Characterization of mosquito CYP6P7 and CYP6AA3: differences in substrate preference and kinetic properties

Cytochrome P450 monooxygenases are involved in insecticide resistance in insects. We previously observed an increase in CYP6P7 and CYP6AA3 mRNA expression in Anopheles minimus mosquitoes during the selection for deltamethrin resistance in the laboratory. CYP6AA3 has been shown to metabolize deltamethrin, while no information is known for CYP6P7. In this study, CYP6P7 was heterologously expressed in the Spodoptera frugiperda (Sf9) insect cells via baculovirus‐mediated expression system. The expressed CYP6P7 protein was used for exploitation of its enzymatic activity against insecticides after reconstitution with the An. minimus NADPH‐cytochrome P450 reductase enzyme in vitro. The ability of CYP6P7 to metabolize pyrethroids and insecticides in the organophosphate and carbamate groups was compared with CYP6AA3. The results revealed that both CYP6P7 and CYP6AA3 proteins could metabolize permethrin, cypermethrin, and deltamethrin pyrethroid insecticides, but showed the absence of activity against bioallethrin (pyrethroid), chlorpyrifos (organophosphate), and propoxur (carbamate). CYP6P7 had limited capacity in metabolizing λ‐cyhalothrin (pyrethroid), while CYP6AA3 displayed activity toward λ‐cyhalothrin. Kinetic properties suggested that CYP6AA3 had higher efficiency in metabolizing type I than type II pyrethroids, while catalytic efficiency of CYP6P7 toward both types was not significantly different. Their kinetic parameters in insecticide metabolism and preliminary inhibition studies by test compounds in the flavonoid, furanocoumarin, and methylenedioxyphenyl groups elucidated that CYP6P7 had different enzyme properties compared with CYP6AA3. © 2011 Wiley Periodicals, Inc.     

Determination of metabolites of pyrethroids in human urine using solid-phase extraction and gas chromatography–mass spectrometry

The described method permits the determination of the five most important metabolites of the pyrethroids permethrin, cypermethrin, deltamethrin, λ-cyhalothrin, fenvalerate, phenothrin and β-cyfluthrin in human urine in one run. The major urinary metabolites of these substances are cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl₂CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br₂CA), fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA). After acidic hydrolysis to release the conjugated carboxylic acid metabolites, the analytes were separated from the matrix by means of solid-phase extraction using a reversed-phase column. The components of the eluate were converted to their methyl esters and extracted in hexane. Separation and quantitative analysis of the pyrethroid metabolites was carried out by capillary gas chromatography and mass selective detection. 2-Phenoxybenzoic acid served as an internal standard. The detection limits lay between 0.3 and 0.5 μg per litre urine. The relative standard deviations of the within-series imprecision were between 1% and 6%. The relative recovery rates ranged between 90% and 98%. Using this method we determined the elimination of pyrethroid metabolites in 24-h urine samples from eight pest controllers after indoor application of permethrin. The detected concentrations ranged from 1 to 70 μg g⁻¹ creatinine.     

Pyrethroid susceptibility and behavioral avoidance in Anopheles epiroticus,a malaria vector in Thailand

The physiological susceptibility to insecticides and the behavioral responses of four wild‐caught populations of female Anopheles epiroticus to synthetic pyrethroids (deltamethrin, permethrin, and alpha‐cypermethrin) were assessed. Test populations were collected from different localities along the eastern coast, Trat (TR), Songkhla (SK), and Surat Thani (ST) and one population from the western coast, Phang Nga (PN). Results showed that all four populations of An. epiroticus were susceptible to all three synthetic pyrethroids tested. Behavioral responses to test compounds were characterized for all four populations using an excito‐repellency test system. TR displayed the strongest contact excitation (‘irritancy’) escape response (76.8% exposed to deltamethrin, 74.1% permethrin, and 78.4% alpha‐cypermethrin), followed by the PN population (24.4% deltamethrin, 35% permethrin, and 34.4% for alpha‐cypermethrin) by rapidly escaping test chambers after direct contact with surfaces treated with each active ingredient compared with match‐paired untreated controls. Moderate non‐contact repellency responses to all three compounds were observed in the TR population but were comparatively weaker than paired contact tests. Few mosquitoes from the SK and ST populations escaped from test chambers, regardless of insecticide tested or type of trial. We conclude that contact excitation was a major behavioral response in two populations of An. epiroticus, whereas two other populations showed virtually no escape response following exposure to the three pyrethroids. The explanation for these large unexpected differences in avoidance responses between pyrethroid‐susceptible populations of the same species is unclear and warrants further investigation.     

