SOME BIOCHEMICAL AND TOXICOLOGICAL STUDIES OF ORGANOPHOSPHATE RESISTANCE IN MYZUS PERSICAE (SULZER-) by Khwaja Ismail Sudderuddin, M.Sc. (Dacca) A thesis submitted for the Degree of Doctor of Philosophy, in the Faculty of Science, University of London. Department of Zoology and Applied Entomology, Imperial College of Science and Technology, Ashurst Lodge, Sunninghill, Ascot, Berkshire, March, 1972. 2 Abstract Insecticide resistance in Mvzus persicae (Sulzer) was investigated from three different aspects. I. The biochemical work was sub-divided into three parts. (a) A histochemical localization of esterases in various tissues of the 'susceptible' aphid. (b) A kinetic study, using titrimetric and colorimetric techniques, to determine the activities of cholinesterases and carboxylesterases from OP-susceptible and OP-resistant strains, towards several speci- fic and general substrates. The in vitro inhibitory effects of organophosphate inhibitors and recovery of these enzymes were also studied. (c) An electrophoretic separation of the esterases on starch and polyacrylamide gels and their subse- quent characterization with specific inhibitors. The RCJ values of the various isoenzymes were calculated and compared. II. A study of the effect of rearing insecticide resistant aphids in an insecticide-free environment and in an environment with gradually increasing doses of insecticide. The carboxylesterases activity of the insects was monitored during this period. III. The toxicological studies included the computation of the toxicities of ten insecticides for the two strains together with the construction of a resistance spectrum for the R-strain. The data are compared with that for other insects and the relationship between toxicity and chemical structure of the compounds is discussed. An appraisal of the work is given with comments on the suggested functions of carboxylesterases in insects. 3 if all the bugs in all the worlds twixt earth and betelgoose should sharpen up their little stings and turn their feelings loose they soon would show all human beans in saturn earth or mars their relative significance Don Marquis, archy's life of mehitabel It 2 ABSTRACT 8 ACHNOWLEDGEMENTS 10 GENERAL INTRODUCTION PART ONE - BIOCHEMICAL STUDIES Section A: Histechemical localization of ester- ase activity in the green peach aphid 19 Introduction 20 Materials and Methods 21 Procedures for fixing, embedding and sectioning material 23 Staining media and procedures for esterase determination 24 Results 31 Discussion Section B(1): Quantitative determinationersome B-esterases of OP-susceptible and OP-resistant Myzus porsicae by a pH-titrimetric method. Sub- strates: acetylcholine chloride and ethyl. buty- rate 34 Introduction 35 Materials and Methods Insect material Preparation of the homogenate Experimental details 39 Results 41 DiSCUSS1011 5 Section B(2): Studies of some B-esterases of susceptible and resistant Mopersicae using Hestrin's colorimetric method. Substrates: acaiylcholine chloride and ethyl butyrate 44 Introduction 45 Materials and Methods Reagents used Preparation of standard curves Basic plan for esterase determination 50 Results Study of substrate concentrations Inhibition of esterases by eserine sulphate and dichlorvos 52 Discussion Section 13(3): Quantitative studies of some B- esterases of susceptible and resistant M.per- sicae by Gomori's colorimetric method. Sub- strates: 1- and 2-naphthyl acetate and 1- and 2-naphthyl butyrate 59 Introduction 61 Materials and Methods Experimental details Calibration curves for 1- and 2-naphthol 64 Results Effect of enzyme concentration Effect of temperature on carboxylesterascs 68 Effect of different substrate concentrations 72 In vitro effect of eserine sulphate, diazinon, d:i.azoxon and dichlorvos 76 Inhibition of carboxylesterases in the presence of the substrate 1-naphthyl acetate 6 83 Study of the instability of OP-resistance and its association with changes in carboxylester- ase level 86 Discussion Section C: Blectrophoretic separation of some B-esterases of susceptible and resistant Mopersicae on polvacrvlamide and starch gels 91 Introduction 94 Materials and Methods Polyacrylamide gel electrophoresis Extraction of enzymes Buffer system Preparation of gels Sample application and electrical conditions 97 Starch gel electrophoresis 98 Treatment of gels Detection of protein bands Dectection of esterase bands 99 Characterization of esterases Phospho4oester hydrolases Carboxylic ester hydrolases 100 Results Polyacrylamide gel electrophoresis Starch gel electrophoresis 105 Discussion PART TWO - TOXICOLOGICAL STUDIES 113 Introduction Historical 114 The phenomenon of dross- and multiple resistance 7 117 Materials and Methods Insect material Insecticides used 122 Topical application