Progress 08/01/03 to 07/31/07
Outputs
The project was initiated to study the nechanism(s) by which Colorado potato beetles (Leptinotarsa decemlineata) have become resistant in the field to neonicotinoid insecticides, and particularly the major insecticide used in their control, imidacloprid. An initial study revealed that resistance levels to topically-applied imidacloprid in adult beetles were over 200-fold. Cross-resistance to all other commercial neonicotinoids was also present though generally at lower levels than imidacloprid. Resistance to nicotine itself was only 2-3x. Extensive pharmacokinetic studies to compare the uptake, metabolism and excretion of [14C]-imidacloprid in the susceptible (S) and resistant(R)insects revealed only minor differences that could not account for resistance. metabolism of imidacloprid was very slow and most of the clearance from the body was through excretion of the parent compound. all of the metabolites were less toxic than imidacloprid. The application of imidacloprid
and other neonicotinoids to the isolated central nervous system with recording from a motor nerve bundle leaving the thoracic ganglion showed a biphasic effect with strong stimulation of neural activity at lower (nM) concentrations but blockage of neurotranmission at higher (uM) concentrations. The S and R insects showed no differences in the excitatory phase but the R insects were about 50-fold less sensitive to the blocking effects of imidacloprid. This difference was not seen with nicotine, to which the R insects are not resistant. The binding of [3H]-imidacloprid to high affinity sites in membranes prepared from beetle heads was assessed. One saturable high affinity site was detected in the S insects (Kd = nM)though the amount of specific binding was quite low (Bmax = ). The results with the R insects were virtually identical indicating that any change in responsiveness of the nervous system was not related to imidacloprid binding to high affinity nicotinic receptors. However,
studies with cockroaches have shown that two types of nicotinic receptors exist, desensitizing and non-desensitizing, and only the desensitizing types are likely to be seen in ligand binding studies usual typical methodology. Non-desensitizing receptors are probably also present in CPBs since the isolated nervous system responds typically to spinosyn A, a compound that is specific for non-desensitizing types in cockroaches. Considerable attempts to develop method to record from isolated CPB neurons to assess the responses of both types of receptors were not successful. Although some records using a single electrode voltage clamp were obtained, the cells were small and fragile and rapidly lost excitability. During this phase of project a parallel study was conducted using isolated neurons from P. americana. These were much more amenable to study
Impacts
Imidacloprid is the primary insecticide used for potato insect control in the US. Resistance to imidacloprid in the Colorado potato beetle continues to increase in the Eastern USA and is now being accompanied by moderate insensitivity to thiamethoxam, another member of the neonicotinoid insecticide group. These compounds act primarily on nicotinic acetylcholine receptors in the central nervous system. Evidence suggests that multiple nicotinic receptor types may be present in insects. We are trying to identify the specific changes within this array of receptors that lead to resistance. This information will assist in detecting and managing resistance as it develops further, and it should provide additional information about the existence and functions of different acetylcholine receptor subtypes in the insect central nervous system.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
In previous work it was discovered that resistance to the neonicotinoid insecticide imidacloprid (IMI) in the Colorado potato beetle (CPB), Leptinotarsa decemlineata, is not due to pharmacokinetic factors but is probably related to site of action changes in the target, nicotinic acetylcholine receptors (nAChRs). However the high affinity binding of labeled imidacloprid to head membranes did not differ between resistant and susceptible insects. Using isolated whole nerve cords it was found that nicotinic agonists have a biphasic effect, initially strongly stimulating axonal firing but at higher doses, blocking neurotransmission. The susceptible and resistant insects did not differ in their sensitivities to the neuroexcitory action but there was a substantial difference in the concentration needed to block neural activity with the resistant insects requiring a 50 to 60-fold higher concentration. The reason for this and its relationship to resistance is unknown. Attempts
to investigate this effect further using voltage clamp recordings from isolated CPB neurons were largely unsuccessful due to their small size and fragility. A second approach involving cloning of the nAChR receptor subunits has been initiated to determine the structural and functional basis for the difference in sensitivity to IMI's neuroblocking action. In the course of methods development for the isolated neuron work, a parallel study was conducted using the larger and more robust neurons from the thoracic ganglia of the cockroach, Periplaneta americana. All seven commercial neonicotinoids and nicotine were applied and all but one caused an inward current followed rapidly by receptor desensitization. The exception was thiamethoxam which neither activated nicotinic receptors nor blocked the effect of other agonists. It appears to act as a propesticide in these cockroaches. The remaining compounds fell into two classes. Compounds with heterocyclic rings as the electronegative moiety
(imidacloprid, thiacloprid and nicotine) were weak partial agonists (efficacy 20-25% of that of acetylcholine) whereas compounds with open chain electronegative group (acetamiprid, nitenpyram, clothiandin and dinotefuran) had efficacies approaching or equal to 100% of that shown by ACh. These two groups also elicited different symptoms in vivo after injection with the former group highly excitatory but the latter group mainly depressive and paralytic. Lethality correlated better with agonist efficacy than with the affinity of these compounds for the receptors. Efficacy and affinity were not correlated. It has been suggested that the toxicity of dinotefuran results from its interaction with a group of nAChRs distinct from those affected by IMI. In competition experiments conducted here between pairs of neonicotinoids applied jointly, no evidence was seen that these compounds interact independently on multiple nAChR subtypes. Such specific interactions might involve nAChRs located
outside the thoracic ganglia or a group of receptors that is small compared to the total pool of receptors and thus hard to detect by this method.