Insecticide resistance monitoring of field‐collected Anopheles gambiae s.l. populations from Jinja,eastern Uganda,identifies high levels of pyrethroid resistance

Insecticide resistance in the malaria vector Anopheles gambiae s.l. (Diptera: Culicidae) threatens insecticide‐based control efforts, necessitating regular monitoring. We assessed resistance in field‐collected An. gambiae s.l. from Jinja, Uganda using World Health Organization (WHO) biosassays. Only An. gambiae s.s. and An. arabiensis (?70%) were present. Female An. gambiae exhibited extremely high pyrethroid resistance (permethrin LT₅₀ > 2 h; deltamethrin LT₅₀ > 5 h). Female An. arabiensis were resistant to permethrin and exhibited reduced susceptibility to deltamethrin. However, while An. gambiae were DDT resistant, An. arabiensis were fully susceptible. Both species were fully susceptible to bendiocarb and fenitrothion. Kdr 1014S has increased rapidly in the Jinja population of An. gambiae s.s. and now approaches fixation (?95%), consistent with insecticide‐mediated selection, but is currently at a low frequency in An. arabiensis (0.07%). Kdr 1014F was also at a low frequency in An. gambiae. These frequencies preclude adequately‐powered tests for an association with phenotypic resistance. PBO synergist bioassays resulted in near complete recovery of pyrethroid susceptibility suggesting involvement of CYP450s in resistance. A small number (0.22%) of An. gambiae s.s. ×An. arabiensis hybrids were found, suggesting the possibility of introgression of resistance alleles between species. The high levels of pyrethroid resistance encountered in Jinja threaten to reduce the efficacy of vector control programmes which rely on pyrethroid‐impregnated bednets or indoor spraying of pyrethroids.     

A metabonomic investigation of the effects of 60 days exposure of rats to two types of pyrethroid insecticides

Type I and II pyrethroid insecticides display different neurotoxicity. To investigate the long-term (60 days exposure) metabolic effect of the two types of pyrethroid insecticides deltamethrin and permethrin, ¹H nuclear magnetic resonance (NMR) spectroscopy-based metabonomics was used to analyze the biochemical composition of urine and serum samples from rats administrated daily with deltamethrin or permethrin for 60 consecutive days, and principal component analysis used to visualize similarities and differences in the resultant biochemical profiles. Rats treated with either deltamethrin or permethrin displayed increased levels of urinary acetate, dimethylamine, dimethylglycine, trimethylamine and serum free amino acids, and decreased urinary 2-oxoglutarate, all of which are indicative of kidney lesions and nephrotoxicity. The reduced excretion of tricarboxylic acid cycle intermediates, together with increased 3-D-hydroxybutyrate, acetate, and lactate in treated rats could suggest disturbance of the energy metabolism, including an increased rate of anaerobic glycolysis, enhanced fatty acid β-oxidation and ketogenesis. These results show that these two types of insecticides have similarities in the urine and serum spectra, indicating that similar metabolic pathways are perturbed by the insecticides, which induced hepatotoxicity and nephrotoxicity. This approach may lead to the discovery of novel biomarkers of pyrethroids toxicity and thereby provide new insights into the toxicological mechanisms of pesticides pyrethroids.     

