Insect Molecular Biology
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Insect Molecular Biology (2014)
doi: 10.1111/imb.12140
Cytochrome P450s from the fall armyworm (Spodoptera frugiperda): responses to plant allelochemicals and pesticides
M. Giraudo*†‡§, F. Hilliou*†‡, T. Fricaux*†‡, P. Audant*†‡, R. Feyereisen*†‡ and G. Le Goff*†‡ *INRA, UMR 1355, Institut Sophia Agrobiotech, Sophia-Antipolis, France; †CNRS, UMR 7254, Sophia-Antipolis, France; ‡Université de Nice Sophia Antipolis, Sophia-Antipolis, France; and §Environment Canada, Centre Saint-Laurent, Montreal, QC, Canada
detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S. frugiperda to its chemical environment. Keywords: cytochrome P450s, Spodoptera frugiperda, gene expression induction, xenobiotics.
Abstract
Introduction
Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S. frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were
The fall armyworm, Spodoptera frugiperda, is a major pest of agriculture, able to feed on more than 40 plant families. These families represent approximately 180 host plants and a wide array of different plant allelochemicals. This phytophagous insect has therefore developed sophisticated mechanisms to either reduce the quantity of or metabolically inactivate some of the potentially toxic xenobiotics that it ingests. These mechanisms range from avoidance of the area of the leaf surface where plant defences have been induced (Perkins et al., 2013), insensitivity to plant protease inhibitors (Jongsma et al., 1995; Brioschi et al., 2007; Dunse et al., 2010; Chikate et al., 2013) to increased excretion of allelochemicals by ABC transporters (Xie et al., 2012; Dermauw et al., 2013a) or active metabolism of toxic compounds (Wadleigh & Yu, 1988; Sasabe et al., 2004; Li et al., 2007). The latter mechanism is of particular importance as it can further lead to insecticide resistance. Dermauw et al. (2013b) for example, found that in the polyphagous spider mite Tetranychus urticae, transition from bean to tomato as the host plant conferred higher tolerance level to acaricides. Some key detoxification enzymes are capable of metabolizing plant secondary compounds and insecticides, amongst which cytochrome P450s (or CYP) play a major role. In the lepidopteran Helicoverpa zea for example, two P450s in particular, CYP6B8 and CYP321A1, have been shown to metabolize plants toxins such as xanthotoxin as well as insecticides including aldrin, cypermethrin and diazinon (Li et al., 2004; Sasabe et al., 2004; Rupasinghe et al.,
Correspondence: Gaëlle Le Goff, INRA, UMR 1355, Institut Sophia Agrobiotech, INRA-CNRS-Université de Nice Sophia-Antipolis, 400 Route des Chappes, 06903 Sophia-Antipolis, France. Tel.: + 33 492385578; fax: + 33 492386401; e-mail: [email protected]
© 2014 The Royal Entomological Society
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2007). The tobacco hornworm, Manduca sexta, a phytophagous insect specialist of Solanaceae, encounters in its diet huge amounts of the well-described plant defence compound, nicotine (Steppuhn et al., 2004). When larvae start feeding on tobacco, the toxicity of nicotine stops them from feeding until the enzymatic machinery of P450s is induced to metabolize this noxious compound (Snyder et al., 1993, 1994; Snyder & Glendinning, 1996). To date, P450 gene numbers in sequenced insect genomes vary from 36 in the body louse to 170 genes in mosquitoes (Arensburger et al., 2010; Kirkness et al., 2010). However, most of the P450s are expressed at low levels and are only expressed when insects have to deal with a toxic compound, mainly because P450 uncoupling produces harmful reactive oxygen species in the cell. In the well-known plant−insect interaction between Papilionidae lepidopterans and the toxic plant secondary metabolites furanocoumarins, several CYP6B transcripts are over-expressed to cope with either linear or angular furanocoumarins. In the specialist caterpillar Papilio polyxenes, CYP6B1 is inducible and able to metabolize linear furanocoumarins, such as xanthotoxin. The promoter region of this P450 contains specific regulation elements (REs) such as a xenobiotic response element to xanthotoxin (XRE-Xan; Petersen et al., 2003). The same RE is also present in the promoter region of CYP6B4 from the generalist Papilio glaucus, even though this caterpillar feeds on a more diverse range of host plants with only a few furanocoumarin-containing plant species (McDonnell et al., 2004). CYP6B4 is inducible and has been demonstrated to metabolize both linear and angular furanocoumarins (Hung et al., 1997; Li et al., 2003). Papilio canadensis, a caterpillar for which furanocoumarins are absent from its host plants, has inducible CYP6Bs, CYP6B25 and CYP6B26 that have low furanocoumarin metabolization abilities (Li et al., 2003). In other lepidopteran species, such as the generalist Helicoverpa zea, CYP6B8 and CYP321A1 have been shown to metabolize several allelochemicals and insecticides (Li et al., 2004; Sasabe et al., 2004; Rupasinghe et al., 2007). In the generalist S. frugiperda, it is well established that P450s can metabolize a broad range of plant secondary metabolites, such as indoles, glucosinolates, flavonoids and coumarins (Yu, 1987). Moreover, it has been demonstrated that when larvae are fed with allelochemicals such as indole 3-carbinol, menthol or flavones, P450 activities are induced (Yu & Ing, 1984; Yu, 1987). These studies however were based on enzymatic activities alone and did not specifically identify which P450 was induced by a given type of compound. The present work aimed to fill this gap by identifying ‘xenobiotic inducer – P450’ combinations and expression patterns in order to better under-
stand the adaptation of this insect to its chemical environment. First, 42 sequences coding for P450s were identified and basal expression levels of the corresponding transcripts were measured in tissues involved in detoxification processes and in the cellular model Sf9. Then, P450 induction profiles were measured after dietary exposure of larvae to 11 chemical compounds including plant secondary metabolites, insecticides and model inducers. In vivo P450 expression patterns were further compared with expression profiles in similarly treated Sf9 cells. Results Identification and phylogeny Thirty sequences coding for S. frugiperda P450s were successfully identified via the lepidopteran comparative genomic project and the screening of a BAC library (d’Alencon et al., 2010). This project was initiated in order to compare the genome of the model Lepidoptera species Bombyx mori and the two noctuid pests Helicoverpa armigera and S. frugiperda. The study was complemented by access to a S. frugiperda expressed sequence tag (EST) database containing eight different libraries built from different tissues and development stages as well as from individuals treated by chemical compounds (http:// www.spodobase.univ-montp2.fr/Spodobase/). Twelve additional sequences were obtained by EST assembly and confirmed by cloning and sequencing. In total, 42 sequences coding for cytochrome P450s were identified and named by David Nelson (http://drnelson.uthsc.edu/ CytochromeP450.html). All of the sequences were deposited in GenBank as BAC or unique P450 sequences (see GenBank accession numbers in Table 1). The 42 P450s were distributed amongst 14 families as shown on the phylogenetic tree in Fig. 1. The majority represented members of the CYP3 (29 sequences) and CYP4 clans (11 sequences) that are mostly involved in xenobiotic metabolism, with only two mitochondrial P450s of the CYP333 family. These sequences represent only part of the full CYPome of S. frugiperda. However they represent a reasonably large sampling of the diversity of CYP3 and CYP4 clan P450s in the fall armyworm. Tissue expression The expression profiles of the 42 P450s previously identified were measured by PCR: specific primers were designed for each sequence and the presence of mRNAs was verified for each of them in the midgut, Malpighian tubules and fat body of larvae as well as in the cellular model Sf9 (Table 1). The results showed that P450 expression profiles can vary amongst members of the same family. For example, in the CYP4L family CYP4L13 © 2014 The Royal Entomological Society
P450 induction in fall armyworm
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Table 1. Cytochrome P450s (CYPs) identified in Spodoptera frugiperda, expression in different tissues Name
GenBank accession no.
P450 size (amino acids)
Midgut
Malpighian tubules
Fat body
Sf9 cells
CYP4G74 CYP4G75 CYP4L12 CYP4L13 CYP4L9 CYP4M14 CYP4M15 CYP4M17 CYP4M18 CYP6AB12 CYP6AE43 CYP6AE44 CYP6AN4 CYP6B38 CYP6B39 CYP6B40 CYP6B41 CYP6B42 CYP6B50 CYP9A24 CYP9A25 CYP9A26 CYP9A27 CYP9A28 CYP9A29 CYP9A30 CYP9A31 CYP9A32 CYP9A58 CYP9A59 CYP9A60 CYP321A10 CYP321A7 CYP321A8 CYP321A9 CYP321B1 CYP332A1 CYP333B3 CYP333B4 CYP337B5 CYP340L1 CYP341A11
KC789745 KC789746 FP340412.1 FP340412.1 FP340412.1 FP340419.1 FP340419.1 FP340419.1 FP340419.1 KC789747 KJ671575 KJ671576 KC789748 FP340416.1 FP340416.1 FP340416.1 FP340416.1 FP340416.1 KC789749 FP340410.1 FP340410.1 FP340410.1 FP340410.1 FP340410.1 FP340410.1 FP340410.1 FP340410.1 FP340410.1 KJ671577 KJ671578 KJ671579 KC789753 KC789750 KC789751 KC789752 KC789754 FP340417.1 FP340412.1 FP340412.1 KJ671580 KC789755 KC789756
563 557 492 493 493 503 502 501 499 514 525 526 517 503 503 504 504 504 501 529 529 530 528 531 531 531 530 530 530 529 528 497 495 496 497 495 503 501 509 492 490 450 partial
ND ND ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ✓ ND
ND ND ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ND ✓ ND ✓ 2A ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ✓ ND
ND ND ND ✓ ND ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ✓ ND ✓ ND 2A ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ✓ ND
ND ND ND ND ND ✓ ✓ ND ND ✓ ✓ ✓ ND ✓ ND ✓ ND ✓ ✓ ✓ ND ✓ ND ✓ ND ✓ ✓ ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ND ✓ 2A ✓ ND ✓ ✓
Expression of the CYP genes was detected in all four tissues except when marked ND (not detected); in some cases, 2 amplicons sizes were detected (2A).
