lable at ScienceDirect

Insect Biochemistry and Molecular Biology 59 (2015) 41e49

Contents lists avai

Insect Biochemistry and Molecular Biology

journal homepage: www.elsevier .com/locate/ ibmb

Epoxide hydrolase activities and epoxy fatty acids in the mosquitoCulex quinquefasciatus

Jiawen Xu, Christophe Morisseau, Jun Yang, Dadala M. Mamatha, Bruce D. Hammock*

Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA 95616, USA

a r t i c l e i n f o

Article history:Received 31 October 2014Received in revised form5 February 2015Accepted 5 February 2015Available online 14 February 2015

Keywords:MosquitoEpoxide hydrolaseEpoxy fatty acidEicosanoidInhibitorSignaling molecule

Abbreviations: EH, epoxide hydrolase; JH, juveepoxide hydrolase; JHEH, juvenile hormone epoxidhydrolase from A. gambiae; HsEH, sEH from H.M. musculus; RsEH, sEH from R. norvegicus; EH 3, eppiens; EH 4, epoxide hydrolase 4 from H.sapiens; CHmEH, mEH from H. sapiens; RmEH, mEH from R. nD. melanogaster; BmJHEH-r1, JHEH-r1 from Bombyx m4-like from Bombyx mori; AmEH, epxoide hydrolaseepoxide hydrolase 4 from T. castaneum.c-SO, cis-stilbeoxide; t-DPPO, trans-diphenylpropene oxide; EETEpOME, epoxy octadecenoic acid; AUDA, 12-(3-adamaacid; t-TUCB, trans-4-{4-[3-(trifluoromethoxy-phebenzoic acid; c-TUCB, cis-4-{4-[3-(trifluoromehexyloxy}-benzoic acid; TPPU, 1-trifluoromethoxyphe4-yl) urea; t-AUCB, trans-4-[4-(3-adamantan-1-yl-uracid; c-AUCB, cis-4-[4-(3-adamantan-1-yl-ureido)-cTPAU, 1-(1-acetylpiperidin-4-yl)-3-[(4-trifluoromethadamantanyl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl]acetypiperidin-4-yl)-3-adamantanylurea; TPAU, 1-tacetylpiperidin-4-yl)urea; Elaidamide, (E)-octadec-9-e* Corresponding author.

E-mail address: bdhammock@ucdavis.edu (B.D. Ha

http://dx.doi.org/10.1016/j.ibmb.2015.02.0040965-1748/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

Culex mosquitoes have emerged as important model organisms for mosquito biology, and are diseasevectors for multiple mosquito-borne pathogens, including West Nile virus. We characterized epoxidehydrolase activities in the mosquito Culex quinquefasciatus, which suggested multiple forms of epoxidehydrolases were present. We found EH activities on epoxy eicosatrienoic acids (EETs). EETs and othereicosanoids are well-established lipid signaling molecules in vertebrates. We showed EETs can be syn-thesized in vitro from arachidonic acids by mosquito lysate, and EETs were also detected in vivo both inlarvae and adult mosquitoes by LC-MS/MS. The EH activities on EETs can be induced by blood feeding,and the highest activity was observed in the midgut of female mosquitoes. The enzyme activities on EETscan be inhibited by urea-based inhibitors designed for mammalian soluble epoxide hydrolases (sEH). ThesEH inhibitors have been shown to play diverse biological roles in mammalian systems, and they can beuseful tools to study the function of EETs in mosquitoes. Besides juvenile hormone metabolism anddetoxification, insect epoxide hydrolases may also play a role in regulating lipid signaling molecules, suchas EETs and other epoxy fatty acids, synthesized in vivo or obtained from blood feeding by femalemosquitoes.

© 2015 Elsevier Ltd. All rights reserved.

nile hormone; sEH, solublee hydrolase; AgEH, epoxidesapiens; MsEH, sEH from

oxide hydrolase 3 from H,sa-eEH1, sEH1 from C. elegans;orvegicus; DmEH, mEH fromori; BmEH, epoxide hydrolase4-like from A.mellifera; TcEH,ne oxide; t-SO, trans-stilbene, epoxyeicosatrienoic acid;ntan-1-yl-ureido) dodecanoicnyl)-ureido]-cyclohexyloxy}-thoxy-phenyl)-ureido]-cyclo-nyl-3-(1-propionylpiperidin-eido)-cyclohexyloxy]-benzoicyclohexyloxy]-benzoic acid;oxy)phenyl]urea; AEPU, 1-}urea; APAU, 1-(1-rifluoromethoxyphenyl-3-(1-namide.

mmock).

1. Introduction

Epoxide hydrolases (EHs) are enzymes that convert a variety ofepoxides into their corresponding diols (Morisseau and Hammock,2005). In insects, epoxide hydrolases are mainly studied as detox-ification enzymes (Dauterman, 1982; Mullin, 1988; Taniai et al.,2003), and enzymes that are involved in the metabolism of juve-nile hormones (Anspaugh and Roe, 2005; Casas et al., 1991; Keiseret al., 2002; Khalil et al., 2006; Seino et al., 2010; Severson et al.,2002; Tsubota et al., 2010; Zhang et al., 2005). It is not knownwhether insect epoxide hydrolases play other important roles ininsect physiology, and what other substrates can be involved.

In mammals, epoxides of fatty acids such as epoxyeicosatrienoicacids (EETs) are a group of eicosanoids that are lipid signalingmolecules. EETs are derived from arachidonic acids, and are mainlyhydrolyzed by the soluble epoxide hydrolase (Yu et al., 2000; Zeldinet al., 1993). Inhibition of soluble epoxide hydrolase revealedtherapeutic effects in several mammalian models, indicating EETsare biologically functional (Morisseau and Hammock, 2013). In in-vertebrates including insects, eicosanoids are also known to playphysiological roles such as ion transport, immunity, reproductionand hostevector interactions, although most studies had focused

J. Xu et al. / Insect Biochemistry and Molecular Biology 59 (2015) 41e4942

on prostaglandins (Stanley, 2006; Stanley and Kim, 2014; Stanleyand Miller, 2006). It remains unknown whether insects produceEETs that are metabolized by epoxide hydrolases, and what thebiological roles are.

Culexmosquitoes are widely distributed around the world, bothin tropical and subtropical areas (Diaz-Badillo et al., 2011). Theyfeed on a variety of hosts and are vectors of many importantmosquito-borne diseases, such as West Nile virus (Bartholomayet al., 2010). Mosquitoes need arachidonic acids as the essentialfatty acids, and replacement of arachidonic acids with prostaglan-dins cannot rescue the mosquitoes, indicating other metabolites ofarachidonic acids may be important (Dadd, 1980; Dadd andKleinjan, 1984). Mosquitoes may oxidize arachidonic acids toform EETs by monooxygenases, such as the cytochrome P450 inmammals (Capdevila et al., 1992; Zeldin, 2001), and femalemosquitos will also ingest xenobiotic EETs during the process ofblood feeding, because EETs and other epoxy fatty acids are regularcomponents in the blood (Jiang et al., 2012, 2005). Many blood-derived molecules have been found and studied. When ingestedby mosquitoes, some are still relatively stable, and can affectmosquitoes' capacity as disease vectors (Pakpour et al., 2013). As aresult, EETs potentially may be among these molecules that haveimpacts on mosquito physiology and hostevector interactions.

