References

Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne, J.D., Amanatides, P.G., Scherer, S.E., Li, P.W., Hoskins, R.A., Galle, R.F., *et al*. (2000). The genome sequence of Drosophila melanogaster. Science **287**, 2185-2195.

Ahmad, S., and Pardini, S. (1990). Antioxidant defense of the cabbage looper, Trichoplusia ni: enzymatic responses to the superoxide-generating flavonoid, quercetin and photodynamic furanocoumarin, xantotoxin. *Photochem Photobiol* **51**, 305-312.

Barbehenn, R.V. (2002). Gut-based antioxidant enzymes in a polyphagous and a graminivorous grasshopper. J Chem Ecol **28**, 1329-1347.

Brown, R.P., McDonnell, C.M., Berenbaum, M.R., and Schuler, M.A. (2005). Regulation of an insect cytochrome P450 monooxygenase gene (CYP6B1) by aryl hydrocarbon and xanthotoxin response cascades. *Gene* **358**, 39-52.

Dhadialla, T.S., Carlson, G.R., and Le, D.P. (1998). New insecticides with ecdysteroidal and juvenile hormone activity. *Annu Rev Entomol* **43**, 545-569.

Dobson, C.M. (2004). Chemical space and biology. *Nature* **432**, 824-828.

Ferry, N., Edwards, M.G., Gatehouse, J.A., and Gatehouse, A.M. (2004). Plant-insect interactions: molecular approaches to insect resistance. *Curr Opin Biotechnol* **15**, 155-161.

Feyereisen, R. (1999). Insect P450 enzymes. *Annu Rev Entomol* **44**, 507-533.

Feyereisen, R. (2005). Insect cytochrome P450. In *Comprehensive molecular insect science* (Elsevier), pp. 1-77.

Gatehouse, J.A. (2002). Plant resistance towards insect hrebivores: a dynamic interaction. *New Phytol* **156**, 145-169.

Gentleman, R.C., Carey, V.J., Bates, D.M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., *et al*. (2004). Bioconductor: open software development for computational biology and bioinformatics. *Genome Biol* **5**, R80.

Girardot, F., Monnier, V., and Tricoire, H. (2004). Genome wide analysis of common and specific stress responses in adult drosophila melanogaster. *BMC Genomics* **5**, 74.

Hartmann, T. (2004). Plant-derived secondary metabolites as defensive chemicals in herbivorous insects: a case study in chemical ecology. *Planta* **219**, 1-4.

Hess, A., and Iyer, H. (2007). Fisher's combined p-value for detecting differentially expressed genes using Affymetrix expression arrays. *BMC Genomics* **8**, 96.

Hu, Z., Lee, K.S., Choo, Y.M., Yoon, H.J., Lee, S.M., Lee, J.H., Kim, D.H., Sohn, H.D., and Jin, B.R. (2010). Molecular cloning and characterization of 1-Cys and 2-Cys peroxiredoxins from the bumblebee Bombus ignitus. *Comp Biochem Physiol B Biochem Mol Biol* **155**, 272-280.
Le Brigand, K., and Barbry, P. (2007). Mediante: a web-based microarray data manager. *Bioinformatics* 23, 1304-1306.

Le Goff, G., Hilliou, F., Siegfried, B.D., Boundy, S., Wajnberg, E., Sofer, L., Audant, P., ffrench-Constant, R.H., and Feyereisen, R. (2006). Xenobiotic response in Drosophila melanogaster: sex dependence of P450 and GST gene induction. *Insect Biochem Mol Biol* 36, 674-682.

Li, X., Berenbaum, M.R., and Schuler, M.A. (2002). Plant allelochemicals differentially regulate Helicoverpa zea cytochrome P450 genes. *Insect Mol Biol* 11, 343-351.

Li, X., Schuler, M.A., and Berenbaum, M.R. (2007). Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. *Annu Rev Entomol* 52, 231-253.

Luque, T., Okano, K., and O'Reilly, D.R. (2002). Characterization of a novel silkworm (Bombyx mori) phenol UDP-glucosyltransferase. *Eur J Biochem* 269, 819-825.

Negre, V., Hotelier, T., Volkoff, A.N., Gimenez, S., Cousserans, F., Mita, K., Sabau, X., Rocher, J., Lopez-Ferber, M., d'Alencon, E., et al. (2006). SPODOBASE: an EST database for the lepidopteran crop pest Spodoptera. *BMC Bioinformatics* 7, 322.

Pauchet, Y., Wilkinson, P., Vogel, H., Nelson, D.R., Reynolds, S.E., Heckel, D.G., and ffrench-Constant, R.H. (2010). Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence. *Insect Mol Biol* 19, 61-75.

Poitout, S., and Bues, R. (1974). [Linolenic acid requirements of lepidoptera Noctuidae Quadrifinae Plusiinae: Chrysodeixis chalcites Esp., Autographa gamma L.' Macdunnoughia confusa Stph., Trichoplusia ni Hbn. reared on artificial diets]. *Ann Nutr Aliment* 28, 173-187.

Poupardin, R., Reynaud, S., Strode, C., Ranson, H., Vontas, J., and David, J.P. (2008). Cross-induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: impact on larval tolerance to chemical insecticides. *Insect Biochem Mol Biol* 38, 540-551.

Ranson, H., Claudianos, C., Ortelli, F., Abgrall, C., Hemingway, J., Sharakhova, M.V., Unger, M.F., Collins, F.H., and Feyereisen, R. (2002). Evolution of supergene families associated with insecticide resistance. *Science* 298, 179-181.

Rausher, M.D. (2001). Co-evolution and plant resistance to natural enemies. *Nature* 411, 857-864.

Richards, S., Gibbs, R.A., Weinstock, G.M., Brown, S.J., Denell, R., Beeman, R.W., Gibbs, R., Bucher, G., Friedrich, M., Grimmelikhuijzen, C.J., et al. (2008). The genome of the model beetle and pest Tribolium castaneum. *Nature* 452, 949-955.

Riskallah, M.R., Dauterman, W.C., and Hodgson, E. (1986). Nutritional effets of the induction of cytochrome P-450 and glutathione transferase in larvae of the tobacco budworm, Heliothis virescens (F.). *Insect Biochem* 16, 491-499.

Smagghe, G., Goodman, C.L., and Stanley, D. (2009). Insect cell culture and applications to research and pest management. *In Vitro Cell Dev Biol Anim* 45, 93-105.
Smyth, G.K. (2004). Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3, Article3.

Snyder, M.J., Stevens, J.L., Andersen, J.F., and Feyereisen, R. (1995). Expression of cytochrome P450 genes of the CYP4 family in midgut and fat body of the tobacco hornworm, Manduca sexta. Arch Biochem Biophys 321, 13-20.

Stevens, J.L., Snyder, M.J., Koener, J.F., and Feyereisen, R. (2000). Inducible P450s of the CYP9 family from larval Manduca sexta midgut. Insect Biochem Mol Biol 30, 559-568.

Tijet, N., Helvig, C., and Feyereisen, R. (2001). The cytochrome P450 gene superfamily in Drosophila melanogaster: annotation, intron-exon organization and phylogeny. Gene 262, 189-198.

Vaughn, J.L., Goodwin, R.H., Tompkins, G.J., and McCawley, P. (1977). The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro 13, 213-217.

Vogel, H., Heidel, A.J., Heckel, D.G., and Groot, A.T. (2010). Transcriptome analysis of the sex pheromone gland of the noctuid moth Heliothis virescens. BMC Genomics 11, 29.

Vontas, J., Blass, C., Koutsos, A.C., David, J.P., Kafatos, F.C., Louis, C., Hemingway, J., Christophides, G.K., and Ranson, H. (2005). Gene expression in insecticide resistant and susceptible Anopheles gambiae strains constitutively or after insecticide exposure. Insect Mol Biol 14, 509-521.

Wang, Q., Chen, K., Yao, Q., Zhao, Y., Li, Y., Shen, H., and Mu, R. (2008). Identification and characterization of a novel 1-Cys peroxiredoxin from silkworm, Bombyx mori. Comp Biochem Physiol B Biochem Mol Biol 149, 176-182.

Wettenhall, J.M., and Smyth, G.K. (2004). limmaGUI: a graphical user interface for linear modeling of microarray data. Bioinformatics 20, 3705-3706.

Willoughby, L., Chung, H., Lumb, C., Robin, C., Batterham, P., and Daborn, P.J. (2006). A comparison of Drosophila melanogaster detoxification gene induction responses for six insecticides, caffeine and phenobarbital. Insect Biochem Mol Biol 36, 934-942.

Yang, Y.H., and Thorne, N.P. (2003). Normalization for two-color cDNA microarray data. In Science and Statistics: A Festschrift for Terry Speed, IMS Lecture Notes - Monograph Series, D.R. Goldstein., ed., pp. 403-418.

Yu, Q.Y., Lu, C., Li, W.L., Xiang, Z.H., and Zhang, Z. (2009). Annotation and expression of carboxylesterases in the silkworm, Bombyx mori. BMC Genomics 10, 553.

Yu, S.J. (1983). Induction of detoxifying enzymes by allelochemicals and host plants in the fall armyworm. Pest Biochem Physiol 19, 330-336.

Yu, S.J. (1986). Consequences of induction of foreign compound-metabolizing enzymes in insects. 153-175.
Yu, S.J., and McCord, E., Jr. (2007). Lack of cross-resistance to indoxacarb in insecticide-
resistant Spodoptera frugiperda (Lepidoptera: Noctuidae) and Plutella xylostella
(Lepidoptera: Yponomeutidae). Pest Manag Sci 63, 63-67.

Zeng, R.S., Wen, Z., Niu, G., Schuler, M.A., and Berenbaum, M.R. (2007). Allelochemical induction of cytochrome P450 monooxygenases and amelioration of xenobiotic toxicity in Helicoverpa zea. J Chem Ecol 33, 449-461.

Zhou, X., Ma, C., Li, M., Sheng, C., Liu, H., and Qiu, X. (2009). CYP9A12 and CYP9A17 in the cotton bollworm, Helicoverpa armigera: sequence similarity, expression profile and xenobiotic response. Pest Manag Sci.
<table><caption>**Table 1 :** primers used in qRT-PCR reactions</caption><thead><tr><th></th><th>Forward primer</th><th>Reverse primer</th></tr></thead><tbody><tr><td>CYP9A24</td><td>GGCACTAAACAACAGAGTGTGG</td><td>ACCAAGCGTTCCTGTACGTC</td></tr><tr><td>CYP9A25</td><td>AATGCAAAGGCTGAGAAGGA</td><td>GAAAAACGATCAGGGTCGAA</td></tr><tr><td>CYP9A26</td><td>TGAAAGGCCAAGAATGGAAG</td><td>GTCATCTGACGCCTCGATTT</td></tr><tr><td>CYP9A27</td><td>CATCAAGTATCGCACGCCTA</td><td>ATTCAAATCTGCCGACGAAC</td></tr><tr><td>CYP9A30</td><td>GTCCTGGTGGCTGTGGTATT</td><td>GTGCGAAAAATGATCGTGTG</td></tr><tr><td>CYP9A31</td><td>ATGCTCGTCTTGGTCTGGTT</td><td>CTGCCCATGTTACCGAAGAT</td></tr><tr><td>CYP9A32</td><td>ATCATTCGTAAGGGCCAGTG</td><td>AAGTGAACGGGACGATTTTG</td></tr><tr><td>CYP6Bp2</td><td>CAATCCAGCACGATGAGAAA</td><td>GTGCGAATTTTGACCAAGG</td></tr><tr><td>CYP6B40</td><td>CTGTATCGGTATGCGGTTTG</td><td>TTCCACCTTTAGGTCCGATG</td></tr><tr><td>CYP4L9</td><td>TCGGTGATGACATGGAAAGA</td><td>AGAACGACAGACGTGCCTTTT</td></tr><tr><td>CYP4L13</td><td>ACGAACGTGAGTCTGCCTATGTGA</td><td>ACGACGTCCGGACCAAAAATC</td></tr><tr><td>CYP4M14</td><td>TGATCTCGGACTTGCACTTG</td><td>GTCCAGCGCTGAAAGGAATA</td></tr><tr><td>CYP4M15</td><td>TGATCTTGGACGTGCATTAT</td><td>GCCCAGCACTGAAGGAATG</td></tr><tr><td>CYP4M17</td><td>AGAGTCGCTGCGCATATACC</td><td>GGGTTCGGGAATAAATCCTC</td></tr><tr><td>CYP4M18</td><td>TCCTACCCGAGAACAGCATC</td><td>ACTTCTGCCACAGCCATCTT</td></tr><tr><td>CYP321A9</td><td>GCGTGGTGTAGCCTTCTACG</td><td>CGGGTCAATGACAAACAGTG</td></tr><tr><td>CYP321A7</td><td>TCCAGACCCAGAAGTTTTCG</td><td>CGGCCTGGACTTGTAATTTG</td></tr><tr><td>CYP333B2</td><td>GAATTATGCCGGTGGTGCT</td><td>TAGCGACATGTCTCGGTGAG</td></tr><tr><td>CYP337B1</td><td>CCGTTTGGTGAAGGAAACA</td><td>GACCGAAGGGACTTCTTTCA</td></tr><tr><td>G6PD</td><td>GGCCCTGTGGCTAACAGAAT</td><td>CATCGTCTCTACCAAAAGGCTTC</td></tr><tr><td>L18</td><td>CGTATCAACCGACCTCCACT</td><td>AGGCACCTTGTAGAGCCTCA</td></tr><tr><td>Rpl4</td><td>CAACAAGAGGGGTTCACGAT</td><td>GCACGATCAGTTCGGGTATC</td></tr></tbody></table>
<table><caption>**Table 2:** induction doses used in *S. frugiperda* larvae *in vivo* exposition for each of the tested compounds. References found in the literature are stated when possible for plant allelochemicals. Doses for insecticide compounds were chosen from 24h toxicity testing with the higher concentrations causing no larvae mortality (<DL05).</caption><thead><tr><td>Compounds</td><td>doses</td><td>references</td></tr></thead><tbody><tr><td>xanthotoxin</td><td>0.05 %</td><td>(Zeng et al., 2007)</td></tr><tr><td>Indole</td><td>0.25 %</td><td>Chosen similar to other allelochemicals</td></tr><tr><td>Indole 3-carbinol</td><td>0.25 %</td><td>(Yu, 1986)</td></tr><tr><td>Quercetin</td><td>0.25 %</td><td>(Yu, 1983)</td></tr><tr><td>2-tridecanone</td><td>0.25 %</td><td>(Riskallah et al., 1986)</td></tr><tr><td>Clofibrate</td><td>0.25 %</td><td>(Stevens et al., 2000)</td></tr><tr><td>Phenobarbital</td><td>0.50 %</td><td>(Snyder et al., 1995)</td></tr><tr><td>Methoxyfenozide</td><td>20 nM</td><td>&lt;DL05 from toxicity testing</td></tr><tr><td>Methoprene</td><td>2 µM</td><td>&lt;DL05 from toxicity testing</td></tr><tr><td>Deltamethrin</td><td>10 nM</td><td>&lt;DL05 from toxicity testing</td></tr><tr><td>Fipronil</td><td>2 µM</td><td>&lt;DL05 from toxicity testing</td></tr></tbody></table>
<table><caption>**Table 3:** induction doses used in Sf9 cells exposition studies for each of the tested compounds. Doses were chosen from 24h MTT cytotoxicity testing at DL10, unless no toxicity was recorded in which case the higher dose that remains soluble in culture medium was chosen.</caption><thead><tr><td>Compounds</td><td>DL10</td><td>Dose chosen</td></tr></thead><tbody><tr><td>Xanthotoxin</td><td>&gt;100 µM</td><td>100 µM (limit of solubility)</td></tr><tr><td>Indole</td><td>250 µM</td><td>250 µM</td></tr><tr><td>Indole 3-carbinol</td><td>250 µM</td><td>250 µM</td></tr><tr><td>Quercetin</td><td>50 µM</td><td>50 µM</td></tr><tr><td>2-tridecanone</td><td>&gt; 500µM</td><td>500 µM (limit of solubility)</td></tr><tr><td>Clofibrate</td><td>100 µM</td><td>100 µM</td></tr><tr><td>Phenobarbital</td><td>250 µM</td><td>250 µM</td></tr><tr><td>Methoxyfenozide</td><td>0.1 µM</td><td>0.1 µM</td></tr><tr><td>Methoprene</td><td>25 µM</td><td>25 µM</td></tr><tr><td>Deltamethrin</td><td>&gt;100µM</td><td>100 µM (limit of solubility)</td></tr><tr><td>Fipronil</td><td>1 µM</td><td>1 µM</td></tr></tbody></table>
<table><caption>**Table 4:** microarray analysis of genes up- and down- regulated in both *S. frugiperda* larvae and Sf9 cells after exposure for 24h to different xenobiotics. Larvae and Sf9 cells were exposed for 24h to sublethal and >DL10 doses of xenobiotic respectively. Only genes found significantly up- or down-regulated in both systems as depicted in figure 1 are shown.</caption><thead><tr><th rowspan="2">compound</th><th rowspan="2">Probe number</th><th rowspan="2">Function</th><th colspan="2">Midgut</th><th colspan="2">Sf9</th></tr><tr><th>Log FC</th><th>p value</th><th>Log FC</th><th>p value</th></tr></thead><tbody><tr><td>Fipronil</td><td>34868</td><td>Pyruvate deshydrogenase</td><td>0.98</td><td>3.6E-2</td><td>-0.81</td><td>3.1E-2</td></tr><tr><td rowspan="3">Xanthotoxin</td><td>34868</td><td>Pyruvate deshydrogenase</td><td>1.75</td><td>6.9E-3</td><td>0.62</td><td>1.3E-2</td></tr><tr><td>40592</td><td>Hypothetical transcript</td><td>1.84</td><td>4.0E-4</td><td>1.24</td><td>9.1E-8</td></tr><tr><td>40600</td><td>Carboxyl cholinesterase</td><td>1.66</td><td>8.0E-4</td><td>1.09</td><td>3.7E-7</td></tr><tr><td rowspan="4">Indole</td><td>27227</td><td>Mitochondrial ribosomal protein</td><td>2.85</td><td>3.8E-9</td><td>1.72</td><td>4.2E-7</td></tr><tr><td>33976</td><td>Oxidase peroxidase</td><td>0.71</td><td>2.9E-2</td><td>2.12</td><td>3.3E-2</td></tr><tr><td>38033</td><td>Ribosomal protein</td><td>-0.81</td><td>5.0E-4</td><td>0.88</td><td>3.3E-2</td></tr><tr><td>40600</td><td>Carboxyl cholinesterase</td><td>0.97</td><td>4.0E-4</td><td>2.05</td><td>4.2E-8</td></tr><tr><td>2-tridecanone</td><td>40999</td><td>Myosin regulatory light chain</td><td>-4.45</td><td>1.0E-2</td><td>2.93</td><td>2.8E-2</td></tr><tr><td rowspan="3">Clofibrate</td><td>39073</td><td>Small nuclear ribonucleoprotein</td><td>-0.82</td><td>5.4E-3</td><td>1.16</td><td>1.0E-4</td></tr><tr><td>39351</td><td>ATP synthase b</td><td>-0.71</td><td>3.4E-2</td><td>0.80</td><td>6.5E-5</td></tr><tr><td>40139</td><td>Hypothetical transcript</td><td>-0.63</td><td>4.3E-2</td><td>0.97</td><td>4.1E-2</td></tr><tr><td rowspan="6">Indole 3-carbinol</td><td>27227</td><td>Mitochondrial ribosomal protein</td><td>2.70</td><td>5.0E-4</td><td>1.94</td><td>4.8E-10</td></tr><tr><td>36136</td><td>Hypothetical protein</td><td>-1.33</td><td>4.6E-3</td><td>-0.80</td><td>4.0E-2</td></tr><tr><td>36395</td><td>Kinesin-like protein</td><td>-1.04</td><td>5.0E-3</td><td>0.87</td><td>7.1E-3</td></tr><tr><td>40592</td><td>Hypothetical transcript</td><td>1.79</td><td>1.7E-6</td><td>2.41</td><td>2.6E-3</td></tr><tr><td>40600</td><td>Carboxyl cholinesterase</td><td>1.65</td><td>4.5E-5</td><td>2.10</td><td>2.8E-8</td></tr><tr><td>40870</td><td>Hsp 70 hsp 90 organizing protein</td><td>3.30</td><td>4.9E-2</td><td>2.93</td><td>1.9E-2</td></tr><tr><td rowspan="2">Methoprene</td><td>40592</td><td>Hypothetical transcript</td><td>-0.77</td><td>7.0E-3</td><td>2.04</td><td>1.5E-8</td></tr><tr><td>40600</td><td>Carboxyl cholinesterase</td><td>-0.71</td><td>2.0E-4</td><td>1.97</td><td>3.7E-7</td></tr><tr><td rowspan="2">Phenobarbital</td><td>27227</td><td>Mitochondrial ribosomal protein</td><td>1.34</td><td>3.7E-8</td><td>0.82</td><td>8.8E-7</td></tr><tr><td>40600</td><td>Carboxyl cholinesterase</td><td>1.25</td><td>3.7E-3</td><td>1.15</td><td>5.2E-7</td></tr><tr><td rowspan="2">Methoxyfenozide</td><td>25793</td><td>Vitellogenin</td><td>0.88</td><td>1.6E-2</td><td>0.77</td><td>1.4E-2</td></tr><tr><td>40151</td><td>Gelsolin</td><td>0.72</td><td>1.9E-5</td><td>0.67</td><td>4.0E-2</td></tr><tr><td>quercetin</td><td>25385</td><td>Peptidyl prolyl isomerase</td><td>0.88</td><td>2.0E-2</td><td>0.68</td><td>2.7E-2</td></tr></tbody></table>
<table><caption>**Table 5:** microarray analysis of selected detoxification genes up- and down-regulated in *S. frugiperda* midgut after exposure for 24h to different xenobiotics.</caption><thead><tr><th>compound</th><th>Gene description</th><th>Probe number</th><th>Log FC</th><th>p value</th></tr></thead><tbody><tr><td rowspan="5">xanthotoxin</td><td>glutathione S-transferase 9</td><td>adhoc-41019</td><td>-1.03</td><td>5.84E-03</td></tr><tr><td>CYP9A25</td><td>adhoc-38527</td><td>-0.80</td><td>3.02E-03</td></tr><tr><td>cytochrome P450 monooxygenase CYP321A1</td><td>adhoc-34609</td><td>-0.67</td><td>4.86E-03</td></tr><tr><td>carboxylesterase</td><td>adhoc-40600</td><td>1.66</td><td>8.41E-04</td></tr><tr><td>glutathione S-transferase 14</td><td>adhoc-27466</td><td>1.53</td><td>1.90E-04</td></tr><tr><td rowspan="10">indole</td><td>glutathione S-transferase 9</td><td>adhoc-41019</td><td>-1.58</td><td>1.57E-04</td></tr><tr><td>glutathione S-transferase 7</td><td>adhoc-39161</td><td>-0.68</td><td>1.29E-02</td></tr><tr><td>CYP6B40</td><td>adhoc-40444</td><td>-0.67</td><td>1.47E-04</td></tr><tr><td>catalase</td><td>adhoc-40325</td><td>1.36</td><td>1.20E-03</td></tr><tr><td>glutathione S-transferase</td><td>adhoc-34379</td><td>1.31</td><td>4.40E-03</td></tr><tr><td>glutathione S-transferase 14</td><td>adhoc-27466</td><td>1.22</td><td>5.45E-07</td></tr><tr><td>carboxylesterase</td><td>adhoc-40600</td><td>0.97</td><td>4.02E-04</td></tr><tr><td>epoxide hydrolase</td><td>adhoc-36011</td><td>0.74</td><td>4.41E-02</td></tr><tr><td>peroxidase</td><td>adhoc-33976</td><td>0.71</td><td>2.89E-02</td></tr><tr><td rowspan="4">Indole 3-carbinol</td><td>S-formylglutathione hydrolase</td><td>adhoc-35696</td><td>-2.43</td><td>3.01E-02</td></tr><tr><td>glutathione transferase zeta</td><td>adhoc-24957</td><td>-2.27</td><td>2.31E-04</td></tr><tr><td>glutathione S-transferase S4</td><td>adhoc-40718</td><td>-0.68</td><td>3.70E-02</td></tr><tr><td>carboxylesterase</td><td>adhoc-40600</td><td>1.65</td><td>4.50E-05</td></tr><tr><td>2-tridecanone</td><td>glutathione S-transferase 14</td><td>adhoc-39621</td><td>-0.60</td><td>3.96E-02</td></tr><tr><td rowspan="10">quercetin</td><td>carboxylesterase-6</td><td>adhoc-40352</td><td>-1.09</td><td>1.30E-02</td></tr><tr><td>glutathione S-transferase 14</td><td>adhoc-39621</td><td>-0.88</td><td>4.09E-04</td></tr><tr><td>phenol UDP-glucosyltransferase</td><td>adhoc-35493</td><td>-0.86</td><td>5.32E-03</td></tr><tr><td>carboxylesterase-6</td><td>adhoc-40797</td><td>-0.83</td><td>2.42E-02</td></tr><tr><td>P450 9A9 [Spodoptera exigua], CYP9A19 [Bombyx mori]</td><td>adhoc-27215</td><td>-0.81</td><td>2.27E-02</td></tr><tr><td>Multidrug-Resistance like Protein 1 CG6214-PE, isoform E</td><td>adhoc-34231</td><td>-0.79</td><td>5.62E-03</td></tr><tr><td>cytochrome p450 6B6 [Helicoverpa armigera]</td><td>adhoc-26019</td><td>-0.75</td><td>2.32E-03</td></tr><tr><td>phenol UDP-glucosyltransferase</td><td>adhoc-41144</td><td>-0.66</td><td>1.60E-03</td></tr><tr><td>CYP4M18</td><td>adhoc-24805</td><td>1.32</td><td>1.24E-02</td></tr><tr><td rowspan="2">deltamethrin</td><td>Reactive mitochondrial oxygen species modulator 1</td><td>adhoc-26149</td><td>0.76</td><td>2.90E-02</td></tr><tr><td>multi drug resistance-associated protein (MRP)</td><td>adhoc-34231</td><td>0.62</td><td>3.77E-02</td></tr><tr><td rowspan="2">fipronil</td><td>phenol UDP-glucosyltransferase</td><td>adhoc-41144</td><td>-0.74</td><td>4.45E-02</td></tr><tr><td>phenol UDP-glucosyltransferase</td><td>adhoc-36536</td><td>2.20</td><td>3.66E-02</td></tr><tr><td rowspan="6">methoxyfenozide</td><td>cytochrome c oxidase, subunit VB</td><td>adhoc-36011</td><td>2.10</td><td>2.22E-02</td></tr><tr><td>glutathione transferase zeta</td><td>adhoc-44426</td><td>1.79</td><td>6.63E-03</td></tr><tr><td>Mn superoxide dismutase</td><td>adhoc-40571</td><td>1.38</td><td>2.55E-02</td></tr><tr><td>carboxylesterase-6</td><td>adhoc-36431</td><td>0.98</td><td>4.99E-02</td></tr><tr><td>glutathione S-transferase 9</td><td>adhoc-40352</td><td>0.73</td><td>1.09E-02</td></tr><tr><td>CYP6B40</td><td>adhoc-41019</td><td>0.59</td><td>1.45E-02</td></tr><tr><td>methoprene</td><td>carboxylesterase</td><td>adhoc-40600</td><td>-0.72</td><td>2.54E-04</td></tr><tr><td rowspan="3">phenobarbital</td><td>glutathione S-transferase 2</td><td>adhoc-26178</td><td>2.97</td><td>1.47E-02</td></tr><tr><td>carboxylesterase</td><td>adhoc-40600</td><td>1.25</td><td>3.71E-03</td></tr><tr><td>phenol UDP-glucosyltransferase</td><td>adhoc-41144</td><td>0.60</td><td>2.75E-02</td></tr><tr><td rowspan="3">clofibrate</td><td>oxidative stress protein</td><td>adhoc-38734</td><td>-2.63</td><td>2.87E-02</td></tr><tr><td>phenol UDP-glucosyltransferase</td><td>adhoc-38067</td><td>-0.63</td><td>1.05E-03</td></tr><tr><td>1-Cys peroxiredoxin</td><td>adhoc-38799</td><td>1.32</td><td>5.26E-03</td></tr></tbody></table>
<table><caption>**Table 6:** microarray analysis of selected detoxification genes up- and down-regulated in Sf9 cells midgut after exposure for 24h to different xenobiotics.</caption><thead><tr><th>compound</th><th>Gene description</th><th>adhoc</th><th>Log FC</th><th>p value</th></tr></thead><tbody><tr><td rowspan="4"><strong>xanthotoxin</strong></td><td>glutathione S-transferase</td><td>adhoc-24868</td><td>-4.22</td><td>2.88E-03</td></tr><tr><td>glutathione S-transferase</td><td>adhoc-26347</td><td>-0.77</td><td>3.99E-02</td></tr><tr><td>peroxidase</td><td>adhoc-33976</td><td>-0.73</td><td>1.32E-02</td></tr><tr><td>Carboxylesterase</td><td>adhoc-40600</td><td>1.09</td><td>3.78E-07</td></tr><tr><td rowspan="4"><strong>indole</strong></td><td>thioredoxin peroxidase</td><td>adhoc-26099</td><td>-0.79</td><td>3.51E-02</td></tr><tr><td>uridine diphosphate glucosyltransferase</td><td>adhoc-35495</td><td>-0.68</td><td>1.69E-02</td></tr><tr><td>peroxidase</td><td>adhoc-33976</td><td>2.12</td><td>3.40E-02</td></tr><tr><td>carboxylesterase</td><td>adhoc-40600</td><td>2.05</td><td>4.27E-08</td></tr><tr><td rowspan="2"><strong>Indole 3-carbinol</strong></td><td>catalase</td><td>adhoc-41112</td><td>-0.61</td><td>2.77E-02</td></tr><tr><td>carboxylesterase</td><td>adhoc-40600</td><td>2.10</td><td>2.84E-08</td></tr><tr><td rowspan="4"><strong>2-tridecanone</strong></td><td>carboxylesterase-6</td><td>adhoc-34509</td><td>-1.26</td><td>1.31E-02</td></tr><tr><td>phenol UDP-glucosyltransferase</td><td>adhoc-38443</td><td>-1.13</td><td>2.39E-03</td></tr><tr><td>glutathione transferase zeta</td><td>adhoc-24957</td><td>2.20</td><td>4.44E-02</td></tr><tr><td>CYP4M15</td><td>adhoc-24793</td><td>1.45</td><td>2.24E-02</td></tr><tr><td><strong>quercetin</strong></td><td>Carboxylesterase</td><td>adhoc-40600</td><td>-0.59</td><td>3.97E-02</td></tr><tr><td rowspan="5"><strong>deltamethrin</strong></td><td>similar to oxidative stress protein</td><td>adhoc-38734</td><td>4.05</td><td>7.84E-03</td></tr><tr><td>cytochrome P450</td><td>adhoc-35655</td><td>-0.94</td><td>3.14E-02</td></tr><tr><td>carboxylesterase</td><td>adhoc-40600</td><td>2.40</td><td>4.28E-10</td></tr><tr><td>CYP4M18</td><td>adhoc-34767</td><td>0.84</td><td>3.96E-03</td></tr><tr><td>cytochrome P450 monooxygenase CYP321A1</td><td>adhoc-34609</td><td>0.72</td><td>3.33E-02</td></tr><tr><td rowspan="3"><strong>fipronil</strong></td><td>gst1</td><td>adhoc-26914</td><td>-0.95</td><td>2.15E-04</td></tr><tr><td>carboxylesterase</td><td>adhoc-34965</td><td>-0.61</td><td>4.81E-02</td></tr><tr><td>carboxylesterase</td><td>adhoc-40600</td><td>0.81</td><td>8.79E-03</td></tr><tr><td><strong>methoxyfenozide</strong></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2"><strong>methoprene</strong></td><td>uridine diphosphate glucosyltransferase</td><td>adhoc-35495</td><td>-0.87</td><td>4.22E-02</td></tr><tr><td>carboxylesterase</td><td>adhoc-40600</td><td>1.97</td><td>3.75E-07</td></tr><tr><td rowspan="2"><strong>phenobarbital</strong></td><td>sulfotransferase</td><td>adhoc-25711</td><td>-1.71</td><td>4.91E-02</td></tr><tr><td>similar to copper-zinc superoxide dismutase</td><td>adhoc-38960</td><td>1.99</td><td>3.78E-02</td></tr><tr><td></td><td>Carboxylesterase</td><td>adhoc-40600</td><td>1.15</td><td>5.22E-05</td></tr><tr><td rowspan="4"><strong>clofibrate</strong></td><td>phenol UDP-glucosyltransferase</td><td>adhoc-38443</td><td>-0.99</td><td>2.63E-05</td></tr><tr><td>CYP6B39</td><td>adhoc-27179</td><td>-0.71</td><td>1.85E-05</td></tr><tr><td>carboxylesterase</td><td>adhoc-40600</td><td>2.00</td><td>4.16E-05</td></tr><tr><td>1-Cys peroxiredoxin</td><td>adhoc-26528</td><td>0.95</td><td>1.96E-03</td></tr></tbody></table>
<table><caption>**Table 7:** real-time quantitative PCR analysis of the differential expression of 19 P450 genes in *S. frugiperda* larvae exposed for 24h to sub-lethal doses of 11 different xenobiotic compounds. Gene expression values are indicated as fold expression in larvae exposed to each xenobiotic comparatively to unexposed larvae (controls). The three reference genes Rpl4, G6PD and L18 were used as internal controls for normalization. Gene expression values in bold are significantly different from the corresponding control as measured by pair-wise t-student test (p<0.05).</caption><thead><tr><th rowspan="2"></th><th colspan="11">indole 3-</th></tr><tr><th>xanthotoxin</th><th>2-tridecanone</th><th>carbinol</th><th>indole</th><th>quercetin</th><th>methoxyfenozide</th><th>methoprene</th><th>deltamethrin</th><th>fipronil</th><th>clofibrate</th><th>phenobarbital</th></tr></thead><tbody><tr><td>CYP4L9</td><td>0.32</td><td>0.79</td><td>0.44</td><td>0.44</td><td>0.46</td><td>0.53</td><td>1.12</td><td>0.86</td><td>1.11</td><td>1.02</td><td>1.25</td></tr><tr><td>CYP4L13</td><td>0.69</td><td>5.09</td><td>0.70</td><td>0.54</td><td>0.29</td><td>0.71</td><td>0.96</td><td>0.91</td><td>1.18</td><td>1.22</td><td>2.90</td></tr><tr><td>CYP4M14</td><td>0.14</td><td>0.56</td><td>0.31</td><td>0.46</td><td>0.47</td><td>0.58</td><td>0.80</td><td>0.60</td><td>0.61</td><td>0.76</td><td>0.71</td></tr><tr><td>CYP4M15</td><td>0.19</td><td>0.78</td><td>0.61</td><td>0.48</td><td>0.45</td><td>1.12</td><td>1.03</td><td>0.98</td><td>0.93</td><td>1.18</td><td>1.08</td></tr><tr><td>CYP4M17</td><td>0.17</td><td>1.01</td><td>0.33</td><td>0.36</td><td>0.41</td><td>0.86</td><td>1.10</td><td>0.86</td><td>0.78</td><td>0.75</td><td>1.25</td></tr><tr><td>CYP4M18</td><td>0.38</td><td>1.04</td><td>0.61</td><td>0.58</td><td>0.69</td><td>1.05</td><td>1.04</td><td>0.92</td><td>1.05</td><td>0.98</td><td>1.08</td></tr><tr><td>CYP6B40</td><td>2.89</td><td>0.72</td><td>2.50</td><td>3.26</td><td>0.42</td><td>1.75</td><td>0.90</td><td>1.14</td><td>1.42</td><td>0.63</td><td>3.26</td></tr><tr><td>CYP6Bp2</td><td>0.26</td><td>0.53</td><td>0.71</td><td>0.53</td><td>0.92</td><td>0.74</td><td>0.59</td><td>0.89</td><td>0.77</td><td>1.04</td><td>1.23</td></tr><tr><td>CYP9A24</td><td>0.99</td><td>0.63</td><td>0.53</td><td>0.53</td><td>0.42</td><td>0.92</td><td>1.07</td><td>1.36</td><td>1.48</td><td>1.00</td><td>0.94</td></tr><tr><td>CYP9A25</td><td>1.85</td><td>0.77</td><td>0.55</td><td>0.45</td><td>0.52</td><td>3.80</td><td>1.14</td><td>1.76</td><td>2.89</td><td>1.16</td><td>0.83</td></tr><tr><td>CYP9A26</td><td>1.41</td><td>0.70</td><td>0.46</td><td>1.43</td><td>0.23</td><td>1.45</td><td>1.29</td><td>0.91</td><td>1.89</td><td>0.56</td><td>0.95</td></tr><tr><td>CYP9A27</td><td>2.92</td><td>0.74</td><td>0.65</td><td>0.77</td><td>0.63</td><td>2.46</td><td>1.56</td><td>1.94</td><td>1.41</td><td>1.16</td><td>0.60</td></tr><tr><td>CYP9A30</td><td>1.53</td><td>0.75</td><td>1.45</td><td>2.10</td><td>0.35</td><td>1.18</td><td>1.11</td><td>1.20</td><td>0.91</td><td>0.86</td><td>1.03</td></tr><tr><td>CYP9A31</td><td>21.04</td><td>0.72</td><td>2.96</td><td>8.13</td><td>0.38</td><td>1.43</td><td>0.94</td><td>1.55</td><td>2.12</td><td>1.01</td><td>2.93</td></tr><tr><td>CYP9A32</td><td>28.10</td><td>0.73</td><td>1.15</td><td>3.28</td><td>0.48</td><td>3.02</td><td>1.70</td><td>3.05</td><td>2.34</td><td>1.42</td><td>0.67</td></tr><tr><td>CYP321A7</td><td>162.11</td><td>0.88</td><td>64.31</td><td>135.95</td><td>0.34</td><td>1.09</td><td>1.13</td><td>1.21</td><td>1.41</td><td>3.62</td><td>3.86</td></tr><tr><td>CYP321A9</td><td>20.97</td><td>0.92</td><td>11.70</td><td>28.59</td><td>0.61</td><td>1.58</td><td>1.49</td><td>1.52</td><td>1.12</td><td>1.22</td><td>3.42</td></tr><tr><td>CYP333B2</td><td>2.66</td><td>0.98</td><td>0.93</td><td>1.64</td><td>0.66</td><td>1.83</td><td>1.31</td><td>2.18</td><td>2.49</td><td>1.33</td><td>1.19</td></tr><tr><td>CYP337B1</td><td>1.23</td><td>1.12</td><td>0.61</td><td>0.74</td><td>1.01</td><td>1.33</td><td>1.08</td><td>1.27</td><td>1.65</td><td>1.04</td><td>1.28</td></tr></tbody></table>
<table><caption>**Table 8:** real-time quantitative PCR analysis of the differential expression of 9 P450 genes in Sf9 cells exposed for 24h to >DL10 doses of 11 different xenobiotic compounds. Gene expression values are indicated as fold expression in cells exposed to each xenobiotic comparatively to unexposed cells (controls). The three reference genes Rpl4, G6PD and L18 were used as internal controls for normalization. Gene expression values in bold are significantly different from the corresponding control as measured by pair-wise t-student test (p<0.05).</caption><thead><tr><th></th><th>xanthotoxin</th><th>2-tridecanone</th><th>indole 3-carbinol</th><th>indole</th><th>quercetin</th><th>methoxyfenozide</th><th>methoprene</th><th>deltamethrin</th><th>fipronil</th><th>clofibrate</th><th>phenobarbital</th></tr></thead><tbody><tr><td>CYP4M14</td><td>1.11</td><td><strong>2.76</strong></td><td>0.53</td><td>1.64</td><td>1.57</td><td>1.04</td><td><strong>3.18</strong></td><td>1.39</td><td>1.01</td><td>1.52</td><td>1.77</td></tr><tr><td>CYP4M15</td><td>0.54</td><td>1.10</td><td>0.28</td><td>0.93</td><td>0.73</td><td>1.22</td><td>2.02</td><td>0.88</td><td>0.64</td><td>0.67</td><td>1.14</td></tr><tr><td>CYP9A24</td><td>0.65</td><td><strong>4.17</strong></td><td>0.96</td><td>1.17</td><td>1.56</td><td><strong>3.06</strong></td><td>1.08</td><td>1.57</td><td>0.87</td><td><strong>5.11</strong></td><td>1.23</td></tr><tr><td>CYP9A26</td><td>1.17</td><td><strong>2.87</strong></td><td>0.76</td><td>2.22</td><td>1.18</td><td>1.32</td><td><strong>3.59</strong></td><td>2.05</td><td>1.32</td><td>2.51</td><td>2.34</td></tr><tr><td>CYP9A30</td><td>6.85</td><td><strong>9.25</strong></td><td>5.12</td><td>5.79</td><td>1.24</td><td>0.47</td><td><strong>8.21</strong></td><td><strong>14.05</strong></td><td>2.60</td><td><strong>22.39</strong></td><td>2.63</td></tr><tr><td>CYP9A31</td><td>2.05</td><td><strong>12.40</strong></td><td>6.46</td><td>6.46</td><td>0.71</td><td>0.86</td><td><strong>9.27</strong></td><td><strong>7.19</strong></td><td>1.77</td><td><strong>13.75</strong></td><td>3.77</td></tr><tr><td>CYP9A32</td><td>1.91</td><td><strong>7.56</strong></td><td>2.07</td><td>2.32</td><td>1.49</td><td>1.20</td><td><strong>3.70</strong></td><td><strong>4.31</strong></td><td>1.52</td><td><strong>6.23</strong></td><td>1.07</td></tr><tr><td>CYP321A9</td><td>1.42</td><td><strong>3.86</strong></td><td><strong>4.03</strong></td><td>1.90</td><td>0.85</td><td>0.92</td><td><strong>5.74</strong></td><td>3.08</td><td>1.00</td><td>1.92</td><td>1.00</td></tr><tr><td>CYP333B2</td><td>2.10</td><td>1.97</td><td><strong>2.71</strong></td><td><strong>5.11</strong></td><td><strong>3.04</strong></td><td>0.17</td><td><strong>4.36</strong></td><td><strong>5.39</strong></td><td><strong>3.91</strong></td><td><strong>4.17</strong></td><td>1.48</td></tr></tbody></table>
Figures legends

