Knockdown resistance to dichlorodiphenyl-trichloroethane and pyrethroid insecticides in the napts mutant of Drosophila melanogaster is correlated with reduced neuronal sensitivity.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 10:293-302 (1989) Knockdown Resistance to Dichlorodiphenyltrichloroethane and Pyrethroid Insecticides in the napfsMutant of Drosophila melanogaster I s Correlated With Reduced Neuronal Sensitivity Jeffrey R. Bloomquist, David M. Soderlund, and Douglas C. Knipple Department of Entomology, New York State Agricultural Experiment Station, Cornell University, Geneva Resistance to pyrethroid insecticides and dichlorodiphenyltrichloroethane (DDT) was investigated in t h e nap” ( n o action potential, temperature sensitive) mutant of Drosophila rnelanogaster. In surface contact bioassays, t h e napfs strain s h o w e d threefold resistance t o deltamethrin a t t h e LC50 level w h e n c o m p a r e d to susceptible Canton-S flies. Cross-resistance was also observed t o DDT a n d t h e pyrethroids NRDC 157 [3-phenoxybenzyl [lR,cis]3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate], fenfluthrin, and MTI -800 [I-(3-phenoxy-4-fluorophenyl)-4-(4-ethoxyphenyl)-4-methylpentane] . The o n s e t of intoxication by pyrethroids in nap’ flies was markedly delayed, a finding that is consistent with t h e existence of a resistance mechanism involving reduced neuronal sensitivity. Resistance at t h e level of t h e nerve was confirmed by electrophysiological recordings of spontaneous and evoked activity in t h e dorsolongitudinal flight muscles of poisoned flies. Preparations from nap” insects treated with fenfluthrin displayed longer latencies to t h e appeara n c e of spontaneous activity a n d also a n absence o r reduction in burst discharges compared to equivalent preparations from susceptible individuals. These results are discussed in light of competing hypotheses concerning t h e mechanism underlying knockdown resistance and reduced nerve sensitivity in insects. Key words: deltamethrin, fenfluthrin, sodium channel, electrophysiology Acknowledgments: These studies were supported in part by CSRS Regional Research Project NE-115. We thank Katherine Vega, an Independent Study participant from William Smith College (Geneva, NY) for her able technical assistance. Received November 16,1988; accepted April 6,1989. Address reprint requests to David M. Soderlund, Department of Entomology, NYS Agricultural Experiment Station, Cornell University, Geneva, NY 14456. Jeffrey R. Bloomquist’s present address is Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. 0 1989 Alan R. Liss, Inc. 294 Bloomquist et al. INTRODUCTION Drosophila rnelanogaster possesses several characteristics that should make it an ideal insect species for studying insecticide resistance. Among these advantages are: the ability to generate a variety of resistant strains by chemical mutagenesis; the ability to transform strains using P-element vectors; and the ability to clone virtually any genetic locus using the well-developed molecular genetic techniques afforded by this system [l]. Despite these advantages, D. rnelanoguster remains an underutilized resource for exploring insecticide resistance problems. The rich variety of defined genetic mutations induced in D. rnelanogaster by chemical mutagenesis includes neurological mutants such as napts (no action potential, temperature sensitive) . Adult flies with the napts phenotype display reversible temperature-sensitiveparalysis at 38°C that is correlated with a reversible, temperature-sensitive blockage of nerve conduction . Embryonic napfSneurons in culture are resistant to the cytotoxic action of the sodium channel activator veratridine . In addition, resistance to veratridine and hypersensitivity to tetrodotoxin, a specific sodium channel blocker, have been observed in feeding experiments with adult flies . In napfSnerve membranes, the density of binding sites for [3H]saxitoxin(a radioligand that specifically labels sodium channels) is lower than in membranes prepared from wildtype flies [5,6]. This reduction in the number of [3H]saxitoxinbinding sites is persuasive evidence for a reduction in the density of voltage-sensitive sodium channels in napts nerve membranes, which has been claimed to be a sufficient mechanism to explain both temperature-sensitive paralysis [71 and altered neurotoxin sensitivity  in naptsflies. A reduction in the number of DDT* and pyrethroid binding sites has been proposed as a mechanism of reduced neuronal sensitivity underlying the kdr resistance phenotype in houseflies .Since the voltage-sensitive sodium channel is the principal site of action of these compounds , the naptSstrain of D. rnelanogaster appears to be an appropriate model for assessing the role of reduced target site density in resistance in vivo and in reduced neuronal sensitivity in physiological assays. Kasbekar and Hall [lo] recently reported that the napts strain is resistant to pyrethroids and that the resistance to fenvalerate in this strain was genetically inseparable from the naptSlocus. In this paper, we report a modest level of cross-resistance to the lethal effects of DDT and a limited group of structurally diverse pyrethroids in this strain. We also document substantial resistance to the rapid paralytic ("knockdown") effects of some pyrethroids that is correlated with reduced sensitivity of the nervous system. MATERIALS AND METHODS Chemicals Deltamethrin and its noncyano analog, NRDC 157*, were provided by J. Martel, Roussel-Uclaf, Romainville, France. DDT was purchased from Chem dichlorodiphenyltrichloroethane; MTI-800 = 1-(3-phenoxy-4= 3-phenoxybenzyl [IR,cisl -3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate. *Abbreviations used: DDT = fluorophenyl)-4-(4-ethoxyphenyl)-4-methylpentane; N R D C 157 Knockdown Resistance in D. melanogaster 295 Service (West Chester, PA). MTI-800 was a gift from N. Janes, Rothamsted Experimental Station (Harpenden, England) and fenfluthrin was supplied by J. Scott, Cornell University (Ithaca, NY). Insects Colonies of D. melunoguster were reared in continuous cultures at 23°C using established procedures. Wildtype (Canton-S)flies obtained from R. MacIntyre, Cornell University, served as the reference susceptible strain. Flies of the nap" strain were the kind gift of S. Benzer, California Institute of Technology (Pasadena, CA). We confirmed that holding these flies at 38°C caused paralysis in both sexes, which was reversed by lowering the temperature. Bioassays Adult females of both strains were exposed to insecticides in a surface contact bioassay used previously to document resistance to DDT in field collected strains of D. melunogusfer [ll]. Solutions of insecticide in acetone (0.5 ml) were applied to the walls and bottom of 50 ml Erlenmeyer flasks and the solvent evaporated to give a uniform residue. Control flasks received identical treatment with solvent only. Twenty adult female flies were introduced into each flask, and the flasks were stoppered with cotton kept moist with sucrose solution. Initial toxicity measurements with deltamethrin were performed after ether anesthesia. Similar results were also obtained with COzanesthesia, which was then used in all subsequent toxicity determinations. Paralysis was assessed at 60 min intervals for the first 4 h of exposure, at 8 h after exposure, and at 24 h after exposure. Those individuals unable to maintain posture and walk normally were considered paralyzed. Flies paralyzed at 24 h did not subsequently recover and were considered dead. Results are presented as the pooled percent paralysis or mortality from at least two replicate experiments in which the responses of the two strains were compared under identical conditions and were corrected for paralysis or mortality in controls (S5% in all experiments). Computerized probit analysis of mortality data for deltamethrin was performed using the POLO program . Electrophysiology Methods for measuring activity in the fibrillar flight muscles of D. melunoguster were essentially those of Tanouye and Wyman . Briefly, adult females were anesthetized by chilling and fixed in soft wax. Sharpened steel stimulating electrodes were placed in the cervical region of the head and thorax near the central nervous system. Stimuli were delivered as rectangular pulses 0.1-0.3 ms in duration at a rate of 0.3 Hz. Responses in single dorsolongitudinal muscle fibers were recorded with a micropipette inserted through the cuticle into the dorsal thoracic insertions of the muscles. Micropipettes were filled with a saline containing (mM) NaCl(130), KC1(5), CaCI2(1.8),MgC1' (4), and HEPES (5), pH 7.2. Flies were treated on the abdomen with 8 ng of fenfluthrin dissolved in 0.25 pl of acetone. Disruption of normal motor nerve function was measured as the latency to both the appearance of evoked burst discharges and the onset of spontaneous activity. 296 Bloornquist et al. TABLE 1. Toxicity of Four Pyrethroids and DTT to Canton-S and napts Flies Dosage, Fglflask Compound Deltamethrin 24 h mortality, % Canton S napfs 0.05 0.15 5.0 50.0 15.0 50.0 0.05 0.5 5.0 50.0 NRDC 157 MTI-800 Fenfluthrin DDT 0 35 3 58 63 93 5 100 12 30 25 72 45 83 87 100 60 100 58 85 RESULTS The napts strain displayed modest levels of cross resistance to the lethal effects at 24 h of all the compounds tested in this study when compared to the susceptible Canton-S strain (Table 1).These studies revealed little response in naptS flies at concentrations of deltamethrin, NRDC 157, fenfluthrin, and DDT that caused appreciable toxicity in the susceptible strain. However, differences between strains in their responses to MTI-800 were small at the concentrations tested. Probit analysis of a more extensive set of toxicity data for deltamethrin (Table 2) showed that responses of these