Insect Antifeedant Benzofurans from Pericallis Species

In this work, we have studied the benzofurans of Pericallis echinata (aerial parts and transformed roots), P. steetzii (aerial parts and transformed roots), P. lanata (aerial parts), and P. murrayi (aerial parts and roots). This work has permitted the isolation of the new benzofurans 10-ethoxy-11-hydroxy-10,11-dihydroeuparin (10), (-)-eupachinin A ethyl ether (12), 11,15-didehydro-eupachinin A (13), 10,12-dihydroxy-11-angelyloxy-10,11-dihydroeuparin (14), 2,4-dihydroxy-5-formyl-acetophenone (15) isolated for the first time as a natural product, 11-angelyloxy-10,11-dihydroeuparin (16), and 12-angelyloxyeuparone (17), along with several known ones (1–9, 11). In addition, the incubation of the abundant component, 6-hydroxytremetone (1), with the fungus Mucor plumbeus has been studied. Benzofurans in the tremetone series (1, 1a, 2–5, 18, 18a), the euparin series (6, 7, 7a, 8–10, 14, 16), and the eupachinin-type (11, 12) were tested for antifeedant effects against the insect Spodoptera littoralis. The antifeedant compounds (1, 4, 6, 11, 12) were further tested for postingestive effects on S. littoralis larvae. The most antifeedant compounds were among the tremetone series, with 3-ethoxy-hydroxy-tremetone (4) being the strongest antifeedant. Glucosylation of 1 by its biotransformation with Mucor plumbeus gave inactive products. Among the euparin series, the dihydroxyangelate 14 was the most active, followed by euparin (6). The eupachinin-type compounds (11, 12) were both antifeedants. Compounds 4, 11, and 12 showed antifeedant effects without postingestive toxicity to orally dosed S. littoralis larvae. Euparin (6) had postingestive toxicity that was enhanced by the synergist piperonyl butoxide.


Introduction
Pericallis, a genus of the tribe Senecioneae (subtribe Senecioninae) in the Asteraceae family, comprises seventeen taxa of woody and herbaceous species which are endemic to Canary Islands, Madeira, and Azores [1]. The name Pericallis was introduced by Don in 1834 for P. tussilaginis [2] but was not used until 134 years later when Pericallis was considered close to the African Cineraria and several Cineraria and Senecio species were included in it [3]. However, a study on the molecular phylogeny of Pericallis found it closer to the American Packera genus [4]. Later, the monophyly of Pericallis was strongly supported and, again, a close relation with the African Cineraria genus was suggested considering morphologic and molecular ITS data [5]. More recently, the influence of geographical isolation, multiple habitat shifts, and hybridization in the evolution of this genus has been studied, using more taxon's sampling and studying both nuclear and chloroplast genomes' data [1,6].
Senecio and Pericallis species produce pyrrolizidine alkaloids (PAs) [7]. PAs are considered feeding deterrents for insect herbivores. A number of insect species from different taxa have evolved adaptations to sequester, store, and utilize plant PAs against their predators and parasitoids [8,9].
Ethanolic extracts of some PA-producing plants from the Canary Islands, including Pericallis (P. appendiculata, P. echinata, P. hansenii, P. multiflora, P. steetzii), were overall more active insect antifeedants than the alkaloidal ones. Considering that the alkaloidal fraction of these ethanolic extracts accounted for a maximum of 2%, the authors concluded that the chemistry of the non-alkaloidal fraction could explain most of their antifeedant effects [7]. Non-alkaloidal fractions of Senecio (and former Senecio such as the Canarian endemism Bethencourtia) species contain insect antifeedant sesquiterpenoids with different skeletons [10][11][12][13][14]. However, little is known on the non-alkaloidal defensive chemistry of Pericallis spp.

Components of Pericallis
All the species studied contained benzofurans ( Figure 1) as the major components, along with triterpenoids and sterols. Most benzofuran-type sesquiterpenes have been isolated from Asteraceae species [15], with a few examples from Senecio [11], but none have been reported from Pericallis.
