We expressed the rat Na(v)1.3 and Na(v)1.6 sodium channel α subunit isoforms in Xenopus oocytes either alone or with the rat β1 and β2 auxiliary subunits in various combinations and assessed the sensitivity of the expressed channels to resting and use-dependent modification by the pyrethroid insecticide tefluthrin using the two-electrode voltage clamp technique. Coexpression with the β1 and β2 subunits, either individually or in combination, did not affecting the resting sensitivity of Na(v)1.6 channels to tefluthrin. Modification by tefluthrin of Na(v)1.6 channels in the absence of β subunits was not altered by the application of trains of high-frequency depolarizing prepulses. By contrast, coexpression of the Na(v)1.6 channel with the β1 subunit enhanced the extent of channel modification twofold following repeated depolarization. Coexpression of Na(v)1.6 with the β2 subunit also slightly enhanced modification following repeated depolarization, but coexpression of Na(v)1.6 with both β subunits caused enhanced modification following repeated depolarization that was indistinguishable from that found with Na(v)1.6+β1 channels. In contrast to Na(v)1.6, the resting modification by tefluthrin of Na(v)1.3 channels expressed in the absence of β subunits was reduced by repeated depolarization. However, tefluthrin modification of the Na(v)1.3 α subunit expressed with both β subunits was enhanced 1.7-fold by repeated depolarization, thereby confirming that β subunit modulation of use-dependent effects was not confined to the Na(v)1.6 isoform. These results show that the actions of pyrethroids on mammalian sodium channels in the Xenopus oocyte expression system are determined in part by the interactions of the sodium channel α subunit with the auxiliary β subunits that are part of the heteromultimeric sodium channel complexes found in neurons and other excitable cells.
In rats expression of the Na(v)1.7 voltage-gated sodium channel isoform is restricted to the peripheral nervous system and is abundant in the sensory neurons of the dorsal root ganglion. We expressed the rat Na(v)1.7 sodium channel α subunit together with the rat auxiliary β1 and β2 subunits in Xenopus laevis oocytes and assessed the effects of the pyrethroid insecticide tefluthrin on the expressed currents using the two-electrode voltage clamp method. Tefluthrin at 100 µM modified of Na(v)1.7 channels to prolong inactivation of the peak current during a depolarizing pulse, resulting in a marked "late current" at the end of a 40-ms depolarization, and induced a sodium tail current following repolarization. Tefluthrin modification was enhanced up to two-fold by the application of a train of up to 100 5-ms depolarizing prepulses. These effects of tefluthrin on Na(v)1.7 channels were qualitatively similar to its effects on rat Na(v)1.2, Na(v)1.3 and Na(v)1.6 channels assayed previously under identical conditions. However, Na(v)1.7 sodium channels were distinguished by their low sensitivity to modification by tefluthrin, especially compared to Na(v)1.3 and Na(v)1.6 channels. It is likely that Na(v)1.7 channels contribute significantly to the tetrodotoxin-sensitive, pyrethroid-resistant current found in cultured dorsal root ganglion neurons. We aligned the complete amino acid sequences of four pyrethroid-sensitive isoforms (house fly Vssc1; rat Na(v)1.3, Na(v)1.6 and Na(v)1.8) and two pyrethroid-resistant isoforms (rat Na(v)1.2 and Na(v)1.7) and found only a single site, located in transmembrane segment 6 of homology domain I, at which the amino acid sequence was conserved among all four sensitive isoform sequences but differed in the two resistant isoform sequences. This position, corresponding to Val410 of the house fly Vssc1 sequence, also aligns with sites of multiple amino acid substitutions identified in the sodium channel sequences of pyrethroid-resistant insect populations. These results implicate this single amino acid polymorphism in transmembrane segment 6 of sodium channel homology domain I as a determinant of the differential pyrethroid sensitivity of rat sodium channel isoforms.
Cytochrome P450 monooxygenase-mediated detoxification is a very important mechanism of resistance to insecticides. In the LPR strain of house fly, the major mechanism of resistance to pyrethroids is monooxygenase-mediated metabolism due to the overexpression of cytochrome P450 6D1 (CYP6D1). Although the role of CYP6D1 in pyrethroid resistance has been demonstrated, there is no information as to whether cytochrome b5 is involved in this reaction and what the major CYP6D1-specific metabolite is. Using a cytochrome b5 antiserum we demonstrate that cytochrome b5 is required for the elevated level of CYP6D1-mediated cypermethrin metabolism observed in microsomes from the resistant LPR strain compared to a susceptible strain. Therefore, cytochrome b5 is directly involved in this monooxygenase-mediated resistance. NADH has no significant effect on this metabolism, suggesting that in vitro the second electron source for CYP6D1 is NADPH when cypermethrin is the substrate. Using GC-MS, the primary CYP6D1 specific metabolite formed in vitro was identified as 4'-OH cypermethrin.
American sloughgrass (Beckmannia syzigachne) is a troublesome weed in winter wheat field rotated with rice in China. Fenoxaprop-p-ethyl and pinoxaden were observed failing to control American sloughgrass in the same filed in Lujiang county in 2011 and 2012, respectively. Whole-plant bioassay was conducted to determine the resistance to fenoxaprop-p-ethyl, pinoxaden and other herbicides in American sloughgrass. Dose-response experiment indicated that Lujiang population was highly resistant to fenoxaprop-p-ethyl (199.8-fold), pinoxaden (76.2-fold), clodinafop-propargyl (334.1-fold) and sethoxydim (15.9-fold); moderately resistant to clethodim (6.3-fold), susceptible to mesosulfuron-methyl, flucarbazone-sodium, pyroxsulam and isoproturon. Partial gene of CT domain was cloned and sequenced to confirm the molecular mechanism of resistance to ACCase-inhibiting herbicides. A Trp2027Cys mutation was found in Lujiang population according to the sequencing result. This mutation is the molecular mechanism of resistance to fenoxaprop-p-ethyl in Lujiang population. Furthermore the Trp2027Cys mutation very likely results in cross resistance to clodinafop-propargyl and pinoxaden in Lujiang population. 103 mutant homozygotes were detected from the 108 plants tested using a rapid dCAPS method developed in this paper. This is the first report of pinoxaden resistance and a mutation at position of 2027 for American sloughgrass.
