Abstract
Cytochrome P450 monooxygenases (P450s) of insects are known to be involved in the metabolism or detoxification of plant allelochemicals and insecticides. Spodoptera litura (Lepidoptera, Noctuidae) is a polyphagous moth responsible for severe yield losses in many crops. In this study, two full-length P450 genes, CYP6B48 and CYP6B58, were cloned from S. litura. The cDNA sequences encode proteins with 503 and 504 amino acids, respectively. Phylogenetic analysis revealed that CYP6B48 and CYP6B58 belong to the CYP6B subfamily of P450s. Quantitative real-time PCR analyses showed that CYP6B48 and CYP6B58 were expressed only at larval stage, but not at pupal and adult stages. The highest levels of transcripts were found in the midguts and fat bodies of the larvae. No expression was detected in the ovary or hemolymph. Feeding with diets containing cinnamic acid, quercetin, or coumarin did not affect expression of CYP6B48. In contrast, diet supplemented with xanthotoxin dramatically increased the levels of CYP6B48 transcript in the midgut and fat bodies. Larvae fed with flavone had high levels of transcript of CYP6B48 in the midgut, whereas only slightly elevated levels were found in the fat bodies. Effects of the tested allelochemicals on CYP6B58 expression were minor. Hence, our findings show that S. litura responds to specific allelochemicals such as xanthotoxin with the accumulation of CYP6B48 transcripts, suggesting that specific signals in the food control the insect’s ability to convert toxic allelochemicals to less harmful forms at the transcriptional level.
Similar content being viewed by others
References
Ahmad M, Iqbal Arif M, Ahmad M (2007) Occurrence of insecticide resistance in field populations of Spodoptera litura (Lepidoptera: Noctuidae) in Pakistan. Crop Prot 26:809–817
Ahmad M, Ghaffar A, Rafiq M (2013) Host plants of leaf worm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) in Pakistan. Asian J Agric Biol 1:23–28
Bhaskara S, Dean ED, Lam V, Ganguly R (2006) Induction of two cytochrome P450 genes, Cyp6a2 and Cyp6a8, of Drosophila melanogaster by caffeine in adult flies and in cell culture. Gene 377:56–64
Bhaskara S, Chandrasekharan MB, Ganguly R (2008) Caffeine induction of Cyp6a2 and Cyp6a8 genes of Drosophila melanogaster is modulated by cAMP and D-JUN protein levels. Gene 415:49–59
Cano-Ramírez C, López MF, Cesar-Ayala AK, Pineda-Martínez V, Sullivan BT, Zúñiga G (2013) Isolation and expression of cytochrome P450 genes in the antennae and gut of pine beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) following exposure to host monoterpenes. Gene 520:47–63
Chandor-Proust A, Bibby J, Régent-Kloeckner M, Roux J, Guittard-Crilat E, Poupardin R, Riaz MA, Paine M, Dauphin-Villemant C, Reynaud S, David J (2013) The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling. Biochem J 455:75–85
Chung H, Sztal T, Pasricha S, Sridhar M, Batterham P, Daborn PJ (2009) Characterization of Drosophila melanogaster cytochrome P450 genes. Proc Natl Acad Sci U S A 106:5731–5736
Cichón LB, Soleño J, Anguiano OL, Garrido SA, Montagna CM (2013) Evaluation of cytochrome P450 activity in field populations of Cydia pomonella (Lepidoptera: Tortricidae) resistant to azinphosmethyl, acetamiprid, and thiacloprid. J Econ Entomol 106:939–944
Feyereisen R (2006) Evolution of insect P450. Biochem Soc Trans 34:1252–1255
Gotoh O (1992) Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J Biol Chem 267:83–90
Grubor VD, Heckel DG (2007) Evaluation of the role of CYP6B cytochrome P450s in pyrethroid resistant Australian Helicoverpa armigera. Insect Mol Biol 16:15–23
Guo FG, Lei JX, Sun YC, Cui YH, Ge F, Patil BS, Koiwa H, Zeng RS, Zhu-Salzman K (2012) Antagonistic regulation, yet synergistic defense: effect of bergapten and protease inhibitor on development of cowpea bruchid Callosobruchus maculatus. PLoS One 7(8):e41877. doi:10.1371/journal.pone.0041877
