Cyclodiene-resistant Strain Research Articles

Mode of action and toxicological effects of the sesquiterpenoid, nootkatone, in insects

Nootkatone, a sesquiterpenoid isolated from Alaskan yellow cedar (Cupressus nootkatensis), is known to possess insect repellent and acaricidal properties and has recently been registered for commercial use by the Environmental Protection Agency. Previous studies failed to elucidate the mechanism of action of nootkatone, but we found a molecular overlay of picrotoxinin and nootkatone indicated a high degree of structural and electrostatic similarity. We therefore tested the hypothesis that nootkatone was a GABA-gated chloride channel antagonist, similar to picrotoxinin. The KD50 and LD50 of nootkatone on the insecticide-susceptible strain of Drosophila melanogaster (CSOR) showed resistance ratios of 8 and 11, respectively, compared to the cyclodiene-resistant strain of RDL1675, indicating significant cross-resistance. Nootkatone reversed GABA-mediated block of the larval CSOR central nervous system; nerve firing of 78 ± 17% of baseline in the CSOR strain was significantly different from 24 ± 11% of baseline firing in the RDL1675 strain (p = 0.035). This finding indicated that the resistance was expressed within the nervous system. Patch clamp recordings on D. melanogaster central neurons mirrored extracellular recordings where nootkatone inhibited GABA-stimulated currents by 44 ± 9% at 100 μM, whereas chloride current was inhibited 4.5-fold less at 100 μM in RDL1675. Taken together, these data suggest nootkatone toxicity in D. melanogaster is mediated through GABA receptor antagonism.

Petuniolides, Natural Insecticides fromPetunia parodii, Are Antagonists of GABAaReceptors

Petuniolide C, an ergostane-type steroidal insecticide isolated fromPetunia parodii,caused immediate uncoordinated activity and salivation in fourth instar nymphs of the desert locust,Schistocerca gregaria,when injected into the hemocoel at doses as low as 0.5 μg insect−1. The LD50for petuniolide C via injection in locust nymphs (14.1 μg insect−1) is nearly identical to that for the convulsant, picrotoxinin (14.0 μg insect−1), a known inhibitor of the GABA (γ-aminobutyric acid)-mediated chloride ionophore. Petuniolide C and picrotoxinin in combination had an additive effect on locust mortality. Following topical administration, petuniolide C caused knockdown and mortality in a cyclodiene-susceptible strain of the German cockroach,Blatella germanica,but significantly reduced effects in a cyclodiene-resistant strain. Using anin vitrorat brain synaptic membrane preparation, both petuniolide C and a natural analog, petuniolide D, were shown to be weak ligands of the GABAareceptor.

DNA sequence and site of mutation of the GABA receptor of cyclodiene-resistant red flour beetle, Tribolium castaneum

Using polymerase chain reactions (PCR) on cDNA, the DNA sequence of a membrane spanning region of a GABA receptor of the red flour beetle, Tribolium castaneum was identified. The deduced amino acid sequence indicates that its basic structure is similar to the GABA receptor of Rdl type subunits of Drosophila melanogaster and of Blattella germanica. Particularly conserved are M 1, M 2 and M 3 segments. Within this 146 amino acid stretch, the GABA receptor from the red flour beetle differed from corresponding ones from Drosophila and Rdl subunit of B. germanica by 12 and eight amino acids, respectively. By using an identical approach, the corresponding DNA region was sequenced from the cDNA of a cyclodiene-resistant strain of T. castaneum. While two points of mutation were found only one mutation in DNA was found to result in an amino acid shift. The site of mutation was at the 5th amino acid of the M 2 cylinder where G to T conversion of the GCT codon resulted in a conversion of alanine to serine. This is qualitatively the same mutational switch of alanine to serine in resistant strains previously reported to have occurred in cyclodiene-resistant Drosophila melanogaster, Aedes aegypti and Blattella germanica, indicating that this amino acid change is the likely cause for evolution of the nerve insensitive type of resistance to cyclodiene insecticides.

Binding and Physiology of 4′-Ethynyl-4- n-propylbicycloorthobenzoate (EBOB) in Cyclodiene-Resistant Drosophila

4′-Ethynyl-4- n-[2,3- 3H 2]propylbicycloorthobenzoate ([ 3H]EBOB) is a novel radioligand for the convulsant binding site of vertebrate and invertebrate γ-aminobutyric acid (GABA) receptors. Previous studies in membranes from house fly heads have shown [ 3H]EBOB to have high affinity for the cyclodiene binding site, which was reduced fourfold in cyclodiene-resistant strains. Following our recent identification of single amino acid replacements in the Drosophila GABA receptor gene Rdl conferring resistance to cyclodienes, we were interested in correlating [ 3H]EBOB binding and physiology with specific replacements of alanine302 in Rdl. Here we report that [ 3H]EBOB binding is not detectable in resistant strains carrying either the resistance-associated alanine302> serine ( Drosophila melanogaster or D. simulans) or the alanine302> glycine ( D. simulans) replacement. Thus, despite high specific binding to membranes from susceptible flies, no binding higher than nonspecific was observed in resistant preparations. EBOB (100 n M) was also shown to functionally block GABA-gated chloride ion currents generated in insect cells infected with a recombinant susceptible Rdl baculovirus, while cells expressing constructs containing the alanine302> serine replacement showed 10-fold insensitivity to block. These results indicate that the presence of alanine302 is central to the binding of [ 3H]EBOB to GABA receptors containing Rdl subunits and confirm the usefulness of this radioligand for the study of this important binding site.