Deltamethrin‐induced feeding plasticity in pyrethroid‐susceptible and ‐resistant strains of the maize weevil,Sitophilus zeamais

Phenotypic plasticity contributes to the adaptative evolution of populations exposed to new or altered environments. Feeding plasticity is a component of phenotypic plasticity not usually considered in insect strains adapted to insecticide‐altered environments, but which may either accentuate or mitigate insecticide resistance. This is a concern in the pyrethroid‐resistant strains of the maize weevil Sitophilus zeamais Motsch. (Col., Curculionidae), and the reason for this study. A pyrethroid‐susceptible and two pyrethroid‐resistant strains of maize weevil were subjected to free‐choice and no‐choice tests with maize grains sprayed with increasing doses of the pyrethroid, deltamethrin. The insects from the pyrethroid‐resistant strains exhibited higher feeding avoidance with increased deltamethrin doses than insects from the susceptible strain when subjected to free‐choice tests. The strains of maize weevil physiologically resistant to pyrethroids were also behaviourally resistant to deltamethrin – an additional management concern. The resistant strains avoid deltamethrin‐sprayed grains and are less nutritionally affected by this compound, with divergent responses from the susceptible strain with increased doses of deltamethrin. Furthermore, the higher relative growth rate and consequently higher efficiency of food conversion observed in the insecticide‐resistant strains were significant even without insecticide exposure, indicating that these traits are stimulus‐independent and may persist even without further insecticide selection, potentially limiting the options available for their management.     

A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Numerous point mutations in sodium channel genes have been identified in pyrethroid-resistant insect species, and many have been confirmed to reduce or abolish sensitivity of channels expressed in Xenopus oocytes to pyrethroids. Recently, several novel mutations were reported in sodium channel genes of pyrethroid-resistant Aedes mosquito populations. One of the mutations is a phenylalanine (F) to cysteine (C) change in segment 6 of domain III (IIIS6) of the Aedes mosquito sodium channel. Curiously, a previous study showed that alanine substitution of this F did not alter the action of deltamethrin, a type II pyrethroid, on a cockroach sodium channel. In this study, we changed this F to C in a pyrethroid-sensitive cockroach sodium channel and examined mutant channel sensitivity to permethrin as well as five other type I or type II pyrethroids in Xenopus oocytes. Interestingly, the F to C mutation drastically reduced channel sensitivity to three type I pyrethroids, permethrin, NRDC 157 (a deltamethrin analogue lacking the ??-cyano group) and bioresemthrin, but not to three type II pyrethroids, cypermethrin, deltamethrin and cyhalothrin. These results confirm the involvement of the F to C mutation in permethrin resistance, and raise the possibility that rotation of type I and type II pyrethroids might be considered in the control of insect pest populations where this particular mutation is present.     

Essential oils enhance the toxicity of permethrin against Aedes aegypti and Anopheles gambiae

Insecticide resistance and growing public concern over the safety and environmental impacts of some conventional insecticides have resulted in the need to discover alternative control tools. Naturally occurring botanically‐based compounds are of increased interest to aid in the management of mosquitoes. Susceptible strains of Aedes aegypti (Linnaeus) (Diptera: Culicidae) and Anopheles gambiae (Meigen) (Diptera: Culicidae) were treated with permethrin, a common type‐I synthetic pyrethroid, using a discriminate dose that resulted in less than 50% mortality. Piperonyl butoxide (PBO) and 35 essential oils were co‐delivered with permethrin at two doses (2 and 10 µg) to determine if they could enhance the 1‐h knockdown and the 24‐h mortality of permethrin. Several of the tested essential oils enhanced the efficacy of permethrin equally and more effectively than piperonyl butoxide PBO, which is the commercial standard to synergize chemical insecticide like pyrethroids. PBO had a strikingly negative effect on the 1‐h knockdown of permethrin against Ae. aegypti, which was not observed in An. gambiae. Botanical essential oils have the capability of increasing the efficacy of permethrin allowing for a natural alternative to classic chemical synergists, like PBO.     

