1

Toxicity and Mode of Action of Avermectin B1 against insects

Terence Simon Corbitt, B.Sc.

A Thesis Submitted for the degree of Doctor of Philosophy of the University of London and for the Diploma of Membership of Imperial College.

Department of Pure and Applied Biology Imperial College Silwood Park Ascot, Berkshire SL5 7PY August 1987 2

ACKNOWLEDGEMENTS

I would like to thank my supervisor Dr. D.J. Wright for his supervision and advice during the course of this work and presentation of this thesis.

I would also like to thank the following: Dr A. Green (Merck Sharp and Dohme, New Jersey) for helpful advice and donation of [3H]AVMB1 and AVMB-j, Drs J. Hardie, D.J.Galley, M. Djamgoz and Mr I. Fosbrook for advice and help given during this study. Thanks also to Ms S.Smith of TDRI, Porton, Wilts, for the Spodoptera 1i ttorali s, and to Mr T.Carty of the Institute of Virology, Oxford for the Heliothis armigera cultures.

This work was carried out with the aid of a grant from the Science and Engineering Research Council. Insect culture was carried out under MAFF licence No. PHF 909/52 (116).

I would like to thank my parents for their interest and continual support during my education. Thanks also to Tom for plant culture, Sarah for typing my tables, Chris Addison and other friends who made my stay at Silwood Park enjoyable. I would like to say a special thank you to Tanya for her help and friendship.

This thesis is dedicated to my wife Anne for her support and understanding during the preparation of this work. 3

TABLE OF CONTENTS

PAGE ACKNOWLEDGEMENTS 2 TABLE OF CONTENTS 3 ABSTRACT 8 1. INTRODUCTION 10 1.1 The Avermectins. 10 1.2 Whole Organism studies. 13 1.3 Cel 1ular 1evel . 21 1.4 Present work. 33 2. GENERAL MATERIALS AND METHODS. 34 2.1 Insect Culture. 34 2.2 Selection of lepidopteran larval instars. 35 2.3 Chemicals. 35 2.4 Topical application of Pesticides. 37 2.5 Calibration of Arnold microapplicator and 37 Agla glass syringe 2.6 Contact/feeding toxicity of AVMB1 and 38 cypermethrin with and without oils. 2.7 Feeding rate of third instar larvae of 38 Spodoptera 1i ttorali s and He!iothi s armigera.

2.8 Effect of AVMB1 on the feeding of third instar 39 Spodoptera 1i ttorali s. 2.9 Analysis of bioassay data. 40 3. TOXICOLOGICAL STUDIES 41 3.1 Materials and Methods. 41 4

PAGE

3.1.1 Toxicity of topically applied AVMB1 and 41 cypermethrin to third, fourth, fifth and sixth larval instars of $.1ittoralis. 3.1.2 Toxicity of topically applied AVMB1 41 to third instar larvae of S.1ittoralis reared on various diets.

3.1.3 Toxicity of topically applied AVMB1 42 to third instar larvae of S.1ittoralis and H.armigera reared on artificial diet. 3.1.4 Injection of AYMB1 into fifth and sixth 42 instar larvae of $.1ittoralis. 3.1.5 Contact/ingestion toxicity of AYMB1 and 42 cypermethrin on Chinese cabbage foliage (with and without oils) against first, third and fourth instar larvae of S.1ittoralis. 3.1.6 Residual (Foliar) toxicity of AVMB1 on cabbage 44 (cv. Flower of Spring) under glass against third instar larvae of S.1ittoralis at 1, 3 and 7 days after spraying. 3.1.7 Contact/ingestion toxicity of AVMB^ on Chinese 44 cabbage and cotton against third instar larvae of S.littoralis and H.armigera. 3.1.8 Contact/ingestion toxicity of AVMB1 on cabbage 45 and cotton against first instar larvae of S.1i ttorali s, H.armi gera and H. vi rescens. 3.1.9 Assessment of feeding rate in third instar 46 larvae of S.1ittoralis and H.armigera reared on cotton. 5

PAGE 3.1.10 Effect of foliar residues of AYMB1 on the 46 feeding of third instar larvae of $.1ittoralis 3.1.11 Effect of foliar residues of AVMB1 on the 46 feeding of third instar larvae of $.1ittoralis. 3.1.12 Distribution ("Choice") of third instar larvae 46 of $.1ittoralis between untreated and AVMB1 -treated Chinese cabbage leaves. 3.2 Results. 48 3.2.1 Toxicity of topically applied AVMB.,and 48 cypermethrin to different larval instars of $.1ittoralis reared on Chinese cabbage 3.2.2 Toxicity of topically applied AVMB1 to third 48 instar larvae of $.1ittoralis reared on various diets. 3.2.3 Toxicity of topically applied AYMB1 to third 52 instar $.1ittoralis and H.armigera reared on artificial diet. 3.2.4 Toxicity of injected AVMB1 to fifth and sixth 52 instar S.1ittoralis larvae. 3.2.5 Contact/ingestion toxicity of AVMB1 and 52 cypermethrin on Chinese cabbage foliage (with and without the addition of oils) against first, third and fourth instar $.1ittoralis. 3.2.6 Residual (foliar) toxicity of AVMB1 on cabbage 56 (cv. Flower of Spring) under glass against third instar larvae of Spodoptera 1ittoralis at 1, 3 and 7 days after spraying. 6

PAGE

3.2.7 Contact/ingestion toxicity of AVMB1 on Chinese 61 cabbage and cotton against third instar

larvae of S.1ittoralis and H.armigera.

3.2.8 Contact/ingestion toxicity of AYMB1 on Chinese- 61 cabbage and cotton leaves against first instar larvae of S.littoralis, H,armigera and H. vireseens. 3.2.9 Assessment of feeding rate in third instar 65

$.1ittoralis and H.armigera.

3.2.10 Effect of AVMB1 on the feeding of third instar 65

larvae of S.1 ittoralis. 3.3 Discussion. 71

3.3.1 Symptoms of poisoning with AYMB1 71

3.3.2 Toxicity of AYMB1 to larval instars. 73

3.3.3 Effect of larval diet on insecticide toxicity. 77

3.3.4 Action of oil enhancers. 78 3.3.5 Larval weight gain. 80

4. UPTAKE OF [3H]AVMB1 BY Peri pianeta americana 83 4.1 Materials and Methods. 83 4.1.1 Localisation of total radioactivity in 83 Peri pianeta americana tissues.

4.1.2 Uptake of radioactivity by P.americana nerve 84 and muscle tissues with time.

4.1.3 Isolation of radioactivity from P.americana 35 nerve and muscle tissues.

4.2 Results. 88

4.2.1 Distribution of radioactivity in P.americana 88 tissues 7

PAGE • v 4.2.2 Uptake of radioactivity by P.americana nerve 88 and muscle tissues with time. 4.2.3 Isolation of radioactivity from nerve and 92 muscle tissues of P.americana.

