Novel polymorphisms in the octopamine receptor gene of amitraz resistant population of Rhipicephalus sanguineus sensu lato, in south India.

The brown dog tick or Rhipicephalus sanguineus sensu lato is an ixodid tick, responsible for the dissemination of pathogens that cause canine infectious diseases besides inflicting the direct effects of tick bite. The hot humid climate of Kerala, a south Indian state, is favorable for propagation of tick vectors and acaricides are the main stay of tick control. Though the resistance against synthetic pyrethroids is reported among these species, the status of amitraz resistance in R. sanguineus s. l. in the country is uncertain due to the lack of molecular characterisation data and scarce literature reports. Hence the present study was focused on the phenotypic detection and preliminary genotypic characterisation of amitraz resistance in the R. sanguineus s. l. A modified larval packet test (LPT) on a susceptible isolate was performed to determine the discriminating dose (DD). Further LPT-DD on 35 tick isolates was carried out to detect amitraz resistance robustly, along with that full dose response bioassays on the resistant isolates were performed. The results indicated that amitraz resistance is prevalent with 49 per cent of the samples being resistant. Amplification of exon 3 of octopamine receptor gene from both the susceptible and resistant larval isolates was carried out. Amplicons of ten pooled amitraz susceptible and ten pooled amitraz resistant representative samples were sequenced and analysed, unveiling a total of three novel non-synonymous mutations in the partial coding region at positions V32A, N41D and V58I in phenotypically resistant larval DNA samples. In silico analysis by homology modelling and molecular docking of the mutated and unmutated receptors showed that these mutations had reduced the binding affinity to amitraz. However, lack of mutations in the octopamine receptor gene in three of the pooled low order resistant R. sanguineus s. l. larval samples could be suggestive of other mechanisms associated with amitraz resistance in the region. Hence, further association studies should be carried out to confirm the association of these mutations with target insensitivity in R. sanguineus s. l. ticks, along with exploring the status of metabolic resistance and other mechanisms of resistance.

Identification and CRISPR-Cas9 validation of a novel ß-adrenergic-like octopamine receptor mutation associated with amitraz resistance in Varroa destructor.

Varroa destructor is widely recognized as a significant contributor to colony collapse disorder. Chemical acaricides, such as amitraz, have been extensively used for Varroa control due to their selectivity within beehives. However, the increasing number of cases of amitraz resistance across global V. destructor populations poses a significant challenge. In this study, we conducted a comprehensive molecular screening of the ß-adrenergic-like octopamine receptor (Octß2R), the target-site of amitraz, across 66 Turkish and 63 Belgian V. destructor populations. Although previously reported amitraz resistance mutations were not detected, the screening revealed a novel Y337F mutation located within transmembrane 7 (TM7) of Octß2R in Turkish Varroa populations. Notably, this mutation was identified in the last residue of the highly conserved NPxxY motif associated with the activation of G-protein coupled receptors (GPCR). Among the 66 Varroa samples from Türkiye, twenty harbored the Y337F mutation, with eight samples exhibiting fixation of the mutation. Subsequent bioassays revealed over 8-fold resistance to amitraz in populations that contain the Y337F mutation. Genotyping of mites after exposure to 10 mg a.i./l amitraz demonstrated that all surviving mites were homozygous for the Y337F mutation, whereas dead mites carried susceptible alleles, providing genetic linkage between mutation and phenotype. Further, we used CRISPR-Cas9 editing to introduce the Y337F mutation in the orthologous Octß2R of the model organism Tetranychus urticae. Crispants exhibited over threefold resistance to amitraz. In conclusion, this study identified and validated a novel amitraz resistance mutation. Additional research is required to further evaluate the phenotypic strength of Y337F in the context of operational resistance with current treatment strategies.

Roles of octopamine neurons in the vertical lobe of the mushroom body for the execution of a conditioned response in cockroaches.

Aminergic neurons mediate reward signals in mammals and insects. In crickets, we showed that blockade of synaptic transmission from octopamine neurons (OANs) impairs conditioning of an odor (conditioned stimulus, CS) with water or sucrose (unconditioned stimulus, US) and execution of a conditioned response (CR) to the CS. It has not yet been established, however, whether findings in crickets can be applied to other species of insects. In this study, we investigated the roles of OANs in conditioning of salivation, monitored by activities of salivary neurons, and in execution of the CR in cockroaches (Periplaneta americana). We showed that injection of epinastine (an OA receptor antagonist) into the head hemolymph impaired both conditioning and execution of the CR, in accordance with findings in crickets. Moreover, local injection of epinastine into the vertical lobes of the mushroom body (MB), the center for associative learning and control of the CR, impaired execution of the CR, whereas injection of epinastine into the calyces of the MB or the antennal lobes (primary olfactory centers) did not. We propose that OANs in the MB vertical lobes play critical roles in the execution of the CR in cockroaches. This is analogous to the fact that midbrain dopamine neurons govern execution of learned actions in mammals.