Behavioural and insecticidal effects of organophosphate‐, carbamate‐ and pyrethroid‐treated mosquito nets against African malaria vectors

Three insecticides – the pyrethroid deltamethrin, the carbamate carbosulfan and the organophosphate chlorpyrifos‐methyl – were tested on mosquito nets in experimental huts to determine their potential for introduction as malaria control measures. Their behavioural effects and efficacy were examined in Anopheles gambiae Giles s.s. (Diptera: Culicidae) and Anopheles funestus Giles s.s. in Muheza, Tanzania, and in Anopheles arabiensis Patton and Culex quinquefasciatus Say in Moshi, Tanzania. A standardized dosage of 25 mg/m² plus high dosages of carbosulfan (50 mg/m², 100 mg/m² and 200 mg/m²) and chlorpyrifos‐methyl (100 mg/m²) were used to compare the three types of insecticide. At 25 mg/m², the rank order of the insecticides for insecticide‐induced mortality in wild An. gambiae and An. funestus was, respectively, carbosulfan (88%, 86%) > deltamethrin (79%, 78%) > chlorpyrifos‐methyl (35%, 53%). The rank order of the insecticides for blood‐feeding inhibition (reduction in the number of blood‐fed mosquitoes compared with control) in wild An. gambiae and An. funestus was deltamethrin > chlorpyrifos‐methyl > carbosulfan. Carbosulfan was particularly toxic to endophilic anophelines at 200 mg/m², killing 100% of An. gambiae and 98% of An. funestus that entered the huts. It was less effective against the more exophilic An. arabiensis (67% mortality) and carbamate‐resistant Cx quinquefasciatus (36% mortality). Carbosulfan deterred anophelines from entering huts, but did not deter carbamate‐resistant Cx quinquefasciatus. Deltamethrin reduced the proportion of insects engaged in blood‐feeding, probably as a consequence of contact irritancy, whereas carbosulfan seemed to provide personal protection through deterred entry or perhaps a spatial repellent action. Any deployment of carbosulfan as an individual treatment on nets should be carried out on a large scale to reduce the risk of diverting mosquitoes to unprotected individuals. Chlorpyrifos‐methyl was inferior to deltamethrin in terms of mortality and blood‐feeding inhibition and would be better deployed on a net in combination with a pyrethroid to control insecticide‐resistant mosquitoes.     

Experimental hut comparisons of nets treated with carbamate or pyrethroid insecticides, washed or unwashed, against pyrethroid-resistant mosquitoes

Abstract. The efficacy against mosquitoes (Diptera: Culicidae) of a bednet treated with carbamate insecticide [carbosulfan capsule suspension (CS) 200 mg/m²] was compared with four types of pyrethroid‐treated nets in veranda‐trap huts at Yaokoffikro near Bouaké, Côte d'Ivoire, where the malaria vector Anopheles gambiae Giles carries the kdr gene (conferring pyrethroid resistance) at high frequency and Culex quinquefasciatus Say is also pyrethroid resistant. Pyrethroids compared were lambdacyhalothrin CS 18 mg/m², alphacypermethrin water dispersible granules (WG) 20 mg/m², deltamethrin 50 mg/m² (Permanet?) and permethrin emulsifiable concentrate (EC) 500 mg/m². Insecticidal power and personal protection from mosquito bites were assessed before and after the nets were used for 8 months and hand washed five times in cold soapy water. Before washing, all treatments except permethrin significantly reduced blood‐feeding and all had significant insecticidal activity against An. gambiae. The carbosulfan net gave significantly higher killing of An. gambiae than all pyrethroid treatments except the Permanet. Against Culex spp., carbosulfan was more insecticidal and gave a significantly better protective effect than any of the pyrethroid treatments. After washing, treated nets retained various degrees of efficacy against both mosquito genera – but least for the carbosulfan net. Washed nets with three types of pyrethroid treatment (alphacypermethrin, lambdacyhalothrin, permethrin) gave significantly higher mortality rates of Culex than in huts with the same pyrethroid‐treated nets before washing. After five washes, the Permanet?, which is sold as a long‐lasting insecticidal product, performed no better than the other nets in our experimental conditions.     