The high-resolution mass spectrum (HREIMS) of compound 11 showed the molecular ion at m/z 274.0834 (C 15 H 14 O 5 ). In its 1 H NMR spectrum were observed signals corresponding to the ring A of a 6-hydroxy-5-acetyl benzofuran grouping. Other resonances present in it were a methyl geminal to an oxygenated function at δ 1.78 and two methylene groups located in contiguous positions. The hydrogens of one of them resonate at δ 2.63 (1H, ddd, J = 17.0, 6.5 and 5.0 Hz, H-15β) and 2.92 (1H, ddd, J = 17.0, 9.0 and 5.0 Hz, H-15α), while that those of the second methylene, coupled with the previous one, appear centred at δ 2.36 (m, 2H-11). In its 13 C NMR spectrum were detected signals of two methyls, two methylenes, two aromatic protons, and two carbonyl groups, one the acetyl group on C-5 to δ 194.1 and another of an α,β-unsaturated carbonyl group at δ 204.6 (C-16). Moreover, a quaternary carbon bearing an oxygen atom at δ 67.4 (C-10) and other six unsaturated quaternary carbons also appear in this spectrum.
The location of the different functional groups was established based on the connectivity observed in the HSQC and HMBC spectra. The correlations in the HMBC experiment of the aromatic protons and the phenolic proton, with their respective geminal and neighbourhood carbons, confirmed that the ring A of the molecule was similar to that of other benzofurans of euparin type. This spectrum also indicated that the two methylene groups were located between the carbonyl group and the quaternary carbon bearing an oxygen atom. Thus, connectivities of H-11 with C-2, C-10, and C-15, of the two H-15 with C-10, C-11, and C-16, and of methyl group (H-12) with C-2, C-10, and C-11 were observed. These spectroscopic data allowed us to assign to our product the structure 11 [37]. To confirm this hypothesis, we submit it to an X-ray diffraction analysis ( Figure 2). centroid distances ranging from 3.696 (3) to 3.721 (4) Å ). The conformation of the molecules A and B are similar showing an envelope conformation for the methyl-cyclohexanone ring. The C molecule has a screw-boat conformation for the methyl-cyclohexanone ring, and the methyl group is in an axial position. The molecule D has a planar conformation (mean torsion angle 5.106 (12)° due to the presence of the 11,15-double bond. Once the relative structure of compound 11 and its 11,15-didehydro derivative 13 had been established by X-ray analysis, a pair of enantiomers (-) and (+)-eupachinin A were isolated from Eupatorium chinense with [α]D -5.1 and +4.8, respectively [30]. Spectroscopic data were identical to those of our compound of optical rotation −8.33, which was consequently identified as (-)-eupachinin A (11). The presence of the undescribed 11,15-didehydro derivative 13 in the crystal fraction of the X-ray analysis was unexpected, because NMR signals of the 11,15-double bond were not observed in the 1 H and 13 C spectra of the fractions studied.
(-)-10-Ethoxy-eupachinin A (12), the corresponding ethyl derivative of 11, was also isolated from P. echinata. With the molecular formula C17H18O5, its 1 H and 13 C NMR spectra were very similar to that of compound 11, except that now the signals corresponding to a 10-ethoxy group also appear. This compound probably is an artefact formed in the extraction of the plant with ethanol. The same applies to the undescribed benzofuran 10-ethoxy-11-hydroxy-euparin (10), also obtained from aerial part of P. echinata together with its known analogues 8 and 9.
The new benzofuran 14 was isolated from the transformed roots of P. echinata and P.steetzzi. Its high-resolution mass spectrum showed the molecular ion at m/z 348.1211 (C18H20O7). The 1 H NMR spectrum displayed the signals of the aromatic protons H-3, H-4, In the crystal, only B molecule is linked to C and D molecules by O-H···O intermolecular hydrogen bonds and π-π stacking interactions between the benzene rings A and C molecules (centroid-centroid distances ranging from 3.696 (3) to 3.721 (4) Å). The conformation of the molecules A and B are similar showing an envelope conformation for the methyl-cyclohexanone ring. The C molecule has a screw-boat conformation for the methyl-cyclohexanone ring, and the methyl group is in an axial position. The molecule D has a planar conformation (mean torsion angle 5.106 (12) • due to the presence of the 11,15-double bond.
