Effects of Ivermectin on -Aminobutyric Acid and ... - ACS Publications

Chapter 16

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Effects of Ivermectin on y-Aminobutyric Acid and Glutamate-Gated Chloride Conductance in Arthropod Skeletal Muscle 1

Ian R. Duce, Narotam S. Bhandal, Roderick H. Scott , and Timothy M. Norris 2

Department of Life Science, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom Ivermectin at low concentrations produced reversible and irreversible increases in Cl conductance in locust skeletal muscle, but had little effect on tick muscle. Two electrode current clamp was used to monitor input conductance in both GABA-sensitive and GABA-insen sitive muscle fibres in locust extensor tibiae muscle. In G A B A -sensitive muscle fibres J V M (100pM-1nM) reversibly increased Cl conductance and antagonised G A B A responses, however higher I V M concentrations (10nM-1μM) induced irreversible increases in Cl conductance which continued to increase during washing. G A B A insensitive muscle fibres produced only irreversible responses to I V M . I V M also inhibited Cl conductance gated by ibotenate-sensitive glutamate receptors in locust muscle fibres. Tick muscle fibres showed little sensitivity to I V M and were insensitive to ibotenate. We conclude that a major component of the I V M response in arthropod muscle is mediated via ibotenate-sensitive Cl channels. -

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Arthropod pests have an enormous impact on mankind. As vectors of viral, bacterial, protozoal and nematode pathogens, blood sucking insects and arachnids are a major threat to human and animal health. Damage by insects and mites to crops and stored products has massive effects on economic welfare and food avilability for humans and domestic stock. These pressures have inevitably lead to strenuous attempts to control arthropod pests. Effective control of both free-living and ectoparasitic arthropods is heavily dependent on chemical control, the scale of which can be seen from sales figures which are projected to reach 7 billion $US by 1995 (1). Although the existing chemical agents in use are valuable both in terms of their ability to boost 1

Current address: Department of Physiology, St. George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, United Kingdom Current address: Department of Entomology, University of California, Riverside, CA 92521

2

0097-6156/95/0591-0251$12.00/0 © 1995 American Chemical Society Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS

agricultural output and to improve public and animal health, a heavy reliance is placed on two major chemical groups: the pyrethroids and the organophosphates. These compounds are neurotoxins affecting respectively: neuronal voltage-sensitive sodium channels, and acetylcholinesterase. However the heavy reliance on actions at these two target sites and the intensive application of these molecules has resulted in a significant problem of resistance in many parts of the world. Historically older commercial pesticides such as D D T and the chlorcycloalkanes, although effective in terms of arthropod control, achieved notoriety as a result of the persistence and toxicity of residues in the natural environment. Thus the search for safer, effective pesticides preferably working at different target sites is driven on by these combined needs. Avermectins. It was in this context that the announcement of a new class of pesticides, the Avermectins derived from a microorganism Streptomyces avermitilis(2), generated considerable interest. These compounds were reported to be very active nematicides(3) insecticides(4,5) and acaricides (S) and it was apparent from early work that the avermectins did not share the same mode of action as existing pesticides(6). The site of action was generally ascribed to 4aminobutyric acid (GABA) receptors, however Turner and Schaeffer(6) in reviewing this topic point to the difficulties in interpreting the many studies carried out in widely different systems using a baffling array of experimental methods. In this chapter we shall review our attempts, using electrophysiological techniques, to determine the mechanism(s) of action of 22,23 dihydroavermectin B (Ivermectin IVM) on the skeletal muscle of two arthropod species: an insect, the locust Schistocerca gregaria; and an acarine ectoparasite, the cattle tick Ambfyomma hebraeum. l a

