Abstract
Chymotrypsins (CTP) are a main type of proteolytic digestive enzymes in Lepidopteran insects. They can play a major role in degrading activated Bt toxin in some insect species, leading to decreased Bt toxicity and inducing insect resistance. Seven CTP-like sequences were obtained by screening transcriptome data of Asian corn borer, Ostrinia furnacalis. The expression analyses revealed that almost all of the CTP genes showed relatively higher transcript levels at the larval stage, and most showed relatively higher transcript levels in the saliva and midgut. We synthesized dsRNA from the seven CTP-like genes and fed it to larvae at the different instar stages. The dsRNAs from all seven CTPs significantly enhanced mortality of larvae from the first to the third instar larval stages, and only the dsRNA from CTP8 had higher lethality to fourth instar larvae. When larvae were fed on a diet containing 0.2 mg/ml dsRNA and Bt (5 μg/g diet), the 5-day mortalities were significantly enhanced, except for larvae treated with dsRNA from CTP5. The dsCTP16 plus Bt treatment resulted in a 5-day mortality of 100 %, and all of the dsCTPs accelerated insect death. These results indicated that members of the CTP-like gene family are effective RNAi targets for pest control. In particular, CTP8 represents an effective target for controlling the whole larval stage, and CTP16 is an effective target to enhance Bt insecticidal efficiency.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abdel-latief M, Meyering-Vos M, Hoffmann KH (2003) Molecular characterization of cDNAs from the fall armyworm Spodoptera frugiperda encoding Manduca sextaallatotropin and allatostatin preprohormone peptides. Insect Biochem Mol Biol 33:467–476. doi:10.1016/S0965-1748(03)00005-5
Alcantara E, Estrada A, Alpuerto V, Head G (2011) Monitoring Cry1Ab susceptibility in Asian corn borer (Lepidoptera: Crambidae) on Bt corn in the Philippines. Crop Prot 30:554–559. doi:10.1016/j.cropro.2010.12.019
Bajgar A, Jindra M, Dolezel D (2013) Autonomous regulation of the insect gut by circadian genes acting downstream of juvenile hormone signaling. Proc Natl Acad Sci USA 110:4416–4421. doi:10.1073/pnas.1217060110
Bhatia V, Bhattacharya R, Uniyal PL, Singh R, Niranjan RS (2012) Host generated siRNAs attenuate expression of serine protease gene in Myzus persicae. PLoS One 7:e46343. doi:10.1371/journal.pone.0046343
Broehan G, Kemper M, Driemeier D, Vogelpohl I, Merzendorfer H (2008) Cloning and expression analysis of midgut chymotrypsin-like proteinases in the tobacco hornworm. J Insect Physiol 54:1243–1252. doi:10.1016/j.jinsphys.2008.06.007
Burand JP, Hunter WB (2013) RNAi: future in insect management. J Invertebr Pathol 112:s68–s74. doi:10.1016/j.jip.2012.07.012
Caldeira W, Dias AB, Terra WR, Ribeiro AF (2007) Digestive enzyme compartmentalization and recycling and sites of absorption and secretion along the midgut of Dermestes maculatus (Coleoptera) larvae. Arch Insect Biochem Physiol 64:1–18. doi:10.1002/arch.20153
Chang X, Liu GG, He KL, Shen ZC, Peng YF, Ye GY (2013) Efficacy evaluation of two transgenic maize events expressing fused proteins to CrylAb-susceptible and -resistant Ostrinia furnacalis (Lepidoptera: Crambidae). J Econ Entomol 106:2548–2556
Chu CC, Sun W, Spencer JL, Pittendrigh BR, Seufferheld MJ (2014) Differential effects of RNAi treatments on field populations of the western corn rootworm. Pestic Biochem Physiol 110:1–6. doi:10.1016/j.pestbp.2014.02.003
Cierpicki T, Bania J, Otlewski J (2000) NMR solution structure of Apis mellifera chymotrypsin/cathepsin G inhibitor-1 (AMCI-1): structural similarity with Ascaris protease inhibitors. Protein Sci 9:976–984. doi:10.1110/ps.9.5.976
Ferre J, Van Rie J (2002) Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annu Rev Entomol 47:501–533. doi:10.1146/annurev.ento.47.091201.145234
Gatehouse JA (2011) Prospects for using proteinase inhibitors to protect transgenic plants against attack by herbivorous insects. Curr Protein Pept Sci 12:409–416
Gatehouse AM, Norton E, Davison GM, Babbe SM, Newell CA, Gatehouse JA (1999) Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleracea; effects of plant protease inhibitors in vitro and in vivo. J Insect Physiol 45:545–558. doi:10.1016/S0022-1910(98)00161-9
Gordon KH, Waterhouse PM (2007) RNAi for insect-proof plants. Nat Biotechnol 25:1231–1232. doi:10.1038/nbt1107-1231
