Imperial College of Science and Technology, Ascot, Berkshire

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SOME BIOCHEMICAL AND TOXICOLOGICAL STUDIES

OF ORGANOPHOSPHATE RESISTANCE IN MYZUS

PERSICAE (SULZER-)

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 specific 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 subsequent 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

2 ABSTRACT

8 ACHNOWLEDGEMENTS

10 GENERAL INTRODUCTION

PART ONE - BIOCHEMICAL STUDIES

Section A: Histechemical localization of esterase 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. butyrate

34 Introduction

35 Materials and Methods Insect material Preparation of the homogenate Experimental details

39 Results

41 DiSCUSS1011

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.persicae 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

83 Study of the instability of OP-resistance and its association with changes in carboxylesterase 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 resistant 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 universality of this hypothesis and it is worth noting that a ChE-mutant tetranychid mite has been reported (Shahtoury and Woodage, 1968). Lord and Potter (1950, 1951, 1954) were the first to suggest that esterases other than ChEs may be involved in organophosphate poisoning. Their idea gained impetus with the discovery by van Asperen and Oppenoorth (1959) and later confirmed by Digley and Plapp (1960), that several strains of

R-houseflies had much less lAliEl than S-flies. The explanation for this discovery was that in R-flies the enzyme hydrolysed methyl butyrate (the substrate used) at a low rate and some organophosphates at a high rate while in the S-flies the enzyme hydrolysed methyl butyrate at a high rate and was poisoned by the organophosphates (Oppenoorth and van Asperen, 1960,

1961). The finding of the Dutch workers led to a spate of research work on insect esterases and their involvement in the the resistance mechanisms. Some of these are summarized below. 1:3

Enzyme kinetics of the housefly, Musca domestica, have been most extensively studied. As already stated workers at Wageningen have established the correlation between resistance and low 'AliE' activity and proved (Oppenoorth, 1959) that both of these were due to a single gene. This gave rise to the "mutant-aliesterase theory" (summarized by van Asperen,

1964) which lately has come under some criticism. O'Brien

(1967) outlines several objections to it and points out that the finding of Smissaert (1964) who showed that though R-strains of the two-spotted spider mite had lower 'AliEl activity, resistance in fact was due to the insensitive ChE in the R--strain. Similar results have been reported for a cattle tick from Australia and will be considered again later. van Asperen and others have also used several substrates to map out the esterase patterns of S- and R-strains of flies electrophoretically. According to van Asperen (1964) " theelectro- phoretic patterns were remarkably strain-specific, but no correlation between organophosphate resistance and patterns could be established". However, Velthius and van Asperen (1963) and van Asperen al., (1965) did show that one esterase band in the resistant Italian strain of houseflies was linked with resistance. Studies of insecticide metabolism in several other organophosphate-resistant strains of houseflies have provided some interesting results. Working on the G and H strains of

M. domestica, Matsum ara and Hogendijk (1964) found that the 14 main interstrain difference was the superior ability of the two malathion R-strains to degrade malathion to its monocar- boxylic acid derivative. Their evidence indicated that an increased carboxyesterase activity was a major cause of malathion resistance in the G and H strains. However, a paradox which they could not resolve was that in spite of the fact that carboxyesterase was 2-fold highef in G, the total degradation in the two strains was about the same. Mengle and Casida (1960) worked out that the ChEsof the R- and S-strains of houseflies were similar but the rate of inhibition of the enzyme in the

S-strain was more than the R-strain when paraoxon or malaoxon was incubated with whole fly homogenates. They also showed that this was not due to the differential breakdown of the inhibitor and suggested an unknown factor which protected the

ChE from inhibition but without hydrolysing the inhibitor. Un- fortunately this observation was never followed by further experiments Some detailed electrophoretic work has been done on housefly esterases (Mengle R± al., 1963; van Asperen and van Mazijk, 1965; Collins and Forgash, 1968 and Ahmad, 1968). Using vertical slab polyacrylamide gel electrophoresis Ahrnad showed that only eight enzymes hydrolysed alpha naphthyl acetate in the SKA strain compared to ten in the S-strain. In the SKA strain "low-aliesterase" was attributed to the absence of 2 esterases (E and E ) and the reduction in the activity level 6 7 of all but one of the other bands in contrast to previous 15 findings (Velthius and van Asperen, 1963 and van Asperen

and van Majizk, 1965) where the absence of a single anodic band was considered significant*. Differences in 'aliesterase' level have been shown for other dipterans. In the blowfly, Chrysomya putoria, it has been demonstrated that the R-strain had only 15 percent of the

tAliEl as compared to the S-strain; reduced eAliET activity and malathion resistance were genetically inseparable (Town- send and Busvine, 1969). Thin layer electrophoresis of Culex

pipiens fatigans indicated a more active esterase band (hy-

drolysing beta naphthyl acetate) and greater acid and alkaline

phosphatase activity in a R-strain than in a S-strain (Stone

and Brown, 1969). However, in an earlier study by Matsumara

and Brown (1961) on the larvae of susceptible and resistant

Culex tarsalis, no differences could be established in the

ChE or 'AliEl activity nor in the sensitivity to malathion,

malaoxon and in the uptake of malathion. In Japan a certain amount of biochemical work has been

done on 3 insect species, viz., Nephottetix cincticeps Uhler, Laodelphax striatellus Fallen and Chile suppressalis Walker,

all of which have acquired widespread resistance to organo-

phosphates. Work on the first two species has been summarized

by Ozaki (1969). In N. cincticeps, ChB sensitivity to malaoxon and phosphatase sensitivity to DDVP were similar in both the

susceptible and the resistant strains (Kojimaa al., 1963d).

However, a methyl butyrate splitting enzyme was found to have 16 a higher activity in the R- than in the S-strain. Ozaki and

Koike (1965) also showed a correlation between high esterase activity and malathion-resistance in these hoppers. Electro- phoresis was also used to show that one esterase band

(hydrolysing beta naphthyl acetate) was more intense in resistant strain (Kassai and Ogita, 1965). A parallel situation occurs in the smaller brown plant hopper, L. striatellus,where there is a close relationship between fenitrothion-resistance and esterase activity of E esterase band (Ozaki, 1969). Ozaki 7 suggested that ' E esterase, which is controlled by 7 co-dominant factors located on the autosomal chromosome, plays an important role in mechanism of resistance to OP'sn. For Chile suppressalis no differences were established between the R- and the S-strains either in the esterase activity, sensitivity to paraoxon or in the penetration of parathion and paraoxon. However, more of the metabolite des- ethyl paraoxon was found in the R-strain and this was suggested as a possible cause of resistance (Kojima et al., 1963a, b).

An interesting variation in the resistance mechanism has been shown for the acarincs Tetranychus urt±cae (Koch) and

Boophilus microplus (Can.). Resistance in both species is well

established (Hello, 1965; Wharton and Roulston, 1970). Smissaert (1964) provided evidence that OP-resistance in

T. urticae is due to decreased sensitivity of ChE to organo-

phosphates-- the ChE activity of the S-strnin being 3 times that of the resistant mites. He also pointed out that since 17

Hello proved that resistance in this mite (both workers used the same R-strain) is determined by only one genetical factor, the difference in the rate of inhibition should also depend on this single factor. However, investigating OP-resistance in T. urtieae Matsumara and Voss (1964) found no such difference in ChE sensitivity but established that their R-strain degraded more malathion and malaoxon, both in vivo and in vitro. Boophilus microplus (Can.) has also been shown to have two different resistance mechanisms rather similar to that found in the two-spotted spider mite (Lee and Batham, 1966 and Roulston et al., 1969).. It is important at this stage to clarify the nomenclature of the esterases investigated in this work.. It is clear from the recently published literature that most entomologists continue to use the name "ali-esterase" to describe carboxylic ester hydrolase. This is an unfortunate misnomer since this enzyme attacks both aliphatic and aromatic neutral esters (Dixon and Webb, 1964) although it is probable that a number of carboxylesterases exist. The Commission on Enzymes of the

International Union of Biochemistry recommended that "aliesterase" be given the systematic name of carboxylic ester hydrolase and a trivial name of carboxylesterase, the Commission number assigned to it being EC 3.1.1.1 (The Commission's report is summarized in Florkin and Stotz, 1964 and in Dixon and Webb, 196L pages 672-785). Therefore, carboxylesterase is 18 used throughout the text except when reference is made to the work of other researchers when tali-esterase' is used within inverted commas. 19

SECTION A: Histochemical Studies

Introduction

The histochemical localization of B-esterases in insect tissue constitutes an important aspect in the study of insecticide metabolism because members of this group of hydrolytic enzymes play an important role in insecticide poisoning. Thus, the cholinesterases are known to represent the biochemical lesion involved in carbamate and organophosphorus inhibition (Metcalf, et al., 1955; Fukuto and

Metcalf, 1956). More recently the isozymes of carboxylesterase ("aliesterase", B.C. 3.1.1.1.1) have been linked with organophosphorus resistance in houseflies (Asperen and Oppenoorth, 1959), Nephotettix cincticeps Uhler (Ozaki and Hiasyoshi, 1965 and Ozaki, 1969), Laodelphax striatellus

Fallen (Ozaki, 1969), Myzus nersicae Sulz. (Needham and

Sawicki, 1971) and Chrysomvia nutoria (Needham and Sawicki, loc. cit.). A great deal of work has been done on the histochemistry of esterases in vertebrate tissues. Nachlas and Selig- man (1949) have demonstrated the distribution of esterases in several tissues of the rat and the dog. Gomori (1952) also showed the placement of esterases in mouse tissue.

Chessick (1953), using alpha naphthyl acetate and naphthol 20

AS acetate as substrates was able to establish the esterase distribution patterns in five mammalian species, namely man, rabbit, cat, rat and mouse. However, work on the histochemical localization of esterases in insect tissues is comparatively meagre. Cholinesterases present in the thoracic ganglion of houseflies have been studied by Lord at. al., 1963), while other insects in whose tissues esterases have been localized include the American cockroach (Iyatomi and Kanehisa, 1958), Rhodnius prelims_ (Wigglesworth, 1958),

Oncopeltus fasciatus (Salkeld, 1959, 1960), Musca domestica

(Ahmad, 1968; Booth and Whitt, .1970) and Culiseta inornata

(Whilder, 1970). Excepting for the work done on Musca domestica there appears to be no published record of the mapping of carboxylesterases in different insect tissues.

Consequently this work was undertaken to map the enzymatic profiles of the carboxylesterases of Myzus persicae Sulz.

Materials and Methods

Adult, apterae (12 - 111 days old) of the 'susceptible'

(unselected) stock were used for all histochemical investigations. Since Myzus is too fragile for frozen sections even when fixed in gum-sucrose after the method of Holt and Hicks

(1961), alternative methods were adapted using sections embedded in paraffin. Attempts to obtain fresh frozen 21 sections of Myzus persicae using Bright's cryostat were unsuccessful.

Because enzymes are easily denatured it was necessary to test the esterase inhibitory effects of several fixatives, i.e., Lillies' 10 percent neutral formalin (see Pearse, 1968, page 601) and 'analar' acetone, at different temperatures and various times of fixation. This was done using the methods of Seligman, et al., 1953, but employing Asperen's (1962) colorimetric technique of estimating esterases. The latter is described in Section B (3). The results will be • presented shortly.

Procedures' usedfixing, embedding and sectioning material.

Two methods used for studying the distribution of esterases included: I. This method was slightly modified from Salkeld (1959). Briefly, it was as follows: whole, live aphids were fixed in 'analar' acetone at -20°C for 211 hours. After fixation the legs and antennae were removed and the specimens cleared in cedar wood oil for 6 hours and placed in xylene for 30 minutes at 0°C. They were then transferred to a 1:1 mixture of xylene and paralTin (M.p. 52°C) at 37°C for half an hour, placed in fresh parafrin at 56°C and held under vacuum at

15 mm Hg pressure, for 30 minutes. Finally, they were em- 22 bedded and sectioned at 7 - 10p. The ribbons were rinsed by carrying them through graded acetone/water mixtures to water and were then floated on the substrate/dye medium

(see below) for 6 - 8 hours. The sections were examined at regular intervals under a micros cope, and when they were adequately stained the ribbons were lifted onto albuminised slides, dried at room temperature and then carried through xylene and mounted in Canada balsam.

II. The second schedule was improvised to circumvent the inhibitory effect of temperature on the thermolabile carboxylesterases. Whole, live apterae were fixed in cold

(4°C) buffered, 10 percent neutral formalin fixative (Lillie, 1954) for 2L hours, after which they were washed in three changes of distilled water for a total of one hour, at

4°C (Wilder, 1970). Then, using a binocular microscope, the legs and antennae of each aphid were removed anda.longi- tudinal incision made along the dorsal side of the thorax and abdomen to enable the dye to penetrate the tissues easily. Care was taken to ensure that the internal organs were not forced out through the incision. Unlike Method I, complete specimens were placed in either of the staining media outlined below and held in vacuum at 10 - 15mm Iig for 5 minutes and then left for a period of 2-3 hours at room temperature (20°C). Subsequently the specimens were carried through graded ethanol/water mixtures to water, left in

xylene for 30 minutes and embedded in vacuum. Sections 23 were cut at 7µ and mounted in Canada balsam in the usual way (Pantin, 1969).

Staining media and procedures for esterase characterization.

Both staining media used incorporated the substrate and the azo dye. The substrate used was alpha naphthyl acetate and the basis for pinpointing the esterases involved the enzymatic conversion of 1-naphthyl acetate to 1-naphthol, which then couples with the diazotised, aromatic amine dye to form a strongly coloured, insoluble pigment - alpha naphthol diazoate. The substrate/staining media were made up as follows: 1. Based on Gurr (1958) this solution consisted of:

1 ml of 1-naphthyl acetate (250 mg in 12.5 ml of analare acetone and 12.5 ml of de-ionised distilled water) 5 ml of disodium phosphate buffer (0.2 M, pH - 7) 500 mg of

Fast Blue RR salt.

2. This medium (after Salkeld, 1959) was made up as follows: 1% solution of 1-naphthyl acetate in 50% lanalarl acetone, 1 ml; napthanil Diazo Blue B, 40 mg; 2M NaC1, 50 ml;

001M barbiturate buffer, pH 708, 20 ml and deionised distilled water, 29 ml. For characterization of the esterases the following treatments were employed:

1. For control sections the substrate was omitted 24 from the medium.

2. To inhibit cholinesterases whole , incised aphids -6 were kept in a 10 m solution of eserine sulphate at room temperature for half an hour, prior to incubation at 37°C.

. 3, Carboxylesterases were easily identified because they were inhibited by a 10-5M solution of DDVP (30 minutes at room temperature).

Results

The tests on the inhibitory effects of the fixatives revealed that maximum enzyme activity (93 - 95%), expressed as a percentage of unfixed, whole Myzus persicae in delonised distilled water at 4°Cfor 24 hours, was retained when Lillies' 10 percent neutral formalin was used at 40C for

24 hours. 'Analar' acetone fixed tissue, at -20°C for 24 hours, gave 88% activity. The results are summarized in

Table 1.1. Consequently, most fiaxtion was done with Lillies' neutral formalin. Both methods of determining esteratic zones produced areas of enzymatic activity which were reddish brown in colour. Method I (after Salkeld) however, failed to indicate carboxylesterases, which are heat sensitive. Method II, devised to avoid this problem, gave good results and was adopted throughout. Both substrate/dye media produced equally good results but medium 1, after Gurr, was preferred because 25

Time Temperature *Percentage Fixative fixed ( oc) activity left (Hours) after fixation

Deionised distilled 12 4 94.20 water 24 4 100.00

Lillie's neutral 12 4 93.02 formalin 24 4 95.50

12 4 40.55 Analar 24 4 64.44 acetone 12 -20 84.84 24 -20 88.22

*mean of triplicate determinations expressed as percentage of values for unfixed, whole M.persicae indeionised dist. water at 400 for 24 hours.

Table 1.1 - Retension of esterase activity

following fixation at different temperatures

and time periods. it was easier to prepare than Salkeld's medium.

Treatments with eserine sulphate and DDVP indicated that the esterase in the brain is a cholinesterase. The main body of the brain, i.e., the protocerebrum and the deuto- cerebrum together with the optic lobes stained deeply (Fig.

1.1). Theesterases in the mid-gut and the hind-gut, the thoracic muscles and the embryos were characterized as 26 carboxylesterases (Figs. 1.3 - 1.8). The mucosal region of the mid-gut and the rectal region of the hind-gut were deeply stained; the muscles stained only slightly. The results are given below in Table 1.2. A system of pluses and minuses has been used to indicate the comparative intensity of the areas stained.

. Intensity of Control i.e., DDVP Eserine Diagnosis Tissue stain (minus stain minus treated treated of ester- inhibitors) substrate tissue tissue ase type. Brain Incl. ++++ _ _ _ Cholin- the esterase lobes

Stomach 4F+ _ - ++4- Carboxyl- or esterase hindgut

Hindgut +++ - _ +4+ Carboxyl- (rectum) esterase

Muscles Possibly in the ++ + _ carboxyl- head aid esterase thorax

_ +4+ Carboxyl- Embryo +4+ - esterase

Table 1.2 - Summary of the results of esterase staining. (CODE: very deep stain = ++++; deep stain = ++4; slight stain = +4; very slight stain = +; no stain = -). 27

Fig. 1.1 - T.S. of head of Myzus persicae showing esterase activity in the brain. (Past Blue RR dye; substrate: 1-naphthyl

acetate)

Fig. 1.2 - T.S. of head of M.persicae showing inhibition of esterases in the brain with eserine. 28

Fig. 1.3 - Horizontal section of mid-gut of M.persicae stained to show carboxylesterase activity.

Fig. 1.4 - Magnified view of mid-gut showing esterase activity in the mucosal region. 29

Fig. 1.5 - Horizontal section of end of hind-gut of M.persicae. Dark areas show carboxylesterase activity.

Fig. 1.6 - Parasagittal section of the hind-gut of M.persicae, showing carboxyl-esterase activity. 30

Fig. 1.7 - Magnified view of hind-gut. (Rest as Fi g,1.5)

Fig. 1.8 - Some muscles of the thoracic region showing slight esterase activity. 31

Dicussion

Although fresh, frozen sections should theoretically be the best to show enzyme activity histochemically, considerable difficulties arise. The most important of these is that good frozen sections of fragile tissues are difficult to obtain; this is of particular relevance to Myzus persicae which even when fixed in buffered formaldehyde and embedded in gum-sucrose as described by Holts and Hicks (1961) failed to give good frozen sections. In addition, as pointed out by

Pearse (1968), prolonged incubation " in the substrate of unfixed material results in loss of enzyme into the medium greater than in fixed material producing false localization of enzyme, widespread deposition on the section of the production of enzymatic activity in the medium and general filthiness of the sections due to partial dissolution of its components". The method described solves this problem by allowing tissues to be fixed and embedded in paraffin which gives excellent results. The deleterious effects of temperature on carboxylesterases is overcome by staining whole, fixed insects before embedding in paraffin, a tech- neque recently used and reported by Wilder (1970).

