CYP9Q-mediated detoxification of in the honey bee (Apis mellifera)

Wenfu Maoa, Mary A. Schulerb, and May R. Berenbauma,1

aDepartment of Entomology and bDepartment of Cell and Developmental Biology, University of Illinois, Urbana, IL 61801

Contributed by May R. Berenbaum, June 27, 2011 (sent for review March 22, 2011) Although Apis mellifera, the western honey bee, has long encoun- is not uniquely vulnerable to (10), its capacity to me- tered pesticides when foraging in agricultural fields, for two dec- tabolize multiple toxins simultaneously may be limited. Syner- ades it has encountered pesticides in-hive in the form of acaricides gistic interactions, for example, have been documented in honey to control Varroa destructor, a devastating parasitic mite. The py- bees between and (11, 12), between pyr- rethroid tau-fluvalinate and the organophosphate coumaphos ethroids and chlordimeform (13), and between the organo- have been used for Varroa control, with little knowledge of honey phosphate and pyrethroid acaricides approved for use against bee detoxification mechanisms. Cytochrome P450-mediated de- Varroa (14). Such synergism of co-occurring pesticides may be of toxification contributes to pyrethroid tolerance in many insects, particular importance in view of the extensive contamination of but specific P450s responsible for detoxification in honey bees, bee products, and wax foundation with in-hive and agri- bees (indeed, in any hymenopteran pollinator) have not been de- cultural pesticides documented in the wake of colony collapse fined. We expressed and assayed CYP3 clan midgut P450s and disorder, a phenomenon associated with extensive honey bee demonstrated that CYP9Q1, CYP9Q2, and CYP9Q3 metabolize colony losses that have been reported annually since 2006 (7, 15). tau-fluvalinate to a form suitable for further cleavage by the car- Understanding how A. mellifera metabolizes pesticides is, there- boxylesterases that also contribute to tau-fluvalinate tolerance. fore, important for maintaining a viable apiculture industry in These in vitro assays indicated that all of the three CYP9Q enzymes the United States; identifying the specific mechanisms contrib- also detoxify coumaphos. Molecular models demonstrate that cou- uting to metabolism is critical for assessing the limits of