Here we characterized the EH activities in the mosquito Culexquinquefasciatus, and demonstrated epoxy fatty acids are endoge-nous substrates. We showed that mosquitoes can produce epoxyfatty acids in vivo, and female mosquitoes will obtain xenobioticepoxy fatty acids during blood feeding. The highest EH activities onEETs in adults were found in female midgut, which is the center ofmany physiological processes, including detoxification, digestionand molecular signaling. We also found urea-based sEH inhibitorscan inhibit EH activities on EETs in mosquitoes. These inhibitorshave been demonstrated to play diverse biological roles inmammalian systems (Shen and Hammock, 2011), which may beuseful tools to evaluate epoxy fatty acid functions in mosquitoes.

2. Materials and methods

2.1. Mosquito rearing

The mosquito C. quinquefasciatus were reared in an insectaryincubator at a constant temperature of 28 ± 1 �C and 80 ± 5%relative humidity. Eggs were hatched in plastic water cups, andlarvae were fed twice a day with grounded fish food (TetraMin,Germany) and cat food (Purina, MO) until pupation. Emerged adultswere transferred to mosquito cages (30 cm � 30 cm � 30 cm) andfed 10% sucrose ad libitum soaked in cotton balls daily. Three or fourdays after eclosion, mosquitoes were fed with defribrinated sheepblood (Quad Five, MT) at 37 �C for 30 min. Parafilm® M (Sigma-eAldrich, MO) was used as the artificial membrane for bloodfeeding. After blood feeding, mosquitoes were provided with 10%sucrose daily, andwater cupswere provided for egg laying two daysafter blood feeding.

2.2. Enzyme preparation

4th instar larvae (8e9 days old after hatch) and adult mosquitoes(4e7 days female after eclosion) were homogenized by ceramicpestle and mortar in cold homogenization buffer (pH 8, 50 mMTriseHCl buffer containing 1 mM phenylmethylsulfonyl fluorideand 1 mM ethylenediaminetetraacetic acids). Because we werespecifically interested in the EH activities in female mosquitoes,only female adults were selected. The whole mosquito extract wassubjected to 100 � g centrifugation for 5 min to remove debris. Thesupernatant was collected as the crude lysate. The mitochondria

fraction was obtained by centrifuging the lysate at 18,000 � g for20 min, and the resulting pellets were resuspended in 50 mM, pH 8TriseHCl buffer. The resulting supernatant was centrifuged again at100,000 � g for 1 h. The supernatant was collected as the cytosolicfraction, and the pellet was resuspended in TriseHCl buffer as themicrosomal fraction. The pellets in each step were washed once byhomogenization buffer before any further processing. All differen-tial centrifugations were processed at 4 �C. Protein concentrationwas measured by BCA assay (Pierce, Rockford, IL) with BSA (Pierce,Rockford, IL) as the standard throughout this study.

2.3. Measurement of epoxide hydrolase activity

Epoxide hydrolase activities on c-SO, t-SO, t-DPPO, c-DPPO, JHIII, 14,15-EET and 9,10-EpOME were measured as previouslydescribed (Morisseau, 2007). To inhibit GST activity, diethyl male-ate was added to the buffer at 1 mM final concentration. In theassays on JH III, 1 ml of 1 mM OTFP (3-octylthio-1, 1, 1-trifluoropropan-2-one) in ethanol was added into 100 ml enzymesolution (10 mM final concentration of OTFP) to inhibit JH esteraseactivity (Abdel-Aal and Hammock, 1985). Assays were done intriplicate, and all enzyme activities were corrected for non-enzymatic hydration. If inhibitors were added, different concen-trations of inhibitors in DMSO were prepared, and 1 ml of inhibitorsolutionwas added into 100 ml of lysate. 1 ml of DMSO was added incontrol assays although inhibition by DMSOwas not observed at 1%(v:v) concentration.

2.4. Separation of epoxide hydrolase activities by ion exchangechromatography

A preliminary small-scale batch adsorption was used to definethe loading and eluting conditions. DEAE Sepharose ion exchanger(1 ml) (GE Healthcare, UK) was added into a 10 � 75 mm borosil-icate glass tube. The DEAE ion exchanger was equilibrated with pH8, 20mM, TriseHCl containing 0.02% CHAPS bywashing the gel fivetimes with 10 ml of buffer. A known amount of enzyme solutionsolubilized by 0.02% CHAPS was added to the pre-equilibratedexchanger. The solution and gel was gently mixed, and the gelwas allowed to settle. Proteins were eluted by step-wise elution byadding 1 ml of pH 8, 20 mM TriseHCl buffer containing increasingconcentration of NaCl, ranging from 0.05 M, 0.1 M, 0.2 M, 0.5 M and1.0 M NaCl. Every fraction was collected, and assayed for epoxidehydrolase activities with t-DPPO, JH III and 14,15-EET as the sub-strates. To achieve better separation, a column and gradient elutionwas used. A batch of DEAE ion exchanger was equilibrated as pre-viously described, and a known amount of enzyme solution wasadded into the exchanger. The whole solution was poured into thecolumn. The outlet valve was opened to allow the gel to pack. Thecolumnwas washed by 5 column volumes of pH 8, 20 mM TriseHClbuffer containing 0.02% CHAPS to elute unbound materials. Boundproteins were eluted by 30 column volumes of NaCl gradient(200 mM to 1M). At last 5 column volumes of 1 M NaCl was used toelute any remaining materials on the column. Every 10 ml elutantwas collected for enzyme activity assays.

2.5. Synthesis of EETs by mosquito crude lysate

The mosquito 4th instar (8e9 days old after hatch) crude lysate(2 mg/ml) may be purged with CO for 2 min (one bubble persecond) or added EH inhibitor AUDA to a final concentration of1 mM. Then 1 ml of 10 mM arachidonic acid in ethanol (100 mMfinal concentration) was added into 95 ml mosquito crude lysate(2 mg/mL) in pH 8, 50 mM TriseHCl buffer with or without pre-mixed 5 ml of NADPH generating system (100 mM glucose-6-

J. Xu et al. / Insect Biochemistry and Molecular Biology 59 (2015) 41e49 43

phosphate, 20 mM NADPþ, 20 units glucose-6-phosphate dehy-drogenase). Borosilicate culture tubes (ThermoFisher Scientific,MA) were used for the assay. The resulting solution was incubatedat 30 �C in a shaking water bath, and the reaction was stopped byadding 400 ml methanol at 5, 10, 20 and 30 min respectively. CUDA(1 ml of 100 mM in methanol) was added into the solution as theinternal standard (200 nM CUDA in a total volume of 500 ml so-lution). The tubes were vortexed for 5 s, and centrifuged at2500 � g for 5 min. The supernatant was collected for LC-MS/MSanalysis.