Harrison TL, Zanger AR, Schule MA, Berenbaum MR (2001) Developmental variation in cytochrome P450 expression in Papilio polyxenes in response to xanthotoxin, a hostplant allelochemical. Arch Insect Biochem Physiol 48:179–189
Hlavica P (2011) Insect cytochromes P450: topology of structural elements predicted to govern catalytic versatility. J Inorg Biochem 105:1354–1364
Hung CF, Berenbaum MR, Schuler MA (1997) Isolation and characterization of CYP6B4, furanocoumarin-inducible cytochrome P450 from a polyphagous caterpillar (Lepidoptera: Papilionidae). Insect Biochem Mol 27:377–385
Ismail HM, O’Neill PM, Hong DW, Finn RD, Henderson CJ, Wright AT, Cravatt BF, Hemingway J, Paine MJ (2013) Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions. Proc Natl Acad Sci U S A 110:19766–19771
Jensen HR, Scott IM, Sims S, Trudeau VL, Arnason JT (2006) Gene expression profiles of Drosophila melanogaster exposed to an insecticidal extract of Piper nigrum. J Agric Food Chem 54:1289–1295
Li X, Berenbaum MR, Schuler MA (2002a) Plant allelochemicals differentially regulate Helicoverpa zea cytochrome P450 genes. Insect Mol Biol 11:343–351
Li X, Schuler MA, Berenbaum MR (2002b) Jasmonate and salicylate induce expression of herbivore cytochrome P450 genes. Nature 419:712–715
Li W, Zangerl AR, Schuler MA, Berenbaum MR (2004a) Characterization and evolution of furanocoumarin-inducible cytochrome P450s in the parsnip webworm, Depressaria pastinacella. Insect Mol Biol 13:603–613
Li X, Berenbaum MR, Schuler MA (2004b) Structural and functional divergence of insect CYP6B proteins: from specialist to generalist cytochrome P450. Proc Natl Acad Sci U S A 101:2939–2944
Li X, Schuler MA, Berenbaum MR (2007) Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Annu Rev Entomol 52:231–253
Liu X, Zhang L, Zhang XT, Gao XW (2013) Molecular cloning and recombinant expression of cytochrome P450 CYP6B6 from Helicoverpa armigera in Escherichia coli. Mol Biol Rep 40:1211–1217
Liu D, Zhou X, Li M, Zhu S, Qiu X (2014) Characterization of NADPH-cytochrome P450 reductase gene from the cotton bollworm, Helicoverpa armigera. Gene 545:262–270
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time tuantitative PCR and the 2-ΔΔCT Method. Methods 25(4):402–408
Mao W, Schuler MA, Berenbaum MR (2007) Cytochrome P450s in Papilio multicaudatus and the transition from oligophagy to polyphagy in the Papilionidae. Insect Biochem Mol Biol 16:481–490
McDonnell CM, Brown RP, Berenbaum MR, Schuler MA (2004) Conserved regulatory elements in the promoters of two allelochemical-inducible cytochrome P450 genes differentially regulate transcription. Insect Biochem Mol Biol 34:1129–1139
Niu G, Rupasinghe SG, Zangerl AR, Siegel JP, Schuler MA, Berenbaum MR (2011) A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella). Insect Biochem Mol Biol 41:244–253
Qin HG, Wang DD, Ding J, Huang RH, Ye ZX (2006) Host plants of Spodop teralitura. Acta Agric Jiangxi 18:51–58
Riga M, Tsakireli D, Ilias A, Morou E, Myridakis A, Stephanou EG, Nauen R, Dermauw W, van Leeuwen T, Paine M, Vontas J (2014) Abamectin is metabolized by CYP392A16, a cytochrome P450 associated with high levels of acaricide resistance in Tetranychus urticae. Insect Biochem Mol Biol 46:43–53
Rupasinghe SG, Wen ZM, Chiu TL, Schuler MA (2007) Helicoverpa zea CYP6B8 and CYP321A1: different molecular solutions to the problem of metabolizing plant toxins and insecticides. Protein Eng Des Sel 20:615–624
Schuler MA (2011) P450s in plant–insect interactions. BBA-Protein Proteomics 1814:36–45