fl fi SCIENCES maphos and tau- uvalinate t into the same catalytic pocket, pro- pesticide tolerance and evaluating the probability of synergistic AGRICULTURAL viding a possible explanation for the synergism observed between and antagonistic interactions in this species, as well as for de- these two compounds. Induction of CYP9Q2 and CYP9Q3 tran- signing less-toxic alternatives for use against pesticide-resistance scripts by honey extracts suggested that diet-derived phytochem- mites and for monitoring exposures to pesticides in agricultural fields. icals may be natural substrates and heterologous expression of fi fl The multifunctional activities of cytochrome P450 mono- CYP9Q3 con rmed activity against quercetin, a avonoid ubiqui- oxgenases (P450s) contribute to the metabolism of natural and tous in honey. Up-regulation by honey constituents suggests that synthetic toxins in most aerobic organisms (16–18). P450-medi- diet may influence the ability of honey bees to detoxify pesticides. ated detoxification is central to tolerance and evolved resistance Quantitative RT-PCR assays demonstrated that tau-fluvalinate to pesticides in many pest insects (19, 20), including tolerance of enhances CYP9Q3 transcripts, whereas the pyrethroid bifenthrin pyrethroid insecticides in honey bees (11, 21, 22). Specific P450s enhances CYP9Q1 and CYP9Q2 transcripts and represses CYP9Q3 responsible for pesticide detoxification in honey bees have not transcripts. The independent regulation of these P450s can be use- been previously defined but, with the availability of the honey ful for monitoring and differentiating between pesticide expo- sures in-hive and in agricultural fields. bee genome sequence, identifying them is greatly facilitated by the reduced inventory of P450 loci in this species (23). The Apidae | miticide | transcriptional regulation complement of 46 full-length P450 genes in this genome is about half the number of P450 genes found in the dipteran Drosophila melanogaster genome (24) and the hymenopteran Nasonia vitri- s a pollinator, the western honey bee Apis mellifera con- pennis genome (25). Of these P450 genes, 28 belong to the CYP3 Asumes nectar and pollen (albeit in processed form as honey clan, a large P450 lineage including many CYP6 and CYP9 and beebread), and in doing so generally encounters dietary family members that are known to mediate detoxificative func- phytochemicals in substantially lower concentrations than do tions in other insects (19). The unique expansion of genes in the insect herbivores that consume chemically well-defended foliage CYP6AS subfamily (relative to other hymenopteran genomes) (1). The polylectic foraging behavior of A. mellifera (2), however, suggests that these proteins play a role in the detoxification of exposes the honey bee to a potentially broad diversity of phy- xenobiotics present in the honey bee’s distinctive diet of honey tochemicals and its habit of concentrating nectar to make honey and beebread. In fact, Mao et al. demonstrated that CYP6AS3 is suggests that honey bees encounter phytochemicals at higher capable of contributing to the detoxification of quercetin, a fla- concentrations than do most nectar-feeding pollinators that do – vonol widely distributed in plant nectars (1). not process their food (3 6). In the late nineteenth century, the To identify the specific P450s contributing to pyrethroid me- chemical milieu of managed honey bees changed profoundly tabolism in the honey bee, we assayed a number of CYP3 clan with the introduction of agrochemicals, especially pesticides; P450s expressed in the honey bee midgut (the principal site of since that time, honey bees have regularly encountered a tre- xenobiotic metabolism) and demonstrated that those in the mendous diversity of synthetic toxins via pesticide drift or resi- CYP9Q subfamily hydroxylate tau-fluvalinate to a form poten- dues in treated crops (7). Since 1990, the chemical environment of the honey bee changed yet again, with the widespread de- liberate use of in-hive acaricides for control of Varroa, a devas- fl Author contributions: W.M., M.A.S., and M.R.B. designed research; W.M. performed re- tating parasitic mite af icting North American apiculture (8). search; M.A.S. contributed new reagents/analytic tools; W.M., M.A.S., and M.R.B. ana- In-hive acaricides, including the