was the only one detected in all three tissues, with the two other members not present in the fat body. CYP6AE43 was only detected in the midgut and Malpighian tubules, whereas CYP6AE44 was solely expressed in the fat body. In the case of the CYP9A family, all members were detected in midgut, fat body and Malpighian tubules. Only four mRNA transcripts were absent in the tissues tested (CYP4G74, CYP4G75, CYP6B41 and CYP321B1). The CYP4G family expression profile confirmed Qiu et al.’s (2012) results that showed that CYP4G expression was restricted to the oenocyte cells, in which CYP4G acts as an oxidative decarbonylase in the biosynthesis of cuticular hydrocarbons. © 2014 The Royal Entomological Society
Induction by allelochemicals and xenobiotics Spodoptera frugiperda is a polyphagous insect that encounters in its diet several plant toxic compounds as well as insecticides. As P450s are known to be expressed at low levels and to be inducible when insects have to deal with toxic compounds, P450 induction profiles were measured in larvae exposed to plant secondary metabolites (xanthotoxin, 2-tridecanone, indole 3-carbinol, indole and quercetin), insecticides (deltamethrin, fipronil, methoprene and methoxyfenozide) and model inducers (clofibrate and phenobarbital). The same battery of compounds was used to treat Sf9 cells and both profiles were compared. Only
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M. Giraudo et al. 100 71
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97 87
100 87 63 100
100
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100 95
75 100
48
80
95 100
67
100 39 100 100
58
75 65
100
70 100
99
64
39 100
18
25 58 100
99
Sf-CYP333B3 Sf-CYP333B4 Sf-CYP340L1 Sf-CYP341A11 Sf-CYP4G74 Sf-CYP4G75 Sf-CYP4M18 Sf-CYP4M17 Sf-CYP4M14 Sf-CYP4M15 Sf-CYP4L12 Sf-CYP4L13 Sf-CYP4L9 Sf-CYP332A1 Sf-CYP337B5 Sf-CYP321B1 Sf-CYP321A7 Sf-CYP321A8 Sf-CYP321A10 Sf-CYP321A9 Sf-CYP6AE43 Sf-CYP6AE44 Sf-CYP6AB12 Sf-CYP6AN4 Sf-CYP6B50 Sf-CYP6B41 Sf-CYP6B42 Sf-CYP6B40 Sf-CYP6B38 Sf-CYP6B39 Sf-CYP9A32 Sf-CYP9A60 Sf-CYP9A28 Sf-CYP9A29 Sf-CYP9A30 Sf-CYP9A31 Sf-CYP9A24 Sf-CYP9A59 Sf-CYP9A26P Sf-CYP9A27 Sf-CYP9A25 Sf-CYP9A58
Figure 1. Phylogenetic analysis of Spodoptera frugiperda cytochrome P450s (SfP450s).
P450s with acceptable quantitative real-time PCR (qRTPCR) efficiencies (>85%, Table 2) were measured, which included 29 P450s in larval midguts and 11 for the Sf9 cell model. All chemical compounds were used at sublethal concentrations (Giraudo et al., 2013), which means that exposure doses might be different between chemicals but with the same effect on insects. Table 3 shows the results for S. frugiperda larvae. Amongst the 29 P450s analysed, 14 were over-expressed in at least one of the treatments, three were down-regulated and 12 were not differentially expressed. Each chemical induced a specific pattern, with xanthotoxin being the most potent inducer, whereas deltamethrin and clofibrate did not significantly regulate the expression of any P450 at the concentration used. Only one P450, CYP9A28 or CYP333B4, was differentially expressed in response to methoprene and fipronil treatment, respectively. CYP6B39 was the gene regulated by the most compounds, with high induction by four of the plant secondary metabolites and phenobarbital, whereas a fifth compound, quercetin, down-regulated its expres-
sion. By contrast, CYP9A28 was significantly repressed by four chemicals, including both plant compounds and insecticides. The Sf9 cell expression results are presented in Table 4. Out of the 11 P450s monitored, eight showed significant over-expression and three were not differentially expressed by any of the treatments. The pattern of induction seems to be specific for each chemical compound. Methoprene and 2-tridecanone were the best inducers of P450s in Sf9 cells, whereas xanthotoxin and phenobarbital did not significantly regulate any of the P450s studied. CYP333B4 was over-expressed in seven out of the 11 treatments. CYP9A30, CYP9A31 and CYP9A32 showed similar induction patterns in exposed cells. The Sf9 cell response shared few similarities with the in vivo response in larval midgut tissue. Only CYP321A9 and CYP333B4 were co-regulated in both tissues and over-expressed in 2-tridecanone and fipronil treatment, respectively.
Identification of putative regulation elements in the promoter region of S. frugiperda P450s Once S. frugiperda P450s that are induced by chemical compounds had been identified along with their corresponding inducers, the next step was to search for specific motifs of expression regulation in the promoter region of these genes. This was only possible for P450s that had been identified in BAC sequences. The analysis was performed on the 2000 nucleotides located upstream of the methionine start codon, sometimes on a smaller region when P450s were in a cluster with shorter intergenic regions. The presence of putative consensus elements for antioxidant response element (ARE), ecdysone response element (EcRE), xenobiotic response element (XRE) from the aryl hydrocarbon receptor (AhR) and for xanthotoxin (Xan) was searched for and reported in Table 5. ARE was found in 10 of the 23 promoter regions analysed, either once or twice and sometimes in reverse orientation. Eight out of the 10 genes found with an ARE motif were differentially regulated by one or several of the compounds tested, including CYP6B39, the most strongly induced P450 in this study. EcRE was present in 12 of the 23 promoters analysed, in both forward and reverse orientations. The XRE-AhR motif was only present in nine out of the 23 promoters analysed, whereas the XRE-Xan element was found in more than half of them.
Discussion Cytochrome P450s have essential endogenous functions and play a major role in the adaptation of insects to their chemical environment (Feyereisen, 2012). In the present study, 42 sequences coding for P450s were identified in the lepidopteran pest S. frugiperda. This number © 2014 The Royal Entomological Society
P450 induction in fall armyworm
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Table 2. Primers used in quantitative real-time PCR (qRT-PCR) PCR efficiencies (%) Name
Primer sequence
Fragment length (bp)
Midgut
Cells
SfCYP4G74-F Sf CYP4G74-R SfCYP4G75-F SfCYP4G75-R SfCYP4L9-F SfCYP4L9-R SfCYP4L12-F SfCYP4L12-R SfCYP4L13-F SfCYP4L13-R SfCYP4M14-F SfCYP4M14-R SfCYP4M15-F SfCYP4M15-R SfCYP4M17-F SfCYP4M17-R SfCYP4M18-F SfCYP4M18-R SfCYP6AB12-F SfCYP6AB12-R SfCYP6AE43-F SfCYP6AE43-R SfCYP6AE44-F SfCYP6AE44-R SfCYP6AN4-F SfCYP6AN4-R SfCYP6B38-F SfCYP6B38-R SfCYP6B39-F SfCYP6B39-R SfCYP6B40-F SfCYP6B40-R SfCYP6B41-F SfCYP6B41-R SfCYP6B42-F SfCYP6B42-R SfCYP6B50-F SfCYP6B50-R SfCYP9A24-F SfCYP9A24-R SfCYP9A25-F SfCYP9A25-R SfCYP9A26-F SfCYP9A26-R SfCYP9A27-F SfCYP9A27-R SfCYP9A28-F SfCYP9A28-R SfCYP9A29-F SfCYP9A29-R SfCYP9A30-F SfCYP9A30-R SfCYP9A31-F SfCYP9A31-R SfCYP9A32-F SfCYP9A32-R SfCYP9A58-F SfCYP9A58-R SfCYP9A59-F SfCYP9A59-R SfCYP9A60-F SfCYP9A60-R SfCYP321A7-F