Once the relative structure of compound 11 and its 11,15-didehydro derivative 13 had been established by X-ray analysis, a pair of enantiomers (-) and (+)-eupachinin A were isolated from Eupatorium chinense with [α] D -5.1 and +4.8, respectively [30]. Spectroscopic data were identical to those of our compound of optical rotation −8.33, which was consequently identified as (-)-eupachinin A (11). The presence of the undescribed 11,15-didehydro derivative 13 in the crystal fraction of the X-ray analysis was unexpected, because NMR signals of the 11,15-double bond were not observed in the 1 H and 13 C spectra of the fractions studied.
(-)-10-Ethoxy-eupachinin A (12), the corresponding ethyl derivative of 11, was also isolated from P. echinata. With the molecular formula C 17 H 18 O 5 , its 1 H and 13 C NMR spectra were very similar to that of compound 11, except that now the signals corresponding to a 10-ethoxy group also appear. This compound probably is an artefact formed in the extraction of the plant with ethanol. The same applies to the undescribed benzofuran 10-ethoxy-11-hydroxy-euparin (10), also obtained from aerial part of P. echinata together with its known analogues 8 and 9.
The new benzofuran 14 was isolated from the transformed roots of P. echinata and P.steetzzi. Its high-resolution mass spectrum showed the molecular ion at m/z 348.1211 (C 18 H 20 O 7 ). The 1 H NMR spectrum displayed the signals of the aromatic protons H-3, H-4, and H-7 at δ 6.75, 6.99, and 7.95, respectively, the phenolic proton associated with the carbonyl group at δ 12.44 and the acetyl group at δ 2.69 (H-14), which are similar to those observed in other euparin derivatives. The main differences with euparin (6) were the disappearance of the methylene double bond and the presence in 14 of singlets corresponding to two hydroxyls and also the characteristic signals of an angelyloxy group. The oxygenated methylenes, H-11 and H-12, resonate as pairs of doublets at δ 4.55 and 4.61 (J = 11.6 Hz) and δ 3.90 and 3.98 (J = 12.7 Hz), respectively, while the signals of the angelate group appear at δ 1.83 (t, H-5 ), 1.91 (dd, H-4 ), and 6.12 (ddd, H-3 ). In its 13 C NMR spectrum (Table 2), eighteen signals were observed, three methyls, two methylenes, four methines, and nine quaternary carbons. The HMBC experiment showed correlations in the angelyloxy group of the H-4 and H-5 methyls with C-1 /C-2 /C-3 , and the location of this ester at C-11, with crosspeaks of H-11 with C-2/C-10/C-12/C-1 , H-12 with C-2 and OH-12 with C-2/C-10/C-12. Thus, the structure 10, 12-dihydroxy-11-angelyloxy-10,11-dihydroeuparin (14) was assigned to this new product. Compound 15 (benzofuran precursor), also isolated from the aerial part of P. echinata, was determined as 2,4-dihydroxy-5-formyl-acetophenone (5-acetyl-2,4-dihydroxy-benzalde hyde) (15). The HRMS showed the molecular ion at m/z 180.0428 in accordance with the formula C 9 H 8 O 4 . In its 1 H NMR spectrum only singlets were observed, the methyl ketone at δ 2.64 and two aromatic protons at δ 6.46 and 7.99 due to H-6 and H-3, respectively, while the aldehyde hydrogen resonated at δ 9.77. Two phenolic protons were displaced at low-field, δ 11.62 and 13.08, due to hydrogen bonds with carbonyl groups, which were assigned to HO-4 and HO-2, respectively, considering the connectivities observed in the HMBC experiment. The 13 C NMR spectrum, described in the experiment, displayed the corresponding signals of carbons bearing hydrogens and those of four substituted aromatic carbons. The position of the substituents was established based on the correlations observed in the HMBC spectrum: H-3 with C-1/C-4; H-6 with C-2/C-4/C-7/C-9; H-8/with C-1/C-7; and H-9 with C-4/C-5. This compound 15 had been obtained as an intermediate in the synthesis of neobavachalcone [31] and now has been isolated for the first time as a natural product.
