Characterization and comparative pharmacological studies of a functional γ-aminobutyric acid (GABA) receptor cloned from the tobacco budworm,Heliothis virescens (Noctuidae:Lepidoptera)

This paper reports the functional expression and pharmacological characterization of a full length complementary deoxyribonucleic acid (cDNA) (pIVY12) cloned from aHeliothis virescens fertilized egg cDNA library that encodes for a γ-aminobutyric acid (GABA) receptor subunit (HVRDL-Ser 285). Two electrode voltage clamp recordings ofXenopus oocytes expressing the HVRDL GABA-gated chloride channel revealed robust chloride ion conductance in response to GABA and the GABA_A receptor agonist, muscimol. Baclofen, a GABA_B agonist had no effect. Phenobarbital showed a positive dose-dependent allosteric modulatory effect, whereas the benzodiazepine, flunitrazepam, had no effect. Chloride conductance was depressed by the novel insecticide, fipronil ((±)-5-amino-1-(2,6 dichloro-α, α, α-trifluoro-p-tolyl)-4-trifluoromethyl-sulfinylpyrazole-3-carbonitrile) and the GABA_A antagonist, picrotoxinin. The HVRDL GABA receptor was insensitive to blockage by dieldrin and the GABA_A antagonist, bicuculline. The comparative actions of fipronil, picrotoxinin and dieldrin were examined on oocytes expressing theH. virescens wild-type (HVRDL-Ser 285), the site-directed mutant (HVRDL-Ala 285), theDrosophila melanogaster Rdl wild-type (DMRDL-Ala 302) and theRdl dieldrin resistant (DMRDL-Ser 302) homo-oligomeric GABA receptors. HVRDL-Ala 285 was 15-fold more sensitive to blockage by fipronil than HVRDL-Ser 285. DMRDL-Ala 302 and DMRDL-Ser-302 showed a similar level of sensitivity to blockage by fipronil. HVRDL-Ser 285 and DMRDL-Ser 302 exhibited a similar level of insensitivity to picrotoxinin. HVRDL-Ala 285 and DMRDL-Ala 302 showed a similar range of picrotoxinin sensitivity. DMRDL-Ala 302 and HVRDL-Ala 285 showed some sensitivity to blockage by dieldrin. Fipronil sensitivity was significantly altered by the serine to alanine mutation at position 285 in the M2 region of the HVRDL subunit, whereas no difference was observed between the DMRDL-Ser 302 and DMRDL-Ala 302 receptors.     

Receptors for L-glutamate and GABA in the nervous system of an insect (Periplaneta americana)

The nervous system of the cockroach Periplaneta americana is well suited to studies of invertebrate amino acid receptors. Using a combination of radioligand binding and electrophysiological techniques, several distinct receptors have now been identified. These include an l-glutamate-gated chloride channel which has no known counterpart in the vertebrate nervous system, and a putative kainate/quisqualate receptor with pharmacological properties different from those of the existing categories of vertebrate excitatory amino acid receptors. GABA receptors have also been characterized in the cockroach nervous system. Bicuculline, benzodiazepines and steroids have revealed important differences between certain insect GABA-gated chloride channels and vertebrate GABA receptors. Identifiable neurones may facilitate the allocation of specific functions to amino acid receptor subtypes. In view of the existence of subtypes of amino acid receptors in insects, it is of interest to examine how this is reflected at the molecular level in terms of receptor subunit composition and amino acid sequence. Preliminary molecular cloning studies on insect GABA receptors are described.     

Functional assay for GABA receptor subtypes of a cockroach giant interneuron.

gamma-Aminobutyric acid (GABA) receptors were examined in the cockroach central nervous system (CNS) using the single fiber-oil gap method applied to an identified giant interneuron. Short-lasting pressure application of 10 mM GABA developed a multiphasic response composed of a fast hyperpolarization followed by a transient depolarizing component and a stable hyperpolarization. This triphasic characteristic shape of the response was modified according to the dose of GABA injected or bath-applied and to the precise localization of the injection within the dendritic area. The transient depolarizing phase showed a negative reversal potential of -70 mV. Both hyperpolarizing phases reversed at a more negative level ranging to -80 mV. A positive shift of these values was caused by a decrease in external chloride concentration. Bath-application of 0.1 mM picrotoxin (Ptx) decreased the depolarizing phase which was progressively replaced by a stable hyperpolarization. The transient depolarizing component desensitized quickly and was the most sensitive phase to Ptx action. The Ptx-resistant response reversed at a mean value of -100 mV close to the equilibrium potential for potassium ions (EK+), suggesting that it was generated by a K(+)-channel coupled receptor. Although baclofen was unable to mimic the Ptx-resistant GABA response, the compound CGA 147823, known to bind with a high specificity to vertebrate GABAB receptors, has been successfully used to reproduce the Ptx-resistant GABA response. It is suggested that, in addition to GABA receptors linked to chloride channels, the insect CNS possesses GABA receptors sharing ionic characteristics of GABAB receptors especially those located in the vertebrate CNS, although they are insensitive to baclofen.     