5′-AGGTCCACATCGACAAGT-3′ 5′-GAATGTGGGAGCGATCATTT-3′ 5′-AGAGCGGTGTTGTCATTTCC-3′ 5′-GGTGGATACCCATCATCGAC-3′ 5′-TCGGTGATGACATGGAAAGA-3′ 5′-AGAACGACAGACGTGCCTTTT-3′ 5′-CTCTGATCGAGCGGAGAATC-3′ 5′-CCAACTGAAGGGGTTTTTCA-3′ 5′-ACGAACGTGAGTCTGCCTATGTGA-3′ 5′-ACGACGTCCGGACCAAAAATC-3′ 5′-TGATCTCGGACTTGCACTTG-3′ 5′-GTCCAGCGCTGAAAGGAATA-3′ 5′-TGATCTTGGACGTGCATTAT-3′ 5′-GCCCAGCACTGAAGGGAATG-3′ 5′-AGAGTCGCTGCGCATATACC-3′ 5′-GGGTTCGGGAATAAATCCTC-3′ 5′-TCCTACCCGAGAACAGCATC-3′ 5′-ACTTCTGCCACAGCCATCTT-3′ 5′-TTCGGGAAAGCCAGTATGAC-3′ 5′-ATTCGTTTCGCAATTTCTGG-3′ 5′-GCTGATTGCGCAGAGTGTTA-3′ 5′-CATGAACTCGTCCACCTCCT-3′ 5′-CTTCACTTACGGCGACAGGT-3′ 5′-CTCGAAGATACGGGCACATT-3′ 5′-ACGTCGTCATCAGCAACAAG-3′ 5′-GCTGATGGAGTCGTAGCACA-3′ 5′-TTCTTCACCAAGTTTTGCAGT-3′ 5′-ATCAAAGAACTTTTTTATGGCG-3′ 5′-AAGTTCCAAGTGGAGCCATCGAGG-3′ 5′-CCTCCTTTGGGCCCGACGAGAAG-3′ 5′-CTGTATCGGTATGCGGTTTG-3′ 5′-TTCCACCTTTAGGTCCGATG-3′ 5′-CAAGGATTTTGATGCGTTCA-3′ 5′-GTAAAGCCCTCCAGGTGTGA-3′ 5′-CTACTACTATGGCCTACCTGA-3′ 5′-TTGTTCGCTTGAACAGTTGC-3′ 5′-CAATCCAGCACGATGAGAAA-3′ 5′-GTGCGAATTTTGACCAAGG-3′ 5′-GGCACTAAACAACAGAGTGTGG-3′ 5′-ACCAAGCGTTCCTGTACGTC-3′ 5′-AATGCAAAGGCTGAGAAGGA-3′ 5′-GAAAAACGATCAGGGTCGAA-3′ 5′-TGAAAGGCCAAGAATGGAAG-3′ 5′-GTCATCTGACGCCTCGATTT-3′ 5′-CATCAAGTATCGCACGCCTA-3′ 5′-ATTCAAATCTGCCGACGAAC-3′ 5′-TCAAGCACATCAAGCCAGTC-3′ 5′-CCGTTGTGAGTCCATCACTGAC-3′ 5′-CAAAGAACATACCAGCGATGA-3′ 5′-AGTTCAAGGTCACGGACTACA-3′ 5′-GTCCTGGTGGCTGTGGTATT-3′ 5′-GTGCGAAAAATGATCGTGTG-3′ 5′-ATGCTCGTCTTGGTCTGGTT-3′ 5′-CTGCCCATGTTACCGAAGAT-3′ 5′-ATCATTCGTAAGGGCCAGTG-3′ 5′-AAGTGAACGGGACGATTTTG-3′ 5′-GCTCTCTGCGAGATGAAGGT-3′ 5′-GAGCCAGTGTCCTCCCTGTA-3′ 5′-GGATACCCACGTATGCCATC-3′ 5′-TCCTAGGACCAGTGCCAAAT-3′ 5′-CATCGTTTGGCCAGAGAACT-3′ 5′-CAGGGTGTACAGCCAACTCA-3′ 5′-TCCAGACCCAGAAGTTTTCG-3′
119
ND
ND
120
ND
ND
178
98
ND
176
87
ND
93
91
ND
126
93
105
126
105
104
150
99
ND
121
90
ND
© 2014 The Royal Entomological Society
131
BE
100
136
BE
BE
115
ND
BE
132
100
ND
102
BE
BE
84
96
ND
146
103
BE
101
ND
ND
97
BE
BE
147
100
BE
147
105
106
125
94
ND
146
92
106
134
103
ND
87
105
BE
113
BE
ND
122
99
104
131
99
97
136
97
102
135
113
BE
138
88
BE
141
106
110
134
106
ND
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Table 2. Continued PCR efficiencies (%) Name
Primer sequence
SfCYP321A7-R SfCYP321A8-F SfCYP321A8-R SfCYP321A9-F SfCYP321A9-R SfCYP321A10-F SfCYP321A10-R SfCYP321B1-F SfCYP321B1-R SfCYP332A1-F SfCYP332A1-R SfCYP333B3-F SfCYP333B3-R SfCYP333B4-F SfCYP333B4-R SfCYP337B5-F SfCYP337B5-R SfCYP340L1-F SfCYP340L1-R SfCYP341A11-F SfCYP341A11-R RpL4-F RpL4-R G6PD-F G6PD-R L18-F L18-R
5′-CGGCCTGGACTTGTAATTTG-3′ 5′-CCAGTAGAAAAGGGGACAAA-3′ 5′-TGGCATACCTCTCCCCTATG-3′ 5′-GCGTGGTGTAGCCTTCTACG-3′ 5′-CGGGTCAATGACAAACAGTG-3′ 5′-AACTTCACTGTGGCATCGA-3′ 5′-GGAATAAGCTGAACATCAACG-3′ 5′-CGTACGATGCAGTCTTGGAA-3′ 5′-CATTGCCTACAGGCAGAACA-3′ 5′-GCATGCATGAAACGCTAAGA-3′ 5′-CCACGTTCACGTAGACTGGA-3′ 5′-AGGCTGCTTGAAACGTATGG-3′ 5′-AGTCCAGTTGCGTGTCCTCT-3′ 5′-GAATTATGCCGGTGGTGTCT-3′ 5′-TAGCGACATGTCTCGGTGAG-3′ 5′-CCGTTTGGTGAAGGAAACA-3′ 5′-GACCGAAGGGACTTCTTTCA-3′ 5′-GTGCATTCCACAATGGGTGT-3′ 5′-CTGGCAAGGCACCAGGATA-3′ 5′-GTGCTCAATATGGGGTGTCC-3′ 5′-CCCATTACTGAAGGGCATGT-3′ 5′-CAACAAGAGGGGTTCACGAT-3′ 5′-GCACGATCAGTTCGGGTATC-3′ 5′-GGCCCTGTGGCTAACAGAAT-3′ 5′-CATCGTCTCTACCAAAAGGCTTC-3′ 5′-CGTATCAACCGACCTCCACT-3′ 5′-AGGCACCTTGTAGAGCCTCA-3′
Fragment length (bp)
Midgut
182
98
172
98
Cells
BE 95
99
BE
BE
126
ND
ND
123
112
BE
197
101
BE
116
101
93
142
101
ND
115
ND
BE
133
ND
BE
149
99
98
142
99
98
126
104
108
BE, bad efficiency in qRT-PCR; ND, not detected; RpL4, ribosomal protein L4; G6PD, glucose 6-phosphate dehydrogenase; L18, ribosomal protein L18.
represents only part of the complete CYPome of this pest insect. It is difficult or nearly impossible to predict the precise number of P450s in a species, as the size of the CYPome and the diversity inside a single P450 family is specific to each species (Feyereisen, 2011). However, based on the number of P450s found in other Lepidoptera such as the polyphagous H. armigera or the oligophagous M. sexta, the estimated number of P450s in S. frugiperda could reach up to 100–120 coding genes (F. Hilliou, pers. comm.). These numbers are slightly higher than the 87 P450 sequences found in the model Lepidoptera B. mori, a monophagous species that feeds exclusively on mulberry leaves (Ai et al., 2011). It is easy but not necessarily correct to hypothesize that the expansion of certain P450 families may be linked to the necessity of polyphagous insects to adapt to the toxic plant secondary metabolites that they encounter in their diet. The same reduction of the number of P450s has been observed in the Drosophila genus, in which the specialist Drosophila sechellia contains fewer P450s, 74 putatively functional genes, than the generalists that have been sequenced, for which the relative number is around 90 (Good et al., 2014). However, related species can have different numbers of P450s and a rapid evolution of this number may occur without a link to adaptation processes
(Feyereisen, 2011; Sezutsu et al., 2013). Hence, correlating the ability of insects to detoxify xenobiotics to the size of their CYPome is a shortcut that can easily lead to misinterpretations. The honeybee, for example, was suggested to be more susceptible to insecticides because of its reduced number of detoxification enzymes, especially P450s and glutathione S-transferases (Claudianos et al., 2006). However, this hypothesis is no longer supported when actual toxicological data are compared between Apis mellifera and other insects (Hardstone & Scott, 2010). Similar observations can be made in the louse (Pediculus humanus), which is highly resistant to many insecticides despite having only 36 genes encoding P450s in its genome (Kirkness et al., 2010). In addition, true one-to-one orthologues in closely related species are relatively rare. The Drosophila genus for example contains only 30 P450 orthologues for an evolution scale between 0.5 to ∼50 Ma (Good et al., 2014). It would therefore be difficult to predict the number of P450s in S. frugiperda when it diverged from its most closely related species H. armigera between 20 and 40 Ma (d’Alencon et al., 2010). More than half of the identified P450 genes (28/42) were expressed in the midgut, Malpighian tubules and fat body, which have essential functions in insect physiology. © 2014 The Royal Entomological Society
Indole
1.12 ± 0.05 1.64 ± 1.09 0.58 ± 0.18 0.36 ± 0.91 0.46 ± 1.10 0.48 ± 0.97 0.69 ± 0.28 0.54 ± 0.17 0.44 ± 0.50 5.69 ± 2.88 0.74 ± 0.75 135.95 ± 182.28 119.20 ± 56.24 28.59 ± 10.79 0.65 ± 0.28 0.53 ± 0.73 3.26 ± 2.99 135.14 ± 52.08 3.28 ± 4.16 1.42 ± 0.87 1.01 ± 0.54 2.10 ± 2.59 8.13 ± 8.47 0.53 ± 0.05 0.23 ± 0.10 1.43 ± 2.37 0.77 ± 0.18 0.45 ± 0.11 1.15 ± 0.90
Xanthotoxin
2.35 ± 0.82 2.66 ± 0.63 0.38 ± 0.15 0.17 ± 0.46 0.14 ± 0.38 0.19 ± 0.50 0.81 ± 0.36 0.69 ± 0.97 0.32 ± 0.49 2.50 ± 1.22 1.23 ± 1.24 162.11 ± 35.09 95.54 ± 20.19 20.97 ± 6.72 0.25 ± 0.12 0.26 ± 0.32 2.89 ± 0.83 181.71 ± 12.98 28.10 ± 9.73 0.73 ± 0.06 0.44 ± 0.03 1.53 ± 1.73 21.04 ± 5.26 0.99 ± 0.66 1.37 ± 0.42 1.41 ± 2.39 2.92 ± 1.73 1.85 ± 1.62 0.44 ± 0.11
