Cyclodiene resistance at the insect GABA receptor/chloride channel complex confers broad cross resistance to convulsants and experimental phenylpyrazole insecticides

This study investigated the pharmacological profile of cyclodiene resistance in Drosophila melanogaster and the mode of action of a phenylpyrazole insecticide, JKU 0422. Toxicological studies were performed with a sucrose bait assay containing the synergist piperonyl butoxide. The Maryland strain of D. melanogaster was resistant to dieldrin, lindane, picrotoxinin, TBPS, p-CN-TBOB, and JKU 0422. In contrast, this strain was susceptible to cypermethrin and the avermectins MK-243, abamectin, and abamectin 8,9-oxide. Neurophysiological studies showed that both TBPS and JKU 0422 reversed the inhibitory action of GABA in central nerve preparations from susceptible D. melanogaster. However, the response to these compounds was attenuated in nerve preparations from the resistant Maryland strain, which indicated that the resistance was expressed at the level of the nerve. Topical toxicity bioassays with JKU 0422 on susceptible (CSMA) and cyclodiene-resistant (LPP) strains of German cockroach revealed a resistance ratio of 553-fold for this compound. These studies demonstrate that cyclodiene resistance in D. melanogaster confers broad cross resistance toward compounds thought to block the GABA-gated chloride channel in a manner similar to the cyclodienes. Moreover, the cross resistance extends to JKU 0422, and resistance to this compound is also present in a strain of cyclodiene-resistant German cockroach. These toxicological results, along with the neurophysiological studies, confirm that JKU 0422 has a mode of action that is similar to the cyclodienes and TBPS. These findings suggest that the introduction and use of new chloride channel antagonists as insecticides should be managed carefully in order to prevent the rapid development of resistance in the field. © 1994 Wiley-Liss, Inc.     

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.     

Gene mapping and cross-resistance in cyclodiene insecticide-resistant Drosophila melanogaster (Mg.)

Resistance to the cyclodiene insecticide dieldrin maps to a single gene (Rdl) on the left arm of chromosome III in Drosophila melanogaster (Meigen). The gene was further mapped by the use of chromosomal deficiencies to a single letter sub-region, 66F, on the polytene chromosome. The cross-resistance spectrum of a backcrossed strain lacking elevated mixed function oxidase activity, a common resistance mechanism, was examined. Levels of resistance similar to those found in other insects were found to dieldrin, aldrin, endrin, lindane, and picrotoxinin. Strong similarity of this single major gene with that found in other cyclodiene resistant insects is suggested by its cross-resistance spectrum and chromosomal location, via homology with other Diptera. The significance of major genes in insecticide resistance is discussed.     

Selection of dieldrin-resistant strains of Lucilia cuprina (Diptera: Calliphoridae) after ethyl methanesulfonate mutagenesis of a susceptible strain.

After ethyl methanesulfonate (EMS) mutagenesis of a susceptible strain (SWT), selective screening of Lucilia cuprina (Wiedemann) resulted in four strains that were resistant to the insecticide dieldrin. Concentrations used for selection were greater than LC99 of susceptible phenotypes. No resistant variants were screened from the standard laboratory strain (SWT) not treated with EMS. The resistance phenotypes of the four resistant strains were similar to each other and to that of a field-selected resistant strain. The genetic basis of resistance is monogenic in all strains and the data are consistent with the same locus, Rdl, determining resistance status in each strain. The Rdl locus maps to chromosome V, approximately 3.5 map units distal to the Sut locus. Dieldrin resistance may be caused by less effective blocking of insect neuronal GABA receptors by the chemical in resistant strains. The data indicate that the evolution of resistance to an insecticide in the field may be constrained by a limited number of genetical and biochemical options if a monogenic response is selected for and that the spontaneous mutation rate to the Rdl allele is less than 1 in 10(6) in the laboratory.     