2.6. Mosquito dissection

Adult mosquitoes (4e7 days after eclosion) were anesthetizedin a 4 �C freezer, and transferred to a Petri dish on ice to keepimmobilized. Mosquito legs were pulled off by hand. A drop ofcold phosphate buffered saline was placed onto a glass slidemounted under the microscope, and mosquitoes were transferredto the slide. To get midguts and Malpighian tubules, a needle-tipprobe was used to hold down the thorax, and a fine-tipped for-cep was used to grasp the end of abdomen to gently pull off theabdomen in a slow motion. The midgut remained intact andattached to the thorax segment. The Malpighian tubules are longand slender tubules that are located between midgut and hindgut.To obtain salivary glands, a probe was used to hold down thethorax, and another probe was used to gently push down theanterior part of thorax, where the salivary gland is located. Thedissected mosquito tissues were homogenized by a Pyrex® 1 mlglass tissue grinder (SigmaeAldrich, MO) in pH 8, 50 mM TriseHClbuffer.

Table 1Substrate selectivity of epoxide hydrolase activities from Culex quinquefasciatus.Enzymes were extracted from 4th instar larva (8e9 days old), and enzyme activitieswere measured in pH 8, 50 mM TriseHCl buffer with 50 mM substrates (5 mM for JHIII), 1% (v:v) DMSO at 30 �C. For JH III assays, potential hydrolysis of the ester of JH IIIby contaminating esterases was prevented by adding 1 ml of 1 mM esterase inhibitorOTFP in ethanol (10 mM final concentration). Values are mean activity ±SD based onthree independent preparations. The detection of limit is shown in parentheses. If noactivity was detected, only the limit of detection is shown.

Substrates Structure of substrates Specific Activity(pmoldiols formed/(min � mg protein))

t-SO <30

c-SO <30

t-DPPO 404 ± 23 (<30)

c-DPPO <30

14, 15-EET 631 ± 25 (<0.4)

9, 10-EpOME 299 ± 3(<0.4)

JH III 75 ± 1 (<2)

2.7. Sampling, lipid extraction and LC-MS/MS

Mosquito 4th instar larvae (8e9 days old after hatch) or 4e7days old adults (female only) were weighed and put in a 1.5 mLeppendorf tube. 10 ml of anti-oxidant solution (0.2 mg/ml ofbutylated hydroxytoluene and EDTA) and 10 ml of deuteratedstandards were added. 400 ml of ice-cold methanol with 0.1% ofacetic acid and 0.1% of butylated hydroxytoluene were also added.Samples were stored in a �80 �C freezer for 30 min. After freezing,samples were homogenized with a plastic pestle and stored ina �80 �C freezer overnight. The next day the samples werecentrifuged at 10,000 � g for 10 min. The supernatant wascollected, and the remaining pellets were washed with 100 ml ofice-cold methanol with 0.1% of acetic acid and 0.1% of butylatedhydroxytoluene. The tubes were centrifuged again. The superna-tant was combined and diluted with 1.5 ml of water. The resultingsolution was loaded onto 60 mg Oasis HLB cartridges (Waters,MA).

The solid phase extraction (SPE) and LC-MS/MS analysis wasfollowed as previously described (Yang et al., 2009). The elutedsamples from SPE were evaporated by vacuum (SpeedVac) andreconstituted with 50 ml of 200 nM CUDA in methanol, which wasused as the internal standard. Agilent 1200 SL liquid chromatog-raphy series (Agilent Corporation, CA) was used for separationwithan Agilent Eclipse Plus C-18 reversed-phase column (2.1 �150 mm,1.8 mMparticle size). Water with 0.1% glacial acetic acid was used asmobile phase A. Acetonitrile: methanol (84:16) with 0.1% glacialacetic acids was used as mobile phase B. The detection was carriedout by monitoring the selected-reaction transitions using a 4000QTrap tandem mass spectrometer (Applied Biosystems InstrumentCorporation, CA) equipped with an electrospray source (Turbo V®).More detailed information of oxylipin analysis can be foundsomewhere else (Yang et al., 2009).

3. Results and discussion

3.1. Biochemical characterization of EH activities

Among the substrates evaluated in the study, c-SO, t-SO, t-DPPOand c-DPPO are tritium-labeled compounds, which were surrogatesubstrates for EH activities. JH III is an endogenous substrate for EHsin insects. 14,15-EET and 9,10-EpOME are epoxides of arachidonicacid and linoleic acid respectively. The structure of the substrates isshown in Table 1. No EH activity on c-SO, t-SO and c-DPPO wasfound in 4th instar larvae (8e9 days old) (Table 1), while activitieson JH III, 14,15-EET and 9,10-EpOMEwere detected. EH activity on t-DPPO was only detected in 4th instar larvae (8e9 days old afterhatch), not in pupa (10e12 days old after hatch) and female adults(4e7 days old after eclosion) (Fig. 1). Activities on JH III, 14,15-EETand 9,10-EpOME were detected in larvae, pupa and adults (Fig. 1).The highest specific activity was found in the 4th instar larvae(Fig. 1), although the physiological significance of the difference isnot known. In lysate of the 4th instar larvae, different levels of EHactivities on four substrates were found in mitochondrial, cytosolicand microsomal fractions (Fig. 2), indicating distribution of EHs inmultiple subcellular fractions. Solubilized EH activities on t-DPPO,JH III and 14,15-EET can all bind to the DEAE-Sepharose (GEHealthcare, UK) equilibrated by pH 8, 20 mM TriseHCl buffer, andthe activities were all eluted by 0.5 M and 1.0 M NaCl gradient bystep elution (Fig. S1). When a NaCl gradient was used for elution,EH activities with different chromatographic characteristics weredetected (Fig. 3). The balance sheet for the purification is shown inTable S1. The activities on t-DPPO, JH III and 14,15-EET overlapped,but the peak for each substrate did not. Activities on four substratesin different developmental stages, subcellular locations and sepa-ration by ion exchange chromatography suggest the presence ofmultiple forms of epoxide hydrolases. They may have overlappingand complementary substrate selectivity, while sharing the samesubcellular location.

Fig. 1. Epoxide hydrolase activities in different developmental stages of Culex quin-quefasciatus. Enzyme activities were measured with triplicate assays. Values are meanactivity ±SD based on three independent preparations. 4th instar (8e9 days old afterhatch), pupa (10e12 days old after hatch) and female adults (4e7 days old aftereclosion) were collected and homogenized for EH activity assays.