**Figure 1:** Venn diagrams representing the number of regulated genes in *S. frugiperda* midgut and Sf9 cells exposed to 11 different xenobiotic compounds for 24h measured by microarray analysis. The number of genes regulated by each of the xenobiotic compound tested is indicated in the upper dark grey circle for the midgut and in the lower light grey circle for Sf9 cells. Numbers in bold represented at the intersection of both circles are genes found regulated in both systems.

**Figure 2:** Correlation between numbers of genes found up- or down-regulated by microarray analysis in the midgut of *S. frugiperda* and Sf9 cells after exposure to 11 different xenobiotics for 24h.

**Figure 3:** Numbers of genes up- and down-regulated in *S. frugiperda* midgut (A) and Sf9 cells (B) after exposure to sub-lethal doses of 11 xenobiotic compounds for 24h measured by microarray analysis. The total number of genes presents on the DNA microarray (9773) is represented in yellow and the respective number of genes up- and down-regulated found after exposure to each of the compound are represented in red and yellow, respectively.
<figure><img src="image_36.png" /><figcaption>figure 1</figcaption></figure>
<figure><img src="image_37.png" /><figcaption>Figure 2</figcaption></figure>
A midgut

<figure><img src="image_38.png" /></figure>

B Sf9 cells

<figure><img src="image_39.png" /><figcaption>Figure 3</figcaption></figure>
CHAPTER 3

Manuscript: Effects of hormone agonists on the Sf9 cells, cellular proliferation and cell cycle arrest.
<figure><img src="image_40.png" /></figure>

We have identified which type of xenobiotic induces which P450 in *S. frugiperda* larvae and Sf9 cells. We will now focus our attention on two specific insecticide inducers: the 20-hydroxyecdysone (20E) agonist, methoxyfenozide and the Juvenile Hormone (JH) analog, methoprene.

20-E and JH are two major hormones that control insect development and metamorphosis. They have therefore been attractive target for the elaboration of effective insecticides that would disrupt insect development. Methoxyfenozide and methoprene belong to these hormone mimic insecticides and present the advantage of being potent to insect pests but less toxic to non-target organisms (Dhadialla et al., 1998).

The mode of action of these insecticides is regulated through nuclear receptors. 20-E agonists exert their activity by binding to the ecdysone receptor complex that comprises the two nuclear receptors EcR and USP. JH analog mode of action however remains to be elucidated as they are several receptor candidates for JH among which MET (methoprene tolerant) (Ashok et al., 1998) and USP (ultraspiracle) (Jones and Sharp, 1997).

We will present here the results of our study on the effects of methoprene and methoxyfenozide in Sf9 cells.
Effects of hormone agonists on the Sf9 cells, cellular proliferation and cell cycle arrest

Maeva Giraudo<sup>a</sup>, Jérôme Califano<sup>a,b</sup>, Nathalie Taquet<sup>a,c</sup>, Frédérique Hilliou<sup>a</sup>, Trang Tran<sup>a,d</sup>, René Feyereisen<sup>a</sup> and Gaëlle Le Goff<sup>a,e</sup>

<sup>a</sup> UMR 1301 IBSV, INRA-CNRS-Université de Nice Sophia Antipolis, 400 Route des Chappes, 06903 Sophia-Antipolis, France

<sup>b</sup> Département des affaires réglementaires (parfum, arômes, matières premières), Robertet SA 37 avenue Sidi-Brahim 06130 Grasse, France

<sup>c</sup> Institute of Developmental Biology and Cancer, Polarized growth in yeast, CNRS UMR6543 Université de Nice – Sophia Antipolis, Faculté des Sciences - Parc Valrose, 06108 Nice Cedex 2, France

<sup>d</sup> Lanaud Gestion-Pôle de Lanaud, 87220 Boisseuil, France

<sup>e</sup> Corresponding author: Gaëlle Le Goff, UMR 1301 IBSV, INRA-CNRS-Université de Nice Sophia-Antipolis, 400 Route des Chappes, 06903 Sophia-Antipolis, FRANCE; phone : 33 492386578, fax : 33 492386401, e-mail : legoff@sophia.inra.fr

Short title : hormone agonist insecticides on Sf9

In preparation for Journal of Insect Physiology
**Abstract:**

Methoxyfenozide and methoprene are two insecticides, which mimic the action of the main hormones involved in the control of insect growth and development, the 20-hydroxyecdysone and the juvenile hormone. We used the *Spodoptera frugiperda* cell line Sf9 to investigate their mode of action. Toxicological tests showed that methoxyfenozide was more potent than methoprene in cell viability tests and in the inhibition of cellular proliferation. Cell growth arrest occured in G1 phase after a methoxyfenozide treatment and in G2/M for methoprene. Microarray experiments and qPCR to follow the expression of receptors were performed to understand the molecular action of these hormone agonists. Twenty-six genes were differentially expressed after methoxyfenozide treatment and 55 genes after methoprene treatment but no gene was shared between the two treatments. Our results suggest two different signalling pathways in Sf9 cells.

**Keywords:** methoxyfenozide, methoprene, Sf9 cells, cell cycle arrest
# 1. Introduction

Growth and development are controlled by two major hormones, the steroid 20-hydroxyecdysone (20E) and the sesquiterpenoid juvenile hormone (JH) insects (Truman and Riddiford, 1999). The cross-talk between these two hormones regulates all stages between egg-larva-pupa to adult. High level of 20E is required to initiate all developmental transitions and JH determines the nature of the molt (Dubrovsky, 2005). JH is necessary for larval molting and growth but metamorphosis occurs in its absence (Riddiford, 1996). The signalling action of these hormones involves nuclear receptors. If the mode of action of 20E is well-known, it remains more enigmatic for JH due to the lack of knowledge of its receptor. 20E exerts its action through the binding to a nuclear receptor heterodimer consisting of an ecdysone receptor (EcR) and ultraspiracle (USP), which is the insect ortholog of retinoid-X-receptor from vertebrate (Yao et al., 1992). The complex will regulate expression of target genes by binding to promoting region. In Drosophila, it was shown that 20E linked to its receptor will activate early genes among which are transcription factor regulators Broad complex (BR-C), E74 and E75 (Karim and Thummel, 1992; Thummel, 2001). It is those transcription factors that in turn will regulate late genes that have a direct role in metamorphosis mechanism (like cell death, cellular proliferation, differentiation, cuticle production...). Several receptor candidates for JH exist among which MET (Methoprene tolerant) is a member of the bHLH-PAS transcription factor family (Ashok et al., 1998) and USP (Jones and Sharp, 1997). MET can bind JH at physiological concentration (Miura et al., 2005) whereas USP was shown to bind JH with low affinity, at least 100 times lower than expected for a nuclear receptor (Jones et al., 2001). However the situation is complex and it is difficult
to generalize finding about Met and USP from one insect group to another. Indeed, Met has a close paralog in Drosophila, germ cell expressed (gce) (Moore et al., 2000) and it is one of the explanation why Met-null mutants are fully viable (Wilson and Ashok, 1998). The Met/gce duplication is recent and the two paralogs are found in the Drosophila genus but is not found in mosquitoes (Baumann et al., 2010). In the other insects, Met has therefore only one ortholog and in *Tribolium castaneum* its depletion by RNAi causes premature pupal morphogenesis (Konopova and Jindra, 2007). A phylogenetic study of USP receptors shows that there are two types of receptor in arthropods, one having lost the ability to bind the ligand in *Bemisia tabaci* (Hemiptera) and *Tribolium castaneum* (Coleoptera) and another still able to bind the ligand in Diptera and Lepidoptera (Iwema et al., 2007). Moreover the molecular signalling mechanism downstream of JH binding to its putative receptor remains limited. Two transcription factors, Broad complex (BR-C) and Krüppel homolog 1 (Kr-h1) seem to play an important role (Konopova and Jindra, 2008; Minakuchi et al., 2008; Parthasarathy et al., 2008; Suzuki et al., 2008). Minakuchi et al. (2009) have proposed a model in the red flour beetle *Tribolium castaneum* where Kr-h1 works downstream of Met in the larval stage and downstream of Met but upstream BR-C in the pupa, allowing metamorphosis inhibition in one case or its initiation in the other (Minakuchi et al., 2009).

These hormonal receptors (EcR, USP and Met) are also the target for insecticides, which act by disrupting insect development. Agonist hormone insecticides are of growing interest because some are shown to have selective toxicity, they are potent against pest insects and less or non toxic for the beneficial insects, mammals, fish and birds (Dhadialla et al., 1998). Among 20E agonists are diacylhydrazines, a non-steroidal agonist family, having insecticide activity by binding to the EcR-USP receptors. This family of compounds provokes a premature molt that leads to the death of the insect; they are only acting on larvae. The
activity spectrum of these compounds varies within insect orders and it is directly correlated
to receptor affinity for the insecticide (Dhadialla et al., 1998; Smagghe et al., 2002). As an
example, methoxyfenozide which we are using in our study is more effective against
Lepidoptera (Carlson et al., 2001). The other insecticides that mimic hormone action are
juvenile agonists (JHA), initially designed to be metabolically stable JH analogs. Their precise
molecular target is less well-known due to the still controversial mode of action of JH. JHA
block insects at an intermediate stage during development making them unable to emerge
normal adults. They also disrupt reproduction in insects where JH is gonadotropic.
Methoprene was the first successfully used JHA (Henrick et al., 1973) and it is more effective
against dipteran insects compared to Lepidoptera.

Cell lines could be a useful tool to understand insecticide mode of action. Several
members of the biacylhydrazines were used on cell lines, showing an inhibition of cellular
proliferation. This is the case for Drosophila Kc cell treated by RH-5849 and tebufenozide
(Mikitani, 1996; Wing, 1988). Similar effects on arrest of cell growth have also been
observed with these compounds in a lepidopteran cell line IAL-PID2 from the imaginal wing
disks of *Plodia interpunctella* (Silhacek et al., 1990), as well as in the epithelial cell line from
*Chironomus tentans* (Quack et al., 1995). Further studies on IAL-PID2 with tebufenozide have
shown a G2/M arrest with an induction of mRNA transcripts for EcR and USP associated with
a decreased in the expression of cyclin B, one of the protein involved in the cell cycle control
(Auzoux-Bordenave et al., 2005). Effects on cell proliferation were also reported for JHA like
on IAL-PID2 (Oberlander et al., 2000) but the molecular mechanism leading to this arrest was
not clarified.
In our study we were interested in the effects of insecticides that mimic hormone
action on *Spodoptera frugiperda* Sf9 cell line. The toxicity of methoxyfenozide and
methoprene was evaluated. As expected, both insecticides inhibit cellular proliferation. Flow
cytometry analysis showed a distinct action between these compounds with a G2/M arrest
after methoprene treatment whereas methoxyfenozide induced a G1 arrest. To investigate
the differential molecular mode of action of these hormone agonists we have performed
microarray experiments and the expression of receptors was followed by qRT-PCR. Our
results suggest two different signalling pathways.

**2. Materials and Methods**

2.1. *Cell culture*

Sf9 cell line was derived from the pupal ovarian tissue of *Spodoptera frugiperda*. Cells were cultured at 27°C in monolayer with the insect-Xpress protein free medium (Lonza). For experiments cells were cultured in 6 well plates, sowed at 5.10⁵ cells/ml. Cell proliferation was estimated by scraping off cells from a well and a cell aliquot added volume/volume of 1mg/ml of methylene blue was counting in a Malassez haemocytometer under the microscope.