GABA receptors on the cell-body membrane of an identified insect motor neuron

The pharmacology of a gamma-aminobutyric acid (GABA) receptor on the cell body of an identified motor neuron of the cockroach (Periplaneta americana) was investigated by current-clamp and voltage-clamp methods. Iontophoretic application of GABA increased membrane conductance to chloride ions, and prolonged application resulted in desensitization. Hill coefficients, determined from dose-response data, indicated that binding of at least two GABA molecules was required to activate the chloride channel. Differences between vertebrate GABAA receptors and insect neuronal GABA receptors were detected. For the GABA receptor of motor neuron Df, the following rank order of potency was observed: isoguvacine greater than muscimol greater than or equal to GABA greater than 3-aminopropanesulphonic acid. The GABAB receptor agonist baclofen was inactive. Of the potent vertebrate GABA receptor antagonists (bicuculline, pitrazepin, RU5135 and picrotoxin), only picrotoxin (10(-7) M) produced a potent, reversible block of the response to GABA of motor neuron Df. Both picrotoxinin and picrotin also blocked GABA-induced currents. Bicuculline hydrochloride (10(-4) M) and bicuculline methiodide (10(-4) M) were both ineffective when applied at resting membrane potential (-65 mV), although at hyperpolarized levels partial block of GABA-induced current was sometimes observed. Pitrazepin (10(-4) M) caused a partial, voltage-independent block of GABA-induced current. The steroid derivative RU5135 was inactive at 10(-5) M. In contrast to the potent competitive blockade of vertebrate GABAA receptors by bicuculline, pitrazepin and RU5135, none of the weak antagonism caused by these drugs on the insect GABA receptor was competitive. Flunitrazepam (10(-6) M) potentiated GABA responses, providing evidence for a benzodiazepine site on an insect GABA-receptor-chloride-channel complex.     

Activation of picrotoxin-resistant GABA receptors by GABA and related compounds induces modulation of cockroach dorsal paired median (DPM) neuron firing

Activation of gamma-aminobutyric acid (GABA) receptors in insect dorsal paired median (DPM) neurons induced two types of response which appeared to be mediated by two different GABA receptor subtypes. When activated by bath application of GABA, one receptor subtype, insensitive to picrotoxin (PTX), mediated a drastic reduction in the firing frequency, leading to a blockade of the spontaneous electrical activity. These effects were accompanied by decreases in the amplitude and duration of the plateau action potential (AP) and the spike after-hyperpolarization (AHP). In most cases, a slight depolarization of the resting membrane potential occurred. Bath application of the vertebrate GABA(B) receptor agonists 3-aminopropyl(methyl)phosphinic acid (SKF 97541) and 3-aminopropylphosphinic acid (CGA 147823/CGP 27492) induced similar responses. Another GABA receptor subtype, less sensitive to GABA, mediated a chloride dependent hyperpolarization that was suppressed by bath application of PTX. The approximate locations of these two GABA receptor subtypes were determined by local pressure microapplications of GABA and vertebrate GABAergic agonists. The PTX-sensitive receptors were located predominantly on the surface of the ganglion where the apical pole of the soma is situated, while the PTX-resistant receptors appeared to be located deeper within the ganglion.These results reveal the existence of two GABA receptor subtypes on the DPM neurons and provide evidence for a functional role for PTX-resistant GABA receptors in the regulation of spontaneous firing.     