strains were statistically different and that the napts strain exhibited threefold resistance to this compound at the LC5,, level. However, the dosage-response curves for the naptS and Canton-S strains were not parallel, so that differences in their responses to deltamethrin were most pronounced at low levels of toxicity but were not evident at mortalities above 90%. In view of the low levels of resistance to other compounds found in our screening assays (Table l),efforts to determine LC50values and resistance ratios for these compounds were not pursued. More dramatic differences between strains were noted in the time course of intoxication. Deltamethrin at 0.15 &flask (Fig. 1)produced extensive knockdown of Canton-S flies during the second hour of exposure. In contrast, paralysis of napts flies were barely detectable during the first 4 h of exposure at the same dosage, although at 24 h this treatment produced approximately 50% mortality (Table 2). Fenfluthrin at 0.1 pg/flask (Fig. 2) also caused a timedependent increase in knockdown that approached 80% in the susceptible Canton S strain after 3 h of exposure, but naptsflies were virtually unaffected during the first 2 h of exposure to fenfluthrin and only 15% knockdown was observed after 4 h. A similar pattern of knockdown resistance was observed TABLE 2. Probit Analysis of the Lethal Effects of Deltamethrin on Adult Female Canton-S and n a d s Flies* Strain Canton-S naps‘” n LC,,,pg/flask 95% conf. limits Slope 300 299 0.06 0.19 0.02-0.13 0.16-0.23 1.73 4.18 “Thedosage-response curves were found to be nonidentical and nonparallel (likelihoodratio tests, P < .05 ). Knockdown Resistance in D. melanogaster 0 1 2 4 3 297 5 Time, hr Fig. 1. Time course of deltamethrin-dependent paralysis of Canton-S and napt' flies. 100 m Canton-S 80 s -6 s E 60 u) p" 40 Fenfluthrln 0.1 pglflask 20 0 0 1 2 3 4 5 Time, h Fig. 2. Time course of fenfluthrin-dependent paralysis of Canton-S and nap" flies. with MTI-800 at 15 pg/flask (Fig. 3), a dosage that produced little difference between strains in assays of lethality at 24 h (Table 1).Knockdown by MTI-800 was approximately linear for the first 4 h of exposure to Canton-S flies, producing about 80% knockdown. The napfSstrain, however, displayed virtually no sensitivity to the knockdown action of MTI-800 when tested under identical conditions. Similar results were also observed with NRDC 157 (data not shown). However, the slow action of DDT resulted in a complete absence of knockdown in either strain during the first 8 h of exposure. Electrophysiological studies showed that the resistance to the knockdown and lethal effects of pyrethroids displayed by the napfsstrain was correlated with reduced sensitivity of the nervous system. Untreated preparations from both napfs and Canton-S flies displayed single muscle action potentials in 298 Bloomquist et al. 100 = Canton-S I MTI 800 15 pg/flask -n-----o- 0 1 2 3 4 5 Time, h Fig. 3. Time course of MTI 800-dependent paralysis of Canton4 and nap" flies response to stimulus pulses (Fig. 4A). We also observed varying amounts of spontaneous firing activity in these preparations immediately after recordings were initiated, but this activity declined with time and was absent at the time of treatment with insecticide solution. After treatment with fenfluthrin, preparations were monitored for the appearance of spontaneous activity and changes in the evoked responses. In Canton-S flies (n=5), evoked burst discharges like those shown in Figure 4B were observed after a variable latency. Four of the preparations displayed consistent bursting 5,5.5,6, and 22 min after treatment, while another preparation showed only double-spike responses after 32 min of poisoning. However, the appearance of spontaneous trains of action potentials in Canton-S preparations was more consistent, appearing 4 2 1min B CFig. 4. Responses in the dorsolongitudinal flight muscles to stimulation of the central nervous system in Canton-S and nap" flies. A: Single-muscle action potential typical of those normally evoked by a single stimulus in Canton-S and nap" preparations before fenfluthrin treatment. B: Typical burst discharges elicited by a single stimulus after treatment of a CantonS fly with 8 ng of fenfluthrin. C: Type of response observed in some nap" preparations treated with fenfluthrin. See text for explanation. Knockdown Resistance in D. melanogaster 299 (mean ? S.D.) after treatment. Identically treated preparations of napts flies either displayed single evoked spikes for at least 30 min after treatment (2 of 4 preparations) or a truncated bursting response like that shown in Figure 4C. These short responses appeared in two preparations 14 min and 17 min after treatment and, unlike the consistent bursting observed in most Canton-S preparations, were interspersed with many single evoked spikes. Spontaneous activity in nap" preparations appeared 9.75 2 3.9 