Superfamily of genes encoding G protein‐coupled receptors in the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae)

G protein‐coupled receptors (GPCRs) are the largest and most versatile superfamily of cell membrane proteins, which mediate various physiological processes including reproduction, development and behaviour. The diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), is one of the most notorious insect pests, preferentially feeding on cruciferous plants. P. xylostella is not only one of the world's most widespread lepidopteran insects, but has also developed resistance to nearly all classes of insecticides. Although the mechanisms of insecticide resistance have been studied extensively in many insect species, few investigations have been carried out on GPCRs in P. xylostella. In the present study, we identified 95 putative GPCRs in the P. xylostella genome. The identified GPCRs were compared with their homologues in Bombyx mori and Drosophila melanogaster. Our results suggest that GPCRs in different insect species may have evolved by a birth‐and‐death process. One of the differences among compared insects is the duplication of short neuropeptide F receptor and adipokinetic hormone receptors in P. xylostella and B. mori. Another divergence is the decrease in quantity and diversity of the stress‐tolerance gene, Mth, in P. xylostella. The evolution by the birth‐and‐death process is probably involved in adaptation to the feeding behaviour, reproduction and stress responses of P. xylostella. Some of the genes identified in the present study could be potential targets for the development of novel pesticides.     

Identification of promoter polymorphisms in the cytochrome P450 CYP6AY1 linked with insecticide resistance in the brown planthopper,Nilaparvata lugens

Imidacloprid resistance in the brown planthopper, Nilaparvata lugens, is primarily the result of the over‐expression of cytochrome P450 monooxygenases. Here, a field‐collected strain of N. lugens was shown to be highly resistant to both imidacloprid and buprofezin. Insecticide exposure and quantitative real‐time PCR revealed that its resistance was mainly associated with a cytochrome P450 gene, CYP6AY1. CYP6AY1 is known to metabolize imidacloprid but its effect on buprofezin is unclear. In the 5′‐untranslated region of CYP6AY1, a novel alternative splicing was detected. After a 1990‐bp promoter region was cloned, its basal luciferase activity was assessed. Furthermore, genotyping studies identified 12 variations in the promoter region that discriminated between the field‐collected and control strain. Finally, survival bioassays revealed a single nucleotide polymorphism and an insertion‐deletion polymorphism linked to buprofezin and imidacloprid resistance. Mutagenesis of these sites enhanced the promoter activity of CYP6AY1. These results suggest that promoter polymorphisms may affect P450‐mediated multiple insecticide resistance of pests.     

Imidacloprid is hydroxylated by Laodelphax striatellus CYP6AY3v2

Laodelphax striatellus (Fallén) is one of the most destructive pests of rice, and has developed high resistance to imidacloprid. Our previous work indicated a strong association between imidacloprid resistance and the overexpression of a cytochrome P450 gene CYP6AY3v2 in a L. striatellus imidacloprid resistant strain (Imid‐R). In this study, a transgenic Drosophila melanogaster line that overexpressed the L. striatellus CYP6AY3v2 gene was established and was found to confer increased levels of imidacloprid resistance. Furthermore, CYP6AY3v2 was co‐expressed with D. melanogaster cytochrome P450 reductase (CPR) in Spodoptera frugiperda 9 (SF9) cells. A carbon monoxide difference spectra analysis indicated that CYP6AY3v2 was expressed predominately in its cytochrome P450 (P450) form, which is indicative of a good‐quality functional enzyme. The recombinant CYP6AY3v2 protein efficiently catalysed the model substrate P‐nitroanisole to p‐nitrophenol with a maximum velocity (V_max) of 60.78 ± 3.93 optical density (mOD)/min/mg protein. In addition, imidacloprid itself was metabolized by the recombinant CYP6AY3v2/nicotinamide adenine dinucleotide 2'‐phosphate reduced tetrasodium salt (NADPH) CPR microsomes in in vitro assays (catalytic constant (K_cat) = 0.34 pmol/min/pmol P450, michaelis constant (K_m) = 41.98 μM), and imidacloprid depletion and metabolite peak formation were with a time dependence. The data provided direct evidence that CYP6AY3v2 is capable of hydroxylation of imidacloprid and conferring metabolic resistance in L. striatellus.     