Characterization of glutathione S‐transferase in the saturniid moth,Samia Cynthia pryeri (Lep.: Saturniidae)

An enzyme, which possesses glutathione S‐transferase (GST) activity, has been found in the midgut of the saturniid moth, Samia cynthia pryeri. The enzyme was initially purified into homogeneity by ammonium sulphate fractionation, affinity chromatography, and ion‐exchange chromatography. The resulting enzyme revealed a single band with a molecular mass of 23 kDa by sodium dodecyl sulfate polyacrylamide electrophoresis under reduced conditions. When tested with 1‐chloro‐2,4‐dinitrobenzene, a universal substrate of GST, the purified remnants had an optimum pH of 8.0 for enzymatic activity, and was fairly stable at pH 5–9 and at temperatures below 40°C. The enzyme was also responsive to 4‐hydroxynonenal, a cytotoxic lipid‐peroxidation product. The present GST was inhibited by organophosphorus and pyrethroid insecticides including fenitrothion, permethrin and deltamethrin.     

Pinpointing P450s associated with pyrethroid metabolism in the dengue vector, Aedes aegypti: developing new tools to combat insecticide resistance

Background

Pyrethroids are increasingly used to block the transmission of diseases spread by Aedes aegypti such as dengue and yellow fever. However, insecticide resistance poses a serious threat, thus there is an urgent need to identify the genes and proteins associated with pyrethroid resistance in order to produce effective counter measures. In Ae. aegypti, overexpression of P450s such as the CYP9J32 gene have been linked with pyrethroid resistance. Our aim was to confirm the role of CYP9J32 and other P450s in insecticide metabolism in order to identify potential diagnostic resistance markers.

Methodology/Principal Findings

We have expressed CYP9J32 in Escherichia coli and show that the enzyme can metabolize the pyrethroids permethrin and deltamethrin. In addition, three other Ae. aegypti P450s (CYP9J24, CYP9J26, CYP9J28) were found capable of pyrethroid metabolism, albeit with lower activity. Both Ae. aegypti and Anopheles gambiae P450s (CYP''s 6M2, 6Z2, 6P3) were screened against fluorogenic and luminescent substrates to identify potential diagnostic probes for P450 activity. Luciferin-PPXE was preferentially metabolised by the three major pyrethroid metabolisers (CYP9J32, CYP6M2 and CYP6P3), identifying a potential diagnostic substrate for these P450s.

Conclusions/Significance

P450s have been identified with the potential to confer pyrethroid resistance in Ae.aegypti. It is recommended that over expression of these enzymes should be monitored as indicators of resistance where pyrethroids are used.     

Modifications of pyrethroid effects associated with kdr mutation in Anopheles gambiae