The occurrence of two acetylcholinesterases, AChE1 and AChE2, in the mosquito Culex pipiens has been recently documented. Resistance to organophosphates and carbamates due to target insensitivity is the result of a qualitative change of only AChE1, encoded by the Ace.1 gene. Because AChE1 and AChE2 differ in their sensitivity to inhibitors, Ace.1 genotypes can be misclassified by previous tests. We describe a new rapid microplate test that allows unambiguous identification of Ace.1 genotypes. This test involves comparing AChE activities in the absence of insecticide and in the presence of two propoxur concentrations: a low concentration that inhibits only the sensitive AChE1 and a higher concentration that inhibits also AChE2 but not the insensitive AChE1 responsible of insecticide resistance. This comparison allows the identification of the three Ace.1 genotypes: resistant (Ace.1RR), susceptible (Ace.1SS) homozygotes, and heterozygotes (Ace.1RS). The similarity of propoxur sensitivity of modified AChE1s found in various resistant strains from the United States, Europe, and Africa indicates that this test is probably suitable for all the Ace.1 alleles described so far in C. pipiens.
The susceptibilities of maize (Zea mays cv. Champ) and two graminicide-resistant grass species, Poa annua (annual meadow grass) and Festuca rubra (red fescue), to two aryloxyphenoxypropionates (quizalofop and fluazifop) and a cyclohexanedione (sethoxydim) graminicide were evaluated in leaf blades and isolated chloroplasts, and by assaying acetyl-coenzyme A carboxylase (ACCase) in desalted leaf homogenates. The graminicide resistance of P. annua and F. rubra appeared to be at the level of ACCase. Festuca rubra ACCase was highly insensitive and P. annua ACCase was partially insensitive to the graminicides that were tested. Fatty acid synthesis in isolated maize chloroplasts was more susceptible to inhibition than was ACCase activity from whole leaves. There was a smaller difference in graminicide sensitivity between these two test systems in P. annua. The developmental pattern of ACCase specific activity and its inhibition by quizalofop was measured in maize and P. annua leaf blades. There was an age-dependent increase in the sensitivity of maize leaf ACCase activity to inhibition by quizalofop. Together with the greater susceptibility of chloroplasts compared with leaf homogenates this could imply that a graminicide-insensitive (extrachloroplastic) ACCase isoform is less highly expressed in older leaves. Poa annua ACCase did not significantly alter in sensitivity as leaves aged, consistent with the smaller difference in the level of inhibition between chloroplasts and leaf homogenates in this species. A small pyruvate carboxylase activity was detected in maize leaves after 9 days. By 38 days, when leaves were senescing, pyruvate carboxylase activity predominated over ACCase.
The peach-potato aphid Myzus persicae is a pest of many commercial crops due to its polyphagous nature of feeding and has a well-documented history of acquiring resistance to insecticides. In 2009 a strain (M. persicae FRC) emerged in southern France with a point mutation (R81T) at the nicotinic acetylcholine receptor (nAChR), the target site for neonicotinoids such as imidacloprid. This point mutation was associated with the loss of the high affinity imidacloprid binding site (pM Kd), with the single remaining binding site (low nM Kd) highly overexpressed compared to laboratory controls (Bass et al., 2011 ). Here we report that after 2years of continuous selection in the glass house environment with neonicotinoids, the total level of IMD-sensitive nAChRs (low nM Kd) in M. persicae FRC is now comparable to laboratory controls (pM and low nM Kd). Interestingly, despite this large reduction in IMD-sensitive nAChRs, this was not associated with any significant alteration in NNIC-lethality. Additionally, sustained absence of neonicotinoid-selection did not alter nAChR protein levels. We suggest that alterations in nAChR protein expression level described in the original characterisation of the field-isolated M. persicae FRC is unlikely to have been a direct consequence of the R81T mutation. Rather, we speculate that nAChR expression in aphids is likely influenced by as yet unknown conditions in the natural field environment that are absent in the laboratory setting.
A serine to glycine point mutation of acetylcholinesterase (AChE, EC 18.104.22.168) was identified in an azinphosmethyl-resistant strain of Colorado potato beetle [Leptinotarsa decemlineata (Say)]. The position of the mutation corresponds to Val 238 of the Torpedo AChE and represents the first amino acid residue to form the alpha-helix, alpha-E'1. The predicted secondary structure of the mutation-containing region of AChE suggested that the transition from the turn to the alpha-helix occurs sooner in the sequence when serine is replaced by glycine. Thus, conformational changes in the AChE due to the alpha-helix deformation were expected to impinge upon both the catalytic and the peripheral binding sites, resulting in the modification of the bindings of organophosphorus insecticides and other ligands to these sites. The mutation appeared to be associated with the fitness of the beetle. The intrinsic rate of increase of the azinphosmethyl-resistant (AZ-R) strain was relatively low when the beetles were reared on the Russet Burbank potato cultivar, but was relatively high when they were reared on the NDA 1725-1 potato cultivar. Because these two potato cultivars contain different amounts of steroidal glycoalkaloids (e.g., alpha-solanine and alpha-chaconine), the different fitness of the AZ-R strain on different potato cultivars may be partially attributed to the increased sensitivity of the azinphosmethyl-resistant form of AChE to the inhibition by alpha-solanine and reduced sensitivity to alpha-chaconine as previously reported.