J. Xu et al. / Insect Biochemistry and Molecular Biology 59 (2015) 41e4944

Recently we have expressed and characterized an epoxide hy-drolase from Anopheles gambiae in insect cells (AgEH) that has ahigh activity on epoxy fatty acids (Xu et al., 2014).When the proteinsequence of AgEH was used as a query for blast in the genome ofC. quinquefasciatus, we found five protein coding sequences withepoxide hydrolase signature elements (Arand et al., 1996;Hopmann and Himo, 2006; Morisseau and Hammock, 2005). Weincluded the five sequences in a phylogeny analysis with reportedmammalian and insect epoxide hydrolases. In the phylogenyanalysis (Fig. 4), the five sequences were evolutionarily more closeto mammalian EH 3, EH 4 and sEHs, a family of EHs that areinvolved in a variety of mammalian physiology by regulating themetabolism of epoxy fatty acids (Decker et al., 2012; Yu et al., 2000).They are remotely homologous to mammalian and insect micro-somal EHs, including reported insect JHEHs (Seino et al., 2010;Severson et al., 2002; Taniai et al., 2003; Touhara et al., 1994;Zhang et al., 2005). Three putative JHEH sequences fromC. quinquefasciatuswere also clustered with insect JHEHs, but had adifferent evolutionary history with the five sequences mentionedpreviously. Interestingly, putative epoxide hydrolase sequenceshomologous to the mosquito AgEH and EHs from

Fig. 2. Subcellular distribution of EH activities in 4th instar larvae (8e9 days old afterhatch) on four substrates. Values are mean activity ±SD based on three independentpreparations. The mitochondria fraction was obtained by centrifuging the lysate at18,000 � g for 20 min, and the resulting pellets were resuspended in 50 mM, pH 8TriseHCl buffer. The resulting supernatant was centrifuged again at 100,000 � g for1 h. The supernatant was collected as the cytosolic fraction, and the pellet wasresuspended in TriseHCl buffer as the microsomal fraction.

C. quinquefasciatuswere also found in the genomes of Bombyx mori(BmEH), Apis mellifera (AmEH) and Tribolium castaneum (TcEH)(Fig. 4), suggesting mammalian EH 3, EH 4 and sEH homologs maybe common in insects. Besides juvenile hormone epoxide hydro-lases, there are other epoxide hydrolases in insects that mayregulate insect physiology by metabolizing other important sub-strates (such as epoxy fatty acids) other than juvenile hormones. Itis widely accepted that insects can synthesize fatty acids or obtainthem from food resources (Dadd, 1981). Insects not only have thecapacity to structurally alter the fatty acids, but also these fatty acidderived molecules may have different biological functions fromtheir parent molecules (Stanley-Samuelson et al., 1988). Thesefunctions include components of insect exoskeleton, synthesis ofpheromones (Stanley-Samuelson et al., 1988), reproduction andimmunity (Stanley and Kim, 2014). Epoxides of fatty acids, as lipidsignaling molecules, have been demonstrated to play a variety ofroles in mammalian physiology and medicinal biology (Morisseauand Hammock, 2013), which are regulated by epoxide hydrolases(Morisseau, 2013). It is fundamental to express and characterize theputative epoxide hydrolases in B. mori, A. mellifera and T. castaneum,and determine whether insects and mammals share anotherconserved signaling pathway regulated by epoxide hydrolases.

The phylogeny analysis is consistent with our biochemicalcharacterization that multiple forms of epoxide hydrolases exist inC. quinquefasciatus. The repertoire of protein-coding genes inC. quinquefasciatuswas reported to be 22% and 52% larger than thatof Aedes aegypti and A. gambiae, respectively (Arensburger et al.,2010). One of the gene-family expansions is the expansion ofgenes associated with xenobiotic detoxification (Arensburger et al.,2010). Epoxide hydrolases historically were studied as detoxifica-tion enzymes, and enzymes that hydrolyze juvenile hormones ininsects (Morisseau and Hammock, 2008). Multiple EHs expressedin C. quinquefasciatus can be a reflection of Culex-specific charac-teristics (Reddy et al., 2012), and EHs may play a role in metabo-lizing epoxy fatty acids, as their mammalian counterparts do.

Fig. 3. Epoxide hydrolase activities with different chromatographic characteristics.Each datum point is the mean of three independent chromatographic runs. Thestandard deviations are within 10% of the mean values and are not shown here. Acolumn of 10 ml DEAE ion exchanger was used and 1 column volume is 10 ml ofeluting buffer. The NaCl gradient was made by mixing 200 mM and 1 M NaCl inTriseHCl buffer in a gradient maker. Gradient elution began at 5th column volume andended at 35th column volume. EH activities in the first five column volumes (unboundenzyme fractions) were not detected. Protein concentration was measured by A280with BSA as the standard. The binding and eluting conditions are shown in Fig. S1. Thebalance sheet for the purification is shown in Table S1.

Fig. 4. Phylogeny analysis of EH sequences in Culex quinquefasciatus and other reported EHs. The full names of abbreviations and corresponding references are: AgEH, epoxidehydrolase from A. gambiae (Xu et al., 2014). HsEH, sEH from H. sapiens (Beetham et al., 1993); MsEH, sEH from M. musculus (Grant et al., 1993); RsEH, sEH from R. norvegicus (Knehret al., 1993); EH 3, epoxide hydrolase 3 from H. sapiens (Decker et al., 2012); EH 4, epoxide hydrolase 4 from H. sapiens (Decker et al., 2012); CeEH1, sEH1 from C. elegans (Harris et al.,2008); HmEH, mEH from H. sapiens (Skoda et al., 1988); RmEH, mEH from R. norvegicus (Falany et al., 1987); DmEH, mEH from D. melanogaster (Taniai et al., 2003); BmJHEH-r1,JHEH-r1 from Bombyx mori (Seino et al., 2010); BmEH, putative epoxide hydrolase 4-like from Bombyx mori; AmEH, putative epxoide hydrolase 4-like from A. mellifera; TcEH,putative epoxide hydrolase 4 from T. castaneum. The accession number of amino acid sequences is shown in the parenthesis. The percentage of replicate trees in which theassociated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

J. Xu et al. / Insect Biochemistry and Molecular Biology 59 (2015) 41e49 45

3.2. EET and EpOME are endogenous substrates for mosquitoepoxide hydrolases

We were particularly interested in the activities on epoxy fattyacids such as EpOMEs and EETs. EETs are epoxides of arachidonicacid, and have not been reported so far in insects to our knowledge.In mammals, EETs are lipid signaling molecules mainly hydrolyzedby soluble epoxide hydrolases (sEHs). The inhibition of sEH wasshown to have therapeutic effects in several disease models, indi-cating EETs are anti-inflammatory, vasodilatory, angiogenic andanalgesic (Morisseau and Hammock, 2013).