Characterization and functional analysis of an α-adrenergic-like octopamine receptor in the small brown planthopper Laodelphax striatellus.

As an important biogenic amine in invertebrates and corresponding to the neurotransmitter norepinephrine in vertebrates, octopamine (OA) regulates diverse physiological and behavioral processes by binding to specific octopamine receptors (OARs) in invertebrates. At present, OARs have been identified and characterized in several insects. However, less is known about the OARs of Laodelphax striatellus, one of the most destructive pests in East Asian rice fields. In the present study, an α1-adrenergic-like OAR (LsOA1) from L. striatellus was cloned. LsOA1 has the typical characteristics of G-protein coupled receptors and is clustered with other insect homologs. The transcript level of LsOA1 varied in various stages and tissues, and was highly expressed at the egg stage and in the brain. Silencing of LsOA1 causes a reduction in vitellogenin (LsVg) and vitellogenin receptor (LsVgR) expression. Although LsOA1 interference did not affect the fecundity and survival of L. striatellus, the hatching rate of L. striatellus was significantly reduced, and the hatching period was prolonged. The decrease in the amount of honeydew excreted after silencing LsOA1 indicates that LsOA1 may be involved in regulating the feeding behavior of L. striatellus. In addition, the interference of LsOA1 significantly reduced the expression of capsid protein (CP) and viral RNA3 segment (RNA3) in rice stripe virus (RSV)-viruliferous L. striatellus, but did not affect the vertical transmission rate of RSV. The present study demonstrated that LsOA1 played a crucial role in the physiological and behavioral processes of L. striatellus, which will provide the basis for developing a new target gene for pest control.

Molecular and Pharmacological Characterization of ß-Adrenergic-like Octopamine Receptors in the Endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae).

Octopamine (OA) is structurally and functionally similar to adrenaline/noradrenaline in vertebrates, and OA modulates diverse physiological and behavioral processes in invertebrates. OA exerts its actions by binding to specific octopamine receptors (OARs). Functional and pharmacological characterization of OARs have been investigated in several insects. However, the literature on OARs is scarce for parasitoids. Here we cloned three ß-adrenergic-like OARs (CcOctßRs) from Cotesia chilonis. CcOctßRs share high similarity with their own orthologous receptors. The transcript levels of CcOctßRs were varied in different tissues. When heterologously expressed in CHO-K1 cells, CcOctßRs induced cAMP production, and were dose-dependently activated by OA, TA and putative octopaminergic agonists. Their activities were inhibited by potential antagonists and were most efficiently blocked by epinastine. Our study offers important information about the molecular and pharmacological properties of ß-adrenergic-like OARs from C. chilonis that will provide the basis to reveal the contribution of individual receptors to the physiological processes and behaviors in parasitoids.

Molecular Characterization of the ß2-like Octopamine Receptor of Helicoverpa armigera.

Helicoverpa armigera is a devastating polyphagous and cosmopolitan crop pest. There are reports of this insect being resistant to a variety of pesticides raising concern worldwide. The Octopamine (OA) binding ß2-like receptor (OAR), a GPCR, is widely distributed in the nervous system of the insect and plays essential roles in the physiology and development and thus is an important target for insecticides. Yet, the molecular characterization of the H. armigera OAR (HarmOAR) and rational design of compounds based on this receptor is lacking. As a first step, we performed multiple sequence alignment of all insect OARs, which revealed that the sequences contained all conserved class A GPCR motifs. Phylogenetic studies showed clade-specific variations in the protein sequences primarily arising owing to differences in the ICL3 loop region. Further, a structural model of HarmOAR was built using the inactive human ß2AR as a template. 0.9 µs atomistic simulations revealed conserved inter helical contacts and water molecules of HarmOAR. The detailed binding of octopamine was studied using molecular docking and 0.3 µs atomistic simulations. Twenty-two insecticides active against octopamine receptors of other insects were compiled and docked to HarmOAR followed by rescoring with binding free energies to prioritize them for H. armigera. Our study suggests α-terpineol to be a good candidate as an insecticide or insect repellent for Helicoverpa armigera.