At this. juncture it is worth saying a few words about the fixatives used. Reference to Table 1.1 shows that when using either buffered formalin or acetone, the percentage 32

activity of esterases left after fixation was higher after

24 hours than after 12 hours. This is not a new finding as

similar results were reported for formol-calcium fixed rat

liver where it was shown that more esterase was retained

when the tissue was fixed for seven days than when it was

fixed for 24 hours (Holts and Hicks, 1961); the difference

though slight (2%) is still valid. This may be explained by

the fact that the essential feature of formaldehyde fixation

is the formation of cross-links between protein end-groups.

This is optimal the longer the fixation (Pearse, 1968) and

helps to preserve proteins as near to life as possible so

that with time better fixation and hence more esterase re-

tention may be expected; unfixed proteins are lost in

subsequent treatments. As already outlined, histochemical demonstration of

esterase activity, using 1-naphthyl acetate as substrate,

has been commonly employed by many workers to study esterases

in vertebrate tissues (Nachlas and Seligman, 1949a; Gomori,

•1952; Chessick, 1953). The latter two and Metcalf et al., (1956), among others, also used inhibitors to characterize

esterases. Using the inhibitor characterization technique

it has been shown, as expected, that in Myzus persicac the

brain and the optic lobes are the zones where the bulk of

cholinesterases is present. The main areas of carboxylesterase

activity were shown to be in the mid-gut, the hind-gut and

the developing embryos. The presence of the enzymes in the 33 mid- and hind-guts parallels the distribution in the housefly. The fact that developing embryos contain a large amount of carboxylesterase may prove to be significant in understanding the function of these enzymes. It may mentioned, that in Myzus persicae, which normally reproduces parthenogenetically, differences in the levels of carboxylesterase activities in susceptible and organophosphate resistant aphids, have been associated with the phenomenon of resistance; more about this in subsequent pages. 34 SECTION B (1): Quantitative determination of B-esterases of susceptible and OP-resistant Myzus persicae by a pH-titrimetric method.

Introduction

The activity of an enzyme, such as cholinesterase, may be measured by titration with a standard alkali (Stedman et al., 1932). The pH-titrimetric method employed in the present work has been described by Glick (1937) and Alles and Hawes

(1940) and adapted by Shahtoury (1963) and Ahmad (1968). The method relies on the fact that fatty acid is produced when an enzyme acts upon an ester. For example, the decomposition of acetylcholine involves the hydrolysis of the ester according to the following reaction:

CH 0 0 CH 3 I 1 AChE 3 CH - N-F-CH -CH -C-CH ------1" + CH -C-0- + CH -N+-CH -CH OH 3 2 2 3 ' 3 3 2 2 + 3 H02 CHH3 (Acetylcholine) fatty acid). (alcohol)

In otharwords the carboxylic acid liberated is directly proportional to the amount of substrate hydrolysed. If the acid is neutralized, using pH as an indicator, by an alkali of known strength then the quantity of alkali used may be recorded as an index of the amount of substrate converted, i.e., the 35

quantity of enzyme present may be indirectly ascertained by

the amount of hydrolysis it can catalyse. This activity may

be expressed as pM of substrate split by an enzyme for a given

period at optimum experimental conditions. Often, it is also

' expressed as microlitres of alkali needed to neutralize the

acid produced. For substrates such as acetylcholine and

ethyl butyrate, the following relationship holds true(Ahmad,

1968) :

100 Fl of 0.05M NaOH 5 pM of the substrate/30 mins.

Materials and Methods

Insect material

Two strains of the green peach aphid, Myzus persicae

Sulz., were used in all determinations. The susceptible (un-

selected) strain was taken from the culture at Silwood Park

while the OP-resistant strain, obtained from Jealott's Hill,

Bracknell, was selected and maintained with dichlorvos. Other

details of the rearing technique are described fully under

Materials and Methods, in Part Two, page 117. Preparation of the homogenate 62 - 63 mg of aphid material,(adult, apterous virgino-

parae were placed in an all glass hand homogenisor with teflon

coated pestle. The homogenisor was partly immersed in crushed o Ice so that all homogenisation was done at or below 4 -u. 4 m1 of de-ionised distilled water were added and after thoroughly

36 homogenising the aphids the volume was brought up to a total of 9 ml with distilled water. Thus, 2 ml of the homogenate contained about 14 mg of insect material.

Experimental details Unlike previous workers (Shahtoury, 1963 for example), the working volume of solution per reaction vessel was modified to give a total of 12 ml instead of 6.5 ml, This was necessary because the reaction vessels used were larger (7.5 cm by 2.5 cm) with a capacity of 15 ml so that the both the electrode and the extension of the burette end could be easily placed inside the vessel during titration (Fig. 1.9). The mixture was initially buffered to pH 7 with phosphate buffer. Acetylcholine chloride was used as the substrate for determination of ChB activity and ethyl butyrate was used as the substrate for carboxylesterase. In both cases two electrolytes,

MgCl and NaC1, were used as activators of the enzymes (Shah- 2 toury, 1963). For a typical run, the concentrations and amounts

of chemicals added per vessel may be listed as follows:

Phosphate buffer (0.1M) - 1.2 ml , final molar conc. - 10-2

1? MgC12 (0.1M) 1.2 ml , tt

NaC1 (1M) 2.4 ml ,

*Acetylcholine Chloride_ - 1,2 ml , tt al U - 1,5x10-2 (0.15M) OR

t -2 *Ethyl butyrate (0.3M) - - 1.2 ml .t► I 3x10

Deionised, dist. H2O - - 4.0 ml

* substrates 37

Homogenate (62 - 63 mg/9 ml) - 2.0 ml, final conc. 14 mg/2 ml

12.0 ml total, initial working volume per vessel

The homogenate was always added last. The vessels were placed in a water bath and the mixture incubated at 37°C with constant shaking. After 5 minutes when the vessels' contents had equilibrated, the pH was adjusted to 7 with 0.05 M NaOH. The vessels were replaced in the water bath and after another

30 minutes incubation, the mixture was titrated once more with 0.0514NaOH to a pH of 7 and the amount of alkali used was recorded. During titration the vessel was turned constantly by the electric motor on which it was mounted vertically ensuring thorough mixing of the contents. Four replications were made of each determination. For experiments with inhibitors the inhibitor was added instead of or in addition to the deionised distilled water, the two together never exceeding a total volume of 4 ml. For -8 ChE inhibition a final molar concentration of 10 eserine sulphate was used. Thus, 1.2 ml of a 10-7M eserine sulphate plus 2.8 ml of deionised distilled water were added to each vessel. For carboxylesterase inhibition a final molar concen- -4 tration of 10 TOCP (triorthocresyl phosphate, purified as outlined by Aldridge, 1954) was used, i.e., 1.2 ml of 10-3M

TOCP (in 0.1M phosphate buffer plus a couple of drops of --Micro-burette (lml )

(0.05 M )

--pH Electrode Reaction ye sse I— ------

--E lectric motor

FIG.1.9— APPARATUS FOR pH T ITRIMETRY 39

Triton-X) and 2.8 ml of water per vessel. In both cases, controls (i.e., substrates minus inhibitors and/or enzymes and in the case of ethyl butyrate plus 2 drops of Triton-X) were run simultaneously. The standard incubation and titration were followed as described above.

Results

All the results are presented in Tables 1.3 to 1.6. It is evident that the ChE levels in both the strains are similar, i.e., 47.0 pl to 45.3 pl. The carboxylesterase activity, on the other hand, differs considerably.

Strain of Activity in pl of 0,05(M) NaOH Myzus persicae Replicates Mean ± S.E. used 1 40

2 50 OP- 47.0 ± 4.8 Susceptible 3 48 4 . 5o 1 40 2 45 OP- 45.3 -1,- 4,1 Resistant 3 50 4 46

Table 1.3 - Cholinesterase activity in OP-

susceptible and OP-resistant Myzus persicae. 110

Strain of Activity in Ill of 0.05 (M) Myzus persicae Replicates NaOH used Mean + S.E. 1 1090

OP- 2 1075 1080.0 ± 9.1 Susceptible 3 1085 4 1070 1 825

OP- 2 850 840.0 ± 10.8 Resistant 3 845 4 840

Table 1.4 - Carboxylesterases, hydrolysing ethyl butyrate, of OP-susceptible and OP-resistant Myzus persicae.

Activity in pl of 0.05 M NaOH Replicates Treated with ChE Control Mean ± S.E. 10-8 Mean Eserine ± S.E. iphi- 1 bited 1 /1-5 15 15.7 2 48 45.0 ± 3.0 17 4. 3.2 65.2 3 42 15

Table 1.5 - In vitro inhibition of cholinesterase activity of whole OP- susceptible Myzus persicae by eserine sulphate. (Substrate:acetylcholine chloride) 41

Activity in p1 of 0.05 M NaOH Treated with.Mean /0 Replicates Control Mean + S.E. 10-h M pure +S.E. of CarE* TOCP inhibit- od

1 1000 645 65 2. 0 2 1120 1048.0+7.5 660 + 7.6 37.8 3 1020 650 •

Table 1,6 - In vitro inhibition of *carboxylesterase activity of whole OP-susceptible Myzus persicae by pure TOCP. (Substrate - ethyl butyrate)

The resistant strain shows a 22.2% decrease in its overall carboxylesterase activity compared to that in the unselected strain (840 pl vs 1080p1). Inhibition experiments with the susceptible strain indicated that at a final molar 8 concentration of 10 eserine could inhibit 65 percent of the

ChE activity, Only 37.8% of carboxylesterase activity was in- -4 hibited when a final molar concentration of pure, 10 M triorthocresyl phosphate (TOCP = TOTP) was used (Table 1.6).

Discussion

That there were no differences in the in vitro activity of esterases of OP-susceptible and OP-resistant Myzus persicae to hydrolyse acetylcholine is comparable to the situation reported for several other insect species such as Musca 112 domestica and Nephotettix cincticeps (see General Introduction).

However, there was a difference in the in vitro esterase activity of the two strains to ethyl butyrate: more activity was noted in the S-strain than in the R-strain. Such a difference has also been reported for the housefly (Ahmed, 1968,e.g.) and for Chrysomya putoria (Townsend and Busvine, 1969). As already recorded for M. persicae (Needham and Sawicki, 1971) and detailed in the work described later in this thesis esterases insensitive to eserine (at 10-7 Mj and hydrolysing alpha naphthyl acetate are in greater quantities in the R-strain than in the S-strain. This discrepancy of esterase activity towards different substrates is an interesting finding and suggested a more thorough study of ethyl butyrate hydrolysis by carboxylesterases of M. persicae.

Although the pH-titrimetric method of esterase study was suitable for work where great accuracy was not required it could not be used in the more sensitive work described in Section B (2) because of several shortcomings of the method which are as follows: (i) The method is generally cumbersome and time consuming because titration involved manual drop by drop determination. Thus the number of samples that could be handled in one day was limited. (ii) It was found that inaccuracies arose when the pH was sometimes buffered beyond the required level of pH 7 as a result of poor droplet control. (iii) Though not a serious problem, for inhibition studies 43

titration may not give the best results (Bernheim and

Bernheim, 1936). (iv) This technique was found to be very insensitive as it required large amounts of insect material per assay. This was by far the greatest disadvantage. In view of the above it was found expedient to use the colorimetric method of Hestrin (1949) with necessary modifications. The details are given in the following pages. The implications of the findings reported in this section will be commented upon fully in Section B (2). 44

SECTION B (2): Studies of some B.eSteraSeS of OP.-susceptible and OP-resistant Myzus'persicae rising Hestrin's colorimetric method.

Introduction

In 1949 Hestrin outlined a procedure by which one could estimate short chain carboxylic acid esters based on their ability to react with hydroxylamine quantitatively in an aqueous alkaline solution. Subsequently, several workers, among them Metcalf (1951), Robbins et al., (1958), Bigley and

Plapp (1960) and Abdallah (1963) have modified this technique to study ChE and carboxylesterase activity especially in houseflies. This hydroxamic acid test for esters relies upon the reaction of hydroxylamine, :NH2OH, with an ester, the summary expression being:

RC OH RC R' OH + :NH2 O R' NHOH

(hydroxamic acid)

The test depends upon the fact that hydroxamic acids give deeply coloured (red to purple) complex salts with ferric ion, ferric chloride being commonly used. Thus the amount of u.nhydrolysed substrate (an ester) is determined colorimetrically and this,substracted from the total amount of substrate 4.5 in the control, provides the amount of the ester hydrolysed by the enzyme under investigation.

The following experiments were based on the above method as modified by Robbins et al., (loc. cit.) and have been used to study the B-esterases of Myzus persicae which act on acetylcholine chloride and ethyl butyrate. As noted earlier this technique has several advantages over the pH-titrimetric method especially in the comparatively smaller amounts of insect material needed per assay. Moreover, it also has an advantage over Gomori's colorimetric technique detailed in

Section B (3), as it enables one to study the action of ChE and carboxylesterase on specific substrates such acetylcholine chloride and ethyl butyrate.

Materials and Methods

Adult, apterae of both OP-susceptible and OP-resistant strains were used. The preparation of the homogenate (enzyme) was as described for the pH-titrimetric method except that in the case of ChE determination each estimation involved only

30-aphids per ml of water whereas for carboxylesterases 15

aphids per assay were adequate.

Reagents used

The reagents used were similar to those described by

Robbins al., with appropriate modifications in the concen- 46 trations and volumes employed (Higley and Plapp, 1960). The reagents used were: 1. Phosphate buffer A 0.04 M phosphate buffer, pH 7, was prepared by diluting a stock solution of 0.4 M concentration made up with K2HPO4 and KH2PO4 in deionised distilled water.

2. Substrates - (a) Acetylcholine chloride - a 3 x 10-2 M stock solution was prepared and diluted as required. (b) Ethyl -2 butyrate - all dilutions required were prepared from a 3 x 10 M solution to which a drop of Triton-X had been added to produce a homogenous mixture. Both substrates were made daily from the stock solutions which were freshly made every fourth day.

3. Hydroxylamine hydrochloride - A 2M. solution was used and this was freshly prepared every week,

4. Sodium hydroxide solution - 305M solution was used,

5. Alkaline hydroxylamine - This solution was freshly prepared every day just before the analyses by mixing equal volumes of 3 and 4. 6, Hydrochloric acid (concentrated, sp. gr. 1,18), diluted with two parts by volume of distilled water, and 7. Ferric chloride - a 0.37M solution made up in 0,1N HC1 was used.

Preparation of standard curves Standard curves were prepared by reacting 1 ml of the appropriate amount of the substrate and 1 ml of phosphate buffer (pH 7, 0.04M) with 2 ml of alkaline hydroxylamine for several minutes when 1 ml of the acid solution (6) was added Fig. 1.10 - Standard curve for the determination

of acetylcholine chloride.

Fig. 1.11 - Standard curve for the determination of ethyl butyrate. 47 48

1 .0

0;6 0 H C

0;2

227.1 454. 2 681.3 908.4 ACETYLCHOLINE CHLORIDE pms)

1.2

to A 0.8

0 o.

174 348 696 1392

ETHYL BUTYRATE (GMs) 49 followed by 1 ml of the ferric chloride solution. After another 10 minutes the solution was filtered and its colour density determined using a Unlearn Colorimeter SP Series 2 at

La,. 540 mil for acetylcholine chloride and ca. 620 mp. for ethyl butyrate. A blank was obtained in exactly the same manner only the order of addition of alkaline hydroxylamine and hydrochloric acid was reversed. The optical densities were then plotted against the p.gm of the substrate present per assay (Figures

1.10 and 1.11).

Basic plan for esterase determination

A typical procedure for esterase determination involved the following: Blank > 1 ml of (2) + lml of (1) + 1 ml HC1 + 2 ml of (5) + 1 ml FeC13.6H20 (A)

Control----> 1 ml of (2) + 1 ml of (1) + 2 ml of (5) + 1 ml HC1 + 1 ml FeC13.6H20 (B)

With enzyme---1 ml of (2) + 1 ml enzyme + 2 ml + 1 ml FeC1 .6H 0 . . . (C) of (5) + 1 ml HC1 3 2

The order in which the solutions were added, from left to right as indicated by arrows, was important as this determined the non-colouration of the blank. The enzyme and substrate and the buffer and substrate were incubated for 30 minutes at 37°C after which the rest of the solutions were added. The control sample provided a correction factor for any non-enzymic hydrolysis of the substrate. ( Note - the addition 50 of each solution was always followed by a vigourous swirling of the mixture). The optical density of the colour produced was converted to micrograms of the substrate with reference to the standard curve. Then substracting (C) from (B) gave the ligms of the substrate hydrolysed. When inhibitors were used they were incorporated with the substrate solution. in such cases a fourth additional test tube was required, thus:

With inhibitor---->1 ml of (2) with appropriate conc. of inhibitor + 1 ml of enzyme + 2 ml of (5) + 1 ml HCl + 1 ml FeC1 . 3 61120 . 00• ...... (D)

(The substrate-inhibitor-enzyme complex was incubated for 30 minutes at 37°C before the addition of the rest of the solutions). Substracting (D) from (B) provided the quantity (in pgms) of substrate hydrolysed in the presence of the inhibitor. Unless otherwise stated all colorimetric readings were based on three replications per sample.

Results

Study of substrate concentrations Two substrates, acetylcholine chloride and ethyl butyrate, were used to evaluate the esterase activities of the susceptible and resistant strains of Myzus persicae. The concentrations employed and the results obtained are given'in Table 1.7.

The data confirmed the titrimetric findings which showed that there was no significant difference between the total ChE 51 of the strains (Table 1.7). Consequently, the Lineweaver-

Durk plot for determining Michaelis constant (Dixon and

Webb, 1964) was not constructed. Data presented in Appendix

Table I ), Because substrate concentration is one of the important factors which determine the velocity of enzyme reactions,knowing the Michaelis constant (i.e., Km value i.e., substrate concentration at which the reaction proceeds at half its maximal initial rate) of an enzyme helps to character- ise it.