Insect Skeletal Muscle The Locust Extensor Tibiae (ET) Muscle The E T muscle in the metathoracic (jumping) leg of grasshoppers and locusts consists of many bundles of muscle fibres. The complex postural movements and powerful jumping response mediated by this muscle are under the control of "fast excitatory", "slow excitatory" and "inhibitory" motoneurons situated in the CNS of the insect. The pattern of innervation and nomenclature of these muscle bundles was described by Hoyle(7), who showed that the smaller proximal and distal muscle bundles received both slow excitatory and inhibitory innervation whereas the large medial bundles were only supplied by fast excitatory axons (Figure 1). The excitatory and inhibitory transmit ters are respectively L-glutamate and GABA(8). Pharmacology of (ET) Muscle Bundles The actions of drugs and neurotransmitters on the mechanical and electrophysiological responses of locust ET have been extensively studied over the last 25 years (8,9). It is now generally agreed that the principal excitatory and inhibitory neurotransmitters are L-glutamate and G A B A respectively. Ionophoresis of L-glutamate demonstrated that receptors occurred not only at neuromuscular junctions but were distributed across the extrajunctional sarcolemma (10); furthermore the extrajunctional receptors were of two types: Depolarising glutamate receptors which were sensitive to the glutamate analogue Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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Effects of Ivermectin in Arthropod Skeletal Muscle

253

quisqualate and gated cationic channels (qGluR) and hyperpolarising glutamate receptors which were sensitive to ibotenate and gated chloride channels (iGluR) (11). As one would predict the excitatory junctional receptors responded to L glutamate by gating cationic channels resulting in depolarisation. Inhibitory post-synaptic potentials and ionophoretic G A B A responses in E T muscle bundles were both found to activate chloride (CI) channels with the same reversal potential (12) and responses were antagonised by picrotoxin providing strong evidence that G A B A was the inhibitory neuromuscular transmitter. ET muscles have also been shown to be innnervated by neurons releasing octopamine (13) and proctolin (14,15) which modulate the contractility of E T muscle fibres, probably via second messenger systems. Two-Electrode Current Clamp When we embarked on our studies of the actions of I V M on insect muscle we were aware of the problematic properties of this pesticide for electrophysiology, it is highly lipophilic, relatively insoluble in water, active at low concentrations and its actions were reported to involve irreversible changes in CI" permeability. The method we employed was to measure the input conductance of muscle cells using a two-electrode current clamp whilst delivering I V M and other drugs via bath perfusion or local microperfusion from a micropipette. Constant current pulses were injected into the muscle through a microelectrode whilst monitoring the membrane potential via a second microelectrode. Alterations in input conductance resulting frrom the opening or closing of ion-channels in the cell membrane result in changes in the size of the electrotonic potential resulting from the injected current pulses (Figure 2). The recording system is very stable permitting experiments to be carried out over several hours without change in the resting input conductance. The bathing physiological saline contained 2% dimethyl sulfoxide (DMSO) to keep I V M in solution. This vehicle is frequently used in studies on I V M and other lipophilic pesticides and its effects on cell physiology are often discounted, however in our studies we found that DMSO altered potassium permeability (16) and in all experiments muscles were equilibrated with saline containing 2 % D M S O which was present in all subsequent bathing solutions and drug applications. GABA-Sensitive Distal Muscle Bundles The distal muscle bundles 32-34 (Figure 1) are innervated by the inhibitory motor nerve(7). Application of G A B A to these muscle fibres resulted in dose-dependent increases in input condcutance (17, 18) (Figure 2) which were abolished in Cl'-free saline. The pharmacology of the G A B A response was characterised using a range of agonists and antagonists as well as modulators of GABA-gated CI" conductance such as pentobarbitone (19). The pharmacology of these locust ET muscle G A B A receptors resembled that of the vertebrate G A B A receptor in several respects, however locust E T muscle G A B A receptors were insensitive to bicuculline. The actions of I V M on these muscle fibres were complex (20). At low concentrations (lOOpM - lOnM) microperusion of I V M induced dose dependent increases in CI" conductance which were fully reversible on washing (Figure 3). Application of I V M (lOOpM - lOnM) during a G A B A activated increase in conductance resulted in a decrease in the G A B A response (Table I). The G A B A antagonism was reversible and non-competitive (Table I). A

Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


254


32

33

34

Figure 1. Diagram of the femur of a locust Schistocerca gregaria metathoracic ("jumping") leg showing the position of the GABAsensitive distal muscle bundles 31-33 and the GABA-insensitive "Fast" muscle bundles of the extensor tibiae muscle.

ImMGABA

-60mV

lOnA

60s Figure 2. Current-clamp recording showing the change in input conductance in muscle bundle 33 due to perfusion of ImM GABA. Downward deflections represent hyperpolarising electrotonic potentials resulting from the injection oflOnA pulses of current. GABA produces a hyperpolarisation of the membrane potential and an increase in input conductance.