The cattle tick, Rhipicephalus (Boophilus) microplus (Bm), and the sand fly, Phlebotomus papatasi (Pp), are disease vectors to cattle and humans, respectively. The purpose of this study was to characterize the inhibitor profile of acetylcholinesterases from Bm (BmAChE1) and Pp (PpAChE) compared to human and bovine AChE, in order to identify divergent pharmacology that might lead to selective inhibitors. Results indicate that BmAChE has low sensitivity (IC50 = 200 μM) toward tacrine, a monovalent catalytic site inhibitor with sub micromolar blocking potency in all previous species tested. Similarly, a series of bis(n)-tacrine dimer series, bivalent inhibitors and peripheral site AChE inhibitors possess poor potency toward BmAChE. Molecular homology models suggest the rBmAChE enzyme possesses a W384F orthologous substitution near the catalytic site, where the larger tryptophan side chain obstructs the access of larger ligands to the active site, but functional analysis of this mutation suggests it only partially explains the low sensitivity to tacrine. In addition, BmAChE1 and PpAChE have low nanomolar sensitivity to some experimental carbamate anticholinesterases originally designed for control of the malaria mosquito, Anopheles gambiae. One experimental compound, 2-((2-ethylbutyl)thio)phenyl methylcarbamate, possesses >300-fold selectivity for BmAChE1 and PpAChE over human AChE, and a mouse oral LD50 of >1500 mg/kg, thus providing an excellent new lead for vector control.
Conventional insecticides targeting acetylcholinesterase (AChE) typically show high mammalian toxicities and because there is resistance to these compounds in many insect species, alternatives to established AChE inhibitors used for pest control are needed. Here we used a fluorescence method to monitor interactions between various AChE inhibitors and the AChE peripheral anionic site, which is a novel target for new insecticides acting on this enzyme. The assay uses thioflavin-T as a probe, which binds to the peripheral anionic site of AChE and yields an increase in fluorescent signal. Three types of AChE inhibitors were studied: catalytic site inhibitors (carbamate insecticides, edrophonium, and benzylpiperidine), peripheral site inhibitors (tubocurarine, ethidium bromide, and propidium iodide), and bivalent inhibitors (donepezil, BW284C51, and a series of bis(n)-tacrines). All were screened on murine AChE to compare and contrast changes of peripheral site conformation in the TFT assay with catalytic inhibition. All the inhibitors reduced thioflavin-T fluorescence in a concentration-dependent manner with potencies (IC50) ranging from 8 nM for bis(6)-tacrine to 159 μM for benzylpiperidine. Potencies in the fluorescence assay were correlated well with their potencies for enzyme inhibition (R(2) = 0.884). Efficacies for reducing thioflavin-T fluorescence ranged from 23-36% for catalytic site inhibitors and tubocurarine to near 100% for ethidium bromide and propidium iodide. Maximal efficacies could be reconciled with known mechanisms of interaction of the inhibitors with AChE. When extended to pest species, we anticipate these findings will assist in the discovery and development of novel, selective bivalent insecticides acting on AChE.
To understand the nematicidal mode of action of phytochemicals derived from plant essential oils against the pinewood nematode (Bursaphelenchus xylophilus), we evaluated 97 compounds (49 monoterpenes, 17 phenylpropenes, 16 sesquiterpenes, and 15 sulfides) for their inhibitory effects on B. xylophilus acetylcholinesterases (BxACEs). In the primary inhibition assay using B. xylophilus crude protein, more than 50% BxACE inhibition activity was observed with 3 monoterpenes, (+)-α-pinene, (-)-α-pinene, and 3-carene; 2 phenylpropenes, ο-anisaldehyde, and coniferyl alcohol; and 1 sesquiterpene, cis-nerolidol. Other compounds showed moderate or weak inhibitory activity. The inhibitory activities against 3 recombinant BxACEs were subsequently estimated using the identified active compounds in a primary inhibition assay. (+)-α-Pinene showed the strongest inhibition of BxACE-1 followed by 3-carene, coniferyl alcohol, (-)-α-pinene, o-anisaldehyde, and cis-nerolidol. The half maximal inhibitory concentration (IC50) values of (+)-α-pinene, 3-carene, o-anisaldehyde, cis-nerolidol, and (-)-α-pinene against BxACE-2 were found to be 0.64, 1.41, 8.18, 8.53, 15.28, and 18.03mM, respectively. Coniferyl alcohol showed the strongest inhibition of BxACE-3 followed by (+)-α-pinene and cis-nerolidol.
Sodium channel inhibitor (SCI) insecticides were discovered almost four decades ago but have only recently yielded important commercial products (eg., indoxacarb and metaflumizone). SCI insecticides inhibit sodium channel function by binding selectively to slow-inactivated (non-conducting) sodium channel states. Characterization of the action of SCI insecticides on mammalian sodium channels using both biochemical and electrophysiological approaches demonstrates that they bind at or near a drug receptor site, the "local anesthetic (LA) receptor." This mechanism and site of action on sodium channels differentiates SCI insecticides from other insecticidal agents that act on sodium channels. However, SCI insecticides share a common mode of action with drugs currently under investigation as anticonvulsants and treatments for neuropathic pain. In this paper we summarize the development of the SCI insecticide class and the evidence that this structurally diverse group of compounds have a common mode of action on sodium channels. We then review research that has used site-directed mutagenesis and heterologous expression of cloned mammalian sodium channels in Xenopus laevis oocytes to further elucidate the site and mechanism of action of SCI insecticides. The results of these studies provide new insight into the mechanism of action of SCI insecticides on voltage-gated sodium channels, the location of the SCI insecticide receptor, and its relationship to the LA receptor that binds therapeutic SCI agents.
