been used to control pests in orchards in China.  To enhance our understanding of the effects of diamides on RyRs, full-length cDNAs were isolated and characterized from the summer fruit tortrix moth, Adoxophyes orana, which is the most severe pest of stone and pome trees worldwide.  In addition, the modulation of AoRyR mRNA expression by diamide insecticides was investigated.  The AoRyR mRNA obtained had an open reading frame (ORF) of 15 402 bp nucleotides encoding 5 113 amino acids, and shared high and low identity with its orthologs in other insects and mammals of 77–92 and 45–47% identity, respectively.  One alternative splice site with two exclusive exons was revealed in AoRyR (a/b).  The usage of exon was more frequent in eggs and larvae than in pupae and adults.  Quantitative real-time reverse transcription PCR (qRT-PCR) showed that AoRyR mRNA was expressed at all developmental stages, especially in eggs, male pupae and male adults.  The expression levels of AoRyR mRNA in the whole body were up-regulated markedly after 3rd instar larvae were treated with chlorantraniliprole at LC10, LC20 and LC50 dosages.  The results could provide the basis for further functional studies of AoRyR and for the development of new chemicals with selective activity against insects. 
Keywords:  ryanodine receptor        Adoxophyes orana        chlorantraniliprole        mRNA expression  
Received: 09 March 2020   Accepted:
Fund: This research was supported by the National Key R&D Program of China (2017YFD0200300), and the Fundamental Research Funds for Central Non-profit Scientific Institution, China (1610182019011).
Corresponding Authors:  Correspondence QIU Gui-sheng, Tel: +86-429-3598266, E-mail:   
About author:  SUN Li-na, E-mail:

Cite this article: 

SUN Li-na, LIU Yan-di, ZHANG Huai-jiang, YAN Wen-tao, YUE Qiang, QIU Gui-sheng. 2021. Molecular characterization of the ryanodine receptor from Adoxophyes orana and its response to lethal and sublethal doses of chlorantraniliprole. Journal of Integrative Agriculture, 20(6): 1585-1595.

Campos M R, Silva T B M, Silva W M, Silve J E, Siqueira H A A. 2015. Susceptibility of Tuta absoluta (Lepidoptera: Gelechiidae) Brazilian populations to ryanodine receptor modulators. Pest Management Science, 71, 537–544.
Cui L, Yang D B, Yan X J, Rui C H, Wang Z Y, Yuan H Z. 2013. Molecular cloning characterization and expression profiling of a ryanodine receptor gene in Asian corn borer Ostrinia furnacalis (Guenée). PLoS ONE, 8, e75825. 
Cordova D, Benner E A, Sacher M D, Rauh J J, Sopa J S, Lahm G P, Selby T P, Stevenson T M, Flexner L, Gutteridge S. 2006. Anthranilic diamides: A new class of insecticides with a novel mode of action ryanodine receptor activation. Pesticide Biochemistry and Physiology, 84, 196–214.
Dinter A, Brugger K E, Frost N M, Woodward M D. 2009. Chlorantraniliprole (Rynaxypyr): A novel DuPontTM insecticide with low toxicity and low risk for honey bees (Apis mellifera) and bumble bees (Bombus terrestris) providing excellent tools for uses in integrated pest management. Julius-Kühn-Archiv, 423, 84–96.
Ebbinghaus-Kintscher U, Luemmen P, Lobitz N, Schulte T, Funke C, Fischer R, Masaki T, Yasokawa N, Tohnishi M. 2006. Phthalic acid diamides activate ryanodine sensitive Ca2+ release channels in insects. Cell Calcium, 39, 21–33.
Ebbinghaus-Kintscher U, Lümmen P, Raming K, Masaki T, Yasokawa N. 2007. Flubendiamide the first insecticide with a novel mode of actionon insect ryanodine receptors. Pflanzenschutz-Nachrichten Bayer, 60, 117–140.
Fill M, Copella J A. 2002. Ryanodine receptor calcium release channels. Physiological Reviews, 82, 893–922.
Gopal M, Mishra E. 2008. Analytical method for estimation of a new insecticide flubendiamide and its safety evaluation for usage in rice crop. Bulletin of Environmental Contamination and Toxicology, 81, 360–364.
Guo L, Liang P, Zhou X G, Gao X W. 2014a. Novel mutations and mutation combinations of ryanodine receptor in a chlorantraniliprole resistant population of Plutella xylostella (L.). Scientific Report, 4, 6924.
Guo L, Tang B Z, Dong W, Liang P, Gao X W. 2012. Cloning characterisation and expression profiling of the cDNA encoding the ryanodine receptor in diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae). Pest Management Science, 68, 1605–1614.