pyrethroid tau-fluvalinate and lyzed data; and W.M., M.A.S., and M.R.B. wrote the paper. the organophosphate coumaphos, have been used for Varroa The authors declare no conflict of interest. control with little knowledge of honey bee detoxification path- 1To whom correspondence should be addressed. E-mail: [email protected]. ways. An extensive literature review documenting pesticide This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sensitivity in the honey bee (9) shows that, although this species 1073/pnas.1109535108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1109535108 PNAS | August 2, 2011 | vol. 108 | no. 31 | 12657–12662 Downloaded by guest on September 27, 2021 tially suitable for further cleavage by carboxylesterases (Fig. S1), P450s expressed in honey bee midguts metabolize λ-cyhalothrin shown by Johnson et al. to contribute to tau-fluvalinate tolerance (11), we set out to determine whether P450s metabolizing tau- (14). This study is thus unique in identifying specific genes fluvalinate are also located in this tissue. Intact midguts of 3-d-old contributing to pesticide metabolism within the order Hyme- worker bees were incubated with tau-fluvalinate in the presence noptera, which includes many of the world’s most important and absence of piperonyl butoxide (PBO), an inhibitor for most pollinator species, which also are facing pesticide exposures in bacterial and eukaryotic P450s. GC-MS data for the products agricultural landscapes. generated in these reactions indicated that P450s expressed in midgut tissue metabolize tau-fluvalinate (Fig. S2). Results Further identification of the P450s responsible for conversion Identification of Tau-Fluvalinate Metabolites in Frass. Although of tau-fluvalinate proceeded by cloning of 10 honey bee P450s Johnson et al. established that P450s are involved in pyrethroid (including CYP6AS3, CYP6AS10, CYP6AQ1, CYP6BD1, metabolism in honey bees, specific inhibitors of carboxylesterase CYP338A1, and all CYP9 family members) (Fig. S3) and coex- enzymes indicated that these enzymes might also be involved pressing them in Sf9 cells with house fly P450 reductase, as (22). As demonstrated for cypermethrin metabolism in Heli- described in Mao et al. (1). Carbon monoxide difference quan- coverpa (Heliothis) armigera, carboxylesterases metabolize not tification of their P450 levels (27) indicated that eight folded only pyrethroids but also the hydroxylated metabolites of pyr- correctly and two (CYP6AQ1 and CYP6BD1) folded incorrectly ethroids generated by P450s (26). Rather than use previously (generating P420 peaks). In vitro metabolism assays conducted described isotopic labeling procedures, we opted to characterize with 60 pmoles of the eight successfully expressed indicated that pyrethroid metabolites in honey bee midgut samples using GC- only CYP9Q1, CYP9Q2, and CYP9Q3 metabolized tau-fluvali- MS analysis after BSTFA [N, O-bis (trimethylsilyl) tri- nate to any detectable extent. GC-MS data for these samples are fluoroacetamide] derivatization. In addition to increasing the shown in Fig. S4. volatility of hydroxylated pyrethroids, these combined proce- Because tau-fluvalinate and coumaphos synergize each other dures replace the active hydrogen of the P450-generated hy- (14), and CYP9Q3 can be induced by honey extract (vide infra), droxyl group with a 73-kDa trimethylsilyl tag, which is extremely the catalytic activity of the CYP9Q enzymes toward pesticides useful in the nontargeted analysis of GC-MS chromatograms. By and honey constituents was analyzed; the synergism experiments using these methods to compare components in frass from of Johnson et al. (22) suggest the possibility that the two dif- control bees and tau-fluvalinate-treated bees, a 4′-hydroxylated ferent acaricides share a common detoxification enzyme, and subfragment of tau-fluvalinate having an atomic mass of 308 was induction by honey suggests that certain compounds in honey are identified (Fig. 1). substrates. These in vitro assays indicated that all of the three CYP9Q enzymes detoxify coumaphos, albeit at different rates Characterization of CYP9Q Subfamily Members Metabolizing Tau- (ranging from 0.96 to 2.21 pmol substrate·pmol P450·min), and Fluvalinate, Coumaphos, and Quercetin. With the knowledge that that CYP9Q1 and CYP9Q3 metabolize quercetin very efficiently