2.2. *Cell viability by MTT assay*

Sf9 cells were seeded in 96-well culture plates and treated for 24, 48 and 72 hours
with increased concentrations of methoprene and methoxyfenozide. Methoxyfenozide was
purchased from Cluzeau and MTT from Sigma. Methoprene was a gift from the former
Zoecon Corp., Palo Alto, California. Cells in culture were then loaded with MTT (5mg/ml) and
incubated at 27°C for 2 hours. Cell homogenates were used to measure absorbance at 570nm using a microplate reader (SpectraMax, Molecular Devices).

## 2.3. Cell cycle analysis

Cellular DNA content was determined by staining cells with propidium iodide and measuring fluorescence (FACSCalibur, Becton Dickinson). The Sf9 cells were incubated during 48 hours with Methoxyfenozide or Methoprene and resuspended and fixed in ice during 30 minutes with 70% ethanol/PBS (10 mM Na2HPO4, 138 mM NaCl, 2.7 mM KCl, pH 7.4). The fixed cells were incubated in a solution containing 50 mg/ml RNAse and 50 mg/ml propidium iodide for 20 min at 37°C. For each cell population, 10,000 cells were analysed by FACS and the percentage of cells in a specific phase of the cell cycle was determined with the propidium iodide DNA staining technique. Cells were classified in G0/G1, S and G2/M phases depending on the intensity of the fluorescence peaks.

## 2.4. RNA extraction

Total RNA was extracted from cells of a well of the 6 wells plate using Trizol Reagent (Invitrogen Life technologies). Extractions were performed on the three independent biological replicates.

## 2.5. Microarray experimental design

Our oligonucleotides were designed from 79148 ESTs sequences of eight different tissues of *Spodoptera frugiperda* (fat body, hemocytes, midgut...). Using the assembly analysis (programme CAP3), we obtained 10092 contigs and singleton from these ESTs. Our *Spodoptera frugiperda* microarray consists of 9773 60-mers oligonucleotides synthesised by
Sygma-Aldrich that were designed to match unique contigs or singleton and to suit our hybridization conditions (GC content average 46% and average Tm of 46°C). Each comparison consisted in six microarrays, three biological replicates hybridised with dye swap (fully balance dye swap design) and duplicate spots. cDNA were synthesized from 7µg of total RNA and labelled with the dyes Cy3-dCTP and Cy5-dCTP (Amersham) using the ChipShot direct labeling system Promega according to the manufacturer's instructions. The microarray were hybridised with cDNA prepared as described in (Le Goff et al., 2006)and scanned using GenePixPro scanner (Axon, version 3.01). Experimental data and associated microarray designs have been deposited in the NBCI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under serie X and platform record X using Mediante database for data transfer (Le Brigand and Barbry, 2007).

## 2.6. Data analysis

We used the Bioconductor suite of statistical packages (Gentleman et al., 2004): *limma* (Wettenhall and Smyth, 2004) for our data analysis. The expression intensity was obtained by subtracting the background intensity from the foreground intensity for each non-flag spot (all flagged spots were eliminated). The expression data were normalised by the use of the within-array normalization with the “loess method” and the between-array normalization using the “quantile method” (Yang and Thorne, 2003). The linear model for series of arrays and empirical Bayes method were then applied for assessing differential expression (Smyth, 2004). The false discovery rate of the p-value for multiple tests was controlled by using Benjamini-Hochberg method. Differentially expressed genes were
selected if the absolute value of log2-fold-change greater than 1 and adjusted p-value below 0.01 and if the average intensity is greater than two times of average background. In order to provide an overall measure of evidence of differential expression, we used the Fisher's method for combining adjusted p-values from independent tests of significance of duplicate spots (Hess and Iyer, 2007).

## 2.7. Quantitative real-time PCR

Total RNA (1µg) was reverse transcribed using the iScript cDNA Synthesis kit (Biorad). QPCR reactions were carried out on an Opticon monitor 2 (Biorad) using the qPCR Mastermix plus for SYBR Green I no ROX (Eurogentec). The PCR conditions were as follows: 50°C for 2 min, 95°C for 10min, followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. Each reaction was performed in triplicate and the mean of the three independent biological replicates was calculated. All results were normalized using mRNA level of three control genes (RpL4, L18 and G6PD) and relative expression values were calculated in R using the RqPCRBase package developed in our laboratory (Hilliou and Tran, manuscript in preparation).

# 3. Results

## 3.1. Toxicological effects of methoxyfenozide and methoprene on the Sf9 cells

Cell viability was determined by MTT test after an insecticide exposure of 24, 48 and 72 hours. Methoxyfenozide had already a marked effect at 100nM, the lowest concentration tested (Fig.1A). Surprisingly, increased insecticide concentrations did not sensibly modify the
cell viability. The IC₅₀ is below 100nM at 72 h and can not be calculated for the time 24 and 72h. Figure 1B shows the results for methoprene. Almost no effects were observed up to 25µM, whatever the time of treatment (90% of cells are viable). For a concentration range between 50 and 75µM, toxicity was low (after 72h, still 70% of viable cells). The calculated IC₅₀ at 48 and 72h was around 200µM and below 100µM respectively.

Thus the two hormone agonists induced cell death of the Sf9 cell line and methoxyfenozide was more potent than methoprene by a toxicological factor of about 1,000.

## 3.2. Cellular proliferation inhibition

The potential effect of these insecticides on the cellular proliferation was monitored. Cells were sowed at 5.10⁵ cells/ml in 6 well plates, then agonist hormone treatment was started 24h after sowing. Treated cells were counted daily during 3 days. Cells in the DMSO control grew to reach to 15 to 17.10⁵ cells/ml at 72h (Fig.2). Cell density at 24h and 72h in DMSO was significantly different, indicating that cells proliferated. A normal and significant growth of the cells treated by methoxyfenozide was observed at the lower concentration used 10nM, however all the other tested concentrations had an antiproliferative effect (Fig.2A). At 50µM of methoxyfenozide, the number of cells remained stable for 3 days. Methoprene had no effect at 1µM with a cell density correlated to that of the DMSO control. An inhibition of cell proliferation was observed at the concentration range between 50 and 100µM, with no significant difference between numbers of cells counted at 24h or 72h (Fig.2B).
Methoprene and methoxyfenozide therefore both caused an arrest of cell proliferation in a dose-dependent manner.

### 3.3. Distinct phases of arrest in the cell cycle

In order to evaluate in which phase of the cell cycle the cells are arrested after insecticide treatment, we turned to flow cytometry. First, the distribution of untreated cells in the different phases was evaluated (Fig. 3A). 1.7% of the cells were found in apoptosis, 26.19% in G1, 15.85% in S and 56.25% in G2/M. No significant difference was observed for cells treated only with the solvent (DMSO) used to solubilise the insecticides (Fig. 3B). The insecticide-treated cells were then analyzed. In cells treated with methoxyfenozide, a shift occurred between the percentage of cells in G2/M compared to the percentage of cells in G1 (Fig.3C). 42.42% of cells are in the G1 phase related to 26.19% of the control. In cells treated with methoprene, cells were arrested in the G2/M phase with 75.28% of cells in this stage (Fig.3D).

### 3.4. Molecular pathways involved in the cell response to hormone agonist treatments

Although the phenotypic effects were identical between Sf9 cells treated by methoprene or methoxyfenozide (arrest of the cell growth but cell morphology identical to control cells, data not shown), nevertheless the insecticides had different molecular effects with an arrest of the cell cycle in G1 for methoxyfenozide and G2/M for methoprene. The molecular genetic regulation leading to this arrest still needs to be elucidated. We compared the transcriptional effect mediated by the signalling transduction pathway of each hormone agonist by using a specifically designed oligonucleotide microarray. We chose the first concentration of insecticide having a significant effect on cell proliferation, i.e 100nM for
methoxyfenozide and 25µM for methoprene, in order to focus on physiological effects and avoid toxicological effects.

Genes were considered as differentially regulated by the insecticide if their expression ratio was >1.5 or <0.66 and P value <0.05. Sequences of these genes were analysed in Blast2Go to assign them Gene Ontology (GO) terms and then classified in biological process level 3. The sequences for which no homology was found by blastx were submitted to a blastn in Butterflybase and classified in "hypothetical protein" category if homology was found with the sequence of another lepidopteran transcript, while the sequences restricted to *Spodoptera frugiperda* were put in a "hypothetical transcripts" category. Table 1 represents these results. After methoprene treatment, 55 genes were differentially expressed with 39 overexpressed and 16 down-regulated. In the case of methoxyfenozide, 26 genes were differentially regulated with 14 over-expressed and 12 down-regulated. We first noted that there was no overlap between the genes regulated by methoprene and methoxyfenozide. The main category of genes up-regulated by methoprene is the hypothetical transcripts category (15 genes). The next categories are the genes involved in cellular metabolic process (9 genes) and transcription/translation (7 genes). These categories are also the most populated for cells treated by methoxyfenozide. Five upregulated genes by methoprene belonged to the response to stress category. One gene could be present in different biological process.

Looking in more detail to the list of differentially regulated gene may help to identify the potential function of genes involved in the molecular and cellular effects of methoxyfenozide (Table2). In the transport proteins, two vacuolar ATPases are regulated, subunit B that is upregulated and subunit C that is downregulated. Insect vacuolar proteins
consist in two functional parts, the peripheral catalytic V1 complex composed of eight
different subunits (from A to H) that hydrolyzes ATP and the integral membrane V0 complex
consisting of four different subunits (a,c,d,e) that transports protons across the membrane
(Beyenbach and Wieczorek, 2006). These two parts can disassemble and reassemble
depending on conditions which regulate V-ATPase activity (Sumner et al., 1995; Wieczorek
et al., 2000). Both subunits can bind actin filament (Holliday et al., 2000; Vitavska et al.,
2003), but subunit C is the only subunit that can be phosphorylated (Voss et al., 2007).
Another specificity of subunit C is its release in the cytosol upon dissociation of the two
complexes (Kane, 2000; Merzendorfer et al., 2000). Clearly, subunit C has its own properties
and can be a good candidate to mediate signalling pathways (Wieczorek et al., 2009). So it
may not be surprising to see opposite regulation of subunit B and C. Moreover promoter
studies have revealed different regulatory elements between the two subunits genes in
*Manduca sexta* (Wieczorek et al., 2000). Down regulation of V-ATPase in apical globelet cell
of *Manduca sexta* during moulting and starvation was also suggested (Graf et al., 1996;
Sumner et al., 1995). These data may indicate a possible involvement of hormone on the
regulation of V-ATPase expression. Calcium may play a role in the signalling because at least
two proteins whose function is calcium-dependent, cadherin and calreticulin are up-
regulated. Cadherin is an essential protein for cell adhesion and calreticulin is a quality-
control chaperone. Furthermore, it was shown on mammalian cell line that over-expression
of calreticulin correlates with increased cell adhesiveness and increased of cadherin
expression (Fadel et al., 2001). These proteins are also known in mammals to be regulated
by steroid hormones. Crossin et al. have shown that inhibition of rat astrocytes proliferation
is mediated through the glucocorticoid receptor pathway and they also observed a level of
mRNA for calreticulin increased (Crossin et al., 1997). Among other genes over-expressed
three are implicated in the translation/transcription process. PolyA binding protein 2 has a
function in translation initiation of polyadenylated mRNA. P27BBP/eIF6 known as eukaryotic
translation initiation factor 6 is a ribosomal anti-association factor (Gartmann et al., 2010).
Dead box RNA helicase catalyzes the ATP-dependent unwinding of double-stranded RNA. In
the spruce budworm (Choristoneura fumiferana) tebufenozide another 20E agonist was
shown to enhance expression of a dead box RNA helicase (Zhang et al., 2004). In human, it
has been suggested that this type of proteins could be involved in cell cycle control and the
regulation of apoptosis (Schroder, 2009). A chymotrypsin gene is up-regulated, in Tribolium
castaneum RNAi of several genes coding for chymotrypsin lead to severe molting defects
(Broehan et al., 2010). Certain genes in the list are known to be regulated by the 20E like
vitellogenin. An over-expression of this gene after treatment by an agonist of the hormone
is consistent. Vitellogenin transcription is activated by 20E in mosquito via direct binding of
the heterodimer receptor EcR-USP (Martin et al., 2001).

Genes differentially regulated by methoprene are listed in the table 3. The two main
category processes are transcription/translation and response to stress with 7 genes (5 up-
regulated and 2 down-regulated) and 8 genes (7 up-regulated and one down) respectively. In
translation several ribosomal proteins were over-expressed, these data can be related to
study on the human HeLa cells where authors have identified that translation-related genes
seem essential for G2/M progression (Mukherji et al., 2006), siRNA of ribosomal protein
reduced cell proliferation. In response to stress different enzyme types are overexpressed,
carboxylesterases already well known in insecticide resistance (Ffrench-Constant, 2007),
apolipoprotein D precursor, in Drosophila correlation exists between overexpression of
apolipoprotein D and increased stress resistance (Walker et al., 2006). In a general way, these enzymes are over-expressed which is understandable after an insecticide treatment, nevertheless uridine diphosphate glucosyltransferase and DNAJ-1 are down-regulated. Overexpression is found for genes implicated in spindle assembly. Microtubule associated protein RP/EB family 3 may be involved in microtubule polymerization and stabilization. Kinesins are class of motor protein and beta-tubulin cofactor E is involved in the formation of the tubulin dimer. All of three can regulate microtubule dynamics and stabilization, in mammals an increased in microtubule stabilization leads to a mitotic G2/M arrest (Gallagher, 2007) which is in agreement with our results. Other gene categories are overexpressed like S-adenosylmethionine decarboxylase, it is an enzyme implicated in the synthesis of polyamines (Larsson and Rasmuson-Lestander, 1997). Polyamines affect the cell cycle progression, alterations in their relative amount can cause an arrest in cell cycle (Ackermann et al., 2003). Among this gene list, we can also cited gene down-regulated such as 14-3-3 epsilon protein. This protein family is involved in a variety of molecular and cellular functions (Darling et al., 2005). However, in Drosophila loss of 14-3-3 epsilon protein induces growth repression (Nielsen et al., 2008).

### 3.5. Expression of hormone receptors

Microarrays were not sensitive enough to detect EcR or USP, and we did not have probes for Met. Indeed we were unable to find the Met sequence on Spodobase (http://www.spodobase.univ-montp2.fr/Spodobase). Therefore, we used RT-qPCR approaches to study the expression of EcR and USP. qPCR primers were designed and expression of EcR and USP was followed in Sf9 cells treated by methoxyfenozide or methoprene and this was compared to cells treated by DMSO. As our results shown in figure
4, the two insecticides significantly induced the expression of both receptors. EcR was induced by a 1.92 fold in methoxyfenozide treated cells when USP expression was increased by 1.82 fold in methoprene treated cells.

## 4. Discussion

Cell lines provide a useful tool to investigate the molecular mode of action of insecticides. The *Spodoptera frugiperda* Sf9 cell line is probably the most widely used for high level expression of recombinant protein (Atmann et al., 1999). However, *Spodoptera frugiperda* from which the Sf9 cell line is derived is a major crop pest, a polyphagous insect able to feed on more than 25 different host plants. We decided to use this cell line in order to determine the effects of two insecticides, which are agonists of major hormones controlling insect development, the 20E and JH.

Methoxyfenozide acts as mimics of 20E and by binding to the EcR/USP receptor complex. Its affinity for the receptor in Lepidoptera is 10000 times higher than for the insect moulting hormone. However the affinity varies within insect orders, more potent on the lepidopteran cell line *Plodia interpunctella* than on the Drosophila Kc cells (Dhadialla et al., 1998). We have shown that methoxyfenozide was able to induce the expression of EcR in Sf9 cells, which is consistent with the affinity of this compound for the receptor. Methoprene has a different spectrum of activity, being very effective against dipteran insects but less so against Lepidoptera (Staal, 1975). In our results, methoprene induced the expression of USP, suggesting that USP might be responding to JH as it was already suggested.
In a second step, we have shown that these two insecticides could inhibit cellular proliferation. Treatment by tebufenozide, another bisacylhydrazine, leads to the same effects in two other lepidoptera cell lines, IAL-PID2 from *Plodia interpunctella* (Auzoux-Bordenave et al., 2005) and Se4 from *Spodoptera exigua* (Decombel et al., 2005). Methoprene and another analog of JH, fenoxycarb, significantly inhibit cell proliferation of the IAL-PID2 cell line (Oberlander et al., 2000). In this same cell line Auzoux-Bordenave et al. reported that tebufenozide arrested the cell cycle in G2/M (Auzoux-Bordenave et al., 2005). However, most studies to date have focused on hormone action. 20E causes an arrest in G2 in Kc cell (Stevens et al., 1980) and in IAL-PID2 cells (Mottier et al., 2004) whereas arrest occurs in the G1 phase in mosquito C7-10 cells (Gerenday and Fallon, 2004). To understand in which phase of the cell cycle the cells are accumulated after our insecticide treatments, flow cytometry experiments were performed. Inhibition of cellular proliferation associated with methoxyfenozide treatment induces an arrest of the cell cycle in G1 phase. In contrast, methoprene blocks the cells in G2/M.

Then finally, we have investigated insecticide mode of action at the molecular level. Which pathway leads to this arrest? Is there any element in common between the two treatments? We used a custom microarray consisting of 9773 probes of *Spodoptera frugiperda*. Very few genes are differentially expressed following a methoxyfenozide or a methoprene treatment, 26 in one case and 55 in the other. The cell cycle consists of four distinct phases: G1 phase where cells grow and cyclin D is expressed, S phase during which DNA replication occurs and cyclins E and A predominate, then the G2 phase, with degradation of the cyclin E and accumulation of the cyclin B. This phase is followed by the mitosis which leads to the cell division (Fallon and Gerenday, 2010). The progression through the cell cycle is controlled by cyclins and cyclin dependent kinases (CDK). The cyclins form
complexes with CDK, CDK4 for cyclin D, CDK2 for cyclin E. Our array only has one probe for
cyclin A, but not for other cyclins. No change in cyclin A levels was detected although
Mottier et al. reported a significant decrease in the expression of cyclin A and B after a 20E
treatment of IAL-PID2 and the level of both cyclins remain very low between 12 and 36h
post-treatment (Mottier et al., 2004). On the other hand, some results are consistent with
an arrest of the cell cycle as we observed overexpression of different genes involved in the
spindle assembly after the methoprene treatment. Indeed a biosynthetic step occurs during
the G2 phase, mainly involving the production of microtubules, which are required during
the process of mitosis. Genes such as cadherin are overexpressed in our methoxyfenozide
experiments and these have been shown to be overexpressed in cells that had stopped to
proliferate (Crossin et al., 1997). Genes and pathways involved in the various stages of the
cell cycle progression were identified in a study on human HeLa cells (Mukherji et al., 2006).
Several concordant observations can be made between genes shown to be essential in that
study and genes differentially regulated in our study. For example, several ribosomal
proteins, kinesin, DNA-J have been shown to be essential for G2/M progression and are
differentially regulated after a methoprene treatment. Similarly, eIF, ATPase and dead box
RNA helicase are essential in G1 phase and are differentially regulated after the
methoxyfenozide treatment. Although an evolutionary conservation of core cell-cycle
regulatory transcripts among species would be expected, there are in fact only few genes
(16) conserved between human and yeast cell-cycle regulatory networks (Mukherji et al.,
2006)
References

Ackermann, J.M., Pegg, A.E., and McCloskey, D.E. (2003). Drugs affecting the cell cycle via actions on the polyamine metabolic pathway. Prog Cell Cycle Res 5, 461-468.

Ashok, M., Turner, C., and Wilson, T.G. (1998). Insect juvenile hormone resistance gene homology with the bHLH-PAS family of transcriptional regulators. Proc Natl Acad Sci U S A 95, 2761-2766.

Atmann, F., Staudacher, E., and Wilson, I.B. (1999). Insect cells as hosts for the expression of recombinant glycoproteins. Glycoconj J 2, 109-123.

Auzoux-Bordenave, S., Solvar, M., Queguiner, I., Bozzolan, F., Mottier, V., Siaussat, D., Porcheron, P., and Debernard, S. (2005). Comparative effects of a non-steroidal ecdysone agonist RH-5992 and 20-hydroxyecdysone in a lepidopteran cell line (IAL-PID2). Insect Biochem Mol Biol 35, 1033-1042.

Baumann, A., Fujiwara, Y., and Wilson, T.G. (2010). Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila. J Insect Physiol.

Beyenbach, K.W., and Wieczorek, H. (2006). The V-type H+ ATPase: molecular structure and function, physiological roles and regulation. J Exp Biol 209, 577-589.

Broehan, G., Arakane, Y., Beeman, R.W., Kramer, K.J., Muthukrishnan, S., and Merzendorfer, H. (2010). Chymotrypsin-like peptidases from Tribolium castaneum: a role in molting revealed by RNA interference. Insect Biochem Mol Biol 40, 274-283.

Carlson, G.R., Dhadialla, T.S., Hunter, R., Jansson, R.K., Jany, C.S., Lidert, Z., and Slawecki, R.A. (2001). The chemical and biological properties of methoxyfenozide, a new insecticidal ecdysteroid agonist. Pest Manag Sci 57, 115-119.

Crossin, K.L., Tai, M.H., Krushel, L.A., Mauro, V.P., and Edelman, G.M. (1997). Glucocorticoid receptor pathways are involved in the inhibition of astrocyte proliferation. Proc Natl Acad Sci U S A 94, 2687-2692.

Darling, D.L., Yingling, J., and Wynshaw-Boris, A. (2005). Role of 14-3-3 proteins in eukaryotic signaling and development. Curr Top Dev Biol 68, 281-315.

Decombel, L., Tirry, L., and Smagghe, G. (2005). Action of 24-epibrassinolide on a cell line of the beet armyworm, Spodoptera exigua. Arch Insect Biochem Physiol 58, 145-156.

Dhadialla, T.S., Carlson, G.R., and Le, D.P. (1998). New insecticides with ecdysteroidal and juvenile hormone activity. Annu Rev Entomol 43, 545-569.

Dubrovsky, E.B. (2005). Hormonal cross talk in insect development. Trends Endocrinol Metab 16, 6-11.
Fadel, M.P., Szewczenko-Pawlikowski, M., Leclerc, P., Dziak, E., Symonds, J.M., Blaschuk, O., Michalak, M., and Opas, M. (2001). Calreticulin affects beta-catenin-associated pathways. *J Biol Chem* 276, 27083-27089.

Fallon, A.M., and Gerenday, A. (2010). Ecdysone and the cell cycle: Investigations in a mosquito cell line. *J Insect Physiol*.

Ffrench-Constant, R.H. (2007). Which came first: insecticides or resistance? *Trends Genet* 23, 1-4.

Gallagher, B.M., Jr. (2007). Microtubule-stabilizing natural products as promising cancer therapeutics. *Curr Med Chem* 14, 2959-2967.

Gartmann, M., Blau, M., Armache, J.P., Mielke, T., Topf, M., and Beckmann, R. (2010). Mechanism of eIF6-mediated inhibition of ribosomal subunit joining. *J Biol Chem* 285, 14848-14851.

Gentleman, R.C., Carey, V.J., Bates, D.M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B. et al. (2004). Bioconductor: open software development for computational biology and bioinformatics. *Genome Biol* 5, R80.

Gerenday, A., and Fallon, A.M. (2004). Ecdysone-induced accumulation of mosquito cells in the G1 phase of the cell cycle. *J Insect Physiol* 50, 831-838.

Graf, R., Harvey, W.R., and Wieczorek, H. (1996). Purification and properties of a cytosolic V1-ATPase. *J Biol Chem* 271, 20908-20913.

Henrick, C.A., Staal, G.B., and Siddall, J.B. (1973). Alkyl 3,7,11-trimethyl-2,4-dodecadienoates, a new class of potent insect growth regulators with juvenile hormone activity. *J Agri Food Chem* 21, 354-359.

Hess, A., and Iyer, H. (2007). Fisher's combined p-value for detecting differentially expressed genes using Affymetrix expression arrays. *BMC Genomics* 8, 96.

Holliday, L.S., Lu, M., Lee, B.S., Nelson, R.D., Solivan, S., Zhang, L., and Gluck, S.L. (2000). The amino-terminal domain of the B subunit of vacuolar H+-ATPase contains a filamentous actin binding site. *J Biol Chem* 275, 32331-32337.

Iwema, T., Billas, I.M., Beck, Y., Bonneton, F., Nierengarten, H., Chaumot, A., Richards, G. et al (2007). Structural and functional characterization of a novel type of ligand-independent RXR-USP receptor. *Embo J* 26, 3770-3782.

Jones, G., and Sharp, P.A. (1997). Ultraspiracle: an invertebrate nuclear receptor for juvenile hormones. *Proc Natl Acad Sci U S A* 94, 13499-13503.

Jones, G., Wozniak, M., Chu, Y., Dhar, S., and Jones, D. (2001). Juvenile hormone III-dependent conformational changes of the nuclear receptor ultraspiracle. *Insect Biochem Mol Biol* 32, 33-49.
Kane, P.M. (2000). Regulation of V-ATPases by reversible disassembly. FEBS Lett 469, 137-141.

Karim, F.D., and Thummel, C.S. (1992). Temporal coordination of regulatory gene expression by the steroid hormone ecdysone. EMBO J 11, 4083-4093.

Konopova, B., and Jindra, M. (2007). Juvenile hormone resistance gene Methoprene-tolerant controls entry into metamorphosis in the beetle Tribolium castaneum. Proc Natl Acad Sci U S A 104, 10488-10493.

Konopova, B., and Jindra, M. (2008). Broad-Complex acts downstream of Met in juvenile hormone signaling to coordinate primitive holometabolan metamorphosis. Development 135, 559-568.

Larsson, J., and Rasmuson-Lestander, A. (1997). Cloning, mapping and mutational analysis of the S-adenosylmethionine decarboxylase gene in Drosophila melanogaster. Mol Gen Genet 256, 652-660.

Le Brigand, K., and Barbry, P. (2007). Mediante: a web-based microarray data manager. Bioinformatics 23, 1304-1306.

Le Goff, G., Hilliou, F., Siegfried, B.D., Boundy, S., Wajnberg, E., Sofer, L., Audant, P., ffrench-Constant, R.H., and Feyereisen, R. (2006). Xenobiotic response in Drosophila melanogaster: sex dependence of P450 and GST gene induction. Insect Biochem Mol Biol 36, 674-682.

Martin, D., Wang, S.F., and Raikhel, A.S. (2001). The vitellogenin gene of the mosquito Aedes aegypti is a direct target of ecdysteroid receptor. Mol Cell Endocrinol 173, 75-86.

Merzendorfer, H., Reineke, S., Zhao, X.F., Jacobmeier, B., Harvey, W.R., and Wieczorek, H. (2000). The multigene family of the tobacco hornworm V-ATPase: novel subunits a, C, D, H, and putative isoforms. Biochim Biophys Acta 1467, 369-379.