Characterization and functional analysis of a ß-adrenergic-like octopamine receptor from the oriental armyworm (Mythimna separata Walker).

The ß-adrenergic-like octopamine receptor (OA2B2), which binds the biogenic amine octopamine, belongs to the class of G-protein coupled receptors and significantly regulates many physiological and behavioral processes in insects. In this study, the putative open reading frame sequence of the MsOA2B2 gene in Mythimna separata was cloned, the full-length complementary DNA was 1191 bp and it encoded a 396-amino acid protein (GenBank accession number MN822800). Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. Real-time quantitative polymerase chain reaction of spatial and temporal expression analysis revealed that the MsOAB2 gene was expressed in all developmental stages of M. separata and was most abundant in egg stages and second and fourth instars compared with other developmental stages, while the expression level during the pupal stage was much lower than that at the other stages. Further analysis with sixth instar M. separata larvae showed that the MsOA2B2 gene was expressed 1.81 times higher in the head than in integument and gut tissues. Dietary ingestion of dsMsOA2B2 significantly reduced the messenger RNA level of MsOA2B2 and decreased mortality following amitraz treatment. This study provides both a pharmacological characterization and the gene expression patterns of OA2B2 in M. separata, facilitating further research for insecticides using MsOA2B2 as a target.

Ion channels and G protein-coupled receptors as targets for invertebrate pest control: from past challenges to practical insecticides.

In the late 1970s, we discovered that toxic bicyclic phosphates inhibit the generation of miniature inhibitory junction potentials, implying their antagonism of γ-aminobutyric acid (GABA) receptors (GABARs; GABA-gated chloride channels). This unique mode of action provided a strong incentive for our research on GABARs in later years. Furthermore, minor structural changes conferred insect GABAR selectivity to this class of compounds, convincing us of the possibility of GABARs as targets for insecticides. Forty years later, third-generation insecticides acting as allosteric modulator antagonists at a distinctive site of action in insect GABARs were developed. G protein-coupled receptors (GPCRs) are also promising targets for pest control. We characterized phenolamine receptors functionally and pharmacologically. Of the tested receptors, ß-adrenergic-like octopamine receptors were revealed to be the most sensitive to the acaricide/insecticide amitraz. Given the presence of multiple sites of action, ion channels and GPCRs remain potential targets for invertebrate pest control.

Knockdown of a ß-Adrenergic-Like Octopamine Receptor Affects Locomotion and Reproduction of Tribolium castaneum.

The neurohormone octopamine regulates many crucial physiological processes in insects and exerts its activity via typical G-protein coupled receptors. The roles of octopamine receptors in regulating behavior and physiology in Coleoptera (beetles) need better understanding. We used the red flour beetle, Tribolium castaneum, as a model species to study the contribution of the octopamine receptor to behavior and physiology. We cloned the cDNA of a ß-adrenergic-like octopamine receptor (TcOctß2R). This was heterologously expressed in human embryonic kidney (HEK) 293 cells and was demonstrated to be functional using an in vitro cyclic AMP assay. In an RNAi assay, injection of dsRNA demonstrated that TcOctß2R modulates beetle locomotion, mating duration, and fertility. These data present some roles of the octopaminergic signaling system in T. castaneum. Our findings will also help to elucidate the potential functions of individual octopamine receptors in other insects.

Assessment of Insecticidal Activity of Benzylisoquinoline Alkaloids from Chilean Rhamnaceae Plants against Fruit-Fly Drosophila melanogaster and the Lepidopteran Crop Pest Cydia pomonella.

The Chilean plants Discaria chacaye, Talguenea quinquenervia (Rhamnaceae), Peumus boldus (Monimiaceae), and Cryptocarya alba (Lauraceae) were evaluated against Codling moth: Cydia pomonella L. (Lepidoptera: Tortricidae) and fruit fly Drosophila melanogaster (Diptera: Drosophilidae), which is one of the most widespread and destructive primary pests of Prunus (plums, cherries, peaches, nectarines, apricots, almonds), pear, walnuts, and chestnuts, among other. Four benzylisoquinoline alkaloids (coclaurine, laurolitsine, boldine, and pukateine) were isolated from the above mentioned plant species and evaluated regarding their insecticidal activity against the codling moth and fruit fly. The results showed that these alkaloids possess acute and chronic insecticidal effects. The most relevant effect was observed at 10 µg/mL against D. melanogaster and at 50 µg/mL against C. pomonella, being the alteration of the feeding, deformations, failure in the displacement of the larvae in the feeding medium of D. melanogaster, and mortality visible effects. In addition, the docking results show that these type of alkaloids present a good interaction with octopamine and ecdysone receptor showing a possible action mechanism.