Fig. 1. Untargeted GC-MS analysis of tau-fluvalinate metabolism. (A) Chromatograms for controls (black) and for tau-fluvalinate treatments (red). These are compounds extracted from frass of control insects and of tau-fluvalinate–treated insects. Products were BFSTA-derivatized and then analyzed by GC-MS. (B)Mass spectrum of the arrow-marked red peak specific for the tau-fluvalinate treatments. The insert represents the fragment of tau-fluvalinate metabolite (338 Da).

12658 | www.pnas.org/cgi/doi/10.1073/pnas.1109535108 Mao et al. Downloaded by guest on September 27, 2021 (8.57 ± 1.08 and 12.19 ± 0.28 pmol substrate·pmol P450·min, SRS5, and CYP9R1 contains three phenylalanine residues and respectively) (Table S1). an alanine replacement in SRS5. In addition, the positively charged Lys221 predicted to contact the acidic group of tau- Structure Predictions for the CYP9Q Proteins. To identify the fluvalinate is replaced by hydrophobic residues in CYP9P1 structural features that enable specific honey bee P450s to (phenylalanine) and CYP6R1 (alanine). metabolize tau-fluvalinate, a molecular model of CYP9Q2 was developed using human CYP3A4 (28) as the main template Induction of CYP9Q Subfamily Enzymes by Honey Constituents and and CYP2C9 (29) as the variable FG region, as detailed in Pyrethroids. To determine if phytochemicals in honey influence Baudry et al. (30). Tau-fluvalinate was docked in the predicted the ability to metabolize tau-fluvalinate, honey bees were treated CYP9Q2 catalytic site using the DOCK function of MOE and its with ethyl acetate and methanol subfractions of an ethyl acetate inherent Monte-Carlo procedures with the MMFF94s force field extract of honey and CYP9Q subfamily transcript levels were (31). From 100 docked conformations analyzed, the lowest en- monitored by qRT-PCR analysis. These comparisons indicated ergy conformation is consistent with the documented hydroxyl- that CYP9Q3 transcripts are strongly induced by the both sub- ation at the 4′-carbon on the phenyl ring (Fig. 2 A and B). In this fractions, whereas CYP9Q2 transcripts are strongly reduced by binding mode, the long, thin tau-fluvalinate molecule is pre- both subfractions (Fig. 3A). dicted to be positioned with its 4′-carbon of tau-fluvalinate at To determine if tau-fluvalinate is both an inducer and sub- 4.94 Å from the heme oxygen and an interaction energy of strate for the CYP9Q transcripts and proteins, honey bees were −73.834 kcal/mol. The alcohol group on this substrate appears to treated with tau-fluvalinate and two other pyrethroids, cyper- be located in a nonpolar ring in the catalytic pocket formed by methrin and bifenthrin, and CYP9Q subfamily transcripts were four phenylalanine residues, including Phe123 in SRS1, Phe305 analyzed by qRTPCR (Fig. 3B, differences inferred from non- in SRS4, Phe374 in SRS5, and Phe491 in SRS6 (Fig. 2A). The overlapping SEs). Whereas tau-fluvalinate and cypermethrin negatively charged acidic group on this substrate associates with have little effect on CYP9Q1 and CYP9Q2 transcript levels the positively charged Lys221 in SRS2 through electrostatic (<0.5-fold induction), tau-fluvalinate enhances CYP9Q3 tran- interactions (Fig. 2 A and B). Overlays of predicted structures for script levels by ∼1.5-fold. In contrast, bifenthrin enhances the three CYP9Q proteins (Fig. 2A)showsignificant conservation CYP9Q2 transcript levels (>1.5-fold) and represses CYP9Q3 in many side chains predicted to contact tau-fluvalinate in the transcript levels (up to 1-fold).

CYP9Q2 model. SCIENCES

Compared with tau-fluvalinate, coumaphos is a small hydro- Discussion AGRICULTURAL phobic molecule that could be accommodated horizontally in the The relatively high tolerance of honey bees to tau-fluvalinate and nonpolar bottom of the catalytic pocket (Fig. 2C), with its eighth the lack of alternative chemical treatments for Varroa led to its carbon 5.52 Å from the heme oxygen and an interaction energy rapid approval for in-hive use as a miticide, despite the absence of −35.244 kcal/mol. The fact that coumaphos and tau-fluvalinate of any information about its metabolic fate in A. mellifera. Sub- can both fit into the same catalytic pocket, albeit in different sequent toxicity analysis of tau-fluvalinate in the presence of positions, provides a possible explanation for the synergism ob- PBO, a general P450 inhibitor, established that one or more served between these two compounds (14); binding of one to the P450s contribute to its metabolism (22). Our biochemical anal- catalytic pocket may partially or completely exclude the other yses of the in vivo derivatives of tau-fluvalinate identified a frag- substrate and reduce the rate at which it can be metabolized. ment of 4′-hydroxylated tau-fluvalinate through comparison of To identify the structural differences between the CYP9Q chromatograms for the treatment without the P450 inhibitor proteins that metabolize tau-fluvalinate and the CYP9P1 and PBO and the control with PBO. This specific fragment served as CYP9R1 proteins that do not, the sequences of CYP9Q1, a marker indicating that P450s in the midgut metabolize tau- CYP9Q2, CYP9Q3, CYP9P1, and CYP9R1 were aligned using fluvalinate; further in vitro experiments with individual midgut the ALIGN function of MOE and evaluated for variations in P450s expressed heterologously demonstrated that CYP9Q sub- their predicted substrate contact residues. These alignments family members are responsible for tau-fluvalinate metabolism. (Fig. S5) indicated that the ring of nonpolar phenylalanines in Moreover, the ability of these P450s to metabolize coumaphos in the CYP9Q2 catalytic site (Fig. 2A) is conserved to varying in vitro experiments, along with molecular models demonstrating extents in these five P450s: CYP9Q1 contains all four phenylal- that both acaricides fit in the catalytic pocket (albeit in different anine residues, CYP9Q3 contains three phenylalanine residues places), provides evidence in support of competitive inhibition as and an isoleucine replacement in SRS6, CYP9P1 contains two the mechanism underlying reported synergism between these two phenylalanine residues and leucine replacements in SRS4 and compounds (14). It remains to be seen whether competitive in-