The comparative efficacy and mechanism of action of the herbicide dymuron and its two optically active monomethyl analogues, (R)-1-(alpha-methylbenzyl)-3-(p-tolylurea) (R-MBTU) and S-MBTU, as safeners of rice against injury from bensulfuron-methyl were investigated. Bioassays using etiolated seedlings of rice (Oryza sativa L., cv. Lemont), grown either in agar or in liquid media containing bensulfuron-methyl, showed that this sulfonylurea herbicide is a potent inhibitor of rice root growth (I50 = 120 nM). Similar studies with the herbicide dymuron and its two optically active monomethyl analogues (R-MTBU and S-MTBU) showed that at 10 μM, dymuron and R-MBTU reduced rice root length by 25 and 17%, respectively, whereas S-MBTU had no effect on root growth of Lemont rice. Combined treatments with bensulfuron-methyl and dymuron or each of its two analogues confirmed that S-MTBU is an excellent safener of rice against bensulfuron-methyl, far more effective than either dymuron or R-MTBU. The protective action of S-MTBU appeared to result mainly from a drastic reduction in the uptake of bensulfuron-methyl by safened rice seedlings. Dymuron and R-MTBU reduced also the uptake of bensulfuron-methyl by rice seedlings, but to a lesser extent than S-MTBU. Quantitative changes in the metabolism of bensulfuron-methyl by safened rice seedlings were also observed, but did not appear to support the involvement of enhanced herbicide metabolism in the safening action of S-MBTU, R-MBTU, and dymuron. The major metabolites of bensulfuron-methyl detected in both safened and unsafened rice seedlings were tentatively identified as methyl-(4-hydroxy-6-methoxypyrimidin-2-yl-carbamoylsulfamoyl)-o-toluate; methyl-(aminosulfonyl)-o-toluate; and 1H-2,3-benzothiazin-4-(3H)-one-2,2-dioxide.
Selectivity to insects over mammals is one of the important characteristics for a chemical to become a useful insecticide. Fipronil was found to block cockroach GABA receptors more potently than rat GABA(A) receptors. Furthermore, glutamate-activated chloride channels (GluCls), which are present in cockroaches but not in mammals, were very sensitive to the blocking action of fipronil. The IC(50)s of fipronil block were 30 nM in cockroach GABA receptors and 1600 nM in rat GABA(A) receptors. Moreover, GluCls of cockroach neurons had low IC(50)s for fipronil. Two types of glutamate-induced chloride current were obswerved: desensitizing and non-desensitizing, with fipronil IC(50)s of 800 and 10 nM, respectively. We have developed methods to separately record these two types of GluCls. The non-desensitizing and desensitizing currents were selectively inhibited by trypsin and polyvinylpyrrolidone, respectively. In conclusion, in addition to GABA receptors, GluCls play a crucial role in selectivity of fipronil to insects over mammals. GluCls form the basis for development of selective and safe insecticides.
In vitro assays were used to determine if organophosphate, carbamate, and synthetic pyrethroid insecticides affected the cytochrome P450 monooxygenase (P450) catalyzed hydroxylation of nicosulfuron, bentazon, cinnamic acid, or lauric acid in maize microsomes. All P450 activities were inhibited approximately 50% by carbaryl, and none were inhibited by permethrin. Hydroxylations of nicosulfuron, bentazon, lauric acid, and cinnamic acid were inhibited by malathion 83, 92, 38, and 0%, respectively. Terbufos was only moderately (36%) inhibitory of in vitro P450 hydroxylation of nicosulfuron. Nicosulfuron hydroxylation was more sensitive than bentazon hydroxylation to inhibition by the insecticides, and both herbicide hydroxylations were more sensitive than lauric acid or cinnamic acid hydroxylations to the insecticides. Since the oxidative metabolites of terbufos were shown to be more potent inhibitors of in vivo nicosulfuron metabolism than terbufos, we examined the effect of terbufos-sulfone on in vivo and in vitro herbicide metabolism. Terbufos-sulfone inhibited metabolism of nicosulfuron and imazethapyr, but not bentazon, in excised corn shoots. Microsomal hydroxylation of nicosulfuron, bentazon, chlorimuron ethyl, and imazethapyr, as well as the desulfuration of malathion, were strongly inhibited (>65%) by terbufos-sulfone. Cinnamic acid hydroxylase appeared to be different from the P450(s) responsible for the pesticide metabolism as it was not inhibited by terbufos-sulfone. However, the data also suggest that malathion, nicosulfuron, bentazon, chlorimuron ethyl, and imazethapyr all share a P450 in common with terbufos-sulfone. Alternatively, there may be separate P450s for the metabolism of the herbicides and malathion, all of which also metabolize terbufos-sulfone. These data show that the inhibition of P450 hydroxylation of nicosulfuron by terbufos-sulfone can explain the injury when maize is exposed to both terbufos and nicosulfuron. However, the insecticides that are the most potent in vitro P450 inhibitors are not necessarily the ones that cause the most herbicide injury in the field.
Imidacloprid, a neonicotinoid the newest class of major insecticide has outstanding potency and systemic action for crop protection against piercing and sucking insects pests and also highly effective for control of flea on cats and dogs. The effect of oral administration of two doses of imidacloprid 10 and 20mg/kg/day for 60days on biochemical parameters, histopathology and protein profile of female albino rat was assessed. Average feed intake was significantly reduced (P<0.01) at 20mg/kg/day. Relative weight of heart and spleen decreased significantly (P<0.05) at higher dose level. Non significant increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), acid phosphatase (ACP), alkaline phosphatase (AKP) activity was observed in both the imidacloprid treated groups. There was significant decrease (P<0.01, P<0.05) in acetyl cholinesterase (AChE) activity in plasma and brain of both the imidacloprid treated groups. Microscopically, liver tissue of rats treated with higher dose of imidacloprid showed marked dilation and congestion of central vein and degeneration of hepatocytes. The exposure to imidacloprid produced histopathological changes that could be correlated with changes in the biochemical profile of female albino rats. The blood plasma proteins were examined by SDS PAGE. There was no diagnostic difference in the pattern of plasma protein profile of control and treated rats. Based on the present physiological, biochemical and histological studies it is evident that imidacloprid did not produce any significant effects at 10mg/kg/day dose but induced toxicological effects at 20mg/kg/day to female rats.
γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [(3)H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [(3)H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [(3)H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [(3)H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [(3)H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of [(3)H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides in insect GABARs.