Impacts
Imidacloprid is the primary insecticide used for potato insect control in the US. Resistance to imidacloprid in the Colorado potato beetle continues to increase in the Eastern USA and is now being accompanied by moderate insensitivity to thiamethoxam, another member of the neonicotinoid insecticide group. These compounds act primarily on nicotinic acetylcholine receptors in the central nervous system. Evidence suggests that multiple nicotinic receptor types may be present in insects. We are trying to identify the specific changes within this array of receptors that lead to resistance. This information will assist in detecting and managing resistance as it develops further, and it should provide additional information about the existence and functions of different acetylcholine receptor subtypes in the insect central nervous system.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
The mechanism of resistance has been investigated in a strain of Colorado potato beetle (CPB; Leptinotarsa decemlineata) that is highly resistant to the neonicotinoid insecticide, imidacloprid. No significant differences were found in the pharmacokinetics (uptake, metabolism and excretion) between the resistant strain and two susceptible ones. A difference was observed in the response of their isolated CNS to the blocking (but not excitatory) action of imidacloprid with the resistant strain about 50-fold less sensitive. However, no differences between the strains were found in the binding of tritiated imidacloprid to high affinity sites in nervous system membranes. It was postulated that a subset of nicotinic acetylcholine receptors that do not desensitize, and thus have lower affinities for imidacloprid, may exist in CPB, as described for the cockroach, and that these may differ in responsiveness to imidacloprid in the different strains. Studies with isolated cell
bodies from the CPB central nervous system were initiated to examine the existence of such non-desensitizing receptors and whether they differ between the R and S strains. However, results with this approach have so far been limited because of the small size of these cells and their short viability under recording conditions compared to those from Periplaneta americana that were used for method development. Efforts to improve this preparation continue using both single electrode clamp and, recently, patch clamp methods in collaboration with Dr. James Galligan (MSU) and Dr Vincent Salgado (BASF Corp.) In the process of developing this preparation, considerable additional information has been gained on the cholinergic actions of neonicotinoid insecticides in insects using single central nerve cell recordings from P. americana. The actions of seven commercial neonicotinoids with varied structures were compared on these cells. Compared to acetylcholine, imidacloprid has a higher affinity
but a much lower efficacy in opening ion channels. On the other hand, clothianidin als has high affinity but is a full agonist. In general, open chain analogs such as clothianidin and dinotefuran show better efficacy as agonists than closed chain analogs such as imidacloprid and thiachloprid. Toxicity does not appear to be closely related to receptor efficacy in this preparation. Further, we have obtained evidence that these compounds vary in the ion-specificity of the nicotinic channels that they open which provides additional confirmation that multiple nicotinic receptor types are present. Future work will include additional efforts to develop stable recordings from CPB single neurons particularly using patch clamp methodologies and the initiation of cloning and comparative sequence studies of nAChR subunits from the R and S strains of CPB.
Impacts
Imidacloprid is the primary insecticide used for potato insect control in the US. Resistance to imidacloprid in the Colorado potato beetle continues to increase in the Eastern USA and is now being accompanied by moderate insensitivity to thiamethoxam, another member of the neonicotinoid insecticide group. These compounds act primarily on nicotinic acetylcholine receptors in the central nervous system. Evidence suggests that multiple nicotinic receptor types may be present in insects. We are trying to identify the specific changes within this array of receptors that lead to resistance. This information will assist in detecting and managing resistance as it develops further, and it should provide additional information about the existence and functions of different acetylcholine receptor subtypes in the insect central nervous system.
Publications
- Mota-Sanchez, D., R.M. Hollingworth, E.J. Grafius and D.D. Moyer (2006) Resistance and cross-resistance to neonicotinoid insecticides and spinosad in the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Pest Management Science 62: 30-37.