Chloride channels as tools for developing selective insecticides

Ligand-gated chloride channels underlie inhibition in excitable membranes and are proven target sites for insecticides. The gamma-aminobutyric acid (GABA(1)) receptor/chloride ionophore complex is the primary site of action for a number of currently used insecticides, such as lindane, endosulfan, and fipronil. These compounds act as antagonists by stabilizing nonconducting conformations of the chloride channel. Blockage of the GABA-gated chloride channel reduces neuronal inhibition, which leads to hyperexcitation of the central nervous system, convulsions, and death. We recently investigated the mode of action of the silphinenes, plant-derived natural compounds that structurally resemble picrotoxinin. These materials antagonize the action of GABA on insect neurons and block GABA-mediated chloride uptake into mouse brain synaptoneurosomes in a noncompetitive manner. In mammals, avermectins have a blocking action on the GABA-gated chloride channel consistent with a coarse tremor, whereas at longer times and higher concentrations, activation of the channel suppresses neuronal activity. Invertebrates display ataxia, paralysis, and death as the predominant signs of poisoning, with a glutamate-gated chloride channel playing a major role. Additional target sites for the avermectins or other chloride channel-directed compounds might include receptors gated by histamine, serotonin, or acetylcholine.The voltage-sensitive chloride channels form another large gene family of chloride channels. Voltage-dependent chloride channels are involved in a number of physiological processes including: maintenance of electrical excitability, chloride ion secretion and resorption, intravesicular acidification, and cell volume regulation. A subset of these channels is affected by convulsants and insecticides in mammals, although the role they play in acute lethality in insects is unclear. Given the wide range of functions that they mediate, these channels are also potential targets for insecticide development.     

The pharmacological profile of GABA receptors on cultured insect neurones

Neuronal cultures of the cockroach, Periplaneta americana, were used to study the pharmacological profile of GABA receptors using the whole-cell-voltage clamp technique. The results indicated that insect GABA receptors are linked to a chloride channel that can be activated by both GABA(A) and GABA(C) receptor agonists. The receptors are blocked by GABA(A) chloride channel blockers and some insecticides but not by competitive GABA(A) receptor antagonists. The GABA(C) receptor competitive antagonists were either full or partial agonists of the cockroach GABA receptors. The receptors were modulated by the enantiomers of lindane. In conclusion, insect GABA receptors appear to have a distinct pharmacological profile that does not conform to either vertebrate GABA(A) or GABA(C) receptors.     

Crayfish sensory terminals and motor neurones exhibit two distinct types of GABA receptors

Motor neurones of the crayfish walking system display inhibitory responses evoked either by γ-amino butyric acid (GABA) or glutamate, possibly involving the same receptor (Pearlstein et al. 1994). In order to test if this sensibility to both GABA and glutamate was a specific property of crayfish GABA receptors, pharmacological characteristics of GABA-evoked responses in both sensory terminals from CB chordotonal organ and motor neurones of the walking system have been compared. Both receptors are GABA-gated Cl⁻ channels activated by specific GABA_A (muscimol, isoguvacine), GABA_B (3-aminopropyl phosphinic acid), and GABA_C (cis-4-amino crotonic acid) agonists, and blocked by competitive (β-guanidino propionic acid) and non-competitive (picrotoxin) antagonists. They were insensitive to specific GABA_A (bicuculline, SR-95531) and GABA_B (phaclofen) antagonists. Furthermore, in both cases, nipecotic acid and the modulatory drug diazepam had no effect. However, our results demonstrate that GABA receptors of sensory terminals are different from those of motor neurones. GABA-induced desensitisation only occurred in sensory terminals. Moreover, glutamate was shown to activate GABA-gated Cl⁻ channels in motor neurones, but not in sensory terminals. Therefore, GABA is likely to be the endogenous neurotransmitter of presynaptic inhibition in sensory terminals, whereas inhibition between antagonistic motor neurones would be achieved by glutamate. Accepted: 10 July 1996     

Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone

The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D_f) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D_f motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.     

RdlDv,a novel GABA-gated chloride channel gene from the American dog tick Dermacentor variabilis

The American dog tick Dermacentor variabilis is a major transmitter of bacterial and viral pathogens in human and animal populations, and compounds active against this species would benefit both human and animal health. Invertebrate GABA-gated chloride channels are validated targets of commonly used insecticides and acaricides. We cloned a novel member of the invertebrate GABA-gated chloride channel gene family from Dermacentor variabilis, RdlDv. The closest homologue of the predicted gene product of RdlDv is the RDL protein encoded by the GABA-gated chloride channel gene Drosophila Rdl (Resistance to Dieldrin), with which it shares 64% amino acid identity. When expressed in Xenopus oocytes, RdlDv produces GABA-activated currents blocked by the known insecticides and RDL antagonists fipronil and picrotoxinin. These results suggest that RdlDv encodes a GABA-gated chloride channel subunit, making it a potential target for compounds active against the tick D. variabilis.     