When arachidonic acids were added into the 4th instar (8e9days old after hatch) crude lysate containing the premixed NADPHgenerating system, 8,9-EET, 11,12-EET, 14,15-EET and their corre-sponding diols were all detected by LC-MS/MS (Fig. 5A). Theregioisomer 5,6-EET is not chemically stable (Fulton et al., 1998),and it is not reported here. The diols were also detected probablybecause of the epoxide hydrolase activities in the lysate. When1 mM EH inhibitor AUDA was added into the reaction, 14,15-DHET(diols of 14,15-EET) detected decreased nine fold, and the amountof 14,15-EET detected was increased two fold (Fig. 5B). Using theregioisomer 14,15-EET as a representative of EETs, the largestamount of 14,15-EET was detected when NADPH generating systemwas added into the lysate (Fig. 5C). Interestingly, when the lysatewas purged with CO or the NADPH generating system was notadded, a lower amount of EET and diols was detected, but thisamount was still significantly higher than the control groups(Fig. 5C). These data indicate cytochrome P450s are involved in thesynthesis of EETs in vitro, but they are probably not the only sourceof EET production. During the 30-min time course, the conversionrate from arachidonic acid to 14,15-EET and diols was 0.01%.

Reasons for the low conversion may be arachidonic acid is thesubstrate for a variety of reactions, including b-oxidation, chainelongation and production of eicosanoids, leading to a smallportion of arachidonic acid had been converted to EETs. Anotherpossibility is that the EETs and their diols had been converted toother metabolites for which we have no standards for analysis.

When mosquito 4th instar larvae (8e9 days after hatch) andadult samples (female only, 4e7 days after eclosion) were extractedfor oxylipin analysis, all epoxides of linoleic acid, arachidonic acidand their corresponding diols were detected (Table 2). The con-centration of EpOMEs was 45 fold and 25 fold higher than EETs inlarvae and adult respectively. The concentration of DiHOMEs was42 fold and 315 fold higher than DHETs in larvae and female adult.

By in vitro EET synthesis and oxylipin analysis (Fig. 5 andTable 2), we demonstrated EETs, as well as EpOMEs, are theendogenous substrates for mosquito epoxide hydrolases. EETs andother eicosanoids have been extensively studied in terms of humanhealth and drug development (Morisseau and Hammock, 2013;Tapiero et al., 2002). EETs have not been identified in insects, andtheir biology has not been reported to our knowledge. Althoughpresent in low concentrations, EETs and the corresponding diolsclearly are endogenous molecules in mosquitoes. EETs and otherepoxy fatty acids may also play physiological roles in mosquitoes asthey do in mammals, which are regulated by epoxide hydrolases.

3.3. EH activities on 14,15-EET are highest in female midgut

To investigate the distribution of EH activities on EETs inmosquitoes, we dissected and homogenized a representative set oforgans from 50male or female (4e7 days old) adult mosquitoes. EHactivities were measured with 14,15-EET as the substrate. There are

Fig. 5. Synthesis of EETs by mosquito 4th instar (8e9 days old after hatch) lysatesin vitro. Student's t test was used to evaluate statistical significance (** indicatesp < 0.01). Error bars represent the standard deviations of the means. A: Differentregioisomers of EETs and corresponding diols detected by LC-MS/MS in 30 min ofincubation. 5,6-EET was not included because its chemical instability. EETs and diolsdetected in the arachidonic acid solution and mosquito 4th instar (8e9 days old larvae)lysate only were regarded as background. In the treatment groups, arachidonic acidand NADPH generating system was added into mosquito 4th instar (8e9 days oldlarvae) lysate. B. Effects of a sEH inhibitor (AUDA) on the in vitro synthesis of 14,15-EETat 30 min of incubation. C: Synthesis of 14,15-EET with or without NADPH or CO in30 min of incubation. The data of two control treatments ‘Buffer þ ARA’ and ‘ Lysateonly’ overlap and therefore only the data of ‘Lysate only’ is shown.

Table 2Detection of epoxy fatty acids in vivo by LC-MS/MS. Mosquito 4th instar larvae (8e9days old larva after hatch) or adults (female, 4e7 days old after eclosion) wereweighted before extraction of lipid by solid phase extraction. Concentrations of C-18and C-20 epoxy fatty acids and corresponding diols are presented as mean ± SDbased on three independent measurements.

Parent fattyacids

Oxylipin pmol/g larva pmol/g adult

Linoleic acid Epoxy fatty acids 9,10-EpOME 9 ± 2 11 ± 112,13-EpOME 6 ± 2 10 ± 1

Dihydroxy fatty acids 9,10-DiHOME 12 ± 4 150 ± 3012,13-DiHOME 16 ± 3 90 ± 20

Arachidonicacid

Epoxy fatty acids 8,9-EET 0.20 ± 0.04 0.25 ± 0.0811,12-EET 0.07 ± 0.02 0.35 ± 0.0914,15-EET 0.08 ± 0.02 0.30 ± 0.05

Dihydroxy fatty acids 8,9-DHET 0.18 ± 0.05 0.10 ± 0.0411,12-DHET 0.15 ± 0.04 0.12 ± 0.0514,15-DHET 0.30 ± 0.10 0.5 ± 0.1

Fig. 6. Epoxide hydrolase activities on 14,15-EET in different tissues of adult mosqui-toes. Mosquitoes aged 4e7 days after eclosion were dissected under a microscope.Student's t test was used to evaluate statistical significance (*p < 0.01, **p < 0.001)between tissues of males and females. Error bars represent the standard deviations ofthe means based on three independent measurements.

Fig. 7. Induction of epoxide hydrolase activities on 14,15-EET after blood feeding. Eachdatum point represents means ± SD unless the SD is smaller than the datum point. Tenblood-ingested females were sampled every 3 h after feeding, Non-ingested femaleswere also sampled as controls. At 6, 9,12,15 h post blood feeding, the differences werestatistically significant (p < 0.01 by Student's t test) from the controls.

J. Xu et al. / Insect Biochemistry and Molecular Biology 59 (2015) 41e4946

sex differences in epoxide hydrolase activities on 14,15-EET in maleand female mosquitoes (Fig. 6). The highest specific activities werefound in the midgut of female mosquitoes. Intermediate enzymeactivities were found in Malpighian tubules. The activities in thesalivary gland, head andwhole fly were relatively low in both sexes.

The sexual difference and tissue distribution suggest that theepoxide hydrolase activities on epoxy fatty acids may serve varying

physiological roles in different tissues and sexes. Since only femalemosquitoes need blood meals, and the midgut is the center fordigestion, detoxification and pathogenic infection, it is tempting tohypothesize that the EH activities in the midgut on epoxy fatty

Table 3Inhibition of epoxide hydrolase activities on 14,15-EET by EH inhibitors. Enzymeswere extracted from 4th instar larvae (8e9 days old). Enzyme activities withoutinhibitors were assigned 0% inhibition, and values are mean ± SD based on threeindependent enzyme assays.

Inhibitor (1 mM) Structure Activity inhibited(% inhibition ±SD)

t-TUCB 30 ± 4

c-TUCB 15 ± 1

TPPU 15 ± 2

t-AUCB 65 ± 1

AEPU 17 ± 3

c-AUCB 18 ± 3

APAU 22 ± 1

TPAU 21 ± 3

AUDA 97.4 ± 0.1

Elaidamide 6 ± 1

Fig. 8. IC50 of AUDA on 14,15-EET. 4th instar (8e9 days old after hatch) lysate wasincubated with different concentrations of AUDA, the most potent inhibitor identifiedin Table 3 for 5 min on ice. The symbols represent the mean of enzyme activity in-hibition in three independent measurements. The IC50 is presented as mean ± SDbased on three independent measurements. 1 ml of 5 mM 14,15 EET in DMSO wasadded (50 mM final concentration) for measuring enzyme activity. The 4 parameterlogistic model describes the sigmoid-shaped response was used to calculate IC50 by thesoftware SigmaPlot.