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Progress 01/01/04 to 12/31/04
Outputs
Studies on the cross-resistance of imidacloprid-resistant Colorado potato beetles (Leptinotarsa decemlineata Say; CPB) to other neonicotinoid insecticides, nicotine, and the nicotinic activator, spinosad, were completed. After topical application to adult insects, lower but significant (10 to 50-fold) levels of resistance to other commercial neonicotinoids were seen compared to about 200-fold for imidacloprid. Interestingly, resistance to nicotine was insignificant but resistance to spinosad was about 10-fold. The mechanism of resistance to imidacloprid and related neonicotinoids was investigated. These compounds are believed to act as agonists on nicotinic acetylcholine receptors (nAChRs) in the insect central nervous system (CNS). Studies of the binding of tritiated imidacloprid to putative nAChRs in membranes prepared from the head and thorax of resistant (R) and susceptible (S) insects revealed no differences between the strains, and electrophysiological
recordings from the whole isolated central nervous system showed no difference between the strains in the concentration of imidacloprid needed to elicit increased neural activity. However, there was a significant difference in the concentration needed to block neurotransmission, with the resistant insects being about 50-fold less sensitive to this action of imidacloprid. The R strain is about 200-fold resistant to imidacloprid but shows little resistance to nicotine itself. No differences were seen between the R and S strains in either the stimulatory or blocking potencies of nicotine on the isolated CNS. Based on published studies with cockroaches, these data can be explained on the basis of the existence of two nAChR receptor subtypes, one having a high (nM) affinity for imidacloprid and the other having a lower (uM) affinity. Only the high affinity receptors are detected in ligand binding assays, and on the basis of the binding studies and the initial stimulatory effect on the CNS,
these nAChRs do not differ between the R and S strains. The lower affinity receptors are postulated to be responsible for the block of neuroactivity in the CNS. Here there is a significant difference in sensitivity between the R and S strains and this action is believed to be closely related to the paralytic state that precedes death in these insects. Further studies are proposed with isolated neurons from the CNS to determine whether the CPB has two distinct types of nAChRs and whether the lower affinity sites for imidacloprid are altered in the R strain. We would like to acknowledge the valuable collaboration of Drs. V. Salgado and R. Nauen (Bayer CropScience) in some of these studies.
Impacts
Resistance to the insecticide imidacloprid in the Colorado potato beetle is an increasing problem for potato growers in the Eastern USA. The discovery of broad cross-resistance to other neonicotinoid insecticides in a field-collected imidacloprid-resistant strain of the beetle suggests that it may not be an effective long-term strategy to switch to these analogs as resistance to imidacloprid spreads. Cross-resistance to a related insecticides, spinosad, was also observed. This may limit options for resistance management. Recent results that reveal a change in sensitivity of a subgroup of acetylcholine receptors in the resistant insects may explain the resistance to imidacloprid and also raise interesting questions about the existence and functions of different acetylcholine receptor subtypes in the insect central nervous system.
Publications
- No publications reported this period
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Progress 08/01/03 to 12/31/03
Outputs
The cross-resistance spectrum to neonicotinoid insecticides was determined in an imidacloprid-resistant strain of Colorado potato beetle (Leptinotarsa decemlineata) collected in Long Island, NY. Topical application to adults revealed a resistance ratio (LD50 resistant/LD50 susceptible) of 310 for imidacloprid. Six other structurally-related neonicotinoids (acetamiprid, clothiandin, dinotefuran, nitenpyram, thiacloprid, thiamethoxam) had resistance ratio ranging from 10 (nitempyram) to 59 (dinotefuran). The ratio for N-methylimidacloprid was 28. N-demethylthiamethoxam was too weakly toxic to the resistant strain to provide a resistance ratio. The pattern of cross-resistance indicates that at least one basic mechanisms of resistance is common to all neoniocotinoids, but does not reveal any basic difference between secondary and tertiary (N-methyl) amine-type neonicotinoids, as has been suggested with aphids. Preliminary studies of the binding of tritated imidacloprid to
putatitive nicotinic acetylcholine receptors from the brains of resistant and susceptible insects revealed no differences in the binding characteristics of imidacloprid or in its displacement by competition with other neonicotinoids.
Impacts
Resistance to the insecticide imidacloprid in the Colorado potato beetle is an increasing problem for potato growers in the Eastern USA. The discovery of broad cross-resistance to other neonicotinoid insecticides in a field-collected imidacloprid-resistant strain of the beetle suggests that it may be difficult to switch to these analogs as resistance to imidacloprid spreads. This may limit options for resistance management.
Publications
- No publications reported this period
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