4.3 Discussion. g8

5. ACTIONS OF AVMB1 ON A NERVE CORD PREPARATION OF . 10O Periplaneta americana

5.1 Materials and Methods. 100

5.1.1 In vitro effects of AVMB1 on spontaneous and 100

evoked activities recorded extracel1ularly from the ventral nerve cord of Peri pianeta americana.

5.1.2 Recording of spontaneousand evoked activities 102 from the ventral nerve cord of P.americana

injected with AVMB1 i_n vivo.

5.1.3 Relationship between spontaneous and evoked 102 activities present in the ventral nerve cord of adult male P.americana pre-injected with

[3H]AVMB1 and the level of radioactivity within nerve and muscle tissues 24 and 96h after injection.

5.2 Results. 104

5.2.1 In vi tro preparations. 104

5.2.2 In vi vo preparations. 110 5.3 Discussion. 115

6 . GENERAL DISCUSSION AND CONCLUSIONS 122

6.1 Toxicity of AVMB1 againstlepidopteran larval instars. 122

6.2 Radiochemical and Neurophysiological studies. 124 REFERENCES 128

APPENDICES 136 8

ABSTRACT

The avermectins are a group of naturally-derived compounds which possess insecticidal, nematicidal and acaricidal properties. Some of the insecticidal effects of Avermectin B1a/1b (80:20) (AVMB1) have been studied in three noctuid species (Lepidoptera): the Eygptian leafworm, Spodoptera 1ittoralis; the cotton budworm, He!iothis armigera, and the tobacco budworm, He!iothis vireseens, while radiochemical and electrophysiological studies with AVMB1 were conducted on the cockroach, Peri pianeta americana. Topical application of AYMB1 or the pyrethroid, cypermethrin to third, fourth, fifth and sixth instar S.littoralis indicated that the fourth and fifth instars were insensitive to AYMB^ this was not found with cypermethrin. Injection of AVMB1 into fifth and sixth instars was found to reduce the concentration of AVMB1 required to give an LD5Q value. This observation, together with the fact that fourth instar larvae of S.1ittoralis were sensitive to AVMB1 residues on leaves suggested that the insensitivity of this instar to topically-applied AYMB1 was due to a reduced rate of penetration of the pesticide when compared with the third instar, for example. Feeding of larvae on AYMB1 or cypermethrin-treated leaves, with and without Sunspray 6E and safflower oil enhancers, was found to be a particularly effective method of controlling first and third instar larvae of S.1ittoralis, H.armigera and H.virescens. The residual toxicity of AYMB1 on cabbage under 9

glasshouse conditions was also determined. A significant reduction in activity was seen fourteen days after spraying. Sunspray 6E and safflower oils were found to enhance the residual activity of AVMB1. The weight gain of third instar $.1ittoralis fed on AVMB1-treated leaves or topically applied with AVMB1 was seen to be reduced compared with controls. However, there was no preference between control and AYMB1-treated leaf discs suggesting that AVMB1 has an anorexic rather than an antifeedant action. The toxicity of AVMB1 towards larval instars of the above insect species was thus shown to be complex, with dose-dependent lethal and sub-lethal effects, causing changes in feeding behaviour which protect the plant from attack by these species. In the second part of this study, an attempt was made to correlate toxicological and physiological effects of AVMB1 on insects. Avermectins are known to interact with chloride ion channels, in particular those associated with the inhibitory neurotransmitter gamma-amino butyric acid. The action of AVMB1 on a nerve preparation of adult, male P.americana was investigated following jjn vitro and in vivo treatment with the pesticide . Spontaneous and evoked activities were recorded extracellularly using a suction electrode. In some preparations, the sixth abdominal ganglion was partially desheathed to facilitate entry of AVMB1 . The distribution and uptake of [3H]AVMB1 in adult; male P.americana was investigated by standard radiochemical techniques. The rate at which symptoms of poisoning appeared was used'to determine the times dissection procedures were carried out. 10

INTRODUCTION

1.1 The Avermectins. The avermectins (AVM) are a group of compounds which were isolated from the actinomycete Streptomyces avermitil is (NRR L8165) during a Merck Sharp and Dohme anthelmintic screening programme in 1976 (Burg et al ., 1979). Structually, the AYM consist of a rigid sixteen membered lactone ring system. A spiroketal group is attached to the lactone ring so as to form two-six membered rings. Additionally, there is present either a cyclohexenediol or methoxy-cyclohexenediol group which is cis fused to a five membered cyclic ether. These compounds are further characterised by the presence of a dissacharide substituent, consisting of two identical a-1-oleandrose monomers coupled to carbon 13 through an oxygen bond. The AVM are closely related to the milbemycins which are a group of sixteen membered macrocyclic lactones, also derived from a Streptomyces strain. However, notable differences between the two groups are the absence of the 13 hydroxy disaccharide substituent and the saturation at the 22,23 position in all the reported milbemycin compounds (Fisher and Mrozik, 1984). AVM may be separated into four major (A1g,A2g,B1a> B2a) and four minor (A1b,A2b,B1b,B2b) homologous components (Fig. 1.1), mixtures of homologous pairs containing at least 80% >of the 'a' and not more than :20% of the ' b' , component being referred to as AVM A1,A2,B1,B2. The 11

■'B1.series are generally more active, the relative toxicity of the B., and B2 groups varying according to the target species and mode of application (Campbel1 et a±., 1983). The large 'A' designation refers to those AVM components in which there is a methoxy group present at carbon 5, whilst a hydroxy group present at this position designates a large 'B1. The number 1 designates those natural products possessing a 22,23 double bond whilst a carbon 23 hydroxy substituent is designated number 2. Both series of components are further character!*sed by the presence of a secondary butyl substituent in the 25 carbon position (F ig .1-1). The AVM have been shown to be active against nematodes, acarines and many insect species, but have been found to be inactive against trematodes, cestodes protozoa, bacteria and fungi (Campbell, 1981; Putter et al-,

1981; Wright, 1986). Avermectin B1 (AVMB^ , MK936 or

Abamectin) has been developed for use as an agricultural acaricide/insecticide (Dybas and Green, 1984; Wright et al_., 1985,a,b) whilst a synthetic derivitive of AVMB1 : 22,23 dihydroavermectin B1 (DHAVMB1# MK933 or Ivermectin) is marketed for veterinary use (Campbell et al., 1983). 12

Fig. 1-1. General structure of avermectins

R1 = C^3 (comPonent A); H (component B) r 2 = CoHr (component a); CHo (component b) • OH X = -CH=CH- (component 1); ^.(component 2) 13

1.2 Whole Organism studies

1.2.1 Systemic and Translamlnar effects. Ostlind et a]_. (1979) demonstrated that a crude extract of AVM's possessed an Insecticidal action against the flour beetle Tribolium confusum. These authors also tested AVM against cuterbrid larval infections of mice, the mice being infected with three larvae and dosed by gavage with AVM. It was found that AVM possessed a systemic action, the 'A' component being effective at 0.078 mg per kg, other AVM's being less active at this level, the minimum for 1B11 was estimated to be 0.1 mg per kg.