of insecticides Holding and measurement and recording of response 126 Systemic application of insecticides 127 Results and Discussion 150 APPRAISAL AND GENERAL COMMENTS 156 GENERAL SUMMARY 160 BIBLIOGRAPHY 182 APPENDIX Simple modification 'of the standard disc electrophoresis apparatus 8 Acknowledgements All research projects tend to rely heavily on the goodwill and intellectual experience of many people . This work is no exception and it is with pleasure that I thank, My supervisor, Dr. G. Murdie for his constant help, support and encouragement all along but especially for his acute and vigilant criticism of the manuscript. Drs. C.T,Lewis and S.Ahmad, Professor M.J.Way and Drs. P.H.Needham and R.M.Sawicki the latter of Rothamsted Experimental Station) for many useful hints and suggestions. Professor T.R.E.Southwood for permitting my stay here and for providing the necessary facilities° Mr. Roger H. Williams for the use of his enormous talent in photography, and The many members of the Field Station who helped indirectly by creating an atmosphere of amiability in and around the laboratory and by providing provocative and stimulating conversation without which research would be drudgery. Finally may I add that any error or weaknesses of judgement that remain are entirely my own. This project was carried out while I was in receipt of an IDA scholarship provided through the courtscy of the East Pakistan Agricultural University, Mymensingh. 9 PART ONE Biochemical Studies 10 General Introduction In spite of the introduction of new chemicals every year insecticide resistance remains a serious threat to the control of insect pests. This is evident by the fact that by 1968, 224 species of insects, ticks and mites had become re- sistant to almost all classes of pesticides in use (Brown,. 1968). Of these the total of organophosphate-resistant species number 54 insects and acarines and the list keeps growing. The problem though immense is not entirely unresolved. Over the years there has been a gradual accumulation of data on the resistance phenomenon as the problem is tackled from several different aspects. Thus, cross-resistance patterns of tolerant species have provided useful information on how degree of resistance may be correlated to the structural make- up of related compounds (Busvine, 1970), This information be- comes more relevant if it is linked with a quantitative study of the detoxifying enzymes involved. A good case in point is that of a malathion-resistant strain of Culex tarsalis. It was shown that the R-strain had 3 times more carboxyesterase than the S-strain, the enzyme being active against the COOC H group of malathion but not against the COOCH group 2 5 3 of carboxymethyl malathion (O'Brien, 1967). Since most insecticides, if not all, arc modified enzymically following their absorption by insects (Winter- 11 ingham, 1965) other angles from which the problem may be studied would be (i) the localization of the degrading enzymes in the tissue components (ii) the kinetics of these enzymes on various substrates and the effect of specific inhi- bitors and (iii) the identification of the different isoen- zymes implicated, by electrophoresis. Consequently, the following work was designed along these lines to help answer a few of the questions raised thus filling some of the gaps in our knowledge and understanding of the resistance phenomenon. The ways by which insects resist chemicals are not completely understood (Agosin, 1963; Brown, 1964). Several mechanisms suggested include 1) a rapid detoxification by the hydrolysis of phosphates or phosphorothionates (Kreuger et al., 1960; Plapp et al., 1961; Bigley and Plapp, 1962; Matsumara and Hogendijk, 1964). In this case a decrease in the level of 'aliesterase' in the resistant(R)-strain has been associated with the occurence of enzymes capable of the said hydrolysis, 2) a slower penetration of insecticides through the integument of the R-strain (Kreuger Ail., loc. cit.; Mengle and Casida, 1960; Forgash et al., 1962; Farnham et al., 1965) and 3) a change or replacement in the 'bensitive mechanism" by some "insensitive mechanism" less susceptible to an insecticide (Hoskins and Gordons, 1956). The potency of organophosphates to inhibit mammalian cholinesterases (ChE) has been known for sometime but their 12 inhibitory properties against ChE and 'aliesterase' (AliE) in insects were reported only within the last 25 years (Chadwick and Hill, 1947; Metcalf and March, 1949). There is general agreement that organophosphate poisoning blocks ChEs leading to acetylcholine accumulation in connective tissues followed by failure of nerve stimuli and conduction (Smallman, 1956; Colhoun, 1959; Metcalf, 1959; Winteringham and Lewis, 1959). However, several workers have doubted the