Identification of the site of mutation within the M2 region of the GABA receptor of the cyclodiene-resistant German cockroach

The DNA and amino acid sequences of the membrane-spanning region of the GABA receptors of the German cockroach have been identified along with information on the nature and the specific site of mutation in the cyclodiene-resistant strains. In this resistant strain, the mutation has occurred at the most conserved, lower M2 cylinder region involving a G to T conversion, resulting in an amino acid change of alanine (GCC) residue to serine (TCC). The site, furthermore, coincides with the most conserved region of all GABA receptor subunits and the expected Cl- transporting segment constituting the innermost surface of the channel opening. The deduced sequence of the German cockroach GABA receptor differs from that of Drosophila mainly in the connecting region between M3 and M4.

Identification of the site of mutation within the M2 region of the GABA receptor of the cyclodiene-resistant German cockroach

The DNA and amino acid sequences of the membrane-spanning region of the GABA receptors of the German cockroach have been identified along with information on the nature and the specific site of mutation in the cyclodiene-resistant strains. In this resistant strain, the mutation has occurred at the most conserved, lower M2 cylinder region involving a G to T conversion, resulting in an amino acid change of alanine (GCC) residue to serine (TCC). The site, furthermore, coincides with the most conserved region of all GABA receptor subunits and the expected Cl− transporting segment constituting the innermost surface of the channel opening. The deduced sequence of the German cockroach GABA receptor differs from that of Drosophila mainly in the connecting region between M3 and M4.

Reduced neuronal sensitivity to dieldrin and picrotoxinin in a cyclodiene-resistant strain of Drosophila melanogaster (Meigen).

Toxicological and neurophysiological studies were performed to characterize the resistance mechanism in a cyclodiene-resistant strain of Drosophila melanogaster (Maryland strain). Dieldrin had an LC50 of 0.058 ppm against the larvae of susceptible D. melanogaster (Oregon-R wild type) when formulated in the rearing media. The LC50 of the resistant Maryland strain was 10.8 ppm, giving a resistance ratio (LC50-Maryland/LC50-susceptible) of 186-fold. Suction electrode recordings were made from peripheral nerves of the larval central nervous system to test whether reduced nerve sensitivity played any role in the observed resistance. In susceptible preparations (n = 5), inhibition of nerve firing by 1 mM gamma-aminobutyric acid (GABA) was effectively antagonized within 3-10 min by 10 microM dieldrin. In contrast, 30 min incubations with 10 microM dieldrin had no effect on preparations from cyclodiene-resistant individuals (n = 5). Similarly, 10 microM picrotoxinin blocked GABA-dependent inhibition in susceptible nerve preparations (n = 3). In recordings from resistant insects (n = 4), picrotoxinin displayed either weak antagonism of GABA or hyperexcitation indistinguishable from susceptible preparations. These results demonstrate that cyclodiene resistance in the Maryland strain of D. melanogaster 1) is expressed in immature stages, 2) is present at the level of the nerve, and 3) extends to picrotoxinin, albeit at a reduced level compared with dieldrin. The possible role of an altered GABA receptor in this resistance is discussed.

House fly brain γ-aminobutyric acid-gated chloride channel: target for multiple classes of insecticides

The γ-aminobutyric acid receptor/chloride ionophore is the proposed target in insects for a wide range of commercial and candidate insecticides, a hypothesis that can be tested directly by developing a toxicologically relevant radioligand binding assay. This was achieved with house fly brain and 1-(4-ethynylphenyl)-4-[2,3- 3H 2]propyl-2,6,7-trioxabicyclo[2.2.2]octane ([ 3H]EBOB) for which the binding site was demonstrated to be identical to or overlapping with that of seven classes of structurally diverse insecticides, i.e., trioxabicyclooctanes, dithianes, silatranes, lindane, toxaphene, chlorinated cyclodienes, and picrotoxinin. The affinity of the binding site for this radioligand is reduced fourfold in a cyclodiene-resistant strain. There is no cross resistance to avermectin B 1a and moxidectin which are potent noncompetitive inhibitors of [ 3H]EBOB binding.