James et al_« (1980) using a crude extract of AVM's on first instar larvae of the blowfly Luci1ia cuprina found an approximate LC5Q of 0.12 pg/ml (ppm). However, the purified components showed even greater activities, 'A1a‘ for instance had a LC5Q of 0.0058 ppm; the 'B' component having a similar value against this species. This compared favourably with the organophosphorus insecticide diazinon which had a LC5Q of 0.03 ppm with L.cuprina . Further studies included the saturation of a patch of sheep wool with a crude mixture of the AVM's, where it was found that up to 31 weeks protection was given against L.cuprina larvae (insecticide susceptible strain); diazinon at a comparable concentration gave only 10 weeks protection. When larvae were implanted into sheep, a mixture of the 'A' AVM's gave up to 13 weeks protection against susceptible larvae, compared with 10 weeks protection using diazinon. With organophosphorus resistant larvae, only 4 weeks protection was afforded by diazinon 14 compared with 12 weeks when 'A' class AVM's were used. The systemic activity of AVMB1 was emphasised by Standfast et aK (1984) working on CulIcoldes brevltarsis, an important vector of cattle arboviruses. Flies feeding on treated cattle (subcutaneous injection of AVMB1 at 200 jug per kg) at 10 and 18 days post-treatment, showed a 48h mortality of 99% and 40% respectively for 10 and 18 days post-treatment. No mortality occurred in those flies that alighted but did not feed. The work of Wright et al_. (1985a) showed that AVMB1 had a translaminar action against the mite species Tetranychus urticae and two aphid species (Aphis fabae and Aphis gossypii). A difference was noticed between the 72h LC-^ values obtained against T.urticae using AVMB1-treated French beans and Chrysanthemum leaves. The latter have waxier leaves, and it was suggested that this was a major factor in determining the degree of penetration of AVM through the leaf cuticle. The addition of safflower or Sunspray oil was found to reduce the translaminar LC for AVM. Translaminar activity was 50 greatest against T.urticae in comparison with the aphid species, which was possibly due to the method of feeding. T.urticae feeds destructively on parenchyma cells and may therefore come into contact with a greater level of AVM than the aphids which feed selectively within the phloem. Another factor involved in the differences in toxicity of AVMB1 towards the above species was body size. AVMB1a has been demonstrated to possess a considerable intrinsic toxicity towards T.urticae (LDgQ of 0.02 to 0.03 ppm) when applied directly onto adult, or 15 nymphal populations (Putter et a U , 1981). When leaves were dipped into AVMB1g solutions (0.5-1.0 ppm) and allowed to dry, it was found that the leaves were toxic to mixed adult and nymphal populations; with persistence of toxicity for up to one month. However, AVMB1g appeared to possess no ovicidal action against mites. Laboratory studies indicated that AVMB1g acted by contact and ingestion; mites being made moribund soon after contact with AVMB1g , with death occurring some 3 to 4 days later. Grafton-Cardwell and Hoy (1983) showed that AVMB1 had no ovicidal action against the predatory mite Metasei1us occidentalis, and it was also found to be less toxic towards the nymphs and adults of this species compared wi th T.urticae or Panonychus u1mi. Thi s would suggest that spraying crops with the correct formulation and dose of AYMB1 would lead to a greater reduction in phytoparasitic mites, compared with the predatory species. The above differences between predatory and phytophagous species may be associated with the distribution and persistence of AVM on foliage. For example, significant AVMB1 activity against T.urticae was found within leaves some fourteen days post spraying (Wright et aH ., 1985b) whereas Bullet aK (1984) found that the half life of toxic AVM residues on the surface of foliage was less than one day. 16

1.2.2 Effects on Metamorphosis and Development. There are several reports of AVM affecting developmental processes In Insects. For example, Wright (1984) found that AYMB1 interfered with the development of pupae of the boll weevil Anthonomus grandis into adults. As the concentration of AYMB1 applied to pupae decreased there was an increasing number of the following sequence of changes: darkening of the abdominal venter, and development of the proboscis, antennae, head, thorax, and appendages. Higher concentrations of AVMB1 completely inhibited development of newly formed pupae. Pupation was completely inhibited in yellowjacket larvae, Vespula maculifrons treated with 10 ppm AVMB1, this was said to be due to the paralysing action of AVMB1. Lower dosages of AYMB1 (0.01 and 0.1 ppm) were found to have no discernible effect on the completion of adult development (Parrish and Roberts, 1984). Third-instar larvae of the noctuid moths He!i othi s vi re seens, H.zea, and Spodoptera frugi perda that survived for 96h after topical application of AVMB1 were able to feed and develop normally up until the time of pupation. However, some larvae subsequently developed into larval-pupal intermediate forms that soon died, while others entered an apparently normal pupal stage but also died. Some fifth instar larvae given an oral dose of AVMB1 formed larval-pupal intermediates, or remained in the larval stage beyond the normal time of pupation; few of these larvae formed pupae that developed into adults (Bull, 1986). 17

1.2.3 Effects on reproduction and related processes. The delayed toxicity of AVM was Investigated by Lofgren and Williams (1982) with the red imported fire ant, Solenopsis invicta. In this species, the destruction of a colony depends upon the death of the queen for the, survival of this morph may mean the establishment of a new colony. The use of a poison with delayed toxicity is therefore important since the workers distribute food containing toxicant via trophylaxis to other members of the colony. If the toxicant were fast acting it would not spread through the colony, and most importantly it might not reach the queen, Lofgren and Williams (1982) found that AVMB1a affected the reproductive capacity of the queen which led to the death of the colony. AVMB1a was found by Glancey et (1982) to inhibit reproduction of the queen at a concentration of 0.0025%. Pathological symptoms included hypertrophy of the squamous epithelium of the ovaries and reduced yolk within eggs, which were also of a smaller size and fewer in number. These types of action were suggested to point to an effect on neurosecretory cells responsible for the control of reproduction which in insects is via neurohormones secreted from cells present in the pars intercerebralis (Chapman, 1969; Wigglesworth, 1972). However, M.S. Blum (unpublished data) found the corpora allata and corpora cardiaca to be normal in AVM treated fire ants. This may mean that AVM either blocked the production of neurohormones or interfered with their activity without causing structural changes in the tissues containing neurosecretory cells. Parrish and Roberts (1984) found that AVMB1 at a 18 concentration of 1 to 10 ppm incorporated into the diet of larval yellowjackets resulted In a significant degree of mortality. A bait delivering AVMB1 at a concentration of at least 1 ppm was said to be sufficient to destroy a colony by eliminating the brood. It was also pointed out that the brood serves as a food reservoir for adult foragers and AVMB1 could reduce the adult population by eliminating their food source, or by direct toxic effects of AYMB1 on the adults. Another study has shown that AVM caused reduced egg laying and stippling by adult females of the Serpentine leafminer, Liriomyza trifoli together with a reduction in egg hatch (Schuster and Everett, 1983). Leaflets dipped in AVMB.| were not found to be repellant to ovipositing female L.trifoli, since when given a choice the females deposited similar numbers of eggs on AVMB1-sprayed and water-sprayed leaflets. However, it was possible that AVMB1 caused irreversible damage to the ovaries of the leafminer, as was found by Glancey et jaK (1982) with fire ants, or perhaps more likely the muscles associated with the ovipositor were affected so that use of the ovipositor was restricted. An 80% mortality was noted in adult leafminers exposed to AYMB-j-dipped leaflets 24h after application. This was possibly due to the reduction in stippling by the flies 'paralysed' ovipositor which would have caused a reduction in food intake and thence starvation. The observed reduction in egg hatch was not found to be due to inhibition of early embryonic development for in nearly every case the sickle shaped mandibles of the larvae were visible through the chorions of the treated eggs. Many. 19

larvae penetrated the chorions but died before complete eelosion, it is therefore possible that the larvae may have been killed by contact with residues in or on the chorions or in the leaf mesophyll.