Cross-resistance between dieldrin and fipronil in German cockroach (Dictyoptera: Blattellidae)

The toxicity of fipronil and dieldrin was determined in one susceptible laboratory strain and seven insecticide-resistant field-collected strains of Blattella germanica (L). The Zo960302 and Ga021001 strains were 1,270- and 2,030-fold resistant to dieldrin and 15- and 14-fold resistant to fipronil. The Su960304 and Od010803 strains were 15- and 13-fold resistant to dieldrin and two- and four-fold resistant to fipronil. Three strains showed no or a low level of resistance to dieldrin and fipronil. Crosses were performed between the susceptible strain Danish Pest Infestation Laboratory (DPIL)-SUS and the resistant strains Zo960302 and Su960304 and resistance to dieldrin and fipronil were intermediate compared with the susceptible and the resistant strains. Backcrosses to both of the parental strains showed cosegregation of dieldrin and fipronil resistance. The toxicity of dieldrin and fipronil was correlated when compared at LD50, and 93% of the observed variation in LD50 of fipronil can be ascribed to variation among predictions based on the value of LD50 of dieldrin. The frequency of the A302S substitution in the resistance to dieldrin (Rdl) gene in the highly dieldrin- and fipronil-resistant strains Zo960302 and Ga021001 and the moderately resistant Su960304 was 0.97, 1.0, and 0.38, respectively. We consider the connection between the frequency of the Rdl mutation and dieldrin and fipronil resistance a causal connection and not merely a coincidence.     

用溴氰菊酯选育抗敌百虫淡色库蚊的研究

将室内选育成功的抗敌百虫淡色库蚊Culex pipiens pallens Coq.品系(RD)分为二个分系,一个不再用敌百虫处理,称之为RD衰退品系(RD_139-x),34代后对敌百虫的敏感度增加了10倍,对溴氰菊酯的敏感度无显著变化.另一分系改用溴氰菊酯选育,命名为Rde品系,53代后对溴氰菊酯抗性达200倍左右,对敌百虫敏感度上升约10倍,对DDT的交互抗性高达118倍,对马拉硫磷、杀螟硫磷的敏感度与敏感品系(SEN,上海昆虫所保存)比较也有上升,呈负交互抗性现象.用高剂量溴氰菊酯处理幼虫、也证明Rde在20分钟内麻痹率比敏感品系低,可见抗性机制主要是抗击倒因子(Kdr).但增效醚(Pb)对溴氰菊酯明显增效,可见mfo酶也起重要作用,推测抗性为多因子遗传.     

Pre- and postsynaptic inhibition mediated by GABA(B) receptors in rat ventrolateral periaqueductal gray neurons

The present study examined the actions of a GABA(B)-receptor agonist, baclofen, on synaptic transmission in rat ventrolateral periaqueductal gray (PAG) neurons of brainstem slices by using whole-cell voltage-clamp recordings. Baclofen (10 microM) induced a slow outward current (peak amplitude: 30.1+/-3.1pA, n=13) at -70mV, which persisted in the presence of tetrodotoxin (0.5 microM) and was diminished in the presence of postsynaptic intracellular K(+)-channel blockers (Cs(+) and TEA) and GDP-beta-S, indicating a direct postsynaptic depression mediated by K(+) channels and G proteins. Baclofen (10 microM) also decreased the frequency of both glutamatergic spontaneous EPSC (by 36+/-7%, n=11) and GABAergic spontaneous IPSC (by 37+/-12%, n=6) without changes in their amplitudes, indicating its presynaptic inhibitions. Taken together, the activation of postsynaptic GABA(B) receptors inhibits ventrolateral PAG neurons directly. At the same time, activating presynaptic GABA(B) receptors on glutamatergic and GABAergic nerve terminals inhibits glutamate and GABA release, respectively. The overall effects might influence an output of ventrolateral PAG neurons that build up the descending pain control system to the spinal dorsal horn.     

Differential sensitivity of two insect GABA-gated chloride channels to dieldrin,fipronil and picrotoxinin

In the central nervous system of both vertebrates and invertebrates inhibitory neurotransmission is mainly achieved through activation of γ-aminobutyric acid (GABA) receptors. Extensive studies have established the structural and pharmacological properties of vertebrate GABA receptors. Although the vast majority of insect GABA-sensitive responses share some properties with vertebrate GABAA receptors, peculiar pharmacological properties of these receptors led us to think that several GABA-gated chloride channels are present in insects. We describe here the pharmacological properties of two GABA receptor subtypes coupled to a chloride channel on dorsal unpaired median (DUM) neurones of the adult male cockroach. Long applications of GABA induce a large biphasic hyperpolarization, consisting of an initial transient hyperpolarization followed by a slow phase of hyperpolarization that is not quickly desensitized. With GABA, the transient hyperpolarization is sensitive to picrotoxinin, fipronil and dieldrin whereas the slow response is insensitive to these insecticides.When GABA is replaced by muscimol and cis-4-aminocrotonic acid (CACA) a biphasic hyperpolarization consisting of an initial transient hyperpolarization followed by a sustained phase is evoked which is blocked by picrotoxinin and fipronil. Exposure to dieldrin decreases only the early phase of the muscimol and CACA-induced biphasic response, suggesting that two GABA-gated chloride channel receptor subtypes are present in DUM neurones. This study describes, for the first time, a dieldrin resistant component different to the dieldrin- and picrotoxinin-resistant receptor found in several insect species.     