Fig. 2. Docking of tau-fluvalinate in the CYP9Q2 catalytic site. (A) The docking mode for tau-fluvalinate (elemental colors in stick format) in the predicted CYP9Q2 catalytic site is shown with substrate contacts within 4.5 Å of this sub- strate. Overlaid with this are the predicted CYP9Q1 and CYP9Q3 catalytic sites with Phe123, Phe305, Phe374, Phe491, and Lys221 conserved in two or three CYP9Q proteins, shown in yellow, and other side chains conserved in all three CYP9Q proteins, shown in green. The heme and its bound oxygen are shown in space-filling format. (B and C) The predicted docking modes for tau-fluvalinate (B)and coumaphos (C) in CYP9Q2 are shown in elemental colors, with amino acid side chains within 4.5 Å of this substrate shown in red for acidic residues, blue for basic residues, green for hydrophobic residues, and cyan for hydrophilic residues.

Mao et al. PNAS | August 2, 2011 | vol. 108 | no. 31 | 12659 Downloaded by guest on September 27, 2021 the possibility exists that honey bees may have naturally en- countered pyrethrins or structurally related compounds over the course of their evolutionary history. Indeed, in an extensive re- view of literature documenting topical pesticide toxicity across a wide range of species, Hardstone and Scott determined that the honey bee is in the bottom 50% of all species surveyed for sensitivity to 5 of 10 pyrethroids surveyed (10). Despite its importance for understanding the metabolism of pesticides, induction of detoxification activity has rarely been examined in the honey bee. Yu et al. failed to detect an increase in midgut “microsomal oxidase” activity after exposure to rep- resentatives of five different classes of insecticides (42), but Kezic et al. administered benzo-(α)-pyrene in sugar syrup to honey bees and observed 5- to 25-fold increases in “benzo- (α)-pyrene monooxidase” activity that peaked after 9 d (43). At a molecular level, Johnson examined xenobiotic responses in honey bees using phenobarbital as a representative inducer and monitoring transcript variations on custom microarrays con- taining probes for 45 P450 genes, 10 carboxylesterase genes, and 7 GST genes, along with 206 chemosensory-related genes, 17 tetraspanins, and numerous houskeeping genes (44). This anal- ysis indicated that only a single gene, tetraspanin 16, was dif- ferentially expressed (P ≤ 0.05, false-discovery rate) in response to phenobarbital; no detoxificative genes were differentially Fig. 3. Induction of CYP9Q subfamily genes. (A) Relative expression levels of CYP9Q subfamily transcripts in midguts of workers fed with ethyl acetate/ expressed in response to phenobarbital. In a more directed methanol fractions (10× concentrated) of ethyl acetate extract of honey analysis of individual P450 genes, Mao et al. demonstrated that using solvent treatments as controls. (B) Relative expression levels of CYP9Q extracts of honey, pollen, and propolis induced several honey bee subfamily transcripts in midguts of workers treated with 15 μg per bee of CYP6AS enzymes mediating metabolism of quercetin, a ubiqui- tau-fluvalinate, 0.1 μg per bee of cypermethrin, and 0.1 μg per bee of tous plant-derived constituent of these hive materials (1). bifenthrin with acetone treatments as controls. The data shown are mean ± Although P450 induction in honey bees appears to be more SEM (three biological replicates). specific than has been demonstrated in other insects, differential induction of the CYP9 transcripts by some pesticides is an at- tribute that honey bees share with other insect species. In Heli- hibition of CYP9 P450s can account for other synergistic inter- coverpa armigera, deltamethrin induces transcription of two actions among pesticides in the honey bee. CYP9A subfamily members, although the functions of these To date, most P450s identified as responsible for or associated have not yet been defined (45). One of