P450 name
CYP333B3 CYP333B4 CYP4M18 CYP4M17 CYP4M14 CYP4M15 CYP4L12 CYP4L13 CYP4L9 CYP332A1 CYP337B5 CYP321A7 CYP321A8 CYP321A9 CYP6AN4 CYP6B50 CYP6B40 CYP6B39 CYP9A32 CYP9A60 CYP9A28 CYP9A30 CYP9A31 CYP9A24 CYP9A59 CYP9A26 CYP9A27 CYP9A25 CYP9A58
1.86 ± 0.99 0.93 ± 0.27 0.61 ± 0.29 0.33 ± 0.22 0.31 ± 0.32 0.61 ± 1.15 0.95 ± 0.36 0.70 ± 0.21 0.44 ± 0.59 0.94 ± 0.37 0.61 ± 0.18 64.31 ± 138.34 28.71 ± 4.93 11.70 ± 0.95 0.83 ± 0.13 0.71 ± 0.95 2.50 ± 1.87 224.13 ± 49.16 1.15 ± 0.59 0.75 ± 0.17 0.51 ± 0.30 1.45 ± 1.74 2.96 ± 1.22 0.53 ± 0.26 0.40 ± 0.22 0.46 ± 0.59 0.65 ± 0.19 0.55 ± 0.36 0.61 ± 0.08
Indole 3-carbinol 0.35 ± 0.05 1.19 ± 0.22 1.08 ± 0.13 1.25 ± 0.17 0.71 ± 0.69 1.08 ± 0.69 0.56 ± 0.05 2.90 ± 0.73 1.25 ± 0.17 2.83 ± 1.00 1.280.39 3.86 ± 3.12 2.04 ± 0.26 3.42 ± 0.79 1.62 ± 0.43 1.23 ± 0.20 3.26 ± 0.54 81.23 ± 9.48 0.67 ± 4.31 0.77 ± 0.33 0.29 ± 0.13 1.03 ± 4.99 2.93 ± 3.70 0.94 ± 0.54 0.12 ± 0.07 0.95 ± 0.82 0.60 ± 0.12 0.83 ± 0.60 0.70 ± 0.15
Phenobarbital 5.91 ± 4.43 1.83 ± 0.79 1.05 ± 0.58 0.86 ± 1.99 0.58 ± 0.82 1.12 ± 3.07 1.38 ± 0.69 0.71 ± 0.53 0.53 ± 0.76 1.26 ± 0.35 1.33 ± 1.49 1.09 ± 0.66 0.70 ± 0.46 1.58 ± 0.99 0.85 ± 0.40 0.74 ± 1.60 1.75 ± 2.21 2.73 ± 1.75 3.02 ± 0.58 3.00 ± 0.71 0.92 ± 0.49 1.18 ± 1.46 1.43 ± 0.995 0.92 ± 0.49 5.60 ± 5.49 1.45 ± 2.29 2.46 ± 2.31 3.80 ± 5.32 2.70 ± 0.47
Methoxyfenozide 0.69 ± 0.36 0.98 ± 0.31 1.04 ± 0.72 1.01 ± 3.12 0.56 ± 1.83 0.78 ± 2.16 1.16 ± 0.68 5.09 ± 8.42 0.79 ± 1.06 0.69 ± 0.18 1.12 ± 0.78 0.88 ± 0.89 1.19 ± 0.85 0.92 ± 0.60 1.26 ± 0.31 0.53 ± 1.00 0.72 ± 0.34 4.23 ± 0.81 0.73 ± 0.14 0.87 ± 0.23 0.22 ± 0.06 0.75 ± 0.89 0.72 ± 0.12 0.63 ± 0.48 0.22 ± 0.17 0.70 ± 1.22 0.74 ± 0.37 0.77 ± 0.73 0.72 ± 0.09
2-Tridecanone 0.77 ± 0.38 2.49 ± 1.67 1.05 ± 0.93 0.78 ± 1.89 0.61 ± 1.36 0.93 ± 2.10 0.34 ± 0.20 1.18 ± 1.07 1.11 ± 1.80 2.13 ± 0.62 1.65 ± 2.09 1.41 ± 1.16 0.88 ± 0.37 1.12 ± 0.74 0.45 ± 0.17 0.77 ± 1.62 1.42 ± 1.18 0.43 ± 0.18 2.34 ± 0.96 0.64 ± 0.15 0.73 ± 0.27 0.91 ± 1.21 2.12 ± 1.42 1.48 ± 0.93 1.46 ± 0.81 1.89 ± 4.23 1.41 ± 0.97 2.89 ± 4.18 0.73 ± 0.09
Fipronil 1.92 ± 0.89 1.31 ± 0.25 1.04 ± 0.68 1.10 ± 3.70 0.80 ± 3.03 1.03 ± 2.98 1.04 ± 0.55 0.96 ± 0.85 1.12 ± 1.45 1.29 ± 0.53 1.08 ± 2.29 1.13 ± 0.95 0.68 ± 0.33 1.49 ± 0.86 0.71 ± 0.18 0.59 ± 0.44 0.90 ± 0.29 0.33 ± 0.11 1.70 ± 0.80 1.57 ± 0.19 0.26 ± 0.11 1.11 ± 1.67 0.94 ± 0.31 1.07 ± 0.64 1.27 ± 1.20 1.29 ± 2.29 1.56 ± 0.85 1.14 ± 1.20 1.22 ± 0.06
Methoprene
1.74 ± 0.71 0.66 ± 0.31 0.69 ± 0.47 0.41 ± 0.92 0.47 ± 0.84 0.45 ± 0.74 0.52 ± 0.11 0.29 ± 0.17 0.46 ± 0.63 0.49 ± 0.13 1.01 ± 1.21 0.34 ± 0.31 0.44 ± 0.26 0.61 ± 0.38 0.36 ± 0.23 0.92 ± 1.24 0.42 ± 0.11 0.12 ± 0.01 0.48 ± 0.32 1.39 ± 0.58 0.10 ± 0.71 0.35 ± 0.34 0.38 ± 0.15 0.42 ± 0.20 0.45 ± 0.40 0.23 ± 0.09 0.63 ± 0.05 0.52 ± 0.29 0.56 ± 0.27
Quercetin
1.74 ± 0.82 1.33 ± 0.61 0.98 ± 0.95 0.75 ± 2.62 0.76 ± 3.21 1.18 ± 4.09 0.98 ± 0.47 1.22 ± 1.64 1.02 ± 1.32 0.57 ± 0.26 1.04 ± 1.16 3.62 ± 9.01 0.29 ± 0.15 1.22 ± 1.06 1.47 ± 1.00 1.04 ± 2.19 0.63 ± 0.41 0.46 ± 0.20 1.42 ± 0.90 1.22 ± 0.63 0.39 ± 0.26 0.86 ± 1.13 1.01 ± 0.11 1.00 ± 1.17 0.66 ± 0.61 0.56 ± 1.60 1.16 ± 0.84 1.16 ± 1.51 1.37 ± 0.40
Clofibrate
4.48 ± 3.75 2.18 ± 1.01 0.92 ± 0.34 0.86 ± 2.21 0.60 ± 1.06 0.98 ± 2.25 0.85 ± 0.15 0.91 ± 0.99 0.86 ± 1.12 1.06 ± 0.47 1.27 ± 0.46 1.21 ± 0.19 0.79 ± 0.53 1.52 ± 0.08 2.18 ± 1.48 0.89 ± 1.63 1.14 ± 1.05 1.01 ± 0.56 3.05 ± 2.49 1.52 ± 0.27 0.70 ± 0.42 1.20 ± 1.55 1.55 ± 1.65 1.36 ± 0.75 3.81 ± 3.58 0.91 ± 0.29 1.94 ± 1.23 1.76 ± 1.78 2.10 ± 0.63
Deltamethrin
Table 3. Quantitative real-time PCR analysis of the differential expression of 29 cytochrome P450 (CYP) genes in Spodoptera frugiperda larvae exposed for 24 h to sublethal doses of 11 different xenobiotic compounds. Gene expression values are indicated as fold expression in larvae exposed to each xenobiotic compared with unexposed larvae (controls). The three reference genes ribosomal protein L4, glucose 6-phosphate dehydrogenase and ribosomal protein L18 were used as internal controls for normalization. Gene expression values in bold are significantly different from the corresponding control as measured by pairwise Student’s t-tests (P < 0.05). Genes highlighted in light grey and dark grey are respectively induced or repressed
P450 induction in fall armyworm
© 2014 The Royal Entomological Society
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1.39 ± 1.15 0.88 ± 1.28 1.52 ± 0.55 1.57 ± 0.54 2.05 ± 0.62 14.05 ± 0.77 7.19 ± 3.42 4.31 ± 0.74 1.64 ± 0.80 3.08 ± 0.63 5.39 ± 1.95 1.52 ± 0.35 0.67 ± 0.18 1.10 ± 0.43 5.11 ± 1.45 2.51 ± 0.26 22.39 ± 15.29 13.75 ± 6.53 6.23 ± 0.90 0.78 ± 0.45 1.92 ± 0.22 4.17 ± 2.62 1.57 ± 0.38 0.73 ± 0.90 1.05 ± 0.34 1.56 ± 0.31 1.18 ± 0.17 1.24 ± 0.86 0.71 ± 0.03 1.49 ± 0.09 0.22 ± 0.03 0.85 ± 0.03 3.04 ± 1.64 3.18 ± 3.27 2.02 ± 3.65 1.57 ± 0.26 1.08 ± 0.61 3.59 ± 3.05 8.21 ± 4.43 9.27 ± 1.30 3.70 ± 0.45 1.90 ± 0.51 5.74 ± 2.84 4.36 ± 2.60 1.01 ± 0.18 0.64 ± 0.56 0.75 ± 0.14 0.87 ± 0.20 1.32 ± 0.58 2.60 ± 0.44 1.77 ± 0.51 1.52 ± 0.13 0.27 ± 0.06 1.00 ± 0.08 3.91 ± 6.62 2.76 ± 1.00 1.10 ± 1.26 0.74 ± 0.17 4.17 ± 0.99 2.87 ± 0.97 9.25 ± 6.49 12.40 ± 3.46 7.56 ± 0.99 0.40 ± 0.08 3.86 ± 0.47 1.97 ± 1.44 1.04 ± 0.41 1.22 ± 1.07 1.48 ± 0.46 3.06 ± 1.25 1.32 ± 0.19 0.47 ± 0.32 0.86 ± 0.42 1.20 ± 0.18 1.39 ± 0.21 0.92 ± 0.36 0.17 ± 0.17 1.77 ± 0.39 1.14 ± 1.52 0.51 ± 0.18 1.23 ± 0.38 2.34 ± 0.87 2.63 ± 2.04 3.77 ± 0.56 1.07 ± 0.19 0.20 ± 0.03 1.00 ± 0.16 1.48 ± 0.72 1.64 ± 0.65 0.93 ± 0.69 0.83 ± 0.14 1.17 ± 0.85 2.22 ± 0.44 5.79 ± 2.46 6.46 ± 2.47 2.32 ± 0.27 0.70 ± 0.29 1.90 ± 0.30 5.11 ± 2.87 1.11 ± 0.20 0.54 ± 1.06 0.63 ± 0.12 0.65 ± 0.09 1.17 ± 0.25 6.85 ± 4.21 2.05 ± 0.35 1.91 ± 0.16 0.24 ± 0.07 1.42 ± 0.17 2.10 ± 2.27 CYP4M14 CYP4M15 CYP6AB12 CYP9A24 CYP9A26 CYP9A30 CYP9A31 CYP9A32 CYP9A60 CYP321A9 CYP333B4
0.53 ± 0.09 0.28 ± 0.34 0.91 ± 0.07 0.96 ± 0.53 0.76 ± 0.47 5.12 ± 3.66 6.46 ± 3.89 2.07 ± 0.32 0.53 ± 0.09 4.03 ± 1.05 2.71 ± 3.35
Indole Xanthotoxin P450 name
Indole 3-carbinol
Phenobarbital
Methoxyfenozide
2-tridecanone
Fipronil
Methoprene
Quercetin
Clofibrate
Deltamethrin
M. Giraudo et al. Table 4. Quantitative real-time PCR analysis of the differential expression of 11 cytochrome P450 (CYP) genes in Sf9 cells exposed for 24 h to
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Insect Molecular Biology (2014)
doi: 10.1111/imb.12140
Cytochrome P450s from the fall armyworm (Spodoptera frugiperda): responses to plant allelochemicals and pesticides
M. Giraudo*†‡§, F. Hilliou*†‡, T. Fricaux*†‡, P. Audant*†‡, R. Feyereisen*†‡ and G. Le Goff*†‡ *INRA, UMR 1355, Institut Sophia Agrobiotech, Sophia-Antipolis, France; †CNRS, UMR 7254, Sophia-Antipolis, France; ‡Université de Nice Sophia Antipolis, Sophia-Antipolis, France; and §Environment Canada, Centre Saint-Laurent, Montreal, QC, Canada
detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S. frugiperda to its chemical environment. Keywords: cytochrome P450s, Spodoptera frugiperda, gene expression induction, xenobiotics.