Guo L, Wang Y, Zhou X G, Li ZY, Liu S Z, Liang P, Gao X W. 2014b. Functional analysis of a point mutation in the ryanodine receptor of Plutella xylostella (L.) associated with resistance to chlorantraniliprole. Pest Management Science, 70, 1083–1089.
Hamilton S L. 2005. Ryanodine receptors. Cell Calcium, 38, 253–260.
Han W S, Zhang S F, Shen F Y, Liu M, Ren C C, Gao X W. 2012. Residual toxicity and sublethal effects of chlorantraniliprole on Plutella xylostella (Lepidoptera: Plutellidae). Pest Management Science, 68, 1184–1190.
Ioriatti C, Anfora G, Angeli G, Mazzoni V, Trona F. 2009. Effects of chlorantraniliprole on eggs and larvae of Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae). Pest Management Science, 65, 717–722.
Kato K, Kiyonaka S, Sawaguchi Y, Tohnishi M, Masaki T, Yasokawa N, Mizuno Y, Mori E, Lnoue K, Hamachi I, Takeshima H, Mori Y. 2009. Molecular characterization of flubendiamide sensitivity in the lepidopterous ryanodine receptor Ca2+ release channel. Biochemistry, 48, 10342–10352.
Lahm G P, Selby T P, Freudenberger J H, Stevenson T M, Myers B J, Seburyamo G, Smith B K, Flesner L, Clark C E, Cordova D. 2005. Insecticidal anthranilic diamides: A new class of potent ryanodine receptor activators. Bioorganic & Medicinal Chemistry Letters, 15, 4898–4906.
Lahm G P, Stevenson T M, Selby T P, Freudenberger J H, Cordova D, Flexner L, Bellin C A, Dubas C M, Smith B K, Hughes K A, Hollingshaus J G, Clarh C E, Benner E A. 2007. RynaxypyreTM: A new insecticidal anthranilic diamide that acts as a potent and selective ryanodine receptor activator. Bioorganic & Medicinal Chemistry Letters, 17, 6274–6279.
Lai F A, Misrai M, Xu L, Smith H A, Meissner G. 1989. The Ryanodine receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum. Journal of Biological Chemistry, 264, 16776–16785.
Lin L Y, Hao Z Y, Cao P, Yuchi Z G. 2019. Homology modeling and docking study of diamondback moth ryanodine receptor reveals the mechanisms for channel activation, insecticide binding and resistance. Pest Management Science, 76, 1291–1301.
Lin L Y, Liu C, Qin J, Wang J, Dong S J, Chen W, He W Y, Gao Q Z, You M S, Yuchi Z G. 2018. Crystal structure of ryanodine receptor N-terminal domain from Plutella xylostella reveals two potential species-specific insecticide-targeting sites. Insect Biochemistry and Molecular Biology, 92, 73–83.
Lin Q S, Jin F L, Hu Z D, Chen H Y, Yin F, Li Z Y, Dong X L, Zhang D Y, Ren S X, Feng X. 2013. Transcriptome Analysis of chlorantraniliprole resistance development in the diamondback moth Plutella xylostella. PLoS ONE, 8, e72314.
Mandal S D, Shakeel M, Prabhakaran V S, Karthi S, Xu X X, Jin F L. 2019. Alternative splicing and insect ryanodine receptor. Archives of Insect Biochemistry and Physiology, 102, e21590.
Ogawa Y, Murayama T. 1998. The Structure and Function of Ryanodine Receptors. Imperial College Press, London. pp. 5–22.
Ogawa Y, Murayama T, Kurebayashi N. 1999. Comparison of properties of Ca2+ release channels between rabbit and frog skeletal muscles. Molecular and Cellular Biology, 190, 191–201.
Ottini L, Marziali G, Conti A, Charlesworth A, Sorrentino V. 1996. Alpha and beta isoforms of ryanodine receptor from chicken skeletal muscle are the homologues of mammalian RyR1 and RyR3. Biochemical Journal, 315, 207–216.
Peng Y C, Shenga C W, Casida J E, Zhao C Q, Han Z J. 2017. Ryanodine receptor genes of the rice stem borer, Chilo suppressalis: Molecular cloning, alternative splicing and expression profiling. Pesticide Biochemistry and Physiology, 135, 69–77.
Pessah I N, Waterhouse A L, Casida J E. 1985. The calcium ryanodine receptor complex of skeletal and cardiac muscle. Biochemical and Biophysical Research Communications, 128, 449–456.