The accumulation of [3H]inositol phosphates derived from phosphoinositide hydrolysis stimulated by gamma- and delta-hexachlorocyclohexane isomers was characterized in primary cultures of cerebellar granule cells. The EC50 for gamma- and delta-hexachlorocyclohexane was 106 and 85 μM, respectively. Stimulatory effects of hexachlorocyclohexane isomers were highly dependent on extracellular Ca2+ but they were not inhibited by classical voltage-sensitive Ca2+ and Na+ channel blockers. The Na+/Ca2+ exchanger blocker amiloride caused a significant inhibition of delta-hexachlorocyclohexane effects. A lack of additive effects on phosphoinositide hydrolysis stimulation between hexachlorocyclohexane isomers and depolarization by high K+ was observed. The effects of each hexachlorocyclohexane isomer on glutamate or carbachol-induced inositol phosphate stimulation were also not additive, whereas that of high K+ was less than additive or synergistic when combined with glutamate or carbachol, respectively. When neuronal cells were exposed to the combination of delta-hexachlorocyclohexane and glutamate or carbachol in the presence of the respective receptor antagonists only the delta-hexachlorocyclohexane stimulatory effect was observed. Thus, the inhibition of glutamate- and carbachol-stimulated phosphoinositide hydrolysis by delta-hexachlorocyclohexane seems to imply a receptor-independent mechanism. It is suggested that both gamma- and delta-hexachlorocyclohexane activate phosphoinositide-specific phospholipase C partly through Ca2+-related mechanisms.
Ecdysteroids cause salivary gland degeneration in female ixodid ticks. We tested the effects of the following compounds on salivary gland degeneration in the ixodid tick Amblyomma hebraeum Koch: HHCS (22S,23S-homocastasterone), SSBR (22S,23S-homobrassinolide), STGM (2alpha,3alpha(OH)2-Delta22-stigmasten-6-one), RH 5849, and RH 5992. The first three are brassinosteroids (putative ecdysone antagonists) and the last two are nonsteroidal mimics of ecdysone in a variety of insects. In vitro, HHCS (up to 4 μg/ml, 8.4 μM) did not attenuate degeneration caused by 20-hydroxyecdysone; on the contrary, it enhanced the degree of salivary gland degeneration. SSBR (up to 4.5 μg/ml, 9 μM) likewise did not reduce 20-hydroxyecdysone mediated degeneration. RH 5849 up to 15 μg/ml (51 μM) and RH 5992 up to 10 μg/ml (28 μM) had no ecdysone-mimicking effect. In vivo, both RH compounds had an ecdysone-mimicking effect. RH 5849 (but not RH 5992) at 10 μg/tick increased ovary wet weight slightly. None of the brassinosteroids displaced a significant amount of [3H]ponasterone A (PoA) from the ecdysone receptor at 6 μg/ml (12-14 μM) or below. RH 5849 (14 μg/ml, 47 μM) displaced 8% of (PoA) binding; at 68 μg/ml (230 μM) displacement was 30%. For RH 5992 inhibition of PoA binding was 16, 33, and 43% at 4.5, 16, and 79 μg/ml (13, 45, and 79 μM), respectively. Overall, the brassinosteroids and the RH compounds do not act on the tick salivary gland ecdysteroid system in a way similar to the way they act on some insect systems.
Szechuan peppers are extensively used as a spice and in traditional medicine in Asia, primarily because of its active compounds, sanshools (S). However, there is only limited mention in agriculture, and there are no papers reporting its use as an herbicide safener. In this study, we provide the first evidence that S can effectively alleviate rice-seedling injury from metolachlor (M). We observed that the M-treated (0.25μM) rice seedlings, which were 56.0%, 66.0%, and 57.0% of the non-treated control in shoot height, root length, and fresh biomass, respectively, were recovered by S to 93.1%, 97.6%, and 94.8%, respectively. The emergence rate was enhanced to over 80% in the M+S treatment, whereas it was below 60% in the M treatment. This M+S mixture elevated the rice-seedling root activity to higher than 87.0% of the value for the non-treated control. The activity of glutathione transferases in the combined treatments approximately doubles that of the M treatment and quadruples that of the non-treated controls. This effect was positively correlated with the induced expression of OsGSTU3. Our results suggest that S may represent a new group of safeners and enable the possibility of using these compounds for improving plant production or protecting rice from the phytotoxicity of metolachlor.
This study aimed to investigate the protective effect of vitamin E (VitE) on cypermethrin (CPM)-induced damages in the ovary. Wistar rats were divided into seven groups (n=6) including; control-sham (c), CPM-received (CPM, 75mg/kg, i.p.), and CPM and VitE-treated (VitE, 150mg/kg, orally) for 7, 14 and 24days. The antioxidant status determination and hormonal assays along with histological and immunofluorescent assessments were performed. The expression of p53 at mRNA level was also examined. The CPM administration affected the ovarian structure and functions as it elevated the follicular atresia and significantly (P<0.05) lowered the estradiol level, time dependently. VitE administration enhanced the CPM-reduced antioxidant capacity, gonadotropins and estradiol levels. Co-administration of VitE and CPM remarkably attenuated the CPM-induced RNA damage in granulosa and theca cells and elevated the deranged angiogenesis. The CPM-reduced micro and macro vessels distribution was significantly (P<0.05) elevated in the VitE-received animals. Expression of p53 at mRNA level was down regulated in the VitE-treated groups completely and relatively following 7 and 14days, respectively. Our data showed that the CPM-induced biochemical and histological damages could be prevented by VitE. Moreover, protective effects of VitE attribute to its potency in enhancing the antioxidant capacity and promoting the gonadotropins secretion, which resulted in down regulation of p53 overexpression and RNA damage in follicular cells accomplished with improved angiogenesis.