Robertson et a]_. (1985) reported that the fertility of adult Western spruce budworm, Choristoneura occidentalis was reduced when sixth instar larvae were reared on AVMB1-sprayed food. In this case, sterility of the egg rather than inhibition of egg hatch appeared to be responsible. The use of baits was emphasised by Cochran (1985) who demonstrated that newly emerged female B1ate!1 a germanica fed on a diet containing AYMB1 at a concentration of 6.5 ppm showed a high level of mortality; survivors of this treatment failing to reproduce. Lower dosages of AVMB1 were found to produce some mortality but also inhibited moulting and reproduction, and in some treated females the ovaries remained in an underdeveloped condition. Only 53% of females fed on 3.0 ppm AVMB1 mated, which was probably due to direct toxic effects of AYMB1 treatment. Progeny derived from the survivors of AVMB1 treatments (0.3 and 3.0 ppm) reproduced normally except that an extra 2-3 days were required to attain maturity compared with controls. This was also true for the oothacal carrying period. Finally, AVMB1 has been reported to reduce pheromone production in 6 day and 12 day old AYMB-,-treated adult males of the boll weevil, Anthonomus grandis. This was thought to be due to a general debilitation of the weevil by AVMB1 rather than a direct effect since high 20

concentrations of AYMB1 were found to reduce feeding and cause paralysis (Wright, 1984).

1.2.4 Antifeedant actions. An antifeedant action was assigned to AVMB-i by Pienkowski and Mehring (1983) working on the weevil Hypera postica. Laboratory studies showed that the feeding of second instar larvae was significantly reduced by 10 and 100 ppm AVMB-j , whilst that of fourth instars was significantly reduced only by 100 ppm. Field work indicated that a spray of AVMB1 applied to alfalfa was moderately effective at low dose levels in reducing weevil populations. However, significant increases in yield were not achieved. A similar antifeedant activity was reported by Beach and Todd ( 1985) working on the soybean looper, Pseudoplusia includens. While Corbitt et aK ( 1985) showed that AYMB1 greatly reduced the extent of feeding and consequently the rate of development in third instar larvae of the leafworm Spodoptera 1ittoralis. In contrast, AVMB1 applied in a diet to nymphal Blatella germanica did not appear to cause unpalatabi1ity or repellancy (Cochran, 1985). 21

1.3 Cellular level

1.3.1 GABA systems The first electrophysiologial investigation into the mode of action of AVMB1a was carried out by Fritz et al. (1979) working on the lobster neuro-muscular junction. AVMB1a was demonstrated to irreversibly eliminate both inhibitory post synaptic [IPSP] and excitatory post synaptic [EPSP] potentials, the latter being reduced more slowly than the former. In the above work the amplitudes of the evoked potentials were greatly affected but not the amplitude of the muscle membrane potential. However, AVMB1a reduced the muscle membrane resistance which in turn led to a reduction in the IPSP or EPSP. These findings suggested that AVMB1a acts to increase membrane permeability to those ions whose equilibrium potentials are close to the resting potential, namely chloride [Cl“] and potassium [K+]. It was shown that AVMB1a affects Cl"and not K+by bathing muscle in K-free ringer. This resulted in the IPSP being depolarising; an effect due to the resting potential being more negative than the IPSP equilibrium potential, and therefore the Cl"equilibrium potential. When AVMB1a was added to the bath, the muscle membrane was depolarised by several millivolts and the IPSP became reduced. That AVMB1a causes hyperpolarisation in standard ringer, and a depolarisation in K-free ringer suggested that AVMB1a increased the permeability of the membrane to Cl“ and not K* The observed rapid reduction in the IPSP and the slow reduction in the £PSP by AVMB1a may be explained by 22 the shift in the membrane potential towards the IPSP equilibrium potential. The IPSP was reduced due to the decreased ionic driving force and the decreased muscle input resistance, whilst the slower reduction in the EPSP was caused solely by a decreased muscle input resistance. Application of picrotoxin to a bath containing AVMB1a -treated muscle resulted in an increase in the EPSP; if the picrotoxin was washed out, the EPSP became reduced again. Picrotoxin though not completely specific to GABA is known to block gamma-amino butyric acid [GABA]-operated Cl ion channels, it would therefore seem to implicate these channels with AVMB1a, and thence GABA action (Fritz et 1979).

The work of Mellin e_t al_. (1983) confirmed that

AVMB1a blocked synaptic transmission in the lobster strecher muscle and this observation was also extended to the opener muscle. These postsynaptic effects were found to be inhibited by bicuculline (a vertebrate GABA-A receptor antagonist; Bowery et al_., 1984) and picrotoxin. Thus, AVMB1a may act postsynaptical ly on Cl ion channels (Fritz et al ., 1979; Mellin et aK, 1983) or, as suggested by Pong et al . ( 1980 ), act presynaptically on GABA receptors to cause a potent and sustained release of GABA. The seemingly irreversible action of AVMB1a (washing was not found to reverse AVMB1g action) may be related to the sustained stimulation of GABA release; the existence of a GABA re-uptake system helping to replenish the GABA supply. Pong et al. (1980 ) found that the replacement of calcium by cobalt in the lobster saline bathing the stretcher muscle preparation did not affect the membrane resistance of the 23

post-synaptic muscle fibres, but did block nerve evoked IPSP's which are known to be caused by calcium dependant presynaptic GABA release. The addition of AVMB1a to the bath caused a reduction in the muscle membrane resistance, which suggested that AVMB1a may be capable of stimulating GABA release in the absence of calcium. This was also found to be true using synaptosomes; a marked and sustained increase in the release of GABA being found from preloaded rat brain synaptosomes in the presence of AVMB1a (Pong et aU, 1980). It was shown by Misler and Hurlburt (1979) that the stimulation of GABA release from the frog cutaneous pectoris nerve by a-latrotoxin was independent of calcium but was dependent upon the presence of at least one other divalent cation such as cobalt, or magnesium. Pong et al. (1980) pointed out that in their experiments magnesium was always present, so it is not known whether there is a common mechanism in the action of a-latrotoxin and AVMB1a on the nervous system. AVMB1a was found to have a presynaptic action on GABA neurons by Pong and Wang (1980); radiolabelled AVMB1a binding specifically and saturably to dog brain synaptosomes. This binding was not found to be competitive with GABA, its agonists or antagonists which suggested that the drug receptors were not associated with GABA postsynaptic receptors.