Pessah I N, Zimanyi I. 1991. Characterization of multiple [3H]ryanodine binding sites on the Ca2+ release channel of sarcoplasmic reticulum from skeletal and cardiac muscle evidence for a sequential mechanism in ryanodine action. Biochemistry, 39, 679–689.
Roditakis E, Skarmoutsou C, Staurakaki M, Martínez-Aguirre M R, García-Vidal L, Bielza P, Haddi K, Rapisarda C, Rison J L, Bassi A, Teixeira L A. 2013. Determination of baseline susceptibility of European populations of Tuta absoluta (Meyrick) to indoxacarb and chlorantraniliprole using a novel dip bioassay method. Pest Management Science, 69, 217–227.
Rogers E F, Koniuszy F R, Shavel J, Folkers K. 1948. Plant insecticides: Ryanodine, a new alkaloid from Ryania speciosa Vahl. Journal of the American Chemical Society, 70, 3086–3088.
Sattelle D B, Cordova D, Cheek T R. 2008. Insect ryanodine receptors: molecular targets for novel pest control chemicals. Invertebrate Neuroscience, 8, 107–119.
Schmittgen T D, Livak K J. 2008. Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3, 1101–1108.
Schmitt M, Turberg A, Londershausen M, Dorn A. 1996. Binding sites for Ca2+-channel effectors and ryanodine in Periplaneta americana - Possible targets for new insecticides. Pest Management Science, 48, 375–385.
Sun L N, Cui L, Rui C H, Yan X J, Yang D B, Yuan H Z. 2012. Modulation of the expression of ryanodine receptor from Plutella xylostella as a result of diamide insecticide application. Gene, 511, 265–273.
Sun L N, Qiu G S, Zhang H J, Yan W T. 2015a. Growth development and reproduction of the summer fruit tortrix moth Adoxophyes orana beijingensis (Lepidoptera: Tortricidae) on four host plants. Acta Entomologica Sinica, 58, 53–59. (in Chinese)
Sun L N, Yan W T, Zhang H J, Yue Q, Li Y Y, Qiu G S. 2015b. Research dynamics on the diamides insecticides by bibliometric approach. Chinese Journal of Pesticide Science, 17, 640–650. (in Chinese)
Sun L N, Yan W T, Zhang H J, Zhang P, Liu C L, Qiu G S. 2014. Evaluation of six insecticides against the summer fruit tortrix moth Adoxophyes orana bejingensis in apple orchards. Plant Protection, 40, 181–184. (in Chinese)
Sun L N, Zhang H J, Quan L F, Yan W T, Yue Q, Li Y Y, Qiu G S. 2016. Characterization of the ryanodine receptor gene with a unique 3’-UTR and alternative splice site from the oriental fruit moth. Journal of Insect Science, 16, 1–9.
Sun L N, Zhang H J, Yan W T, Ma C S, Qiu G S. 2015c. Molecular cloning and expression profling of a ryanodine receptor gene in the peach fruit moth (Carposina ssakii). Scientia Agricultura Sinica, 48, 1971–1981. (in Chinese)
Tao Y, Gutteridge S, Benner E A, Wu L H, Rhoades D, Sacher M, Rivera M A. 2013. Identification of a critical region in the Drosophila ryanodine receptor that confers sensitivity to diamide insecticides. Insect Biochemistry and Molecular Biology, 43, 820–828.
Teixeira L A, Andaloro G T. 2012. Diamide insecticides: Global efforts to address insect resistance stewardship challenges. Pesticide Biochemistry and Physiology, 106, 76–78.
Tiwari S, Stelinski L L. 2012. Effects of cyantraniliprole, a novel anthranilic diamide insecticide, against Asian citrus psyllid under laboratory and field conditions. Pest Management Science, 69, 1066–1072.
Tohnishi M, Nakao H, Furuya T, Seo A, Kodama H, Tsubata K. 2005. Flubendiamide, a novel insecticide highly active against Lepidopterous insect pests. Journal of Pesticide Science, 30, 354–360.
Troczka B, Zimmer C T, Elias J, Schorn C, Bass C, Emyr Davies T G, Field L M, Williamson M S, Slater R, Nauen R. 2012. Resistance to diamide insecticides in diamondback moth Plutella xylostella (Lepidoptera: Plutellidae) is associated with a mutation in the membrane-spanning domain of the ryanodine receptor. Insect Biochemistry and Molecular Biology, 42, 873–880.
Wang J, Liu Y P, Gao J K, Xie Z J, Huang L, Wang W L, Wang J J. 2013. Molecular cloning and mRNA expression of a ryanodine receptor gene in the cotton bollworm Helicoverpa armigera. Pesticide Biochemistry and Physiology, 107, 327–333.