Effects of knockdown resistance (kdr) were investigated in three pyrethroid‐resistant (RR) strains of the Afrotropical mosquito Anopheles gambiae Giles (Diptera: Culicidae): Kou from Burkina Faso, Tola and Yao from Côte d'Ivoire; compared with a standard susceptible (SS) strain from Kisumu, Kenya. The kdr factor was incompletely recessive, conferring 43‐fold resistance ratio at LD₅₀ level and 29‐fold at LD₉₅ level, as determined by topical application tests with Kou strain. When adult mosquitoes were exposed to 0.25% permethrin‐impregnated papers, the 50% and 95% knockdown times (KdT) were 23 and 42 min for SS females, compared with 40 and 62 min for RS (F₁ Kou × Kisumu) females. On 1% permethrin the KdT₅₀ and KdT₉₅ were 11 and 21 min for SS compared with 18 and 33 min for RS females. Following 1 h exposure to permethrin (0.25% or 1%), no significant knockdown of Kou RR females occurred within 24 h. Permethrin irritancy to An. gambiae was assessed by comparing ‘time to first take‐off’ (TO) for females. The standard TO₅₀ and TO₉₅ values for Kisumu SS on untreated paper were 58 and 1044 s, respectively, vs. 3.7 and 16.5 s on 1% permethrin. For Kou RR females the comparable values were 27.3 s for TO₅₀ and 294 s for TO₉₅, with intermediate RS values of 10.1 s for TO₅₀ and 71.9 s for TO₉₅. Thus, TO values for RS were 2.7–4.4 times more than for SS, and those for RR were 7–18 times longer than for SS. Experiments with pyrethroid‐impregnated nets were designed to induce hungry female mosquitoes to pass through holes cut in the netting. Laboratory ‘tunnel tests’ used a bait guinea‐pig to attract mosquitoes through circular holes (5 × 1 cm) in a net screen. With untreated netting, 75–83% of laboratory‐reared females passed through the holes overnight, 63–69% blood‐fed successfully and 9–17% died, with no significant differences between SS and RR genotypes. When the netting was treated with permethrin 250 mg ai/m² the proportions that passed through the holes overnight were only 10% of SS vs. 40–46% of RR (Tola & Kou); mortality rates were 100% of SS compared with 59–82% of RR; bloodmeals were obtained by 9% of Kou RR and 17% of Tola RR, but none of the Kisumu SS females. When the net was treated with deltamethrin 25 mg ai/m² the proportions of An. gambiae that went through the holes and blood‐fed successfully were 3.9% of Kisumu SS and 3.5% of Yaokoffikro field population (94% R). Mortality rates were 97% of Kisumu SS vs. 47% of Yaokoffikro R. Evidently this deltamethrin treatment was sufficient to kill nearly all SS and half of the Yaokoffikro R An. gambiae population despite its high kdr frequency. Experimental huts at Yaokoffikro were used for overnight evaluation of bednets against An. gambiae females. The huts were sealed to prevent egress of mosquitoes released at 20.00 hours and collected at 05.00 hours. Each net was perforated with 225 square holes (2 × 2 cm). A man slept under the net as bait. With untreated nets, only 4–6% of mosquitoes died overnight and bloodmeals were taken by 17% of SS vs. 29% of Yaokoffikro R (P < 0.05). Nets treated with permethrin 500 mg/m² caused mortality rates of 95% Kisumu SS and 45% Yao R (P < 0.001) and blood‐feeding rates were reduced to 1.3% of SS vs. 8.1% of Yao R (P < 0.05). Nets treated with deltamethrin 25 mg/m² caused mortality rates of 91% Kisumu SS and 54% Yao R (P < 0.001) and reduced blood‐feeding rates to zero for SS vs. 2.5% for Yao R (P > 0.05). Pyrethroid‐impregnated bednets in experimental huts and ‘tunnel tests’ gave equivalent results, showing that nets impregnated with permethrin or deltamethrin provided good levels of protection against kdr homozygous strains of An. gambiae (Kou and Tola), and against the field population at Yaokoffikro with 94% kdr frequency. The explanation seems to be that (a) high proportions of kdr females are killed by prolonged contact with pyrethroids through diminished sensitivity to the usual irritant and repellent effects, and (b) relatively few kdr females take advantage of this prolonged contact to ingest a bloodmeal.     

Organophosphate and pyrethroid resistances in the tomato leaf miner Tuta absoluta (Lepidoptera: Gelechiidae) from Iran

The tomato leafminer, Tuta absoluta (Meyrich) (Lepidoptera: Gelechiidae), is a serious pest of tomato crops worldwide. The intensive use of chemical pesticides to control it has led to the selection of resistant populations. This study investigated the resistance of T. absoluta populations to pyrethroid and the organophosphate insecticides from ten regions of Iran. The resistance ratios at LC₅₀ for chlorpyrifos and diazinon varied among populations from 4.3 to 12 and from 1.4 to 9.0, respectively. The resistance ratios of the pyrethroids cypermethrin, deltamethrin and permethrin varied from 1.3 to 3.7, 2.7 to 13 and 1.2 to 4.3, respectively. Inclusion of synergists in toxicological bioassays and the variation observed in the activity of esterases, glutathione S‐transferase and cytochrome P450‐dependent monooxygenase suggest the existence of metabolically based resistance. Esterase and P450 biochemical assays were positively correlated with deltamethrin, and cypermethrin tolerance and diazinon tolerance correlated with esterase activity. The genes encoding the organophosphate and pyrethroid target sites acetylcholinesterase (ace1) and sodium channel (kdr) were partly sequenced. The genotyping revealed mutations in high frequencies in all populations leading to an A201S substitution in ace1 and three substitutions in the sodium channel gene L1014F, M918T, T929I. In summary, our results indicate the presence of organophosphate and pyrethroid resistance in Iranian T. absoluta populations with involvement of both detoxification enzymes and target site alterations. Most likely the populations of T. absoluta imported to Iran were resistant upon arrival.     