AVMB-|a was found to possess an EC5q value of 2 to 3 pM for stimulating mammalian synaptosomal GABA release (Pong ejt^[., 1980). The value required to paralyse the nematode C.elegans was found to be of the same order of magnitude (0.4 to 1.0 jjM). This evidence suggested that the 24 action of avermectin on nematodes could be via the stimulation of GABA release. However, DHAVMB1 was found to be effective in paralysing C.elegans whereas it caused only small increases in the release of GABA from brain synaptosomes. The latter may reflect differences in the pharmacological properties of vertebrate and invertebrate GABA systems. Lees and Beadle (1986) reported that the GABA receptors present on cultured neurones taken from the central nervous system (CNS) of Periplaneta americana had inhibitory effects which were mediated by a rapid, transient increase in Cl ion permeabi1ity, but these could be blocked by picrotoxin, as in GABA-A receptors which have widespread distribution in vertebrates. However, the insect cells were insensitive to bicuculline. This finding would seem to confirm the observation of Pong et (1980) that GABA receptors differ in their pharmacological specificity across phylogenetic boundaries. Lummis and Sattelle (1985) showed that specific binding sites for [3H]GABA were present in nerve cord extracts taken from the cockroach Peri pianeta americana. This binding was not inhibited by the vertebrate GABA-A receptor antagonist bicuculline, or by the vertebrate GABA-B receptor agonist baclofen (Bowery et 1980; Cain and Simmonds, 1982) which indicated that the binding component possessed a different pharmacological profile to that of [3H]GABA binding sites found in vertebrates. It was shown by Pong et a]_. (1981) that there were different binding sites for AVMB1a , GABA and benzodiazepines on the rat brain synaptic membrane. These 25

sites were thought to be at the GABA post synaptic receptor-chloride ion channel complex. Pong et aK (1982) further showed that the recognition sites for GABA, benzodiazepines, AVMB1a , tracazolate, pentobarbitol and picrotoxin were all associated with the GABA receptor-chloride ion channel complex. The potentiating effects of AVM, tracazolate and pentobarbitol on GABA and benzodiazepine receptor binding could lead to a prolonged opening of the Cl ion channel which would allow an increase in the Cl ion influx into the post synaptic nerve terminal and block neurotransmission. AVMB1a plus tracazolate or pentobarbitol did not increase GABA binding further than AVMB1a alone, which suggested that the binding site for AVMB1a may be shared with pentobarbitol and tracazolate. It was also found that picrotoxin blocked GABA binding due to AVMB1a by 80 to 85% but only 9 to 12% of GABA binding due to tracazolate was blocked; indicating that the AYMB-ja and picrotoxin sites may interact to some extent. A preparation taken from the CNS of P.americana was s^wn to specifically bind [3H]f1 unitrazepam; binding being enhanced by low concentrations of [3H]GABA. This indicated that the benzodiazepine binding component was linked to a GABA binding site (Lummis and Sattelle, 1985). Drexler and Sieghart (1984) found that [3H]AVMB1a showed specific, high affinity binding to membranes taken from several rat brain regions which was fully reversible. This was in apparent contrast to the findings of Fritz et al . ( 1979) and Paul et£l«(1980). One explanation could be due to the hydrophobicity of AVMB1a. It is possible that 26

[3H]AVMB1a can dissociate from it's binding site in exchange for cold AVMB1g , when the latter is present in aqueous solution in a micellar form. In the absence of the micelles, [3H]AVMB-ja dissociating from it's specific binding sites might immediately and non-specifically precipitate on the membrane in aqueous solution (Drexler and Sieghart, 1984). The high affinity [3H]AVMB1a binding site in rat brain membranes was found to be partially inhibited by GABA agonists; this inhibition being blocked by Cl ions (Drexler and Sieghart, 1984). The presence of Cl ions in the incubation medium could have been one reason why Pong and Wang (1980 ) showed there to be no action of GABA on [3H]AVMB1a binding. Bicuculline was also found to block the action of GABA agonists on [3H]AVMB1g binding (Drexler and Sieghart, 1984) and this indicated that at least part of the specific high affinity binding sites for [3H]AVMB1a was associated with a GABA receptor. Furthermore, there was a small inhibitory effect of AYMB1a on [3H]muscimol binding, which also suggested that AVMB1a binding was associated with the GABA receptor. An increased level of

C 3H”)muscimol binding was seen when AVMB1g was present at higher concentrations, the presence of a low affinity AVM binding site or the exposure of new GABA binding sites possibly explaining this effect (Drexler and Sieghart, 1984)

In this connection, Pongand wang(1982) reported that the Bmax of GABA binding sites was increased by AVMB1g; while Lummis and Sattelle (1985) found that DHAVMB. enhanced the 1 a binding of [3H]GABA to a P.americana CNS membrane preparation. A putative GABA receptor has been Identified In the honey bee brain (Apis mel11fera L.) by Aballs and Eldefrawi (1986) where [3H]musc1mol was found to bind with a high and low affinity; binding being totally inhibited by

GABA agonists at a concentration of IOOjjM. This suggested that both the high and low affinity sites are GABA receptor specific. The binding was said to be either to two sites on a receptor molecule, or to two kinds of GABA receptors. The GABA uptake inhibitors [DL-nipocotic acid, 1-pipecolic acid and DL-2,4,diaminobutyric acid] were not found to inhibit [3H]muscimol binding which suggested that the high affinity site was not an uptake site but was on a GABA receptor (Abalis and Eldefrawi, 1986). Abalis and Eldefrawi (1986) believed the honey bee brain receptor was more like a GABA-A than a GABA-B receptor (Bowery e_t £l_* » 1984) due to the effectiveness of

GABA agonists and the ineffectiveness of the GABA-b receptor agonist baclofen on [3H]muscimol binding, even though bicuculline was found to have no effect on the high affinity [3H]muscimol binding. AVMB1a was found to inhibit [3H]muscimol binding to honey bee brain; having a more potent effect against the high affinity binding site [IC5O=10pM]. Abalis and Eldefrawi (1986) also referred to work in press which showed that AVMB1g inhibited binding of [3H]muscimol to mammalian brain tissues unlike the potentiation of [3H]GABA binding to mammalian GABA receptors found by Pong et (1982). The inhibition of [3H]muscimol binding to honey bee brain by AVMB1a may result from its action as an antagonist or agonist of GABA. The finding that AVMB1a potentiated the binding of 28