these and another with pyrethroid metabolism have been characterized in in- CYP9A subfamily member are constitutively overexpressed in secticide-resistant strains [Plutella xylostella (32, 33), Musca laboratory strains selected for resistance to the pyrethroid fen- domestica (34), Culex quinquefasciatus (35), Anopheles gambiae valerate (46). That CYP9Q2 in the honey bee is selectively in- (36), Lygus lineolaris (37)]. In these cases, specific P450s linked to duced by bifenthrin means that P450 transcription can be useful pyrethroid metabolism are generally constitutively overexpressed in developing strategies to monitor honey bee exposure to spe- in resistant strains. In contrast, in the psocid Liposcelis bos- cific pesticides and to differentiate between in-hive exposures to trychophila, CYP6CE1 and CYP6CE2 are constitutively ex- acaricides applied by beekeepers and nontarget exposures to pressed at low levels but up-regulated two- to threefold by del- pesticides applied to agricultural fields where bees forage. Such tamethrin exposure; the role of these specific P450s in monitoring can be of critical importance in evaluating con- metabolism, however, has not yet been established (38). With tributions of pesticide exposures to local and regional bee respect to inducibility, the honey bee CYP9 proteins in this study declines (9). Moreover, the fact that honey bees metabolize resemble the honey bee xenobiotic-metabolizing CYP6 proteins acaricides with enzymes that also metabolize dietary flavonoids more than they do the constitutively overexpressed CYP4, raises the possibility of synergistic or antagonistic interactions CYP6, and CYP9 proteins in pyrethroid-resistant strains of other between diet and pesticides that may be important in establishing species. CYP9Q2 and CYP9Q3 are constitutively expressed at low optimal protocols for feeding and maintaining colonies. levels and their transcriptional responses to pyrethroids or to extracts of honey indicate that they are independently regulated. Materials and Methods CYP9Q2 transcripts are induced by bifenthrin and CYP9Q3 μ fl Chemicals. Cloned Pfu DNA polymerase (2.5 U/ L), SuperScript III reverse transcripts are induced by tau- uvalinate and repressed by transcriptase, Sf9 insect cells, SF-900 serum-free medium and FBS were bifenthrin. The observation that all three of these proteins me- purchased from Invitrogen. StuI, KpnI, and XbaI restriction enzymes were tabolize tau-fluvalinate indicates that inducibility of insect P450s from New England Biolabs. Penicillin/streptomycin came from Bio-Whit- does not necessarily correspond precisely with their metabolic taker. Tau-fluvalinate, bifenthrin, α-cypermethrin, coumaphos, bis (trime- activities, as is often assumed from mammalian studies (39, 40). thylsilyl) trifluoroacetamide, D-glucose-6-phosphate, glucose-6-phosphate This independent regulation and generally low constitutive dehydrogenase (715 U/mg protein), and candidate flavonoids were obtained expression suggest that the pyrethroid-metabolizing activity of from Sigma-Aldrich. these P450s is not the result of intensive pesticide selection (e.g., Insects. Frames of late-stage capped brood were supplied by the University of 20 y of in-hive tau-fluvalinate application for Varroa control) but Illinois Bee Research Facility near Urbana, IL, and adult bees were allowed to rather is the likely result of the fortuitous resemblance of pyre- emerge in a dark 32 to 34 °C incubator. Newly enclosed bees were brushed throid insecticides to natural substrates of these enzymes. In that from frames daily and placed in groups of 30 in waxed paper cups (177 cm3; Hymenoptera are the principal pollinators of pyrethrum flowers Sweetheart) covered with cotton cheesecloth. Bees were provisioned with (Tanacetum cinerariifolium) (41) and related asteraceous plants, 1:1 sugar-water in punctured 1.5-mL microcentrofuge tubes.