16.

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Effects oj Ivermectin in Arthropod Skeletal Muscle

255


Table I. Microperfusion of I V M Induces Reversible Conductance Increase and Inhibition of GABA-induced Conductance In Muscle Bundle 33 of Locust E T Muscle

Dose I V M

Conductance Increase (nS) ± S . E . M . of (n) observations

% Inhibition of Conduct ance produced by I m M G A B A ± S.E.M.(n)

75pM

16 ± 6 (5)

17.3 ± 5.1 (3)

lOOpM

50 ± 7 (6)

63.5 ± 3.3 (7)

InM

105 ± 9 (5)

68.3 ± 1.5 (5)

7.5nM

258 ± 35 (6)

82.0 ± 3.7 (5)

When higher concentrations of I V M (lOnM - ljiM) were perfused over muscle bundles 31-34 an increase in input conductance was produced which was both irreversible on washing with saline containing DMSO and continued to increase for 60 mins after I V M was washed from the bath(Figure 4; Table II). During these irreversible IVM-induced conductances, application of G A B A produced a potentiated response (Table II).

Table n. Microperfusion of I V M Induces Irreversible Conductance Increases Which Continue for 60 Mins After Washing, and Potentiates GABA-induced Conductance in Locust E T Muscle Bundle 33 Dose I V M G*M)

Irreversible Increase in Conduct ance (jiS) ± S . E . M . (n)

% Increase in G A B A Response Compared with ImM G A B A ± S . E . M (n)

3-5 Minutes After I V M

60 Minutes After IVM

10 n M

0.63 ± 0.14

1.79 ± 0.45 (6)

60.8 + 8.1 (25)

lOOnM

3.33 ± 0.69

5.62 ± 0.97 (7)

170.4 ± 24 (13)

500nM

4.2 ± 0.58

8.25 ± 0.65 (3)

Not Tested

1/xM

9.11 ± 0.78

36.0 ± 5.9 (8)

334.1 ± 79.7 (16)

However the picture was further complicated by the following experiment: I V M (lOOnM) was applied and induced an irreversible conductance increase; it was then washed from the bath and G A B A (ImM) was perfused over the muscle and as expected the G A B A response was potentiated. If I V M (lOOnM) was now reapplied Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


256


InM IVM

-64mV

lOmV

60s Figure 3. Reversible increase in input conductance in muscle bundle 33 produced by microperfusion of InM IVM.

120s

-47mVi

lOmV

lOnMIVM Figure 4. Irreversible increase in input conductance in muscle bundle 33 produced by microperfusion of lOnM IVM . The conductance continues to increase after IVM application ends.


16. DUCE ET AL.

257



during the G A B A perfusion, the potentiated G A B A response was antagonised. We interpret these complex responses to I V M on G A B A sensitive muscle fibres in locust E T as being due to effects on both GABA-gated C I channels and other CI" channels which are not associated with G A B A receptors. This hypothesis could be readily tested using the ET muscle where the majority of the fast muscle bundles (Figure 1) do not respond to G A B A . GABA-Insensitive Fast Muscle Bundles Microperfusion of I V M (InM-l/xM) onto fibres which were insensitive to G A B A evoked an irreversible increase in CI" permeability which continued to develop for up to 60 mins following washing of the pesticide from the bath (20) (Table m).

Table HI. Microperfusion of I V M onto GABA-insensitive Muscle Bundles of Locust E T Muscle Induces Irreversible Conductance Increases Which Continue for 60 Minutes Following Washing Dose I V M

Irreversible Increase in Conductance QtS) ± S . E . M . (n) 3-5 Minutes After I V M

60 Minutes After I V M

InM

0.18 ± 0.05

0.65 ± 0.15 (5)

7.5nM

0.47 ± 0.13

1.31 ± 0.04 (3)

lOnM

1.36 ± 0.33

4.74 ± 0.99 (5)

lOOnM

2.81 ± 0.34

7.66 ± 0.25 (3)

1/tM

11.1 ± 1.49

47.4 ± 8.44 (3)