Abstract
Introduction
Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S. frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were
The fall armyworm, Spodoptera frugiperda, is a major pest of agriculture, able to feed on more than 40 plant families. These families represent approximately 180 host plants and a wide array of different plant allelochemicals. This phytophagous insect has therefore developed sophisticated mechanisms to either reduce the quantity of or metabolically inactivate some of the potentially toxic xenobiotics that it ingests. These mechanisms range from avoidance of the area of the leaf surface where plant defences have been induced (Perkins et al., 2013), insensitivity to plant protease inhibitors (Jongsma et al., 1995; Brioschi et al., 2007; Dunse et al., 2010; Chikate et al., 2013) to increased excretion of allelochemicals by ABC transporters (Xie et al., 2012; Dermauw et al., 2013a) or active metabolism of toxic compounds (Wadleigh & Yu, 1988; Sasabe et al., 2004; Li et al., 2007). The latter mechanism is of particular importance as it can further lead to insecticide resistance. Dermauw et al. (2013b) for example, found that in the polyphagous spider mite Tetranychus urticae, transition from bean to tomato as the host plant conferred higher tolerance level to acaricides. Some key detoxification enzymes are capable of metabolizing plant secondary compounds and insecticides, amongst which cytochrome P450s (or CYP) play a major role. In the lepidopteran Helicoverpa zea for example, two P450s in particular, CYP6B8 and CYP321A1, have been shown to metabolize plants toxins such as xanthotoxin as well as insecticides including aldrin, cypermethrin and diazinon (Li et al., 2004; Sasabe et al., 2004; Rupasinghe et al.,
Correspondence: Gaëlle Le Goff, INRA, UMR 1355, Institut Sophia Agrobiotech, INRA-CNRS-Université de Nice Sophia-Antipolis, 400 Route des Chappes, 06903 Sophia-Antipolis, France. Tel.: + 33 492385578; fax: + 33 492386401; e-mail: [email protected]
© 2014 The Royal Entomological Society
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2007). The tobacco hornworm, Manduca sexta, a phytophagous insect specialist of Solanaceae, encounters in its diet huge amounts of the well-described plant defence compound, nicotine (Steppuhn et al., 2004). When larvae start feeding on tobacco, the toxicity of nicotine stops them from feeding until the enzymatic machinery of P450s is induced to metabolize this noxious compound (Snyder et al., 1993, 1994; Snyder & Glendinning, 1996). To date, P450 gene numbers in sequenced insect genomes vary from 36 in the body louse to 170 genes in mosquitoes (Arensburger et al., 2010; Kirkness et al., 2010). However, most of the P450s are expressed at low levels and are only expressed when insects have to deal with a toxic compound, mainly because P450 uncoupling produces harmful reactive oxygen species in the cell. In the well-known plant−insect interaction between Papilionidae lepidopterans and the toxic plant secondary metabolites furanocoumarins, several CYP6B transcripts are over-expressed to cope with either linear or angular furanocoumarins. In the specialist caterpillar Papilio polyxenes, CYP6B1 is inducible and able to metabolize linear furanocoumarins, such as xanthotoxin. The promoter region of this P450 contains specific regulation elements (REs) such as a xenobiotic response element to xanthotoxin (XRE-Xan; Petersen et al., 2003). The same RE is also present in the promoter region of CYP6B4 from the generalist Papilio glaucus, even though this caterpillar feeds on a more diverse range of host plants with only a few furanocoumarin-containing plant species (McDonnell et al., 2004). CYP6B4 is inducible and has been demonstrated to metabolize both linear and angular furanocoumarins (Hung et al., 1997; Li et al., 2003). Papilio canadensis, a caterpillar for which furanocoumarins are absent from its host plants, has inducible CYP6Bs, CYP6B25 and CYP6B26 that have low furanocoumarin metabolization abilities (Li et al., 2003). In other lepidopteran species, such as the generalist Helicoverpa zea, CYP6B8 and CYP321A1 have been shown to metabolize several allelochemicals and insecticides (Li et al., 2004; Sasabe et al., 2004; Rupasinghe et al., 2007). In the generalist S. frugiperda, it is well established that P450s can metabolize a broad range of plant secondary metabolites, such as indoles, glucosinolates, flavonoids and coumarins (Yu, 1987). Moreover, it has been demonstrated that when larvae are fed with allelochemicals such as indole 3-carbinol, menthol or flavones, P450 activities are induced (Yu & Ing, 1984; Yu, 1987). These studies however were based on enzymatic activities alone and did not specifically identify which P450 was induced by a given type of compound. The present work aimed to fill this gap by identifying ‘xenobiotic inducer – P450’ combinations and expression patterns in order to better under-
stand the adaptation of this insect to its chemical environment. First, 42 sequences coding for P450s were identified and basal expression levels of the corresponding transcripts were measured in tissues involved in detoxification processes and in the cellular model Sf9. Then, P450 induction profiles were measured after dietary exposure of larvae to 11 chemical compounds including plant secondary metabolites, insecticides and model inducers. In vivo P450 expression patterns were further compared with expression profiles in similarly treated Sf9 cells. Results Identification and phylogeny Thirty sequences coding for S. frugiperda P450s were successfully identified via the lepidopteran comparative genomic project and the screening of a BAC library (d’Alencon et al., 2010). This project was initiated in order to compare the genome of the model Lepidoptera species Bombyx mori and the two noctuid pests Helicoverpa armigera and S. frugiperda. The study was complemented by access to a S. frugiperda expressed sequence tag (EST) database containing eight different libraries built from different tissues and development stages as well as from individuals treated by chemical compounds (http:// www.spodobase.univ-montp2.fr/Spodobase/). Twelve additional sequences were obtained by EST assembly and confirmed by cloning and sequencing. In total, 42 sequences coding for cytochrome P450s were identified and named by David Nelson (http://drnelson.uthsc.edu/ CytochromeP450.html). All of the sequences were deposited in GenBank as BAC or unique P450 sequences (see GenBank accession numbers in Table 1). The 42 P450s were distributed amongst 14 families as shown on the phylogenetic tree in Fig. 1. The majority represented members of the CYP3 (29 sequences) and CYP4 clans (11 sequences) that are mostly involved in xenobiotic metabolism, with only two mitochondrial P450s of the CYP333 family. These sequences represent only part of the full CYPome of S. frugiperda. However they represent a reasonably large sampling of the diversity of CYP3 and CYP4 clan P450s in the fall armyworm. Tissue expression The expression profiles of the 42 P450s previously identified were measured by PCR: specific primers were designed for each sequence and the presence of mRNAs was verified for each of them in the midgut, Malpighian tubules and fat body of larvae as well as in the cellular model Sf9 (Table 1). The results showed that P450 expression profiles can vary amongst members of the same family. For example, in the CYP4L family CYP4L13 © 2014 The Royal Entomological Society
P450 induction in fall armyworm
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Table 1. Cytochrome P450s (CYPs) identified in Spodoptera frugiperda, expression in different tissues Name
GenBank accession no.
P450 size (amino acids)
Midgut
Malpighian tubules
Fat body
Sf9 cells
CYP4G74 CYP4G75 CYP4L12 CYP4L13 CYP4L9 CYP4M14 CYP4M15 CYP4M17 CYP4M18 CYP6AB12 CYP6AE43 CYP6AE44 CYP6AN4 CYP6B38 CYP6B39 CYP6B40 CYP6B41 CYP6B42 CYP6B50 CYP9A24 CYP9A25 CYP9A26 CYP9A27 CYP9A28 CYP9A29 CYP9A30 CYP9A31 CYP9A32 CYP9A58 CYP9A59 CYP9A60 CYP321A10 CYP321A7 CYP321A8 CYP321A9 CYP321B1 CYP332A1 CYP333B3 CYP333B4 CYP337B5 CYP340L1 CYP341A11
KC789745 KC789746 FP340412.1 FP340412.1 FP340412.1 FP340419.1 FP340419.1 FP340419.1 FP340419.1 KC789747 KJ671575 KJ671576 KC789748 FP340416.1 FP340416.1 FP340416.1 FP340416.1 FP340416.1 KC789749 FP340410.1 FP340410.1 FP340410.1 FP340410.1 FP340410.1 FP340410.1 FP340410.1 FP340410.1 FP340410.1 KJ671577 KJ671578 KJ671579 KC789753 KC789750 KC789751 KC789752 KC789754 FP340417.1 FP340412.1 FP340412.1 KJ671580 KC789755 KC789756
563 557 492 493 493 503 502 501 499 514 525 526 517 503 503 504 504 504 501 529 529 530 528 531 531 531 530 530 530 529 528 497 495 496 497 495 503 501 509 492 490 450 partial
ND ND ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ✓ ND
ND ND ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ND ✓ ND ✓ 2A ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ✓ ND
ND ND ND ✓ ND ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ✓ ND ✓ ND 2A ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ✓ ✓ ✓ ND
ND ND ND ND ND ✓ ✓ ND ND ✓ ✓ ✓ ND ✓ ND ✓ ND ✓ ✓ ✓ ND ✓ ND ✓ ND ✓ ✓ ✓ ✓ ✓ ✓ ✓ ND ✓ ✓ ND ✓ 2A ✓ ND ✓ ✓
Expression of the CYP genes was detected in all four tissues except when marked ND (not detected); in some cases, 2 amplicons sizes were detected (2A).