12660 | www.pnas.org/cgi/doi/10.1073/pnas.1109535108 Mao et al. Downloaded by guest on September 27, 2021 In Vivo Metabolism of Tau-Fluvalinate via Frass Analysis. After anesthetization Phylogenetic Tree Construction. Phylogenetic analyses of the CYP9 proteins with carbon dioxide, 1 μLof10μg/μL tau-fluvalinate in acetone stock solu- from A. mellifera were conducted using the MEGA version 4.1 program (48) tion was delivered to the thorax of each 3-d-old worker bee using a micro- with the alignment generated using the built-in CLUSTALW implementa- liter syringe mounted on a PB-600 repeating dispenser (Hamilton); for tion and a neighbor-joining tree created with 1,000 bootstrap replications control bees, 1 μL of acetone was delivered in the same manner. The (Fig. S3). hindguts of honey bees were dissected 24 h after treatment and homoge- nized in 3× volumes of methanol using a Dounce tissue grinder (7 mL, 13 × Molecular Modeling and Substrate Recognition Sites Domain Determination. 82 mm). The homogenates were centrifuged in an Eppendorf centrifuge at Development of the CYP9Q2 model and tau-fluvalinate/coumaphos docking 11,750 × g for 5 min and the supernatants were dehydrated with anhydrous were carried out with the methods used for CYP6B33 from Papilio multi- MgSO4 and then dried under nitrogen. caudatus (47) using human CYP3A4 [PDB 1TQN (46)] as the main template and human CYP2C9 [PDB 1OG5 (29)] for the variable FG region, as detailed Assays of Midgut Metabolism of Tau-Fluvalinate. For each reaction, 10 intact in Baudry et al. (30). Protein sequence alignments of the CYP9 proteins from midguts of 3-d-old worker bees were placed in 1 mL 0.1 M phosphate A. mellifera were performed using the CLUSTALW version 1.6 program (49) buffer (pH 7.5). To control reactions with the P450 inhibitor PBO, 1 μL 250 mM and the SRS (substrate recognition sites) in these proteins were determined PBO dissolved in ethanol was added; to all other reactions, 1 μL ethanol was by evaluation of amino acids in the active site of the CYP9Q2 model and added. All reactions, including those with and without tau-fluvalinate, were reference to aligned eukaryotic P450 templates. incubated in a shaking water bath at 35 °C for 10 min before adding 2 μL25 mM tau-fluvalinate (dissolved in ethanol) to reactions with PBO serving as Induction of CYP9Q Subfamily Transcripts. For preparation of a honey extract controls and to tau-fluvalinate reactions; to reactions without tau-fluvalinate, to be used for transcript induction, 100 mL of honey from the University of 2 μL ethanol were added to compensate for volume changes. All reactions Illinois Bee Research Facility was diluted with 900 mL warm distilled water. were incubated at 35 °C for 90 min, quenched by adding 100 μL concentrated After extracting with three 300-mL volumes of petroleum ether, the water hydrochloric acid, homogenized, and extracted three times with 1 mL diethyl phase was extracted with three 300-mL volumes of ethyl acetate. The ethyl ether. The combined organic layers were pooled, dehydrated with anhydrous acetate extracts were pooled, dried with a rotary evaporator (Buchi Rota-

MgSO4, and dried under nitrogen. vapor), and resuspended in 1 mL ethyl acetate. This ethyl acetate extract was loaded on a normal phase column packed with 10-g silica gel (Merck; 32–63 Assays of in Vitro Metabolism of Tau-Fluvalinate, Coumaphos, and Quercetin μm). The column was washed with 50 mL ethyl acetate followed by 50 mL

with Individually Expressed P450s. Using an oligo(dT)18 primer and 1 μg total methanol. The ethyl acetate and methanol fractions were concentrated with RNA extracted from honey bee midguts, the first-strand cDNAs were syn- a rotary evaporator and resuspended in 1 mL methanol. thesized as described in the Invitrogen protocol for SuperScript III reverse After 3-d-old worker bees were fed with bee “candy” containing the ethyl