Azinphosmethyl-selected tufted apple bud moths were compared to susceptible and reverted strains with respect to possible metabolic mechanisms of resistance within the third instar, fifth instar, and adults. Based upon bioassays conducted by topical application with azinphosmethyl, LD50s were as high as 867-fold in the selected strain as compared to that seen in susceptible bud moths. The LD50 of the reverted strain was intermediate to that of the susceptible and selected insects at all stages studied. Glutathione transferase activity measured with 1-chloro-2,4-dinitrobenzene was elevated in the selected strain 1.6- and 2.2-fold as compared to third and fifth stadium susceptible bud moths, respectively. No consistent strain differences were noted for 1,2-dichloro-4-nitrobenzene. Cytochrome P450 content and P450 mRNA was not significantly different in fifth instars of the susceptible and selected strain. However, there was a 2.7- and 1.9-fold increase in benzphetamine and p-nitroanisole metabolism, respectively, in the guts of azinphosmethyl-selected fifth instars. Benzo[a]pyrene metabolism was elevated 2.4-fold in the carcass of selected bud moths and no differences were noted for methoxyresorufrin in either gut or carcass. Susceptible fifth instars demonstrated a reduced rate of metabolism of azinphosmethyl to the oxon and methyl benzazamide. Piperonyl butoxide failed to synergize azinphosmethyl toxicity. Esterase activity measured with 1-naphthyl acetate and p-nitrophenyl acetate was elevated in selected larvae compared to that seen in susceptible tufted apple bud moths in both larvae and adults. This increased esterase activity was attributed to several isoforms as resolved by analytical isoelectric focusing. One of these forms was consistently overexpressed in all of the life stages examined. Pretreatment of selected fifth instars with S,S,S-tributylphosphorotrithioate increased the toxicity of azinphosmethyl 400-fold and had minimal effect on toxicity in susceptible insects. It appears that multiple hydrolases are the primary metabolic factor in azinphosmethyl resistance in the tufted apple bud moth.
Ryanodine receptors (RyRs) are the targets of novel diamide insecticides. The cotton bollworm, Helicoverpa armigera, is one of the most important cotton pests in the world. In this study, we report the full-length RyR cDNA sequence (named as HaRyR) of H. armigera. The 16,083-bp contiguous sequence encoded 5, 142 amino acid residues, which shares 80% and 78% overall identities with its homologues in Nilaparvata lugens (NlRyR) and Drosophila melanogaster (DmRyR), respectively. All hallmarks of RyR proteins are conserved in the HaRyR, including the GXRXGGGXGD motif conserved in the Ca(2+) release channels and four copies of RyR domain unique to RyR channels. The previously identified seven lepidopteran-specific RyR residues were also found in HaRyR (N(4977), N(4979), N(4990), L(5005), L(5036), N(5068) and T(5119)). An amino acid sequence alignment showed that the N-terminal region of HaRyR (residues 188-295) shared high sequence identity with NlRyR (94%) and DmRyR (92%), and moderate sequence identity (47-50%) with three rabbit RyR isoforms, while the short segment of the C-terminal transmembrane region of HaRyR (residues 4632-4676) exhibited moderate sequence identity with NlRyR (69%) and DmRyR (67%), and low sequence identity (19-28%) with three rabbit RyR isoforms. In addition, expression analysis of HaRyR revealed that the mRNA expression level in eggs was significantly lower than in third instar larvae, pupae and adults, and anatomical regulation of HaRyR expression was also observed with the highest expression level in head compared with thorax and abdomen. Our results lay a foundation for comprehensive structural and functional characterization of HaRyR and for understanding of the molecular mechanisms of toxicity selectivity of diamide insecticides among different species.
Agricultural chemicals can induce genetic alterations on aquatic organisms that have been associated with effects on growth, reproduction and population dynamics. The evaluation of DNA damage in fish using the comet assay (CA) frequently involves the utilization of erythrocytes. However, epithelial gill cells (EGC) can be more sensitive, as they are constantly dividing and in direct contact with potentially stressing compounds from the aquatic environment. The aim of the present study was to evaluate (1) the sensitivity and suitability of epithelial gill cells of Prochilodus lineatus in response to different genotoxic agents through the application of the CA, (2) the induction of DNA damage in this cell population after in vivo exposure to cypermethrin. Baseline value of the CA damage index (DI) for EGC of juvenile P. lineatus was 144.68±5.69. Damage increased in a dose-dependent manner after in vitro exposure of EGC to methyl methanesulfonate (MMS) and H2O2, two known genotoxic agents. In vivo exposure of fish to cypermethrin induced a significant increase in DNA DI of EGC at 0.150μg/l (DI: 239.62±6.21) and 0.300μg/l (270.63±2.09) compared to control (150.25±4.38) but no effect was observed at 0.075μg/l (168.50±10.77). This study shows that EGC of this species are sensitive for the application of the CA, demonstrating DNA damage in response to alkylation (MMS), oxidative damage (H2O2), and to the insecticide cypermethryn. These data, together with our previous study on DNA damage induction on erythrocytes of this species, provides useful information for future work involving biomonitoring in regions where P. lineatus is naturally exposed to pesticides and other genotoxic agents.
Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.
Based on a Leptinotarsa decemlineata transcriptome dataset and the GenBank sequences, a total of 74 cytochrome P450 monooxygenase genes (Cyps) were identified. These genes fell into CYP2 clan, mitochondrial clan, CYP3 clan and CYP4 clan, and were classified into 19 families and 35 subfamilies according to standard nomenclature. Two new families were discovered in CYP4 clan, and were named CYP412 and CYP413 respectively. Four new families that were recently discovered in Tribolium castaneum, including mitochondrial family CYP353, CYP3 clan families CYP345 and CYP347, and CYP4 clan family CYP350, were also found in L. decemlineata. The phylogenetic trees of CYPs from L. decemlineata and other representative insect species were constructed, and these trees provided evolutionary insight for the genetic distance. Our results facilitate further researches to understand the functions and evolution of L. decemlineata Cyp genes. In order to find cyhalothrin-inducible Cyp genes, the expression levels of Cyps belonging to CYP12, CYP6, CYP9 and CYP4 families were determined by quantitative reverse transcriptase-PCR in cyhalothrin-treated and control fourth-instar larvae. Nine Cyp genes, i.e., Cyp12H2, Cyp6BH2, Cyp6BJ1, Cyp6BQ17, Cyp6EG1, Cyp6EH1, Cyp6EJ1 Cyp4BN13v1 and Cyp4BN15, were highly expressed in cyhalothrin-treated larvae. These CYPs are the candidates that are involved in cyhalothrin detoxification.