The difference between the quantities of the ethyl butyrate splitting esterase in the two strains was also confirmed although the percentage differences between the two strains are not consistent for the two methods (titrimetric

22.2%; colorimetric 13.5%). This will be discussed later. A double-reciprocal plot for the carboxylesterases showed that theLla value for this enzyme of the OP-susceptible strain was

9.7 x 10 5M while that for the R-strain was 8 x 10-5M.

Inhibition of some of the B-esterases using eserine sulphate and dichlorvos.'

The in vitro susceptibility of the two B-esterases to different concentrations of specific inhibitors, viz., eserine sulphate and dichlorvos, is presented in Fig. 1.13 and 1.14, -8 10 M eserine sulphate gave a 50% inhibition of the CIXs from ' both strains indicating similar in vitro susceptibility to this carbamate. The carboxylesterases in both strains also appeared to be equally affected by given concentrations of

Substrates and *Activity in ligms of acetylcholine chlo- concentrations 'ride or ethyl butyrate hydrolysed. (M)

**Acetylcholine OP-susceptible OP-resistant chloride

-3 0,31 75 x 10 2.3 1.7 0,75 x 10-3 18.0 19.4 1,50 x 10-3 26.1 25.9 1.75 x 10-3 27.o 26.3 3,00 x 10-3 27.4 26.8

***Ethyl butyrate -2 0;1875 x 10 8,2 6.4 0,3750 x 10-2 15,0 12.8 -2 0,75 x 10 22,2 19.1 -2 1.5 x 10 24,3 21.2

*mean of 3 replicates; ** per 30 aphids; *** per aphid

Table 1.7 - The influence of substrate Con-

centrations on the esterase activities of OP-susceptible and OP-resistant Myzus

persicae.

dichlorvos,

Discussion

Both cholinesterase and carboxylesterase are known to 53 0-0 S-strain (KM = 0.97 x 10-1M) a—UR-strain (KM = 0.8 x 10 4M)

Fig. 1.12 - Double reciprocal (Lineweaver-Burk) plot of 1/v° vs 1/S for the reaction catalysed by carboxylesterases of M. persicae. (Substrate-ethyl butyrate; see Appendix Table - I ). 54 Fig. 1.13 - In vitro inhibition of whole aphid ChE activity by different concentrations of eserine sulphate. (Substrate-acetylcholine chloride: 1.5x10-3M; temp. 37°C)

Fig. 1.14 - In vitro inhibition of whole aphid carboxylesterase activity by different concentrations of dichlorvos(Substrate- ethyl butyrate: 3.75x10-3M)

55 Control 20 0 0/11111.111 • 0101•016 01101.1M• • MEMO • .1=•n•B

A TS

0 04 10 o ao z- 1-4 0 0 Fig. 1.13

9 8 7 pi (-log molar concentration)

Control 30.0

P a 22.5

rr3 W H ■ 15.0

705

pI (-log molar concentration) 56 catalyse a variety of substrates (Dixon and Webb, 1964).

However, it has been shown that acetylcholine chloride and ethyl butyrate are selectively hydrolysed by these two enzymes respectively.(van Asperen, 1959; Shahtoury, 1963).

Except for the isolated example of the work done on Macro- siphon pisi (Kltb.) by Casida (1955) study of aphid esterases hydrolysing the above esters has not been reported so far. In the experiments described in Sections BM and (2) it has been demonstrated that esterases hydrolysing acetylcholine chloride occurred in equal amounts in both the 'susceptible' and the resistant strains of M. persicae. On the contrary, ethyl butyrate splitting esterases in the unselected strain were more active than those of the R-strain. Thus the R-strain -2 showed 13.5% less activity than the S-strain at 1.5x10 molar concentration of ethyl butyrate. Although this confirms the observations noted earlier with the titrimetric method,an apparent discrepancy lies in the percentage differences between the two strains for the two methods used. i.e., 22.2% vs

13.5%. It is difficult to explain this difference but two possible reasons will be considered. Perhaps the most acceptable is that the total esterase content of insects tends to fluctuate over a period of time. Such variations have been noted in houseflies (Cook and Forgash, 1968) and is further borne out by the findings for M.persicae reported in Section

B(3) (see Fig. 1.27). Second, as indicated earlier, the titrimetric method has many shortcomings which tend to give vari- 57 able results and this makes its findings less reliable.

However, the direction of the difference is important since esterase activity towards ethyl butyrate is less in the OP- resistant strain than in the unselected strain. The implication of this becomes more striking when it is compared with the

1-naphthyl splitting esterases of the two strains. This will be discussed fully at the end of Section B(3).

Inhibition studies did not indicate differences between the strains. ChEs from both strains were equally sensitive to different concentrations of eserine sulphate. Since the total ChB content was also similar for both strains of

M.persicae the types of resistance mechanisms,involving ChB, shown for Tetranychus urticae and Boophilus microplus (see

General Introduction page ) may be ruled out for this particular resistant strain of Myzus. Of the two phosphoric esters used to test the sensitivity of ethyl butyrate hydrolysing carboxylesterases, DDVP proved more potent than TOCP. The latter has been reported by several workers as producing in vivo inhibition of 'aliesterase' activity (Stegwee, 1959,

1960; Myers and Mendel, 1953; O'Brien, 1960; Abdallah, 1963).

In vivo inhibition of 'aliesterase' in Musca domestica, at

200 pg/insect, was 100% according to Stegwee (1959, 1960) but

Abdallah (loc. cit.) at similar doses failed to get more than

50% inhibition.

As suggested by Myers at Al., (1955) in vivo inhibition of 'aliesterase' in insects is not due to TOCP itself but due 58 to one of its toxic metabolites. From this fact alone it is easy to see why in vitro inhibition of esterases with TOCP is less absolute. In the experiments reported only about 38% 4 of carboxylesterase was inhibited at 10 m. This is less than the percentage inhibition obtained by Myers and Mendel (1953) for rat liver slices and by Cook and Forgash (1968) for housefly 'aliesterase'. The observed differences could be due to a TOCP metabolite inhibitor of carboxylesterases which was absent from the purified TOCP used in the present study. As

Aldridge (1954) has shown, in vitro esterase inhibition by TOCP is often due to its impurities. Because of the incomplete inhibitory effect of TOCP further studies with it were not undertaken. Dicblorvos, on the other hand, was quite satisfactory and the ethyl butyrate hydrolysing carboxylesterases of both strains of Myzus persicae were found to be equally sensitive to given concentrations of this organophosphate. 59 SECTION B(3): Quantitative studies of some of the B-esterases of OP- susceptible and OP-resistant Myzus persicae by a colorimetric method.

Introduction

The B-esterases are indubitably important in the mechanism of insecticide resistance (O'Brien, 1967). It is well established that (i) organic phosphorus esters inactivate ChEs and so interfere with the neuro-muscular junction causing stimulation, twitching and finally paralysis (Braunholtz, 1968) and, (ii) that insecticide resistance may involve the presence of modified esterases, which hydrolyse the toxic agent rendering it harmless. Hence it is obvious that a knowledge of the B-esterases is essential in the study of resistance. In 1953, Gomori published a sensitive colorimetric method for studying human esterases. Later van Asperen (1962) using the same technique with slight modifications, presented a fairly detailed study of housefly esterase hydrolysis of 1- and 2-naphthyl acetates and the interaction of these enzymes with some inhibitors. The following work employs the same method with small modification.

Briefly, the method involves the enzymatic breakdown of phenyl- or naphthyl esters to phenol or naphthol. These. may be coupled with diazonium salts (E-41==.4*--9[4E.E.R] ) to produce stable dyes whose optical density may then be measured,

A typical reaction is as follows:

OH CH COOH + Enzyme 3

(1-naphthyl acetate) (1-naphthol)

Cl +N==N N==N+ Cl

ocH3 oc 113

(Diazo blue B)

OH OH N=N

1-naphthol diazoate .(blue colour)

Thus, a measure of the product of hydrolysis, i.e., alpha naphthol, serves as an index of the amount of enzyme which catalysed the reaction.

*coupling may also occur at the,.l►th position 61

Materials and Methods

Insect material

In all experiments both the OP-susceptible and the

• OP-resistant strains of Myzus persicae were used. The aphids

used were adult apterae which had been removed from the host

plant at least six hours prior to homogenization. A number of ,aphids of a given strain, to give a final

concentration of one aphid per ml of water, was thoroughly

homogenized in a Potter-Elvehjem hand homogenizor with a teflon

plunger. The homogenizor was partly immersed in crushed ice

to prevent high temperature denaturing the enzymes. In all

cases the homogenate was freshly made up and used within hours

of preparation.

Experimental details

Reagents used The concentration of the different chemicals used

basically followed those listed by van Asperen (1962) with

slight modifications as required:

(1)Enzyme preparations - As described above.

(2)Phosphate buffer - A stock solution of 0.4M phosphate buffer, pH 7,was stored at 4°C and diluted to give a 0.04M

solution as and when required.

(3)Substrate solutions - -.These were prepared by diluting

a stock solution of 1- or 2-naphthyl acetate or butyrate in acetone and storing it at -20°C. Fresh stock solutions were 62 made regularly. .1. ml of the stock solution was made up to

100 ml with 0.04M phosphate buffer , pH 7, to give the required concentration in 4 ml of the solution instead of 5 ml as used by van Asperen. A typical example would be 1 ml of the stock solution containing 55.75mg of 1-naphthyl acetate in

10 ml acetone, diluted to 100 ml with phosphate buffer so that

It ml of this would give 3 x 10 414. When a series of different concentrations were used appropriate dilutions were made from the highest concentration. For example, to get a concentration 4 of 6 x 10 DI solution from a 100 ml solution of 12 x 10-4m,

50 ml of the latter was diluted with 0.5 ml acetone and 49.5 ml of phosphate buffer (0.04M, pH 7) to give the required concentration. (4)Inhibitors used - The inhibitors used (eserine sulphate, dichlorvos, diazinon and diazoxon - see Part Two for details) were made from stock solutions and diluted with phosphate buffer,to give the required concentration(s), and then stored at 4°C. These were utilized within 24 hours of preparation. In case of diazinon and diazoxon, a couple of drops of Tween 20 was added to the solutions and to the controls) to produce a homogeneous emulsion.

(5)Diazo blue - sodium laurylsulphate solution (=DBLS solution) - A 1% w/v water solution of Diazo blue B (tetrazotised di-o-anisidine; also known as Blue BN salt or Fast Blue 13) and a 5% w/v water solution of sodium laurylsulphate were made as stock solutions. When required, 1 part of the former was mixed

63 with 5 parts of the latter to give the working solution.

A typical experiment, using inhibitors but without replicates, would involve three test tubes, labelled B,C, and

I, containing the following:

B(blank) C(control) I(with inhibitor) 4 ml 4 ml 4 ml . . . . . (3 ) 1 ml 1 ml - . . . . . (2) 1 ml - 1 ml . . . . . (11) - 1 ml 1 ml . . . . . (1) 6 mi 6 ml 6 ml

The homogenate. (1) is added after the mixtures are equilibrated at 37°C for several minutes. Then the whole is incubated for 30 minutes, with constant shaking, after which 1 ml of the DBLS solution is added to stop the reaction. 5 - 10 minutes later a stable blue (or red) colour develops and the optical density is measured against the blank with a Unlearn SP Series 2 colorimeter using a 1 cm cell with filters 3 or 6 - the maximum absorption lying between 540 and 620 mia (depending on whether 1- or 2- naphthol is involved). Calibration curves for 1- and 2-naphthol

Standard curves were obtained by reacting 6 ml of 1- or 2-naphthol solution of appropriate concentrations (in 1% acetone 0.04 M phosphate buffer, pH 7) with 1 ml of DBLS solution. The naphthol concentrations were plotted against the 64 optical density (Figures 1.15 and 1.16). The optical densities from various experiments were referred to the standard curves and converted into micrograms of 1- or 2-naphthol pro- dUced.

Results

Effect of enzyme concentration

To study the influence of homogenate concentration on whole aphid carboxylesterase activity, different quantities of aphid material, of both OP-susceptible and OP-resistant strains, viz,, 0.25, 0.50, 1.0, 2.0, 2.25 and 2.50 aphids per assay, were used to evaluate the amount of 1- or 2-naphthol acetate, at 3 x 10-4M and 1.2 x 10-5M concentrations respectively, that were hydrolysed to 1- or 2-naphthol. In order to block all ChE activity 2 x 10-7M eserine sulphate was added in each assay, to give a final molar concentration of 10-7M.

The results are given in Figures 1.17 and 1.18.

It is evident that an increase in enzyme concentration leads to an increase in the amount of naphthol produced in both strains approaching an asymptote at about 2 aphids. It may also be noted here that whereas there is no appreciable difference in the carboxylesterase activity of susceptible and resistant strains towards 2-naphthyl acetate, considerable difference exists when 1-naphthyl acetate is the substrate. Because of this most later assays were done with 1-naphthyl acetate only.

1.0

Y 0.8 SIT 0.6 0 AL DEN

IC 0.4 OPT Fig. 1.15 - Standard curve for the 0.2 determination of 1-naphthol. 0

3.6 28.84 57.68 115. 36 1-NAPHTHOL (ligms)

1.2

1 .0 Y 0.8 NSIT DE

L 0.6 ICA PT

O 0.4 F.ig. 1.16 - Standard curve for the 0.2 determination of 2-naphthol.

0.9 3.6 14.4 28.8 2-NAPHTHOL (pLus) NAPHTHOL(T IGMS ) 0---00P-susceptible M---00P-resistant Fig. 1.17-Theinfluenceofhomogenate 0.50

30 minutes). acetate: activity oftwostrains ofMyzus concentration on carboxylesterase persicae (Substrate: 1-naphthyl 10 HOMOGENATE CONCENTRATION - 1 7M; temp.37 .0

(aphids/sample) 3 x 10 ° -4 C; incubationtime: M; escrine:2x 2.0

2.50 66 67

0---00P-susceptible CI—MOP-resistant

8.0

4.0

0.50 1.00 2.00 2.50 HOMOGENATE CONCENTRATION (aphids/sample) Fig. 1.18 - The influence of homogenate concentration on the carboxylesterase activity of two strains of Myzus persicae (Substrate: 2-naphthyl acetate- 1.2 x 10-5M; rest as Fig.1.17) 68

Effect of temperature on ox activit y

The carboxylesterase activity of both strains towards

1-naphthyl acetate was studied at five different temperatures, o o i.e., at 13°, 27°, 37°, 45 and 54 C. For both strains carboxylesterase activity was least at the extreme temperatures, i.e., 13° and 540C and maximum at 37°C (Fig. 1.19) and accordingly the temperature for incubation was maintained o at 37 for all further experiments.

Effect of different concentrations of 1- and 2-naphthyl acetate and 1- and 2-naphthyl butyrate on the carboxylesterase activity of susceptible and resistant strains of Myzus persicae.

The influence of substrates and their concentrations was studied to determine the rate of the hydrolysis and to estimate Michaelis constant for the substrates used. The substrates used and their concentrations,together with the carboxylesterase activity (pgms of substrate hydrolysed to naphthol/aphid in 30 minutes at 37°C), are presented in Table 1.8. In each case ChE activity was eliminated by the use of an appropriate amount of eserine sulphate. The difference between the carboxylesterase activity of the two strains toward 1- and 2-naphthyl butyrate and 2- naphthyl acetate was small. Although double reciprocal trans- formations were made for a Lineweaver-Burk plot (Appendix

Table III) graphs were not drawn because the differences were a W NAPHTHOL(p gms/aphid) Fig. 1.19 -Effectoftemperature oncarboxyl- 13 OP-resistant Myzus persicae esterase activity ofOP-susceptible and 1-naphthyl acetate: 3 x10-1M) 27 TEMPERATURE ( 37 ° C) 0-0 Cl'-0 l OP-resistant OP-susceptible (Substrate: 69 70

Substrate and *Activity in pgms of 1- and 2- concentrations naphthol produced per aphid (N) 1-Naphthyl acetate OP-susceptible OP-resistant -4 0.75 x 10 6.6 244 -4- 1.5 x lo 11.0 34.7 -4 3.00 x 10 17.2 43.9 -4 6,00 x 10 23.1 48.7 1.20 x 10-3 26.4 48.3 2-Naphthyl acetate -6 1.50 x 10 1.0 0.9 -6 3.00 x 10 1.9 1.9 -6 6.00 x 10 3,8 307 -5 1,20 x 10 7,6 7.5 2.40 x 10-5 9.8 9.8 1-Naphthyl butyrate 0.75 x 10-5 3.o 2.9 1.5o x 10-5 6,9 6.6 3.00 x 10-5 13.3 13.2 6.00 x 10-5 22,3 24.7 -4 1,20 x 10 37.2 37.6 2-Naphthyl butyrate -6 3.00 x 10 0.8 1.0 -6 6.00 x 10 1,5 1.7 1020 x 10-5 3.2 3,1 2.4o x 10-5 5.8 6.o 4,80 x 10-5 7.9 8,3 * mean of three replicates Table 1,8 - The influence of substrate concentrations on the carboxylesterases of OP-susceptible and OP-resistant Myzus persicae. 71

OP-resistant (Km = 1.25 x 10 4M) \ 0.---01 OP-susceptible (Km = 3.15 x 10-4M)

1 ,,(mm -1 S • Fig. 1.20 - Double reciprocal (Lineweaver- Durk) plot of 1/v0 vs 1/(S) for the reaction catalysed by carboxylesterases of Myzus persicae at 37°C, for 30 mins. (Substrate: 1-naphthyl acetate; see Appendix Table - II ) . 72

insignificant. However, the rates of hydrolysis of 1-naph-

thyl acetate by the two strains were very different (Table

1.8 and Fig. 1.20). The Km value for 1-naphthyl acetate for

the R-strain was 1.2 x 10 4M whereas that for the S-strain -4 was 3.15 x 10 m.

In vitro effect of inhibitors on the carboxylesterase activity of the strains of Myzus persicae. Eserine (physostigmine) sulphate For all inhibition studies the controls run have been

plotted in the respective graphs to help evaluate the rate

and percentage of inhibition. For eserine sulphate a range / -8 of six concentrations was used 00 to 10-3 M final concen-

tration). The results (Fig. 1.21) indicate some interesting differences. It is evident that at 10-7M concentration eserine

sulphate proportionately inhibited the esterase activity in both the susceptible and resistant strains. However, as the

concentration of the inhibitor was increased more esterases

were inhibited in the S-strain but the inhibition rate tended -3 to level off at 10 M. Thus it is apparent that the R-strain

esterases hydrolysing 1-naphthyl acetate were less sensitive

to eserine sulphate inhibition than those of the S-strain and

this difference was more marked at the higher concentrations.