The new benzofurans 11-angelyloxy-10,11-dihydroeuparin (16) and 12-angelyloxyeuparone (17) were isolated from the transformed roots of P. steetzii. Compound 16 was isolated as an oil. Its HRMS was in accordance with the formula C 18 H 20 O 5 . The 1 H NMR spectrum showed the characteristic signals of a benzofuran ring, which were similar to those observed in euparin (6), but now the isopropylene group of this molecule has been substituted in 16 by a -CH(CH 3 )-CH 2 OAng group. Thus, the two H-11 protons of the oxymethylene group resonates as a pair of double doublets at δ 4.33 and 4.40, with 12.0 and 7.0 Hz coupling, respectively, while the H-12 methyl and the H-10 appears at δ 1.41 (d, J = 7.0 Hz) and 3.32 (m). In the 13 C NMR spectrum the corresponding carbons were detected at δ 66.2 (C-11), 15.5 (C-12) and 33.3 (C-10). The typical signals of the angelate group in both spectra were similar to those described for 14. Therefore, the structure of this compound was determined as 11-angelyloxy-10,11-dihydroeuparin (16). This compound has not been previously described in the chemical literature.
The HRMS of 12-Angelyloxyeuparone (17) was in accordance with the structural formula C 17 H 16 O 6 . The 1 H NMR spectrum showed singlet resonances of H-3, H-4, and H-7 at 7.57, 8.20, and 7.10, which were similar to those observed for euparone (7) at δ 7.50, 8.23, and 7.10, respectively. The same occurs with the methyl group at δ 2.74 (H-14) and the associated proton of the hydroxyl group at δ 12.57, in comparison with 2.70 and 12.63 described for 7 [27]. Moreover, in 17 the oxymethylene group appears as a singlet at δ 5.34, and the signals of the angelate ester were similar to those observed in compounds 14 and 16. The presence of this angelate ester was confirmed in the EIMS with fragments at m/z 217 and 203, which were originated from the molecular ion by cleavage of the C-10, C-11 bond and loss of the angelyloxy group, respectively.
appears at δ 1.41 (d, J = 7.0 Hz) and 3.32 (m). In the 13 C NMR spectrum the corresponding carbons were detected at δ 66.2 (C-11), 15.5 (C-12) and 33.3 (C-10). The typical signals of the angelate group in both spectra were similar to those described for 14. Therefore, the structure of this compound was determined as 11-angelyloxy-10,11-dihydroeuparin (16). This compound has not been previously described in the chemical literature.
The HRMS of 12-Angelyloxyeuparone (17) was in accordance with the structural formula C17H16O6. The 1 H NMR spectrum showed singlet resonances of H-3, H-4, and H-7 at 7.57, 8.20, and 7.10, which were similar to those observed for euparone (7) at δ 7.50, 8.23, and 7.10, respectively. The same occurs with the methyl group at δ 2.74 (H-14) and the associated proton of the hydroxyl group at δ 12.57, in comparison with 2.70 and 12.63 described for 7 [27]. Moreover, in 17 the oxymethylene group appears as a singlet at δ 5.34, and the signals of the angelate ester were similar to those observed in compounds 14 and 16. The presence of this angelate ester was confirmed in the EIMS with fragments at m/z 217 and 203, which were originated from the molecular ion by cleavage of the C-10, C-11 bond and loss of the angelyloxy group, respectively.

Biotransformation of 6-Hydroxytremetone (1)
Two benzofuran derivatives, 6-hydroxytremetone β-D-glucoside (18) and 6,10,11-trihydroxytremetone (19), have been obtained from the biotransformation of 6-hydroxytremetone (1) with Mucor plumbeus (Figure 3). This fungus has a broad specificity of substrate and has been used in the biotransformation of sesquiterpenes and diterpenes [38][39][40].  The major biotransformation compound was 6-hydroxytremetone β-D-glucoside (18). Its high-resolution mass spectrum showed the molecular ion at m/z 380, which corresponds to the molecular formula C 19 H 24 O 8 . The 1 H NMR spectrum was similar to that of the substrate, but with additional signals of a glucose moiety, with proton signal between δ 3.44 and 5.05. In the HMBC spectrum, a correlation between the anomeric proton H-1'and C-6 was observed. The coupling constant of H-1 (J = 7.3 Hz) indicated an axial β-configuration for this substituent, which was confirmed by the resonance of the anomeric carbon at δ 103.4 [41]. Acetylation of this compound led to the acetyl derivative 18a, which showed the molecular ion at 548.1874 m/z, corresponding to the molecular formula C 27 H 32 O 12 . Their NMR spectra confirmed this