transcriptase. The cDNAs for CYP9Q1, CYP9Q2, CYP9Q3, CYP9P1, CYP9R1, acetate or methanol fractions of the honey extract for 3 d (10× concen- SCIENCES CYP9S1, CYP6AQ1, CYP6BD1, and CYP336A1 were amplified with Pfu DNA trated) or with unamended control bee candy, six midguts per treatment AGRICULTURAL − fl polymerase using transcript-specific primer sets with suitable restriction were dissected and stored at 80 °C. For tau- uvalinate, cypermethrin, and enzyme sites incorporated on their 5′-ends (Table S2). After inserting the bifenthrin induction assays, honey bees were treated as they were for in vivo μ fl μ cDNAs into the pFastBac vector, the positive clones were selected by se- metabolism assays, except that 15 g of tau- uvalinate per bee, 0.1 gof μ quencing the entire length of each insert using vector primers. Each P450 cypermethrin per bee, or 0.1 g of bifenthrin per bee were used. Six midguts − protein was coexpressed in Sf9 cells with house fly P450 reductase at per treatment were dissected after 48 h and stored at 80 °C. Each treat- a multiplicity of infection ratio of 1:0.5 (P450: P450 reductase) as described ment was independently replicated three times. for CYP6B33 expression (47). The levels and folding of each heterologously expressed P450 were monitored by reduced CO difference analysis (27). The Quantitative PCR Analysis. Using an oligo (dT18) primer and SuperScript III CYP6AS3 and CYP6AS10 proteins were coexpressed with P450 reductase at reverse transcriptase, first-strand cDNA was synthesized with 1 μg total RNA a multiplicity of infection ratio of 1:0.5 as described in an earlier study (1). extracted from midguts of worker bees subjected to different treatments. For For each of the coexpressed P450s, tau-fluvalinate metabolism was analyzed qRTPCR analysis, 10-μL reactions were set up with 2 μL of each cDNA sample as described for in vitro metabolism assays with midgut proteins, except that diluted 100 times, 5 μL of FastSYBR Green Master Mix (Applied Biosystems), 60 pmol heterologously expressed P450 were used in each reaction instead 0.25 μL20μM of each forward and reverse primer (Table S2), and 3.5 μLof of the protein extracted from 10 midguts; metabolism of quercetin and distilled water. All reactions were performed in triplicate. Comparative qPCR coumaphos was analyzed as described in the study of CYP6AS enzymes (1), analysis was performed using the StepOnePlus system (Applied Biosystems) ’ except that a mobile phase of acetonitrile-water (85:15) was used for cou- following the manufacturer s protocol, with the program beginning with a maphos, and coumaphos was monitored at 313 nm. single cycle for 10 min at 95 °C and 40 cycles of 15 s at 95 °C and 30 s at 56 °C. Afterward, the PCR products were heated to 95 °C for 15 s, cooled to 60 °C for Derivatization and Untargeted GC-MS Analysis. Dried extracts were derivatized 1 min, and heated to 95 °C for 15 s to measure the dissociation curves. The with 80 μL BSTFA at 60 °C for 60 min. All derivatized extracts were analyzed on relative levels of CYP9Q transcripts in midguts induced with ethyl acetate and fl a GCMS-QP2010 PLUS (Shimadzu), fitted with a SHRXI-5MS column (30 m × methanol extracts of honey, tau- uvalinate, cypermethrin, and bifenthrin 0.25 mm I.D. × 0.25-μm film). Helium was used as a carrier gas and the injector, were analyzed using StepOneTM Software V2.0 with solvent treatments and interface, and ion source temperatures were held at 250 °C, 280 °C, and 200 °C, eIF3-S8 as reference samples and endogenous control, respectively. respectively. The derivatized extracts were injected at an initial temperature of 60 °C and, after 5 min the oven temperature was raised to 300 °C at 5 °C/min ACKNOWLEDGMENTS. We thank Dr. Gene Robinson and Charley Nye of the University of Illinois Bee Research Facility for access to honey bees; Dr. Arthur and then held for 5 min. The scan range of the mass spectrometer was set from Zangerl for assistance with chemical analyses; Drs. Barry Pittendrigh and 40 to 600 m/z. Comparative analysis of the resulting total ion chromatograms Weilin Sun for advice on qRT-PCR; and Dr. Reed Johnson, Dr. Jeffrey Scott, fl for controls and tau- uvalinate treatments was performed manually by Dr. Marion Ellis, and Dr. Blair Siegfried for valuable discussion and advice on generating extracted ion chromatograms in 5-Da increments to identify this manuscript. This project was funded by US Department of Agriculture specific peaks for the tau-fluvalinate treatment. Grant AFRI 2008-35302-18831.

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