Wang J, Xie Z J, Gao J K, Liu Y P, Wang W H, Huang L, Wang J J. 2014. Molecular cloning and characterization of a ryanodine receptor gene in brown planthopper (BPH) Nilaparvata lugens (Stål). Pest Management Science, 70, 790–797.
Wang J J, Li Y Q, Han Z J, Zhu Y L, Xie Z J, Wang J, Liu Y P, Li X C. 2012. Molecular characterization of a ryanodine receptor gene in the rice leaffolder Cnaphalocrocis medinalis (Guenée). PLoS ONE, 7, e36623.
Wang X L, Wu S W, Yang Y H, Wu Y D. 2012. Molecular cloning characterization and mRNA expression of a ryanodine receptor gene from diamondback moth Plutella xylostella. Pesticide Biochemistry and Physiology, 102, 204–212.
Wang X X, Khakame S K, Ye C, Yang Y H, Wu Y D. 2013. Characterisation of field-evolved resistance to chlorantraniliprole in the diamondbackmoth, Plutella xylostella, from China. Pest Management Science, 69, 661–665.
Wu S F, Zhao D D, Huang J M, Zhao S Q, Zhou L Q, Gao C F. 2018. Molecular characterization and expression profiling of ryanodine receptor gene in the pink stem borer, Sesamia inferens (Walker). Pesticide Biochemistry and Physiology, 146, 1–6.
Xu X H, Bhat M B, Nishi M, Takeshima H, Ma J J. 2000. Molecular cloning of cDNA encoding a Drosophila ryanodine receptor and functional studies of the carboxyl-terminal calcium release channel. Biophysical Journal, 78, 1270–1281.
Yan H H, Xue C B, Li G Y, Zhao X L, Che X Z, Wang L L. 2014. Flubendiamide resistance and Bi-PASA detection of ryanodine receptor G4946E mutation in the diamondback moth (Plutella xylostella L.). Pesticide Biochemistry and Physiology, 115, 73–77.
Yang Y, Wan P J, Hu X X, Li G Q. 2014. RNAi mediated knockdown of the ryanodine receptor gene decreases chlorantraniliprole susceptibility in Sogatella furcifera. Pesticide Biochemistry and Physiology, 108, 58–65.
Yuan G R, Shi W Z, Yang W J, Jiang X Z, Dou W, Wang J J. 2014. Molecular characteristics, mRNA expression and alternative splicing of a ryanodine receptor gene in the oriental fruit fly Bactrocera dorsalis (Hendel). PLoS ONE, 9, e95199.
Zhang H J, Qiu G S, Yan W T, Chen H J, Zhang P, Zheng Y C, Liu C L. 2011. Effectiveness of Ryanxypye on controlling main pests and its impact on the predatory insects in apple orchard. Journal of Environmental Entomology, 33, 493–501. (in Chinese)
Zhang X, Xu Q J, Lu W W, Liu F. 2015. Sublethal effects of four synthetic insecticides on the generalist predator Cytorhinus lividipennis. Journal of Pest Science, 88, 383–392.
Zhao M C, Li P, Li X L, Zhang L, Winkfein R J, Chen S R W. 1999. Molecular identification of the ryanodine receptor pore-forming segment. The Journal of Biological Chemistry, 274, 25971–25974.
Zorzato F, Fujii J, Otso K, Phillips M, Green N M, Lai F A, Meissner G, Maclennan D H. 1990. Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. Journal of Biological Chemistry, 265, 2244–2256.
[1] SHAN Yan-ju, JI Gai-ge, ZOU Jian-min, ZHANG Ming, TU Yun-jie, LIU Yi-fan, JU Xiao-jun, SHU Jing-ting. PGC-1α differentially regulates the mRNA expression profiles of genes related to myofiber type specificity in chicken[J]. >Journal of Integrative Agriculture, 2020, 19(8): 2083-2094.
[2] LU Yun-feng, CHEN Ji-bao, ZHANG Bo, LI Qing-gang, WANG Zhi-xiu, ZHANG Hao, WU Ke-liang . Cloning, expression, and polymorphism of the ECI1 gene in various pig breeds[J]. >Journal of Integrative Agriculture, 2017, 16(08): 1789-1799.
[3] HU Zhen-di, FENG Xia, LIN Qing-sheng, CHEN Huan-yu, LI Zhen-yu, YIN Fei, LIANG Pei, GAO Xi-wu. cDNA cloning and characterization of the carboxylesterase pxCCE016b from the diamondback moth, Plutella xylostella L.[J]. >Journal of Integrative Agriculture, 2016, 15(05): 1059-1068.
No Suggested Reading articles found!