The implication of this observation will be discussed later.

Diazinon and diazoxon

The in vitro effects of diazinon and diazoxon on carboxylesterases (ChE activity being suppressed with eserine -4. NAPHTHOL(1 1gms/aphid) 0—OOP-susceptible U--30P-resistant Fig. 1.21- 8

esterases bydifferent concentrationsof acetate - 3x10 eserine sulphate. (Substrate:1-naphthyl pI (-logmolarconcentration) 7

In xi - 6r° 6 inhibitionofwhole aphid

-4 M) 5

73 3 71t

hormo llmbsomme 11.1,2101 101.1•1110.1.1.11:1

101---400P-resistant 0.---000P-susceptible 0--10 Control ) d hi /ap ms ig (t OL NAPHTH

HA 20 ALP

5 pi (-log molar, concentration)

Fig. 1.22 - In vitro inhibition of whole aphid carboxylesterasc activity by different concentrations of diazinon. 75

....-•...... •• ct_ .....•• *won atm • • INOig OMB • ••••••• • •MO•13 Olnl• • ••••• @mama • ammo • c.•••■•

CI-0 OP-resistant

35 0.0 OP-susceptible 0.---0Control

O •—•-0—•• ••••• ••••••10—• ONSO • a . ila••••11 • •I'Mee • MVO • •••••■• • ...DM • •7=111 • 01=W 0 0 O 15

9 8 7 6 5 t. pi (-log x 3 molar concentration) Fig. 1.23 -In vitro inhibition of whole- aphid carboxylesterase activity by diazoxon. (Substrate: 3 x 10-4 M ) 76 sulphate) of the two strains were as expected with diazoxon

(an oxygen analogue),the more potent inhibitor of the two. -9 Inhibition is higher at 3 x 10 M for diazoxon than it is for diazinon (Fig. 1.22). As diazoxon concentration increases there is rapid suppression of enzyme activity (Fig. 1.23) the curves -5 levelling off at 3 x 10 M. For diazinon the increase in inhibition with concentration is comparatively gradual, maxi- -1 mum inhibition being approached at 10 M. By interpolation in Figures 1.22 and 1.23 it can be estimated that the I50

(50% inhibition) for diazinon is approximately 10-3M for both -7 strains while for diazoxon it is about 3 x 10 M for the S- -8 strain and slightly more than 3 x 10 M for the R-strain. Dichlorvos

Of the several chemicals used dichlorvos was the most effective inhibitor of in vitro activity of carboxylesterases acting on 1-naphthyl acetate. Fifty percent inhibition was attained at a concentration of less than 10 5M in both strains but the R-strain enzyme is slightly more resistant to inhibi- -7 tion up to 10 M. Complete inhibition would perhaps require -3 a concentration of 10 M. It is worth noting as Needham and

Sawicki (1971) have already pointed out, that "AliB" of aphids seems less sensitive than "AliE" of houseflies to inhibition by organophosphorus insecticides.

Inhibition of carboxylesterases in the presence of the substrvto: 1-naphthyl acetate.

The object of this experiment was to determine whether 77 ...... -- Control OP-susceptible OP-resistant

7 5 pI (-log molar concentration)'

Fig. 1.24 - In yitro inhibition of whole aphid carboxylesterases by different concentrations of dichlorvos. 78 the in vitro protection of the enzyme varied when it was mixed with the substrate and allowed to react with it several minutes before the addition of inhibitors. The design of the experiment was similar to that of van Asperen (1960) though the technique adopted was the same as that described earlier on in the thesis. As before eserine sulphate was added to block ChB activity. Briefly, the procedure was as follows: the enzyme was allowed to react with the substrate , 1-naphthyl -h acetate,3 x 10 M, for 15 minutes. At the end of this period the reaction in two of the control test tubes was stopped and the amount of 1-naphthol produced, estimated. Inhibitors were added to the remaining test tubes. Subsequently, at 15 minute intervals (for a total of 75 minutes) some of the reactions were stopped and the amounts of 1-naphthol produced, determined. Because of the complexity of the operation and the need to maintain the time schedules strictly, only two replicates per reading were used. From previous experience it was found that the technique used produced only marginal variations between sets of readings so that 2 replicates were considered adequate. The results are presented in Table 1.9 and in

Figures 1.25 and 1.26. The addition of 1-naphthyl acetate

afforded good protection to the carboxylesterases especially

against dichlorvos. The protection obtained decreased in the

order dichlorvos> diazinon'> diazoxon. In the dichlorvos ex-

periment the differences between the two strains were very

marked since at the end of the 75 minutes about 50% recovery Final Strain inhibitor of Alpha naphthol produced per aphid Treatments cones. Myzus Time (minutes) (NO persicae 15 30 45 • 6o 75 *s 10.6 18.1 22.5 27.8 31,8 Control - R 34.3 42.4 49.8 54.2 55.7 S inhibitor 15.2 20,4 23.5 25.3 Dichlorvos 10-7 R added 39.8 45.4 49.5 50.1 s 11.6 12.9 15.o 15.5 ft 71 105 R 36.3 37.4 42.o 44.4 plus a S 10.3 18.8 25.1 28.1 30,1 Control drop of Tween 20 R 31,0 38.8 46.1 50.0 51,5 S inhibitor 15.6 19.1 21.5 24.3 Diazinon 10- 4 R added 34.4 38.6 42.8 44.0 s 13.1 14.8 16.8 17.5 Diazoxon 10-6 n R 32.4 34.0 36.0 38.8

*S = susceptible; R = resistant Table 1.9 - In vitro inhibition of whole aphid carboxylesterase in the presence of the substrate, 1-naphthyl acetate \I) (3 x 10-4M),by three different inhibitors. 80

(at 10-5M) was evident in the R-strain as compared with less than 25% in the S-strain. Such a difference was not found with diazinon and diazoxon. The full implication of this finding will be considered in the Discussion.

Dichlorvos (10-7M) (10.-5M)

It (10.-7M) (10-5M) C0—.0 OP-susceptible - —0 OP-resistant Control

50 0'

..•••..0 0* •

000•••••••

30 0 inhibitor added

...... tk.lw■w■W.....eimwmo

1 10

-15 0 1 5 30 45 60

TIME (minutes)

Fig. 1.25 -Da vitro inhibition of whole aphid carboxylesterase in the presence of the substrate 1-naphthyl acetate (3x10 M). Temp. 370C; inhibitor added at "zero" time.

0.-0 OP-susceptible 1Control 0. —.0 OP-resistant 4m) Diazinon (10 Diazoxon (10-6M) ) d ko i / 0 h /ap

ms woo

41 ••••11111• GO • • .1" 114,40" SIP ••• • • . ••••• ••• ..10 *•■ (ig

OL / inhibitor added H

HT ..•

NAP / 20 / (/' WINO .110 11..110 / / „. 0 / r fm.• i ie )e. ...,.. .--- To I •••••4•1•01111••••••11 00000 ••••••••••••••••• • ••••• a,* I / 1 A / inhibitor added i e /.d,,

15 30 45

TINE (minutes) Pig. 1.26 - In vitro inhibition of whole aphid carboxylesterase in the presence of the substrate, 1-naphthyl acetate (3x10-4M). Rest as Fig. 1.25. 83

Study of the instability of organophosphate resistance and its association with changes in carboxylesterase levels.

There are several known cases of resistant insects losing their resistance to insecticides when bred in the absence of any selective insecticidal pressure (Kojima et al.,

1963c; Forgash and Hansens, 1967). To study this phenomenon

a batch of resistant Myzus persicae, henceforth referred to as

the IF-strain, was separated from the parent stock and bred in

an insecticide-free environment. The IF-clone was begun on

August 20, 1970, when its resistance factor for dichlorvos stood at 11.5. Stringent measures were taken to ensure that individuals from wild Myzus populations were not accidentally introduced into the IF-cultures. For this reason turnip plants,

on which these insects were reared, were grown with special

care and all soil used was thoroughly sterilized (see Materials

and Methods , Part Two).

Commencing from the date of establishing the IF culture, the levels of carboxylesterase, in both the susceptible and IF-strain, were determined at intervals of 30 days (about 3

generations) using the modified technique of Gomori as out-

lined before (page 61). The resistance factor for dichlorvos,

for the IF-strain, was also determined every month by topical

application as described in Part Two. The results are

summarized in Table 1.10 and Fig. 1.27.

84 Carbon,!esterase Activity Month ,_ ogiopllid a- nopht ho I produced) Resistance (1970-71) OP-Susceptible OP-Resistant • Factor August 20.20 37.00 11.50

September 17.72 37.50 11.80

October 17.98 34.20 10.41.

November 20.00 37.46 10.75

December 17.42 36.31 11.60

January 19.50 36.60 11.10

February 18.00 19.60 2.30

March 19.31 20.42 2.60

TABLE - 1.10 Data indicating a drop in the R.Factor corresponding with a drop in the carboxylesterase level in the R - strain reared in an insecticide-free environment. R-Strain

id) h ap OL (pgi HTH NA P

Aug. Oct. Dec. Feb. Fig.1.27 - Graph illustrating a drop in the carboxylesterase of R-straini reared in an insecticide- free environment after about 18 generations. 85

It is clear that resistance in the IF-strain continued at x11 up to January (i.e., about 18 generations after the culture was removed from insecticidal pressure) and the differences between the carboxAesterase levels of the two strains were not very marked. The monthly variations in the enzyme levels were typical of the strain and probably due to random sampling errors. However, between the January and February analyses there was a marked and sudden decrease in the carboxylesterase level. The test for resistance also showed a decrease from an average of about 11-fold to about 2.3-fold.

A further bioassay in March showed the resistance to have more or less stabilized at 2.6-fold. At the time the IF-culture was started a second batch of resistant Myzus persicae was also separated from the parent stock. This group was subjected to a gradually increasing dose of dichlorvos to determine whether a higher resistance to dichlorvos could be induced and whether this increase would be reflected in the carboxylesterase level of the strain. Unfor- tunately however, the number of aphids surviving the increased pressure was too small to allow adequate experimentation. This was taken as an indication that resistance had not changed; but it must be remembered that the increased dosage regime covered about six months only and that a long-term experiment might well have produced .a more resistant strain. 86

Discussion

In the present series of experiments carboxylesterases from both the 'unselected' normal strain and the OP-resistant strain of M. persicae have been studied in some detail. In a few preliminary experiments optimum temperature conditions and homogenate (enzyme) concentrations for. Myzus carboxylesterase were established. One important point is that carboxylesterase activity was greater at 37°C than at 27C, so unlike others, among them van Asperen (1962) and Needham and Sawicki (1971), the former temperature was used throughout.

To evaluate the characteristics of the carboxylesterases the alpha and beta isomers of two naphtholic esters were used as substrates. 1-naphthyl acetate has frequently been used for esterase determination of several insects especially after van Asperen's (1962) introduction of a modified version of

Gomorits colorimetric technique. It is well established that

ChEs attack naphtholic esters but because experiments described earlier in Section B(1) and (2), showed no difference in ChE activity and sensitivity of the two strains (this was further confirmed in preliminary experiments using 1-naphthyl acetate as substrate and eserine sulphate as the specific inhibitor) details of this enzyme have not been included in this section. This serves to concentrate attention on the carboxylesterases which provide the greatest enzyme differences between the two aphid strains studied. 87 Interesting results emerge from the use of naphtholic

esters as substrates. No significant differences were found

in the carboxylesterase activity between the two strains when

2-naphthyl acetate and 1- and 2-naphthyl butyrate were used

' (Table 1.8). This contrasts strongly with data obtained for

leafhoppers by several Japanese workers (Ozaki and Koike, 1965; Ozaki and Kassai, 1968; Ozaki, 1969) where high activity of

2-naphthyl acetate splitting carboxylesteraes was related to

high resistance. However, in the case of Myzus persicae, when

1-naphthyl acetate was used high carboxylesterase activity was recorded for the R-strain (more than 3 times the S-strain at

1 -4 7.5 x 10-3M ) , the Km value for it being 1.25x10 M compared -4 to 3.15 x 10 M for the S-strain. Unlike van Asperen (1962) it

is not accepted that this anomaly can be explained by assuming

that Myzus carboxylesterase has a remarkable specificity for alpha isomers. Obviously a more complicated situation exists

and this is emphasised further if one considers that ethyl

butyrate splitting esterases were less active in the R- than

the S-strain of M.persicae (Section B(1) and (2)). This find-

ing for M. persicae introduces a totally new facet of es-

terase activity in insects and will be discussed further. Many workers (see General Introduction) have established

that in houseflies carboxylesterase ("AliE") activity towards 1-naphthyl acetate and ethyl or methyl butyrate is consider-

ably less in most OP-resistant strains than in S-strains. The

reverse situation holds in leafhoppers since esterases hydro- 88 lysing methyl-n-butyrate and 2-naphthyl acetate are in greater abundance in the R-strain. Between these two extremes are several resistant species (for example, Aedes aegypti, Matsu- mare and Brown, 1961a and Cimex lectularius, Feroz, 1970) which no show inter-strain differences in carboxylesterase activity.

Myzus persicae presents a unique example in that depending upon the substrate involved carboxylesterase activity varies between the 'unselected' normal strain and the OP-resistant strain. This strongly supports the view held by Augustinsson

(1968) that all work on esterases must involve at least two substrates. Furthermore, as he points out, when the ester used is a non-specific substrate for several forms of esterases (1-naphthyl acetate. is one such ester), it must be stressed that one substrate rarely has the same affinity for various esterase forms; as such a true picture of the esterases may not emerge. It is worth quoting his concluding sentence from the same paper. It may not be valid to state that a tissue is characterized by a high or low content of a specific esterase when activity has been assayed with this type of substrate only". Results of inhibition studies of esterases are of particular interest for the effect of eserine sulphate on esterases hydrolysing 1-naphthyl acetate. Easson and Stedman (1937),

Richter and Croft 0942) and van Asperen (1959) noted that

"AliEs" were very little influenced even by 10-3M eserine sulphate (10-5M according to van Asperen). Contrary to these 89

-7 reports the present work shows that 10 M eserine sulphate more or less completely blocks ChE activity (see Section B(1) and (2)) and therefore higher concentrations of the carbamate must inhibit the carboxylesterases (Fig. 1.21). The important point is that carboxylesterases from the OP-resistant strain of M.persicae are considerably more tolerant of eserine sulphate inhibition than those from the S-strain. A similar situation exists for diazoxon and dichlorvos inhibition, especially at lower concentrations. Since absolute inhibition with organophosphates is not the case, there being a tendency of the curves to level off at the higher concentrations, one is forced to assume that a small amount of arylesterase (aryl ester hydrolase, formerly known as A-esterase), hydrolysing

1-naphthyl acetate, is present. This assumption is corroborated by the findings of the inhibition studies on electrophoreti- eally separated esterases (see Section C). As mentioned -earlier it is known that insects may lose resistance to insecticides when reared in an insecticide- free environment. This has been recorded in Myzus persicae also (Dunn and Kempton, 1966 and Needham and Sawicki, 1971).

Needham and Sawicki also showed that when resistance was lost at the 14th generation, it was sudden and was reflected by a corresponding loss of carboxylesterase activity. The results described here parallel their findings differing only in the minor detail that resistance was lost after about 18 generations.

Two further comments are in order. The findings of Dunn and 90

ICempton (1966) differ from these reports in that loss of resistance was gradual and over 30 generations. This could be explained by the fact that their original stock of resistant insects was from a wild population which could easily have involved non-resistant individuals, which might then slowly increase in numbersand so dilute the resistant stock.

This was not possible in the present case since the resistant population had been maintained with OP-pressure in the laboratory for a considerable period of time.

It is difficult to explain how a population of parthenogenetic reproducing individuals can suddenly lose resistance, i.e. over a period of a month (ca. 3 generations). One possible explanation could be that, provided contamination of the "insecticide-free" (IF) R-stock with lsusceptible individuals is ruled out, some members of the IF-stock mutated to produce

susceptible' individuals. Since during the experiment large numbers of the IF-stock were discarded when fresh host plants were introduced into the stock cultures, it is possible that by chance the 'susceptible' progeny remained while mostly resistant ones were discarded. Subsequent multiplication of the former could explain the sudden loss of resistance but does not provide a very acceptable explanation. 91

SECTION C: Electrophoretic separation of sortie Beeterases of OP-susceptible and ' resistant Mopersicae on polvacryl- amide and starch gels.

Introduction

Since its introduction in 1937 by the Swedish biochem- ist Tiselius electrophoresis has been developed continuously as a valuable tool of the molecular biologist. The basic prin- ciple of electrophoresis is simple: a charged particle (ion) will move in an electrical field, according to the number and type of charges it possesses. Other factors which exert an influence on the separation and rate of migration of the particles include: molecular weight and shape of the molecule; vis- cosity and type-of supporting medium used; the amount of current applied and the concentration and pH of the buffering medium.

Electrophoretic studies have been done on tissues of various types of animals. Augustinsson (1958) studied blood plasma esterases of several mammals. A few years later Hunter and Burstone (1960) pointed out the effectiveness of zymograms as a tool for enzyme substrate specificity and in 1961, Hunter and Strachan published a detailed study of mouse blood esterases,

Ecobichon (1967) and Ecobichon and Israel (1967) investigated liver carboxylesterases of large domestic animals and in the electric eel. There are many examples from varied sources of 92 material but most of the work has been done on starch gels and confined to large animals.

Heterogeneity of insect carboxylesterases has also been demonstrated by starch and agar gel electrophoresis (Laufer, 1960, 1961; Menzle et al., 1963; Eguchi and Sugimoto and Eguchi and Yoshitake, 1965 and 1966 respectively Yoshitake et al.,

1966). With reference to insecticide resistance,electrophoresis has been used to examine the esterases of Musca domestica (Vel- thius and van Asperen, 1963; van Asperen et.al., 1965; van

Asperen and van Mazijk, 1965; Ahmad, 1968; Collins and Forgash,

1968), the green rice leafhopper Nephotettix cincticeps and the brown planthopper, Laodelphax striatellus (work summarized by Ozaki, 1969). Esterase patterns determined on starch or agar gels, either of whole insects or theirsecretions, have also been investigated for Drosophila melanogaster (Meigen) (Ogita,

1963); Oncopeltus fasciatus (Dallas) (Salkeld, 1965); Peripla- neta americana L. (Cook and Forgash, 1965; Matsumara and Sakai, 1968 and Cook at al., 1969); Apis mellifera (Benton, 1967); Pieris brassicae L. (Clements, 1967) and the silkmoth Antherea polyphemus (Katzenellenbogen and Kafatos, 1971). Oncopeltus fasciatus was shown to have 19 1-naphthyl acetate splitting enzymes (Salkeld loc. cit.)