Neuronal RNA‐binding protein HuD regulates addiction‐related gene expression and behavior

The neuronal RNA‐binding protein HuD is involved in synaptic plasticity and learning and memory mechanisms. These effects are thought to be due to HuD‐mediated stabilization and translation of target mRNAs associated with plasticity. To investigate the potential role of HuD in drug addiction, we first used bioinformatics prediction algorithms together with microarray analyses to search for specific genes and functional networks upregulated within the forebrain of HuD overexpressing mice (HuD_OE). When this set was further limited to genes in the knowledgebase of addiction‐related genes database (KARG) that contains predicted HuD‐binding sites in their 3′ untranslated regions (3′UTRs), we found that HuD regulates networks that have been associated with addiction‐like behavior. These genes included Bdnf and Camk2a, 2 previously validated HuD targets. Since addiction is hypothesized to be a disorder stemming from altered gene expression causing aberrant plasticity, we sought to test the role of HuD in cocaine conditioned placed preference (CPP), a model of addiction‐related behaviors. HuD mRNA and protein were upregulated by CPP within the nucleus accumbens of wild‐type C57BL/6J mice. These changes were associated with increased expression of Bdnf and Camk2a mRNA and protein. To test this further, we trained HuD_OE and wild‐type mice in CPP and found that HuD_OE mice showed increased cocaine CPP compared with controls. This was also associated with elevated expression of HuD target mRNAs and proteins, CaMKIIα and BDNF. These findings suggest HuD involvement in addiction‐related behaviors such as cocaine conditioning and seeking, through increased plasticity‐related gene expression.     

Plant essential oils synergize various pyrethroid insecticides and antagonize malathion in Aedes aegypti

Pyrethroid resistance is a significant threat to agricultural, urban and public health pest control activities. Because economic incentives for the production of novel active ingredients for the control of public health pests are lacking, this field is particularly affected by the potential failure of pyrethroid‐based insecticides brought about by increasing pyrethroid resistance. As a result, innovative approaches are desperately needed to overcome insecticide resistance, particularly in mosquitoes that transmit deadly and debilitating pathogens. Numerous studies have demonstrated the potential of plant essential oils to enhance the efficacy of pyrethroids. The toxicity of pyrethroids combined with plant oils is significantly greater than the baseline toxicity of either oils or pyrethroids applied alone, which suggests there are synergistic interactions between components of these mixtures. The present study examined the potential of eight plant essential oils applied in one of two concentrations (1% and 5%) to enhance the toxicity of various pyrethroids (permethrin, natural pyrethrins, deltamethrin and β‐cyfluthrin). The various plant essential oils enhanced the pyrethroids to differing degrees. The levels of enhancement provided by combinations of plant essential oils and pyrethroids in comparison with pyrethroids alone were calculated and synergistic outcomes characterized. Numerous plant essential oils significantly synergized a variety of pyrethroids; type I pyrethroids were synergized to a greater degree than type II pyrethroids. Eight plant essential oils significantly enhanced 24‐h mortality rates provided by permethrin and six plant essential oils enhanced 24‐h mortality rates obtained with natural pyrethrins. By contrast, only three plant essential plants significantly enhanced the toxicity of deltamethrin and β‐cyfluthrin. Of the plant essential oils that enhanced the toxicity of these pyrethroids, some produced varying levels of synergism and antagonism. Geranium, patchouli and Texas cedarwood oils produced the highest levels of synergism, displaying co‐toxicity factors of > 100 in some combinations. To assess the levels of enhancement and synergism of other classes of insecticide, malathion was also applied in combination with the plant oils. Significant antagonism was provided by a majority of the plant essential oils applied in combination with this insecticide, which suggests that plant essential oils may act to inhibit the oxidative activation processes within exposed adult mosquitoes.     