Guo Z et al (2015) Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.). Insect Biochem Mol Biol 59:30–40. doi:10.1016/j.ibmb.2015.01.009
Hakim RS, Baldwin K, Smagghe G (2010) Regulation of midgut growth, development, and metamorphosis. Annu Rev Entomol 55:593–608. doi:10.1146/annurev-ento-112408-085450
He K, Wang Z, Zhou D, Wen L, Song Y, Yao Z (2003) Evaluation of transgenic Bt corn for resistance to the Asian corn borer (Lepidoptera: Pyralidae). J Econ Entomol 96:935–940. doi:10.1093/jee/96.3.935
Hedstrom L (2002) Serine protease mechanism and specificity. Chem Rev 102:4501–4524. doi:10.1021/cr000033x
Herrero S, Combes E, Van Oers MM, Vlak JM, de Maagd RA, Beekwilder J (2005) Identification and recombinant expression of a novel chymotrypsin from Spodoptera exigua. Insect Biochem Mol 35:1073–1082. doi:10.1016/j.ibmb.2005.05.006
Huvenne H, Smagghe G (2010) Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review. J Insect Physiol 56:227–235. doi:10.1016/j.jinsphys.2009.10.004
Jayachandran B, Hussain M, Asgari S (2013) An insect trypsin-like serine protease as a target of microRNA: utilization of microRNA mimics and inhibitors by oral feeding. Insect Biochem Mol Biol 43:398–406. doi:10.1016/j.ibmb.2012.10.004
Katoch R, Sethi A, Thakur N, Murdock LL (2013) RNAi for insect control: current perspective and future challenges. Appl Biochem Biotechnol 171:847–873. doi:10.1007/s12010-013-0399-4
Kikuchi Y, Hayatsu M, Hosokawa T, Nagayama A, Tago K, Fukatsu T (2012) Symbiont-mediated insecticide resistance. Proc Natl Acad Sci USA 109:8618–8622. doi:10.1002/arch.20153
Kim BY et al (2013) Anti-elastolytic activity of a honeybee (Apis cerana) chymotrypsin inhibitor. Biochem Biophys Res Commun 430:144–149. doi:10.1016/j.bbrc.2012.11.056
Koch A, Kogel KH (2014) New wind in the sails: improving the agronomic value of crop plants through RNAi-mediated gene silencing. Plant Biotechnol J 12:821–831. doi:10.1111/pbi.12226
Liu F, Xu Z, Chang J, Chen J, Meng F, Zhu YC, Shen J (2008) Resistance allele frequency to bt cotton in field populations of Helicoverpa armigera (Lepidoptera: Noctuidae) in China. J Econ Entomol 101:933–943. doi:10.1093/jee/101.3.933
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. doi:10.1006/meth.2001.1262
Lorito M, Hayes CK, Zoina A, Scala F, Del Sorbo G, Woo SL, Harman GE (1994) Potential of genes and gene products from Trichoderma sp. and Gliocladium sp. for the development of biological pesticides. Mol Biotechnol 2:209–217. doi:10.1007/BF02745877
Mahon RJ, Olsen KM, Downes S, Addison S (2007) Frequency of alleles conferring resistance to the Bt toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa armigera (Lepidoptera: Noctuidae). J Econ Entomol 100:1844–1853. doi:10.1093/jee/100.6.1844
McGaughey WH (1985) Insect resistance to the biological insecticide Bacillus thuringiensis. Science 229:193–195. doi:10.1126/science.229.4709.193
Milne R, Kaplan H (1993) Purification and characterization of a trypsin-like digestive enzyme from Spruce budworm (Choristoneura fumiferana) responsible for the activation of δ-endotoxin from Bacillus thuringiensis. Insect Biochem Mol Biol 23:663–673
Monteiro EC, Tamaki FK, Terra WR, Ribeiro AF (2014) The digestive system of the “stick bug” Cladomorphus phyllinus (Phasmida, Phasmatidae): a morphological, physiological and biochemical analysis. Arthropod Struct Dev 43:123–134. doi:10.1016/j.asd.2013.11.005
Oppert B (1999) Protease interactions with Bacillus thuringiensis insecticidal toxins. Arch Insect Biochem Physiol 42:1–12. doi:10.1002/(SICI)1520-6327(199909)42:1<1::AID-ARCH2>3.0.CO;2-#
Oppert B, Kramer KJ, Beeman RW, Johnson D, McGaughey WH (1997) Proteinase-mediated insect resistance to Bacillus thuringiensis toxins. J Biol Chem 272:23473–23476
Oppert B, Martynov AG, Elpidina EN (2012) Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts. Comp Biochem Phys D 7:233–242. doi:10.1016/j.cbd.2012.03.005
Payne CD (1987) The GLIM system relaease 3.77 manual(2nd). Oxford: Numeriacl Algorithms Group
Polgar L (2005) The catalytic triad of serine peptidases. Cell Mol Life Sci 62:2161–2172. doi:10.1007/s00018-005-5160-x
Price DR, Gatehouse JA (2008) RNAi-mediated crop protection against insects. Trends Biotechnol 26:393–400. doi:10.1016/j.tibtech.2008.04.004