Electrophoresis of insect esterases on polyacrylamide

gel (a starch gel 'substitute first suggested by Raymond and Weintraub, 1959) has been relatively less widely used. Sims

(1965) used it to study esterases of 2 species of Drosophila. 93

A year later, in 1966, Price and Bosnian employed the medium to investigate non-esteratic protein of the blowfly Calliphora erythrocephala. Arurkar and Knowles (1967 and 1968) conducted a more extensive research on esterases using polyacrylamide gel when they studied the enzymes in homogenates of 7 insect species, namely: the blister beetle Epicauta lemniscata (Stur with 14 "Ali-Es" and E.pennsylvannica (De Geer) and Periplaneta americana, each with 10 "Ali-Es"; the house cricket Acheta domesticus L., the fall webworm, Hyphantria cunea (Drury), the yellow-striped armyworm, Prodenia ornithogalli (Guenee), each with 8, 6 and 9 "AliEs" respectively and the bollworm Heliothis zea (Boddie) with 6 "Ali-Es" and 2 arylesterases. Ahmad (1968) used polyacrylamide gel and demonstrated 10 "Ali-E" bands in an OP-susceptible Musca domestics, Two of these enzymes also behaved as phesphomonoesterases. However, only 8 "Ali-Es" were present in the R-strain and all but one showed a comparatively reduced level of activity. A similar study was also conducted by Collins and Forgash (1968). Two interesting points which emerged from their work were (i) esterase patterns varied a lot between and within strains and

(ii) "no clear-cut consistent differences were observed between resistant and susceptible flies." It is interesting that the latter point contradicts some of the works mentioned hither- t There appears to be no published record of aphid esterase zymograms. The only known cases involve 'susceptible' 94 insects and include Aphis fabae (Scop.) and Acvrthosiphon pisum (Harris) (Patsalcos, 1971, personal communication) and the vetch aphid Megoura vicia (Halt.) (Sudderuddin, 1970, un- published data). In view of the above the following work was conceived to map the general protein and esterase patterns of the 'susceptible' and OP-resistant strains of Myzus persicae. It was further sought to characterize the enzymes with speific substrates and inhibitors and to discuss the findings in the light of the data available from other studies.

Materials and Methods

Insect materials

Adult apterae of a 'susceptible' and an OP-resistant strain of Myzus persicae were used. Further details of the strains are given elsewhere (page117). Prior to homogenization the insects were starved overnight (for about 10 to 12 hours) to ensure homogenity of the extract. Electrophoretic method

1. Polyacrylamide gel electrophoresis

The vertical electrophoretic technique described by Raymond (1964) and used with modifications by Price (1968) and Ahmad (1968) was tried but discarded because of several disadvantages. The most important of these was that the apparatus was cumbersome and required 5* hours for a complete run. 95

In addition a concentrated extract of aphids was required, which, under the circumstances, proved extravagant. Instead, a standard disc electrophoresis unit, made by Shandons, was used with modifications as described by Sudderuddin (1971).

Extraction of the enzyme

Preliminary experiments indicated that satisfactory aphid esterase patterns could be obtained when 12 mg of aphid material was homogenized in 1 ml of distilled water. After homogenization in a glass, hand homogenizor (with teflon coated pestle) immersed in crushed ice, the homogenate was centrifuged at 17,000G for 30 minutes at 4°C and the supernatant used for electrophoresis. In this way at least 50% of soluble "AliE" may be recovered (Ahmad, 1968). Fresh extracts were preferred for all experiments and therefore the homogenate was prepared daily.

Buffer system

Raymond's (1964) recommended buffer system was used and was made up as follows: 5.5 gm of trishydroxymethyl amino methane + 2.5 gin of boric acid 4- 1.0 gm of ethylenediamine tetra acetic acid (EDTA), disodium salt, made up to 1 litre with deionised, distilled water. This gave a pH of 8.5. For each electrophoretic run a total of 380 ml of buffer was needed per unit, i.e., 210 ml for the lower buffer reservoir and 170 ml for the upper buffer reservoir.

Preparation of gels

Gels were cast in rectangular running "tubes" 96

(plugged at one end with plasticene) by polymerization of acrylamide monomers as suggested by Orstein (1964). Since a 6 cm standard, uniform gel of 5% failed to separate esterases of very similar molecular weights a variable gel system was employed. This consisted of an initial 1 cm portion of 5% gel and a main body of 5 cm of 10% gel. The latter was cast first and was prepared by dissolving 2 gm of 'Cyanogum 41, BDH' (a mixture of acrylamide monomer and N:N-methylene-bis-acrylamide) and a few crystals of potassium ferricyanide (to delay gelling) in 20 ml of buffer and pouring this into a beaker containing

180 mg of ammonium persulphate at one side and 0.2 ml of 2- dimethyl-amino-ethyl-cyanide at the other. The mixture was gently but completely mixed and then pipetted into the running tubes up to the 2 cm mark. Then a few drops of water was gently layered onto the surface of the solution in each "tube".

Several minutes later when gelling was complete the surfaces of the gels were blotted dry leaving a flat meniscus. A 5% gel prepared by halving the quantities of the chemicals used for the 10% gel was then poured over the 10% gel and as before several drops of water Ware added and later blotted dry to give a flat meniscus. The gels were allowed to mature for at least two hours; later the plasticene plugs were removed and the system (unit) was ready for sample application and subsequent electrophoresis.

Sample application and electrical conditions

0.4 ml of the supernatant was pipetted onto folded 97 filter papers (0.5 cm x 2.5 cm) in each "tube". The remaining space was filled up with buffer taking care to avoid air bubbles.

The whole unit was then moved into a cold room (800 and the appropriate amounts of buffer were poured in the lower and upper buffer reservoirs. Finally the unit was connected to a

Shandon Vokam constant voltage/current DC power supply with cathode at the top and anode at the base. Since two units were used simultaneuosly the current was kept constant at 80 mA, i.e., 5 mA per running tube. The initial voltage stood at 85 volts increasing to 220 volts at the end of the two-hour run.

2. Starch gel electrophoresis

As an alternative to polyacrylamide gel, horizontal starch gel zone electrophoresis was used to determine whether better esterase separations could be obtained. The technique used has been fully described by Smith (1968). The gel was made up of 11 gm of hydrolysed starch mixed with 100 ml of

0.03 M borate buffer, pH 8.5 (boric acid 1.86 gm 12 ml of

1 M NaOH made up to 1 litre with distilled water and adjusted to pH 8.5 with 0.1 M.HC1). The 11% gel (a modification after

Tan, personal communication) showed less shrinkage at the anode

end unlike gels of higher concentrations. The electrode buffer comprised of 3 M borate at pH 8.0 (18.55 gin of boric acid

50 ml of 1 M NaOH made up to 1 litre with distilled water) The sample (25 mg of aphid material homogenized in 1 ml

of water) was applied on filter papers (1 cm x 0.5 cm) in 1 cm slots at the cathode end of the gel. The system was then 98 hooked onto a constant current/voltage DC power supply unit

the voltage applied was 5 volts per cm of the starch gel; the current fluctuated during the run) and run at 4°C for 16 hours.

Later the gels were sliced and the esterase zones identified as described below.

Treatment of the gels

Detection of protein bands of the 'susceptible strain

Attempts to stain proteins were made only for proteins separated on polyacrylamide gels. Several protein dyes were tested (including light green stain, Nigrosin and Coo- massie Brilliant Blue - the latter after Chrambach et al.,

1967) but naphthalene black 10B also known as amide black or amido sohwartz 10B) gave the best results. The solution was made up and used as follows: 100 mg of naphthalene black was dissolved in a fixer solution of acetic acid, ethanol and water in the ratio of 1:5:5. The gels strips were immersed in this solution and left overnight. Later, excessive stain was removed by washing the gels in fixer solution for a total of

3 hours at 50°C, changing.the solution every hour. The destained gels were then stored in 7% acetic acid.

Detection of esterase bands

Several methods using various stains have been elaborated for the detection of esterases separated by electrophoresis. Unkike the method used by Hunter and Durstone (1960) which incorporated both substrate and dye in the same solution, the method used here utilized separate preparations of the 99 the substrate and the dye (Ahmad, 1968). For polyacrylamide Ifie subArate, gels,,consisted of 250 mg of alpha naphthyl acetate 25 ml 'analar' acetone + 75 ml of deionised distilled water, and the dye of 22 mg of Fast Blue RR salt 50 ml of 0.1M phosphate buffer, pH 7 made up to 50 ml with deionised distilled water.

The mixture was then filtered. Both solutions were always freshly prepared and kept away from light. 2 - 3 ml of the substrate were sprayed onto the gels and left for 20 minutes at room temperature (ca. 20°C)0 They were then rinsed with deionised distilled water and incubated with 4-5 ml of the dye until satisfactory coloration was obtained and usually required about 15 minutes. For starch gels the technique was exactly the same but for the composition of the substrate and the dye solutions which were as suggested by Smith (1968, page 335). Characterization of the esterases

After the total number of esterase bands were established each band was then identified by using specific inhibitors and, in one case, a specific substrate. Phosphomonoester hydrolases

Characterization of two of these enzymes was initially based on their specific activity towards sodium 1- naphthyl phosphate (Ahmad, 1968). Attempts to locate alkaline phosphatases were made by incubating gels in a substrate solution consisting of.0.4% sodium 1-naphthyl phosphate in 0.1M tris-HC1 buffer, pH 9.3 and subsequently staining them with a buffered solution of Garnet GBC salt (after Benton, 1967). 100

For acid phosphatases the same substrate was used but a 0.005M

Tris-citrate buffer, pH 6.2, was used instead (Sur et al., 1962).

In both cases the gels were incubated with the substrate for 30 minutes at room temperature and then stained at 37°C and

stored in 7 % acetic acid. The phosphatase bands were con-

firmed by treating a separate set of gels with KCN solution

prior to incubation.

Carboxylic ester hydrolases

The carboxylic ester hydrolases were identified

by their sensitivity to organophosphates, viz., DDVP. Prior to

incubation in the substrate medium and subsequent staining in

the usual way, a set of gels was treated with a 10 4M solution of eserine sulphate (at room temperature for 20 minutes) while

another set was treated with a 10-3M solution of DDVP.(The

inhibitor solutions were stronger than necessary because of the

diluting effect of the gel matrix). Bands which were relatively

insensitive to eserine but were inhibited by DDVP were identified as carboxylesterases (Stegwee, 1959; Arurkar and Knowles,

1968). Esterases inhibited.by both DDVP and eserine were labelled

as ChEs (Richter and Croft, 1942; Augustinsson, 1958; Arurkar

and Knowles, loc. cit.). Those regions of esterase activity

which weretinaffected by DDVP were considered arylesterases

(Oosterbaan and Jansz, 1 965).

Results

1. Polyacrylamide gel electrophoresis Of the several stains used for the general proteins of 101

M. persicae, naphthalene black 10B and Coomassie Brilliant

Blue gave best results; the former was more effective although the latter gave quicker results since gels did not need to be destained. The variable gel system separated 8 distinct protein zones with several others faintly visible (Fig. 1.28) Zymogram of the water soluble proteins when matched with those of the esterases did not correspond with each other except for El and parts of E2 and E. This lack of correspondence has been reported in houseflies also (Menzel, et al., 1963; Arurkar and

Knowles, 1968 and Ahmad, 1968).and may be due either to a lower concentration of the proteins at the esteratic sites or to the fact that 1-naphthyl diazoate is more intense as a pigment than naphthalene black 10B precipitate.

Carboxyl ester hydrolases

Using a variable gel system, i.e., an initial large portion of y/ and a main body of 10%, a total number of 6 esterase bands, all hydrolysing 1-naphthyl acetate, were dis- cerned. These stained as dark brown areas of varying intensi- ties (Fig. 1.28). El was the fastest moving band and on visual and E3, lying 5.2 and comparison was the most intense. E2 3.3 • cm from the top respectively, were only slightly intense in was fairly intense and occu- the S-strain. In the R-strain E2 pied a larger area. Bit was a broad zone of esterase activity and almost merged with E5. E6 was intensely coloured but the slowest moving only travelling a few millimetres from the origin. The average mobilities (If values) of the different bands

Colour intensity code:

Flinitia1 large pore 1 1 slightly intense intense gel (57) 0 faint fairly intense very intense t I A

S S R S R S R p•Iww•mmima. r, -----; 7 I I 1 I CM 1 . I I E6 Eg E6 E5 E E ,5 5 3 E4

E E E2 3 3 5.8 cm

•• • •..... E E2 E l 2

BEM E l Ei :tET:11.:

Fig. 1,28 - Zymograms of esterases of S- and R-strains of Myzus persicae, spearated on polyacrylamide gel, (Actual size) A. Water soluble proteins of whole susceptible aphids. B. Esterases separated on non-variable, standard, 5% gel. C. Esterases separated on variable gel, i.e., 5% and 10%. D. Esterases treated with 10-4M eserine sulphate prior to incubation with the substrate. E. Esterases treated with 10-3 M DDVP. 103 are given in Table 1.11.

Characterization with inhibitors gave interesting results. Treatment with 10-3M DDVP completely inhibited E2, E3 and E E persisted as a faint zone while E was just barely 4° 6 5 visible. Inhibition with eserine sulphate proved that Eit was 1 , E a ChE. The fact that E 5 and E6 were still active though rather weakly after DDVP and eserine sulphate treatment suggested arylesterase activity. Similar results were obtained for both the S- and the R-strains though it may be noted that E in 3 the R-strains was less affected than the corresponding band in the S-strain and substantiates the quantitative findingsdes- scribed earlier.

Summarizing the reults obtained it is possible to say E comprised of a carboxylesterase and an arylesterase; E and 1 2 E3 of carboxylesterases and Eit predominantly of a CYO. E5 and E6 showed arylesterase and possibily carboxylesterase activities (Fig. 1.29b). Phosphomonoester hydrolases

Using sodium 1-naphthyl phosphate, buffered at

pH 6.2, indicated only one zone of activity in both strains (Fig, 1,29b). This appeared as an orange-brown band and was identified as an acid phosphatase (Sur et. al., 1962; Price,

1968). P1 was not very intense and roughly coincided with the band. No bands were evident when the position of the denser E1 substrate was buffered at pH 9.3. Colour code as in Fig.1.28

)X.i.Car-E?

Car-E3

Car-E2 Car- E1 X (d) (a ) (b) (c)

Fig. 1.29 - Diagrammatic representation of whole Myzus esterases separated in polyacrylamide and starch gels. a Acid phosphatases of .5- and R-strains (polyacrylamide gel). b Complete esterase zymogram derived from Fig. 1.28 (Polyacry1amide gel) o\( Esterases of S- and R-strains separated in Starch gel (115). d) Complete esterase zymogram after characterization with eserine & DDVP. (Car-E = carboxylesterase; X = arylesterase; P1 = acid phosphatase). 105

2. Starch gel electrophoresis

After electrophoresis the treatment of esterases was the same as for polyacrylamide gel.

A. total of 6 bands was produced (Fig. 1.29c and d). Of these E comprised of a ChE and an arylesterase while E was 6 4 mostly carboxylesterase and possibily some arylesterase. The

Rf values of these enzymes are given in Table 1.11 together with those separated on polyacrylamide gel. However, comparisons between the two would be only superficial at best as the two gels exhibit quite different characteristics. The esterases of the R-strain differed on two points: E4 was broader and more active than those of the S-strain though E2 of the R-strain was slightly less active.

Discussion

Because there are no reports of an electrophoretic study of Myzus persicae esterases the present work involved a more or less comprehensive investigation of the esterases from a 'susceptible' and resistant strain of this pest. Two methods have been used employing different types of gel media and as such comparisons can be made in terms of efficiency of the methods and gels involved. A gelling medium, polyacrylamide for instance, acts as a sieve for protein molecules separating them into different components according to the size and charge of the particles POLYACRYLAMIDE GEL STARCH GEL Il Maximum Minimum Maximum Minimum Esterase distance distance Rf Value distance distance Esterase band from from from from origin origin Ic:IM/*SF origin origin band (mm) (mm) 2 (mm) (mm) x y y x

• *SF 55 55 1.00 1.00 6o 60 SF E 50 0.94 0.85 50 E1 53 52 1 E 42 45 0.71 44 41 E 2 0.79 2 E 23 28 0.46 0.60 38 34 E 3 1(20) (27) (0.43) 3 E 8 0;18 E 4 12 0.46 25 20 4 (7) (0.17) (0.39) (27) E 3 0.073 0.15 10 8 E5 5 5 E 0 E 6 0 1.5 0.027 0.017 2 6

*SF = solvent front denoted by marker dye Table 1.11 - Average mobilities of esterase bands of S-strain separated in a variable, polyacrylamide gel sytem (5% and 10%) and in starch gel(11%).Where different the mobilities of the R-strain are given within brackets. 107

(Raymond, 1964). Dy selecting the appropriate medium concentration, the pore size of the gels suitable for particular requirements, may be adjusted. Thus when the separation of esterases in 5% polyacrylamide gel was compared with that obtained with a variable gel system (5% and 10%) it was evident that a band which appeared as one using the non-variable gel was really several bands tightly packed together (Fig. 1.28b).

As Orstein (1964) points out this occurs because separation proceeds as a combination of electrophoresis and gel filtra- tion so that a gradation of gel pores provides a sharper resolution.

Polyacrlamide gel electrophoresis using a variable system resolved 6 esterase bands in both strains of Mvzus persicae though the activities and the Rf values differed in some cases (Table 1.11 and Figs. 1.28 and 1.29). The obvious difference involved band E3 which was shown to be a carboxylesterase being insensitive to eserine but quite susceptible to DDVP. This agrees with the findings described earlier;using the colorimetric method it was shown that 1-naphthyl hydrolysing carboxylesterases were more active in the R-strain. ChE was easily defined also though the enzyme designated E1 , E5 and E 6 could not be completely categorized. It is possible that E5 and E6 bear traces of ChE activity since they shown some sensitivity to eserine. However, this could be.due to the fact that eserine at high concentrations also inhibits carboxylesterases as shown earlier (page 73). This would suggest traces 108

of carboxylesterases at E5 and E6. One point which still remains to be resolved is that all

3 bands i.e., E1 , E5 and E6 still retain some enzyme activity -3 after 10 M DDVP treatment. This would indicate one of two

• possible explanations. If the classification of Oosterbaan and Jansz (1965) is to be followed then the DDVP-insensitive en-

zymes must be labelled as C-type esterases (also called aryl-

esterases) which neither hydrolyse nor are inhibited by or-

ganophosphates. Alternatively, like some of the esterases from

the yellow-striped armyworm, the uninhibitableparts of E1 , E5r and E6 may also represent carboxylesterases which are DDVP-insensitive but are readily inhibited by other organophosphates

(Arurkar and Knowles, 1968). The first interpretation is favoured although the need for further study is indicated.