Toxicity and neurophysiological effects of fipronil and fipronil sulfone on the western corn rootworm (Coleoptera: Chrysomelidae)

Fipronil is a member of the relatively new phenylpyrazole insecticide class that is active at the neuro‐inhibitory gamma amino butyric acid (GABA)‐gated chloride channel/ionopore complex. The toxicity and neurophysiological effects of fipronil and its oxidative sulfone metabolite [5‐amino‐1‐(2,6‐dichloro‐4‐trifluoromethylphenyl)‐3‐cyano‐4‐trifluoromethylsulfonylpyrazole] were investigated by using an insecticide‐susceptible western corn rootworm population. In topical bioassays using adult rootworms, fipronil was toxic at very low doses (LD₅₀ = 0.07; LD₉₀ = 0.33 ng/mg). At the LD₉₀, pre‐treatment with the cytochrome P450 monooxygenase inhibitor piperonyl butoxide led to mild antagonism of fipronil toxicity (LD₉₀ = 0.42 ng/mg), while the sulfone analog had greater toxicity (LD₉₀ = 0.22 ng/mg). In neurophysiological studies of spontaneous electrical activity, adult and larval rootworms were equally affected by fipronil and the sulfone analog at 10 μM (in the presence of 5 mM GABA) in comparison to GABA‐treated baselines. Using larval rootworms, insensitivity of the GABA receptor to binding by picrotoxinin or dieldrin (10 μM ) was not apparent in the presence of 5 mM GABA. Further neurophysiological investigation using a range of concentrations (0.625–20.0 μM) on larval rootworms indicated concentration‐dependent effects on bursting activity for both fipronil and the sulfone analog; however, subtle differences were observed between these two compounds. Results indicate that both fipronil and its oxidative sulfone metabolite have similar toxicological and neurological effects on rootworms. Arch. Insect Biochem. Physiol. 40:150–156, 1999. © 1999 Wiley‐Liss, Inc.     

Knockdown resistance to dichlorodiphenyl-trichloroethane and pyrethroid insecticides in the napts mutant of Drosophila melanogaster is correlated with reduced neuronal sensitivity

Resistance to pyrethroid insecticides and dichlorodiphenyltrichloroethane (DDT) was investigated in the nap^ts (no action potential, temperature sensitive) mutant of Drosophila melanogaster. In surface contact bioassays, the nap^ts strain showed threefold resistance to deltamethrin at the LC₅₀ level when compared to susceptible Canton-S flies. Cross-resistance was also observed to DDT and the pyrethroids NRDC 157 [3-phenoxybenzyl [1R,cis]-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate], fenfluthrin, and MTI-800 [1-(3-phenoxy-4-fluorophenyl)-4-(4-ethoxyphenyl)-4-methylpentane]. The onset of intoxication by pyrethroids in nap^ts flies was markedly delayed, a finding that is consistent with the existence of a resistance mechanism involving reduced neuronal sensitivity. Resistance at the level of the nerve was confirmed by electrophysiological recordings of spontaneous and evoked activity in the dorsolongitudinal flight muscles of poisoned flies. Preparations from nap^ts insects treated with fenfluthrin displayed longer latencies to the appearance of spontaneous activity and also an absence or reduction in burst discharges compared to equivalent preparations from susceptible individuals. These results are discussed in light of competing hypotheses concerning the mechanism underlying knockdown resistance and reduced nerve sensitivity in insects.     