Mikitani, K. (1996). A new nonsteroidal chemical class of ligand for the ecdysteroid receptor 3, 5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide shows apparent insect molting hormone activities at molecular and cellular levels. Biochem Biophys Res Commun 227, 427-432.

Minakuchi, C., Namiki, T., and Shinoda, T. (2009). Kruppel homolog 1, an early juvenile hormone-response gene downstream of Methoprene-tolerant, mediates its anti-metamorphic action in the red flour beetle Tribolium castaneum. Dev Biol 325, 341-350.

Minakuchi, C., Zhou, X., and Riddiford, L.M. (2008). Kruppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster. Mech Dev 125, 91-105.

Miura, K., Oda, M., Makita, S., and Chinzei, Y. (2005). Characterization of the Drosophila Methoprene -tolerant gene product. Juvenile hormone binding and ligand-dependent gene regulation. FEBS J 272, 1169-1178.

Moore, A.W., Barbel, S., Jan, L.Y., and Jan, Y.N. (2000). A genomewide survey of basic helix-loop-helix factors in Drosophila. Proc Natl Acad Sci U S A 97, 10436-10441.
Mottier, V., Siaussat, D., Bozzolan, F., Auzoux-Bordenave, S., Porcheron, P., and Debernard, S. (2004). The 20-hydroxyecdysone-induced cellular arrest in G2 phase is preceded by an inhibition of cyclin expression. Insect Biochem Mol Biol 34, 51-60.

Mukherji, M., Bell, R., Supekova, L., Wang, Y., Orth, A.P., Batalov, S., Miraglia, L., Huesken, D., Lange, J., Martin, C., et al. (2006). Genome-wide functional analysis of human cell-cycle regulators. Proc Natl Acad Sci U S A 103, 14819-14824.

Nielsen, M.D., Luo, X., Biteau, B., Syverson, K., and Jasper, H. (2008). 14-3-3 Epsilon antagonizes FoxO to control growth, apoptosis and longevity in Drosophila. Aging Cell 7, 688-699.

Oberlander, H., Leach, C.E., and Shaaya, E. (2000). Juvenile hormone and juvenile hormone mimics inhibit proliferation in a lepidopteran imaginal disc cell line. J Insect Physiol 46, 259-265.

Parthasarathy, R., Tan, A., Bai, H., and Palli, S.R. (2008). Transcription factor broad suppresses precocious development of adult structures during larval-pupal metamorphosis in the red flour beetle, Tribolium castaneum. Mech Dev 125, 299-313.

Quack, S., Fretz, A., Spindler-Barth, M., and Spindler, K.D. (1995). Receptor affinities and biological responses of non-steroidal ecdysteroid agonists on the epithelial cell line from *Chironomus tentans* (Diptera: Chironomidae). Eur J Entomol 92, 341-347.

Riddiford, L.M. (1996). Juvenile hormone: the status of its "status quo" action. Arch Insect Biochem Physiol 32, 271-286.

Schroder, M. (2009). Human DEAD-box protein 3 has multiple functions in gene regulation and cell cycle control and is a prime target for viral manipulation. Biochem Pharmacol 79, 297-306.

Silhacek, D.L., Oberlander, H., and Porcheron, P. (1990). Action of RH 5849, a non-steroidal acdysteroid mimic, on Plodia interpunctella (Hubner) in vivo and in vitro. Arch Insect Biochem Physiol 15, 201-212.

Smagghe, G., Dhadialla, T.S., and Lezzi, M. (2002). Comparative toxicity and ecdysone receptor affinity of non-steroidal ecdysone agonists and 20-hydroxyecdysone in *Chironomus* *tentans*. Insect Biochem Mol Biol 32, 187-192.

Smyth, G.K. (2004). Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3, Article3.

Staal, G.B. (1975). Insect growth regulators with juvenile hormone activity. Annu Rev Entomol 20, 417-460.

Stevens, B., Alvarez, C.M., Bohman, R., and O'Connor, J.D. (1980). An ecdysteroid-induced alteration in the cell cycle of cultured Drosophila cells. Cell 22, 675-682.
Sumner, J.P., Dow, J.A., Earley, F.G., Klein, U., Jager, D., and Wieczorek, H. (1995). Regulation of plasma membrane V-ATPase activity by dissociation of peripheral subunits. *J Biol Chem* 270, 5649-5653.

Suzuki, Y., Truman, J.W., and Riddiford, L.M. (2008). The role of Broad in the development of Tribolium castaneum: implications for the evolution of the holometabolous insect pupa. *Development* 135, 569-577.

Thummel, C.S. (2001). Molecular mechanisms of developmental timing in C. elegans and Drosophila. *Dev Cell* 1, 453-465.

Truman, J.W., and Riddiford, L.M. (1999). The origins of insect metamorphosis. *Nature* 401, 447-452.

Vitavska, O., Wieczorek, H., and Merzendorfer, H. (2003). A novel role for subunit C in mediating binding of the H+-V-ATPase to the actin cytoskeleton. *J Biol Chem* 278, 18499-18505.

Voss, M., Vitavska, O., Walz, B., Wieczorek, H., and Baumann, O. (2007). Stimulus-induced phosphorylation of vacuolar H(+)-ATPase by protein kinase A. *J Biol Chem* 282, 33735-33742.

Walker, D.W., Muffat, J., Rundel, C., and Benzer, S. (2006). Overexpression of a Drosophila homolog of apolipoprotein D leads to increased stress resistance and extended lifespan. *Curr Biol* 16, 674-679.

Wettenhall, J.M., and Smyth, G.K. (2004). limmaGUI: a graphical user interface for linear modeling of microarray data. *Bioinformatics* 20, 3705-3706.

Wieczorek, H., Beyenbach, K.W., Huss, M., and Vitavska, O. (2009). Vacuolar-type proton pumps in insect epithelia. *J Exp Biol* 212, 1611-1619.

Wieczorek, H., Grber, G., Harvey, W.R., Huss, M., Merzendorfer, H., and Zeiske, W. (2000). Structure and regulation of insect plasma membrane H(+)-V-ATPase. *J Exp Biol* 203, 127-135.

Wilson, T.G., and Ashok, M. (1998). Insecticide resistance resulting from an absence of target-site gene product. *Proc Natl Acad Sci U S A* 95, 14040-14044.

Wing, K.D. (1988). RH 5849, a nonsteroidal ecdysone agonist: effects on a Drosophila cell line. *Science* 241, 467-469.

Yang, Y.H., and Thorne, N.P. (2003). Normalization for two-color cDNA microarray data. *In Science and Statistics: A Festschrift for Terry Speed, IMS Lecture Notes - Monograph Series*, D.R. Goldstein., ed., pp. 403-418.

Yao, T.P., Segraves, W.A., Oro, A.E., McKeown, M., and Evans, R.M. (1992). Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. *Cell* 71, 63-72.

Zhang, D.Y., Zheng, S.C., Zheng, Y.P., Ladd, T.R., Pang, A.S., Davey, K.G., Krell, P.J., Arif, B.M., Retnakaran, A., and Feng, Q.L. (2004). An ecdysone-inducible putative "DEAD box" RNA
helicase in the spruce budworm (Choristoneura fumiferana). Insect Biochem Mol Biol 34, 273-281.
<table><caption>Table 1. Classification of genes regulated in Sf9 cells after treatment by methoxyfenozide or methoprene according to GO terms, blast2Go annotation level 3</caption><thead><tr><th rowspan="2">Biological process</th><th colspan="2">methoxyfenozide</th><th colspan="2">methoprene</th></tr><tr><th>up-regulated</th><th>down-regulated</th><th>up-regulated</th><th>down-regulated</th></tr></thead><tbody><tr><td>biosynthetic process</td><td>2</td><td>1</td><td>4</td><td>3</td></tr><tr><td>cellular component organisation</td><td>3</td><td>0</td><td>1</td><td>3</td></tr><tr><td>cell cycle</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>cellular metabolic process</td><td>8</td><td>4</td><td>9</td><td>4</td></tr><tr><td>establishment of localization</td><td>2</td><td>1</td><td>3</td><td>1</td></tr><tr><td>multicellular organismal development</td><td>1</td><td>1</td><td>2</td><td>2</td></tr><tr><td>regulation of biological process</td><td>2</td><td>1</td><td>2</td><td>3</td></tr><tr><td>reproductive process</td><td>2</td><td>0</td><td>1</td><td>2</td></tr><tr><td>response to stress</td><td>0</td><td>2</td><td>5</td><td>2</td></tr><tr><td>transcription/translation</td><td>4</td><td>1</td><td>7</td><td>1</td></tr><tr><td>transport</td><td>2</td><td>1</td><td>2</td><td>1</td></tr><tr><td>hypothetical protein</td><td>0</td><td>2</td><td>4</td><td>0</td></tr><tr><td>hypothetical transcript</td><td>2</td><td>3</td><td>15</td><td>7</td></tr><tr><td>unknown function</td><td>0</td><td>1</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>Table 2. Microarray data for selected genes after Sf9 treatment by methoxyfenozide</caption><thead><tr><th>Gene description and putative function</th><th>adhoc</th><th>ratio</th><th>P value</th></tr></thead><tbody><tr><td>Actin cytoskeleton<br>gelsolin</td><td>adhoc-40151</td><td>1.59</td><td>0.0406</td></tr><tr><td>Carbohydrate metabolism<br>6-phosphogluconolactonase</td><td>adhoc-27223</td><td>2.39</td><td>0.0102</td></tr><tr><td>Catabolism process<br>3-hydroxyisobutyryl Coenzyme A hydrolase</td><td>adhoc-25654</td><td>0.64</td><td>0.0083</td></tr><tr><td>hydroxyphenylpyruvate dioxygenase</td><td>adhoc-39567</td><td>1.64</td><td>0.0486</td></tr><tr><td>Cell adhesion<br>cadherin</td><td>adhoc-26462</td><td>6.32</td><td>0.0098</td></tr><tr><td>Chaperone proteins<br>calreticulin</td><td>adhoc-40650</td><td>4.47</td><td>0.0168</td></tr><tr><td>Proteolysis<br>von Hippel-Lindau tumour suppressor protein</td><td>adhoc-24976</td><td>0.18</td><td>0.0014</td></tr><tr><td>chymotrypsin</td><td>adhoc-25557</td><td>2.9</td><td>0.0386</td></tr><tr><td>Reproductive protein<br>vitellogenin</td><td>adhoc-25793</td><td>1.69</td><td>0.0149</td></tr><tr><td>Response to stress/Detoxification<br>heat shock protein 90</td><td>adhoc-39008</td><td>0.57</td><td>0.0440</td></tr><tr><td>aldehyde oxidase</td><td>adhoc-34664</td><td>0.14</td><td>0.0436</td></tr><tr><td>Sugar synthesis<br>chondroitin sulfate synthase</td><td>adhoc-44313</td><td>3.55</td><td>0.0254</td></tr><tr><td>Translation/Transcription<br>polyadenylate binding protein 2</td><td>adhoc-36158</td><td>6.19</td><td>0.0102</td></tr><tr><td>p27BBP/eIF6</td><td>adhoc-33876</td><td>3.58</td><td>0.0252</td></tr><tr><td>dead box RNA helicase</td><td>adhoc-25789</td><td>3.11</td><td>0.0422</td></tr><tr><td>Transport<br>vacuolar ATPase subunit B</td><td>adhoc-34648</td><td>9.44</td><td>0.0059</td></tr><tr><td>vacuolar ATPase subunit C</td><td>adhoc-40849</td><td>0.64</td><td>0.0131</td></tr></tbody></table>
<table><caption>Table 3. Microarray data for selected genes after Sf9 treatment by methoprene</caption><thead><tr><th>Gene description and putative function</th><th>adhoc</th><th>ratio</th><th>P value</th></tr></thead><tbody><tr><td>Amino acids biosynthetic process<br>phosphoserine phosphatase</td><td>adhoc-25880</td><td>0.62</td><td>0.0022</td></tr><tr><td>Extracellular matrix protein<br>hemicetin like protein 1</td><td>adhoc-25674</td><td>1.7</td><td>0.0355</td></tr><tr><td>Immune protein<br>scolexin B like</td><td>adhoc-34835</td><td>1.77</td><td>0.0116</td></tr><tr><td>Mitotic cell cycle checkpoint<br>14-3-3 epsilon protein</td><td>adhoc-41120</td><td>0.62</td><td>0.0022</td></tr><tr><td>Phospholipid biosynthetic process<br>choline/ethanolamine kinase</td><td>adhoc-36601</td><td>0.54</td><td>0.0273</td></tr><tr><td>Polyamine synthesis<br>S-adenosylmethionine decarboxylase</td><td>adhoc-44296</td><td>1.71</td><td>0.0151</td></tr><tr><td>Regulation of Rab GTPase activity<br>Tbc1 domain family</td><td>adhoc-39160</td><td>0.57</td><td>0.0026</td></tr><tr><td>Response to stress/Detoxification<br>carboxylesterase</td><td>adhoc-40600</td><td>3.89</td><td>3.75E-07</td></tr><tr><td>prophenoloxidase activating factor</td><td>adhoc-26333</td><td>2.55</td><td>0.0086</td></tr><tr><td>aldehyde dehydrogenase 7 family member A1</td><td>adhoc-25971</td><td>1.89</td><td>0.0019</td></tr><tr><td>pheromone degrading enzyme 2</td><td>adhoc-34999</td><td>1.59</td><td>0.0172</td></tr><tr><td>apolipoprotein D precursor</td><td>adhoc-35472</td><td>1.55</td><td>0.0119</td></tr><tr><td>DNAJ-1</td><td>adhoc-38331</td><td>0.65</td><td>0.0456</td></tr><tr><td>uridine diphosphate glucosyltransferase</td><td>adhoc-35495</td><td>0.55</td><td>0.0422</td></tr><tr><td>Spindle assembly<br>microtubule associated protein RP/EB family 3</td><td>adhoc-44445</td><td>2.86</td><td>0.0441</td></tr><tr><td>kinesin like protein</td><td>adhoc-36395</td><td>2.75</td><td>0.0110</td></tr><tr><td>beta-tubulin cofactor E</td><td>adhoc-36883</td><td>2.07</td><td>0.0152</td></tr><tr><td>Structural constituent of cuticle<br>cuticle protein 1 like</td><td>adhoc-38620</td><td>0.61</td><td>0.0462</td></tr><tr><td>Translation/Transcription<br>mitochondrial ribosomal protein L49</td><td>adhoc-27227</td><td>4.62</td><td>3.83E-06</td></tr><tr><td>60S ribosomal protein L31</td><td>adhoc-25976</td><td>1.77</td><td>0.0019</td></tr><tr><td>coiled-ciol-helix-coiled-coil helix domain containing 8</td><td>adhoc-34522</td><td>1.67</td><td>7.20E-05</td></tr><tr><td>ribosomal protein L10</td><td>adhoc-38355</td><td>1.55</td><td>0.0139</td></tr><tr><td>spt3 associated factor 42</td><td>adhoc-41154</td><td>1.52</td><td>0.0044</td></tr><tr><td>mitochondrial translational release factor 1 like</td><td>adhoc-40890</td><td>0.43</td><td>0.0035</td></tr><tr><td>tRNA splicing endonuclease 2</td><td>adhoc-35120</td><td>1.9</td><td>1.29E-05</td></tr><tr><td>bip2 like</td><td>adhoc-41062</td><td>1.5</td><td>0.0050</td></tr><tr><td>Transport<br>phosphate transport protein</td><td>adhoc-38062</td><td>1.85</td><td>0.0017</td></tr><tr><td>translocase of inner mitochondrial membrane</td><td>adhoc-40155</td><td>0.61</td><td>0.0079</td></tr><tr><td>Vesicle trafficking<br>exocyst complex component 6</td><td>adhoc-36460</td><td>1.5</td><td>0.0340</td></tr></tbody></table>
Figure Legends

<caption>**Figure 1.** Methoxyfenozide and methoprene toxicity on Sf9 cells.</caption>

Cell viability is followed by MTT test at 24, 48 and 72h post-treatment of Sf9 cells by methoxyfenozide (A) or methoprene (B). Data calculated are a mean of three independent experiments.

<caption>**Figure 2.** Effects of methoxyfenozide and methoprene on Sf9 cellular proliferation.</caption>

Cells were numbered 24, 48 and 72h post-insecticide treatment, (A) methoxyfenozide, (B) methoprene. Data calculated are a mean of three independent experiments. A t-test was performed to determine result significance.

<caption>**Figure 3.** Distribution of Sf9 cells in the different phases of the cell cycle.</caption>

Cellular DNA content was determined by staining cells with propidium iodide and measuring fluorescence.

(A) Control, cells untreated.

(B) DMSO control, cells were treated during 48h by the solvent used for insecticide.

(C) Methoxyfenozide treatment, cells were treated during 48h at 10nM methoxyfenozide.

(D) Methoprene treatment, cells were treated during 48h at 10µM methoprene.

<caption>**Figure 4.** Gene expression levels of hormone nuclear receptors after insecticide treatment.
Expression levels are normalized with the three reference genes G6PD, Rpl4 and L18</caption>
A

<figure><img src="image_41.png" /></figure>

B

<figure><img src="image_42.png" /><figcaption>Figure 1</figcaption></figure>
A

<figure><img src="image_43.png" /></figure>

B

<figure><img src="image_44.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_45.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_46.png" /><figcaption>Figure 4</figcaption></figure>
CHAPTER 4

Manuscript: Molecular cloning and expression of nuclear receptors in *Spodoptera frugiperda* midgut ans Sf9 cells.
<figure><img src="image_47.png" /></figure>

In order to elucidate detoxification mechanisms involved in insects, we have identified patterns of gene induction in the model organism *Drosophila* and the polyphagous pest *Spodoptera frugiperda* in response to different types of xenobiotic compounds. By focusing gene expression studies on P450 induction, we have now a good overview of the “catalogue” of effector genes induced in response to xenobiotics in a polyphagous species.

As we have discussed in the previous chapters, we found that only a small subset of P450s were induced in *Drosophila* and *Spodoptera* in response to xenobiotics and that they are different subsets of inducers/induced genes suggesting multiple xenobiotic transduction mechanisms (see chapter 1 and 2 in results). We have therefore studied as a first step in the understanding of molecular mechanisms allowing signal transduction between inducers and effector genes, the response of two well characterised nuclear receptors in insect, EcR and USP, to respective agonists and analog of the two major hormones edcysone and juvenile hormone (chapter 3).

As a final step, we have chosen to identify potential xenosensors in *S. frugiperda* by a candidate gene approach focusing on the only known ortholog of CAR and PXR in *Drosophila*, DHR96. We have discussed earlier that CAR and PXR were two members of the nuclear hormone superfamily that play a central role in the detoxification of xenobiotics by notably inducing the expression of P450s (see section 4.6 of the introduction). Moreover, DHR96 was shown to be involved in xenobiotic response in *Drosophila* by increasing phenobarbital-
sedative effects and decreasing DDT survival in DHR96 mutants (King-Jones et al., 2006). We
therefore expect the same regulation mechanisms of HR96 in *Spodoptera*.

We will now present our results on the cloning of the nuclear receptors SfHR96 and USP in S. *frugiperda*, with the study of temporal and tissue specific expression of these receptors and discuss the role of SfHR96 in xenobiotic response of insects.
Molecular cloning and expression of nuclear receptors in *Spodoptera frugiperda* midgut and Sf9 cells.

Maeva Giraudoª, René Feyereisenª*, Frédérique Hilliouª, Trang Tranᵇ and Gaëlle Le Goffª.

*Corresponding author: René Feyereisen, UMR 1301 IBSV, INRA-CNRS-Université de Nice Sophia-Antipolis, 400 Route des Chappes, 06903 Sophia-Antipolis, FRANCE; phone : 33 492386450, fax : 33 492386401, e-mail : rfeyer@sophia.inra.fr

Short title: cloning of Spodoptera frugiperda HR96 and USP.

<footnote>ª UMR 1301 IBSV, INRA-CNRS-Université de Nice Sophia Antipolis, 400 Route des Chappes, 06903 Sophia-Antipolis, France</footnote>

<footnote>ᵇ Lanaud Gestion-Pôle de Lanaud, 87220 Boisseuil, France</footnote>
**Abstract**

Full-length sequence of HR96 and USP were cloned from *Spodoptera frugiperda* using a PCR-based approach employing degenerate primers designed on the basis of conserved regions of nuclear receptors, together with 5'- and 3'-RACE. The sequence of SfHR96 showed 70% and 66% identity in the DNA and ligand binding domain respectively, with its ortholog in Drosophila, DHR96. Cloning of USP revealed the presence of two isoforms, SfUSP-1 and SfUSP-2 in this species, that differ in their N-terminal region. SfHR96 was constitutively expressed in Sf9 cells and in larval midgut, fat body and Malpighian tubules throughout the last two instars and pupation. However, SfHR96 expression was lower than the other nuclear receptors EcR (Ecdysone receptor) and USP (ultraspiracle). EcR and SfUSP-2 showed peaks of expression before larval molts and during metamorphosis, whereas SfUSP-1 was mainly expressed in the pre-pupal stage. Exposure of cells and larvae to different xenobiotics did not induce the expression of SfHR96. EcR was significantly induced by the 20-hydroxyecdysone agonist, methoxyfenozide, and SfUSP showed an increase expression when exposed to the juvenile hormone analog, methoprene.

**Keywords: Spodoptera frugiperda, Sf9, nuclear receptors, HR96, USP.**
# 1. Introduction

The Nuclear receptor (NR) superfamily comprises transcription factors in metazoans in which they regulate functions as diverse as reproduction, differentiation, metabolism, metamorphosis and homeostasis (Escriva et al., 2000; Mangelsdorf et al., 1995; Robinson-Rechavi et al., 2001). These proteins are characterized by a common structure among family members containing two functional domains, the highly-conserved DNA Binding Domain (DBD) consisting of two C4 type zinc-fingers, and the less conserved Ligand Binding Domain (LBD), which contains a ligand binding pocket, dimerization domain and activation domain (Germain et al., 2006; Robinson-Rechavi and Laudet, 2003). The number of NRs identified in sequenced genomes varies considerably. To date 48 members have been identified in humans (Robinson-Rechavi et al., 2001), 21 in the genome of the fruit fly *Drosophila melanogaster* (Adams et al., 2000) and over 270 are found in *Caenorhabditis elegans* (Sluder and Maina, 2001). Recently sequenced insect genomes of *Anopheles gambiae* revealed 20 NRs (Holt et al., 2002), 22 in the honey bee *Apis mellifera* (Velarde et al., 2006), 21 in the silkworm *Bombyx mori* genome (Cheng et al., 2008), 20 in the yellow fever mosquito *Aedes aegypti* (Cruz et al., 2009) and 21 in the red flour beetle *Tribolium castaneum* (Bonneton et al., 2008; Tan and Palli, 2008). In vertebrates, NRs are able to bind endogenous compounds such as steroids and thyroid hormones and play therefore a key role in a variety of biological pathways from embryonic development to metabolic processes. Similar regulation mechanisms occur in insects, where 20-hydroxyecdysone (20E), the major moulting hormone, binds to an ecdysteroid receptor that regulates the expression of target genes involved in development and metamorphosis (for review, see Thummel, 2002 and Nakagawa and Henrich, 2009). The functional ecdysteroid receptor in insects is a heterodimer of EcR (Ecdysone receptor) and USP (ultraspiracle), two members of the NR family that are
structurally conserved among different species (Mangelsdorf et al., 1995). The identification of both of these receptors in many insect species has revealed the presence of three different isoforms for EcR, EcRA, EcRB1 and EcRB2 (Talbot et al., 1993) and in some cases the presence of two distinct isoforms for USP, USP-1 and USP-2, with USP-2 being 50 amino acids shorter than the other isoform.

The ability of nuclear receptors to bind small lipophilic ligands has triggered the study of NR functions in xenobiotic metabolism and the identification of NRs as drug targets. Detailed studies have defined a central role for two vertebrate NRs in xenobiotic metabolism, the human steroid and xenobiotic receptor (SXR, PXR in mice) and the constitutive androstane receptor (CAR) (Willson and Kliewer, 2002). PXR and CAR are able to bind a wide range of xenobiotics and regulate the expression of overlapping set of genes, including genes encoding Phase I detoxification enzymes such as cytochrome P450s (Maglich et al., 2002; Moore et al., 2006; Pascussi et al., 2005; Willson and Kliewer, 2002). In contrast to these studies in human and mice, relatively little data are available on the regulation of insect xenobiotic responses. Recently, a single ortholog of PXR and CAR has been identified in *Drosophila melanogaster*, DHR96, and has been shown to be involved in xenobiotic response of this insect by playing a role in the regulation of many phenobarbital-regulated genes, including some P450s (King-Jones et al., 2006). Moreover, further studies have reported that DHR96 could be activated by a CAR-selective agonist, suggesting that it may be regulated in a manner similar to that of the vertebrate xenobiotic receptors (Palanker et al., 2006). However, evidence of the ability of DHR96 to bind xenobiotics and transactivate the expression of detoxification enzymes such as P450s still remains to be clearly established.
It is now well known that insect P450s are involved in the detoxification of xenobiotics that insect encounter in their diet, such as plant allelochemicals and insecticides (for a review, see (Feyereisen, 2005). We have recently identified patterns of gene expression induced by different types of xenobiotics in the polyphagous lepidopteran pest, *Spodoptera frugiperda*, with a focus on cytochrome P450 induction (Giraudo et al., unpublished). As a first step in understanding how expression of these P450s is regulated, we have cloned HR96 in *S. frugiperda* (SfHR96) using a PCR-based cloning method employing degenerate primers based on the highly conserved domains of the insect nuclear receptors. In order to compare expression patterns of SfHR96 with other nuclear receptors, we have also cloned two isoforms of SfUSP, for which only a partial sequence was available in public databases. Here we report the results of this cloning and the patterns of expression of SfHR96 in comparison with EcR and SfUSP in *S. frugiperda* larval tissues and Sf9 cells.

## 2. Material and Methods

### 2.1 Reagents

The analytical grade plant allelochemicals xanthotoxin, quercetin, 2-tridecanone, indole, indole 3-carbinol and the herbicide clofibrate used in this study, as well as the drug phenobarbital were purchased from Sigma-Aldrich (St Quentin Fallavier, France). The insecticides deltamethrin, methoxyfenozide and fipronil were purchased from Cluzeau (CIL, France). Methoprene was a gift from the former Zoecon Corp., Palo Alto, California.

### 2.2 Insect rearing and treatments
Larvae of *Spodoptera frugiperda* were fed ad libitum on artificial medium (Poitout and Bues, 1974) and were reared at 25,5°C ±1°C, and 70% relative humidity under a L 14: D 10 photoperiod. Under these conditions, the 5th (penultimate) and 6th (ultimate) larval stages last about 2 to 3 days each, with additional two pre-pupal days characterized by the arrest of wandering and beginning of weight lost towards pupal formation.