structure. The second product, structure 19, was obtained in very low yield. Its mass spectrum showed the molecular ion at m/z: 252.1008 (C 13 H 16 O 5 ), which indicates the introduction of two new oxygen atoms into the molecule with respect to the substrate 1. In addition to the signals observed in the starting benzofuran, an AB system appears, at δ 3.55 and 3.77 due to the two methylene protons (H-11) and the signal of a methyl at δ 1.12, which was assigned as a substituent on C-2, based on the correlations observed in the HMBC experiment. In the 13 C NMR spectrum (Table 3), the disappearance of the signals of the vinyl carbons of the substrate and the presence of two new resonances at δ 67.0 and 73.5 ppm, were typical of C-10 and C-11 respectively, which are linked to oxygens. Previously, this product had been isolated from Helianthopsis stuebelii [42]. It could be originated by opening of the corresponding epoxide during the isolation procedure, as also occurred in the formation of 10,11-dihydroxyeuparin (8).  Table 4 shows the antifeedant and postingestive effects of the tested compounds. The most antifeedant compounds were among the tremetone series (1, 1a, 2-5, 18, 18a), with 3-ethoxy-hydroxy-tremetone (4) being the strongest antifeedant (1st in EC 50 ranking), followed by 6-hydroxy-tremetone (1) (2nd in EC 50 ranking) and 1a (6th in EC 50 ranking). Glucosylation of 1 by its biotransformation with Mucor plumbeus gave inactive products (18 and 18a). Among the euparin series (6, 7, 7a, 8-10, 14, 16), the dihydroxyangelate 14 (third in the EC 50 ranking) was the most active, followed by euparin (6) and euparin angelate (16) (sixth and seventh in the EC 50 ranking). The eupachinin-type compounds (11,12) were both antifeedants (fourth and fifth in the EC 50 ranking) and this is the first report on the antifeedant effects of this type of benzofurans. The antifeedant compounds (1, 4, 6, 11, 12) were further tested for postingestive effects on S. littoralis larvae (Table 4). At a dose of 40 µg/larvae, 4, 11, and 12 showed antifeedant effects without postingestive toxicity (pANCOVA2 > 0.05). Compound 6 (euparin) had postingestive toxicity at 40 µg/larvae (pANCOVA2 = 0.035) that was lost at 20 µg/larvae, except when the larvae were pre-treated with PBO (piperonyl butoxide, a synergist), resulting in a significant antifeedant postingestive effect and indicating the involvement of a PSMO (polysubstrate monoxygenases) detoxification mechanism for 6 in S. littoralis.

Antifeedant and Postingestive Effects
Insect antifeedant effects of benzofurans have been previously described. Natural dihydrobenzofurans such as remirol and aurones were antifeedants to Spodoptera litura [43][44][45]. Euparin (6) was antifeedant to S. litura, but methyleuparin showed stronger effects [43]. Inclusion of an acetyl or methoxy on the aromatic group of benzofurans gave effective antifeedant activity, while hydroxylation or methylation decreased this effect [43]. In this work, ethoxylation of the C3 hydroxy group in the furan ring gave the strongest antifeedant effect (4), while acetylation of the aromatic hydroxyl group reduced or eliminated the antifeedant activity in the tremetone series (1 vs. 1a). Modifications of the side chain also affected the activity. For example, the oxidation of euparin 6 in C10 eliminated the activity (in 7 and 7a), and the presence of a C11 angelate substituent in the side chain (16,14) increased the activity (8).
Previously, methyleuparin showed a moderate growth inhibition effect on Peridroma saucia [45], and 6-hydroxy tremetone derivatives and euparin affected the ratio of Tenebrio molitor larval pupation [46]. In this work, we have shown growth inhibitory effects on S. littoralis larvae for the tremetone derivative 4 and eupachinin compounds 11 and 12, while euparin 6, with an unsaturated benzofuran ring, was the most toxic compound. The oxidation of the unsaturated furan ring turns benzofurans in alkylating agents, becoming cytotoxic and mutagenic [47]. This will explain the larval postingestive toxicity of euparin (6), with an unsaturated furan ring. The larval toxicity of 6 was enhanced by the application of the synergist PBO. PBO is a specific inhibitor of microsomal oxidases and resistance-associated esterases [48] and has shown insecticidal synergistic effects with synthetic unsaturated benzofurans [49], indicating that insects can detoxify these compounds as shown here.