benzodiazepines to mammalian GABA receptors, and inhibited the binding of [35S] t-butyl bicycl ophosphorothi onate to the channel site of the mammalian GABA receptor indicated that AVMB1a was probably acting as an agonist when bound to the C3H]muscimol binding site on the GABA receptor (Aba!is et aK (1987). Tanaka and Matsumura (1985) reported that the application of AVMB1 [IOjjM] to leg muscles of P.americana resulted in the failure of the muscles to respond to stimuli within 30 min. However, the magnitude of contraction was not affected. This suggested that AVMB-j blocked the transmission of stimuli from the CNS to the muscles without affecting the muscles tension strength. This finding was in agreement with those of Fritz et al_. ( 1979) and Mel 1 i n et £l_* (1983) who found that AVMB1a blocked postsynaptic transmission in the lobster stretcher and opener muscles. It was shown that the binding of [3H]muscimol to P.americana muscle and brain membranes was not increased by AYMB1 (Tanaka and Matsumura, 1985), but was inhibited by bicuculline methiodide [1 OpM]. However, AVMB1aat a similar concentration was found to stimulate [3H]muscimol binding to rat brain membranes (Pong et al_., 1982). Similarly, the binding of [3H]GABA to rat brain membranes was stimulated by AVMB1a . AVMB1 at a concentration of IOjuM or lOOnM was not found to increase [3H]benzodiazepine binding to P.americana brain membranes, whereas this binding was increased in a rat brain preparation. The latter was also found by Pong et a]_. (1981). These findings led Tanaka and Matsumura (1985) to suggest that the benzodiazepine 29

receptors have little involvement with the action of AVMB-j in P.americana. This is in contrast to the findings of Pong et aK. (1981), Pong et aK (1982) and Paul et al . (1980) working on mammalian tissues. The binding of [3H]dihydropicrotoxinin to the membranes of the brain and the muscle of P.americana was not found to be affected by AVMB1 , except at a concentration of IOjjM whereupon binding was partially inhibited (Tanaka and Matsumura, 1985). From chloride ion influx studies, it was shown that the influx of Cl ions into muscle tissue was increased by AVMB-j when compared with controls; this increase being concentration dependent. AVMB-j [lOOnM] was found to be more potent than GABA in increasing this influx, and GABA in a combination of GABA and AYMB-, could not increase or decrease the 36C1“ i nf 1 ux induced by AVMB1 alone. This suggests an identical site for their actions. Milbemycin [lOOnM] was found to increase 36C1' influx to the same extent as AVMB1, whilst picrotoxinin

[IOOjjM] and bicuculline [100/jM] blocked the AVMB1 [lOOnM] enhanced 36C1“ influx, by 50% and 20% respectively. If all sodium ion [Na+], except in sodium phosphate [2mM] was replaced by the cholinium cation, it was found that AYMB1 enhanced 36C1 ion influx, but to a lesser extent to that observed in normal saline. Whereas in K ion-free saline the AVMB1-i nduced 36C1" i nf 1 ux was suppressed to 60% of that of the control. The inhibition of 36C1 ion influx into AVMB1 treated muscle bathed in K ion-free saline can be explained by the electrophysiological findings of Fritz et (1979) where the efflux of intracellular Cl ion increased 30 sufficiently for a while in K ion-free saline to cause depolarisation to occur. The evidence was said to suggest that AVMB1 selectively increased Cl ion permeability across the muscle membrane (Tanaka and Matsumura,1985). AVMB1 may therefore act as a presynaptic inhibitor by opening Cl ion channels at the GABA synapse in such a manner that the presynaptic membrane is unable to release an excitatory transmitter, or as a postsynaptic inhibitor by opening the Cl ion channel in the post synaptic region. A study on the ventral nerve cord of P.americana by Tanaka and Matsumura (1985) revealed that nerve excitation induced by the insecticide gamma-BHC [lpM] was eliminated within 15 to 20 min of AVMB1 [lOOnM] application; the nerve being made completely calm whereas controls still gave a few random spikes. It was noticed that the gamma-BHC treated nerve showed severe, but transient excitation before blockage by AVMB-j . Similarly, Lees and Beadle (1986) reported that about 10% of the cells in embryonic neuronal culture from the brain of P.americana which responded to DHAYMB1 depolarised prior to hyperpol ari si ng; at a concentration of 1pm DHAVMB-| caused a large irreversible increase in conductance. Tanaka and Matsumura (1985) concluded that the action of AVMB1 on P.americana was by the opening of Cl ion channels. The latter not being mediated through GABA, benzodiazepine or picrotoxinin receptors in the leg muscle and ventral nerve cord. 1.3.2 Non-GABA systems A dual action in insects was ascribed to avermectin by Duce and Scott (1985a). They found that 31 microperfusion of DHAVM-B1a [90pM to 9nM] onto the GABA-sensitive muscle bundle 33 in the extensor tibiae muscle of the locust Schistocerca gregaria induced a reversible, dose dependent increase in the input conductance which was abolished in Cl ion-free saline, and partially blocked GABA-induced increases in input conductance. DHAYM-B1a at a concentration of 12 nM to 1.2 pM resulted in an irreversible increase in the Cl ion conductance. At these high concentrations DHAVM-B1a potentiated GABA-induced Cl ion conductance, although a further application of pesticide produced a reversible reduction in the GABA response. When DHAVM-B1a [1.2nM to 1.2 pM] was applied to GABA-insensitive fast muscles [bundles 21 to 26], irreversible changes in the Cl ion conductance were induced. Changes which were similar in magnitude and time course to those found in muscle bundle 33. The reversible actions of DHAVM-B1a on locust muscle were said to involve the GABA receptor-chloride ion complex, but it seemed unlikely that this was the site of action of the irreversible effects of DHAVM-B1g on GABA-sensitive and insensitive muscle bundles (Duce and Scott, 1985a). Ibotenic acid was found to selectively increase the permeability of locust muscle bundles towards Cl ions by activating extrajunctional glutamate H receptors (Cull-Candy, 1976). Ibotenic acid at a concentration of 1pm to ImM induced dose dependent increases in conductance in both GABA sensitive and insensitive bundles. This response became rapidly desensitised in the presence of ibotenate.

The changes in input conductance induced by ibotenate in 32

GABA sensitive and GABA insensitive bundles were reduced in a dose dependent manner by 12nM-1.2/jM and 0.6-1.2pM of DHAVM-B1a respectively; concentrations similar to those found causing irreversible effects on GABA sensitive and insensitive muscle bundles (Duce and Scott, 1985b). The characteristic irreversible increases in input conductance could still be induced after glutamate H receptors were desensitised by ibotenate. This work suggested that the action of DHAVM-B1a was at the Cl ion channel of the extrajunctional glutamate H receptor-chloride ion channel complex given that the responses to DHAYM-B-|a were induced despite desensitisation of the H receptor by ibotenate (Duce and Scott, 1985b; Scott and Duce, 1985). The DHAVM-B1a induced irreversible increase in conductance was suggested by Scott and Duce (1986) to involve an ion channel with a greater selectivity for Cl ions, than that activated by GABA, although changes in the filtering properties of a single type of channel were not ruled out.