Activities of the frog skin peptide, ascaphin-8 and its lysine-substituted analogs against clinical isolates of extended-spectrum beta-lactamase (ESBL) producing bacteria

Extended-spectrum beta-lactamase (ESBL)-producing Gram-negative bacteria are becoming increasingly prevalent and their antibiotic resistance necessitates novel therapeutic intervention. Ascaphin-8 is a cationic alpha-helical peptide that shows broad-spectrum antibacterial activity but is also toxic to human erythrocytes (LC(50)= 55 microM). This study assesses the activity of ascaphin-8, and a series of l-lysine-substituted analogs, against a range of clinical isolates of ESBL-producing bacteria. All ESBL-producing Escherichia coli (MIC=1.5-6 microM) and Klebsiella pneumoniae (MIC=12.5-25 microM) strains tested were susceptible to ascaphin-8, as well as a group of miscellaneous ESBL-producing strains (Citrobacter, Salmonella, Serratia, Shigella spp.) (MIC< or = 25 microM). The Lys4- and Lys8-substituted analogs were generally the most potent against bacteria but showed the highest hemolytic activity. However, the Lys10, Lys14, and Lys18 analogs also displayed potent antibacterial activity while showing very low hemolytic activity (LC50> 500 microM). An unexpected finding was the susceptibility of ESBL-producing Proteus mirabilis strains to ascaphin-8 (MIC=12.5-25 microM) and its Lys4-substituted analog (MIC= 6 microM), although non-ESBL isolates of this organism were resistant to these peptides (MIC> 100 microM).     

Development of endosulfan tolerant strain of an egg parasitoid Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae)

A strain of T. chilonis, an egg parasitoid of lepidopteran pests tolerant to the most commonly used cyclodiene insecticide--endosulfan was developed in the laboratory. Tolerance to endosulfan was induced by exposing adult parasitoids sequentially from a sub-lethal concentration (0.004%) to the field recommended concentration (0.09%). The strain acquired tolerance to the insecticide after 341 generation of continuous exposure with LC50 values of 1074.96 ppm as compared to LC50 of (70.91 ppm) in susceptible strain. The genetical study showed that F1 crosses exhibited a semi-dominant response to endosulfan with degree of dominance value (D) of 0.58. The resistant factor of tolerant strain was 15.1 folds and of F1 cross were 8.53 folds over susceptible strain. Under net house conditions, the tolerant strain parasitised 56% Helicoverpa armigera eggs on potted cotton plants immediately after an insecticide spray, compared to 3% by the susceptible strain. High percentage survival of the immature stages of the tolerant strain proved their ability to withstand the insecticide load. Breakdown of insecticide tolerance in the strain occurred after four generations in absence of insecticide load. Use of the tolerant strain as a component of bio-intensive IPM in various crops where insecticide use is higher is discussed.     

The Picrotoxinin Binding Site and Its Relationship to the GABA Receptor Complex

The binding characteristics of [3H] alpha-dihydropicrotoxinin to the picrotoxinin binding site were investigated in membrane preparations of adult rat forebrain and living cultures of rat cerebral cortex. The binding of [3H]alpha-dihydropicrotoxinin to rat forebrain was decreased by lysing, treating with Triton X-100, and heating. Coincubation with gamma-aminobutyric acid (GABA), benzodiazepines, or alterations in the Na+ or Cl- composition of the media had no effect on the binding to the rat brain preparation. However, in the living neurons in tissue culture both GABA and diazepam significantly decreased the binding of [3H]alpha-dihydropicrotoxinin. The dose-response relationships for GABA antagonism of [3H]alpha-dihydropicrotoxinin binding and for picrotoxinin antagonism of the GABA enhancement of [3H]flunitrazepam binding in cultured cortical neurons were also investigated. The Hill coefficients for these actions were reciprocal, suggesting that they result from complementary interactions between the binding sites for GABA and picrotoxinin. These data support the association of the picrotoxinin binding site with the postsynaptic GABA receptor complex.     

Mechanisms of insecticide resistance in the aphid Nasonovia ribisnigri (Mosley) (Homoptera: Aphididae) from France.

Nasonovia ribisnigri, a main pest of salad crops, has developed resistance to various insecticides in southern France, including the carbamate pirimicarb and the cyclodiene endosulfan, two insecticides widely used to control this aphid. Here we have investigated the mechanisms of resistance to these two insecticides by studying cross-resistance, synergism, activity of detoxifying enzymes, and possible modifications of the target proteins. Resistance to pirimicarb was shown to be mainly due to a decreased sensitivity of the target acetylcholinesterase; this modification conferred also, resistance to propoxur but not to methomyl and the two tested organophosphates (acephate and paraoxon). Endosulfan resistance was associated with a moderate level of resistance to dieldrin, and resistance to both insecticides was due, in part, to increased detoxification by glutathione S-transferases (GST). The endosulfan resistant strain displayed the same amino acid at position 302 of the Rdl gene (GABA receptor) as susceptible aphids (e.g. Ala), indicating that the Ala to Ser (or to Gly) mutation observed among dieldrin resistant strains of other insect species was not present.     