Menthol Increases Bendiocarb Efficacy Through Activation of Octopamine Receptors and Protein Kinase A.

Great effort is put into seeking a new and effective strategies to control insect pests. One of them is to combine natural products with chemical insecticides to increase their effectiveness. In the study presented, menthol which is an essential oil component was evaluated on its ability to increase the efficiency of bendiocarb, carbamate insecticide. A multi-approach study was conducted using biochemical method (to measure acetylcholinesterase enzyme activity), electrophysiological technique (microelectrode recordings in DUM neurons in situ), and confocal microscopy (for calcium imaging). In the electrophysiological experiments, menthol caused hyperpolarization, which was blocked by an octopamine receptor antagonist (phentolamine) and an inhibitor of protein kinase A (H-89). It also raised the intracellular calcium level. The effect of bendiocarb was potentiated by menthol and this phenomenon was abolished by phentolamine and H-89 but not by protein kinase C inhibitor (bisindolylmaleimide IX). The results indicate that menthol increases carbamate insecticide efficiency by acting on octopamine receptors and triggering protein kinase A phosphorylation pathway.

Pharmacological characterization of a ß-adrenergic-like octopamine receptor in Plutella xylostella.

The ß-adrenergic-like octopamine receptor (OA2B2) belongs to the class of G-protein coupled receptors. It regulates important physiological functions in insects, thus is potentially a good target for insecticides. In this study, the putative open reading frame sequence of the Pxoa2b2 gene in Plutella xylostella was cloned. Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. PxOA2B2 was transiently expressed in HEK-293 cells. It was found that PxOA2B2 could be activated by both octopamine and tyramine, resulting in increased intracellular cyclic AMP (cAMP) levels, whereas dopamine and serotonin were not effective in eliciting cAMP production. Further studies with series of PxOA2B2 agonists and antagonists showed that all four tested agonists (e.g., naphazoline, clonidine, 2-phenylethylamine, and amitraz) could activate the PxOA2B2 receptor, and two of tested antagonists (e.g., phentolamine and mianserin) had significant antagonistic effects. However, antagonist of yohimbine had no effects. Quantitative real-time polymerase chain reaction analysis showed that Pxoa2b2 gene was expressed in all developmental stages of P. xylostella and that the highest expression occurred in male adults. Further analysis with fourth-instar P. xylostella larvae showed that the Pxoa2b2 gene was mainly expressed in Malpighian tubule, epidermal, and head tissues. This study provides both a pharmacological characterization and the gene expression patterns of the OA2B2 in P. xylostella, facilitating further research for insecticides using PxOA2B2 as a target.

Molecular Targets for Components of Essential Oils in the Insect Nervous System-A Review.

Essential oils (EOs) are lipophilic secondary metabolites obtained from plants; terpenoids represent the main components of them. A lot of studies showed neurotoxic actions of EOs. In insects, they cause paralysis followed by death. This feature let us consider components of EOs as potential bioinsecticides. The inhibition of acetylcholinesterase (AChE) is the one of the most investigated mechanisms of action in EOs. However, EOs are rather weak inhibitors of AChE. Another proposed mechanism of EO action is a positive allosteric modulation of GABA receptors (GABArs). There are several papers that prove the potentiation of GABA effect on mammalian receptors induced by EOs. In contrast, there is lack of any data concerning the binding of EO components in insects GABArs. In insects, EOs act also via the octopaminergic system. Available data show that EOs can increase the level of both cAMP and calcium in nervous cells. Moreover, some EO components compete with octopamine in binding to its receptor. Electrophysiological experiments performed on Periplaneta americana have shown similarity in the action of EO components and octopamine. This suggests that EOs can modify neuron activity by octopamine receptors. A multitude of potential targets in the insect nervous system makes EO components interesting candidates for bio-insecticides.

Accelerated molecular dynamics simulations of the octopamine receptor using GPUs: discovery of an alternate agonist-binding position.