was the only one detected in all three tissues, with the two other members not present in the fat body. CYP6AE43 was only detected in the midgut and Malpighian tubules, whereas CYP6AE44 was solely expressed in the fat body. In the case of the CYP9A family, all members were detected in midgut, fat body and Malpighian tubules. Only four mRNA transcripts were absent in the tissues tested (CYP4G74, CYP4G75, CYP6B41 and CYP321B1). The CYP4G family expression profile confirmed Qiu et al.’s (2012) results that showed that CYP4G expression was restricted to the oenocyte cells, in which CYP4G acts as an oxidative decarbonylase in the biosynthesis of cuticular hydrocarbons. © 2014 The Royal Entomological Society
Induction by allelochemicals and xenobiotics Spodoptera frugiperda is a polyphagous insect that encounters in its diet several plant toxic compounds as well as insecticides. As P450s are known to be expressed at low levels and to be inducible when insects have to deal with toxic compounds, P450 induction profiles were measured in larvae exposed to plant secondary metabolites (xanthotoxin, 2-tridecanone, indole 3-carbinol, indole and quercetin), insecticides (deltamethrin, fipronil, methoprene and methoxyfenozide) and model inducers (clofibrate and phenobarbital). The same battery of compounds was used to treat Sf9 cells and both profiles were compared. Only
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M. Giraudo et al. 100 71
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100 87 63 100
100
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100 95
75 100
48
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100 39 100 100
58
75 65
100
70 100
99
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39 100
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25 58 100
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Sf-CYP333B3 Sf-CYP333B4 Sf-CYP340L1 Sf-CYP341A11 Sf-CYP4G74 Sf-CYP4G75 Sf-CYP4M18 Sf-CYP4M17 Sf-CYP4M14 Sf-CYP4M15 Sf-CYP4L12 Sf-CYP4L13 Sf-CYP4L9 Sf-CYP332A1 Sf-CYP337B5 Sf-CYP321B1 Sf-CYP321A7 Sf-CYP321A8 Sf-CYP321A10 Sf-CYP321A9 Sf-CYP6AE43 Sf-CYP6AE44 Sf-CYP6AB12 Sf-CYP6AN4 Sf-CYP6B50 Sf-CYP6B41 Sf-CYP6B42 Sf-CYP6B40 Sf-CYP6B38 Sf-CYP6B39 Sf-CYP9A32 Sf-CYP9A60 Sf-CYP9A28 Sf-CYP9A29 Sf-CYP9A30 Sf-CYP9A31 Sf-CYP9A24 Sf-CYP9A59 Sf-CYP9A26P Sf-CYP9A27 Sf-CYP9A25 Sf-CYP9A58
Figure 1. Phylogenetic analysis of Spodoptera frugiperda cytochrome P450s (SfP450s).
P450s with acceptable quantitative real-time PCR (qRTPCR) efficiencies (>85%, Table 2) were measured, which included 29 P450s in larval midguts and 11 for the Sf9 cell model. All chemical compounds were used at sublethal concentrations (Giraudo et al., 2013), which means that exposure doses might be different between chemicals but with the same effect on insects. Table 3 shows the results for S. frugiperda larvae. Amongst the 29 P450s analysed, 14 were over-expressed in at least one of the treatments, three were down-regulated and 12 were not differentially expressed. Each chemical induced a specific pattern, with xanthotoxin being the most potent inducer, whereas deltamethrin and clofibrate did not significantly regulate the expression of any P450 at the concentration used. Only one P450, CYP9A28 or CYP333B4, was differentially expressed in response to methoprene and fipronil treatment, respectively. CYP6B39 was the gene regulated by the most compounds, with high induction by four of the plant secondary metabolites and phenobarbital, whereas a fifth compound, quercetin, down-regulated its expres-
sion. By contrast, CYP9A28 was significantly repressed by four chemicals, including both plant compounds and insecticides. The Sf9 cell expression results are presented in Table 4. Out of the 11 P450s monitored, eight showed significant over-expression and three were not differentially expressed by any of the treatments. The pattern of induction seems to be specific for each chemical compound. Methoprene and 2-tridecanone were the best inducers of P450s in Sf9 cells, whereas xanthotoxin and phenobarbital did not significantly regulate any of the P450s studied. CYP333B4 was over-expressed in seven out of the 11 treatments. CYP9A30, CYP9A31 and CYP9A32 showed similar induction patterns in exposed cells. The Sf9 cell response shared few similarities with the in vivo response in larval midgut tissue. Only CYP321A9 and CYP333B4 were co-regulated in both tissues and over-expressed in 2-tridecanone and fipronil treatment, respectively.
Identification of putative regulation elements in the promoter region of S. frugiperda P450s Once S. frugiperda P450s that are induced by chemical compounds had been identified along with their corresponding inducers, the next step was to search for specific motifs of expression regulation in the promoter region of these genes. This was only possible for P450s that had been identified in BAC sequences. The analysis was performed on the 2000 nucleotides located upstream of the methionine start codon, sometimes on a smaller region when P450s were in a cluster with shorter intergenic regions. The presence of putative consensus elements for antioxidant response element (ARE), ecdysone response element (EcRE), xenobiotic response element (XRE) from the aryl hydrocarbon receptor (AhR) and for xanthotoxin (Xan) was searched for and reported in Table 5. ARE was found in 10 of the 23 promoter regions analysed, either once or twice and sometimes in reverse orientation. Eight out of the 10 genes found with an ARE motif were differentially regulated by one or several of the compounds tested, including CYP6B39, the most strongly induced P450 in this study. EcRE was present in 12 of the 23 promoters analysed, in both forward and reverse orientations. The XRE-AhR motif was only present in nine out of the 23 promoters analysed, whereas the XRE-Xan element was found in more than half of them.
Discussion Cytochrome P450s have essential endogenous functions and play a major role in the adaptation of insects to their chemical environment (Feyereisen, 2012). In the present study, 42 sequences coding for P450s were identified in the lepidopteran pest S. frugiperda. This number © 2014 The Royal Entomological Society
P450 induction in fall armyworm
5
Table 2. Primers used in quantitative real-time PCR (qRT-PCR) PCR efficiencies (%) Name
Primer sequence
Fragment length (bp)
Midgut
Cells
SfCYP4G74-F Sf CYP4G74-R SfCYP4G75-F SfCYP4G75-R SfCYP4L9-F SfCYP4L9-R SfCYP4L12-F SfCYP4L12-R SfCYP4L13-F SfCYP4L13-R SfCYP4M14-F SfCYP4M14-R SfCYP4M15-F SfCYP4M15-R SfCYP4M17-F SfCYP4M17-R SfCYP4M18-F SfCYP4M18-R SfCYP6AB12-F SfCYP6AB12-R SfCYP6AE43-F SfCYP6AE43-R SfCYP6AE44-F SfCYP6AE44-R SfCYP6AN4-F SfCYP6AN4-R SfCYP6B38-F SfCYP6B38-R SfCYP6B39-F SfCYP6B39-R SfCYP6B40-F SfCYP6B40-R SfCYP6B41-F SfCYP6B41-R SfCYP6B42-F SfCYP6B42-R SfCYP6B50-F SfCYP6B50-R SfCYP9A24-F SfCYP9A24-R SfCYP9A25-F SfCYP9A25-R SfCYP9A26-F SfCYP9A26-R SfCYP9A27-F SfCYP9A27-R SfCYP9A28-F SfCYP9A28-R SfCYP9A29-F SfCYP9A29-R SfCYP9A30-F SfCYP9A30-R SfCYP9A31-F SfCYP9A31-R SfCYP9A32-F SfCYP9A32-R SfCYP9A58-F SfCYP9A58-R SfCYP9A59-F SfCYP9A59-R SfCYP9A60-F SfCYP9A60-R SfCYP321A7-F
5′-AGGTCCACATCGACAAGT-3′ 5′-GAATGTGGGAGCGATCATTT-3′ 5′-AGAGCGGTGTTGTCATTTCC-3′ 5′-GGTGGATACCCATCATCGAC-3′ 5′-TCGGTGATGACATGGAAAGA-3′ 5′-AGAACGACAGACGTGCCTTTT-3′ 5′-CTCTGATCGAGCGGAGAATC-3′ 5′-CCAACTGAAGGGGTTTTTCA-3′ 5′-ACGAACGTGAGTCTGCCTATGTGA-3′ 5′-ACGACGTCCGGACCAAAAATC-3′ 5′-TGATCTCGGACTTGCACTTG-3′ 5′-GTCCAGCGCTGAAAGGAATA-3′ 5′-TGATCTTGGACGTGCATTAT-3′ 5′-GCCCAGCACTGAAGGGAATG-3′ 5′-AGAGTCGCTGCGCATATACC-3′ 5′-GGGTTCGGGAATAAATCCTC-3′ 5′-TCCTACCCGAGAACAGCATC-3′ 5′-ACTTCTGCCACAGCCATCTT-3′ 5′-TTCGGGAAAGCCAGTATGAC-3′ 5′-ATTCGTTTCGCAATTTCTGG-3′ 5′-GCTGATTGCGCAGAGTGTTA-3′ 5′-CATGAACTCGTCCACCTCCT-3′ 5′-CTTCACTTACGGCGACAGGT-3′ 5′-CTCGAAGATACGGGCACATT-3′ 5′-ACGTCGTCATCAGCAACAAG-3′ 5′-GCTGATGGAGTCGTAGCACA-3′ 5′-TTCTTCACCAAGTTTTGCAGT-3′ 5′-ATCAAAGAACTTTTTTATGGCG-3′ 5′-AAGTTCCAAGTGGAGCCATCGAGG-3′ 5′-CCTCCTTTGGGCCCGACGAGAAG-3′ 5′-CTGTATCGGTATGCGGTTTG-3′ 5′-TTCCACCTTTAGGTCCGATG-3′ 5′-CAAGGATTTTGATGCGTTCA-3′ 5′-GTAAAGCCCTCCAGGTGTGA-3′ 5′-CTACTACTATGGCCTACCTGA-3′ 5′-TTGTTCGCTTGAACAGTTGC-3′ 5′-CAATCCAGCACGATGAGAAA-3′ 5′-GTGCGAATTTTGACCAAGG-3′ 5′-GGCACTAAACAACAGAGTGTGG-3′ 5′-ACCAAGCGTTCCTGTACGTC-3′ 5′-AATGCAAAGGCTGAGAAGGA-3′ 5′-GAAAAACGATCAGGGTCGAA-3′ 5′-TGAAAGGCCAAGAATGGAAG-3′ 5′-GTCATCTGACGCCTCGATTT-3′ 5′-CATCAAGTATCGCACGCCTA-3′ 5′-ATTCAAATCTGCCGACGAAC-3′ 5′-TCAAGCACATCAAGCCAGTC-3′ 5′-CCGTTGTGAGTCCATCACTGAC-3′ 5′-CAAAGAACATACCAGCGATGA-3′ 5′-AGTTCAAGGTCACGGACTACA-3′ 5′-GTCCTGGTGGCTGTGGTATT-3′ 5′-GTGCGAAAAATGATCGTGTG-3′ 5′-ATGCTCGTCTTGGTCTGGTT-3′ 5′-CTGCCCATGTTACCGAAGAT-3′ 5′-ATCATTCGTAAGGGCCAGTG-3′ 5′-AAGTGAACGGGACGATTTTG-3′ 5′-GCTCTCTGCGAGATGAAGGT-3′ 5′-GAGCCAGTGTCCTCCCTGTA-3′ 5′-GGATACCCACGTATGCCATC-3′ 5′-TCCTAGGACCAGTGCCAAAT-3′ 5′-CATCGTTTGGCCAGAGAACT-3′ 5′-CAGGGTGTACAGCCAACTCA-3′ 5′-TCCAGACCCAGAAGTTTTCG-3′