J. Xu et al. / Insect Biochemistry and Molecular Biology 59 (2015) 41e49 47

acids are involved in midgut function, and may be important interms of disease transmission.

3.4. EH activities on 14,15-EET can be induced by blood feeding

It was reported that blood feeding could modulate the expres-sion of a variety genes (Dana et al., 2005; Sanders et al., 2003) inmosquitoes. To test whether the EH activities on epoxy fatty acidscan be affected by blood feeding, we blood-fed the mosquitoes andsampled 10 mosquitoes every 3 h for the measurement of EH ac-tivities. The epoxy fatty acid composition and EH activities of thesheep blood on epoxy fatty acids are shown in supplementaryinformation (Table S2 and Fig. S2). Because 10 mosquitoes werehomogenized in 1 mL pH 8, 50 mM TriseHCl buffer, and onemosquito usually takes 1e2 ml of blood, the fatty acid componentsand EH activity from the blood (Fig. S2) would not interfere the EHactivities measured in our experiments.

Because blood in the gut will interfere with protein concentra-tion determination, the activity was measured as pmol diol formedper mosquito per minute. Non-ingested female mosquitoes were

also sampled at every time point as controls. When not blood fed,the EH activities on 14,15-EET remained consistent (Fig. 7) duringthe course of sampling. After blood feeding, EH activities on 14,15-EET were induced 2 fold at 12 h post blood feeding, and reduced tothe same level as in the non-ingested females at 18 h post bloodfeeding.

3.5. EH activities on epoxy fatty acids are inhibited by urea-derivedcompounds designed as mammalian sEH inhibitors

In mammalian systems, the soluble epoxide hydrolase domi-nates the metabolism of epoxy fatty acids. Although EETs arerelatively stable, establishing their roles in vivo as signaling mole-cules was delayed due to their rapid hydrolysis by the sEH. Becausewe are interested in the biology of EHs and epoxy fatty acids, wetested a representative of EH inhibitors at relevant concentrationswith 14,15-EET as the substrate. The structure of the inhibitors isshown in Table 3. At 1 mM concentration (Table 3), a potent mEHinhibitor (elaidamide) and most sEH inhibitors showed inhibitionno greater than 30%, expressed by assigning the activities in controlsample (only DMSO was added at 1% final concentration) as 0%inhibition. The inhibitor t-AUCB inhibited 65% activity, while AUDAshowed a 97% inhibition. Both compounds were designed as EETmimics to inhibit the mammalian sEH. AUDA is the most closemimics of the EETs and has structural similarity on only one side ofthe urea central pharmacophore (Shen and Hammock, 2011). Weselected AUDA and determined its IC50, which was around 86 nM(Fig. 8). We thus propose AUDA as an inhibitor for the epoxidehydrolases from C. quinquefasciatus to facilitate the investigation ofthe biological roles of epoxy fatty acids. It can also serve as a leadmolecule for the synthesis of more powerful inhibitors of the EHsacting on epoxy fatty acids in insects.

Acknowledgment

This study was funded by NIEHS (R01 ES002710 and P42ES004699), the West Coast Metabolomics Center at UC Davis (NIH/

J. Xu et al. / Insect Biochemistry and Molecular Biology 59 (2015) 41e4948

NIDDK U24 DK097154), the UC Davis Jastro-Shields GraduateResearch Award and the China Scholarship Council. We thank Dr.Ahmet Inceoglu and Dr. Shizuo Kamita for detailed discussions andsuggestions. We also thank Dr. Kin Sing Stephen Lee for the syn-thesis of 14,15-EET substrates.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ibmb.2015.02.004.

References

Abdel-Aal, Y.A.I., Hammock, B.D., 1985. 3-octylthio-1,1,1-trifluoro-2-propanone, ahigh affinity and slow binding inhibitor of juvenile hormone esterase fromTrichoplusia ni (hübner). Insect Biochem. 15, 111e122.

Anspaugh, D.D., Roe, R.M., 2005. Regulation of JH epoxide hydrolase versus JHesterase activity in the cabbage looper, Trichoplusia ni, by juvenile hormone andxenobiotics. J. Insect Physiol. 51, 523e535.

Arand, M., Wagner, H., Oesch, F., 1996. Asp333, Asp495, and His523 form the cat-alytic triad of rat soluble epoxide hydrolase. J. biol. Chem. 271, 4223e4229.

Arensburger, P., Megy, K., Waterhouse, R.M., Abrudan, J., Amedeo, P., Antelo, B.,Bartholomay, L., Bidwell, S., Caler, E., Camara, F., Campbell, C.L., Campbell, K.S.,Casola, C., Castro, M.T., Chandramouliswaran, I., Chapman, S.B., Christley, S.,Costas, J., Eisenstadt, E., Feschotte, C., Fraser-Liggett, C., Guigo, R., Haas, B.,Hammond, M., Hansson, B.S., Hemingway, J., Hill, S.R., Howarth, C., Ignell, R.,Kennedy, R.C., Kodira, C.D., Lobo, N.F., Mao, C., Mayhew, G., Michel, K., Mori, A.,Liu, N., Naveira, H., Nene, V., Nguyen, N., Pearson, M.D., Pritham, E.J., Puiu, D.,Qi, Y., Ranson, H., Ribeiro, J.M., Roberston, H.M., Severson, D.W., Shumway, M.,Stanke, M., Strausberg, R.L., Sun, C., Sutton, G., Tu, Z.J., Tubio, J.M., Unger, M.F.,Vanlandingham, D.L., Vilella, A.J., White, O., White, J.R., Wondji, C.S.,Wortman, J., Zdobnov, E.M., Birren, B., Christensen, B.M., Collins, F.H., Cornel, A.,Dimopoulos, G., Hannick, L.I., Higgs, S., Lanzaro, G.C., Lawson, D., Lee, N.H.,Muskavitch, M.A., Raikhel, A.S., Atkinson, P.W., 2010. Sequencing of Culexquinquefasciatus establishes a platform for mosquito comparative genomics.Science 330, 86e88.

Bartholomay, L.C., Waterhouse, R.M., Mayhew, G.F., Campbell, C.L., Michel, K.,Zou, Z., Ramirez, J.L., Das, S., Alvarez, K., Arensburger, P., Bryant, B.,Chapman, S.B., Dong, Y., Erickson, S.M., Karunaratne, S.H., Kokoza, V.,Kodira, C.D., Pignatelli, P., Shin, S.W., Vanlandingham, D.L., Atkinson, P.W.,Birren, B., Christophides, G.K., Clem, R.J., Hemingway, J., Higgs, S., Megy, K.,Ranson, H., Zdobnov, E.M., Raikhel, A.S., Christensen, B.M., Dimopoulos, G.,Muskavitch, M.A., 2010. Pathogenomics of Culex quinquefasciatus and meta-analysis of infection responses to diverse pathogens. Science 330, 88e90.