Application of DHAVM-B1 [O.I-I.OjjM] to cultured neurones taken from the cockroach P.americana resulted in slow increases in conductance (Lees and Beadle, 1986); these increases being accompanied by a progressive reduction in membrane time constants. However, the conductance response to the application of DHAYM-B-j was not confined to neurones which responded to GABA. Thus, DHAVM-B1 can influence Cl ion conductance in GABA-sensitive and insensitive cells (Duce and Scott, 1985b). Similarly, application of AVMB-| to neurones in the 33

suboesophageal ganglionic mass of the snail, He!ix aspersa resulted in an increase in Cl ion conductance in certain neurones. This effect occurred irrespective of whether the neurones were sensitive to GABA or not and was generally irreversible at higher concentrations

(0.01-1 .OjliM) of AVMB1 (Bokisch and Walker, 1986).

1.4 Present work The aim of the present investigation was to determine the toxicological effects of AVMB1 towards Spodoptera 1ittoralis, He!iothis armigera and Heliothis virescens (Noctuidae) lepidopteran pests of agricultural importance. The action of AVMB1 on spontaneous and evoked activities within the ventral nerve cord of P.americana was also investigated as was the localisation and uptake of [3H]AVMB1 by P.americana tissues. 34

GENERAL MATERIALS AND METHODS

2.1 Insect culture. All the insects were cultured and tested at 26'C under a 16:8 L:D cycle.

Spodoptera 1i ttorali s (Boi sd) Freshly laid batches of Spodoptera 1ittoralis eggs (Canary island strain) obtained from TDRI, Porton, Wiltshire were placed in covered plastic No.11 dishes (10 x 5cm) until they hatched three to four days after oviposition, whereupon Chinese cabbage leaves (cv. Pe Tsai), cotton (cv. Delta Pine 61), or artificial diet (Appendix2*1 ) were provided as a food source. At the second instar stage the larvae were transferred to plastic containers (24 x 24 x 10cm) fitted with net lids, or to covered plastic containers (27 x 15 x 10cm) in the case of those larvae reared on artificial diet. Some fifty larvae were placed on vermiculite lining the base of each container, food being added as required. Pupation occurred about fifteen days after hatching. On emergence, some fourteen days later, the adults were placed within a lidded, plastic column (23 x 12cm) lined with blotting paper onto which eggs could be laid following pairing. The adults were provided with a sucrose/vitamin solution as nutrient source (Appendix 2-1). The adults lived around ten days, the complete life cycle taking about forty days under the above conditions. 35

He11 othis armigera (Hubn.) and Heliothis virescens (F.). Eggs and larvae of Heliothis armigera and Heli othi s vi rescens were treated as for $.1ittorali s. The adults were provided with tissue paper as an oviposition site; the oviposition chamber being covered with a black plastic bag to reduce adult activity. H.armigera were obtained from the Institute of Virology, Oxford whilst H.virescens were obtained from ICI, Jeallots Hil 1, Berks.

Periplaneta americana (L.) Adults and nymphs obtained from a culture at Royal Holloway College, Egham were kept in a lidded plastic bin. Corrugated cardboard was provided as a resting site, water and rabbit pellets being given as required.

2.2 Selection of lepidopteran larval instars. Larval instars were separated by means of their body weight and head capsule width. Since body weight between instars was found to overlap, only those larvae of body weight falling within the ranges detailed in Table 2.1

were used in bioassays (See Appendix 3*1).

2.3 Chemicals. Test solutions of technical grade Cypermethrin, (RS)-alpha-cyano-3-phenoxybenzyl(IRS)-cis,trans-3-(2,2,-dic hiorovinyl)-2,2-dimethyl-cyclopropanecarboxylate (93% v/v) (Ripcord) and Avermectin B1g/B1b (80:20) (91% w/v) (Abamectin, AVMB1) were prepared in ethyl methyl ketone (EMK) and stored at -20 °C. 36

Table 2.1 Spodoptera littoralis larval head capsule and body weight ranges.

______X (95% C.L.)______Instar Head capsule (mm) Weight (mg)

III 0.72 (0.68-0.99) 10.5 (6.5- 14.2) IV 1.17 (1.06-1.28) 40.9 (28.4- 53.5.) V 1.70 (1.61-1.77) 162.3 (119.4-205.2) VI 2.41 (2.30-2.52) 550.3 (444.8-655.8) n=50

Table 2.2 Calibration of Arnold microappllcator.

X Volume del ivered ± S .E . (jul ) Marked volume (ul) Ethyl Methyl Ketone Aqueous solution

1.0 0.46 ± 0.02 0.60 ± 0.03 n=5 37

For residue studies, aqueous solutions of a formulation of AYMB1 (MK936 113M; 1.8% w/v EC) and a commercial formulation of cypermethrin (Cymbush,25% w/v EC) were prepared in distilled water containing 50pg Triton-X 100 per ml. In some experiments, safflower vegetable oil or Sunspray 6E mineral oil was also present at 2500/jg per ml.

For radiochemical and neurophysiological studies, a formulation of [3H]AVMB1 (MK936; 1.8% v/v EC; [5-3H ];

Specific activity : 213 Ci/mol; 9.67 mCi, 358 MBq in 2.5 ml) was prepared in distilled water, or in a 0.1% (v/v) solution of Triton X-100. 2.4 Topical application of Pesticides. One jul of test solution (uncalibrated, marked volume) was applied to the mid-dorsal abdominal region of each larva using an Arnold microapplicator fitted with an Agla all glass syringe, the needle of which was bent at an angle of 90°. The larvae were allowed to dry before being placed within covered plastic No.11 dishes (10 x 5cm) containing food. Ten third instar larvae were placed in each dish whereas only five larvae of the later three instars were used per dish. A minimum of forty larvae were treated per test solution. Mortality was assessed after 96h; any larva that responded when prodded was recorded as ali ve.

2.5 Calibration of Arnold microapplicator and Agla glass syringe. Solutions of [14C] Malathion in EMK or l%(v/v) acetone with 100 jug/ml Triton X-100 containing 1,387 and 1,337 disintegrations (dpm) per pi respectively were 38

dispensed by Arnold microapplicator onto pieces of aluminium-foil. These were placed into scintillation vials with 10ml of Cocktail 1t1 scintillation fluid (BDH Ltd) and radioactivity determined using a Beckman LS-250 liquid scintillation counter calibrated against [14C]hexadecane. Counting was for 50 minutes or until a 2 sigma error of 0-5% was achieved. The volumes actually dispensed by the application equipment are given in Table 2.2 and all subsequent dose calculations were ammended accordingly.

2.6 Contact/feeding toxicity of AVMB1 and cypermethrin with and without oils. Chinese cabbage (cv. Pe Tsai) or cotton (cv. Delta Pine 61) plants were sprayed to the point of incipient run-off with test solution using a Devilbiss hand-spraygun at 13 p.s.i. (0.9 Bar). The leaves were then allowed to dry for at least 3h. Larvae were placed on control or treated leaves within a No.11 plastic dish (Section 2.1). The number of first, third and fourth instar larvae per dish was twenty, ten and five respectively. A minimum of forty larvae were tested per treatment. Mortality was assessed after 96h.