The regulation of alpha-MSH release by GABA is mediated by a chloride-dependent [Ca2+]c increase in frog melanotrope cells

In frog melanotrope cells, gamma-aminobutyric acid (GABA) induces a biphasic effect, i.e. a transient stimulation followed by a more sustained inhibition of alpha-MSH release, and both phases of the GABA effect are mediated by GABAA receptors. We have previously shown that the stimulatory phase evoked by GABAA receptor agonists can be accounted for by calcium entry. In the present study, we have investigated the involvement of the chloride flux on GABA-induced [Ca2+]c increase and alpha-MSH release. We show that GABA evokes a concentration-dependent [Ca2+]c rise through specific activation of the GABAA receptor. The GABA-induced [Ca2+]c increase results from opening of voltage-activated L- and N-type calcium channels, and sodium channels. Variations of the extracellular Cl- concentration revealed that GABA-induced [Ca2+]c rise and alpha-MSH release both depend on the Cl- flux direction and driving force. These observations suggest for the first time that GABA-gated Cl- efflux provokes an increase in [Ca2+]c increase that is responsible for hormone secretion.     

Two novel GABAA receptor subunits exist in distinct neuronal subpopulations

Two cDNAs encoding novel GABAA receptor subunits were isolated from a rat brain library. These subunits, gamma 2 and delta, share approximately 35% sequence identity with alpha and beta subunits and form functional GABA-gated chloride channels when expressed alone in vitro. The gamma 2 subunit is the rat homolog of the human gamma 2 subunit recently shown to be important for benzodiazepine pharmacology. Cellular localization of the mRNAs encoding the gamma 2 and delta subunits in rat brain revealed that largely distinct neuronal subpopulations express the two subunits. The delta subunit distribution resembles that of the high affinity GABAA receptor labeled with [3H]muscimol; the gamma 2 subunit distribution resembles that of GABAA/benzodiazepine receptors labeled with [3H]flunitrazepam. These findings have implications for the composition of two different GABAA receptor subtypes and for information processing in networks using GABA for signaling.     

Bicuculline-insensitive GABA-gated Cl<Superscript>−</Superscript> channels in the larval nervous system of the moth<Emphasis Type="Italic"> Manduca sexta</Emphasis>

GABA-gated Cl^– channels were studied in the nervous system of the larval tobacco hawk moth, Manduca sexta, using electrophysiology, ³⁶Cl^– uptake into membrane microsacs and immunocytochemistry. A GABA-induced increase in Cl^– conductance was recorded from a visually identifiable neurone (fg1) in the desheathed frontal ganglion. The response was insensitive to the vertebrate GABA_A receptor antagonist, bicuculline, but was blocked by picrotoxinin. Bicuculline-insensitive, picrotoxinin-sensitive, GABA-stimulated ³⁶Cl^– uptake was also detected in membrane microsacs prepared from the isolated larval M. sexta nervous system. Such receptors appear to be the major type of GABA receptor in larval nervous system membrane microsac preparations. An antibody raised against a 17 amino acid peptide, based on the predicted C-terminus of the Drosophila GABA receptor subunit (RDL), stained not only cell bodies, including that of fg1, but also the neuropile in the frontal ganglion, indicating the existence of RDL-like GABA receptor subunits in neurones of this ganglion. Thus, bicuculline-insensitive GABA-gated Cl^– channels are present in the larval nervous system of M. sexta.     

On the mechanism by which dopamine inhibits prolactin release in the anterior pituitary

An $\text{in}$$\text{vitro}$ perfusion system was used to assess the effects of chloride channel blockers, dopamine (DA) receptor agonists and antagonists, and GABA receptor agonists and antagonists on prolactin release from the mouse anterior pituitary. Dopamine and muscimol inhibited prolactin release ($\text{IC}{}_{50}{}^1\text{= 6 × 10}{}^{\mathrm{?8}}\text{M and}\text{10}{}^{\mathrm{?5}}\text{M}$ respectively). The GABA receptor antagonist bicuculline blocked the inhibition of prolactin release by muscimol but not dopamine. The dopamine receptor antagonist chlorpromazine blocked the dopamine- but not muscimol-induced inhibition of prolactin release. Haloperidol, however, reversed both the muscimol and dopamine induced inhibition of prolactin release. Furthermore, the chloride channel blocker picrotoxinin blocked the inhibition of prolactin release elicited by both dopamine and muscimol. These later results suggest that the anterior pituitary dopamine receptor which mediates the inhibition of prolactin release may be coupled to a picrotoxinin sensitive chloride ionophore and that haloperidol may affect the function of both DA and GABA receptors in the anterior pituitary.     