Overcoming super‐knock down resistance (super‐kdr) mediated resistance: multi‐halogenated benzyl pyrethroids are more toxic to super‐kdr than kdr house flies

Target site insensitivity because of mutations in the voltage‐sensitive sodium channel gene (Vssc) is a major mechanism of resistance to pyrethroid insecticides in the house fly, Musca domestica. There are three known Vssc alleles that confer resistance to pyrethroids in the house fly: knock down resistance (kdr; L1014F), super‐kdr (M918T + L1014F) and kdr‐his (L1014H), but there has been no side‐by‐side comparison of the resistance levels that they confer. We compared the levels of resistance conferred by the three Vssc alleles in congenic strains to 19 structurally diverse pyrethroids, and compared the full‐length Vssc cDNA sequences from each strain. Generally, the levels of resistance conferred were kdr‐his < kdr < super‐kdr. However, there was significant variation in this pattern, especially for super‐kdr, for which both high and low resistance ratios were observed for several pyrethroids. We also examined the levels of resistance in heterozygotes. Resistance in each of the hybrids was generally inherited as an incompletely recessive trait, except for the kdr‐his/kdr hybrids, which showed incompletely to completely dominant resistance (ie had resistance levels comparable to kdr homozygotes). The importance of these results to understanding the frequencies of these resistance alleles in natural populations, the evolution of insecticide resistance and resistance management strategies are discussed.     

Drug‐Target Residence Time—A Case for G Protein‐Coupled Receptors

A vast number of marketed drugs act on G protein‐coupled receptors (GPCRs), the most successful category of drug targets to date. These drugs usually possess high target affinity and selectivity, and such combined features have been the driving force in the early phases of drug discovery. However, attrition has also been high. Many investigational new drugs eventually fail in clinical trials due to a demonstrated lack of efficacy. A retrospective assessment of successfully launched drugs revealed that their beneficial effects in patients may be attributed to their long drug‐target residence times (RTs). Likewise, for some other GPCR drugs short RT could be beneficial to reduce the potential for on‐target side effects. Hence, the compounds’ kinetics behavior might in fact be the guiding principle to obtain a desired and durable effect in vivo. We therefore propose that drug‐target RT should be taken into account as an additional parameter in the lead selection and optimization process. This should ultimately lead to an increased number of candidate drugs moving to the preclinical development phase and on to the market. This review contains examples of the kinetics behavior of GPCR ligands with improved in vivo efficacy and summarizes methods for assessing drug‐target RT.     

Silencing of P‐glycoprotein increases mortality in temephos‐treated Aedes aegypti larvae

Re‐emergence of vector‐borne diseases such as dengue and yellow fever, which are both transmitted by the Aedes aegypti mosquito, has been correlated with insecticide resistance. P‐glycoproteins (P‐gps) are ATP‐dependent efflux pumps that are involved in the transport of substrates across membranes. Some of these proteins have been implicated in multidrug resistance (MDR). In this study, we identified a putative P‐glycoprotein in the Ae. aegypti database based on its significantly high identity with Anopheles gambiae, Culex quinquefasciatus, Drosophila melanogaster and human P‐gps. The basal ATPase activity of ATP‐binding cassette transporters in larvae was significantly increased in the presence of MDR modulators (verapamil and quinidine). An eightfold increase in Ae. aegypti P‐gp (AaegP‐gp) gene expression was detected in temephos‐treated larvae as determined by quantitative PCR. To analyse the potential role of AaegP‐gp in insecticide efflux, a temephos larvicide assay was performed in the presence of verapamil. The results showed an increase of 24% in temephos toxicity, which is in agreement with the efflux reversing effect. RNA interference (RNAi)‐mediated silencing of the AaegP‐gp gene caused a significant increase in temephos toxicity (57%). In conclusion, we have demonstrated for the first time in insects that insecticide‐induced P‐gp expression can be involved in the modulation of insecticide efflux.     