119
ND
ND
120
ND
ND
178
98
ND
176
87
ND
93
91
ND
126
93
105
126
105
104
150
99
ND
121
90
ND
© 2014 The Royal Entomological Society
131
BE
100
136
BE
BE
115
ND
BE
132
100
ND
102
BE
BE
84
96
ND
146
103
BE
101
ND
ND
97
BE
BE
147
100
BE
147
105
106
125
94
ND
146
92
106
134
103
ND
87
105
BE
113
BE
ND
122
99
104
131
99
97
136
97
102
135
113
BE
138
88
BE
141
106
110
134
106
ND
6
M. Giraudo et al.
Table 2. Continued PCR efficiencies (%) Name
Primer sequence
SfCYP321A7-R SfCYP321A8-F SfCYP321A8-R SfCYP321A9-F SfCYP321A9-R SfCYP321A10-F SfCYP321A10-R SfCYP321B1-F SfCYP321B1-R SfCYP332A1-F SfCYP332A1-R SfCYP333B3-F SfCYP333B3-R SfCYP333B4-F SfCYP333B4-R SfCYP337B5-F SfCYP337B5-R SfCYP340L1-F SfCYP340L1-R SfCYP341A11-F SfCYP341A11-R RpL4-F RpL4-R G6PD-F G6PD-R L18-F L18-R
5′-CGGCCTGGACTTGTAATTTG-3′ 5′-CCAGTAGAAAAGGGGACAAA-3′ 5′-TGGCATACCTCTCCCCTATG-3′ 5′-GCGTGGTGTAGCCTTCTACG-3′ 5′-CGGGTCAATGACAAACAGTG-3′ 5′-AACTTCACTGTGGCATCGA-3′ 5′-GGAATAAGCTGAACATCAACG-3′ 5′-CGTACGATGCAGTCTTGGAA-3′ 5′-CATTGCCTACAGGCAGAACA-3′ 5′-GCATGCATGAAACGCTAAGA-3′ 5′-CCACGTTCACGTAGACTGGA-3′ 5′-AGGCTGCTTGAAACGTATGG-3′ 5′-AGTCCAGTTGCGTGTCCTCT-3′ 5′-GAATTATGCCGGTGGTGTCT-3′ 5′-TAGCGACATGTCTCGGTGAG-3′ 5′-CCGTTTGGTGAAGGAAACA-3′ 5′-GACCGAAGGGACTTCTTTCA-3′ 5′-GTGCATTCCACAATGGGTGT-3′ 5′-CTGGCAAGGCACCAGGATA-3′ 5′-GTGCTCAATATGGGGTGTCC-3′ 5′-CCCATTACTGAAGGGCATGT-3′ 5′-CAACAAGAGGGGTTCACGAT-3′ 5′-GCACGATCAGTTCGGGTATC-3′ 5′-GGCCCTGTGGCTAACAGAAT-3′ 5′-CATCGTCTCTACCAAAAGGCTTC-3′ 5′-CGTATCAACCGACCTCCACT-3′ 5′-AGGCACCTTGTAGAGCCTCA-3′
Fragment length (bp)
Midgut
182
98
172
98
Cells
BE 95
99
BE
BE
126
ND
ND
123
112
BE
197
101
BE
116
101
93
142
101
ND
115
ND
BE
133
ND
BE
149
99
98
142
99
98
126
104
108
BE, bad efficiency in qRT-PCR; ND, not detected; RpL4, ribosomal protein L4; G6PD, glucose 6-phosphate dehydrogenase; L18, ribosomal protein L18.
represents only part of the complete CYPome of this pest insect. It is difficult or nearly impossible to predict the precise number of P450s in a species, as the size of the CYPome and the diversity inside a single P450 family is specific to each species (Feyereisen, 2011). However, based on the number of P450s found in other Lepidoptera such as the polyphagous H. armigera or the oligophagous M. sexta, the estimated number of P450s in S. frugiperda could reach up to 100–120 coding genes (F. Hilliou, pers. comm.). These numbers are slightly higher than the 87 P450 sequences found in the model Lepidoptera B. mori, a monophagous species that feeds exclusively on mulberry leaves (Ai et al., 2011). It is easy but not necessarily correct to hypothesize that the expansion of certain P450 families may be linked to the necessity of polyphagous insects to adapt to the toxic plant secondary metabolites that they encounter in their diet. The same reduction of the number of P450s has been observed in the Drosophila genus, in which the specialist Drosophila sechellia contains fewer P450s, 74 putatively functional genes, than the generalists that have been sequenced, for which the relative number is around 90 (Good et al., 2014). However, related species can have different numbers of P450s and a rapid evolution of this number may occur without a link to adaptation processes
(Feyereisen, 2011; Sezutsu et al., 2013). Hence, correlating the ability of insects to detoxify xenobiotics to the size of their CYPome is a shortcut that can easily lead to misinterpretations. The honeybee, for example, was suggested to be more susceptible to insecticides because of its reduced number of detoxification enzymes, especially P450s and glutathione S-transferases (Claudianos et al., 2006). However, this hypothesis is no longer supported when actual toxicological data are compared between Apis mellifera and other insects (Hardstone & Scott, 2010). Similar observations can be made in the louse (Pediculus humanus), which is highly resistant to many insecticides despite having only 36 genes encoding P450s in its genome (Kirkness et al., 2010). In addition, true one-to-one orthologues in closely related species are relatively rare. The Drosophila genus for example contains only 30 P450 orthologues for an evolution scale between 0.5 to ∼50 Ma (Good et al., 2014). It would therefore be difficult to predict the number of P450s in S. frugiperda when it diverged from its most closely related species H. armigera between 20 and 40 Ma (d’Alencon et al., 2010). More than half of the identified P450 genes (28/42) were expressed in the midgut, Malpighian tubules and fat body, which have essential functions in insect physiology. © 2014 The Royal Entomological Society
Indole
1.12 ± 0.05 1.64 ± 1.09 0.58 ± 0.18 0.36 ± 0.91 0.46 ± 1.10 0.48 ± 0.97 0.69 ± 0.28 0.54 ± 0.17 0.44 ± 0.50 5.69 ± 2.88 0.74 ± 0.75 135.95 ± 182.28 119.20 ± 56.24 28.59 ± 10.79 0.65 ± 0.28 0.53 ± 0.73 3.26 ± 2.99 135.14 ± 52.08 3.28 ± 4.16 1.42 ± 0.87 1.01 ± 0.54 2.10 ± 2.59 8.13 ± 8.47 0.53 ± 0.05 0.23 ± 0.10 1.43 ± 2.37 0.77 ± 0.18 0.45 ± 0.11 1.15 ± 0.90
Xanthotoxin
2.35 ± 0.82 2.66 ± 0.63 0.38 ± 0.15 0.17 ± 0.46 0.14 ± 0.38 0.19 ± 0.50 0.81 ± 0.36 0.69 ± 0.97 0.32 ± 0.49 2.50 ± 1.22 1.23 ± 1.24 162.11 ± 35.09 95.54 ± 20.19 20.97 ± 6.72 0.25 ± 0.12 0.26 ± 0.32 2.89 ± 0.83 181.71 ± 12.98 28.10 ± 9.73 0.73 ± 0.06 0.44 ± 0.03 1.53 ± 1.73 21.04 ± 5.26 0.99 ± 0.66 1.37 ± 0.42 1.41 ± 2.39 2.92 ± 1.73 1.85 ± 1.62 0.44 ± 0.11
P450 name
CYP333B3 CYP333B4 CYP4M18 CYP4M17 CYP4M14 CYP4M15 CYP4L12 CYP4L13 CYP4L9 CYP332A1 CYP337B5 CYP321A7 CYP321A8 CYP321A9 CYP6AN4 CYP6B50 CYP6B40 CYP6B39 CYP9A32 CYP9A60 CYP9A28 CYP9A30 CYP9A31 CYP9A24 CYP9A59 CYP9A26 CYP9A27 CYP9A25 CYP9A58
