Toxicity and Mode of Action of Avermectin B1 Against Insects Terence Simon Corbitt, B.Sc. a Thesis Submitted for the Degree of D
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1 Toxicity and Mode of Action of Avermectin B1 against insects Terence Simon Corbitt, B.Sc. A Thesis Submitted for the degree of Doctor of Philosophy of the University of London and for the Diploma of Membership of Imperial College. Department of Pure and Applied Biology Imperial College Silwood Park Ascot, Berkshire SL5 7PY August 1987 2 ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. D.J. Wright for his supervision and advice during the course of this work and presentation of this thesis. I would also like to thank the following: Dr A. Green (Merck Sharp and Dohme, New Jersey) for helpful advice and donation of [3H]AVMB1 and AVMB-j, Drs J. Hardie, D.J.Galley, M. Djamgoz and Mr I. Fosbrook for advice and help given during this study. Thanks also to Ms S.Smith of TDRI, Porton, Wilts, for the Spodoptera 1i ttorali s, and to Mr T.Carty of the Institute of Virology, Oxford for the Heliothis armigera cultures. This work was carried out with the aid of a grant from the Science and Engineering Research Council. Insect culture was carried out under MAFF licence No. PHF 909/52 (116). I would like to thank my parents for their interest and continual support during my education. Thanks also to Tom for plant culture, Sarah for typing my tables, Chris Addison and other friends who made my stay at Silwood Park enjoyable. I would like to say a special thank you to Tanya for her help and friendship. This thesis is dedicated to my wife Anne for her support and understanding during the preparation of this work. 3 TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS 2 TABLE OF CONTENTS 3 ABSTRACT 8 1. INTRODUCTION 10 1.1 The Avermectins. 10 1.2 Whole Organism studies. 13 1.3 Cel 1ular 1evel . 21 1.4 Present work. 33 2. GENERAL MATERIALS AND METHODS. 34 2.1 Insect Culture. 34 2.2 Selection of lepidopteran larval instars. 35 2.3 Chemicals. 35 2.4 Topical application of Pesticides. 37 2.5 Calibration of Arnold microapplicator and 37 Agla glass syringe 2.6 Contact/feeding toxicity of AVMB1 and 38 cypermethrin with and without oils. 2.7 Feeding rate of third instar larvae of 38 Spodoptera 1i ttorali s and He!iothi s armigera. 2.8 Effect of AVMB1 on the feeding of third instar 39 Spodoptera 1i ttorali s. 2.9 Analysis of bioassay data. 40 3. TOXICOLOGICAL STUDIES 41 3.1 Materials and Methods. 41 4 PAGE 3.1.1 Toxicity of topically applied AVMB1 and 41 cypermethrin to third, fourth, fifth and sixth larval instars of $.1ittoralis. 3.1.2 Toxicity of topically applied AVMB1 41 to third instar larvae of S.1ittoralis reared on various diets. 3.1.3 Toxicity of topically applied AVMB1 42 to third instar larvae of S.1ittoralis and H.armigera reared on artificial diet. 3.1.4 Injection of AYMB1 into fifth and sixth 42 instar larvae of $.1ittoralis. 3.1.5 Contact/ingestion toxicity of AYMB1 and 42 cypermethrin on Chinese cabbage foliage (with and without oils) against first, third and fourth instar larvae of S.1ittoralis. 3.1.6 Residual (Foliar) toxicity of AVMB1 on cabbage 44 (cv. Flower of Spring) under glass against third instar larvae of S.1ittoralis at 1, 3 and 7 days after spraying. 3.1.7 Contact/ingestion toxicity of AVMB^ on Chinese 44 cabbage and cotton against third instar larvae of S.littoralis and H.armigera. 3.1.8 Contact/ingestion toxicity of AVMB1 on cabbage 45 and cotton against first instar larvae of S.1i ttorali s, H.armi gera and H. vi rescens. 3.1.9 Assessment of feeding rate in third instar 46 larvae of S.1ittoralis and H.armigera reared on cotton. 5 PAGE 3.1.10 Effect of foliar residues of AYMB1 on the 46 feeding of third instar larvae of $.1ittoralis 3.1.11 Effect of foliar residues of AVMB1 on the 46 feeding of third instar larvae of $.1ittoralis. 3.1.12 Distribution ("Choice") of third instar larvae 46 of $.1ittoralis between untreated and AVMB1 -treated Chinese cabbage leaves. 3.2 Results. 48 3.2.1 Toxicity of topically applied AVMB.,and 48 cypermethrin to different larval instars of $.1ittoralis reared on Chinese cabbage 3.2.2 Toxicity of topically applied AVMB1 to third 48 instar larvae of $.1ittoralis reared on various diets. 3.2.3 Toxicity of topically applied AYMB1 to third 52 instar $.1ittoralis and H.armigera reared on artificial diet. 3.2.4 Toxicity of injected AVMB1 to fifth and sixth 52 instar S.1ittoralis larvae. 3.2.5 Contact/ingestion toxicity of AVMB1 and 52 cypermethrin on Chinese cabbage foliage (with and without the addition of oils) against first, third and fourth instar $.1ittoralis. 