For xenobiotic induction of nuclear receptor gene expression, newly molted 6th instar larvae were fed for 24h on artificial medium containing either 0.5% phenobarbital, 0.05% xanthotoxin, 0.25% 2-tridecanone, indole 3-carbinol, indole, clofibrate or quercetin, 20nM methoxyfenozide, 2µM methoprene, 10nM deltamethrin, 2µM fipronil or the corresponding concentration of DMSO.

## 2.3 cell culture and treatments

Sf9 cells derived from the pupal ovarian tissue of *Spodoptera frugiperda* were cultured and maintained in insect-Xpress serum free medium (Lonza) at 27°C in suspension spinner flasks with an agitation rate of 100 rpm and passaged routinely every third day. Cell density was determined by Malassez hemocytometer counts and cell viability was evaluated by methylene blue (1mg/mL, v/v) staining. Prior to experiments, cells were sowed in 6 well plates (TPP) at 5.10⁵ cells/ml and left at 27°C for adhesion. For induction of gene expression studies, attached cells were treated for 24h with 500µM 2-tridecanone, 250µM phenobarbital, indole and indole 3-carbinol, 100µM xanthotoxin, clofibrate, and deltamethrin, 50µM quercetin, 25µM methoprene, 1µM fipronil or 0.1µM methoxyfenozide or the same volume of DMSO.

## 2.4 RNA extraction
For tissue and stage specific gene expression studies, midguts, fat bodies and Malpighian tubules were dissected in Phosphate buffer (100mM potassium phosphate buffer, pH7.2, 1mM EDTA) from last day 5th instars larvae and at different time points during the last larval stage: three times a day for the two first days of the 6th instar, and then once per day for the 2 pre-pupal larval stages. Early (~2 days) and Late (~10 days) pupae were ground to powder in liquid nitrogen.

For xenobiotic induction of gene expression studies, midguts were dissected from larvae fed for 24h on artificial medium containing each of the tested compounds at the concentration stated earlier.

Total RNA was extracted from the different tissues using TRIzol Reagent (Invitrogen Life technologies) according to the manufacturer's protocol. RNAs were stored at -80°C until use.
RNA extractions were performed on 4 independent biological replicates for tissue and stage specific gene expression measurements and on 3 biological replicates of 5-pooled midguts for the induction of gene expression studies.

Total RNA was extracted from cells of a well of the 6 wells plate also using Trizol Reagent and RNA were analysed and stored the same way than for tissue samples. Extractions were performed on three independent biological replicates.

## 2.5 PCR amplification and sequencing

Degenerate primers were designed based on the sequences of DHR96 and putative HR96 sequences found in *Bombyx mori*, *Anopheles gambiae*, *Apis mellifera* and *Tribolium castaneum*. These sequences were first aligned in the web-based program Block-Maker (http://bioinformatics.weizmann.ac.il/blocks/blockmkr/www/make_blocks.html), which
finds conserved blocks in a group of two or more aligned protein sequences. The conserved
blocks obtained were further analyzed by CODEHOP
(http://bioinformatics.weizmann.ac.il/blocks/codehop.html), which designs degenerate
primers with a clamp region specific to the conserved blocks. SfHR96 degenerate primers
were found in the LBD region and were as follows: (5'-
GACCAAGTCGCTCTGCTGAARGGNGGNTG-3') for SfHR96-F1 forward primer and (5'-
CAGGTAGTAGTAGGAGTTTTGTTCCAGTYKDATNACRT-3') for SfHR96-R1 reverse primer. The
conditions for the initial PCR amplification were as follows: 1 cycle of 94°C for 1 min,
followed by 35 cycles of 94°C for 15 sec, 60°C for 30 sec and 68°C for 30 sec, using the
Accuprime Taq polymerase (invitrogen). The 320pb fragment obtained was purified using
the Minelute PCR purification kit (Qiagen) and subsequently cloned into pcR2.1 vector (TA
cloning kit Invitrogen) and sequenced.

## 2.6 Rapid Amplification of cDNA 5'- and 3'- ends (5'/3' RACE)

5' and 3' RACE were carried out to obtain the 5' and 3' ends of the cDNA for SfHR96 and SfUSP from *S. frugiperda* midgut using the Marathon cDNA amplification kit (Clontech). Briefly, first and second strand cDNA are synthesized from polyA+ mRNA isolated from *S. frugiperda* midgut with the Illustra Quickprep micromRNA purification kit (GE Healthcare). Specific adaptors are then ligated to the obtained ds cDNA generating a library of adaptor-ligated ds cDNA from which 5'- and 3'- end are amplified using the following gene specific primers SfHR96-F2 (5'-TGGACAGAGGAAGCAGTGGAAGATCC-3') and SfHR96-R2 (5'-ATTACATCGGGATGCACGACCTTGG-3') for SFHR96 designed from the 320bp fragment obtained during the initial PCR amplification (figure 1) and SfUSP-F1 (5'-ATGTCTCGCGTGCGGGATGAAGAGG-3') and SfUSP-R1 (5'-CCCACACCACCAACGCGGCTATCTG-
3') for SfUSP designed from the partial *Spodoptera frugiperda* USP sequence available in Genbank aligned with the complete sequence of USP in *S. littoralis*. Gene specific primers are used together with the adaptor specific primer AP1 supplied with the kit to obtain 5'- and 3'- RACE fragments. A second round of PCR amplification is then realized on the resulting 5'- and 3'- fragments using the following nested gene specific primers: SfHR96-F3 (5'-AAGCTGGCCAAGGGCAACATCTACC-3') and SfHR96-R3 (5'-GGTAGATGTTGCCCTTGGCCAGCTT-3') for SfHR96 and SfUSP-F2 (5'-AGGAGCGACAAAGGGCAGCCAGAGG-3') and SfUSP-R2 (5'-GGGTCAGCCACCAGCGACTCCATCT-3') for SfUSP. These nested gene specific primers are used together with adaptor specific primer AP2 supplied with the kit to generate 5' and 3'- end specific fragments. The 5'- and 3'- RACE products are then cloned in pCR2.1 vector (TA cloning kit, Invitrogen) and sequenced. Cloning of the full-length cDNA/ORF based on the 5'- and 3'-RACE sequenced were realised to confirm by sequencing that the sequence obtained is not a chimera.

## 2.6 Quantitative real-time PCR

Total RNA (1µg) was reverse transcribed using the iScript cDNA Synthesis kit (Biorad) and resulting cDNA were diluted 10 times for quantitative real time PCR reactions. qRT-PCR reactions were carried out on an Opticon monitor 2 (Biorad) using the qPCR Mastermix plus for SYBR Green I no ROX (Eurogentec). PCR primer pairs for *S. frugiperda* HR96 (SfHR96), USP-1, USP-2 and the common part of USP isoforms (SfUSP-1, SfUSP-2, SfUSPcom), EcR and the three control genes (*G6PD*, *L18* and *Rpl4*) were as follows: (5'-ACAAGGCGGAAAAGAGACGGAAAT-3'/5'-TGCAGCGGTATGAGCCCAGTAT-3') for SfHR96, (5'-TGACGGCACTTATCAACTGG-3'/5'-CCAGTGAACAGTCAACAGTCG-3') for SfUSP-1, (5'-GGAGCCCTCGAGAGATTCAG-3'/5'-GGGGGTAGTTCTTGAATGCAG-3') for SfUSP-2 and (5'-
ATAGCGAGGCTGGTCTGGTA-3'/5'-GGCATGTCCGACTCTTCTTC-3') for SfEcR. Final concentration of primers was 20nM. qRT-PCR efficiency of each primer pair was determined by absolute standard curves for the different gene transcripts and their controls by serial (5x) dilutions of 6th instar midgut and Sf9 cells cDNA. The PCR conditions were as follows: 50°C for 2 min, 95°C for 10min, followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. Each reaction was performed in duplicate and the mean of the four (tissue-stage-specific induction) or the three (xenobiotic induction of gene expression and Sf9 cells) independent biological replicates was calculated. All results were normalized using mRNA level of three control genes (RpL4, L18 and G6PD) and relative expression values were calculated in R using the RqPCRBase package developed in our laboratory (Hilliou and Tran, unpublished)

## 2.7 Phylogenetic analysis

Twenty two Nuclear Receptors were compared including NR1IJK sequences: eight vertebrate PXR/CAR/VDR sequences, six insect HR96 sequences and NR1H sequences: three arthropod EcR sequences and four chordate LXR/FXR sequences (rat, mouse and the tunicate *Ciona intestinalis*). Drosophila DHR3 (NR1F) was used as outgroup.

The analysis was performed on the Phylogeny.lirmm.fr platform and comprised the following steps. Sequences were aligned with MUSCLE (v3.7) configured for highest accuracy (MUSCLE with default settings). Two methods of tree reconstruction were used. (1) Using the neighbor joining method implemented in the BioNJ program (settings gamma 1, 1000 bootstraps) and (2) using the maximum likelihood method implemented in the PhyML program (v3.0 aLRT). Reliability of the internal branches was assessed using the aLRT test.
Graphical representation and edition of the phylogenetic trees were performed with TreeDyn (v198.3).

## 3. Results

### 3.2. Cloning of SfHR96 and SfUSP

We have designed degenerate primers based from the amino acid sequence of DHR96 and other putative HR96 sequences that allowed us to amplify a 320bp fragment from *S. frugiperda* midgut cDNA. This fragment shares 63% identity with DHR96 LBD as shown in figure 1. This LBD fragment allowed us to design specific primers for the amplification of 5'- and 3-' end by RACE PCR reactions. Both 5' and 3' RACE reactions did not give specific results from the first round of amplification as many non specific bands were obtained for both reactions. A second round of PCR amplification using nested primer pairs designed in the inner sequence of the first round PCR reaction was needed to obtain both 3' and 5' fragments of SfHR96 of approximately 1700bp for 5'-end and 600bp for the 3'-end. The sequence of both fragments shared a long enough overlapping region that allowed the reconstruction of a putative whole sequence of 1722bp for SfHR96. From this reconstituted sequence, we have cloned the full-length cDNA/ORF of SfHR96 and confirmed by sequencing that the sequence obtained was not a chimera. Alignment of the deduced amino-acid sequence of SfHR96 with DHR96 showed that both sequences share >50% of identity in their whole sequence, with respectively 79% and 66% identity within the DNA and ligand binding domain of the receptor as shown in figure 2.

For SfUSP, the design of specific primers based on the sequence of USP in *S. littoralis* allowed us to obtain two fragments of approximately 900bp for the 5' end and 450bp for the 3' end.
The sequencing of these fragments showed actually some consistent differences in the 5' region of different clones. Indeed, the deduced amino acid sequence of some clones showed a 50 residues shorter 5' sequence. The alignment of our SfUSP with other insect USP amino acid sequences showed the presence of two isoforms that differ in their 5' end in S. frugiperda as it is shown in figure 3, consistently with the presence of two isoforms of this receptor in some insect species.

### 3.3 Phylogenetic analysis of SfHR96

The deduced amino-acid sequence of SfHR96 that we obtained by RACE-PCR cloning was compared to other insect HR96 ortholog and related nuclear receptors as shown in figure 4. Two phylogenetic trees were built using neighbour joining and maximum likelihood methods.

The two trees are relatively consistent showing that our *S. frugiperda* sequence is most closely related to its Bombyx ortholog obtained from the complete genome sequence. In both trees, the VDR/PXR/CAR sequences were monophyletic and most related to the HR96 sequences. The trees differed in the relative position of LXR, FXR and EcR sequences, were the neighbor-joining tree has more support than the maximum likelihood tree.

### 3.3 Tissue- and stage- specific expression of nuclear receptors in *S. frugiperda*

Using qRT-PCR method, we examined gene expression of the *S. frugiperda* nuclear receptors SfHR96, USP-1, USP-2 and EcR during development from the last fifth and 6th instars to the pupae. Although EcR presents three different isoforms in other insect species, we have measured the level of expression using primers designed on the common part of these three isoforms, without differentiating respective contribution of each of the isoforms. Our results
show that EcR is expressed in midgut, fat bodies and Malpighian tubules with a peak of expression in the late 5th instar, just before the 6th instar moult and in the second day of the pre-pupal stage, with consistent high levels of expression in pupal stages (figure 5). SfUSP-1 and SfUSP-2 are also expressed in the three tissues examined, with SfUSP-1 showing a higher level of expression in Malpighian tubules during the 6th instar (figure 7) than SfUSP-2. SfUSP-1 also shows a peak of expression in the last pre-pupal stage, consistently with SfUSP-2 expression. However, levels of gene expression of SfUSP-1 are much lower in pupae than the second isoform (figure 8). SfHR96 is expressed in all tissues examined with higher levels in midguts and Malpighian tubules compare to fat bodies (figure 6). This receptor did not show the same peak of expression in the pre-pupal and pupae stages as it was seen for EcR and USP and seems to be constitutively expressed throughout the development stages, with a slight increase in the midgut during the late 5th instar and in Malpighian tubules during the first day of the 6th instar.

### 3.4 Induction of nuclear receptors expression by xenobiotics

Specific induction of the nuclear receptors SfHR96, EcR, USP-1 and USP-2 were monitored in *S. frugiperda* midgut fed for 24h with sub-lethal doses of different xenobiotic compounds. Our results show that out of the four receptors studied, only EcR and USP-2 were significantly induced respectively by the ecdysone agonist methoxyfenozide (p=7E-03) and the juvenile hormone analog methoprene (p=5E-02), with USP-2 also showing an induction when larvae were exposed to the insecticide fipronil (table 1A). None of the tested compounds were able to affect the expression of USP-1, as it was also the case for SfHR96 except for two xenobiotics, phenobarbital and xanthotoxin. Interestingly, these two compounds reduced significantly the expression of SfHR96 in *S. frugiperda* midguts.
We also measured the induction level of the three receptors in Sf9 cells exposed to the same battery of chemicals (table 1B). We were not able to use specific qRT-PCR primers to differentiate the two USP isoforms with acceptable efficiency in Sf9 cells and we therefore used primer designed on the common sequence of the two isoforms. Similarly to midgut, SfHR96 was not induced by any of the compounds tested. EcR and USP showed however the same induction patterns in response to methoxyfenozide and methoprene as it was seen in the midgut, with additional compounds found to induce the expression of these receptors. 2-tridecanone and clofibrate were able to induce both EcR and USP expression, and USP expression was also higher in response to indole.

## Discussion

The structural features of nuclear receptors, notably the highly conserved domains (DNA and ligand binding domains), enabled us to design degenerate primers, in conjunction with PCR, for cloning SfHR96 and two isoforms of SfUSP, SfUSP-1 and SfUSP-2 from the polyphagous pest *Spodoptera frugiperda*. Our result show that USP isoforms shares high identity with the known USP isoforms identified in *Manduca sexta* (Jindra et al., 1997) and *Bomby mori* (Cheng et al., 2008) and we have found that the partial sequence of SfUSP available in Genbank corresponds in fact to our SfUSP-2. We show that both isoforms are expressed throughout developmental stages and tissues in *S. frugiperda*, with a peak of expression in fat bodies for SfUSP-2 in the final days of 5^th^ and 6^th^ instars just before the moult, as it has been shown in the epidermis for *Manduca sexta* USP-2 (Jindra et al., 1997). In this study, it was found that MsUSP-1 disappears during larval and pupal moults when ecdysteroid titers are high, and Ms-USP2 increases, suggesting that MsUSP-2 could be the isoform that
interacts with EcR for new cuticle production during the insect moult. Similar results were observed in *Bombyx mori* were USP-2 expression appeared to be coordinate with the pulse of ecdysone during metamorphosis, suggesting that this isoform of USP may be the actual component of the EcR/USP receptor complex (Cheng et al., 2008). We found similar increase of expression of SfUSP-2 before larval moult, concomitant with the first peak of expression of EcR. However, SfUSP-1 was the predominantly expressed isoform in the prepupal stage, when EcR expression reaches its second peak. Insects have different isoforms of EcR. EcRA is mainly expressed in the prepupal and pupal stage in Drosophila, when EcRB appears just before larval and pupal moult (King-Jones and Thummel, 2005). In our results, we only studied the common isoform of EcR and we cannot differentiate which isoform is associated with respective peaks of expression. It is therefore possible that SfUSP-1 and 2 interact with one or another EcR isoform depending on the developmental stage, which could explain the difference in SfUSP expression.

We obtained the sequence of HR96 in *Spodoptera frugiperda*. It shares high identity with its Drosophila ortholog and phylogenetic analysis revealed that it was indeed clustered with other identified HR96 sequences in insects, being most closely related to the *Bombyx mori* HR96. The first identification of HR96 in Drosophila showed that its DBD shared high identity with the human vitamin D receptor and EcR (Fisk and Thummel, 1995) and that this receptor was closely related to the vertebrate receptors CAR and PXR (Escriva et al., 2000; Laudet and Bonneton, 2005), representing the single ortholog of these receptors (King-Jones et al., 2006). Our result confirm these data as SfHR96 in both phylogenetic trees showed that HR96 cluster was most related to the monophyletic group containing VDR/PXR/CAR sequences.
We found that this receptor is expressed constitutively at low levels in all developmental stages and tissues examined with a light increase before and after the last instar moult. These results are consistent with the expression of DHR96 in all digestive tissues of Drosophila (Palanker et al., 2006). However, temporal expression studies in *B. mori* showed that BmHR96 was only expressed at specific time points co-ordinately with ecdysone peaks and along with the expression of BmUSP-2 (Cheng et al., 2008). Our results support these observations with the increase of expression of SfHR96 before the last larval moult, concomitant with the first peak of ecdysone and the expression of SfUSP-2 but we did not find an increase of expression during the second ecdysone peak before pupation.

HR96 belong to the large family of orphan receptors for which no ligand has been identified, nor potential hetero-dimerization partner. NRs indeed function mainly as homo or heterodimer, for example the functional ecdysone receptor consists of a heterodimer of EcR and USP (Yao et al., 1992). USP has also been shown to form a heterodimer with DHR38 (Sutherland et al., 1995), suggesting that it can bind to other nuclear receptors. In vertebrate, USP ortholog RXR binds to the nuclear receptors CAR and PXR, which are the ortholog of HR96 in insects. We can therefore suggest that USP could be a good dimerization candidate for HR96. This hypothesis is particularly attractive as the recruitment of USP by HR96 would make it less available for EcR and thus interfere with ecdysteroid signaling mechanisms. Our results support this hypothesis with a concomitant expression of SfHR96 and SfUSP-2 before the larval moult. However, interactions between SfHR96 and USP still remain to be elucidated.

In vertebrate, CAR and PXR regulate the expression of detoxification genes, such as P450s, when they are activated by xenobiotics (Willson and Kliewer, 2002). In Drosophila, DHR96
has been identified as a potential xenosensors involved in the regulation of many
phenobarbital-regulated genes (King-Jones et al., 2006) and was shown to be activated by
CAR agonists suggesting similar regulation mechanisms than its vertebrate ortholog
(Palanker et al., 2006). Regulation of CAR and PXR involves transcriptional expression, with
compounds such as dexamethasone inducing the expression of both receptors (Pascussi et
al., 2000a; Pascussi et al., 2000b). We have therefore tested the ability of different plant
allelochemicals and xenobiotics to induce the expression of SfHR96 in S. frugiperda larvae
and Sf9 cells. Our results show that none of the compounds were inducers, with xanthotoxin
showing a repression of the expression of this receptor. This may suggest that SfHR96 might
regulate the expression of detoxification genes after activation by xenobiotics without
increasing its own expression.

As its vertebrate orthologs CAR and PXR regulate the expression of a number of
detoxification genes, including P450s (Willson and Kliewer, 2002), we could suggest that
SfHR96 act as a xenosensor by regulating P450s in S. frugiperda in a similar manner. We have
recently identified patterns of P450 induction in response to different plant chemicals and
xenobiotics in S. frugiperda larvae and Sf9 cells (Giraudo et al., manuscript in preparation).
The next logical step will therefore be the study of the effect of SfHR96 silencing on the
expression of P450s in these systems. Extinction of the expression of SfHR96 by RNAi will be
tried in order to validate its function as a xenosensors and make the link between the
ingestion of the toxic compound and the induction of detoxification genes in S. frugiperda..
References

Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne, J.D., Amanatides, P.G., Scherer, S.E., Li, P.W., Hoskins, R.A., Galle, R.F., et al. (2000). The genome sequence of Drosophila melanogaster. Science 287, 2185-2195.

Bonneton, F., Chaumot, A., and Laudet, V. (2008). Annotation of Tribolium nuclear receptors reveals an increase in evolutionary rate of a network controlling the ecdysone cascade. Insect Biochem Mol Biol 38, 416-429.

Bujold, M., Gopalakrishnan, A., Nally, E., and King-Jones, K. Nuclear receptor DHR96 acts as a sentinel for low cholesterol concentrations in Drosophila melanogaster. Mol Cell Biol 30, 793-805.

Cheng, D., Xia, Q., Duan, J., Wei, L., Huang, C., Li, Z., Wang, G., and Xiang, Z. (2008). Nuclear receptors in Bombyx mori: insights into genomic structure and developmental expression. Insect Biochem Mol Biol 38, 1130-1137.

Cruz, J., Sieglaff, D.H., Arensburger, P., Atkinson, P.W., and Raikhel, A.S. (2009). Nuclear receptors in the mosquito Aedes aegypti: annotation, hormonal regulation and expression profiling. FEBS J 276, 1233-1254.

Escriva, H., Delaunay, F., and Laudet, V. (2000). Ligand binding and nuclear receptor evolution. Bioessays 22, 717-727.

Feyereisen, R. (2005). Insect cytochrome P450. In Comprehensive molecular insect science (Elsevier), pp. 1-77.

Fisk, G.J., and Thummel, C.S. (1995). Isolation, regulation, and DNA-binding properties of three Drosophila nuclear hormone receptor superfamily members. Proc Natl Acad Sci U S A 92, 10604-10608.

Germain, P., Staels, B., Dacquet, C., Spedding, M., and Laudet, V. (2006). Overview of nomenclature of nuclear receptors. Pharmacol Rev 58, 685-704.

Holt, R.A., Subramanian, G.M., Halpern, A., Sutton, G.G., Charlab, R., Nusskern, D.R., Wincker, P., Clark, A.G., Ribeiro, J.M., Wides, R., et al. (2002). The genome sequence of the malaria mosquito Anopheles gambiae. Science 298, 129-149.

Horner, M.A., Pardee, K., Liu, S., King-Jones, K., Lajoie, G., Edwards, A., Krause, H.M., and Thummel, C.S. (2009). The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis. Genes Dev 23, 2711-2716.

Jindra, M., Huang, J.Y., Malone, F., Asahina, M., and Riddiford, L.M. (1997). Identification and mRNA developmental profiles of two ultraspiracle isoforms in the epidermis and wings of Manduca sexta. Insect Mol Biol 6, 41-53.

King-Jones, K., Horner, M.A., Lam, G., and Thummel, C.S. (2006). The DHR96 nuclear receptor regulates xenobiotic responses in Drosophila. Cell Metab 4, 37-48.
King-Jones, K., and Thummel, C.S. (2005). Nuclear receptors--a perspective from Drosophila. *Nat Rev Genet* 6, 311-323.

Laudet, V., and Bonneton, F. (2005). evolution of nuclear hormone receptors in insects. In *Comprehensive molecular insect science* (Elsevier), pp. 287-318.

Maglich, J.M., Stoltz, C.M., Goodwin, B., Hawkins-Brown, D., Moore, J.T., and Kliewer, S.A. (2002). Nuclear pregnane X receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification. *Mol Pharmacol* 62, 638-646.

Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., et al. (1995). The nuclear receptor superfamily: the second decade. *Cell* 83, 835-839.

Moore, D.D., Kato, S., Xie, W., Mangelsdorf, D.J., Schmidt, D.R., Xiao, R., and Kliewer, S.A. (2006). International Union of Pharmacology. LXII. The NR1H and NR1I receptors: constitutive androstane receptor, pregnene X receptor, farnesoid X receptor alpha, farnesoid X receptor beta, liver X receptor alpha, liver X receptor beta, and vitamin D receptor. *Pharmacol Rev* 58, 742-759.

Nakagawa, Y., and Henrich, V.C. (2009). Arthropod nuclear receptors and their role in molting. *FEBS J* 276, 6128-6157.

Palanker, L., Necakov, A.S., Sampson, H.M., Ni, R., Hu, C., Thummel, C.S., and Krause, H.M. (2006). Dynamic regulation of Drosophila nuclear receptor activity in vivo. *Development* 133, 3549-3562.

Pascussi, J.M., Drocourt, L., Fabre, J.M., Maurel, P., and Vilarem, M.J. (2000a). Dexamethasone induces pregnane X receptor and retinoid X receptor-alpha expression in human hepatocytes: synergistic increase of CYP3A4 induction by pregnane X receptor activators. *Mol Pharmacol* 58, 361-372.

Pascussi, J.M., Gerbal-Chaloin, S., Fabre, J.M., Maurel, P., and Vilarem, M.J. (2000b). Dexamethasone enhances constitutive androstane receptor expression in human hepatocytes: consequences on cytochrome P450 gene regulation. *Mol Pharmacol* 58, 1441-1450.

Pascussi, J.M., Robert, A., Nguyen, M., Walrant-Debray, O., Garabedian, M., Martin, P., Pineau, T., Saric, J., Navarro, F., Maurel, P., et al. (2005). Possible involvement of pregnane X receptor-enhanced CYP24 expression in drug-induced osteomalacia. *J Clin Invest* 115, 177-186.

Poitout, S., and Bues, R. (1974). [Linolenic acid requirements of lepidoptera: Noctuidae Quadrifinae Plusiinae: Chrysodeixis chalcites Esp., Autographa gamma L.' Macdunnoughia confusa Stph., Trichoplusia ni Hbn. reared on artificial diets]. *Ann Nutr Aliment* 28, 173-187.

Robinson-Rechavi, M., Carpentier, A.S., Duffraisse, M., and Laudet, V. (2001). How many nuclear hormone receptors are there in the human genome? *Trends Genet* 17, 554-556.
Robinson-Rechavi, M., and Laudet, V. (2003). Bioinformatics of nuclear receptors. In Methods in enzymology, Elsevier, ed., pp. 95-118.

Sluder, A.E., and Maina, C.V. (2001). Nuclear receptors in nematodes: themes and variations. *Trends Genet* **17**, 206-213.

Sutherland, J.D., Kozlova, T., Tzertzinis, G., and Kafatos, F.C. (1995). Drosophila hormone receptor 38: a second partner for Drosophila USP suggests an unexpected role for nuclear receptors of the nerve growth factor-induced protein B type. Proc Natl Acad Sci U S A **92**, 7966-7970.

Talbot, W.S., Swyryd, E.A., and Hogness, D.S. (1993). Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell **73**, 1323-1337.

Tan, A., and Palli, S.R. (2008). Identification and characterization of nuclear receptors from the red flour beetle, Tribolium castaneum. Insect Biochem Mol Biol **38**, 430-439.