At this juncture it is worth noting briefly the esterase patterns of 3 other 'susceptible' aphids. Patsakos (1971, per-

sonal communication), using a variable gel system has demonstrated a total of 5 and 6 esterase bands in Acyrthosiphon pisum

Harris, and Aphis fabae Scop. respectively. In each case only

one band was. characterized as a ChE and one as a carboxylesterase. The remaining bands, he believes, to be arylesterases. In

Megoura vicia Buckton, a total of 4 esteratic zones has been

established. Of these E was typified as a ChE, E and E as 4 3 2 and part of E as arylesteraso (Sudder- earboxylesterase_ and E1 2 uddin, 1970). Comparing the esterase profiles in all four

'susceptible' aphids it is not difficult to see the variability 109 that exists in different genera.

Starch gel electrophoresis also gave 6 esteratic zones.

Two zones of ChE activity could be defined in both Myzus strains. The differences in the esterase activity of the two strains centred around E and E In the latter case consi- 3 4' derably more activity was found in the R-strain though E 3 showed a slightly less activity in the same strain. As with polyacrylamide gel separation, C-type esterases (arylesterases) and E were evident at E6 4' Of the several resistant insects whose esterases have been studied electrophoretically three will be considered briefly to provide a basis for comparison with the results described here.

Most work on resistant houseflies (Musca domestica)has been done by van Asperen and co-workers (1964), Collins and Porgash, 1968 and Ahmad (1968). Their results do not agree completely although they do agree on some basic points. Ahmad found a total of 10 "Ali-Es" in the normal strain as against 8 in the SKA (OP-resistant) strain; all the bands in the latter showing reduced levels of activity. However, his findings differed from those of Velthius and van Asperen (1963), van

Asperen and Majizk (1965) and Menzel et al., (1963) because he showed that the difference in the "Ali-E"level of the R-strain was not due to the absence of an anodic band but due to the absence of two bands midway between the cathode and anode. To further complicate matters Collins and Forgash (1968) reported 110 considerable variation in esterase patterns between and within strains and they failed to observe any clear-cut, consistent differences between S- and R-flies.

Ozaki (1969) has reviewed esterase patterns of R- and

S-strains of Nephotettix cincticeps and Laodelphax striatellus as ascertained by several workers. In both these insects esterases hydrolysing 2-naphthyl acetate were more pronounced in the R-strainsthan in the S-strains. In resistant N.cincticeps most individuals showed a high activity of E2, E and /or E 14 3 bands whereas in L.striatellus, E7 was more active in the R- strain.

Qualitatively speaking, the esterase zymograms of /Cpersicae in the present work are reasonably constant though it must be pointed out, as is evident from the data in Section

B, that quantitative fluctuations over a period of time are quite common. One reason for the reasonable homogenity of zymograms would be that samples prepared for electrophoretic studies involved many individuals so bands observed represented esterases of a composite of large numbers. This would tend to average out discrepancies evident with small samples.

It is difficult to say whether the different carboxylesterases are polymeric forms of the same molecules; but following Wilkinson's (1965) idea that they are , "multiple enzyme forms occuring in a single species" they may be called 'tendency isoenzymes although the recent has been to refer them as individual 'Ali-Es", i.e., carboxylesterases. Reference to the 111 mobilities of the esterases of Myzus persicae show they follow the classical patterns as outlined by Augustinnson (1958): the ChEs moving the least followed by the car boxylesterases and the arylesterases. Small amou is of material giving a posi- tive reaction for esterases were retained at the origin. This may be due to several reasons the most probable being that they were bound or desmo-esterases not sedimented at the cen- trifugal force applied. Bratkowski (1967, quoted from Arurkar and Knowles, 1968) has shown that addition of a surfactant often increases the solubility of this material so that migrates into the gel.

Finally, a few words about the comparative efficiency of the methods :used. Both techniques gave satisfactory results and each had its own merits. Polyacrylamide gel electrophoresis was quicker and allowed considerable flexibility in choosing the gel concentration. In addition, gels could be handled easily without fear of breakage. One disadvantage lay in the fact that equal layering of gels in the variable system was difficult to achieve so ,slight variations in gel lengths had to be considered. Starch gels did not have this problem as variable systems were not involved. However, starch gels are very fragile and handling them during staining and photo- recto...2s graphy Acareful handling. Furthermore, unlike polyacrylamide gels, starch gels can only be preserved for a short period of time. Though esterases were resolved satisfactorily in both media, protein separation was superior in polyacrylamide gels. 112

PART T W 0

Toxicological Studies 113

Introduction

Historical Insecticide resistance in aphids is fairly well documen-

, ted. Boyce (1928) was the first to induce resistance in Aphis

gossvpii (Glover) by artificial selection with hydrocyanic acid.

But it was twenty six years later that resistance in the field

was reported by Michelbacher et a1”(1954) when they found that

Chromaphis juglandicola (Kalt.) was resistant to parathion.

This was followed by a series of reports on resistance in field

populations of Myzus persicae (Sulz.) from different parts of

the world. From U.S.A. came evidence of resistance to parathion,

malathion and DDT (Anthon, 1955); to demeton, schradan and para-

thion (Klostermeyer et al.,1956); to parathion (Shirck, 1960

and Georghiou, 1963) and to parathion, thiodan, malathion and

diazinon (Wolfenberger, 1960 and Shorey, 1961). Resistance was

also reported from Switzerland - to parathion (Weismann, 1955);

from Vest Germany - to E605f (Baerecke, 1962); from Great

Britain - to dimethoate and demeton-methyl (Dunn and Kempton,

1965) and to other organophosphates (Gould, 1966 and Wyatt, 1966);

from Sweden - to methyl demeton-0 and parathion (Bjorling et al"

1966); from Czechoslovakia - to thiometon, malathion, diazinon

and parathion (Harkova, 1970) and from several other parts of

Asia (FAO Reports,.1965 and 1968 and Baranyovits and Gosh,

1969). Resistance in aphids is not confined to Myzus persicae 114

alone. There are records of organophosphate resistance in

Therioaphis maculata (Buckton) - 113-fold to parathion (Stern

and Reynolds, 1958); in the damson-hop aphid, Phorodon humuli

(Schrank) - Anon, 1966, HrdS;- and Zeleny, 1968; in Aphis gossypii (Glover) to BHC and organophosphates; in the wooly aphid, Eriosoma lanigerum (Hausmann); in the rosy aphid, Dysaphis plantiginea (Pass.); in Aphis pomi (De Geer) etc. (FAO. Reports,

1965 and 1968). Thus it is apparent from the above review that

within the last decade resistance of aphids to insecticides

has become recognised as a serious problem.

The Phenomenon of Cross- and Multiple-resistance.

Ever since Busvine (1953, 1954) noted that strains of

insects resistant to one compound often showed resistance to

other compounds, cross resistance and miltiple resistance have

-provided important clues to the study of resistance mechanisms.

Several excellent reviews of the subject have appeared (Met-

calf, 1955; March, 1959; liinteringham and Hewlett, 1964; Busvine, 1967, 1968; Grayson and Cochran, 1968 and Brown and

Pal, 1971) which give comprehensive tables outlining cross-

resistance in various insects. This data, some of which are

given in the Discussions clearly indicate that exposure of an insect population to one insecticide may induce cross-

resistance to chemically different compounds as well as to

those with different modes of action. There appears to have been no concerted effort to 115 collate the information on the resistance profiles of aphids.

Many workers have used resistant aphid strains for toxicological work but only a handful seemed to have assayed the same strain against a range of insecticides. DjOrling et al.,

(1966) tested nine compounds against a resistant strain of

Myzus persicae referred to as R5, cultured from a field population apparently resistant to demeton-0-methyl and parathion. They have shown that the R5-strain had a 10 to 3 fold resistance in the descending order to the following compounds: parathion, demeton, thiometon, phosphamidon and phosphorothio- ate. However, the strain had no resistance to isolan, methyl- carbamate, endosulphan and nicotine. Multiple resistance in four different populations of Myzus persicae to thiometon, diazinon, parathion and malathion has been recorded (Hurkova, 1970) while Hr0 at al., 1970, have shown Phorodon humuli to be resistant to thiometon, teration (2.9x) and Terra sytam

(3.3x). More recently, Needham and Sawicki (1971) worked out the resistance factors for a resistant strain (GR) of Myzus persicae that had been exposed to many organophosphorus and carbamate insecticides. The GR strain was 212 times resistant to dimethoate, 171 times to parathion, 398 times to "d:Lsul- photon sulphone", 457 times to oxydisulphoton but only about

10 times to Demeton-S and 12 times to pirimicarb.

Finally,' perhaps it is worthwhile to briefly outline the extensive work done in Japan (by Kojima et al., 1963 c and d and by Ozaki and Korosu, 1967) on two other homopterans 116 of the jassid family. Ozaki (1969) has reviewed these findings and regarding cross-resistance, he traces out how a strain of Nephotettix cincticeps (Uhler) with a 98-fold resistance to malathion was also resistant to dimethoate (137x) and cidal

(37x) but, only slightly, to malaoxon. Members of this clone also showed resistance to parathion (17x), diazinon and fenitrothion (8x), methyl parathion (4x), fenthion (6x), EPN (10x) and mitimate (8x). However, the R-strain remained susceptible to saligenin cyclic phosphorus and carbamate insecticides except for carbaryl (sevin) to which a slight resistance was noted. Interestingly enough DDT and BHC were more toxic to the R-strain than to the 'susceptible' one. A more or less similar pattern of resistance was also observed in a malathion-

12 and fenitrothion-R strain of Laodelphax striatellus (Fallen),

As Busvine (1968) points out, the resistance spectra of these hoppers suggest that they possess more than one resistance mechanism.

The Aim of this Investigation

The main purpose of the following toxicological work was to draw up a "resistance spectrum" of a resistant strain of Myzns persicae selected continuously with dichlorvos, for a range of different compounds and to compare this with the resistance data available for some other species of insects.

It was further endeavoured to determine whether the differences 117 in the structures of the chemical compounds used would help to indicate the resistance pattern in this particular resistant strain of Myzus persicae.

Materials and Methods

Insect material

Adult, apterous, virginoparous Myzus persicae were used in all toxicological experiments. As already mentioned, the resistant strain, primarily selected with DDVP, was obtained from Jealott's Hill, Bracknell. A constant insecticidal pressure, (that i , spraying 15 ml of 250 ppm of dichlorvos per plant, once approximately every twelve days) was maintained throughout to keep up the resistance of the insects.

The 'susceptible' (unselected) strain used was derived from the colony cultured at Silwood Park and has been known to be free from any insecticide contamination for atleast five years. The aphids were reared on turnip plants (Brassica rang_ var. depressa) sown in partially sterilized (one and a half hour at 180°C) soil (loam, sand and peat in the ratio of 3:2:1, plus natural fertilizers) and grown in muslin covered cages in the greenhouse. The plants were kept free from aphids and other parasites. In the laboratory the aphid culture was maintained in a controlled temperature room at 118

Fig. 2.1(a) - One of the perspex cages

used for rearing Myzus perseae in

C. T. rooms

Fig. 2.1(b) - Galley's leaf-disc unit

employed for toxicological assay of insecticides. 119 200 -I-1°C and 47% (t 5%) R.H., in perspex cages (30 cm x 30 cm x 30 cm) with circular openings (20 cm diameter) , at one end (Fig. 2.1). The openings were covered with fine muslin lined with "velcro" strips so that the muslin flaps could be easily removed and "stuck" on again with slight pressure.

Insecticides Used

The ten insecticides used in the toxicological work are detailed in the tables below (Tables 2.1 a and b and . their structural formulae are given in Pig. 2.2.. They have been grouped under two broad headings depending on how they were applied to the aphids.

Table 2.1(a) - Insecticides applied topically

Common and alternative *Chemical Names Purity names

1.DDT (pp'-iso- 1,1,1-trichloro-2,2-di-(4-chloro- 100% mer) phenyl) ethane (recrystallised)

2. Aldrin 1,2,3,4,10,10-hexachloro-1,4,4a, Analyti- (Octalene) 5,8,8a-hexahydro-exo-1, 4-endo- 5,8-dimethanonaphthalene cal grade

3. Dichlorvos 2,2-dichlorovinyl dimethyl 98.6% (DDVP;Nogos; . phosphate vapona; DDVP- USSR)

*BSI nomenclature used throughout except for diazoxon 120 Table 2.1(a) contd.

4. Diazoxon 0,0-diethyl,0-(2-isopropyl-6- l00% methyl-4-pyrimidyl) phosphate

5. Diazinon diethyl 2-isopropyl-6-methyl-4- 95.2% (Basudin; pyrimidinyl phosphorothionate G-24480)

6. Ethyl para- diethyl 4-nitrophenyl phospho- 98.7% thion rothionate (Thiophos- USSR)

Table 2.1(b) - Insecticides used systemically

Common and alternative *Chemical names Purity name

1 9 Dimethoate dimethyl S-(N-methylearbamoyl- (Rogor; Fos- methyl) phosphorothiolothionate 99% tion M,M;/ Per- fekthion-. Roxion)

diethyl S-(ethyl thiomethyl) 2. Phorate 98.9% (Thimet) phosphorothiolothioate

3. Demeton a mixture of Demeton-0 i.e., di- (Systox) ethyl 2-(ethylthio) ethyl phosphorothionate and Demeton-S i.e., 95.5% diethyl 2-(ethylthio) ethyl phosphorothiolate

4. Pirimicarb 2-dimethylamino-5, 6-dimethyl 100 d (PP 062 - ICI) pyrimidin-4y1 dimethyl carbamate * BSI nomenclature used throughout except for diazinon 12·1

CI CI

H· CI ~ ~/--- CI CI< )-{-~CI H ti__ -t~'1 ../ C~- LJ \~H 3 H 'H

DDT ALDRIN

DDVP DIAZOXON

CH I 3 ~C N ~ 'CH S I II II OC H ( CH )CH-C C-O-p ..... 2 5 3 2 ~N/ 'OC2H5

PARATHION DIAZINON

DIMETHOATE PHORATE

(I)

( II)

DEMETON

Fig. 2.2 - Structural formulae of the insecticides tested agai.nst Myzus persicae (Sulz.) 122 Topical Application of Insecticides

The FAO (1970) recommended method for topical application of insecticide to Mvzus persicae was modified only in the micro-applicator employed. This modification was found necessary because 0.25 111 droplets tended to flood the aphids so that some insecticide was invariably lost. Instead, micro- capillary glass tubes similar to those described by Hewlett and Lloyd (1960), who showed that tubes capable of producing reproducible volumes of 0.03 Ill were possible, were used.

Briefly, the micro-capillaries used were made as follows: Glass tubing with an internal diameter about half that of the external was evenly heated over a bunsun flame and drawn out into fine capillaries. The internal diameter of the drawn out tubing was then measured under a microscope with x8 eyepieCe and x10 objective, and its volume was estimated using 2 V = 'Tr x 1, where, r = the internal radius of the precision-bore tube, and 1 its length. Only those tubes delivering a volume of approximately 0.08 pl were selected and used with a "Drummond microcap". To minimize inaccuracies which might arise from irregularities in the internal bores of long tubes, the length of tubes chosen was restricted to the range 4.5 - 7.5 mm. Table 2.2 summarizes the details of the specifications necessary to obtain the volume of liquid required. 123

Internal diameter Length of mi- Approximate I 2 % of micro-caps. in Radiuskr ) cro-cap reqd. volume of mm = (-)2 when vol.=0.08p1 micro-caps. *MD x 0,0139 = Y 1 = 0.08 3.143x r2

0.1181 0.0035 7.27 0.08 pl 0.1251 0G0040 6.35 tt 0.1320 0p0044 5.80 n 0.1390 0.0049 5.20 in 0.1460 0.0053 4.80 H

* MD = micrometre division = 0.0139 mm Table 2.2 - Table for determining lengths of micro- capillaries capable of delivering 0.08 pl approximately (internal diameter known)

The volume delivered by a given micro-capillary was confirmed by calibration: a set of ten deliveries of a densely coloured dye solution (a saturated solution of methylene blue at room temperature, centrifuged at 5000 rpm for 15 minutes to remove undissolved particles) from each micro-capillary was collectively added to a known volume of water and the optical density was then measured colorimetrically. Reference to a standard curve (Fig. 2.3) giving the optical densities of appropriate dilutions of methylene blue provided the total volume delivered by each micro-capillary which divided by 10 gave the average volume of the micro-capillary in question. OPTICAL DENSITY (Filter no. 0.5 Fig. 2.3-Astandard curveforthedeter-

mination ofmethylene bluedelivered 1.0 TIL OFMETHYLENEBLUEIN by micro-capillaries 5 MLOFWATER 2.0 124 125

Application of insecticidest holdipgand measurement and recording of response

For topical applications insecticides were dissolved in acetone (analar grade) and appropriate dilutions were prepared after preliminary bioassays had been used to determine the ranges of dosages required. Turnip leaves infested with aphids were excised 24 hours before the assay whence subsequent wilting of the leaves forced aphids to withdraw their stylets so that they could be collected without dam- aging their mouthparts. Young adults and fourth (late) in- star larvae were selected for the experiments. Groups of 10 insects were anaesthetised with carbon dioxide and 0.07 -

0.08 111 (depending upon the volume of the particular micro- capillary employed) of the insecticide/acetone solution was applied dorsally on each aphid. Three replicates of 10 aphids at each of 4/5 dosage levels, Baranyovits and Muir,

1969 (plus batches of acetone treated controls) were held on turnip leaves in Galley's (1968) leaf-disc units (Fig. 2.2) for 24 hours at 20°C after which mortalities were recorded. Paralysed and moribund aphids were counted as dead. In two cases the control mortalities exceeded 10 percent possibly due to contamination of the perspex leaf-disc unit and the assays were repeated after the units had been cleaned with overnight soaking.in a solution of Decon 75 followed by a thorough rinsing with water. 126

Systemic application of insecticides

Here the basic procedure of collecting and selecting aphids was the same as described in the preceding page.