CNS depressants accelerate the dissociation of 35S-TBPS binding and GABA enhances their displacing potencies

The specific binding of 35S-t-butylbicyclophosphorothionate (TBPS) was studied in synaptosomal membranes of rat cerebral cortex. The displacing potencies of eleven CNS depressants and three convulsants were determined in the presence of 1 microM GABA and 10 nM R 5135. GABA enhanced the displacing potencies of depressants of most diverse chemical structures: diaryltriazine (LY 81067), pyrazolopyridine (etazolate), cinnamide, glutarimide, 2,3-benzodiazepine (tofizopam) and alcohol derivatives, barbiturates, (+)etomidate, methaqualone and meprobamate. In contrast, the IC50 values of convulsants (picrotoxinin, pentetrazol and the barbiturate enantiomer S(+) MPPB) were not significantly affected. The depressants accelerated either basal or GABA-augmented dissociation of 35-TBPS mainly by increasing the contribution of its rapid first phase.     

Antagonist actions of bicuculline methiodide and picrotoxin on extrasynaptic gamma-aminobutyric acid receptors

The effects of picrotoxin and bicuculline methiodide to block depolarizing responses of extrasynaptic receptors for gamma-aminobutyric acid (GABA) are compared using excitability testing of myelinated axons in amphibian peripheral nerve. The actions of the antagonists appear both complex and dissimilar. Picrotoxin (10-1000 microM) produces large reversible depressions of the maximal response to GABA (0.01-10mM) and increases the EC50 from 0.33 to 12.6 mM. With high concentrations of agonist and antagonist an insensitive component is apparent. The action of picrotoxin is not classically noncompetitive: it may represent a mixed antagonism (competitive and noncompetitive) or a noncompetitive one, masked by the presence of receptor reserve and (or) secondary depolarizing influences (e.g., GABA-evoked [K+] o accumulation). Bicuculline methiodide (10-200 microM) shifts the GABA concentration-response curve to the right; maximal responses persist and are even enhanced. The impression that bicuculline methiodide has a competitive action is supported by analysis of its inhibition of responses to low concentrations of the agonist. It is suggested that the enhancement of GABA responses by bicuculline methiodide and their apparent resistance to block by picrotoxin may be due to a common secondary effect of the antagonists such as a decrease in membrane conductance to K+ and (or) block of transmitter uptake.     

Permethrin Induction of Multiple Cytochrome P450 Genes in Insecticide Resistant Mosquitoes,Culex quinquefasciatus

The expression of some insect P450 genes can be induced by both exogenous and endogenous compounds and there is evidence to suggest that multiple constitutively overexpressed P450 genes are co-responsible for the development of resistance to permethrin in resistant mosquitoes. This study characterized the permethrin induction profiles of P450 genes known to be constitutively overexpressed in resistant mosquitoes, Culex quinquefasciatus. The gene expression in 7 of the 19 P450 genes CYP325K3v1, CYP4D42v2, CYP9J45, (CYP) CPIJ000926, CYP325G4, CYP4C38, CYP4H40 in the HAmCqG8 strain, increased more than 2-fold after exposure to permethrin at an LC50 concentration (10 ppm) compared to their acetone treated counterpart; no significant differences in the expression of these P450 genes in susceptible S-Lab mosquitoes were observed after permethrin treatment. Eleven of the fourteen P450 genes overexpressed in the MAmCqG6 strain, CYP9M10, CYP6Z12, CYP9J33, CYP9J43, CYP9J34, CYP306A1, CYP6Z15, CYP9J45, CYPPAL1, CYP4C52v1, CYP9J39, were also induced more than doubled after exposure to an LC50 (0.7 ppm) dose of permethrin. No significant induction in P450 gene expression was observed in the susceptible S-Lab mosquitoes after permethrin treatment except for CYP6Z15 and CYP9J39, suggesting that permethrin induction of these two P450 genes are common to both susceptible and resistant mosquitoes while the induction of the others are specific to insecticide resistant mosquitoes. These results demonstrate that multiple P450 genes are co-up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, providing additional support for their involvement in the detoxification of insecticides and the development of insecticide resistance.     