Rodriguez-Cabrera L, Trujillo-Bacallao D, Borras-Hidalgo O, Wright DJ, Ayra-Pardo C (2010) RNAi-mediated knockdown of a Spodoptera frugiperda trypsin-like serine-protease gene reduces susceptibility to a Bacillus thuringiensis Cry1Ca1 protoxin. Environ Microbiol 12:2894–2903. doi:10.1111/j.1462-2920.2010.02259.x
Ryan CA (1981) Proteinase inhibitors. The Biochemistry of plants:351-370
Scott JG et al (2013) Towards the elements of successful insect RNAi. J Insect Physiol 59:1212–1221. doi:10.1016/j.jinsphys.2013.08.014
Shao Z, Cui Y, Liu X, Yi H, Ji J, Yu Z (1998) Processing of delta-endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 in Heliothis armigera midgut juice and the effects of protease inhibitors. J Invertebr Pathol 72:73–81. doi:10.1006/jipa.1998.4757
Spit J, Zels S, Dillen S, Holtof M, Wynant N, Broeck JV (2014) Effects of different dietary conditions on the expression of trypsin- and chymotrypsin-like protease genes in the digestive system of the migratory locust, Locusta migratoria. Insect Biochem Mol 48:100–109. doi:10.1016/j.ibmb.2014.03.002
Tabashnik BE, Cushing NL, Finson N, Johnson MW (1990) Field Development of Resistance to Bacillus-Thuringiensis in diamondback moth (Lepidoptera, Plutellidae). J Econ Entomol 83:1671–1676
Terenius O et al (2011) RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design. J Insect Physiol 57:231–245. doi:10.1016/j.jinsphys.2010.11.006
Terra WR, Ferreira C (1994) Insect digestive enzymes: properties, compartmentalization and function. Comp Biochem Physiol Part B 109:1–62. doi:10.1016/0300-9629(94)90307-7
Terra WR, Ferreira C, Jordao BP, Dillon RJ (1996) Digestive enzymes. In: Lehane MJ, Billingsley PF (eds) Biology of the insect midgut. Chapman and Hall, London, pp 153–193
Wan H et al (2013) A spider (Araneus ventricosus) chymotrypsin inhibitor that acts as an elastase inhibitor and a microbial serine protease inhibitor. Comp Biochem Physiol Part B 165:36–41. doi:10.1016/j.cbpb.2013.03.004
Wang Y, Zhang H, Li H, Miao X (2011) Second-generation sequencing supply an effective way to screen RNAi targets in large scale for potential application in pest insect control. PLoS One 6:e18644. doi:10.1371/journal.pone.0018644
Yazdani E, Zibaee A, Sendi JJ (2014) Digestive proteases of Papilio demoleus: compartmentalization and characterization. Phytoparasitica 42:121–133. doi:10.1007/s12600-013-0323-z
Zhang C et al (2010) A chymotrypsin-like serine protease cDNA involved in food protein digestion in the common cutworm, Spodoptera litura: cloning, characterization, developmental and induced expression patterns, and localization. J Insect Physiol 56:788–799. doi:10.1016/j.jinsphys.2010.02.001
Zhang T, He M, Gatehouse AM, Wang Z, Edwards MG, Li Q, He K (2014) Inheritance patterns, dominance and cross-resistance of Cry1Ab- and Cry1Ac-selected Ostrinia furnacalis (Guenée). Toxins 6:2694–2707. doi:10.3390/toxins6092694
Zhou DR, He KL, Wang ZY, Ye ZH, Wen LP, Gao YX, Song YY (1995) In: Zhou DR (ed) Asian corn borer and its integrated management, Golden Shield Press, Beijing, pp. 65–72
Acknowledgments
This work was supported by the National Basic Research Program of China (2015CB755703), the National Transgenic Great Subject from the Ministry of Agriculture of China (2014ZX08009-003-001), the Key Deployment Project from Chinese Academy of Sciences (KSZD-EW-Z-021-2-1), and the National Natural Science Foundation of China (31172152). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by M. Traugott.
Electronic supplementary material
Below is the link to the electronic supplementary material.
10340_2016_797_MOESM1_ESM.docx
Supplementary Figure S1. Seven CTP gene expression levels of Asian corn borer (Ostrinia furnacalis) after feeding with different dsRNAs (DOCX 305 kb)
10340_2016_797_MOESM5_ESM.xlsx
Supplementary Table S4. Pairwise identity values of chymotrypsin sequences from Lepidopteran insect species (XLSX 10 kb)
10340_2016_797_MOESM6_ESM.docx
Supplementary Table S5. Larval mortality rates of Asian corn borer (Ostrinia furnacalis) after feeding with dsRNA synthesized from seven CTPs post 5 days (DOCX 15 kb)
Rights and permissions
About this article
Cite this article
Guan, R., Li, H. & Miao, X. RNAi pest control and enhanced BT insecticidal efficiency achieved by dsRNA of chymotrypsin-like genes in Ostrinia furnacalis . J Pest Sci 90, 745–757 (2017). https://doi.org/10.1007/s10340-016-0797-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10340-016-0797-9