1 | |

If these responses are not due to the action of I V M on G A B A receptor CI' channels the obvious question is what type of CI" might they be? In Ascaris muscle patchclamp studies (21) showed that I V M depressed GABA-activated channel currents, but also opened non-GABA-activated CI" channels with long average open times. In locust ET muscle ionophoresis (11) and patch-clamping (22) revealed that iGluR are widely distributed across the sarcolemma (Figure 5). They gate CI" currents and can be selectively activated by the glutamate analogue [2-(3-hydroxyisoxazol5yl)glycine] (ibotenic acid) (23,11). Using two-electrode current-clamp we found that perfusion of ibotenic acid on to E T muscle bundles (Figure 6) produced a dosedependent increase in CI" conductance over the range 1/xM - I m M (24). Responses to ibotenic acid (lOOjiM) were reduced by I V M (500pM-100nM) (Table IV). These results demonstrate that I V M does interact at low concentrations with iGluR, however to gain a more precise understanding of the interaction of I V M with iGluR it will be necessary to use a method with a higher resolution. Recent developments in methodology have now enabled recordings of membrane currents resulting from the activation of small populations of iGluR using a liquid filament switch to apply brief pulses of ibotenate to an outside-out patch of membrane excised from E T muscles in a patch-clamp micropipette (Figure 7). This method Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Distal Muscle Bundles 32-34

+

Na K*

iGluR (

Glu

)

Cl

1

G , u R

+

Na K

+

Fast Muscle Bundles Figure 5. Cartoon of the putative receptors for neurotransmitter amino acids on ET muscle fibres. The presence of extrajunctional qGluR and GABA receptors on distal muscle fibres is likely but has not been shown experimentally.

lOnA

lOmV

100/xM Ibotenate

10s

Figure 6. Perfusion of lOOuM ibotenic acid induces an increase in input conductance in a GABA-insensitive fast muscle bundle. The response desensitises during ibotenate application.


16. DUCE ET AL.


259

should allow us to gain a fuller understanding of the actions of I V M on locust muscle.


Table IV. I V M Inhibits Increases in Input Conductance Produced by Perfusion of Ibotenic Acid (100/xM) in Non-GABA Sensitive Muscle Bundles of Locust E T Muscle

Dose I V M GxM)

Mean % Reduction in Response to Ibotenic Acid

500pM

51 ± 7.8 (4)

InM

59 ± 4.1 (13)

lOnM

100(3)

(IOOJXM) Caused by Microperfusion of I V M ± S E M (n)

Tick Skeletal Muscles Treatment of domestic stock using I V M has been shown to control a range of ectoparsites including cattle ticks of the genus Boophilus (24). Injection of I V M into the cattle tick Ambfyomma hebraeum caused paralysis which affected movement and feeding (25). Harrow et al (26) found that I V M at concentrations in excess of IfiM produced an increase in muscle membrane CI" conductance and inhibited spontaneous activity recorded from the synganglion (CNS) of Ambtyomma hebraeum. Our findings on the actions of I V M on insect muscle CI" channels encouraged us to extend our studies to examine the skeletal muscles of the cattle tick Ambtyomma hebraeum. The size and anatomy of cattle ticks restricts the range of skeletal muscles which are accessible to electrophysiological studies. For the work described here an incision was made around the dorsal/lateral edge of the cuticle (shield). The shield was folded anteriorly and the viscera were dissected away to reveal four sets of coxal muscles. The preparation was continuously perfused with tick saline (mM: NaCl 203, KC16, CaCl 10, HEPES 10, M g C l 1 ; p H 6.9) and drugs were applied via the bath perfusion or microperfiised onto the target muscle. Coxal muscle fibres were impaled with two microelectrodes and two electrode current-clamp was used to investigate changes in input conductance produced by drug perfusion. 2

2

Pharmacology of tick coxal muscle Coxal muscle fibres responded to G A B A (luM - ImM) with dose-dependent increases in CI" permeability (Figure 8). Muscimol was a more potent agonist with an E C approximately 10 fold lower than that for G A B A . G A B A responses were antagonised by picrotoxin but not by bicuculline and were potentiated in the presence of pentobarbitone and the benzodiazepine flurazepam (27). This pharmacological profile closely resembles that described above for locust skeletal muscle G A B A receptors. Application of L-glutamate resulted in depolarisation accompanied by a dose dependent increase in input conductance (Figure 9) which is consistent with glutamate induced increases in cation permeability described in other arthropod 5 0



260


10s

100/xM Ibotenate 100/iM

Figure 7. Pulses of ibotenate applied using the liquid filament technique activate transmembrane Ct currents in outside-out patches of membrane excised from locust fast muscle fibres.