Geographical variation in larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) to Bacillus thuringiensis spore-crystal mixtures and purified crystal proteins and associated resistance development in India

The susceptibility of larvae of the diamondback moth, Plutella xylostella Linnaeus to purified crystal proteins and spore-crystal preparations of Bacillus thuringiensis was investigated for 13 populations from seven states in India. The LC50 (microg ml(-1), 48 h) values of Cry proteins for different populations of P. xylostella ranged from 0.14-3.74 (Cry1Aa), 0.007-1.25 (Cry1Ab), 0.18-2.47 (Cry1Ac) and 0.12-3.0 (Cry1C). The LC50 (mg (ai) l(-1), 48 h) of spore-crystal preparations ranged from 0.02-0.98 (HD-1) and 0.06-2.14 (HD-73). Significantly higher LC50 values for all tested toxins and strains were obtained with populations collected from Iruttupallam and Ottanchathiram in the southern state of Tamil Nadu, whereas some of the populations collected from the northern part of India were more susceptible than the susceptible IARI 17-65 population. The high levels of resistance in the Iruttupallam and Ottanchathiram populations to Cry1Ab suggested selection pressure by Cry1Ab, which is the predominant toxin in B. thuringiensis formulations used in India. Cry1Ab was found to be more toxic than the other toxins. The population from Iruttupallam showed increased resistance following selection with Cry1Ab in the laboratory (LC50 from 1.25 to 4.31 microg ml(-1) over two generations) and also showed cross resistance to CrylAa and CrylAc. The resistance to Biobit in the field population from Iruttupallam declined slowly; requiring c. 33 generations for an overall 10-fold decline in LC50 when the insects were reared in the laboratory without exposure to B. thuringensis.     

Comparison of the capsaicin- and amino acid-sensitivity of dorsal root C fibres in the rat and the toad.

1. The C elevation of the compound action potential (CAP) was recorded with suction electrodes from dorsal roots of rats at 25 degrees C and toads (Bufo bufo) at 10 degrees C. The C fibre CAP had a conduction velocity of 0.5 +/- 0.07 SE M per sec (N = 10) and 0.25 +/- 0.04 M per sec (N = 8) in the rat and toad nerves respectively. 2. The depressant effect of applied drugs on the amplitude of the C fibres CAP was measured. Nerves from both species had similar sensitivities to GABA. EC50 5.0 microM +/- 0.5 SEM (N = 3) and 5.5 microM +/- 1.4 (N = 3) for the rat and toad respectively. Maximum depressant effects of GABA produced in rat and toad nerves were 35% +/- 5 SEM and 17% +/- 2.5 respectively. 3. In five out of ten of the rat nerves tested kainate had a clear depressant effect (maximum 36% +/- 4.3 SEM, EC50 6.8 microM +/- 0.9 SEM, N = 3) on the C fibre CAP. Kainate, at concentrations from 100 to 500 microM, had no effect on seven toad nerves. 4. Toad nerves were about 100 times less sensitive, than rat nerves, to capsaicin (ED50 values 430 microM +/- 190 SEM and 0.7 microM +/- 0.2 respectively, N = 4). 5. The similar sensitivity of nerves in both species to GABA and differing sensitivities to kainate and capsaicin suggests that amphibian C fibres specifically lack sensitivity to capsaicin and kainate.     

35S]t-butylbicyclophosphorothionate binding sites in susceptible and cyclodiene-resistant houseflies.

4-aminobutyric acid (GABA)-gated chloride ion channels are important molecular targets for a number of polychlorocycloalkane compounds including cyclodiene insecticides. Previous radioligand binding studies have indicated that cyclodiene insecticides are potent inhibitors of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to housefly thorax and abdomen membranes. In the present study, a laboratory-reared, cyclodiene-resistant (CYW) housefly strain (Musca domestica) showed resistance to a number of cyclodiene insecticides. Specific, saturable [35S]TBPS binding was detected in thorax and abdomen membranes prepared from housefly strains susceptible (CSMA) and resistant (CYW) to cyclodienes. Scatchard analysis of [35S]TBPS binding data from CSMA and CYW membranes revealed no significant differences between the two strains in either the affinity (Kd) or the density (Bmax) of specific, saturable binding sites. There were no differences in the comparative effectiveness of a range of polychlorocycloalkanes, including cyclodiene insecticides, as inhibitors of specific [35S]TBPS binding to CSMA and CYW thorax and abdomen membranes. Therefore, if an alteration in target site is a mechanism for resistance to cyclodienes in the CYW strain, it is not readily measurable using [35S]TBPS.