To apply the insecticide 1.0 pl of the appropriate dilution of each insecticide/acetone solution was injected (by means of a Shandon 10 pl syringe) into the petioles of leaf-discs

(2.54 cm in diameter) cut from healthy turnip plants. Control leaf-discs were injected with 1,0 pl of acetone only. Once the discs were placed on the perspex tray, their petioles aligned with their water-wicks, 10 aphids, with a total of atleast 30 per dosage level, were placed on each disc. The whole unit was then removed to a controlled temperature room

(20° ± 1°C) and mortalities were recorded 24 and 48 hours after the injection of the insecticides.

Data Analysis

The data were analysed using the London University

CDC 6600 computer (through the computer terminal at Silwood

Park) and a program for probit analysis developed by the

Canadian Department of Agriculture. The highlights of the program included an independent line analysis for each of the series in an experiment, a joint analysis for any specified subset(s) of series and a test for parallelism using the likelihood ratio statistic. 127 Results and Discussion

For all the dosage levels tested, probit mortalities

(after correcting for control deaths using Abbott's formula,

1925) were plotted against the log dose of each insecticide multiplied by a factor of 10 to avoid negative values. Then using the data supplied by the computer analyses the "best- fit" regression lines were drawn for both the 'susceptibe'

(unselected) and the resistant strains (Figs. 2.4 - 2.13)

A summary of the results after statistical evaluations is given in Tables 2.3(a) and 2.3(b). The resistant factor

(ratio) was calculated using the formula : LD of resistant strain 50 LD of 'susceptible' strain 50

From the Tables it is evident that the order of toxicity for the Silwood 'susceptible' strain to the topically applied insecticides was as follows : parathion > diazoxon> diazinon > aldrin > dichlorvos > DDT while the resistance levels for these compounds were, in descending order, parathion, dichiorvos, diazinon, diazoxon, DDT and aldrin. Among the systemics tested pirimicarb was most toxic followed by dimethoate, phorate and demeton. Resistance to

dimethoate 'at x51.3 was the highest while it was similar for phorate and demeton (11.4 and 11.3 fold respectively) values for each and lowest for pirimicarb (x6.6). The LD50 128

Fig. 2i$- - Mortality/response curves for DDT applied topically to two strains of Myzus persicae (Selz.)

2.5 - Mortality/response curves for aldrin

applied topically to two strains or myzus

persicae (Sulz.) PROBITMO RTA LITY 6.5 3.5 4.5 505 1.o 1.5

—0 LOG DOSE x10 (pg LOG DOSEx10 S-otrain 1.9

2.o

kpg/aphid) 4 4

2.3

/ aphid) . 2.7 3.o 1:29 130 Fig. 2.6 - Mortality/response curves for dichlorvos applied topically to two strains of Myzus

2=1E19. (sulz.)

Pia. 2.7 - Mortality/response curves for diazoxon

appUi.ccl topically to two strains of Myzus

persleae (Sulz.) 131

0---0S-strain 0 0 R-strain 7.0

Y 6.o LIT

MORTA 5.0 IT OB

PR 11.o

1.7 2.5 3.3

4 / LOG DOSE x10 kmaphid)

7.0 Y 6.0 ALIT MORT 5.0 IT PROB 4.0

1.0 1.3 1.6 1.9 2.2 LOG DOSE x104 (pg/aphid) 132 Fig. 2.8 - Mortality/response curves for diazinon applied topically to two strains of Myzus porsicae (Selz.)

Pic. 2.9 - Mortality/response curves for parathion

appLiect topicaLly to two strains of Hyzus

persleao (Solz.) PROBITMORTA LITY PROBITMORTALITY 7.0 4.0 6.o 5.0 0.8 0.8 o---0 S-strain

LOG DOSEx10(vg/aphid) LOG DOSE x10 (pg/aphid) 1.6 2.0

5 2i 3.2 133 134 Fig. 2.10 - Mortality/response regression for two

strains of Myzus persicae to the systemic

action of dimethoate (Symbols as in Fig. 2.12)

Fig. 2.11 - Mortality/response ror;ression Cor two

strains oC M.porsicne to the systemic action

or phovato (Symhot:1 as in Pia. 2.12) PROBITMOR TA LITY PROBITMORT ALITY 6.o 4.0 7.0 5. 0 1.3 1.7

LOG DOSEx10(pg/leafdisc) LOG DOSE x10 2.5

2.1

3.3 2

(Fig/leaf disc) 401

2.9 409 135 136 Fig. 2.12 - Mortality/response regression for two

strains of Monersicae to the systemic action

of demeton

Fig, 2.13 — Mortnlity/responso regression Cor two

strains oC M.persicne to i;ho systemic action

or pirimicarb (Symbols as in 'ip; 2.12)

137 S-strain after 24 hours A— AR-strain 0—.-0 S-strain " 48 " / 0— o R-strain 7.0 / / Y IT AL RT MO IT OB R P

FIGURE - 2.12 3.0

t.3 2.1 2.9 LOG DOSE x102 (lig/leaf disc)

7.0 / / ./ A / .,,•/ 0 ./ ./ / ./ E-1 6.0 • H •/ .41/ / / .c4 / ./. gE-1 .0/ , ./I 0 A c., 5.0 / A / H el. / P /- ./ 0a / / Ii /. ..? 4 . 0 9" .41° / A FIGURE - 2.13

1.2 2.0 2 . 8 LOG' DOSE x103 (pg./leaf disc) STRAIN OF PARAMETERS OF 95% CONFIDENCE HETEROGENEITY LD 50 S.E. RESISTANCE INSECTICIDE MYZUS FROBIT LINE LIMITS OF LD50 (p.g/aphid) FACTOR PERSICAE a b ± S.E. X2 D.P. p LOWER UPPER S 0.7621 1.8728 t .584 6.261 2 • 0.0183 ±.061 -- -- DDT 1.8 R -2.7812 3.0856 t .490 0.200 1 > .50 0.0333 ±.042 0.0268 0.0401 S 2.6140 1.1673 ± .180 0.371 1 > .50 0.0111 ±.100 0.0068 0.0174 ALDRIN 1.50 R 2.6530 1.0559 ± .200 2.080 3 > .70 0.0167 +.057 0.0093 0.0289 S -0.6140 2.5310 + .423 0.326 1 > .50 0.0165 ±.052 0.0127 0.0208 DICHLORVOS 11.5 R -3.7897 2.6815 ± .475 0.310 , 1 > .50 0.1896 1.053 0.1433 0.2384 S 1.7785 2.5249 t .580 0.494 1 > .30 0.0019 *.059 0.0013 0.0024 DIAZOXON 2.1 R 0.9385 2.5601 * .449 1.960 1 > .10 0.0039 ±.052 0.0029 0.0048 S " . 2.3408 1.7265 * .287 2.250 2 >.30 0.0035 *.067 0.0026 0.0049 DIAZINON 2.8 R 0..6414 2.1925 i .336 0.694 2 > .70 0.0097 1.059 0.0072 0.0125 S 1.9383 2.1069 t .328 0.375 1 > .50 0.0003 ±.059 0.0002 0.0004 PARATHION 182.4 R -5.8336 2.9169 t .548 3.586 2 > .10 0.0518 ±.048 0.0405 0.0644

Table 2.3(a) - Probit analyses data for Myzus persicae (Sulz.) for the topically applied insecticides (° regression not significant ) STRAIN OF PARAMETERS OF 95% CONFIDENCE HETEROGENEITY LD t S.E. RESISTANCE 50 LIMITS OF LD INSECTICIDE MYZUS PROBIT LINE 50 (pg/leaf disc) FACTOR PERSICAE a b t S.E. "X2 D.F. P UPPER S -1.0774 2.7485 ± .448 0.174 1 >.50 0.1626 1.055 0.1219 0.2068 DIMETHOATE 51.3 R -18.6647 6.0372 11.029 2.775 2 >.10 8.3140 +.033 6.8980 9.4900 S -0.7826 3.7211 1 .737 0.589 1 >.30 0.3581 1.044 0.2754 0.4287 PHORATE 11.4 R 0.2464 1.8197 + .403 2.09? 2 <.50 4.0960 1.068 2.8000 5.5770 S 0.2604 2.8875 + .478 0.693 1 >.30 0.4379 +.055 0.3257 0.5536 DE ETON 11.3 R -2.7498 2.8753 + .483 1.389 2 .50 4.9580 ±.049 3.8660 6.2070 S 1.2210 2.2810 t .445 0.281 1 >.50 0.0454 +.061 0.0333 0.0606 PIRIMICARB 6.6 R -3.0011 3.2339 t .508 0.387 1 ;>.50 0.2980 ±.044 0.2382 0.3626

Table 2.3 (b) - Probit analyses data for Myzuspersicae (Sulz.) for four systemic insecticides. 4o insecticide and the histogram of the resistance levels are presented in Fig. 2.14.

It is difficult to compare the LD50 values of the insecticides used here with those published by other workers especially because of the different methods of assays involved. Besides, Myzus persicae Populations may show variations in their susceptibilities to insecticides as has been shown for dimethoate (Needham and Dunning, 1965).

However, the LD50 values for parathion and diazinon are comparable with those reported by the FAO (1970) and those for phorate and pirimicarb are quite close to those worked out by Ahmad (1970); but the FAO report shows diazinon to be more toxic than parathion contrary to the present work and to the findings of Guthrie et al.,(1956). At this point it is worth introducing the data obtained by Ludvik and Decker (1947, 1951), on the relationship between chemical structure and toxicity, for a large number of chemicals tested against several insects including Myzus persicae. Altogether they :tested well over

one hundred and fifty esters of phosphorus acids as contact insecticides (as water solutions .sprayed on infested leaves) and concluded that for high toxicity the following requirements were essential : (i) The compounds should have pentaval.ent

phosphorus atoms with two of the valencies linked with 141

an 0- or a S-atom and two others occupied by OR groups, the limits of R being ethyl or isopropyl, and (ii) for the highest toxicity, the fifth place should be occupied by P-OR' (R' could be a large alkyl group, an acyl group, an aryl group or an arylacyl group). According to them the general formula for a high toxicity compound would be (RO) P(0) OR'. It is interesting that parathion, 2 diazoxon and diazinon which were found to be most toxic among the compounds tested in the present study, agree with

their findings reasonably well.

The resistance pattern that emerges, for the insecticides used, is not easily explained at first with

reference to the .chemical groups of the insecticides

involved. The small number of coumpounds tested is an

,obvious limitation. However, the results become more meaning-

ful if one draws upon the data obtained by other workers

for several different resistant species. Consequently,

histograms of the resistance levels for Muses domestica,

Chrysomyia putoria and Nephotettix cincticeps together

with one for Myzus persicae as recorded by others, is

given in Fig. 2.15. Superimposing the histogram from Fig.

2.14 on to Fig. 2.15 presents the results in a better

perspective. LD50 INSECTICI DES pgi aphid LEVEL OF RESISTAN C E ( Topically applied ) S -STRAIN 10 30 50 I /....180. 1 1 3 OR GANO DDT (p.--e'-isomer) .0183

CHLORINES Aldrin .0111

Di ch I o rvos .0165 0 R P=0 G Diazoxon .0019 A N o Diazinon .0035 P H P=S 0 Parathion .0 0 03 S P H *Demeton .4379 A T P=S Dimethoate .1626 E S P ho r a t e .3581

CARBAMATE Pirimicarb .0454

LD I NSECTI CIDES 50 Ng/leaf disc (Systemically applied) S-STRAIN

FIG.2.14— RESISTANCE SPECTRUM OF• A STRAIN OF MYZUS PERSICAE

TO A RANGE OF 1 NSECT ICI DES. (LEVEL OF RESISTANCE = RATIO OF 1050 OF RESISTANCE STRAIN TO LD50 OF SUSCEPTIBLE STRAIN )-

(* = interchange of S to 0) 143

To a limited extent the resistance spectrum of

Mopersicae for some organophosphates and for one carbamate,‘ parallels that given by Needham and. Sawicki (1971) for the

GR strain, which in the glasshouse was exposed to .both groups of compounds. Thus,

GR-strain I "Silwood-R" strain

Resistance Factor (ratio)

Parathion 171 183

Demeton-S 10 NT

Demeton NT * 11.3

Dimethoate 212 * 51.3

Pirimicarb 12 * 6.6

NT = not tested; * = applied syste-

mically; rest topically applied.

The obvious difference lies in the rather high resistance of the GR-strain to. dimethoate. It is interesting to note that the two strains came from different sources but both were exposed to dichlorvos and selected continuously during experimentation. As a generalization it may be said that the OP-resistance spectrum of M.persicae provides two

points which might be significant. First, resistance to organophosphates, with the ex-

ception of dichlorvos, seems to be confined to the phosphoro- 144 Fig. 2.15 - Resistance spectrum of some insect species to several insecticides. (Insecticides: Pa-parathion; Da-diazinon; Do-diazoxon; Ph-phorate; De-demeton; DeS- demeton-S; Rg-dimethoate; Di-dichlorvos; Ma-malathion; Th-thiometon and Pi-pirimicarb)

A. Ne hotettix cincticeps, Nakagahawara strain F I Selected in the field with methyl parathion and EPN - Ozaki and Kurosu, 1967) N.cincticeps, Fujita strain (malathion and methyl parathion resistant - Ozaki & Kurosu, loc. cit.) B. Musca domestica (data from Bell & Busvine, see Busvine, WHO Publications, 1967) C. Chrysomyia putoria (data from Busvine et al., 1 963) D. Myzus persica°, "Silwood-R" strain FT rr rt "Gr-strain" (Needham & Sawicki, 1 971 ) rr "Genetic garden" strain, Harkov;), 1970.

-37 - indicates the insecticides used for select- 4 ing the particular strain

nt - not tested

146 thionates (P=S) - a feature in common with several other insects. However, it is difficult to explain the difference between the resistance factors for parathion and diazinon.

It has been shown that compounds with strong electron attracting substituents, like NO2 for example, on the phenyl ring (as in parathion) are less toxic to a resistant strain

of bed-bug (Feroz, 1970). This is understandable as it is known that phosphorus atoms with NO 2 or Cl sustituents tend to be more easily hydrolyse=d (Metcalf, 19551). This could, perhaps, partly explain the high resistance of M.perstne to

parathion (183-fold) rather than to diazinon (2.8-fold) in

the present studies. Though similar results have been shown for other insects (viz., the Nakagawahara population of

Nephotettix cincticeps which had a 41-fold resistance to

parathion but only 5-fold to diazinon) and mites (Jeppson,

et al., 1958) the reverse situation also prevails as indica-

ted by Oppenoorth (1959a) for strain A of Musca domestica

collected from Denmark. HArkova (1970) who noted a 73.5-fold

resistance to diazinon and only a 5.3-fold resistance to

parathion in two different field populations of M. persicae,

unfortunately did not assay each clonal line against both

insecticides. However, she did show that clones from both

populations were fairly resistant (x30 and x90) to the

phosphorothionate, thiometon.

Secondly, unlike the situation reported for other

insects, for example, Culex tarsalis (Matsumara and Drown, 147

(1961b), Cimex lectularius (Feroz, 1970) and strains of

Musca domestica (Oppenoorth, 1959b; Busvine, 1959; Forgash and Hansens, 1960 and Bell, 1968), the OP- resistance spectrum so far known for M.persicae suggests that the resistance mechanism(s) in this aphid works equally well against diethoxy (parathion) and dimethoxy (dimethoate) phosphorothionates. This of course could just be fortuitous because the number of insecticides, containing the two groups,

tested is small and the compounds chosen could represent

exceptions rather than the rule'.

One point that seems to assert itself is that for all

the cases of OP-resistance reported so far for M.persicae,

resistance to parathion has been most widespread in the field whereas resistance to malathion appears to be restricted

even in tests conducted in the laboratory (BArkovh, 1970).

This suggests that a resistance spectrum for M.persicae

needs to be worked out for compounds containing the carboxy-

ester group. It would be interesting to see if the unique

resistance shown specifically to malathion by some malathion-

resistant strains of Culex tarsalis, Musca domestica and the

German cockroach (van den Heuval and Cochran, 1965) also

exists in Myzus persicae. From the information available on the OP-resistance

spectrum of Myzus persicae it seems feasible to theorize that

the pattern indicates more than one resistance mechanism as

has been suggested for other insects, which show resistance to 1118 unrelated compounds.

Finally a brief comment on the absence of resistance. to DDT and aldrin, resistance to the carbamate pirimicarb and on the genetics of resistance in Myzus persicae. That the "Silwood-R" strain was only slightly tolerant of DDT and aldrin is not surprising as susceptibilities to organo- chlorines have been reported in other OP_ redstant insects, for example Cimex lectularius (Feroz, 1970) and two leafhopper species, Nephotettix cincticeps and Laodelphax striatellus. The last two showed a wide range of cross- resistance to many organophosphates and a few carbamates but remained susceptible to DDT and BHC (Kimura, 1965 and

Ozaki and Kassai, 1968 - as quoted by Ozaki, 1969). However, it may be mentioned here that Anthon (1955) did note that in North Central 'Washington, Myzus persicae showed resistance

to malathion, parathion and parathion DDT.

Little can be said about pirimicarb resistance in

Myzus though it is worth observing the interesting coin-

cidence that the three compounds namely, diazinon, diazoxon

and pirimicarb, to which resistance was rather low were all

structurally constructed round a pyrimidinyl ring.

The genetics of asexually reproducing insects is complex

and not fully understood. In aphids this is more so because

of the difficulties encountered in inducing aphids to lay fertile eggs under laboratory conditions (Sharma, 1971).

Stern and Reynolds (1958) noted that the developmental 149 pattern of resistance in parthenogenetic insects was unlike those in multivoltine, bisexual species. Shirck (1960) shares this view and all three assume that the mechanism of segregation and recombination of genes are absent in aphids reproducing parthenogenetically and therefore the diploid larvae (daughters) must be identical with the parent.