Anthranilic and phthalic diamides exemplified by chlorantraniliprole (Chlo) or cyantraniliprole (Cyan) and flubendiamide (Flu), respectively, are the newest major chemotype of insecticides with outstanding potency, little or no cross resistance with other classes and low mammalian toxicity. They are activators of the ryanodine (Ry) receptor (RyR)-Ca(2+) channel, based on Ca(2+) flux and electrophysiology investigations. The goal of this study is to define species differences in the degree and mechanisms of diamide selective action by radioligand specific binding studies at the [(3)H]Ry, [(3)H]Chlo and [(3)H]Flu sites. The [(3)H]Ry site is observed in muscle of lobster, rabbit and four insect species (Musca domestica, Apis mellifera, Heliothis virescens and Agrotis ipsilon) whereas the [(3)H]Chlo site is evident in the four insects and the [(3)H]Flu site in only the two lepidoptera (Agrotis and Heliothis). [(3)H]Ry binding is significantly stimulated by Chlo, Cyan and Flu with the insects (except Flu with Musca) but not the lobster and rabbit. [(3)H]Chlo binding is stimulated by Ry and Flu in Musca and Apis but not in the lepidoptera, while Flu and Cyan are inhibitory. [(3)H]Flu binding is strongly inhibited by Chlo and Cyan in Agrotis and Heliothis. [(3)H]Chlo and [(3)H]Flu binding are not dependent on added Ca(2+) or ATP in Heliothis and Agrotis whereas the other radioligand-receptor combinations are usually enhanced by Ca(2+) and ATP. More generally, there are species differences in the Ry, Chlo and Flu binding sites of the RyR that may confer selective toxicity and determine target site cross resistance mechanisms.
Electrophysiological methods were used to characterize the modulatory effects of hexachlorocyclohexane isomers on GABA-activated chloride currents in dissociated locust neurones. Conductance changes associated with hyperpolarising responses (or outward voltage-clamped currents), evoked by brief GABA pulses, were inhibited by the gamma-isomer at 0.01-1 μM. The antagonistic action was noncompetitive and voltage-independent: the concentration required for half-maximal block was <0.01 μM. The rate of equilibration with the membrane/receptor was inversely proportional to the concentration applied and was not dependent upon agonist-induced channel gating: effects were only partially reversible with extensive washing. In contrast the noninsecticidal beta-isomer was a low-affinity (>/=10 μM) positive modulator of the inhibitory chloride channels. The modulatory action was voltage-dependent (more pronounced for inward currents at hyperpolarised potentials), did not reflect changes in chloride equilibrium potential, and was completely reversible. Nicotinic acetylcholine receptors were consistently antagonised by the beta-isomer at 10 μM. The above results were interpreted by computer-assisted modelling and are discussed in the context of a pharmacophore model for picrotoxinin and convulsant insecticides. The gamma-isomer overlays well with picrotoxinin and a convulsant butyrolactone whereas the beta-isomer and anticonvulsant butyrolactones conform to a different template. Liganding mechanisms and comparative/species differences in chloride channel pharmacology are discussed in the context of potential for development of invertebrate-selective pesticides.
A dicarboximide-susceptible (DS) isolate and a dicarboximide-resistant (DR) isolate of Botrytis cinerea were compared with regard to spore germination, mycelial growth, cellular leakage, and lipid peroxidation upon treatment with the dicarboximide fungicide vinclozolin. The fungicide inhibited spore germination and mycelial growth of the DS isolate, but not those of the DR isolate. The inhibitory effect of the fungicide was greater on mycelial growth than on spore germination of the DS isolate. Significant cellular leakage from the fungicide-treated DS or DR isolate began to increase after 24 hr incubation, depending on the concentration of the fungicide and the duration of incubation time. However, the magnitude of cellular leakage was much greater from the DS isolate than from the DR isolate. Vinclozolin caused considerable lipid peroxidation in the DS isolate, whereas little or no lipid peroxidation occurred in the DR isolate treated with the fungicide. Lipid peroxidation preceded cellular leakage from the DS isolate following fungicide treatment. The effects of the fungicide on mycelial growth, cellular leakage, and lipid peroxidation of the DS isolate were all reversed by the addition of alpha-tocopherol to the incubation medium. These results demonstrate that vinclozolin causes significant lipid peroxidation and subsequent cellular leakage from a DS isolate, but not from a DR isolate. Furthermore, they suggest that the mechanism of action of dicarboximide fungicides is associated with membrane lipid peroxidation.
A previous study demonstrated that the flight capacity of Nilaparvata lugens adults treated with triazophos was enhanced significantly. However, the physiological and regulative mechanisms of the flight enhancement are not well understood. Trehalose is a primary blood sugar in insects, and the enzyme trehalase is involved in energy metabolism. The present study investigated the effects of triazophos on the trehalose content, trehalase activity (soluble trehalase and membrane-bound trehalase) and the mRNA transcript levels of their corresponding genes (NlTre-1 and NlTre-2) in fifth instar nymphs, as well as in the brachypterous and macropterous N. lugens adult females. Our findings showed that the trehalose content in fifth instar nymphs as well as in the brachypterous and the macropterous adults significantly decreased following triazophos treatment. However, the glucose content, soluble trehalase activity and expression level of NlTre-1 mRNA increased significantly compared to the controls. No significant enhancement of NlTre-2 expression was found, indicating that regulation of energy metabolism of triazophos-induced flight capacity in N. lugens was not associated with NlTre-2 expression. In addition, soluble trehalase activity and the expression level of NlTre-1 mRNA in the macropterous females was significantly higher than that in the brachypterous females. The present findings provide valuable information on the molecular and regulative mechanisms of the increased flight capacity found in adult N. lugens after treatment with triazophos.