Octopamine receptors (OARs) perform key biological functions in invertebrates, making this class of G-protein coupled receptors (GPCRs) worth considering for insecticide development. However, no crystal structures and very little research exists for OARs. Furthermore, GPCRs are large proteins, are suspended in a lipid bilayer, and are activated on the millisecond timescale, all of which make conventional molecular dynamics (MD) simulations infeasible, even if run on large supercomputers. However, accelerated Molecular Dynamics (aMD) simulations can reduce this timescale to even hundreds of nanoseconds, while running the simulations on graphics processing units (GPUs) would enable even small clusters of GPUs to have processing power equivalent to hundreds of CPUs. Our results show that aMD simulations run on GPUs can successfully obtain the active and inactive state conformations of a GPCR on this reduced timescale. Furthermore, we discovered a potential alternate active-state agonist-binding position in the octopamine receptor which has yet to be observed and may be a novel GPCR agonist-binding position. These results demonstrate that a complex biological system with an activation process on the millisecond timescale can be successfully simulated on the nanosecond timescale using a simple computing system consisting of a small number of GPUs. Proteins 2016; 84:1480-1489. © 2016 Wiley Periodicals, Inc.

Characterization of a ß-Adrenergic-Like Octopamine Receptor in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel).

The biogenic amine octopamine plays a critical role in the regulation of many physiological processes in insects. Octopamine transmits its action through a set of specific G-protein coupled receptors (GPCRs), namely octopamine receptors. Here, we report on a ß-adrenergic-like octopamine receptor gene (BdOctßR1) from the oriental fruit fly, Bactrocera dorsalis (Hendel), a destructive agricultural pest that occurs in North America and the Asia-Pacific region. As indicated by RT-qPCR, BdOctßR1 was highly expressed in the central nervous system (CNS) and Malpighian tubules (MT) in the adult flies, suggesting it may undertake important roles in neural signaling in the CNS as well as physiological functions in the MT of this fly. Furthermore, its ligand specificities were tested in a heterologous expression system where BdOctßR1 was expressed in HEK-293 cells. Based on cyclic AMP response assays, we found that BdOctßR1 could be activated by octopamine in a concentration-dependent manner, confirming that this receptor was functional, while tyramine and dopamine had much less potency than octopamine. Naphazoline possessed the highest agonistic activity among the tested agonists. In antagonistic assays, mianserin had the strongest activity and was followed by phentolamine and chlorpromazine. Furthermore, when the flies were kept under starvation, there was a corresponding increase in the transcript level of BdOctßR1, while high or low temperature stress could not induce significant expression changes. The above results suggest that BdOctßR1 may be involved in the regulation of feeding processes in Bactrocera dorsalis and may provide new potential insecticide leads targeting octopamine receptors.

Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito.

Studying insecticide resistance in mosquitoes has attracted the attention of many scientists to elucidate the pathways of resistance development and to design novel strategies in order to prevent or minimize the spread and evolution of resistance. Here, we tested the synergistic action of piperonyl butoxide (PBO) and two octopamine receptor (OR) agonists, amitraz (AMZ) and chlordimeform (CDM) on selected novel insecticides to increase their lethal action on the fourth instar larvae of Aedes aegypti L. However, chlorfenapyr was the most toxic insecticide (LC50 = 193, 102, and 48 ng/ml, after 24, 48, and 72 h exposure, respectively) tested. Further, PBO synergized all insecticides and the most toxic combinatorial insecticide was nitenpyram even after 48 and 72 h exposure. In addition, OR agonists significantly synergized most of the selected insecticides especially after 48 and 72 h exposure. The results imply that the synergistic effects of amitraz are a promising approach in increasing the potency of certain insecticides in controlling the dengue vector Ae. aegypti mosquito.

Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae).

We recently reported that formamidine pesticides such as amitraz and chlordimeform effectively synergize toxic actions of certain pyrethroid and neonicotinoid insecticides in some insect species on the 4th instar larvae of Aedes aegypti. Here we studied the biochemical basis of the synergistic actions of the formamidines in amplifying the toxicity of neonicotinoids and pyrethroids such as dinotefuran and thiamethoxam, as well as deltamethrin-fenvalerate type of pyrethroids. We tested the hypothesis that their synergistic actions are mediated by the octopamine receptor, and that the major consequence of octopamine receptor activation is induction of trehalase to increase glucose levels in the hemolymph. The results show that formamidines cause a significant up-regulation of the octopamine receptor and trehalase mRNA expressions. Furthermore, formamidines significantly elevate levels of free glucose when co-treated with dinotefuran, deltamethrin and fenvalerate, but not with permethrin or fenitrothion, which showed no synergistic toxic effects with formamidines. These results support the conclusion that the main mode of synergism is based on the ability to activate the octopamine receptor, which is particularly effective with insecticides causing hyperexcitation-induced glucose release and consequently leading to quick energy exhaustion.