Molecular characterization,expression pattern and metabolic activity of flavin‐dependent monooxygenases in Spodoptera exigua

Enhanced detoxification is one of the important mechanisms for insecticide resistance. Most research in this field to date has focused on the role of cytochrome P450s. Our previous work revealed that flavin‐dependent monooxygenases (FMOs) were involved in metabolic resistance of Spodoptera exigua. In the present study we investigated the molecular characteristics, expression patterns and oxidative activities of SeFMO on insecticides. Three FMO genes, which encode proteins with the typical FMO motifs, were cloned from S. exigua. The oxidative activities of eukaryotically expressed SeFMO enzymes were verified with the model substrate of FMO. Importantly, the SeFMOs had significantly higher oxidative activities on metaflumizone and lambda‐cyhalothrin than on model substrates and other insecticides tested. The three SeFMOs were mainly expressed in the midgut, fat body and Malpighian tubules. The tissues responsible for xenobiotic metabolism and their expression characteristics were similar to those of P450s acting as detoxification genes. The study also revealed that the expression of SeFMOs could be induced by insecticide exposure, and that SeFMOs were over‐expressed in a metaflumizone‐resistant strain of S. exigua. These results suggest that SeFMOs are important insecticide detoxifying enzymes, and that over‐expression of FMO genes may be one of the mechanisms for metabolic resistance in S. exigua.     

Modular cis‐regulatory logic of yellow gene expression in silkmoth larvae

Colour patterns in butterflies and moths are crucial traits for adaptation. Previous investigations have highlighted genes responsible for pigmentation (ie yellow and ebony). However, the mechanisms by which these genes are regulated in lepidopteran insects remain poorly understood. To elucidate this, molecular studies involving dipterans have largely analysed the cis‐regulatory regions of pigmentation genes and have revealed cis‐regulatory modularity. Here, we used well‐developed transgenic techniques in Bombyx mori and demonstrated that cis‐regulatory modularity controls tissue‐specific expression of the yellow gene. We first identified which body parts are regulated by the yellow gene via black pigmentation. We then isolated three discrete regulatory elements driving tissue‐specific gene expression in three regions of B. mori larvae. Finally, we found that there is no apparent sequence conservation of cis‐regulatory regions between B. mori and Drosophila melanogaster, and no expression driven by the regulatory regions of one species when introduced into the other species. Therefore, the trans‐regulatory landscapes of the yellow gene differ significantly between the two taxa. The results of this study confirm that lepidopteran species use cis‐regulatory modules to control gene expression related to pigmentation, and represent a powerful cadre of transgenic tools for studying evolutionary developmental mechanisms.     

Succinate independently stimulates full platelet activation via cAMP and phosphoinositide 3‐kinase‐β signaling

Summary. Background: The citric cycle intermediate succinate has recently been identified as a ligand for the G‐protein‐coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most highly expressed GPCRs in human platelets. Objective: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signaling pathways of this receptor in platelets. Methods and Results: Using real‐time‐PCR, we demonstrated that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y₁ receptor. Light transmission aggregation experiments showed dose‐dependent aggregation induced by succinate, reaching a maximum response at 0.5 mm . The effect of succinate on platelet aggregation was confirmed with flow cytometry, showing increased surface expression of activated glycoprotein IIb–IIIa and P‐selectin. Intracellular SUCNR1 signaling was found to result in decreased cAMP levels, Akt phosphorylation mediated by phosphoinositide 3‐kinase‐β activation, and receptor desensitization. Furthermore, succinate‐induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A₂, and ATP release. Platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y₁₂ receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Conclusions: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate‐induced platelet aggregation depends on thromboxane A₂ generation, ATP release, and P2Y₁₂ activation.