min (mean S.D.) after treatment. The absent or reduced bursting in napts flies, along with the greater latency for the onset of elevated spontaneous activity, provide evidence of reduced sensitivity of the napts nervous system to exogenously applied pyrethroid. * DISCUSSION Our findings confirm and extend those of Kasbekar and Hall [lo], demonstrating that the napts strain of D. melanogaster exhibits low levels of resistance to DDT as well as to a group of structurally diverse pyrethroids. The compounds used in our experiments were chosen to exemplify the diversity of insecticidal compounds known to act at the sodium channel. This group included examples of compounds producing both type I (e.g., NRDC 157) and type I1 (e.g., deltamethrin) symptomology , compounds having rapid (e.g., fenfluthrin) or slow (e.g., DDT) action, and compounds expected to be resistant to detoxicationby cytochrome P-450-dependentmonooxygenases(e.g., fenfluthrin  or carboxylesterases (e.g., DDT, M1'1800). We conclude from our findings and those of other workers [lo] that the napts strain, like kdr strains of the housefly [ 151, exhibits broad cross-resistance to insecticides acting at the sodium channel. The levels of resistance found in this study are consistent with both the reduction in sodium channel density observed in the nap" strain [5,6] and the results of biophysical studies of pyrethroid-sodium channel interactions, which suggest that less than 1%of sodium channels must be modified to compromise normal function . In nerves having reduced sodium channel density, the modification of an equal number of target sites per unit area of nerve membrane would require modification of a greater fraction of the total complement of sodium channels. This in turn would require a higher concentration of insecticide, which would be reflected both at the level of the nerve and at the level of the whole organism as resistance. However, if the fraction of sodium channels that must be modified under normal circumstances is small , the twofold reduction in sodium channel density evident in [3H]saxitoxinbinding assays with napts insects should confer approximately twofold resistance. This predicted value is in excellent agreement with low levels of resistance observed in our bioassays. The fact that resistance is directly related to the number or density of sodium channels provides further evidence that the pyrethroid/DDT recognition site is intimately associated with the voltage-sensitive sodium channel itself. This finding validates the use of specific sodium channel radioligands, such as [3H]saxitoxin, to assess whether alterations in binding site density are involved in pyrethroid resistance in other species. Although differences in the responses of the naptSand Canton-S strains to the lethal effects of these insecticideswere small, much larger differences were 300 Bloomquist et al. observed between these strains in their responses to the rapid paralytic actions of some of the compounds tested. With deltamethrin, fenfluthrin, and MTI 800, the onset of paralysis in Canton-S flies was rapid, whereas little or no paralysis occurred in parallel assays of napfSflies exposed to the same dosage of toxicant. Striking differences in the onset of paralysis were evident even in cases (e.g., MTI 800 at 15 pgflask) where differencesin mortality at 24 h between strains were very small. The extent of resistance to the rapid paralytic effects of these two compounds in the napfS strain is much greater than that previously reported for fenvalerate [lo]. This difference may simply result from our use of rapidly-acting compounds for these assays. Alternatively, it is possible that our bioassay method, which involved exposure of flies to a uniform residue on a glass surface [ll], provides a more precise estimate of rapid paralysis than that involving exposure to a treated filter paper disc [lo]. The delay in the onset of intoxication in the napfSstrain of D. melanogaster is similar to that observed in kdr house flies . This result is consistent with a mechanism of resistance involving reduced target sensitivity but does not, in itself, rule out the possible involvement of other mechanisms. However, two other lines of evidence also point to a mechanism involving altered response of the target tissue. First, resistance to fenvalerate in the napfSstrain is genetically inseparable from the napfSlocus itself [lo]. Since the napts mutation is known to affect both the density of sodium channels and the normal functioning of nerves, this finding clearly implicates a neuronal mechanism rather than one involving differences in penetration or metabolism of the insecticide. Second, the fact that resistance extends to compounds such as MTI 800, DDT, and fenfluthrin also provides indirect evidence against the existence of a mechanism in nap" flies that involves increased metabolic detoxication. Crossresistance to MTI 