Effects of Essential Oils on Biological Characteristics and Potential Molecular Targets in Spodoptera frugiperda.

Spodoptera frugiperda control methods have proved to be inefficient, which justifies the search for new control measures. In this search for botanical insecticides for controlling S. frugiperda, the following were evaluated: (i) the toxicity of essential oils (EOs) from Cinnamodendron dinisii, Eugenia uniflora, and Melaleuca armillaris; (ii) the effect of EOs on life table parameters against S. frugiperda; (iii) the chemical characterization of EOs; and (iv) the in silico interaction of the chemical constituents present in the three EOs with the molecular targets of S. frugiperda. The EO from E. uniflora had the lowest LD50 (1.19 µg of EO/caterpillar). The major compounds bicyclogermacrene (18.64%) in C. dinisii and terpinolene (57.75%) in M. armillaris are highly predicted to interact with the octopamine receptor (OctpR). The compound 1,8-cineole (21.81%) in M. armillaris interacts mainly with a tolerant methoprene receptor (MET) and curzerene (41.22%) in E. uniflora, which acts on the OctpR receptor. Minor compounds, such as nerolidol in C. dinisii and ß-elemene in E. uniflora, are highly ranked for multiple targets: AChE, MET, OctpR, and 5-HT1. It was concluded that the EO from E. uniflora negatively affects several biological parameters of S. frugiperda development and is promising as an active ingredient in formulations for controlling this insect pest.

Expression and potential regulatory functions of Drosophila octopamine receptors in the female reproductive tract.

Aminergic signaling is known to play a critical role in regulating female reproductive processes in both mammals and insects. In Drosophila, the ortholog of noradrenaline, octopamine, is required for ovulation as well as several other female reproductive processes. Two octopamine receptors have already been shown to be expressed in the Drosophila reproductive tract and to be required for egg-laying: OAMB and Octß2R. The Drosophila genome contains 4 additional octopamine receptors-Octα2R, Octß1R, Octß3R, and Oct-TyrR-but their cellular patterns of expression in the reproductive tract and potential contribution(s) to egg-laying are not known. In addition, the mechanisms by which OAMB and Octß2R regulate reproduction are incompletely understood. Using a panel of MiMIC Gal4 lines, we show that Octα2R, Octß1R, Octß3R, and Oct-TyrR receptors are not detectable in either epithelium or muscle but are clearly expressed in neurons within the female fly reproductive tract. Optogenetic activation of neurons that express at least 3 types of octopamine receptors stimulates contractions in the lateral oviduct. We also find that octopamine stimulates calcium transients in the sperm storage organs and that its effects in spermathecal, secretory cells, can be blocked by knock-down of OAMB. These data extend our understanding of the pathways by which octopamine regulates egg-laying in Drosophila and raise the possibility that multiple octopamine receptor subtypes could play a role in this process.

Contrasting roles of cytochrome P450s in amitraz and chlorfenapyr resistance in the crop pest Tetranychus urticae.

The molecular mechanisms of amitraz and chlorfenapyr resistance remain only poorly understood for major agricultural pests and vectors of human diseases. This study focusses on a multi-resistant field strain of the crop pest Tetranychus urticae, which could be readily selected in the laboratory to high levels of amitraz and chlorfenapyr resistance. Toxicity experiments using tralopyril, the active toxophore of chlorfenapyr, suggested decreased activation as a likely mechanism underlying resistance. Starting from the same parental strain, transcriptome profiling revealed that a cluster of detoxifying genes was upregulated after amitraz selection, but unexpectedly downregulated after chlorfenapyr selection. Further functional validation associated the upregulation of CYP392A16 with amitraz metabolism and the downregulation of CYP392D8 with reduced activation of chlorfenapyr to tralopyril. Genetic mapping (QTL analysis by BSA) was conducted in an attempt to unravel the genetic mechanisms of expression variation and resistance. This revealed that chlorfenapyr resistance was associated with a single QTL, while 3 QTLs were uncovered for amitraz resistance. Together with the observed contrasting gene expression patterns, we argue that transcriptional regulators most likely underly the distinct expression profiles associated with resistance, but these await further functional validation.