Schuler MA, Berenbaum MR (2013) Structure and function of cytochrome P450S in insect adaptation to natural and synthetic toxins: insights gained from molecular modeling. J Chem Ecol 39:1232–1245
Scully ED, Hoover K, Carlson JE, Tien M, Geib SM (2013) Midgut transcriptome profiling of Anoplophora glabripennis, a lignocellulose degrading cerambycid beetle. BMC Genomics 14:850
Shad SA, Sayyed AH, Fazal S, Saleem MA, Zaka SM, Ali M (2012) Field evolved resistance to carbamates, organophosphates, pyrethroids, and new chemistry insecticides in Spodoptera litura Fab. (Lepidoptera: Noctuidae). J Pestic Sci 85:153–162
Wang B, Shahzad MF, Zhang Z, Sun H, Han P, Li F, Han Z (2014) Genome-wide analysis reveals the expansion of Cytochrome P450 genes associated with xenobiotic metabolism in rice striped stem borer, Chilo suppressalis. Biochem Biophys Res Commun 443:756–760
Wen Z, Pan L, Berenbaum MR, Schuler MA (2003) Metabolism of linear and angular furanocoumarins by Papilio polyxenes CYP6B1 co-expressed with NADPH cytochrome P450 reductase. Insect Biochem Mol Biol 33(9):937–947
Wen Z, Berenbaum MR, Schuler MA (2006) Inhibition of CYP6B1-mediated detoxification of xanthotoxin by plant allelochemicals in the black swallowtail (Papilio polyxenes). J Chem Ecol 2:507–522
Wen Z, Zeng RS, Niu G, Berenbaum MR, Schuler MA (2009) Ecological significance of induction of broad-substrate cytochrome P450s by natural and synthetic inducers in Helicoverpa zea. J Chem Ecol 5:183–189
Willoughby L, Chung H, Lumb C, Robin C, Batterham P, Daborn PJ (2006) A comparison of Drosophila melanogaster detoxification gene induction responses for six insecticides, caffeine and phenobarbital. Insect Biochem Mol Biol 36:934–942
Zeng RS, Wen Z, Niu G, Schuler MA, Berenbaum MR (2007) Allelochemical induction of cytochrome P450 monooxygenases and amelioration of xenobiotic toxicity in Helicoverpa zea. J Chem Ecol 33:449–461
Zeng RS, Wen Z, Niu G, Berenbaum MR (2013) Aflatoxin B1: toxicity, bioactivation and detoxification in the polyphagous caterpillar Trichoplusia ni. Insect Sci 20:318–328
Zhang C, Luo X, Ni X, Zhang Y, Li X (2010) Functional characterization of cis-acting elements mediating flavone-inducible expression of CYP321A1. Insect Biochem Mol Biol 40:898–908
Zhang YE, Ma HJ, Feng DD, Lai XF, Chen ZM, Xu MY, Yu QY, Zhang Z (2012) Induction of detoxification enzymes by quercetin in the silkworm. J Econ Entomol 105:1034–1042
Zhou XJ, Sheng CF, Li M, Wan H, Liu D, Qiu XH (2010) Expression responses of nine cytochrome P450 genes to xenobiotics in the cotton bollworm Helicoverpa armigera. Pestic Biochem Physiol 97:209–213
Zhou JL, Shu YH, Zhang GR, Zhou Q (2012a) Lead exposure improves the tolerance of Spodoptera litura (Lepidoptera: Noctuidae) to cypermethrin. Chemosphere 88:507–513
Zhou JL, Zhang GR, Zhou Q (2012b) Molecular characterization of cytochrome P450 CYP6B47 cDNAs and 5′-flanking sequence from Spodoptera litura (Lepidoptera: Noctuidae): Its response to lead stress. J Insect Physiol 58:726–736
Acknowledgments
This research was supported by the National Natural Science Foundation of China (31070388, 31100286, 31470576), National 973 Program of China (2011CB100400), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2010), Guangdong Natural Science Foundation of China (S2011040004336), and Ph.D. Foundation of the Ministry of Education of China (20104404110004). The authors thank Dr. David Nelson for naming the P450 genes.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wang, RL., Li, J., Staehelin, C. et al. Expression Analysis of Two P450 Monooxygenase Genes of the Tobacco Cutworm Moth (Spodoptera litura) at Different Developmental Stages and in Response to Plant Allelochemicals. J Chem Ecol 41, 111–119 (2015). https://doi.org/10.1007/s10886-014-0540-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10886-014-0540-z