Mode of Action of Insecticidal Compounds Acting at Inhibitory Synapse

All cyclodiene-resistant strains of the German cockroach, the house fly and the mosquito are also resistant to picrotoxinin, a well-known GABA antagonist. By using electrophysiological techniques, picrotoxinin was confirmed to act at a presynaptic region, resulting in a stimulation of the release of an excitatory transmitter. The electrophysiological symptoms caused by picrotoxinin were quite similar to those of γ-BHC and dieldrin. It was determined by using a 3H-dihydropicrotoxinin binding method that the nerve membrane in CNS of the American cockroach contained a component having a high affinity toward picrotoxinin and heptachlor epoxide, which can be regarded as a picrotoxinin receptor. By using a sucrose density centrifugation technique, it was determined that the fraction sedimented at the interphase of 1.0 and 1.2M sucrose at 100, 000×g contained the highest level of specific binding site. The 3H-dihydropicrotoxinin binding on the receptor was inhibited by all cyclodienes tested and γ-BHC. Toxaphene, bicyclic phosphate and TETS also bound with the receptor. Dieldrin resistant German cockroach showed cross-resistance to the chemicals having binding potencies to picrotoxinin receptor. Milbemycin and avermectin were found to stimulate Cl- uptake by the leg muscles of the American cockroach within 4min at 10-7M. This stimulatory action could be antagonized by picrotoxinin (10-4M). It was concluded that the action of milbemycin and avermectin is to open the chloride channel on the plasma membrane. Their actions do not seem to be mediated through GABA receptor, picrotoxinin receptor and benzodiazepam receptor, suggesting their direct attack on the chloride channel proper both in the central nervous and the neuromuscular systems.

Difference in the picrotoxinin receptor between the cyclodiene-resistant and susceptible strains of the german cockroach

As a result of toxicity tests, it was established that all cyclodiene-resistant strains of the German cockroach are also resistant to picrotoxinin, a plant-origin neurotoxicant. Two of the cockroach strains which exhibit a distinct cross-resistance pattern to picrotoxinin (i.e., LPP and FRP) are the ones that have been purified genetically by backcrossing against the susceptible (CSMA) strain. This cross-resistance pattern appears to be specific to picrotoxinin and does not extend to other neuroexcitants such as bicuculline, beta-bungarotoxin, and DDT. The nervous system of the resistant cockroach was found to be less sensitive to picrotoxinin. Furthermore, it was determined that nerve components from the resistant cockroaches have significantly lower binding capacity to [ 3H]α-dihydropicrotoxinin. The most likely explanation for the above phenomenon is that these cockroaches have developed the cyclodiene resistance by altering the nerve receptor for picrotoxinin.

Enzyme induction in the housefly: The specificity of the cyclodiene insecticides

Eleven cyclodiene compounds, including the insecticides aldrin, dieldrin, heptachlor, and endrin, were compared as inducers of microsomal oxidases in a cyclodieneresistant strain of the housefly. All of the compounds except dihydroisodrin caused increases in enzyme activity. The epoxide members of the group appeared to be more effective inducers than their olefin precursors. Isodrin and endrin were effective at approximately 1 300 th the dose of the other compounds tested although neither compound enhanced enzyme activity as much as the others in the group. The maximum increase in enzyme activity was approximately 100% for aldrin and about 30% for isodrin and endrin. There was no additive effect when each of the compounds was combined with dieldrin in the induction treatments. In addition to microsomal epoxidation and hydroxylation, O-demethylation was enhanced by the cyclodiene treatments. Furthermore, DDT dehydrochlorinase, a nonmicrosomal enzyme, was increased as much as 137% by the treatments. The results indicate that within this group of compounds there are some structural requirements for activity but that the overall effect on enzyme activity is rather nonspecific.

Genetics of cyclodiene-insecticide resistance in the seed-corn maggot.

Base-line toxicity data obtained on a susceptible and homozygous cyclodiene-resistant strain of Hylemya Platura (Meigen) indicated that the pattern of resistance was similar to other species of root maggots in that there was a high degree of resistance to aldrin (727X) but no crossresistance to either diazinon (1.1X) or DDT (0.8X). With the exception of the F1 generation, results obtained in various crosses and backcrosses indicated that cyclodiene-resistance was monofactorial. However, the F1 population was not homogeneous; approximately 25% of the population was more resistant than predicted. It is suggested that the inheritance of cyclodiene-resistance in H. platura is due primarily to a single gene which is incompletely dominant, but that a modifying gene may be present at a very low level in either or both of the strains, which fact would make its presence known only in the hybrid individual.

Contact Toxicity of 13 Insecticides to Cyclodiene-Susceptible and Cyclodiene-Resistant Strains of the Onion Maggot1

Spray-tower applications of heptachlor, aldrin, dieldrin, endrin, and lindane to both cyclodiene-susceptible and cyclodiene-resistant strains of onion maggot, Hylemya antiqua (Meigen), adults showed resistance levels of ×647.1, ×591.2, ×356.6, ×27.2, and ×14.3, respectively. There was no cross-resistance to DDT or diazinon. Similar applications with 6 other organophosphate insecticides showed no significant difference in susceptibility between the 2 strains. When compared at the LD50, naled was the most toxic material followed by diazinon, parathion, carbophenothion, malathion, Nemacide® (O- (2–4-dichlorophenyl) O,O-diethyl phosphorothioate), and ethion. Diazinon was equitoxic to aldrin when tested against the susceptible strain. The slope of the regression line, LD50, and fiducial limits of the LD50 for 13 insecticides on both strains are presented.