Plant Material
All Pericallis species were collected at their flowering stage (aerial parts) in the Canary Islands (Spain) and identified by Dr. Arnoldo Santos. All voucher specimens (ORT) have been deposited at the Herbarium del Jardín de Aclimatación de La Orotava, Tenerife, Spain.

Plant Material
Agrobacterium rhizogenes ATCC-15834 was inoculated with a needle to the stem of aseptic plantlets germinated from seeds and cultured on agar medium containing 30 g/mL sucrose and half-strength MS medium [50]. The induced hairy roots were excised and cultured on hormone-free half-Gamborg B5 solid medium [51] supplemented with 30 g/L sucrose and 0.5 mg/mL ampicillin to eliminate bacteria. The axenic hairy roots obtained were subcultured every 25-30 days in the dark at 25 • C on the same solid medium without antibiotics. Then, they were cultivated in the dark at 25 • C in 250 mL Erlenmeyer flasks containing 100 mL of half-GB liquid medium supplemented with 30 g/L sucrose and shaken on a rotary shaker at 90 rpm. After five weeks, the hairy roots were harvested and separated from the culture medium by filtration through filter paper under vacuum.

DNA Extraction and Analysis
Total genomic DNA was extracted from transformed root tissue and from the untransformed root plants by using a "GenElute TM Plant Genomic DNA Miniprep Kit" (Sigma-Aldrich, St. Louis, MO, USA). Plasmid DNA from A. rhizogenes strain ATCC-15834 was used as a positive control. Polymerase chain reaction was performed using REDExtract-N-Amp Plant PCR Kit (Sigma) to detect the insertion of T L -DNA of A. rhizogenes ATCC-15834 in the transformed roots. The oligonucleotide primers for T L -DNA were 5 -ATGGATCCCAAATTGCTATTCCTTCCA-3 and 5 -TTAGGCTTCTTTCTTCA GG TTTA-3 which amplify a segment complementary to the 5 coding sequence of rol B to the 3 coding sequence of rol C on the T L -DNA region. PCR amplification was performed in a DNA thermal cycler (Applied Biosystems 2700, Foster City, CA) under the following conditions: initial denaturation at 94 • C for 2 min, followed by 30 cycles of 90 • C for 30 s, annealing at 55 • C for 1 min, with a final extension at 72 • C for 5 min. The PCR reaction mixture was electrophoresed on a 1.2% agarose gel using tris-acetate-EDTA buffer and visualized by ethidium bromide staining under ultraviolet light at 260 nm.

Extraction and Isolation of Compounds from Pericallis
Pericallis plant parts (aerial and roots) were dried in open air in the shade and grinded to give dry plant materials (P. echinata, Pe, 4.700 g; P. lanata, Pl, 980 g; P. steetzii, Ps, 1800 g; P. murrayi aerial, Pma, 1251.0 g; P. murrayi roots, Pmr, 687.5 g). Hairy roots were freezedried and powdered to give transformed roots dried materials (P. echinata, PeTR, 112 g and P. steetzii, PsTR, 49.7 g). The dry materials were exhaustively extracted with EtOH in a Soxhlet. The solvent was evaporated at reduced pressure to give crude extracts (Pe,120 g, PeTR 41 g, Pl 175 g, Ps 180 g, PsTR 11.4 g Pma 137 g, Pmr 90.2 g).
The ethanolic extracts were chromatographed with silica gel vacuum liquid chromatography columns (VLC column, 10 × 25 cm) eluted with a hexane-EtOAc-MeOH gradients. Further, the main fractions were chromatographed on Si gel columns, Sephadex LH-20 and/or preparative normal phase HPLC eluted with different solvents and proportions of hexane-EtOAc and/or CH 2 Cl 2 /MeOH.
Modifications of the side chain also affected the activity. For example, the oxydation of euparin 6 in C10 eliminated the activity (in 7 and 7a) and the presence of a C11 angelate substituent in the side chain (16,14) increased the activity (8).
Compounds 4, 11, and 12 showed antifeedant effects without postingestive toxicity to orally dosed S. littoralis larvae. In this work, we have shown growth inhibitory effects on S. littoralis larvae for the tremetone derivative 4 and eupachinin compounds 11 and 12, while euparin 6, with an unsaturated benzofuran ring, was the most toxic compound. The larval toxicity of 6 was enhanced by the application of the synergist PBO, indicating that insects can detoxify these compounds.