O Muscimol

Concentration M Figure 8. Dose-response curves for GABA and Muscimol application to tick coxal musclefibres.Each point is the mean change in conductance ± S.E.M (n=3).


16. DUCE ET AL.



10pM Glu

SO/iM Glu

lOOpMGlu

500/xMGlu

261

ImMGlu

lOmV 60s Figure 9. L-glutamate induces dose-dependent depolarisation and increased input conductance in tick coxal muscle fibres. skeletal muscles (28). Glutamate has also been shown to produce muscle contractions in ticks (29). The actions of G A B A and Glutamate were closely parallel to those found in locust muscle, however in 85% of tick muscles tested perfusion of I V M (InM - 50JAM) had no effect on either the input conductance or membrane potential. Very high doses of I V M (> lOO^M) produced irreversible increases in conductance. 15% of fibres tested produced small reversible increases in input conductance at concentra tions above 50 n M . These results were surprising considering the high sensitivity of insect muscle and the knowledge that I V M is a potent paralytic acaricide. In view of the evidence presented above that I V M may interact with ibotenate-sensitive glutamate-gated CI" channels in insect muscle and the recent findings of Arena et al and Rohrer et al (this volume) that the high affinity binding site for I V M in nematodes and insect CNS is also an ibotenate sensitive glutamategated Cl~ channel, we decided to test whether tick muscle was sensitive to ibotenate. Figure 10 shows that perfusion of G A B A 100/xM over tick muscle produced a clear increase in input conductance, whereas the same muscle fibre is insensitive to both ibotenate and I V M . This strongly suggests that the sensitivity of arthropod skeletal muscles is associated significantly with iGluR channels. The acaricidal actions of I V M are probably mediated through sites in the CNS. We obtained electrophysiological data confirming the findings of Harrow et al (26) that I V M potently inhibits spontaneous electrical activity in the CNS of Amblyomma hebraeum. Conclusion Our results show that I V M has several sites of action and more than one pharmacological effect in arthropod muscles. The actions on insect muscle appear to involve CI' channels gated by both G A B A and iGluR. It is likely that the widespread distribution of iGluR, estimated from patch-clamp studies to be 100 per fim (22), is responsibility for the high sensitivity of locust muscle to I V M . The low sensitivity of tick muscle to both ibotenate and I V M presumably implies a paucity 2


262



lOO/zM GABA

lOOpM Ibotenate

lOOnM IVM

60s Figure 10. Perfusion oflOOuM GABA produces hyperpolarisation and a large increase in input conductance in a tick coxal muscle fibre. However the same muscle fibre is insensitive to lOOuM ibotenate and lOOnMTVM.

of glutamate-gated CI" channels in tick muscles, however without single channel studies this will remain speculative. I V M was found to produce conductance increases in tick muscle at very high concentrations, however this result may a non-specific effect of I V M as at uM concentrations it has been shown to produce unitary currents in planar lipid bilayers which are cation specific (30). Although a picture is emerging from studies on nematodes and insects of I V M opening CI" channels associated with amino acid receptors, the precise action of I V M on these molecules is unknown and awaits detailed molecular and electrophysiological studies. Acknowledgments ERD thanks the SERC for grant support. NSB thanks Pfizer Central Research (UK) for support through the SERC C A S E studentship scheme. Literature Cited (1) Voss,G.; Neumann,R. In Neurotox '91: Molecular Basis of Drug and Pesticide Action; Duce,I.R. Ed.; Elsevier Applied Science: London and New York, 1992; pp vii-xx (2) Burg,R.W.; M i l l e r , B . M . ; Baker,E.E.; Birnbaum,J.; Currie,J.A.; Hartman,R.; Kong,Y-L; Monaghan,R.L.; Olson,G.; Putter,I.; Tunac,J.P.; Wallick,H.; Stanley,E.O.; Oiwa,R.; Omura,S.; Antimicrob. Agents Chemother. 1979, 15, 361367. (3) Egerton,J.R.; Ostlind,D.A.; Blair,L.S.; Eary,C.H.; Suhayda,D.; Cifelli, S.; Riek,R.F.; Campbell,W.C.; Antimicrob.Agents Chemother. 1979, 15, 372-378.