Cogneti's (1961) work contradicts this hypothesis. He has been able to show a special type of meiosis (he calls it endomeiosis) in three species of aphids including Myzus

(Myzodes) nersicae, where crossing over and hence recombination of genes occurs thus introducing genetical variations

in parthenogenetic clones. Whether Cogneti's work is beyond

censure only further research will confirm. But accepting it as it stands could help explain the loss of insecticide resistance in asexually reproducing insects when insecticidal

pressure is removed. 150

Appraisal and General Comments

As Perry (1964) points out physiological resistance to organaphosPhates may be characterized by the degree of inhibitor of cholinesterase and/or "ali-esterase" enzymes, and by differences in rates of activation and inactivation of the

0P-compound. Generally, the ability of insects to resist chemical poisoning will depend upon the balance between the rates of intoxication and detoxication, although other mechanisms such as insensitivity of the OP receptor at the site of action (Mengle and Casida, 1960) or the production of altered detoxifying enzymes (van Asperen and Oppenoorth, 1959) may also play some part. In the series of experiments described in

Part One Section B, it was clearly shown that, within the limits of the experiments, in the strains of My-zus persicae used, activity and insensitivity of cholinesterases could not be implicated in the resistance mechanism.

One of the most significant findings has been the differential ability of R-strain carboxylesterases to act on different substrates. Thus a high carboxylesterase activity towards 1-naphthyl acetate was recorded for the R-strain though an opposite results was obtained when ethyl butyrate was used.

Moreover, no differences in activity were evident for 2-naphthyl acetate and 1- and 2-naphthyl butyrates. This is important since it seems that of all resistant insects studied 151 resistant Mepersicae is the only one which combines high and low carboxylesterase activity depending on the ester being hydrolysed. Obviously a new factor in the resistance mechanism has now to be considered. The apparently anomalous behaviour of carboxylesterases strongly suggests the presence of different isoenzymes each acting differently on different substrates.

That carboxylesterase isoenzymes are present were amply demonstrated when these enzymes were separated electrophoretically on starch and polyacrylamide gels. Although electrophoretic separation was restricted to 1-naphthyl acetate-

splitting enzymes it was possible, by selective inhibition, to pinpoint particular isoenzymes responsible for differential activity towards the naphthyl ester.

Further insight into the role of carboxylesterases in the resistance mechanism was afforded by inhibition studies on

the enzymes. In vitro inhibition of carboxylesterases with

several organophosphates (DDVP, diazinon and diazoxon) and a

carbamate (eserine sulphate) demonstrated that enzymes from

the R-strain were not only more resistant to these chemicals

but also when incubated with the substrate before the addition

of the toxin, recovery was more complete in the R- than in the S-strain. The way in which these findings relate to the various

theories on resistance phenomenon has been discussed elsewhere.

However, the position of carboxylesterases in the general

physiological processes of an insect is not completely under-

stood and requires further consideration. PROBITM ORTA LITY 6.5 4.5 5.5 305 1.0 105 0

0 LOG DOSE x10 (lig/aphid) LOG DOSEx10 S.-strain 1.9

2 .0

4 4 (pg/aphid) 2.3 /

2.7 3.0 1:29 152

Many workers have successfully associated carboxylesterases with insecticide resistance yet very little is known about the precise function(s) of these enzymes. In a series of articles Wan and Hooper (1967, 1969) and

Hooper and Wan (1969); have demorftrated that at least in Musca domestica and Blattella germanica "Ali-E" activity is cyclical and highest in mated females. They also suggest that "Ali-E"

plays a role in reproduction (eg., in the production of oothe-

cae and in vitellogenesis in B. germanica) part of which may be mobilization of fats. The latter finding is given further

substance by the work of Tan (1972). He found that in the fat

body of the cave roach, Pvcnoscelus striatus (Kirby) lipases,

which are normally responsible for the breakdown of triglycer- ides to diglycerides, had very low activity whereas carboxyl-

esterases were very active: In starvation experiments he showed that lipids are very heavily drawn on for energy and he

postulates that lipases alone could not cope with this demand

and that carboxylesterases (non-specific) might play an important role. van Asperen.(1959) pointed out that high lipase

activity in the fat body of Schistocerca gregaria reported by

George and Eapen (1959) was due to carboxylesterases. It is

worth noting that (i) in higher animals liver "aliesterase" is

known to display activity towards fats in solution (Oosterbaan

and Jansz, 1965) and that (ii) most insect carboxylesterases

are located in the abdomen where large quantities of fat are

also stored. 153

The present evidence strongly suggests that in whatever way carboxylesterases may function in the resistance mechanism they are also certainly involved in insect reproduction and fat mobilization. If the latter two . are established beyond doubt as general functions in most insects it would be interesting to see how this could be linked with insecticide resistance in parthenogenetic insects.

The toxicological studies described indicate that the strain of resistant M.persicae used, in common with other insect species, shows cross-resistance to a. number of insecticides. However, the important point to note is that the resistance mechanisms seemed to work equally well against diethoxy and dimethoxyphosphorothionates and were almost absent against

DDT and aldrin. To suggest that more than one resistance mechanism is involved in M.persicae would be based on circumstantial

- evidence. However, the hypothesis becomes more credible if

one takes into account the following facts: (1) the existence

of carboxylesterases isoenzymes which show different degrees of substrate preference (2) the in vitro insensitivity of these

enzymes to several organophosphates (3) the involvement of multi-function oxidases in the resistance mechanism as indica-

ted by "sesamex" treatment (Needham and Sawicki, 1971) and

(4) the fairly wide cross-resistance spectrum of the aphid to

insecticidal compounds. A further suggested mechanism of 154. resistance in Myzus needs mention. Eastop and Banks (1970) noted that the siphunculii (modified lateral abdominal tuber- cles of aphids concerned with wax or triglyceride secretion) of R-Myzus persicae were relatively longer in more resistant clones and that the mean siphunculii length for the II-population dropped when it lost its resistance. This led them to suggest that the mechanism of resistance could involve the excretion of insecticides in association with wax or lipid, through the siphunculii. How much credence may be attached to this hypothesis will depend on the results of a more thorough investigation on the excretory mechanisms and the excretory products of aphids. Finally a few comments on the future of the resistance problem in green peach aphids. Because of the limited work done on the resistance mechanism of Myzus persicae there is

scope for further work. Foremost would be an investigation of

the degradation of organophosphorus compounds by the enzymes

in question, leading to an accurate recording of the metabolites produced. Detailed in vivo studies involving knock-

down and enzyme recovery are clearly indicated although the

small size of this aphid will introduce some difficulties. The

loss of insecticide resistance following the removal of insecti-

cide pressure certainly needs more attention especially since

this fact could be exploited in the control of these pests.

This would include working out a cross-resistance spectrum

using more insecticides and checking whether the loss of 155

resistance applies to all insecticides employed. The control possibilities from such investigations would be almost limitless. T56

General Summary

PART ONE

Section A

1. A method has been described which makes it possible to cut sections of fragile tissues by embedding them in paraffin without destroying the esterases present. This method was used to find out which tissues of the green peach aphid, Myzus persicae, were zones of esterase activity.

2. Using different inhibitors, the brain was found to be the main centre of ChE activity whereas the gut and the developing embryos were the main regions of carboxylesterase activity.

3. The advantages of fixing tissues for enzyme study are discussed and it has been shown that 10% buffered formalin, at 4oC for twenty four hours is the best fixative for retaining esterase activity of Myzus persicae.

Section B (1), (2) and (3)

1. Using titrimetric and colorimetric techniques it has been demonstrated that ChEs hydrolysing acetylcholine chloride occur in equal amounts in both the 'unselected' normal and the

OP-resistant strains of Myzus persicae (Sulz.). ChEs from both strains were equally sensitive to eserine sulphate and DDVP inhibition. Quantitative and kinetic differences between the ethyl butyrate-splitting carboxylesterases of the two 157 strains were established. The R-strain carboxylesterase hydrolysing ethyl butyrate had a Km value of 8 x 10-5M compared to 9.7 x 10-5M for the S-strain.

2. An investigation of carboxylesterase hydrolysis of alpha and beta isomers of naphthyl acetate and naphthyl butyrate was made. No differences were recorded in the in vitro carboxylesterase activity of the 2 strains toward beta naphthyl acetate and alpha and beta naphthyl butyrate. Carboxyl- esterase activity towards alpha naphthyl acetate was consider- -4 ably higher in the R-strain with a Km value of 1.2 x 10 M -4 compared to 3014 x 10 M for the S-strain. It was shown that 1-naphthyl acetate-splitting carboxylesterases from the R-strain were not only more resistant to in vitro inhibition with eserine sulphate and DDVP but recovery from the inhibitory

effects of the latter was greater in the R- than the S-strain. Because•of the unspecificity of naphthyl esters it has been suggested that esteratic studies involving only such substrates

may not always provide an accurate understanding of these

enzymes.

3. Loss of organophosphate insecticide resistance in

green peach aphids reared in an insecticide-free environment was accompanied by a corresponding decrease in carboxylesterase

activity.

Section C 1. An electrophoretic study of whole Myzus nersicae 158 esterases was carried out in polyacrylamide and starch gels.

Starch gel electrophoresis, using standard equipemnt, resolved

6 esterase bands in both strains. E4 showed more activity in the R-strain. Variable polyacrylamide gel electrophoresis was done with a modified disc electrophoretic apparatus. In this case also 6 bands of esterases were established of which E was more pronounced in the R-strain. The Rf values of the 3 esterases have been calculated and some differences between the two strains have been recorded. Acid phosphatase was characterized and found to be similar in both strains. No alkaline phosphatase activity could be demonstrated. 3. The relative efficiencies of the two gel media have been discussed and the results obtained compared.

PART TWO

Of the ten insecticides assayed for toxicity against

the 'susceptible' (unselected) and the resistant strains of

Myzus persicae it was found that the order of toxicity for

the topically applied insecticides was as follows: parathion

diazoxon>diazinon>aldrin> dichlorvos> DDT while the resis-

tance levels for these compounds were, in descending order,

parathion, dichlorvos, diazinon, diazoxon, DDT and aidrin.

Similarly, of the systemic insecticides tested, pirimicarb

was most tozic followed by dimethoate, phorate and demeton.

Resistance ratios were in the order dimethoate> phorate> 159 demeton> pirimicarb.

From the OP-resistance spectrum obtained two things were indicated. (i) There appears to be more than one resistance mechanism involved in Myzus persicae towards organophosphates insecticides and (ii) unlike the cases reported for several other insects resistance in Myzus seems to work equally well against both diethoxy and dimethoxy compounds. 160

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0.3175 3.15 2.29 4.37 1.72 5.81 0.7500 1.33 18.03 0.55 19.40 0.52 Acetyl- 26.13 0.38 25.90 Choline 1.5000 0.67 0.39 26.25 0.38 Chloride i 1.7500,0.57 27.0 0.37 3.0000 0.33 27.4 0.36 26.81 0.37

1.875 0.53 8.16 1.23 6.48 1.54 Ethyl 3.750 0.27 15.04 0.66 12.75 0.78 butyrate 7.500 0.13 22.2 0.45 19.06 0.52 15.000 0.07 24.33 0.41 21.15 0.47

Appendix Table I - Data for the double- reciprocal plot of 1/v0 vs 1/S for the reaction catalysed by whole aphid esterases at 370C.(Per 30 aphid for acetylcholine chloride and per aphid for ethyl butyrate) *See G. Morris' A Biologist's Physical Chemistry (1971) page 269. (S) 1 OP-SUSCEPTIBLE OP-RESISTANT Substrate (S) v° 1 ,vo 1 vo x 10 x 10 (S) mm mm-1 v0 (ugm/30 min) (pgm/30 min)-1 (pgm/30 min) (pgm/30 min)-1

0.075 13.33 6.61 1.51 24.37 0.41 0.150 6.67 11.02 0.91 34.71 0.29 1-naph- 0.58 43.88 0.23 thyl 0.300 3.33 17.24 Acetate 0.600 1.67 23.13 0.43 48.66 0.20 1.200 0.83 26.44 0.38 48.25 0.21

0.0015 666.7 0.96 10.42 0.93 10.75 0.0030 333.3 1.89 5.29 1.86 5.37 2-naph- 2.64 thyl 0.0060 166.7 3.83 2.61 3.78 Acetate 0.0120 83.3 7.62 1.31 7.57 1.32 0.0240 41.7 9.80 1.02 9.78 1.02

Appendix Table II - Data for the double reciprocal (Lineweaver-Burk) plot of 1/v° vs 1/(S) for the reaction catalysed by whole aphid carboxylesterases at 3700. (S) 1 OP-SUSCEPTIBUR OP-RESISTANT vo vo Substrate (S) 1/v° x 10 1/v° x 10 (S) mM mm-1 (pgm/30 min) (1.1gm/30 min)-1 (ligm/30 min) (ligm/30 min)-1

0.0075 133.3 3.00 3.33 2.90 3.45 0.0150 66.7 6.88 1.45 6.61 1.51 1-naphthyl 0.030o 33.3 13.31 0.75 13.22 0.76 butyrate 0.0600 16.7 22.30 0.45 26.66 0.40 0.1200 8.3 37.21 0.27 37.62 0.26

0.003 333.3 0.83 12.05 1.03 9.71 0.006 166.7 1.53 6.53 1.7o 5.88 2-naphthyl 0.012 83.3 3.17 3.15 3.12 3.21 butyrate 0.024 41.7 5.83 1.72 5.98 1.67 0.048 20.8 7.89 1.27 8.28 1.21

Appendix Table III - Data calculated for the double reciprocal (Lineweaver-Burk) plot of 1/v° vs 1/(S) for the reaction catalysed by whole aphid carboxylesterases at 37°C. 809

LABP 20-106 APPARATUS AND DEVICES From: Lab. Practice, Oct. 1971, Vol. 20. No. 10 Simple modification of the standard disc electrophoresis apparatus. by K. I. Sudderuddin Department of Zoology & Applied Entomology, Imperial College Field Station, Sunninghill, Ascot, Berkshire

Ever since the work of Orstein and Davis (1964) disc This paper describes a simple modification of the standard, electrophoresis has been extensively used in most fields of cylindrical running tubes which overcomes the above dis- biological and medical sciences. It provides quick and advantages while still retaining all the merits of the standard reproducible bands of high resolution using micro quantities equipment. of proteins and sera. With the suggestion of Raymond and Apparatus Weintraub (1959) to use polyacrylarnide gels as a substitute Instead of the traditional cylindrical, glass running tubes for starch gels in electrophoresis, the former, in combination (5mm bore x 75 mm long) rectangular, perspex tubes are with disc electrophoresis, has proved extremely versatile. used. Figure 1 (a) and (b) shows both the standard and the However the standard disc electrophoretic apparatus has modified versions. The latter is constructed from a perspex certain shortcomings, which if eliminated would enhance its sheet, 1.5 mm thick, which is cut into two sizes: one measur- value as a tool for reseach. The disadvantages may be listed ing 7.8 cm by 0.7 cm and the other measuring 7.8 cm by as follows: (1) because it is not always easy to adjust the 0.6 cm. Thus each rectangular tube comprises four pieces running tubes (Figure 1(a)) vertical to the base of the upper (two measuring 7.8 cm by 0.7 cm and two measuring 7.8 cm buffer reservoir during an electrophoretic run, the protein by 0.6 cm) joined together with chloroform (Figure 1 (d)). bands (discs) do not always spread uniformly round the Each tube is then marked crosswise at three points: the first gel rods; (2) the cylindrical gel rods are difficult to slice one 1 cm from one end (henceforth called the top end), longitudinally if one desires to have two identical halves; the second 2 cm and the third 7 cm from the top end respect- (3) photography of the gels is inconvenient; and (4) densito- ively (Figure 1 (c)). The base plate of the upper buffer reser- meter tracings are not always easy (Smith, 1968). voir of the standard apparatus is removed and a new plate upper buffer resevoir

4- base 4c) pia to 7.8

,running tube

(c) (b) (a) 0:7 `c) 0 Figure 1. Parts of a standard (a) and modified (b) disc electrophoretic apparatus. (c) Expanded view of a rectangular running tube. (d) Cross section of (c). All dimensions in centimetres. (d) 810 Sudderuddin — Modification to the Disc electrophoresis apparatus LABORATORY PRACTICE

(2 mm thick) is attached. Eight rectangular tubes are now halves, one of which could then be used as a control while permanently fixed to the base plate so that (1) the tubes are the other could be treated with an inhibitor, for example. 1.5 cm away from the inner wall of the reservoir and equi- This duplicity is necessary when one wishes to study isozymes, distant from each other, (2) 4 mm of each tube projects into (2) Because the rectangular perspex tubes are permanently the upper buffer reservoir, and (3) each tube is vertical to fixed at right angles to the base plate the protein bands or the plane of the base plate. "plates" are evenly spread all around so that better and Whcn the apparatus is to be used the bases of the tubes clearer separations are achieved. (3) Photography and are plugged • with Plasticene. The polyacrylamide gel is dcnsitometry of flat gels are easier, especially the former prepared as described by Price (1968). When the gel is set the which now requires no sandwiching of the gels between glass plugs are removed and electrophoresis is carried on in the plates and an X-ray viewing box as with round gel rods. usual way as described by Smith (1968). In short this modified system incorporates the advantages If a variable gel system is required then the main body of the of disc electrophoresis and the vertical slab technique des- gel may be made up of a 71% of polyacrylamide solution scribed by Bosman (1966) and Price (1968). (up to the 2 cm mark from the top) and the initial portion may Perhaps the only disadvantage of having permanently fixed be made up of a 3% solution of polyacrylamide (up to the running tubes is that they are somewhat difficult to clean after 1 cm mark). The remaining 1 cm is used for the sample, use. This is easily overcome if the upper buffer reservoir Folded filter papers (3 cm by 0.7 cm) may be used in the together with the tubes are soaked in a warm solution of sample slot to prevent the sample from flowing back into the either Extran or Decon 75 for several hours and then thor- reservoir. oughly rinsed with distilled water. Gels are removed from the tubes by a rectangular Perspex plunger, which cloSely fits the tubes. If the plunger is pressed Acknowledgements gently in from the top end of the tubes the gels slide out I would like to record my sincere thanks to my supervisor Dr G. easily. Mttrdie for his support and encouragement during this work. References Discussion Bosman, T. (1966). Lab. Practice, 15, 435-439. The modifications of the standard apparatus described Orstein, L. and Davies, B. J. (1964). Annals N.Y. Acad. Sd., 121, 321-404. are simple and can be carried in an average laboratory. Price, G. M. (1968). Lab. Practice, 17, (4) 467-470. While retaining the accuracy anddan sensitivity of the original Raymond, S. and Weintraub, L. (1959). Science, 130, 711. apparatus the modifications allow for the following advan- Smith, I. (1968). Chromatographic and Electrophoretic Techniques. tages. (1) The rectangular gels can be easily sliced with a Vol. 11 — Zone Electrophoresis. William Heinemann Medical sharp knife or a thin piece of wire. This provides two identical Books Ltd.