800 and DDT, compounds that lack carboxylester moieties, unequivocally rules out the involvement of enhanced hydrolytic activity in resistance, but the lack of an effect of oxidative detoxication is less clearly delineated. Kasbekar and Hall [lo] obtained contradictory results using combined treatments of piperonyl butoxide (an inhibitor of cytochrome P450-dependent monooxygenases)and fenvalerate. This combination completely counteracted the resistance to the lethal effects of fenvalerate in the napfSstrain but did not completely remove the resistance to the rapid paralytic effects of this compound. Nevertheless, resistance of the napfS strain of D. melanogaster to fenfluthrin, a compound not attacked by the cytochrome P450-dependent monooxygenases that contribute to high levels of resistance to a variety of other pyrethroids in the Learn-PyR strain of the housefly , provides additional evidence that a mechanism other than enhanced oxidation may be involved in this strain. Direct evidence for reduced neuronal sensitivity to insecticides in the naptS strain was obtained in physiological assays of the onset of altered neuronal function in flies individually treated with fenfluthrin. Fenfluthrin was chosen for physiological assays because it produces consistent burst discharges in equivalent dorsolongitudinal muscle preparations of the housefly 1171. The increased latency of abnormal spontaneous and evoked activity in napfSflies was qualitatively similar to the type of responses observed when this assay is used to define reduced neuronal sensitivity in kdr houseflies  and provides the Knockdown Resistance in D. melanogaster 301 first physiological documentation of reduced neuronal sensitivity to pyrethroids in D. rnelanogaster. However, the differences between wildtype (Canton-S)and napfs strains in D. melanogaster are more subtle than those observed between susceptible and kdr strains in the house fly, reflecting the low levels of resistance at the organismal level conferred by the napts mutation. The abnormal evoked multiple potentials (e.g., Fig. 4C) that were observed in some napfS preparations may represent the highest frequency bursting responses measurable for this strain, reflecting the prolonged refractory period of nap" nerves . Support for this interpretation also comes from our failure to observe high-frequency bursting responses, such as that shown in Figure 48, to fenfluthrin or any other pyrethroid in dorsolongitudinal muscles of intoxicated napfs flies treated either by surface contact or topical application (J. R. Bloomquist, unpublished observations). Although our findings identify the mechanism of resistance conferred by the napfs mutation as "kdr-like", the napts strain does not appear to be an appropriate mechanistic model for the kdr mechanism of the housefly. If a reduction in target site density were the principal mechanism underlying the kdr phenotype, the high levels of resistance observed in some allelic variants at the kdr locus  should be correlated with substantial reductions in sodium channel density. Although a reduction in sodium channel density in kdr houseflies has been reported , recent studies in this laboratory  and elsewhere [22,23] failed to document any differences in the density of binding sites for [3H]saxitoxinin well-characterized susceptible and kdr or super-kdr housefly strains. We conclude from these findings that a reduction in sodium channel density is not specifically associated with the kdr mechanism. These considerations argue against the reliance on any single well-characterized case of reduced neuronal sensitivity as a mechanistic model for similar phenomena in other strains and species. Although our study was restricted to the napfsstrain, three other temperature-sensitive paralytic mutants of D. rnelanogaster have been described that also exhibit altered sodium channel properties: parafs (paralysis), seits (seizure), and tip-E" (temperature-induced paralysis, locus E) . Of these mutant strains, parats is of particular interest because the cloning and sequencing of this locus has revealed a significant structural homology with vertebrate sodium channel structural genes 1251. Moreover, a preliminary report  suggests that different alleles of parats may exhibit either resistance or hypersensitivity to pyrethroids. These findings suggest that strains of D. rnelanogaster exhibiting altered neuronal function will continue to be valuable resources for characterizing target site-mediated mechanisms of insecticide resistance. LITERATURE CITED 1. Wilson TG: Drosophila melanogaster (Diptera: Drosophilidae): A model insect for insecticide resistance studies. J Econ Entomol82,22 (1988). 2. Wu C-F, Ganetzky B, Jan LY, Jan Y-N, Benzer S: A Drosophila mutant with a temperaturesensitive block in nerve conduction. Proc Natl Acad Sci USA 75,4047 (1978). 3. 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