1.86 ± 0.99 0.93 ± 0.27 0.61 ± 0.29 0.33 ± 0.22 0.31 ± 0.32 0.61 ± 1.15 0.95 ± 0.36 0.70 ± 0.21 0.44 ± 0.59 0.94 ± 0.37 0.61 ± 0.18 64.31 ± 138.34 28.71 ± 4.93 11.70 ± 0.95 0.83 ± 0.13 0.71 ± 0.95 2.50 ± 1.87 224.13 ± 49.16 1.15 ± 0.59 0.75 ± 0.17 0.51 ± 0.30 1.45 ± 1.74 2.96 ± 1.22 0.53 ± 0.26 0.40 ± 0.22 0.46 ± 0.59 0.65 ± 0.19 0.55 ± 0.36 0.61 ± 0.08
Indole 3-carbinol 0.35 ± 0.05 1.19 ± 0.22 1.08 ± 0.13 1.25 ± 0.17 0.71 ± 0.69 1.08 ± 0.69 0.56 ± 0.05 2.90 ± 0.73 1.25 ± 0.17 2.83 ± 1.00 1.280.39 3.86 ± 3.12 2.04 ± 0.26 3.42 ± 0.79 1.62 ± 0.43 1.23 ± 0.20 3.26 ± 0.54 81.23 ± 9.48 0.67 ± 4.31 0.77 ± 0.33 0.29 ± 0.13 1.03 ± 4.99 2.93 ± 3.70 0.94 ± 0.54 0.12 ± 0.07 0.95 ± 0.82 0.60 ± 0.12 0.83 ± 0.60 0.70 ± 0.15
Phenobarbital 5.91 ± 4.43 1.83 ± 0.79 1.05 ± 0.58 0.86 ± 1.99 0.58 ± 0.82 1.12 ± 3.07 1.38 ± 0.69 0.71 ± 0.53 0.53 ± 0.76 1.26 ± 0.35 1.33 ± 1.49 1.09 ± 0.66 0.70 ± 0.46 1.58 ± 0.99 0.85 ± 0.40 0.74 ± 1.60 1.75 ± 2.21 2.73 ± 1.75 3.02 ± 0.58 3.00 ± 0.71 0.92 ± 0.49 1.18 ± 1.46 1.43 ± 0.995 0.92 ± 0.49 5.60 ± 5.49 1.45 ± 2.29 2.46 ± 2.31 3.80 ± 5.32 2.70 ± 0.47
Methoxyfenozide 0.69 ± 0.36 0.98 ± 0.31 1.04 ± 0.72 1.01 ± 3.12 0.56 ± 1.83 0.78 ± 2.16 1.16 ± 0.68 5.09 ± 8.42 0.79 ± 1.06 0.69 ± 0.18 1.12 ± 0.78 0.88 ± 0.89 1.19 ± 0.85 0.92 ± 0.60 1.26 ± 0.31 0.53 ± 1.00 0.72 ± 0.34 4.23 ± 0.81 0.73 ± 0.14 0.87 ± 0.23 0.22 ± 0.06 0.75 ± 0.89 0.72 ± 0.12 0.63 ± 0.48 0.22 ± 0.17 0.70 ± 1.22 0.74 ± 0.37 0.77 ± 0.73 0.72 ± 0.09
2-Tridecanone 0.77 ± 0.38 2.49 ± 1.67 1.05 ± 0.93 0.78 ± 1.89 0.61 ± 1.36 0.93 ± 2.10 0.34 ± 0.20 1.18 ± 1.07 1.11 ± 1.80 2.13 ± 0.62 1.65 ± 2.09 1.41 ± 1.16 0.88 ± 0.37 1.12 ± 0.74 0.45 ± 0.17 0.77 ± 1.62 1.42 ± 1.18 0.43 ± 0.18 2.34 ± 0.96 0.64 ± 0.15 0.73 ± 0.27 0.91 ± 1.21 2.12 ± 1.42 1.48 ± 0.93 1.46 ± 0.81 1.89 ± 4.23 1.41 ± 0.97 2.89 ± 4.18 0.73 ± 0.09
Fipronil 1.92 ± 0.89 1.31 ± 0.25 1.04 ± 0.68 1.10 ± 3.70 0.80 ± 3.03 1.03 ± 2.98 1.04 ± 0.55 0.96 ± 0.85 1.12 ± 1.45 1.29 ± 0.53 1.08 ± 2.29 1.13 ± 0.95 0.68 ± 0.33 1.49 ± 0.86 0.71 ± 0.18 0.59 ± 0.44 0.90 ± 0.29 0.33 ± 0.11 1.70 ± 0.80 1.57 ± 0.19 0.26 ± 0.11 1.11 ± 1.67 0.94 ± 0.31 1.07 ± 0.64 1.27 ± 1.20 1.29 ± 2.29 1.56 ± 0.85 1.14 ± 1.20 1.22 ± 0.06
Methoprene
1.74 ± 0.71 0.66 ± 0.31 0.69 ± 0.47 0.41 ± 0.92 0.47 ± 0.84 0.45 ± 0.74 0.52 ± 0.11 0.29 ± 0.17 0.46 ± 0.63 0.49 ± 0.13 1.01 ± 1.21 0.34 ± 0.31 0.44 ± 0.26 0.61 ± 0.38 0.36 ± 0.23 0.92 ± 1.24 0.42 ± 0.11 0.12 ± 0.01 0.48 ± 0.32 1.39 ± 0.58 0.10 ± 0.71 0.35 ± 0.34 0.38 ± 0.15 0.42 ± 0.20 0.45 ± 0.40 0.23 ± 0.09 0.63 ± 0.05 0.52 ± 0.29 0.56 ± 0.27
Quercetin
1.74 ± 0.82 1.33 ± 0.61 0.98 ± 0.95 0.75 ± 2.62 0.76 ± 3.21 1.18 ± 4.09 0.98 ± 0.47 1.22 ± 1.64 1.02 ± 1.32 0.57 ± 0.26 1.04 ± 1.16 3.62 ± 9.01 0.29 ± 0.15 1.22 ± 1.06 1.47 ± 1.00 1.04 ± 2.19 0.63 ± 0.41 0.46 ± 0.20 1.42 ± 0.90 1.22 ± 0.63 0.39 ± 0.26 0.86 ± 1.13 1.01 ± 0.11 1.00 ± 1.17 0.66 ± 0.61 0.56 ± 1.60 1.16 ± 0.84 1.16 ± 1.51 1.37 ± 0.40
Clofibrate
4.48 ± 3.75 2.18 ± 1.01 0.92 ± 0.34 0.86 ± 2.21 0.60 ± 1.06 0.98 ± 2.25 0.85 ± 0.15 0.91 ± 0.99 0.86 ± 1.12 1.06 ± 0.47 1.27 ± 0.46 1.21 ± 0.19 0.79 ± 0.53 1.52 ± 0.08 2.18 ± 1.48 0.89 ± 1.63 1.14 ± 1.05 1.01 ± 0.56 3.05 ± 2.49 1.52 ± 0.27 0.70 ± 0.42 1.20 ± 1.55 1.55 ± 1.65 1.36 ± 0.75 3.81 ± 3.58 0.91 ± 0.29 1.94 ± 1.23 1.76 ± 1.78 2.10 ± 0.63
Deltamethrin
Table 3. Quantitative real-time PCR analysis of the differential expression of 29 cytochrome P450 (CYP) genes in Spodoptera frugiperda larvae exposed for 24 h to sublethal doses of 11 different xenobiotic compounds. Gene expression values are indicated as fold expression in larvae exposed to each xenobiotic compared with unexposed larvae (controls). The three reference genes ribosomal protein L4, glucose 6-phosphate dehydrogenase and ribosomal protein L18 were used as internal controls for normalization. Gene expression values in bold are significantly different from the corresponding control as measured by pairwise Student’s t-tests (P < 0.05). Genes highlighted in light grey and dark grey are respectively induced or repressed
P450 induction in fall armyworm
© 2014 The Royal Entomological Society
7
1.39 ± 1.15 0.88 ± 1.28 1.52 ± 0.55 1.57 ± 0.54 2.05 ± 0.62 14.05 ± 0.77 7.19 ± 3.42 4.31 ± 0.74 1.64 ± 0.80 3.08 ± 0.63 5.39 ± 1.95 1.52 ± 0.35 0.67 ± 0.18 1.10 ± 0.43 5.11 ± 1.45 2.51 ± 0.26 22.39 ± 15.29 13.75 ± 6.53 6.23 ± 0.90 0.78 ± 0.45 1.92 ± 0.22 4.17 ± 2.62 1.57 ± 0.38 0.73 ± 0.90 1.05 ± 0.34 1.56 ± 0.31 1.18 ± 0.17 1.24 ± 0.86 0.71 ± 0.03 1.49 ± 0.09 0.22 ± 0.03 0.85 ± 0.03 3.04 ± 1.64 3.18 ± 3.27 2.02 ± 3.65 1.57 ± 0.26 1.08 ± 0.61 3.59 ± 3.05 8.21 ± 4.43 9.27 ± 1.30 3.70 ± 0.45 1.90 ± 0.51 5.74 ± 2.84 4.36 ± 2.60 1.01 ± 0.18 0.64 ± 0.56 0.75 ± 0.14 0.87 ± 0.20 1.32 ± 0.58 2.60 ± 0.44 1.77 ± 0.51 1.52 ± 0.13 0.27 ± 0.06 1.00 ± 0.08 3.91 ± 6.62 2.76 ± 1.00 1.10 ± 1.26 0.74 ± 0.17 4.17 ± 0.99 2.87 ± 0.97 9.25 ± 6.49 12.40 ± 3.46 7.56 ± 0.99 0.40 ± 0.08 3.86 ± 0.47 1.97 ± 1.44 1.04 ± 0.41 1.22 ± 1.07 1.48 ± 0.46 3.06 ± 1.25 1.32 ± 0.19 0.47 ± 0.32 0.86 ± 0.42 1.20 ± 0.18 1.39 ± 0.21 0.92 ± 0.36 0.17 ± 0.17 1.77 ± 0.39 1.14 ± 1.52 0.51 ± 0.18 1.23 ± 0.38 2.34 ± 0.87 2.63 ± 2.04 3.77 ± 0.56 1.07 ± 0.19 0.20 ± 0.03 1.00 ± 0.16 1.48 ± 0.72 1.64 ± 0.65 0.93 ± 0.69 0.83 ± 0.14 1.17 ± 0.85 2.22 ± 0.44 5.79 ± 2.46 6.46 ± 2.47 2.32 ± 0.27 0.70 ± 0.29 1.90 ± 0.30 5.11 ± 2.87 1.11 ± 0.20 0.54 ± 1.06 0.63 ± 0.12 0.65 ± 0.09 1.17 ± 0.25 6.85 ± 4.21 2.05 ± 0.35 1.91 ± 0.16 0.24 ± 0.07 1.42 ± 0.17 2.10 ± 2.27 CYP4M14 CYP4M15 CYP6AB12 CYP9A24 CYP9A26 CYP9A30 CYP9A31 CYP9A32 CYP9A60 CYP321A9 CYP333B4
0.53 ± 0.09 0.28 ± 0.34 0.91 ± 0.07 0.96 ± 0.53 0.76 ± 0.47 5.12 ± 3.66 6.46 ± 3.89 2.07 ± 0.32 0.53 ± 0.09 4.03 ± 1.05 2.71 ± 3.35
Indole Xanthotoxin P450 name
Indole 3-carbinol
Phenobarbital
Methoxyfenozide
2-tridecanone
Fipronil
Methoprene
Quercetin
Clofibrate
Deltamethrin
M. Giraudo et al. Table 4. Quantitative real-time PCR analysis of the differential expression of 11 cytochrome P450 (CYP) genes in Sf9 cells exposed for 24 h to