Thummel, C.S. (2002). Ecdysone-regulated puff genes 2000. Insect Biochem Mol Biol **32**, 113-120.

Velarde, R.A., Robinson, G.E., and Fahrbach, S.E. (2006). Nuclear receptors of the honey bee: annotation and expression in the adult brain. Insect Mol Biol **15**, 583-595.

Willson, T.M., and Kliewer, S.A. (2002). PXR, CAR and drug metabolism. Nat Rev Drug Discov **1**, 259-266.

Yao, T.P., Segraves, W.A., Oro, A.E., McKeown, M., and Evans, R.M. (1992). Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. Cell **71**, 63-72.
<table><caption>**Table 1:** qRT-PCR analysis of nuclear receptors gene induction in *S.frugiperda midgut* (A) and Sf9 cells (B) by xenobiotics and insecticides. Larvae and cells were exposed for 24h to sublethal concentrations of the different compounds as described in material and methods. Gene expression values are indicated as fold expression in larvae and cells exposed to each xenobiotic comparatively to unexposed larvae and cells (controls). The three reference genes Rpl4, G6PD and L18 were used as internal controls for normalization. Gene expression values in bold are significantly different from the corresponding control as measured by pair-wise t-student test (p<0.05).</caption><thead><tr><th rowspan="2">A<br>xenobiotics</th><th colspan="2">SfHR96</th><th colspan="2">EcR</th><th colspan="2">USP-1</th><th colspan="2">USP-2</th></tr><tr><th>ratio</th><th>P value</th><th>ratio</th><th>P value</th><th>ratio</th><th>P value</th><th>ratio</th><th>P value</th></tr></thead><tbody><tr><td>Phenobarbital</td><td>0.58</td><td>2.0E-02</td><td>0.45</td><td>6.0E-02</td><td>0.84</td><td>5.5E-01</td><td>0.68</td><td>1.4E-01</td></tr><tr><td>Xanthotoxin</td><td></td><td></td><td>0.60</td><td>2.2E-01</td><td>0.73</td><td>3.7E-01</td><td>0.63</td><td>1.8E-01</td></tr><tr><td>2-tridecanone</td><td>0.78</td><td>2.5E-01</td><td>0.57</td><td>1.8E-01</td><td>0.70</td><td>3.2E-01</td><td>0.75</td><td>3.5E-01</td></tr><tr><td>Indole 3-carbinol</td><td>0.81</td><td>3.2E-01</td><td>0.64</td><td>2.7E-01</td><td>1.07</td><td>8.0E-01</td><td>0.91</td><td>7.4E-01</td></tr><tr><td>Clofibrate</td><td>0.92</td><td>6.9E-01</td><td>0.84</td><td>6.2E-01</td><td>0.90</td><td>7.5E-01</td><td>0.95</td><td>8.6E-01</td></tr><tr><td>Quercetin</td><td>0.81</td><td>3.3E-01</td><td>0.71</td><td>3.7E-01</td><td>0.77</td><td>4.4E-01</td><td>1.31</td><td>2.5E-01</td></tr><tr><td>Indole</td><td>0.80</td><td>3.0E-01</td><td>0.67</td><td>3.0E-01</td><td>1.05</td><td>8.6E-01</td><td>0.97</td><td>7.3E-01</td></tr><tr><td>Methoxyfenozide</td><td>0.99</td><td>9.6E-01</td><td></td><td></td><td>1.10</td><td>7.4E-01</td><td>1.44</td><td>1.1E-01</td></tr><tr><td>Methoprene</td><td>0.91</td><td>6.4E-01</td><td>0.92</td><td>7.9E-01</td><td>0.73</td><td>3.8E-01</td><td></td><td></td></tr><tr><td>Deltamethrin</td><td>0.92</td><td>6.6E-01</td><td>0.88</td><td>7.1E-01</td><td>1.00</td><td>8.2E-01</td><td>1.22</td><td>4.2E-01</td></tr><tr><td>Fipronil</td><td>0.85</td><td>7.3E-01</td><td>1.09</td><td>7.7E-01</td><td>1.07</td><td>8.1E-01</td><td>1.69</td><td>5.0E-02</td></tr></tbody></table>

<table><thead><tr><th rowspan="2">B<br>xenobiotics</th><th colspan="2">SfHR96</th><th colspan="2">EcR</th><th colspan="2">USP-com</th></tr><tr><th>ratio</th><th>P value</th><th>ratio</th><th>P value</th><th>ratio</th><th>P value</th></tr></thead><tbody><tr><td>Phenobarbital</td><td>1.05</td><td>8.6E-01</td><td>1.22</td><td>8.5E-01</td><td>1.48</td><td>1.6E-01</td></tr><tr><td>Xanthotoxin</td><td>2.29</td><td>4.9E-01</td><td>1.35</td><td>1.4E-02</td><td>1.42</td><td>1.8E-01</td></tr><tr><td>2-tridecanone</td><td>1.78</td><td>1.6E-01</td><td></td><td></td><td></td><td></td></tr><tr><td>Indole 3-carbinol</td><td>2.36</td><td>1.9E-02</td><td>1.06</td><td>4.6E-03</td><td>1.45</td><td>1.5E-01</td></tr><tr><td>Clofibrate</td><td>1.90</td><td>1.1E-01</td><td></td><td></td><td></td><td></td></tr><tr><td>Quercetin</td><td>1.80</td><td>1.9E-01</td><td>1.28</td><td>2.1E-03</td><td>1.78</td><td>2.7E-02</td></tr><tr><td>Indole</td><td>2.05</td><td>7.9E-01</td><td>1.56</td><td>3.5E-01</td><td></td><td></td></tr><tr><td>Methoxyfenozide</td><td>0.75</td><td>6.7E-01</td><td></td><td></td><td>1.60</td><td>5.7E-02</td></tr><tr><td>Methoprene</td><td>2.15</td><td>7.0E-01</td><td>1.98</td><td>1.1E-01</td><td></td><td></td></tr><tr><td>Deltamethrin</td><td>2.32</td><td>2.2E-02</td><td>1.61</td><td>3.1E-01</td><td>1.63</td><td>4.7E-02</td></tr><tr><td>Fipronil</td><td>1.36</td><td>1.1E-01</td><td>1.36</td><td>5.5E-01</td><td>1.36</td><td>2.1E-01</td></tr></tbody></table>
Figure legends

**Figure 1:** alignment of the deduced amino-acid sequence of SfHR96 LBD obtained in the initial PCR amplification with the protein sequence of DHR96 using CLUTALW2. DHR96 LBD is indicated by the frame.

**Figure 2:** alignment of SfHR96 and DHR96. The amino-acid sequence deduced from the full length SfHR96 that we cloned by RACE-PCR was aligned with the protein sequence of DHR96 using CLUSTALW2. Highly conserved DBD and LBD are indicated in frames. Poorly conserved hinge region between the LBD and the DBD showed weak alignment and was therefore not shown (indicated by double slashes).

**Figure 3:** alignment of the deduced amino-acid sequences obtained from the cloning of SfUSP-1 (A) and SfUSP-2 (B) with USP-1 and -2 orthologs in insects. Ms: *Manduca sexta*, Bm: *Bombyx mori*, SfUSP-G: partial sequence of *S. frugiperda* USP (Genbank acc. Number AF411255).

**Figure 4:** Phylogenetic analysis of SfHR96. Trees were built with the neighbour joining method (A) of maximum likelihood method (B). Bootstrap values are indicated for each branch. Dmel: *Drosophila melanogaster*; Agam: *Anopheles gambiae*; Bmor: *Bombyx mori*; Sfru: *Spodoptera frugiperda*; Amel: *Apis mellifera*; Tcas: *Tribolium castaneum*.

**Figures 4-5-6-7:** qRT-PCR analysis of nuclear receptors gene expression in different tissues and developmental stages of *S. frugiperda* larvae. Expression was monitored in last day 5th instar larvae (L5), at different time points of the first and second days of 6th instars (L6-1 to 3 and L6-4 to 6 respectively), in first and second pre-pupal stages (PP1 and PP2) and in early (~2days) and late (~10 days) pupae. EcR levels of expression are shown in figure 2, SfHR96 in
figure 3, SfUSP-1 in figure 4 and SfUSP-2 in figure 5. Expression levels are normalized with the three reference genes G6PD, Rpl4 and L18.
<table><tbody><tr><td>DHR96</td><td>MSPPKNAVCVGDKALGYNFNAVTGESCKAFFRRNALAKKQFTCPFNQNCDITVVTRRFCQ</td><td>60</td></tr><tr><td>SfHR96</td><td></td><td></td></tr><tr><td>DHR96</td><td>KCRLRKCLDIGMKSENIMSEEDKLIKRRKIETNRAKRRLMENGTDACDADGGEERDHKAP</td><td>120</td></tr><tr><td>SfHR96</td><td></td><td></td></tr><tr><td>DHR96</td><td>ADSSSSNLDHYSGSQDSQSCGSADSGANGCSGRQASSPGTQVNPLQMTAEKIVDQIVSDP</td><td>180</td></tr><tr><td>SfHR96</td><td></td><td></td></tr><tr><td>DHR96</td><td>DRASQAINRLMRTQKEAISVMEKVISSQKDALRLVSHLIDYPGDALKIISKFMNSPFNAL</td><td>240</td></tr><tr><td>SfHR96</td><td></td><td></td></tr><tr><td>DHR96</td><td>TVFTKFMSSPTDGVEIISKIVDSPADVVEFMQNLMHSPEDAIDIMNKFMNTPAEALRILN</td><td>300</td></tr><tr><td>SfHR96</td><td></td><td></td></tr><tr><td>DHR96</td><td>RILSGGGANAAQQTADRKPLLDKEPAVKPAAPAERADTVIQSMLGNSPPISPHDAAVDLQ</td><td>360</td></tr><tr><td>SfHR96</td><td></td><td></td></tr><tr><td>DHR96</td><td>YHSPGVGEQPSTSSSHPLPYIANSPDFDLKTFMQTNYNDEPSLDSDFSINSIESVLSEVI</td><td>420</td></tr><tr><td>SfHR96</td><td></td><td></td></tr><tr><td>DHR96</td><td>RIEYQAFNSIQQAASRVKEEMSYGTQSTYGGCNSAANNSQPHLQQPICAPSTQQLDRELN</td><td>480</td></tr><tr><td>SfHR96</td><td></td><td></td></tr><tr><td>DHR96</td><td>EAEQMKLRELRLASEALYDPVDEDLSALMMGDDRIKPDDTRHNPKLLQLINLTAVAIKRL</td><td>540</td></tr><tr><td>SfHR96</td><td></td><td></td></tr><tr><td>DHR96</td><td>IKMAKKITAFRDMCQEDQVALLKGGCTEMMIMRSVMIYDDDRAAWKVPHTKENMGNIRTD</td><td>600</td></tr><tr><td>SfHR96</td><td>QVALLKGGCIEMMVLRSTMTYDGQRKQWKIPHCQEQFGSIRTD</td><td>43</td></tr><tr><td></td><td>********** **:*.** **:.* **:.* *:*.**</td><td></td></tr><tr><td>DHR96</td><td>LLKFAEGNIYEEHQKFITTFDEKWRMDENIILIMCAIVLFTSARSRVHKDVIRLEQNSY</td><td>660</td></tr><tr><td>SfHR96</td><td>VLKLAKGNIYRSHDSFIRSTFEARWRTDEHVILIMSAILLFTPDRPKVVHPDVIQLEQNSY</td><td>103</td></tr><tr><td></td><td>**:*:****.**:** :*: **: :** :****.**:*** .*::* ***:*****</td><td></td></tr><tr><td>DHR96</td><td>YYLLRRYLESVYSGCEARNAFIKLIQKISDVERLNKFIIINVYLNVNPSQVEPLLREIFDL</td><td>720</td></tr><tr><td>SfHR96</td><td>YYL-------------------------------------------- 106</td><td></td></tr><tr><td>DHR96</td><td>KNH 723</td><td></td></tr><tr><td>SfHR96</td><td>---</td><td></td></tr></tbody></table>

LBD

<caption>Figure 1</caption>
SfHR96 MDNNSDKKDDASAQKKEVLSSLOKICLVCGDKALGYNFNAISCESCKAFFRRNALASKEF 60
DHR96 ------------------MSPFKNCAVCGDKALGYNFNAVTGESCKAFFRRNALAKKQF 41
* * ****************:***************.:*:

SfHR96 KCPFTNNCVITVTRRFCQKCRLREKCLSIGMVKEFIMSEEDKAEKRRKIEENRARKRQRD 120
DHR96 TCPFNQNCDITVVTRRFCQKCRLRKCLDIGMKSEFIMSEEDKLIKRRKIE TNRAKRRLME 101
*** .*** ******************* .*** *** * *:*** :* :

SfHR96 //
DHR96 //

SfHR96 ------------------SSRELNEVERAKLNELIVANKALHAFIDDDVTVLGGETASSAGFKG 364
DHR96 PHLQQPICAPSTQQLDRELNEAEQMKLRELRLASEALYDFVDEDLSALM---MGDDRIKP 517
.*****.*: **.** :*.:**: *:**:*.:**: *: .. :*

SfHR96 GDGKHDPRLITIVNLTAVAIRRFIKMAKKINAFKNMCEEDQVALLKGGCIEMMVLRSTMT 424
DHR96 DDTRHNPKLLQLINLTAVAIKRLIKMAKKITAFRDMCQEDQVALLKGGCTEMMIMRSVMI 577
.* :*:*:*: : :********:.* *********.**: :**:************** ***:***.*

SfHR96 YDGQRKQWKIPHCQEQFGSIRTDVLKLAKGNIYRSHDSFIRSFEARWRTDEHVILIMSAI 484
DHR96 YDDDRAAWKVPHTKENMGNIRTDLLKFAEGNIYEEHHQKFITTFDEKWWRMDENIILIMCAI 637
**.*: **:**: *:**:******:**:******.*: **.*: **:**:******

SfHR96 LLFTPDRPKVVHPDVIKLEQNSYYYLLRRYLESVYPGCEAKSTFLKLIQKILELRKLAEE 544
DHR96 VLFTSARSRVIHKDVIRLEQNSYYYLLRRYLESVYSGCEARNAFIKLIQKISDVERLNKF 697
:*** .*:.*:*: ***:*************************** .***: .: :***** :..:.* :

SfHR96 VTGVYLDVNP--IEPPLLMEIFDLKHHA 570 LBD
DHR96 IINVYLVNVPSQVEPLLREIFDLKNH-- 723
: .***:*** :**** ***:***:*

<caption>Figure 2</caption>
A

<table><tbody><tr><td>MsUSP-1</td><td>MSSVAKKDKRTMSVTALINRAWPLTPAPHQQQ-SMPSSQPSNFLQPLATPSTTPSVE--L</td><td>57</td></tr><tr><td>BmUSP-1</td><td>MSSVAKKDKRTMSVTALINRAWPMTPSPQQQQQMVPSTQHSNFLHAMATPSTTPNVVE--L</td><td>58</td></tr><tr><td>SfUSP-1</td><td>-MSVAKKDKPTMSVTALINWARPLPG-QQQQPMTPTS-FGSMLQPMATPSNIPTVDCSL</td><td>57</td></tr><tr><td>MsUSP-1</td><td>DIQWLNIEPGFMSPMSPPEMKPDTAMLDGLRDDSTPPPAFKNYPPNHPLSGSKHLCSICG</td><td>117</td></tr><tr><td>BmUSP-1</td><td>DIQWLNIESGFMSPMSPPEMKPDTAMLDGFRDDSTPPPPFKNYPPNHPLSGSKHLCSICG</td><td>118</td></tr><tr><td>SfUSP-1</td><td>DIQWLNLESGFMSPMSPPEMKPDTAMLDGLRDDSTPPPAFKNYPPNHPLSGSKHLCSICG</td><td>117</td></tr><tr><td>MsUSP-1</td><td>DRASGKHYGVYSSEGCKGFFKRTVRKDLTYACREDRNCIIDKQRNRQCQYCRYQKCLACG</td><td>177</td></tr><tr><td>BmUSP-1</td><td>DRASGKHYGVYSSEGCKGFFKRTVRKDLTYACREDRNCIIDKQRNRQCQYCRYQKCLACG</td><td>178</td></tr><tr><td>SfUSP-1</td><td>DRASGKHYGVYSSEGCKGFFKRTVRKDLTYACREERNCIIDKQRNRQCQYCRYQKCLACG</td><td>177</td></tr><tr><td>MsUSP-1</td><td>MKREAVQEERQRAARGTEDAHPSS-SVQELSIERLLEIESLVADPPEEFQFLRVGPESG</td><td>235</td></tr><tr><td>BmUSP-1</td><td>MKREAVQEERQRAARRTEDAHPSS-SVQELSIERLLELEALVADSAEELQILRVGPESG</td><td>236</td></tr><tr><td>SfUSP-1</td><td>MKREAVQEERQRAARGTEDAHPSSSVQVQELSIERLLEMESLVADPTEEYQFLRVGDSN</td><td>237</td></tr><tr><td>MsUSP-1</td><td>VPAKYRAPVSSLCQIGNKQIAALVVARDIPHFGQLELEDQILLIKNSWNELLFAIAWR</td><td>295</td></tr><tr><td>BmUSP-1</td><td>VPAKYRAPVSSLCQIGNKQIAALIVWARDIPHFGQLEIDDQILLIKGSWNELLFAIAWR</td><td>296</td></tr><tr><td>SfUSP-1</td><td>VPPKFRAPVSSLCQIGNKQIAALVVARDIPHFSLELEDQMLLIKGSWNELLFAIAWR</td><td>297</td></tr><tr><td>MsUSP-1</td><td>SMEYLTERENVDS---RSTAPQLMCLMPGMTLHRNSALQAGVGQIFDRVLSELSLKMR</td><td>352</td></tr><tr><td>BmUSP-1</td><td>SMEFLNDERENVDS---RNTAPQLICLMPGMTLHRNSALQAGVGQIFDRVLSELSLKMR</td><td>353</td></tr><tr><td>SfUSP-1</td><td>SMEYLTEREERVVDDSGNRTTSPQLMCLMPGMTLHRNSALQAGVGQIFDRVLSELSLKMR</td><td>357</td></tr><tr><td>MsUSP-1</td><td>TLRMDQAEYVALKAIILLNPDVKGLKNKPEVVVLREKMFSCLDEYVRRSRCAEGRFAAL</td><td>412</td></tr><tr><td>BmUSP-1</td><td>SLRMDQAEVVALKAIILLNPDVKGLKNKQEVDVLREKMFLCLDEYCRRSGGEEGRFAAL</td><td>413</td></tr><tr><td>SfUSP-1</td><td>ALRVDQAEYVALKAIILLNPDVKGLKNRPEVEILREKMFSCLDEYVRRSRGGEEGRFAAL</td><td>417</td></tr><tr><td>MsUSP-1</td><td>LLRLPALRSISLKCFEHLYFFHVLADTSIASYIHDALRNHAPSIDTSIL</td><td>461</td></tr><tr><td>BmUSP-1</td><td>LLRLPALRSISLKSEHLYFFHVLADTSIASYIREALRSHAPPIDANLM</td><td>462</td></tr><tr><td>SfUSP-1</td><td>LLRLPALRSISLKSEHLFFHVLADTSIASYIREALRSHAPPIDANLM</td><td>466</td></tr></tbody></table>

B

<table><caption>Figure 3</caption><tbody><tr><td>SfUSP-G</td><td>MMEPSRDSGLNLESGFMSPMSPPEMKPDTAMLDGLRDDSTPPPAFKNYPPNHPLSGSKHL</td><td>60</td></tr><tr><td>SfUSP-2</td><td>MMEPSRDSGLNLESGFMSPMSPPEMKPDTAMLDGLRDDSTPPPAFKNYPPNHPLSGSKHL</td><td>60</td></tr><tr><td>BmUSP-2</td><td>-MEPSRESGLNIESGFMSPMSPPEMKPDTAMLDGFRDDSTPPPPFKNYPPNHPLSGSKHL</td><td>59</td></tr><tr><td>SfUSP-G</td><td>CSICGDRASGKHYGVYSSEGCKGFFKRTVRKDLTYACREERNCIIDKQRNRQCQYCRYQK</td><td>120</td></tr><tr><td>SfUSP-2</td><td>CSICGDRASGKHYGVYSSEGCKGFFKRTVRKDLTYACREERNCIIDKQRNRQCQYCRYQK</td><td>120</td></tr><tr><td>BmUSP-2</td><td>CSICGDRASGKHYGVYSSEGCKGFFKRTVRKDLTYACREDKNCIIDKQRNRQCQYCRYQK</td><td>119</td></tr><tr><td>SfUSP-G</td><td>CLACGMKREAVQEERQRAARGTEDAHPSSSVQVQELSIERLLEMESLVADPTEEYQFLRV</td><td>180</td></tr><tr><td>SfUSP-2</td><td>CLACGMKREAVQEERQRAARGTEDAHPSSSVQVQELSIERLLEMESLVADPTEEYQFLRV</td><td>180</td></tr><tr><td>BmUSP-2</td><td>CLACGM</td><td>125</td></tr><tr><td>SfUSP-G</td><td>GPDSNVPPKFRAPVSSLCQIGNKQIAALVWARDIPHFSSLELEDQMLLIKGSWNELLLE</td><td>240</td></tr><tr><td>SfUSP-2</td><td>GPDSNVPPKFRAPVSSLCQIGNKQIAALVWARDIPHFSSLELEDQMLLIKGSWNELLLE</td><td>240</td></tr><tr><td>BmUSP-2</td><td></td><td></td></tr><tr><td>SfUSP-G</td><td>AIAWRSMEYLTEEREVVDSFGNRTTSPPQLMCLMPG</td><td>276</td></tr><tr><td>SfUSP-2</td><td>AIAWRSMEYLTEEREVVDDSGNRTTSPPQLMCLMPGMTLHRNSALQAGVGQIFDRVLSEL</td><td>300</td></tr><tr><td>BmUSP-2</td><td></td><td></td></tr><tr><td>SfUSP-G</td><td></td><td></td></tr><tr><td>SfUSP-2</td><td>SLKMRALRVDQAEYVALKAIILLNPDVKGLKNRPEVEILREKMFSCLDEYVRRSRGGEEG</td><td>360</td></tr><tr><td>BmUSP-2</td><td></td><td></td></tr><tr><td>SfUSP-G</td><td></td><td></td></tr><tr><td>SfUSP-2</td><td>RFAALLRLPALRSISLKSEHLFFHVLADTSIASYIREALRSHAPPIDANLM</td><td>414</td></tr><tr><td>BmUSP-2</td><td></td><td></td></tr></tbody></table>
A

<figure><img src="image_48.png" /></figure>

B

<figure><img src="image_49.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_50.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_51.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_52.png" /><figcaption>Figure 7</figcaption></figure>
<figure><img src="image_53.png" /><figcaption>Figure 8</figcaption></figure>
Additional results:

# 1. RNAi impairment of SfHR96

We have successfully cloned SfHR96, the ortholog of DHR96 in *Spodoptera frugiperda* and shown that it was expressed in the three major tissues involved in detoxification, the midgut fat body and Malpighian tubules. Of relevance to the use of Sf9 cells as tools to study induction, we found that SfHR96 was expressed in those cells. This receptor seemed to be constitutively expressed independently of xenobiotic exposure. In an attempt to identify the function of SfHR96 in the detoxification response of this insect, we have tried to knock-down the expression of the gene encoding the receptor by RNAi experiments *in vivo* in *S. frugiperda* larvae and *in vitro* in Sf9 cells and to study the effect of this impairment on P450 gene induction.

## 1.1 *in vivo* experiments

dsRNA were synthesized using MEGAscript RNAi kit (Ambion) from *S. frugiperda* midgut cDNA. Specific primers were designed based on the sequence of SfHR96 that we have cloned and a fragment of 334bp was amplified by PCR using these primers and was used as a template to synthesize the dsRNA. 3µg of dsRNA were then injected to newly molted 5^th^ instar larvae and newly molted dsRNA 6^th^ instar larvae were then fed for 48h with artificial medium containing 0.05% of xanthotoxin or the equivalent amount of DMSO. In parallel, we have also injected larvae with dsRNA prepared from the Phenylalanine ammonia lyase (PAL) gene from *Arabidopsis thaliana* as a control. RNA was then extracted from the midguts of larvae and the expression of specific P450s was measured by qRT-PCR analysis in dsHR96 xanthotoxin treated larvae, dsPAL xanthotoxin treated larvae and uninjected xanthotoxin treated larvae. Primers for P450 genes and SfHR96, qRT-PCR conditions, and data analysis were the same as described in the material and methods of chapter 2. We have chosen xanthotoxin as inducer as it was identified as the stronger inducer of P450 genes in *S. frugiperda* midgut, with induction levels of 162.1 for CYP321A7 and 20.97 for CYP321A9.

Tabel 5 presents our results of CYP321A7 and CYP321A9 expression levels in dsRNA and control larvae measured in 5 independent biological replicates
<table><caption>**Table 5** : levels of P450 expressionª measured by qRT-PCR analysis in *S. frugiperda* larvae injected with dsRNA targeted against SfHR96 and exposed to xanthotoxin. Larvae injected with dsPAL from *A. thaliana* served as control.</caption><thead><tr><th></th><th>CYP321A9</th><th>CYP321A7</th><th>SfHR96</th></tr></thead><tbody><tr><td><strong>Replicat 1</strong></td><td></td><td></td><td></td></tr><tr><td>dsPAL - DMSO</td><td>4.2</td><td>1.9</td><td>1.2</td></tr><tr><td>dsPAL - Xantho 0.05%</td><td>33.2</td><td>572.1</td><td>1.0</td></tr><tr><td>dsHR96 - DMSO</td><td>1.0</td><td>1.0</td><td>2.1</td></tr><tr><td>dsHR96 - Xantho 0.05%</td><td>70.0</td><td>1289.1</td><td>1.3</td></tr><tr><td><strong>Replicat 2</strong></td><td></td><td></td><td></td></tr><tr><td>dsPAL - DMSO</td><td>1.7</td><td>1.0</td><td>6.7</td></tr><tr><td>dsPAL - Xantho 0.05%</td><td>13.0</td><td>777.5</td><td>1.0</td></tr><tr><td>dsHR96 - DMSO</td><td>1.0</td><td>3.0</td><td>9.1</td></tr><tr><td>dsHR96 - Xantho 0.05%</td><td>6.3</td><td>334.0</td><td>1.2</td></tr><tr><td><strong>Replicat 3</strong></td><td></td><td></td><td></td></tr><tr><td>dsPAL - DMSO</td><td>13.1</td><td>16.5</td><td>3.5</td></tr><tr><td>dsPAL - Xantho 0.05%</td><td>6.9</td><td>79.2</td><td>1.7</td></tr><tr><td>dsHR96 - DMSO</td><td>1.0</td><td>1.0</td><td>1.0</td></tr><tr><td>dsHR96 - Xantho 0.05%</td><td>69.81</td><td>3612.7</td><td>1.6</td></tr><tr><td><strong>Replicat 4</strong></td><td></td><td></td><td></td></tr><tr><td>dsPAL - DMSO</td><td>1.0</td><td>1.0</td><td>1.6</td></tr><tr><td>dsPAL - Xantho 0.05%</td><td>25.3</td><td>363.8</td><td>1.0</td></tr><tr><td>dsHR96 - DMSO</td><td>4.0</td><td>23.0</td><td>1.1</td></tr><tr><td>dsHR96 - Xantho 0.05%</td><td>5.8</td><td>59.4</td><td>1.3</td></tr><tr><td><strong>Replicat 5</strong></td><td></td><td></td><td></td></tr><tr><td>dsPAL - DMSO</td><td>3.3</td><td>1.2</td><td>2.1</td></tr><tr><td>dsPAL - Xantho 0.05%</td><td>19.5</td><td>253.0</td><td>1.0</td></tr><tr><td>dsHR96 - DMSO</td><td>1.0</td><td>1.0</td><td>1.2</td></tr><tr><td>dsHR96 - Xantho 0.05%</td><td>3.1</td><td>78.6</td><td>1.2</td></tr></tbody></table>

Our results show that we could observe a significant gene expression decrease in 3 replicates, with levels of CYP321A7 being between 2 and 6 times less induced in dsRNA treated larvae exposed to xanthotoxin, compared to uninjected larvae. However, the exact same pattern was also observed in 2 replicates where the expression of CYP321A7 was induced by a 2 and 45 fold in dsRNA treated larvae exposed to xanthotoxin.