Beetham, J.K., Tian, T.G., Hammock, B.D., 1993. cDNA cloning and expression of asoluble epoxide hydrolase from Human Liver. Arch. Biochem. Biophys. 305,197e201.

Capdevila, J.H., Falck, J.R., Estabrook, R.W., 1992. Cytochrome P450 and the arach-idonate cascade. FASEB J. off. Publ. Fed. Am. Soc. Exp. Biol. 6, 731e736.

Casas, J., Harshman, L.G., Hammock, B.D., 1991. Epoxide hydrolase activity on ju-venile hormone in Manduca sexta. Insect Biochem. 21, 17e26.

Dadd, R.H., 1980. Essential fatty acids for the mosquito Culex pipiens. J. Nutr. 110,1152e1160.

Dadd, R.H., 1981. Essential Fatty Acids for Mosquitoes, Other Insects andVertebrates.

Dadd, R.H., Kleinjan, J.E., 1984. Prostaglandin synthetase inhibitors modulate theeffect of essential dietary arachidonic acid in the mosquito Culex pipiens.J. Insect Physiol. 30, 721e728.

Dana, A.N., Hong, Y.S., Kern, M.K., Hillenmeyer, M.E., Harker, B.W., Lobo, N.F.,Hogan, J.R., Romans, P., Collins, F.H., 2005. Gene expression patterns associatedwith blood-feeding in the malaria mosquito Anopheles gambiae. BMC Genomics6, 5.

Dauterman, W.C., 1982. The role of hydrolases in insecticide metabolism and thetoxicological significance of the metabolites. J. Toxicol. Clin. Toxicol. 19,623e635.

Decker, M., Adamska, M., Cronin, A., Di Giallonardo, F., Burgener, J., Marowsky, A.,Falck, J.R., Morisseau, C., Hammock, B.D., Gruzdev, A., Zeldin, D.C., Arand, M.,2012. EH3 (ABHD9): the first member of a new epoxide hydrolase family withhigh activity for fatty acid epoxides. J. Lipid Res. 53, 2038e2045.

Diaz-Badillo, A., Bolling, B.G., Perez-Ramirez, G., Moore, C.G., Martinez-Munoz, J.P.,Padilla-Viveros, A.A., Camacho-Nuez, M., Diaz-Perez, A., Beaty, B.J., MunozMde, L., 2011. The distribution of potential West Nile virus vectors, Culex pipienspipiens and Culex pipiens quinquefasciatus (Diptera: Culicidae), in Mexico City.Parasit. Vectors 4, 70.

Falany, C.N., McQuiddy, P., Kasper, C.B., 1987. Structure and organization of themicrosomal xenobiotic epoxide hydrolase gene. J. Biol. Chem. 262, 5924e5930.

Fulton, D., Falck, J.R., McGiff, J.C., Carroll, M.A., Quilley, J., 1998. A method for thedetermination of 5,6-EET using the lactone as an intermediate in the formationof the diol. J. Lipid Res. 39, 1713e1721.

Grant, D.F., Storms, D.H., Hammock, B.D., 1993. Molecular cloning and expression ofmurine liver soluble epoxide hydrolase. J. Biol. Chem. 268, 17628e17633.

Harris, T.R., Aronov, P.A., Jones, P.D., Tanaka, H., Arand, M., Hammock, B.D., 2008.Identification of two epoxide hydrolases in Caenorhabditis elegans thatmetabolize mammalian lipid signaling molecules. Arch. Biochem. Biophys. 472,139e149.

Hopmann, K.H., Himo, F., 2006. Theoretical study of the full reaction mechanism ofhuman soluble epoxide hydrolase. Chem. a Eur. J. 12, 6898e6909.

Jiang, H., Anderson, G.D., McGiff, J.C., 2012. The red blood cell participates inregulation of the circulation by producing and releasing epoxyeicosatrienoicacids. Prostagl. Other Lipid Mediat. 98, 91e93.

Jiang, H.L., Quilley, J., Reddy, L.M., Falck, J.R., Wong, P.Y.K., McGiff, J.C., 2005. Redblood cells: reservoirs of cis- and trans-epoxyeicosatrienoic acids. Prostagl.Other Lipid Mediat. 75, 65e78.

Keiser, K.C.L., Brandt, K.S., Silver, G.M., Wisnewski, N., 2002. Cloning, partial puri-fication and in vivo developmental profile of expression of the juvenile hor-mone epoxide hydrolase of Ctenocephalides felis. Arch. Insect Biochem. Physiol.50, 191e206.

Khalil, S.M.S., Anspaugh, D.D., Roe, R.M., 2006. Role of juvenile hormone esteraseand epoxide hydrolase in reproduction of the cotton bollworm, Helicoverpa zea.J. Insect Physiol. 52, 669e678.

Knehr, M., Thomas, H., Arand, M., Gebel, T., Zeller, H.D., Oesch, F., 1993. Isolation andcharacterization of a cDNA encoding rat liver cytosolic epoxide hydrolase andits functional expression in Escherichia coli. J. Biol. Chem. 268, 17623e17627.

Morisseau, C., 2007. Measurement of soluble epoxide hydrolase (sEH) activity. Curr.Protoc. Toxicol. 33, 4.23.1e4.23.18.

Morisseau, C., 2013. Role of epoxide hydrolases in lipid metabolism. Biochimie 95,91e95.

Morisseau, C., Hammock, B.D., 2005. Epoxide hydrolases: mechanisms, inhibitordesigns, and biological roles. Annu. Rev. Pharmacol. Toxicol. 45, 311e333.

Morisseau, C., Hammock, B.D., 2008. Gerry Brooks and epoxide hydrolases: fourdecades to a pharmaceutical. Pest Manag. Sci. 64, 594e609.

Morisseau, C., Hammock, B.D., 2013. Impact of soluble epoxide hydrolase andepoxyeicosanoids on human health. Annu. Rev. Pharmacol. Toxicol. 53, 37e58.

Mullin, C.A., 1988. Adaptive relationships of epoxide hydrolase in herbivorous ar-thropods. J. Chem. Ecol. 14, 1867e1888.

Pakpour, N., Akman-Anderson, L., Vodovotz, Y., Luckhart, S., 2013. The effects ofingested mammalian blood factors on vector arthropod immunity and physi-ology. Microbes Infect. 15, 243e254.

Reddy, B.P.N., Labbe, P., Corbel, V., 2012. Culex genome is not just another genomefor comparative genomics. Parasites Vectors 5.

Sanders, H.R., Evans, A.M., Ross, L.S., Gill, S.S., 2003. Blood meal induces globalchanges in midgut gene expression in the disease vector, Aedes aegypti. InsectBiochem. Mol. Biol. 33, 1105e1122.