3.2.6 Residual (foliar) toxicity of AVMB1 on cabbage 56 (cv. Flower of Spring) under glass against third instar larvae of Spodoptera 1ittoralis at 1, 3 and 7 days after spraying. 6 PAGE 3.2.7 Contact/ingestion toxicity of AVMB1 on Chinese 61 cabbage and cotton against third instar larvae of S.1ittoralis and H.armigera. 3.2.8 Contact/ingestion toxicity of AYMB1 on Chinese- 61 cabbage and cotton leaves against first instar larvae of S.littoralis, H,armigera and H. vireseens. 3.2.9 Assessment of feeding rate in third instar 65 $.1ittoralis and H.armigera. 3.2.10 Effect of AVMB1 on the feeding of third instar 65 larvae of S.1 ittoralis. 3.3 Discussion. 71 3.3.1 Symptoms of poisoning with AYMB1 71 3.3.2 Toxicity of AYMB1 to larval instars. 73 3.3.3 Effect of larval diet on insecticide toxicity. 77 3.3.4 Action of oil enhancers. 78 3.3.5 Larval weight gain. 80 4. UPTAKE OF [3H]AVMB1 BY Peri pianeta americana 83 4.1 Materials and Methods. 83 4.1.1 Localisation of total radioactivity in 83 Peri pianeta americana tissues. 4.1.2 Uptake of radioactivity by P.americana nerve 84 and muscle tissues with time. 4.1.3 Isolation of radioactivity from P.americana 35 nerve and muscle tissues. 4.2 Results. 88 4.2.1 Distribution of radioactivity in P.americana 88 tissues 7 PAGE • v 4.2.2 Uptake of radioactivity by P.americana nerve 88 and muscle tissues with time. 4.2.3 Isolation of radioactivity from nerve and 92 muscle tissues of P.americana. 4.3 Discussion. g8 5. ACTIONS OF AVMB1 ON A NERVE CORD PREPARATION OF . 10O Periplaneta americana 5.1 Materials and Methods. 100 5.1.1 In vitro effects of AVMB1 on spontaneous and 100 evoked activities recorded extracel1ularly from the ventral nerve cord of Peri pianeta americana. 5.1.2 Recording of spontaneous and evoked activities 102 from the ventral nerve cord of P.americana injected with AVMB1 i_n vivo. 5.1.3 Relationship between spontaneous and evoked 102 activities present in the ventral nerve cord of adult male P.americana pre-injected with [3H]AVMB1 and the level of radioactivity within nerve and muscle tissues 24 and 96h after injection. 5.2 Results. 104 5.2.1 In vi tro preparations. 104 5.2.2 In vi vo preparations. 110 5.3 Discussion. 115 6 . GENERAL DISCUSSION AND CONCLUSIONS 122 6.1 Toxicity of AVMB1 against lepidopteran larval instars. 122 6.2 Radiochemical and Neurophysiological studies. 124 REFERENCES 128 APPENDICES 136 8 ABSTRACT The avermectins are a group of naturally-derived compounds which possess insecticidal, nematicidal and acaricidal properties. Some of the insecticidal effects of Avermectin B1a/1b (80:20) (AVMB1) have been studied in three noctuid species (Lepidoptera): the Eygptian leafworm, Spodoptera 1ittoralis; the cotton budworm, He!iothis armigera, and the tobacco budworm, He!iothis vireseens, while radiochemical and electrophysiological studies with AVMB1 were conducted on the cockroach, Peri pianeta americana. Topical application of AYMB1 or the pyrethroid, cypermethrin to third, fourth, fifth and sixth instar S.littoralis indicated that the fourth and fifth instars were insensitive to AYMB^ this was not found with cypermethrin. Injection of AVMB1 into fifth and sixth instars was found to reduce the concentration of AVMB1 required to give an LD5Q value. This observation, together with the fact that fourth instar larvae of S.1ittoralis were sensitive to AVMB1 residues on leaves suggested that the insensitivity of this instar to topically-applied AYMB1 was due to a reduced rate of penetration of the pesticide when compared with the third instar, for example. Feeding of larvae on AYMB1 or cypermethrin-treated leaves, with and without Sunspray 6E and safflower oil enhancers, was found to be a particularly effective method of controlling first and third instar larvae of S.1ittoralis, H.armigera and H.virescens. The residual toxicity of AYMB1 on cabbage under 9 glasshouse conditions was also determined. A significant reduction in activity was seen fourteen days after spraying. Sunspray 6E and safflower oils were found to enhance the residual activity of AVMB1. The weight gain of third instar $.1ittoralis fed on AVMB1-treated leaves or topically applied with AVMB1 was seen to be reduced compared with controls. However, there was no preference between control and AYMB1-treated leaf discs suggesting that AVMB1 has an anorexic rather than an antifeedant action. The toxicity of AVMB1 towards larval instars of the above insect species was thus shown to be complex, with dose-dependent lethal and sub-lethal effects, causing changes in feeding behaviour which protect the plant from attack by these species. In the second part of this study, an attempt was made to correlate toxicological and physiological effects of AVMB1 on insects.