We have tried to improve our results by injecting dsRNA at different instars or with different quantity of dsRNA but could not get a sufficient reproducibility showing a consistent decrease in P450 gene expression in dsRNA larvae. Expression levels of SfHR96 did not show

<footnote>ª for each replicate and each P450, relative expression values were calculated by qBase, which rescale the lowest relative quantity to 1 for each gene in every sample. Expression values in red show genes for which the level of induction is decreased in dsHR96 treated cells exposed to xanthotoxin, in comparison to dsPAL transfected cells.</footnote>
a significant decrease in dsRNA treated larvae either but as the basal level of expression of this gene is very low, it might be difficult to measure accurately a decrease in expression, which however do not mean that RNAi did not work.

1.2 *in vitro* experiments

We have therefore tried to knock-down SfHR96 in Sf9 cells by transfection of either dsRNA or siRNA. dsRNA were the same used for *in vivo* experiments and siRNA were designed and synthesized by Eurogentec based on our cloned sequence of SfHR96.

2µg of dsRNA were transfected into Sf9 cells with three different transfection reagents according to the manufacturer's protocol: Insect GeneJuice (Novagen), polyethylenimin and TransIT-siQuest (Mirus Bio). Cells were then treated for 24h with xanthotoxin, 24h after transfection. Expression levels of CYP9A30, 9A31 and 9A32 were followed in dsRNA xanthotoxin induced cells and untransfected xanthotoxin treated cells, with dsPAL transfected cells used as control. We have chosen to follow the expression of the CYP9A members as they were identified as the most induced P450s in Sf9 cells by xanthotoxin in preliminary studies (data not shown). However, we have observed in chapter 2 that actually xanthotoxin did not induce significantly the expression of any of these P450s. Our preliminary results indicating the opposite could therefore have been false positive resulting from experimental biases. This observation could therefore explain why we did not observe any significant decrease in P450 induction nor SfHR96 expression in the cells treated with dsHR96 (data not shown).

We have therefore tried to silence the expression of SfHR96 by transfecting Sf9 cells with siRNA at different concentrations, with TranIT-siQuest (Mirus Bio) transfection reagents. Our first results are depicted in figure 26.
<figure><img src="image_54.png" /><figcaption>**Figure 26** : Preliminary results of gene expression levels of SfHR96 (A), CYP9A31 (B) and CYP9A30 (C) in Sf9 cells transfected with 3 concentrations (10, 25 and 50 nM) of siRNA targeted against SfHR96 (siHR96) and untransfected cells (ctrl) exposed to 50µM deltamethrin (delta), 250µM indole (Ind) or 25µM methoprene (Mtp). Gene expression values are represented as the ratio between xenobiotic treated cells and untreated cells, in percentage of control (untransfected exposed cells)</figcaption></figure>

Our results show that we could efficiently silence the expression of SfHR96 in Sf9 cells with an extinction ratio between 20 and 60% (figure 26A). A concomitant decrease of P450 gene expression was observed for CYP9A31 (figure 26B) and CYP9A30 (figure 26C), the latter showing the strongest decrease with only 20% expression remaining in siHR96 transfected cells exposed to deltamethrin.

These preliminary results suggest that SfHR96 is necessary for the induction of CYP9A30 and CYP9A31 in Sf9 cells, in contrary to the results we obtained with dsRNA transfections.
However, like the situation in *S. frugiperda* larvae injected with dsRNA, we were unable to reproduce these results. We have tried different time pre- and post transfection, different concentrations of siRNA, different P450s, different time-course of xenobiotic exposition but could never reproduce twice the results that we obtained in the first place. However encouraging, these results need to be validated and raise the question about RNAi efficiency in *S. frugiperda*.

RNAi is a powerful technique for unveiling gene functions in many organisms and has been used on numerous insect species. Its use in non-drosophilids is reviewed in (Belles, 2010). *In vivo* injection of dsRNA in *Tribolium castaneum* for example has allowed the functional identification of 19 nuclear receptors (Tan and Palli, 2008) and injection of dsRNA targeted against aminopeptidase N in *Spodoptera litura* gave evidence of the function of this protein as a receptor for *Bacillus thuringiensis* insecticidal proteins (Rajagopal et al., 2002). Moreover, RNAi techniques have also been used in insect cell line such as in *S. frugiperda* Sf21 cell line through siRNA transfection to study the effect of baculovirus-expressed aminopeptidase (Agrawal et al., 2004), in Sf9 cells by stable expression of dsRNA through baculovirus-mediated expression vector (Lin et al., 2006) and in *Bombyx mori* cells using long-hairpin RNA-expressing plasmid DNA (Fujita et al., 2009).

In *Spodoptera frugiperda*, there are only two recorded studies in the literature showing evidence of successful RNAi silencing by injection and feeding of dsRNA to *S. frugiperda* larvae to study the role of allatostatin in JH signaling mechanisms (Griebler et al., 2008; Meyering-Vos et al., 2006). This suggests that this species is likely to be non sensitive to RNAi *in vivo* techniques as negative results are often not reported. Species sensitivity is a major drawback in RNAi functional analysis and it has been hypothesized that less derived species such as *Tribolium castaneum* are more sensitive to systemic RNAi than more derived species such as Lepidoptera (Belles, 2010). In addition, some tissues might be more resistant to respond to RNAi resulting from poor penetration and transmission of the interfering signal through cells and tissues, and RNAi can also have gene-specific sensitivity (Palli, R., personal communication). All these observations might help to understand our lack of reproducible results in *S. frugiperda* larvae and cells.
## 2. Bombyx mori transgenic construction

In an attempt to circumvent pitfalls encountered in *S. frugiperda* using RNAi techniques, we have worked in collaboration with the National Institute of Agrobiological Sciences in Tsukuba, Japan, to construct transgenic *Bombyx mori* that would express non-functional HR96 receptors in order to study the effect of HR96 impairment in this species. Dr. Tamura's team at the NIAS has developed a robust protocol to achieve stable genetic transformation of *Bombyx mori* larvae, this insect being used as a model organism to represent lepidopteran insects. The entire genomic sequence of *B. mori* was recently published (International Silkworm Genome Consortium, 2008). In *B. mori*, the techniques of piggyBac transposon-mediated transgenesis were established by the injection of DNA into preblastodermal embryos (Tamura et al., 2000). The binary GAL4/UAS system was constructed in *B. mori* (Imamura et al., 2003). In the system, the yeast transcriptional activator, GAL4, activates the gene downstream of UAS target sequence (see figure 27). Using the GAL4/UAS system, enhancer trap system in the silkworm has been developed recently (Uchino et al., 2008). The enhancer trap lines allow the study of gene expression or repression by RNAi in specific tissues/organ and at specific stages. Indeed, the lines that expressed GAL4 gene in the midgut were successfully used to determine the function of the yellow brood gene Y (Sakudoh et al., 2007) and densovirus-resistance gene nsd-2 (Ito et al., 2008).

<figure><img src="image_55.png" /><figcaption>**Figure 27** : the GAL4/UAS system principle. GAL4 transcription produces the GAL4 transcription factor that will bind to the specific UAS promoting sequence upstream of reporter gene, e.g. EGFP, inducing its transcription.</figcaption></figure>

We have therefore taken benefit from NIAS technical knowledge to knockdown HR96 in *B. mori* by two approaches.
First, we have conducted transgenic RNAi with GAL4/UAS system using a transgene of UAS-driven inverted repeat (UAS-IR) constructed by cloning the gene fragment of *BmHR96* as an inverted repeat, using *piggyBac* as a vector and *EGFP* gene as an eye marker (see figure 28 for the plamid map). The DNA of the UAS-IR transgene is then injected into the eggs of the silkworm with helper plasmid and mRNA of the transposase for germline transformation. When the UAS-IR transgenic line is obtained, it will be crossed with a midgut-GAL4 line. By this approach, we will knock-down the messenger of *BmHR96* in transgenic silkworms by expressing *BmHR96* IR under the control of UAS-GAL4 promotor.

<figure><img src="image_56.png" /><figcaption>(1-3)pBacMCS/UAS-SV40, 3#1B88B9</figcaption></figure>

**Figure 28:** restriction map of the plasmid DNA used as a vector for generating transgenic *B. mori*. ORF of *BmHR96* inserted into BlnI site.

Second, the same methodology was used to clone dominant negative (ΔAF-2) fragments of *BmHR96* into the *piggyBac* vector. Indeed, Kocarek and colleagues (2002) have shown that the deletion of the N-terminal AF-2 region of nuclear receptors is sufficient to prevent the formation of active transcription factor complex (Kocarek et al., 2002). They constructed expression plasmid carrying PXR, CAR and LXR nuclear receptor truncated from 8 to 10 amino acids in their AF-2 domain. When co-transfected with reporter plasmids, they showed a decrease in the expression of receptors-regulated genes, such as P450s. We have therefore conducted the same approach in *B. mori* with *BmHR96* receptor. First, we have successfully cloned the full sequence of *BmHR96*, which shows 79% identity with its *S. frugiperda* ortholog (figure 29). Then, we designed specific primers that allowed the deletion of 18 amino acids from the C-terminal part of the receptor by PCR technique, as it is shown in figure 29. The full-length and dominant negative ΔAF-2 fragments were then cloned into the *piggyBac* vector the same way than for IR of *BmHR96*. By this approach, we will knock-down the function of the protein by expressing non-functional dominant negative receptors under the control of UAS/GAL4 promotor into transgenic silkworms.
The resulting G0 moths are intercrossed to produce the G1 silkworm. The transgenic insects are obtained in the G1 silkworm by the marker screening. The UAS-IR strain is then crossed with the GAL4 strains that express GAL4 gene in the midgut.

<figure><img src="image_57.png" /><figcaption>**Figure 29**: alignment of the deduced amino acid sequence of BmHR96 with SfHR96. Conserved DBD (pink) and LBD (green) domains are indicated. The red boxed sequence of 18 amino acids at the C-terminal part represent the truncated part in dominant negative ΔAF-2 BmHR96.</figcaption></figure>

The cloning and construction of the full length and dominant negative mutants was realized during my 3 months stay in Dr Shinoda's laboratory at NIAS. Transgenic silkworms will be available in the upcoming weeks and experiments on P450 induction in dominant negative and RNAi transgenic *B. mori* will be conducted by the NIAS team members, in a continuing collaboration with our INRA laboratory.
As results, we would expect to see a decrease in induction level of P450 in dominant negative and RNAi transgenic *B. mori*. Such results will validate the function of HR96 as a key regulator in detoxification gene induction in insects.
# GENERAL CONCLUSION
The overall objective of this work was to study xenobiotic detoxification mechanisms in *Spodoptera frugiperda* and in particular to identify and characterize potential xenosensors in this polyphagous lepidopteran pest.

Phytophagous insect species encounter in their diet many plant allelochemicals and insecticides used in pest management. They have therefore developed sophisticated mechanisms to detoxify these potentially toxic compounds and survive in this chemically adverse environment. Such mechanisms are mainly based on the activity of detoxification enzymes, one of the best known being the cytochrome P450s. It is now well established that insect P450s are induced by and metabolize toxic plant allelochemicals and have been involved in insecticide resistance. However, a link is still missing between the ingestion of the toxin and the induction of P450s. Nuclear receptors have been shown in many vertebrate studies to be key transcription factors that would recognize xenobiotics and induce the expression of P450s. However, unlike the situation in vertebrate, to date there has been no clear evidence of such xenosensing receptor in insects. Our work aimed to fill this gap in our knowledge.

## The detoxification gene response

As a first step in studying the xenobiotic response in insects, we have identified patterns of gene expression in the model species *Drosophila melanogaster* by studying the expression of a specific P450, CYP6A2 in larval and adult tissues of *Drosophila* and in response to various xenobiotics. Our results compared to the available literature P450 induction that could be gleaned from microarray studies indicate that only a third of the "CYP-ome" is inducible by xenobitocis and that there are distinct subsets of inducers/induced genes, suggesting multiple xenobiotic transduction mechanisms.

We have then extended our study to the xenobiotic response of a polyphagous pest, *S. frugiperda* by identifying patterns of gene expression in response to various plant allelochemicals and xenobiotics using microarray analysis and measuring specific P450 gene induction by qRT-PCR analysis. Our results confirm the first study in *Drosophila* as only a small subset of P450s were induced by the different xenobiotics, showing different patterns of induction in *S. frugiperda* larvae and Sf9 cells. Overall, even though Sf9 cells could not recreate what is happening *in vivo*, both systems showed detoxification gene induction
responses as microarray analysis revealed with the expression of several detoxification
genes being affected by the different treatments.

Our results allowed us to identify which type of xenobiotic induced which type of P450. With this information being catalogued, it would now be particularly interesting to study the ability of these P450s to metabolize effectively the inducing compounds. Heterologous expression of P450s has been largely used to study the ability of these enzymes to metabolize insecticide and plant compounds. For example, CYP6B1 from *Helicoverpa zea* expressed in Sf9 cells through baculovirus-mediated expressing vector is able to metabolize different plant allelochemicals, such as quercetin and xanthotoxin as well as insecticide such as cypermethrin (Li et al., 2004). We have already undertaken the construction of yeast heterologous expression of the most induced P450s in *S. frugiperda* larvae. The analysis of their ability to efficiently metabolize each of their inducing compounds may show that the xenobiotic response of *S. frugiperda* is based on the induction of P450 metabolizing enzymes that allow this species to feed on toxic plant hosts.

The xenosensing nuclear receptors

In the second step of this work, we have studied the role of nuclear receptors in the xenobiotic response of *S. frugiperda* by first looking at the effects of two hormone agonists on Sf9 cells, methoprene and methoxyfenozide. Methoprene is an analog of the Juvenile Hormone (JH) and methoxyfenozide is an agonist of the moulting hormone 20-hydroxyecdysone (20E). Both compounds have been widely used in pest management controls as they mimic the action of the two major hormones controlling metamorphosis and development in homometabolous insects, thus interfering with hormone signaling and leading to defect in development of insects. Our results show that both insecticides induce cell growth arrest in the G1 phase for the methoxyfenozide and in the G2/M phase for the methoprene. Microarray analysis showed that the regulation of specific genes involved in cell cycle progression was affected by both of the insecticides.

If the mode of action of 20E is well-known, it remains more enigmatic for JH due to
the lack of knowledge of its receptor. 20E exerts it action through the binding to a nuclear
receptor heterodimer consisting of an ecdysone receptor (EcR) and ultraspiracle (USP). We
found that EcR was induced by the 20E agonist methoxyfenozide and by methoprene, the JH
analog induced the expression of USP. Our results suggets therefore two different signalling
pathways involved in Sf9 cells.

We showed that Sf9 cells respond to insecticide compounds that mimic the action of two
main hormones by stopping cell cycle progression and that nuclear receptors EcR and USP
might be involved in this signaling mechanism.

In the last step, we have therefore tried to identify nuclear receptors that would act as
xenosensors in *S. frugiperda* and regulate the expression of P450 genes in response to
xenobiotic rather than hormonal exposure. We have chosen a candidate gene approach by
focusing on the ortholog of DHR96, the vertebrate ortholog of CAR and PXR, for which
xenosensing function is well established and involvement in P450 expression well identified.
We have cloned the full-length sequence of SfHR96. We have shown that it was expressed
both in *S. frugiperda* midgut, fat body and Malpighian tubules, as well as in Sf9 cells. In the
latter case, this is not surprising as Sf9 cells have been shown to express both EcR and USP as
well as other nuclear receptors (Chen et al., 2002). However, SfHR96 expression was found
very low, with no changes throughout the developmental stages, except for a light peak of
expression just before the last instar moult, concomitant with a high expression of SfUSP-2.
In addition, SfHR96 expression was not induced by any of the xenobiotics tested, suggesting
that this receptor might be constitutively expressed and therefore able to be constantly
activated by xenobiotic compounds.

Our attempts to silence the expression of SfHR96 by RNAi experiments failed both in vitro
and in vivo, still raising the question of whether this receptor is involved in the P450 gene
induction. Further RNAi experiments should be undertaken in Sf9 cells with stable DNA
vector based expression of dsRNA as it has already been proven to be successful (Lin et al.,
2006). Moreover, dsRNA transfection presents advantages over siRNA as in the latter case
the silencing effects are often transient and the transfection efficiency of siRNA would
influence the silencing effects in target cells (Agrawal et al., 2004). In addition, RNAi effects
should be tested on many different xenobiotic compounds and different induced P450 genes
as RNAi could be gene specific and we could have missed effects on P450 gene expression by
focusing on only a few responding genes.
The work on transgenic *Bombyx mori* in collaboration with NIAS in Japan will bring new insights into the functional identification of HR96 in the detoxification response of Lepidoptera. Although *B. mori* is a specialist species in contrats to *S. frugiperda*, there is no doubt that we will find potent compounds that will induce the expression of some P450 genes in a similar way that in *Spodoptera* and study the effect of BmHR96 silencing on this induction. In addition, NIAS teams have developed technical knowledge to express DNA vector-based long hairpin RNA in *B. mori* cell line, allowing stable RNAi silencing of different genes (Fujita et al., 2009). We could therefore use this model to study the effect of HR96 impairment in *B. mori* cells if Sf9 cells remain resistant to that technique.

We think that SfHR96 is a good candidate for xenosensors in insects. Further characterization of the genes regulated by this receptor will help to validate our hypothesis.

DHR96 has already been shown to be involved on the regulation of phenobarbital-regulated genes (King-Jones et al., 2006) and despite the fact that this receptor has been involved as a sterol sensor in the regulation of cholesterol metabolism (Horner et al., 2009). The fact that HR96 mutants are fully viable in *Tribolium castaneum* (Tan and Palli, 2008) show that the presence of this receptor is actually not essential for metamorphosis and development and support the fact that this receptor might have two non-exclusive functions, not necessary only related to steroid mechanisms.

The characterization of this orphan receptor would also have to go through the identification of potential ligands as well as potential hetero-dimerization partners. Indeed, most nuclear receptors function as homo- or heterodimer with another member of the NR superfamily. This is for example the case for the EcR/USP complex involved in ecdysteroid mechanisms. In the case of HR96, no binding partners have yet been identified. It is known that USP can interact with another nuclear receptor, DHR38 (Baker et al., 2000), suggesting that USP might as well be involved as a heterodimerization partner for other nuclear receptors. USP is the vertebrate ortholog of the Retinoid X receptor (RXR), which is known to bind to CAR and PXR. As HR96 is the insect ortholog of these receptors, we can assume that USP would bind HR96 in similar manner. We have found that the expression of SfUSP-2 was somehow correlated with the peak of expression of SfHR96 in late 5^th^ instar larvae, as it has already been shown in *B. mori* (Cheng et al., 2008). However further experiments are needed to
validate our hypothesis of USP being a dimerization partner for SfHR96. RNAi technique could for example be used against SfUSP together with the extinction of SfHR96 in order to monitor if the presence of USP is necessary for the induction of P450 genes by SfHR96. This hypothesis is particularly attractive, as the recruitment of USP by HR96 upon xenobiotic exposure would implicate that USP is less available for binding with EcR. Thus xenobiotic effects would interfere with ecdysteroid signaling mechanisms and that could explain the developmental and metamorphic effects of some of these xenobiotics. Protein binding assay is another technique that could be used to measure the ability of USP to bind to SfHR96 as well as identifying potential ligands for the latter receptor. High-throughput techniques have been made available with the development of Surface Plasmon resonance (SPR) analysis known as the BIAcore technology, which allow the study of real-time protein-protein interactions and binding affinity. The use of such technology could help to find potential ligands for SfHR96 by measuring the binding affinities of a virtually infinite number of molecules, in addition to testing the ability of USP and SfHR96 to form a heterodimer.

Overall, the identification of SfHR96 as a xenosensor involved in the regulation of P450s in S. frugiperda will help to understand xenobiotic responses in insects by making the link between the ingestion of the toxin and the induction of detoxification mechanisms. In addition to unveiling how phytophagous insects survive in a chemically adverse environment, our findings could have potential implications in pest management. Indeed, xenosensing by intracellular receptors offers an innovative target as interfering with an insect's ability to induce the detoxification of plant secondary chemicals can have a beneficial impact. Lack of induction (antagonists) can cause toxicity or feeding arrest (and thus increased exposure to natural enemies), whereas enhanced induction (agonists) can favor the activation of plant pro-toxins. Furthermore, the sustainability of host plant resistance programs, often based on the genetic improvement of the plant chemical defenses, could be enhanced by xenosensor antagonists that would act as synergists. Elaboration on such strategies (plant-based or spray-based) is pointless without a thorough understanding of the insect xenosensors.
REFERENCES
Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne, J.D., Amanatides, P.G., Scherer, S.E., Li, P.W., Hoskins, R.A., Galle, R.F., et al. (2000). The genome sequence of *Drosophila melanogaster*. *Science* **287**, 2185-2195.

Agrawal, N., Malhotra, P., and Bhatnagar, R.K. (2004). siRNA-directed silencing of transgene expressed in cultured insect cells. *Biochem Biophys Res Commun* **320**, 428-434.

Antebi, A., Yeh, W.H., Tait, D., Hedgecock, E.M., and Riddle, D.L. (2000). daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age in *C. elegans*. *Genes Dev* **14**, 1512-1527.

Aranda, A., and Pascual, A. (2001). Nuclear hormone receptors and gene expression. *Physiol Rev* **81**, 1269-1304.

Ashok, M., Turner, C., and Wilson, T.G. (1998). Insect juvenile hormone resistance gene homology with the bHLH-PAS family of transcriptional regulators. *Proc Natl Acad Sci USA* **95**, 2761-2766.

Baker, K.D., Warren, J.T., Thummel, C.S., Gilbert, L.I., and Mangelsdorf, D.J. (2000). Transcriptional activation of the *Drosophila* ecdysone receptor by insect and plant ecdysteroids. *Insect Biochem Mol Biol* **30**, 1037-1043.

Belles, X. (2010). Beyond *Drosophila*: RNAi in vivo and functional genomics in insects. *Annu Rev Entomol* **55**, 111-128.

Berenbaum, M.R. (1990). Evolution of specialization in insect-umbellifer associations. *Annu Rev Entomol* **35**, 319-343.

Berenbaum, M.R. (2002). Postgenomic chemical ecology: from genetic code to ecological interactions. *J Chem Ecol* **28**, 873-896.

Berenbaum, M.R., Favret, C., and Schuler, M.A. (1996). On defining "key innovations" in an adaptive radiation: cytochrome P450s and papilionidae. *Am Nat* **148**, S139-S155.

Bitra, K., and Palli, S.R. (2009). Interaction of proteins involved in ecdysone and juvenile hormone signal transduction. *Arch Insect Biochem Physiol* **70**, 90-105.

Bonneton, F., Chaumot, A., and Laudet, V. (2008). Annotation of *Tribolium* nuclear receptors reveals an increase in evolutionary rate of a network controlling the ecdysone cascade. *Insect Biochem Mol Biol* **38**, 416-429.

Bourguet, W., Germain, P., and Gronemeyer, H. (2000). Nuclear receptor ligand-binding domains: three-dimensional structures, molecular interactions and pharmacological implications. *Trends Pharmacol Sci* **21**, 381-388.

Brattsten, L.B. (1979). Ecological significance of mixed-function oxidations. *Drug Metab Rev* **10**, 35-58.

Brattsten, L.B., Evans, C.K., Bonetti, S., and Zalkow, L.H. (1984). Induction by carrot allelochemicals of insecticide-metabolising enzymes in the southern armyworm *Spodoptera eridania*. *Comp Biochem Physiol C* **77**, 29-37.
Brown, R.P., McDonnell, C.M., Berenbaum, M.R., and Schuler, M.A. (2005). Regulation of an insect cytochrome P450 monooxygenase gene (CYP6B1) by aryl hydrocarbon and xanthotoxin response cascades. *Gene* **358**, 39-52.

Brun, A., Cuany, A., Le Mouel, T., Berge, J., and Amichot, M. (1996). Inducibility of the *Drosophila melanogaster* cytochrome P450 gene, CYP6A2, by phenobarbital in insecticide susceptible or resistant strains. *Insect Biochem Mol Biol* **26**, 697-703.

Bujold, M., Gopalakrishnan, A., Nally, E., and King-Jones, K. Nuclear receptor DHR96 acts as a sentinel for low cholesterol concentrations in *Drosophila melanogaster*. *Mol Cell Biol* **30**, 793-805.

Bull, D.L., Ivie, G.W., Beier, R.C., and Pryor, N.W. (1986). *In vitro* metabolism of a linear furanocoumarin (8-methoxypsoralen, xanthotoxin) by mixed function oxidases of larvae of black swallowtail butterfly and fall armyworm. *J Chem Ecol* **12**, 885-892.

Chen, J.H., Turner, P.C., and Rees, H.H. (2002). Molecular cloning and induction of nuclear receptors from insect cell lines. *Insect Biochem Mol Biol* **32**, 657-667.

Cheng, D., Xia, Q., Duan, J., Wei, L., Huang, C., Li, Z., Wang, G., and Xiang, Z. (2008). Nuclear receptors in *Bombyx mori*: insights into genomic structure and developmental expression. *Insect Biochem Mol Biol* **38**, 1130-1137.

Claessens, F., and Gewirth, D.T. (2004). DNA recognition by nuclear receptors. *Essays Biochem* **40**, 59-72.

Cohen, M.B., Berenbaum, M.R., and Schuler, M.A. (1989). Induction of cytochrome P450-mediated detoxification of xanthotoxin in the black swallowtail. *J Chem Ecol* **15**, 2347-2355.