Pyrethroids disrupt nerve function by altering the rapid kinetic transitions between conducting and nonconducting states of voltage-gated sodium channels that underlie the generation of nerve action potentials. Recent studies of pyrethroid action on cloned insect and mammalian sodium channel isoforms expressed in Xenopus laevis oocytes show that in some cases pyrethroid modification is either absolutely dependent on or significantly enhanced by repeated channel activation. These use-dependent effects have been interpreted as evidence of preferential binding of at least some pyrethroids to the open, rather than resting, state of the sodium channel. This paper reviews the evidence for state-dependent modification of insect and mammalian sodium channels expressed in oocytes by pyrethroids and considers the implications of state-dependent effects for understanding the molecular mechanism of pyrethroid action and the development and testing of models of the pyrethroid receptor.
The diamide insecticides activate ryanodine receptors (RyRs) to release and deplete intracellular calcium stores from the sarcoplasmic reticulum of muscles and the endoplasmic reticulum of many types of cells. They rapidly interrupt feeding of the target pest and eventually kill the pest due to starvation. However, information about the structure and function of insect RyRs is still limited. In this study, we isolated a 15,985bp full-length cDNA (named SfRyR) from Sogatella furcifera, a serious rice planthopper pest throughout Asia. SfRyR encodes a 5140-amino acid protein, which shares 78-97% sequence identities with other insect homologues, and less than 50% identities with Homo sapiens RyR1-3. All hallmarks of the RyR proteins are conserved in SfRyR. In the N-terminus, SfRyR has a MIR domain, two RIH domains, three SPRY domains, four copies of RyR repeated domain and a RIH-associated domain. In the C-terminus, SfRyR possesses two consensus calcium ion-binding EF-hand motifs, and six transmembrane helices. Temporal and spatial expression analysis showed that SfRyR was widely found in all development stages including egg, first through fifth instar nymphs, macropterous adult females and males. On day 2 fifth-instar nymphs, SfRyR was ubiquitously expressed in the head, thorax and abdomen. Dietary ingestion of dsSfRyR1 and dsSfRyR2 significantly reduced the mRNA level of SfRyR in the treated nymphs by 77.9% and 81.8% respectively, and greatly decreased chlorantraniliprole-induced mortality. Thus, our results suggested that SfRyR gene encoded a functional RyR that mediates chlorantraniliprole toxicity to S. furcifera.
Tobacco cytochrome P450 (CYP) 71AH11 metabolized the herbicide chlorotoluron, and its mRNA level was increased in tobacco culture cells by the treatment of 2,4-D. In order to clarify molecular mechanisms of induced gene expression of CYP71AH11 by herbicide treatment, a 1574-bp 5'-flanking region of CYP71AH11 was cloned, ligated to the reporter β-glucuronidase (GUS) gene, and then transformed into tobacco plants. The GUS activity in the transgenic tobacco plants was highly induced by bromoxynil treatment, followed by 2,4-D. Chlorotoluron was slightly increased the GUS activity. The bromoxynil-increased GUS activity was partially repressed by the antioxidants, suggesting that reactive oxygen species may be involved in activation of the 5'-flanking region of CYP71AH11 by bromoxynil treatment. Deletion and mutation assays showed that the region CD (-1281 to -770bp from the start codon of CYP71AH11) was important, but not sufficient, for response to bromoxynil. Electrophoretic mobility shift assays and southwestern blotting revealed that the sequences AAAAAG, and GAACAAAC and GAAAATTC in the CD region were important for interaction to the nuclear proteins of <30 and ≈75kDa, respectively. Particularly, interaction between AAAAAG and <30kDa proteins was increased by bromoxynil treatment. These results gave a cue for understanding the bromoxynil-induced gene expression of CYP71AH11, which may contribute to herbicide tolerance and selectivity in crop plants.
Flupoxam, an herbicide representative of a powerful new class of herbicides intended for control of broad-leaved weeds in cereals, has been identified as a mitotic disrupter herbicide. Flupoxam was evaluated on watercress seedlings for evidence of antimicrotubule effects characteristic of mitotic disrupter herbicides. At 1 μM, flupoxam inhibited root elongation and induced club root morphology. However, examination by light microscopy and tubulin immunofluorescent microscopy revealed that flupoxam is not a mitotic disrupter herbicide.
House fly (Musca domestica) CYP6D1 is a cytochrome P450 involved in metabolism of xenobiotics. CYP6D1 is located on chromosome 1 and its expression is inducible in response to the prototypical P450 inducer phenobarbital (PB) in insecticide susceptible strains. Increased transcription of CYP6D1 confers resistance to permethrin in the LPR strain, and this trait maps to chromosomes 1 and 2. However, the constitutive overexpression of CYP6D1 in LPR is not further increased by PB and the non-responsiveness to PB maps to chromosome 2. It has been suggested that a single factor on chromosome 2 could be responsible for both the constitutive overexpression and lack of PB induction of CYP6D1 in LPR. We examined the PB inducibility of CYP6D1v1 promoter from LPR using dual luciferase reporter assays in Drosophila S2 cells and found the CYP6D1v1 promoter was able to mediate PB induction, similar to the CYP6D1v2 promoter from the insecticide susceptible CS strain. Therefore, variation in promoter sequences of CYP6D1v1 and v2 does not appear responsible for the lack of PB induction of CYP6D1v1 in LPR; this suggests an unidentified trans acting factor is responsible. HR96 has been implicated in having a role in PB induction in Drosophila melanogaster and M. domestica. Therefore, house fly HR96 cDNA was cloned and sequenced to examine if this trans acting factor is responsible for constitutive overexpression of CYP6D1v1 in LPR. Multiple HR96 alleles (v1-v10) were identified, but none were associated with resistance. Expression levels of HR96 were not different between LPR and CS. Thus, HR96 is not the trans acting factor responsible for the constitutive overexpression of CYP6D1 in LPR. The identity of this trans acting factor remains elusive.