Seino, A., Ogura, T., Tsubota, T., Shimomura, M., Nakakura, T., Tan, A., Mita, K.,Shinoda, T., Nakagawa, Y., Shiotsuki, T., 2010. Characterization ofjuvenile hormone epoxide hydrolase and related genes in the larval develop-ment of the silkworm Bombyx mori. Biosci. Biotechnol. Biochem. 74, 1421e1429.

Severson, T.F., Goodrow, M.H., Morisseau, C., Dowdy, D.L., Hammock, B.D., 2002.Urea and amide-based inhibitors of the juvenile hormone epoxide hydrolase ofthe tobacco hornworm (Manduca sexta: Sphingidae). Insect Biochem. Mol. Biol.32, 1741e1756.

Shen, H.C., Hammock, B.D., 2011. Discovery of inhibitors of soluble epoxide hy-drolase: a target with multiple potential therapeutic indications. J. Med. Chem.55, 1789e1808.

Skoda, R.C., Demierre, A., McBride, O.W., Gonzalez, F.J., Meyer, U.A., 1988. Humanmicrosomal xenobiotic epoxide hydrolase. Complementary DNA sequence,complementary DNA-directed expression in COS-1 cells, and chromosomallocalization. J. Biol. Chem. 263, 1549e1554.

Stanley, D., 2006. Prostaglandins and other eicosanoids in insects: biological sig-nificance. Annu. Rev. Entomol. 25e44.

Stanley, D., Kim, Y., 2014. Eicosanoid signaling in insects: from discovery to plantprotection. Crit. Rev. Plant Sci. 33, 20e63.

Stanley, D.W., Miller, J.S., 2006. Eicosanoid actions in insect cellular immune func-tions. Entomol. Exp. Appl. 119, 1e13.

Stanley-Samuelson, D.W., Jurenka, R.A., Cripps, C., Blomquist, G.J., Derenobales, M.,1988. Fatty acids in insects - composition, metabolism, and biological signifi-cance. Arch. Insect Biochem. Physiol. 9, 1e33.

Taniai, K., Inceoglu, A.B., Yukuhiro, K., Hammock, B.D., 2003. Characterization andcDNA cloning of a clofibrate-inducible microsomal epoxide hydrolase inDrosophila melanogaster. Eur. J. Biochem. 270, 4696e4705.

Tapiero, H., Nguyen Ba, G., Couvreur, P., Tew, K.D., 2002. Polyunsaturated fatty acids(PUFA) and eicosanoids in human health and pathologies. Biomed. Pharmac-other. 56, 215e222.

Touhara, K., Soroker, V., Prestwich, G.D., 1994. Photoaffinity-labeling of juvenilehormone epoxide hydrolase and JH-binding proteins during ovarian and eggdevelopment in Manduca sexta. Insect Biochem. Mol. Biol. 24, 633e640.

Tsubota, T., Nakakura, T., Shiotsuki, T., 2010. Molecular characterization and enzy-matic analysis of juvenile hormone epoxide hydrolase genes in the red flourbeetle Tribolium castaneum. Insect Mol. Biol. 19, 399e408.

Xu, J., Morisseau, C., Hammock, B.D., 2014. Expression and characterization of anepoxide hydrolase from Anopheles gambiae with high activity on epoxy fattyacids. Insect Biochem. Mol. Biol. 54, 42e52.

J. Xu et al. / Insect Biochemistry and Molecular Biology 59 (2015) 41e49 49

Yang, J., Schmelzer, K., Georgi, K., Hammock, B.D., 2009. Quantitative profilingmethod for oxylipin metabolome by liquid chromatography electrospray ioni-zation tandem mass spectrometry. Anal. Chem. 81, 8085e8093.

Yu, Z.G., Xu, F.Y., Huse, L.M., Morisseau, C., Draper, A.J., Newman, J.W., Parker, C.,Graham, L., Engler, M.M., Hammock, B.D., Zeldin, D.C., Kroetz, D.L., 2000. Solubleepoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids.Cir. Res. 87, 992e998.

Zeldin, D.C., 2001. Epoxygenase pathways of arachidonic acid metabolism. J. Biol.Chem. 276, 36059e36062.

Zeldin, D.C., Kobayashi, J., Falck, J.R., Winder, B.S., Hammock, B.D., Snapper, J.R.,Capdevila, J.H., 1993. Regiofacial and enantiofacial selectivity of epoxyeicosa-trienoic acid hydration by cytosolic epoxide hydrolase. J. Biol. Chem. 268,6402e6407.

Zhang, Q.R., Xu, W.H., Chen, F.S., Li, S., 2005. Molecular and biochemical charac-terization of juvenile hormone epoxide hydrolase from the silkworm, Bombyxmori. Insect Biochem. Mol. Biol. 35, 153e164.

Fig. S1. Binding and elution of epoxide hydrolase activities from DEAE with step elution by NaCl.

0

250

500

750

1000S

pec

ific

Act

ivit

y p

mol

/(m

in ×

mg

pro

tein

)t-DPPO

0

50

100

150

200JH III

0

300

600

900

1200 14,15-EET

Fig. S2. EH activity on 14,15-EET in the blood. A: Specific activity. B. Total activity.

B

0

1

2

3

Lysed RBC Serum

pm

ol/(

min

×m

g p

rote

ins)

0

50

100

150

200

Lysed RBC Serum

pm

ol/(

min

×m

L)

A

a Ther

valuesdeviat

Tableactivi

re are tws are the tions are

e S1. Thities from

wo majormean vawithin 5

he balancm Culex

r peaks foalues of t5% of th

ce sheet quinque

for EH actriplicate

he mean

for the sefasciatu

ctivities e enzymvalues a

separatious.

on the sme assaysand are n

on with t

substrates. All thenot show

the DEA

e 14,15-Ee standar

wn in the

AE colum

EET. Therd table.

mn on EH

e

H

Table S2. C18 and C20 epoxy fatty acids and corresponding diols in the serum and red blood cells of the sheep blood. Values represent mean ± standard deviations based on three independent measurements.

Parent Fatty

Acids

Oxylipin

Serum (nM)

RBCs

(pmol/1010 cells)

Linoleic acid Epoxy fatty acids

9,10-EpOME 12,13-EpOME

6±2 6±2

1.2±0.3 1.2±0.3

Arachidonic acid

Dihydroxy

fatty acids

Epoxy fatty

acids

Dihydroxy

fatty acids

9,10-DiHOME

12,13-DiHOME

8,9-EET

11,12-EET

14,15-EET

8,9-DHET

11,12-DHET

14,15-DHET

35±2

34±2

1.8±0.2

1.5±0.4

0.7±0.3

11±2

5.2±0.2

8.6±0.3

2.4±0.1

2.9±0.1

0.40±0.08

0.4±0.1

0.23±0.04

0.78±0.09

0.42±0.04

0.60±0.03