Cohen, M.B., Schuler, M.A., and Berenbaum, M.R. (1992). A host-inducible cytochrome P-450 from a host-specific caterpillar: molecular cloning and evolution. *Proc Natl Acad Sci U S A* **89**, 10920-10924.

Cruz, J., Sieglaff, D.H., Arensburger, P., Atkinson, P.W., and Raikhel, A.S. (2009). Nuclear receptors in the mosquito *Aedes aegypti*: annotation, hormonal regulation and expression profiling. *FEBS J* **276**, 1233-1254.

Daborn, P., Boundy, S., Yen, J., Pittendrigh, B., and Ffrench-Constant, R. (2001). DDT resistance in *Drosophila* correlates with *Cyp6g1* over-expression and confers cross-resistance to the neonicotinoid imidacloprid. *Mol Genet Genomics* **266**, 556-563.

Daborn, P.J., Lumb, C., Boey, A., Wong, W., Ffrench-Constant, R.H., and Batterham, P. (2007). Evaluating the insecticide resistance potential of eight *Drosophila melanogaster* cytochrome P450 genes by transgenic over-expression. *Insect Biochem Mol Biol* **37**, 512-519.

Daborn, P.J., Yen, J.L., Bogwitz, M.R., Le Goff, G., Feil, E., Jeffers, S., Tijet, N., Perry, T., Heckel, D., Batterham, P., et al. (2002). A single p450 allele associated with insecticide resistance in *Drosophila*. *Science* **297**, 2253-2256.
Denison, M.S., Fisher, J.M., and Whitlock, J.P., Jr. (1988). The DNA recognition site for the dioxin-Ah receptor complex. Nucleotide sequence and functional analysis. *J Biol Chem* **263**, 17221-17224.

Denison, M.S., and Nagy, S.R. (2003). Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. *Annu Rev Pharmacol Toxicol* **43**, 309-334.

Despres, L., David, J.P., and Gallet, C. (2007). The evolutionary ecology of insect resistance to plant chemicals. *Trends Ecol Evol* **22**, 298-307.

Dhadialla, T.S., Retnakaran, A., and Smagghe, G. (2005). Insect growth- and development-disrupting insecticides. In *Comprehensive molecular insect science* (Elsevier), pp. 55-115.

Dicke, M. (2000). Chemical ecology of host-plant selection by herbivorous arthropods: a multitrophic perspective. *Biochem Syst Ecol* **28**, 601-617.

Dobson, C.M. (2004). Chemical space and biology. *Nature* **432**, 824-828.

Duncan, D.M., Burgess, E.A., and Duncan, I. (1998). Control of distal antennal identity and tarsal development in *Drosophila* by spineless-aristapedia, a homolog of the mammalian dioxin receptor. *Genes Dev* **12**, 1290-1303.

Dunkov, B.C., Guzov, V.M., Mocelin, G., Shotkoski, F., Brun, A., Amichot, M., Ffrench-Constant, R.H., and Feyereisen, R. (1997). The *Drosophila* cytochrome P450 gene Cyp6a2: structure, localization, heterologous expression, and induction by phenobarbital. *DNA Cell Biol* **16**, 1345-1356.

Emmons, R.B., Duncan, D., Estes, P.A., Kiefel, P., Mosher, J.T., Sonnenfeld, M., Ward, M.P., Duncan, I., and Crews, S.T. (1999). The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in *Drosophila*. *Development* **126**, 3937-3945.

Enmark, E., and Gustafsson, J.A. (2001). Comparing nuclear receptors in worms, flies and humans. *Trends Pharmacol Sci* **22**, 611-615.

Escriva, H., Delaunay, F., and Laudet, V. (2000). Ligand binding and nuclear receptor evolution. *Bioessays* **22**, 717-727.

Escriva, H., Safi, R., Hanni, C., Langlois, M.C., Saumitou-Laprade, P., Stehelin, D., Capron, A., Pierce, R., and Laudet, V. (1997). Ligand binding was acquired during evolution of nuclear receptors. *Proc Natl Acad Sci U S A* **94**, 6803-6808.

Estabrook, R.W., Cooper, D.Y., and Rosenthal, O. (1963). The Light Reversible Carbon Monoxide Inhibition of the Steroid C21-Hydroxylase System of the Adrenal Cortex. *Biochem Z* **338**, 741-755.

Feyereisen, R. (1999). Insect P450 enzymes. *Annu Rev Entomol* **44**, 507-533.

Feyereisen, R. (2005). Insect cytochrome P450. In *Comprehensive molecular insect science* (Elsevier), pp. 1-77.
Feyereisen, R. (2006). Evolution of insect P450. *Biochem Soc Trans* **34**, 1252-1255.

Feyereisen, R., Koener, J.F., Farnsworth, D.E., and Nebert, D.W. (1989). Isolation and sequence of cDNA encoding a cytochrome P-450 from an insecticide-resistant strain of the house fly, *Musca domestica*. *Proc Natl Acad Sci U S A* **86**, 1465-1469.

Fisk, G.J., and Thummel, C.S. (1995). Isolation, regulation, and DNA-binding properties of three *Drosophila* nuclear hormone receptor superfamily members. *Proc Natl Acad Sci U S A* **92**, 10604-10608.

Fujii-Kuriyama, Y., and Mimura, J. (2005). Molecular mechanisms of AhR functions in the regulation of cytochrome P450 genes. *Biochem Biophys Res Commun* **338**, 311-317.

Fujita, K., Sagisaka, A., Tomimoto, K., Ishibashi, J., Imanishi, S., Yamakawa, M., and Tanaka, H. (2009). DNA vector-based RNA interference in cell lines derived from *Bombyx mori*. *Biosci Biotechnol Biochem* **73**, 2026-2031.

Garfinkel, D. (1958). Studies on pig liver microsomes. I. Enzymic and pigment composition of different microsomal fractions. *Arch Biochem Biophys* **77**, 493-509.

Gatehouse, J.A. (2002). Plant resistance towards insect herbivores: a dynamic interaction. *New Phytol* **156**, 145-169.

Germain, P., Staels, B., Dacquet, C., Spedding, M., and Laudet, V. (2006). Overview of nomenclature of nuclear receptors. *Pharmacol Rev* **58**, 685-704.

Giguere, V. (1999). Orphan nuclear receptors: from gene to function. *Endocr Rev* **20**, 689-725.

Gilbert, L.I., and Warren, J.T. (2005). A molecular genetic approach to the biosynthesis of the insect steroid molting hormone. *Vitam Horm* **73**, 31-57.

Girardot, F., Monnier, V., and Tricoire, H. (2004). Genome wide analysis of common and specific stress responses in adult *Drosophila melanogaster*. *BMC Genomics* **5**, 74.

Gould, F., and Hodgson, E. (1980). Mixed function oxidase and glutathione transferase activity in last instar *Heliothis virescens* larvae. *Pest Biochem Physiol* **13**, 34-40.

Graham, S.E., and Peterson, J.A. (1999). How similar are P450s and what can their differences teach us? *Arch Biochem Biophys* **369**, 24-29.

Griebler, M., Westerlund, S.A., Hoffmann, K.H., and Meyering-Vos, M. (2008). RNA interference with the allatoregulating neuropeptide genes from the fall armyworm *Spodoptera frugiperda* and its effects on the JH titer in the hemolymph. *J Insect Physiol* **54**, 997-1007.

Gronemeyer, H., Gustafsson, J.A., and Laudet, V. (2004). Principles for modulation of the nuclear receptor superfamily. *Nat Rev Drug Discov* **3**, 950-964.

Gueguen, Y., Mouzat, K., Ferrari, L., Tissandie, E., Lobaccaro, J.M., Batt, A.M., Paquet, F., Voisin, P., Aigueperse, J., Gourmelon, P., et al. (2006). [Cytochromes P450: xenobiotic metabolism, regulation and clinical importance]. *Ann Biol Clin (Paris)* **64**, 535-548.
Guengerich, F.P. (2001). Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. *Chem Res Toxicol* **14**, 611-650.

Gunderson, C.A., Brattsten, L.B., and Fleming, J.T. (1986). Microsomal oxidase and glutathione transferase as factors influencing the effects of pulegone in southern and fall armyworm larvae. *Pest Biochem Physiol* **26**, 238-249.

Hahn, M.E. (2002). Aryl hydrocarbon receptors: diversity and evolution. *Chem Biol Interact* **141**, 131-160.

Handschin, C., and Meyer, U.A. (2003). Induction of drug metabolism: the role of nuclear receptors. *Pharmacol Rev* **55**, 649-673.

Hartmann, T. (2004). Plant-derived secondary metabolites as defensive chemicals in herbivorous insects: a case study in chemical ecology. *Planta* **219**, 1-4.

Helvig, C., Koener, J.F., Unnithan, G.C., and Feyereisen, R. (2004). CYP15A1, the cytochrome P450 that catalyzes epoxidation of methyl farnesoate to juvenile hormone III in cockroach corpora allata. *Proc Natl Acad Sci U S A* **101**, 4024-4029.

Holt, R.A., Subramanian, G.M., Halpern, A., Sutton, G.G., Charlab, R., Nusskern, D.R., Wincker, P., Clark, A.G., Ribeiro, J.M., Wides, R., et al. (2002). The genome sequence of the malaria mosquito *Anopheles gambiae*. *Science* **298**, 129-149.

Honkakoski, P., and Negishi, M. (2000). Regulation of cytochrome P450 (CYP) genes by nuclear receptors. *Biochem J* **347**, 321-337.

Honkakoski, P., Zelko, I., Sueyoshi, T., and Negishi, M. (1998). The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. *Mol Cell Biol* **18**, 5652-5658.

Horner, M.A., Pardee, K., Liu, S., King-Jones, K., Lajoie, G., Edwards, A., Krause, H.M., and Thummel, C.S. (2009). The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis. *Genes Dev* **23**, 2711-2716.

Hung, C.F., Harrison, T.L., Berenbaum, M.R., and Schuler, M.A. (1995a). CYP6B3: a second furanocoumarin-inducible cytochrome P450 expressed in *Papilio polyxenes*. *Insect Mol Biol* **4**, 149-160.

Hung, C.F., Prapaipong, H., Berenbaum, M.R., and Schuler, M.A. (1995b). Differential induction of cytochrome P450 transcripts in *Papilio polyxenes* by linear and angular furanocoumarins. *Insect Mol Biol* **25**, 89-99.

Imamura, M., Nakai, J., Inoue, S., Quan, G.X., Kanda, T., and Tamura, T. (2003). Targeted gene expression using the GAL4/UAS system in the silkworm *Bombyx mori*. *Genetics* **165**, 1329-1340.

International Silkworm Genome Consortium (2008). The genome of a lepidopteran model insect, the silkworm *Bombyx mori*. *Insect Biochem Mol Biol* **38**(12):1036-45.

Ito, K., Kidokoro, K., Sezutsu, H., Nohata, J., Yamamoto, K., Kobayashi, I., Uchino, K., Kalyebi, A., Eguchi, R., Hara, W., et al. (2008). Deletion of a gene encoding an amino acid transporter
in the midgut membrane causes resistance to a *Bombyx* parvo-like virus. Proc Natl Acad Sci U S A **105**, 7523-7527.

Jakoby, W.B., and Ziegler, D.M. (1990). The enzymes of detoxication. J Biol Chem **265**, 20715-20718.

Jindra, M., Huang, J.Y., Malone, F., Asahina, M., and Riddiford, L.M. (1997). Identification and mRNA developmental profiles of two ultraspiracle isoforms in the epidermis and wings of *Manduca sexta*. Insect Mol Biol **6**, 41-53.

Johnson, E.F., Wester, M.R., and Stout, C.D. (2002). The structure of microsomal cytochrome P450 2C5: a steroid and drug metabolizing enzyme. Endocr Res **28**, 435-441.

Jones, G., Jones, D., Teal, P., Sapa, A., and Wozniak, M. (2006). The retinoid-X receptor ortholog, ultraspiracle, binds with nanomolar affinity to an endogenous morphogenetic ligand. Febs J **273**, 4983-4996.

Jones, G., and Sharp, P.A. (1997). Ultraspiracle: an invertebrate nuclear receptor for juvenile hormones. Proc Natl Acad Sci U S A **94**, 13499-13503.

Kawamoto, T., Sueyoshi, T., Zelko, I., Moore, R., Washburn, K., and Negishi, M. (1999). Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the *CYP2B* gene. Mol Cell Biol **19**, 6318-6322.

King-Jones, K., Horner, M.A., Lam, G., and Thummel, C.S. (2006). The DHR96 nuclear receptor regulates xenobiotic responses in *Drosophila*. Cell Metab **4**, 37-48.

King-Jones, K., and Thummel, C.S. (2005). Nuclear receptors - a perspective from *Drosophila*. Nat Rev Genet **6**, 311-323.

Kliewer, S.A., Moore, J.T., Wade, L., Staudinger, J.L., Watson, M.A., Jones, S.A., McKee, D.D., Oliver, B.B., Willson, T.M., Zetterstrom, R.H., et al. (1998). An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell **92**, 73-82.

Klingenberg, M. (1958). Pigments of rat liver microsomes. Arch Biochem Biophys **75**, 376-386.

Kocarek, T.A., Shenoy, S.D., Mercer-Haines, N.A., and Runge-Morris, M. (2002). Use of dominant negative nuclear receptors to study xenobiotic-inducible gene expression in primary cultured hepatocytes. J Pharmacol Toxicol Methods **47**, 177-187.

Koch, M.A., Schuffenhauer, A., Scheck, M., Wetzel, S., Casaulta, M., Odermatt, A., Ertl, P., and Waldmann, H. (2005). Charting biologically relevant chemical space: a structural classification of natural products (SCONP). Proc Natl Acad Sci U S A **102**, 17272-17277.

Koelle, M.R., Talbot, W.S., Segraves, W.A., Bender, M.T., Cherbas, P., and Hogness, D.S. (1991). The *Drosophila* EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell **67**, 59-77.

Kretschmer, X.C., and Baldwin, W.S. (2005). CAR and PXR: xenosensors of endocrine disrupters? Chem Biol Interact **155**, 111-128.
Krust, A., Green, S., Argos, P., Kumar, V., Walter, P., Bornert, J.M., and Chambon, P. (1986). The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors. EMBO J **5**, 891-897.

Kuhnel, K., Ke, N., Cryle, M.J., Sligar, S.G., Schuler, M.A., and Schlichting, I. (2008). Crystal structures of substrate-free and retinoic acid-bound cyanobacterial cytochrome P450 CYP120A1. *Biochemistry* **47**, 6552-6559.

Kumar, R., and Litwack, G. (2009). Structural and functional relationships of the steroid hormone receptors' N-terminal transactivation domain. *Steroids* **74**, 877-883.

Kumar, R., and Thompson, E.B. (1999). The structure of the nuclear hormone receptors. *Steroids* **64**, 310-319.

Laudet, V., and Bonneton, F. (2005). evolution of nuclear hormone receptors in insects. In *Comprehensive molecular insect science* (Elsevier), pp. 287-318.

Lavery, D.N., and McEwan, I.J. (2005). Structure and function of steroid receptor AF1 transactivation domains: induction of active conformations. *Biochem J* **391**, 449-464.

Le Goff, G., Boundy, S., Daborn, P.J., Yen, J.L., Sofer, L., Lind, R., Sabourault, C., Madi-Ravazzi, L., and ffrench-Constant, R.H. (2003). Microarray analysis of cytochrome P450 mediated insecticide resistance in *Drosophila*. *Insect Biochem Mol Biol* **33**, 701-708.

Le Goff, G., Hilliou, F., Siegfried, B.D., Boundy, S., Wajnberg, E., Sofer, L., Audant, P., ffrench-Constant, R.H., and Feyereisen, R. (2006). Xenobiotic response in *Drosophila melanogaster*: sex dependence of P450 and GST gene induction. *Insect Biochem Mol Biol* **36**, 674-682.

Lehmann, J.M., McKee, D.D., Watson, M.A., Willson, T.M., Moore, J.T., and Kliewer, S.A. (1998). The human orphan nuclear receptor PXR is activated by compounds that regulate *CYP3A4* gene expression and cause drug interactions. *J Clin Invest* **102**, 1016-1023.

Li, W., Petersen, R.A., Schuler, M.A., and Berenbaum, M.R. (2002). CYP6B cytochrome p450 monooxygenases from *Papilio canadensis* and *Papilio glaucus*: potential contributions of sequence divergence to host plant associations. *Insect Mol Biol* **11**, 543-551.

Li, W., Schuler, M.A., and Berenbaum, M.R. (2003). Diversification of furanocoumarin-metabolizing cytochrome P450 monooxygenases in two papilionids: Specificity and substrate encounter rate. *Proc Natl Acad Sci U S A* **100** Suppl 2, 14593-14598.

Li, X., Baudry, J., Berenbaum, M.R., and Schuler, M.A. (2004). 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, M.A., and Berenbaum, M.R. (2007). Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. *Annu Rev Entomol* **52**, 231-253.

Lin, C.C., Hsu, J.T., Huang, K.L., Tang, H.K., and Lai, Y.K. (2006). Stable RNA interference in *Spodoptera frugiperda* cells by a DNA vector-based method. *Biotechnol Lett* **28**, 271-277.

Lindblom, T.H., Pierce, G.J., and Sluder, A.E. (2001). A *C. elegans* orphan nuclear receptor contributes to xenobiotic resistance. *Curr Biol* **11**, 864-868.
Lipinski, C., and Hopkins, A. (2004). Navigating chemical space for biology and medicine. *Nature* **432**, 855-861.

Ma, R., Cohen, M.B., Berenbaum, M.R., and Schuler, M.A. (1994). Black swallowtail (*Papilio polyxenes*) alleles encode cytochrome P450s that selectively metabolize linear furanocoumarins. *Arch Biochem Biophys* **310**, 332-340.

Maglich, J.M., Stoltz, C.M., Goodwin, B., Hawkins-Brown, D., Moore, J.T., and Kliewer, S.A. (2002). Nuclear pregnane X receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification. *Mol Pharmacol* **62**, 638-646.

Mangelsdorf, D.J., and Evans, R.M. (1995). The RXR heterodimers and orphan receptors. *Cell* **83**, 841-850.

Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., et al. (1995). The nuclear receptor superfamily: the second decade. *Cell* **83**, 835-839.

Mansuy, D. (1998). The great diversity of reactions catalyzed by cytochromes P450. *Comp Biochem Physiol C Pharmacol Toxicol Endocrinol* **121**, 5-14.

Mao, W., Rupasinghe, S.G., Johnson, R.M., Zangerl, A.R., Schuler, M.A., and Berenbaum, M.R. (2009). Quercetin-metabolizing CYP6AS enzymes of the pollinator *Apis mellifera* (Hymenoptera: Apidae). *Comp Biochem Physiol B Biochem Mol Biol* **154**, 427-434.

McDonnell, C.M., Brown, R.P., Berenbaum, M.R., and Schuler, M.A. (2004). Conserved regulatory elements in the promoters of two allelochemical-inducible cytochrome P450 genes differentially regulate transcription. *Insect Biochem Mol Biol* **34**, 1129-1139.

McEwan, I.J. (2004). Molecular mechanisms of androgen receptor-mediated gene regulation: structure-function analysis of the AF-1 domain. *Endocr Relat Cancer* **11**, 281-293.

Meyering-Vos, M., Merz, S., Sertkol, M., and Hoffmann, K.H. (2006). Functional analysis of the allatostatin-A type gene in the cricket *Gryllus bimaculatus* and the armyworm *Spodoptera frugiperda*. *Insect Biochem Mol Biol* **36**, 492-504.

Miura, K., Oda, M., Makita, S., and Chinzei, Y. (2005). Characterization of the *Drosophila* Methoprene -tolerant gene product. Juvenile hormone binding and ligand-dependent gene regulation. *FEBS J* **272**, 1169-1178.

Moore, D.D., Kato, S., Xie, W., Mangelsdorf, D.J., Schmidt, D.R., Xiao, R., and Kliewer, S.A. (2006). International Union of Pharmacology. LXII. The NR1H and NR1I receptors: constitutive androstane receptor, pregnene X receptor, farnesoid X receptor alpha, farnesoid X receptor beta, liver X receptor alpha, liver X receptor beta, and vitamin D receptor. *Pharmacol Rev* **58**, 742-759.

Moreau, A., Maurel, P., Vilarem, M.J., and Pascussi, J.M. (2007). Constitutive androstane receptor-vitamin D receptor crosstalk: consequence on CYP24 gene expression. *Biochem Biophys Res Commun* **360**, 76-82.
Murataliev, M.B., Guzov, V.M., Walker, F.A., and Feyereisen, R. (2008). P450 reductase and cytochrome b5 interactions with cytochrome P450: effects on house fly *CYP6A1* catalysis. *Insect Biochem Mol Biol* **38**, 1008-1015.

Nakagawa, Y., and Henrich, V.C. (2009). Arthropod nuclear receptors and their role in molting. *FEBS J* **276**, 6128-6157.

Neal, J.J. (1987). Metabolic costs of mixed-function oxidase induction in *Heliothis zea*. *Entomol exp appl* **43**, 175-179.

Nebert, D.W., Adesnik, M., Coon, M.J., Estabrook, R.W., Gonzalez, F.J., Guengerich, F.P., Gunsalus, I.C., Johnson, E.F., Kemper, B., Levin, W., et al. (1987). The P450 gene superfamily: recommended nomenclature. *DNA* **6**, 1-11.

Nebert, D.W., Dalton, T.P., Okey, A.B., and Gonzalez, F.J. (2004). Role of aryl hydrocarbon receptor-mediated induction of the CYP1 enzymes in environmental toxicity and cancer. *J Biol Chem* **279**, 23847-23850.

Nebert, D.W., and Nelson, D.R. (1991). P450 gene nomenclature based on evolution. *Methods Enzymol* **206**, 3-11.

Nebert, D.W., Nelson, D.R., Adesnik, M., Coon, M.J., Estabrook, R.W., Gonzalez, F.J., Guengerich, F.P., Gunsalus, I.C., Johnson, E.F., Kemper, B., et al. (1989). The P450 superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci. *DNA* **8**, 1-13.

Nebert, D.W., and Russell, D.W. (2002). Clinical importance of the cytochromes P450. *Lancet* **360**, 1155-1162.

Nelson, D.R. (1999). Cytochrome P450 and the individuality of species. *Arch Biochem Biophys* **369**, 1-10.

Nelson, D.R., Kamataki, T., Waxman, D.J., Guengerich, F.P., Estabrook, R.W., Feyereisen, R., Gonzalez, F.J., Coon, M.J., Gunsalus, I.C., Gotoh, O., et al. (1993). The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. *DNA Cell Biol* **12**, 1-51.

Nelson, D.R., Koymans, L., Kamataki, T., Stegeman, J.J., Feyereisen, R., Waxman, D.J., Waterman, M.R., Gotoh, O., Coon, M.J., Estabrook, R.W., et al. (1996). P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. *Pharmacogenetics* **6**, 1-42.

Nuclear Receptors Nomenclature Committee (1999). A unified nomenclature system for the nuclear receptor superfamily. *Cell* **97**, 161-163.

Olefsky, J.M. (2001). Nuclear receptor minireview series. *J Biol Chem* **276**, 36863-36864.

Omura, T., and Sato, R. (1964). The Carbon Monoxide-Binding Pigment of Liver Microsomes. *I. Evidence for Its Hemoprotein Nature*. *J Biol Chem* **239**, 2370-2378.

Oppenoorth, F.J. (1984). Biochemistry of insecticide resistance. *Pest Biochem Physiol* **22**, 187-193.
Oro, A.E., McKeown, M., and Evans, R.M. (1990). Relationship between the product of the *drosophila* ultraspiracle locus and the vertebrate retinoid X receptor. *Nature* **347**, 298-301.

Palanker, L., Necakov, A.S., Sampson, H.M., Ni, R., Hu, C., Thummel, C.S., and Krause, H.M. (2006). Dynamic regulation of Drosophila nuclear receptor activity *in vivo*. *Development* **133**, 3549-3562.

Pascussi, J.M., Gerbal-Chaloin, S., Duret, C., Daujat-Chavanieu, M., Vilarem, M.J., and Maurel, P. (2008). The tangle of nuclear receptors that controls xenobiotic metabolism and transport: crosstalk and consequences. *Annu Rev Pharmacol Toxicol* **48**, 1-32.

Petersen, R.A., Niamsup, H., Berenbaum, M.R., and Schuler, M.A. (2003). Transcriptional response elements in the promoter of CYP6B1, an insect P450 gene regulated by plant chemicals. *Biochim Biophys Acta* **1619**, 269-282.

Petersen, R.A., Zangerl, A.R., Berenbaum, M.R., and Schuler, M.A. (2001). Expression of CYP6B1 and CYP6B3 cytochrome P450 monooxygenases and furanocoumarin metabolism in different tissues of *Papilio polyxenes* (Lepidoptera: Papilionidae). *Insect Biochem Mol Biol* **31**, 679-690.

Petryk, A., Warren, J.T., Marques, G., Jarcho, M.P., Gilbert, L.I., Kahler, J., Parvy, J.P., Li, Y., Dauphin-Villemant, C., and O'Connor, M.B. (2003). Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone. *Proc Natl Acad Sci U S A* **100**, 13773-13778.

Poulos, T.L., Finzel, B.C., and Howard, A.J. (1986). Crystal structure of substrate-free *Pseudomonas putida* cytochrome P-450. *Biochemistry* **25**, 5314-5322.

Prapaipong, H., Berenbaum, M.R., and Schuler, M.A. (1994). Transcriptional regulation of the *Papilio polyxenes* CYP6B1 gene. *Nucleic Acids Res* **22**, 3210-3217.

Rajagopal, R., Sivakumar, S., Agrawal, N., Malhotra, P., and Bhatnagar, R.K. (2002). Silencing of midgut aminopeptidase N of *Spodoptera litura* by double-stranded RNA establishes its role as *Bacillus thuringiensis* toxin receptor. *J Biol Chem* **277**, 46849-46851.

Rates, S.M. (2001). Plants as source of drugs. *Toxicon* **39**, 603-613.

Rausher, M.D. (2001). Co-evolution and plant resistance to natural enemies. *Nature* **411**, 857-864.

Ravichandran, K.G., Boddupalli, S.S., Hasermann, C.A., Peterson, J.A., and Deisenhofer, J. (1993). Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450's. *Science* **261**, 731-736.

Ray, J.W. (1967). The epoxidation of aldrin by housefly microsomes and its inhibition by carbon monoxide. *Biochem Pharmacol* **16**, 99-107.

Remmer, H., and Merker, H.J. (1963). Drug-Induced Changes in the Liver Endoplasmic Reticulum: Association with Drug-Metabolizing Enzymes. *Science* **142**, 1657-1658.

Rendic, S. (2002). Summary of information on human CYP enzymes: human P450 metabolism data. *Drug Metab Rev* **34**, 83-448.