Sensitivities of Achatina giant neurones to putative amino acid neurotransmitters

1. GABA receptors in Achatina identifiable giant neurones were classified into the muscimol I, muscimol II and baclofen types. Muscimol I and II type GABA receptors were sensitive to GABA and muscimol but insensitive to baclofen, whereas baclofen type receptors were sensitive to GABA and baclofen but insensitive to muscimol. Muscimol I and baclofen types were associated with the inhibition caused by GABA, while muscimol II type with the GABA excitation.2. GABA, muscimol and TACA produced a transient outward current (I_out) with an increase in membrane conductance (g) of an Achatina neurone, TAN, having the muscimol I type GABA receptors. Their relative potency values (RPV) at GABA ed₅₀ (approximately 10⁻⁴ M) were: GABA: muscimol: TACA = 1:0.6:0.3. The GABA effects were potentiated by pentobarbitone, antagonized competitively by pitrazepin and non-competitively by picrotoxin and diazepam, and unaffected by bicuculline. The ionic mechanism of effects of GABA and its two analogues was the increase in membrane Cl⁻ conductance (g_Cl).3. GABA and (±)-baclofen produced a slow I_out with a g increase of another Achatina neurone, RPeNLN, having the baclofen type GABA receptors. The two compounds were almost equipotent (ed₅₀: approximately 3 × 10⁻⁴ M). The ionic mechanism of their effects was the increase in g_k. The two compounds hardly affected the voltage-gated and slowly inactivating calcium current. I_out produced by GABA and (±)-baclofen were reduced by TEA, but unaffected by 4-AP, bicuculline, pitrazepin and picrotoxin.4. β-hydroxy-l-glutamic acid (l-BHGA) showed the marked effects on the Achatina giant neurones; the two neurones were excited by the compound, whereas the three inhibited. D-BHGA, l-Glu, d-Glu and NMDA were less effective than l-BHGA or almost ineffective. Erythro-l-BHGA was more or less effective than threo-l-BHGA according to the neurones tested.5. α-Kainic acid and domoic acid excited the two neurones, which were excited by l-BHGA. l-Quisqualic acid showed the similar effects to l-BHGA, which were mostly much stronger than l-BHGA. Erythro-l-tricholomic acid and dl-ibotenic acid showed the effects similar to l-BHGA selectively on some neurones.6. It was pointed out that the pharmacological features of GABA on the Achatina neurones are simpler than those of l-BHGA, due to the simpler structure of the former compound having less binding sites than the latter.     

Pharmacology of insect GABA receptors

A GABA-operated Cl^– channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl^– channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABA_A receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl^– channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active att-butylbicy-clophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.Special issue dedicated to Dr. Eugene Roberts     

Activation of gamma-aminobutyric acid insensitive chloride channels in mouse brain synaptic vesicles by avermectin B1a.

The interaction of avermectin B1a (AVMB1a) with mouse brain chloride channels was characterized using a radiochloride efflux assay. The loss of intravesicular chloride from synaptoneurosomes preloaded with 36Cl involved an initial rapid phase followed by a slower phase that approached equilibrium within 10 min. AVMB1a stimulated a 30% loss of intravesicular chloride within the first 2 s of exposure; however, AVMB1a had no effect on the rate of the slower phase of chloride loss. Experiments with lysed synaptoneurosomes showed that both chloride loading and basal and AVMB1a-stimulated chloride release required the presence of intact vesicles. The efflux of 36Cl from mouse brain synaptosomes and the stimulation of efflux by AVMB1a were qualitatively similar to the results obtained with synaptoneurosomes but involved much lower overall levels of chloride loading and release. AVMB1a produced half-maximal stimulation of chloride efflux from synaptoneurosomes at a concentration of 2.1 +/- 0.3 microM and a 35.4 +/- 1.4% maximal loss of intravesicular chloride at saturating concentrations. gamma-Aminobutyric acid (GABA), bicuculline, or the chloride channel blockers picrotoxinin, t-butylbicyclophosphorothionate (TBPS) 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene 9-carboxylic acid (9-CA) had little or no effect on the loss of chloride from synaptoneurosomes either in the presence or the absence of AVMB1a. However, the chlorinated cycloalkane insecticides dieldrin and lindane were equally effective as inhibitors of GABA-dependent chloride uptake and AVMB1a-stimulated chloride efflux. These data demonstrate that AVMB1a-stimulated chloride efflux from mouse brain synaptic vesicles results from the activation of GABA-insensitive chloride channels and that this action is distinct from their previously documented effects on GABA-gated chloride channels in mouse brain preparations. Our findings imply that both GABA-gated and GABA-insensitive chloride channels may be toxicologically significant targets for the action of avermectins.     