Toxicity of the new pyrethroid insecticide,deltamethrin, to Daphnia magna

The toxicity of deltamethrin, a synthetic pyrethroid insecticide, was determined under standardized conditions (ISO, 1982) in neonates and juveniles of Daphnia magna. Neonates (6 to 24 h old) were more sensitive than juveniles (48 to 72 h old). The 24- and 48-h EC₅₀s (immobilization) in neonates were 0.113 and 0.031 µg l^–1, respectively. The toxicity of deltamethrin was highly toxic. The 96-h EC₅₀ was in the ppt (µg l^–1) range. Toxicity tests with Daphnia may be used to detect toxic residues in water and sediment in areas treated with deltamethrin and other highly toxic pyrethroid pesticides.     

Efficacy of various synthetic pyrethroid-impregnated encasement materials against house dust mite under laboratory conditions

The acaricidal activity of synthetic pyrethroid and benzyl benzoate against Dermatophagoides pteronyssinus was examined in the laboratory, using a specially designed test set up. On the basis of median lethal dose (LD₅₀) values, the compound found to be most toxic to D. pteronyssinus was benzyl benzoate (LD₅₀ = 50 mg/m²), followed by permethrin (LD₅₀ = 76.7 mg/m²), deltamethrin (LD₅₀ = 146.7 mg/m²), esbioallenthrin (LD₅₀ = 186.6 mg/m²) and lamdacyhalothrin (LD₅₀ = 756.6 mg/m²). Very low toxicity was observed with bifenthrin (LD₅₀ = 5157.8 mg/m²). A laboratory control trial was also carried out to compare the acaricidal activity (residual effect) of four pyrethroids impregnated on woven and non-woven encasement materials against house dust mites during a 4-month period. Of the pyrethroids used in this study, esbioallenthrin demonstrated the highest acaricidal activity, and of the pyrethroid impregnated materials, the non-woven encasement material was more effective than the woven encasement material.     

Glutathione S-transferase detoxification as a potential pyrethroid resistance mechanism in the maize weevil, Sitophilus zeamais

Insecticide resistance patterns among 16 Brazilian populations of the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), were recognized by surveying resistance to three organophosphates (chlorpyrifos‐methyl, malathion, and pirimiphos‐methyl) and three pyrethroids (cypermethrin, deltamethrin, and permethrin). Two population clusters were obtained: one with three populations (Bragança Paulista, Cristalina, and Nova Andradina) showing low frequency of cypermethrin resistance (13–36%) and negligible frequency of deltamethrin resistance (2–9%); and another with six populations (Campos dos Goytacazes, Ivinhema, Patos de Minas, Penápolis, Uberlândia, and Venda Nova) showing low to negligible levels of pyrethroid resistance (0–23%). The remaining seven populations, including a susceptible, and a DDT‐ and pyrethroid‐resistant reference populations (Sete Lagoas and Jacarezinho, respectively), were significantly different from each other and from the two recognized clusters. In contrast with pyrethroid resistance, organophosphate resistance was negligible except for chlorpyrifos‐methyl in two populations (Fátima do Sul and Penápolis). There was no correlation between geographic distance and the Mahalanobis distance estimated from the resistance pattern ordination of the populations by canonical variate analysis, suggesting local selection and/or broad dispersal of resistant populations by grain trade. The results of biochemical in vitro studies measuring the activity of detoxification enzymes (esterases and glutathion S‐transferases) in conjunction with canonical correlation analysis suggest a major involvement of enhanced conjugation by glutathione S‐transferases (> 2‐fold increase) in pyrethroid resistance and, in the case of cypermethrin resistance, enhanced phosphotriesterase activity.