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Effects of Ivermectin in Arthropod Skeletal Muscle 263

(4) Ostlind,D.A.; Cifelli,S.; Lang,R.; Vet. Record 1979, 105, 168. (5) Putter,I.; MacConnell,J.G.; Preiser,F.A.; Haidri,A.A.; Ristich,S.S.; Dybas,R.A.; Experientia 1981, 37, 963-964. (6) Turner,M.J.; Schaeffer, J.M. In Ivermectin and Abamectin; Campbell,W.C. Ed.; Springer-Verlag: New York, 1989; pp 73-88 (7) Hoyle,G.; J. Exp. Biol. 1978, 73, 205-233. (8) Usherwood,P.N.R.; Cull-Candy,S.G. In Insect Muscle; Usherwood,P.N.R. Ed.; Academic Press: New York, 1975, pp207-280. (9) Piek,T. In Comprehensive Insect Physiology, Biochemistry and Pharmacology; Kerkut,G.A.; Gilbert,L.I. Eds.; PergamonPress: Oxford, 1985, Vol 11;pp 55-118. (10) Cull-Candy,S.G.; Usherwood,P.N.R.; Nature New Biol. 1973, 246, 62-64. (11) Cull-Candy,S.G.; J. Physiol. 1976, 255, 449-464. (12) Usherwood,P.N.R.; Grundfest,H.; J.Neurophysiol. 1965, 28, 497-518. (13) Evans,P.D.;O'Shea,M.; Nature (Lond.) 1977, 270, 257-259. (14) Piek,T.;Mantel,P.; J.Insect Physiol. 1977, 23, 321-325. (15) O'shea,M; Adams,M.E.; Adv.Insect Physiol. 1986, 19, 1-27. (16) Scott,R.H.; Duce,I.R.; Pesticide Sci. 1985, 16, 695. (17) Duce,I.R.; Scott,R.H.; J.Physiol (Lond.) 1983, 343, 31-32p. (18) Scott,R.H.; Duce,.I.R.; J.Insect Physiol. 1987, 33, 183-189. (19) Scott,R.H.; Duce,I.R.; Comp. Biochem. Physiol. 1987, 86C, 305-311. (20) Duce,I.R.; Scott, R.H.; Brit. J. Pharmacol. 1985, 85, 395-401. (21) Martin,R.J.; Pennington,A.J.; Brit. J. Pharmacol. 1989, 98, 747-756. (22) Dudel,J; Franke,Ch.; Hatt, H.; Usherwood,P.N.R.; Brain Res. 1989, 481, 215-220. (23) Lea,T.J.; Usherwood,P.N.R.; Comp. Gen. Pharmacol. 1973, 4, 333-350. (24) Benz,G.W.; Roncalli,R.A.; Gross S.J. In Ivermectin and Abamectin; Campbell,W.C. Ed.; Springer-Verlag; New York, 1989; pp 215-229. (25) Kaufman,W.R.; Ungarian,S.G.; Noga,A.E.; Exp. Applied. Acarology 1986, 2, 1-18. (26) Harrow,I.D.; Gration,K.A.F.; Evans, N.A.; Parasitology 1991, 102, S59-S69. (27) Bhandal,N.S.; Duce,I.R.; Pesticide Sci. 1991, 32, 509-510. (28) Anis,N.A.; Clark,R.B.; Gration,K.A.F.; Usherwood,P.N.R.; J.Physiol. (Lond.) 1981, 312, 345-364. (29) Hart,R.J.; Potter,C.; Wright,R.A.; Lea,P.J.; Physiological Entomol. 1978, 3, 289-295. (30) Bhandal,N.S.; Duce,I.R.; Pesticide Sci. 1991, 33, 509-510. RECEIVED August 15, 1994


Effects of Ivermectin on -Aminobutyric Acid and ... - ACS Publications

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