Influence of GABA-agonists and antagonists on neuroleptic-induced catalepsy in rats.

Direct (muscimol) or indirect (aminooxyacetic acid, diaminobutyric acid, pyrrolidinone) GABA-mimetic compounds significantly potentiate neuroleptic induced catalepsy in rats. In contrast at subconvulsant doses, direct (bicuculline, picrotoxinin and indirect (allylglycine) GABA antagonists antagonized haloperidol-induced catalepsy. The effect of bicuculline and picrotoxinin was biphasic with the lowest doses increasing catalepsy. These results indicate that GABA mechanisms are involved in the induction of catalepsy by neuroleptics.     

KNOCKDOWN OF THE IONOTROPIC γ‐AMINOBUTYRIC ACID RECEPTOR (GABAR) RDL GENE DECREASES FIPRONIL SUSCEPTIBILITY OF THE SMALL BROWN PLANTHOPPER,Laodelphax striatellus (HEMIPTERA: DELPHACIDAE)

Insect γ‐aminobutyric acid receptors (GABARs) are important molecular targets of cyclodiene and phenylpyrazole insecticides. Previously GABARs encoding rdl (resistant to dieldrin) genes responsible for dieldrin and fipronil resistance were identified in various economically important insect pests. In this study, we cloned the open reading frame cDNA sequence of rdl gene from fipronil‐susceptible and fipronil‐resistant strains of Laodelphax striatellus (Lsrdl). Sequence analysis confirmed the presence of a previously identified resistance‐conferring mutation. Different alternative splicing variants of Lsrdl were noted. Injection of dsLsrdl reduced the mRNA abundance of Lsrdl by 27–82%, and greatly decreased fipronil‐induced mortality of individuals from both susceptible and resistant strains. These data indicate that Lsrdl encodes a functional RDL subunit that mediates susceptibility to fipronil. Additionally, temporal and spatial expression analysis showed that Lsrdl was expressed at higher levels in eggs, fifth‐instar nymphs, and female adults than in third‐instar and fourth‐instar nymphs. Lsrdl was predominantly expressed in the heads of 2‐day‐old female adults. All these results provide useful background knowledge for better understanding of fipronil resistance related ionotropic GABA receptor rdl gene expressed variants and potential functional differences in insects.     

The novel isoxazoline ectoparasiticide fluralaner: Selective inhibition of arthropod γ-aminobutyric acid- and l-glutamate-gated chloride channels and insecticidal/acaricidal activity

Isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and l-glutamate-gated chloride channels (GluCls). In this study, the effects of the isoxazoline drug fluralaner on insect and acarid GABACl (RDL) and GluCl and its parasiticidal potency were investigated. We report the identification and cDNA cloning of Rhipicephalus (R.) microplus RDL and GluCl genes, and their functional expression in Xenopus laevis oocytes. The generation of six clonal HEK293 cell lines expressing Rhipicephalus microplus RDL and GluCl, Ctenocephalides felis RDL-A₂₈₅ and RDL-S₂₈₅, as well as Drosophila melanogaster RDLCl-A₃₀₂ and RDL-S₃₀₂, combined with the development of a membrane potential fluorescence dye assay allowed the comparison of ion channel inhibition by fluralaner with that of established insecticides addressing RDL and GluCl as targets. In these assays fluralaner was several orders of magnitude more potent than picrotoxinin and dieldrin, and performed 5-236 fold better than fipronil on the arthropod RDLs, while a rat GABACl remained unaffected. Comparative studies showed that R. microplus RDL is 52-fold more sensitive than R. microplus GluCl to fluralaner inhibition, confirming that the GABA-gated chloride channel is the primary target of this new parasiticide. In agreement with the superior RDL on-target activity, fluralaner outperformed dieldrin and fipronil in insecticidal screens on cat fleas (Ctenocephalides felis), yellow fever mosquito larvae (Aedes aegypti) and sheep blowfly larvae (Lucilia cuprina), as well as in acaricidal screens on cattle tick (R. microplus) adult females, brown dog tick (Rhipicephalus sanguineus) adult females and Ornithodoros moubata nymphs. These findings highlight the potential of fluralaner as a novel ectoparasiticide.