2.7 Feeding rate of third instar larvae of Spodoptera littoral is and He!iothis armigera. Ten, pre-weighed third instar larvae of H.armigera or S.1ittoralis were placed onto a pre-weighed cotton leaf (cv. Delta Pine 61) within a covered plastic No.11 dish. After 24h the larvae and the leaf (cleaned of detritus) were reweighed. Before and after the experiment, 39 each of the leaves used was also photocopied and cutouts of the photocopies were weighed and compared.

2.8 Effect of AVMB^ on the feeding of third instar Spodoptera littoral is

[a] Residue studies. Four, preweighed third instar larvae of S.1ittoralis were placed on AVMB1-treated or control leaves (Section 2.6) within a plastic Petri dish (5cm dia). The larvae were re-weighed after 24h; the skin of any larva that had moulted being included in the re-weighing. Forty larvae were treated per test solution.

[b] Topical application. One ul of test solution was applied to pre-weighed third instar larvae as in section 2.4 except that the test solution was applied to the dorsal-posterior abdominal area in order that chemoreceptors located on the mouthparts were not contaminated. The larvae were then placed on unsprayed Chinese cabbage leaves (cv. Pe Tsai) and re-weighed after 24h. Ten larvae were treated per test solution.

[c] 'Choice* experiment. A Petri dish (9cm dia) was marked into two equal halves; one half containing five AVMB1-sprayed leaf discs (2.5cm dia), the other half containing unsprayed leaf discs (section 2.6). A Petri dish containing only unsprayed leaf discs was also set up. Ten third instar larvae were placed along the dividing line in each dish and their distribution 40 recorded after 24h. Any dead larvae were then removed; the remaining larvae being left for a further 24h and their subsequent distribution re-recorded. Forty larvae were used per treatment.

2.9 Analysis of bioassay data. Probit regression lines were fitted to bioassay data using a computer programme based on Finney (1971). The dose expected to produce mortality in 50% and 95% of the population (LD5Q and LD95 respectively) were calculated together with the 95% confidence limits. The slope, standard error of the slope and chi-square value were also obtained for each set of data. A value of chi-square less than tabulated indicated homogeneity, whilst a value greater than tabulated indicated heterogeneity. In the latter case, the data was discarded. Data was also tested for parallelism. The potency of each test solution relative to a pre-indicated standard being deduced along with the individual LD50 and LDQ5 values and 95% confidence limits. The test requires that the single lines under investigation be parallel. If the latter is true the value of the slope for each of the single lines will not be significantly different from the joint analysis estimated value (P>0.05). The weight gain experiment was subject to ANOVA and individual 95% confidence intervals were compared. 41

TOXICOLOGICAL STUDIES

3.1 Materials and Methods

3.1.1 Toxicity of topically applied AVMB1 and cypermethrin to third, fourth, fifth and sixth larval instars of S.littoralis.

1. AVMB1 Third, fourth, fifth and sixth instar larvae of S. 1 i ttoral i s were treated topically (section 2.4) with 392, 549, 1079 and 2139; 4581 and 16,186; 16,186; and 153, 200, 392 and 549 jjg AVMB1 per ml respectively.

2. Cypermethrin Third, fourth, fifth and sixth instar larvae of S.1ittoralis were treated topically (section 2.4) with 0.5, 2, 5 and 10; 5, 10, 25 and 48; 25, 48, 90, 181 and 1023; and 90, 452, 1023 and 2018 jug cypermethrin per ml respectively.

3.1.2 Toxicity of topically applied AVMB^ to third instar larvae of S.littoralis reared on various diets. Larvae reared on artificial diet, Chinese cabbage (cv. Pe Tsai) and cotton (cv. Delta Pine 61) were treated topically (section 2.4) with 186, 394, 831 and 1871; 392, 549, 1079 and 2139; and 80, 186, 394 and 831 pg AVMB1 per ml respectively. 42

3.1.3 Toxicity of topically applied AVMB^ to third instar larvae of S.littoralis and H.armigera reared on artificial diet. Third instar larvae of S.1ittoralis and H. armigera were treated topically (section 2.4) with 186, 394, 831 and 1871; and 392, 549, 831 and 1871 pg AYMB1 per ml respectively.

3.1.4. Injection of AVMB1 into fifth and sixth instar larvae of S.littoralis. Larvae were injected with ljj 1 (uncalibrated, marked volume) of test solution using a 33 gauge needle fitted ontoan Agla all-glass syringe in an Arnold microapplicator. Fifth and sixth instar larvae of $♦1ittoralis were treated with 186, 392 and 549; and 80, 186 and 394 pg AYMB-, per ml respecti vely. Five larvae were placed within a plastic No. 11 dish containing Chinese cabbage (cv. Pe Tsai) leaves. A minimum of thirty larvae were treated per test solution. Mortality was assessed after 96h.

3.1.5 Contact/ingestion toxicity of AVMB^ and cypermethrin on Chinese cabbage foliage (with and without the addition of oils) against first, third and fourth instar larvae of S.littoralis.

1. AVMB First instar larvae were placed on Chinese cabbage (cv. Pe Tsai) leaves which had been sprayed (section 2.6) with 0.45, 0.7, 0.9, 1.25 and 1.5 pg AYMB1 per ml; 0.15, 43

0.45, 0.7 and 0.9 pg AVMB-j per ml with safflower oil; 0.072, 0.18, 0.36 and 0.81 pg AVMB1 per ml with Sunspray 6E oil respectively. Third instar larvae were placed on Chinese cabbage (cv. Pe Tsai) leaves which had been sprayed (section 2.6) with 9, 18, 36 and 72 jug per ml AYMB1 ; 1.5, 3, 4.5 and 18 pg AVMB1 per ml with safflower oil; 0.9, 1.8, 3, 4.5, 6 and 9 jug AVMB-j per ml with Sunspray 6E oil respectively. Fourth instar larvae were placed on Chinese cabbage (cv. Pe Tsai) leaves which had been sprayed (section 2.6) with 18, 27, 36 and 72 jug AYMB1 per ml.

2. Cypermethrin First instar larvae were placed on Chinese cabbage (cv. Pe Tsai) leaves which had been sprayed (section 2.6) with 0.045, 0.15, 0.45 and 1.5 jjg cypermethrin per ml; 0.15, 0.45, 0.7 and 0.9 pg cypermethrin per ml with safflower oil; 0.009, 0.015, 0.045 and 0.09 pg cypermethrin per ml with Sunspray 6E oil respectively. Third instar larvae were placed on Chinese cabbage (cv. Pe Tsai) leaves which had been sprayed (section 2.6) with 0.5, 1.0, 1.5, 2.0 and 2.5 pg cypermethrin per ml; 0.09, 0.15, 0.3 and 0.5 pg cypermethrin per ml with safflower oil; 0.15, 0.3, 0.45, 0.75 and 0.9 pg cypermethrin per ml with Sunspray 6E oil respectively. 44

3.1-6 Residual (Foliar) toxicity of AVMB on cabbage (cv. Flower of Spring) under glass against third instar larvae of S.littoralis at 1, 3 and 7 days after spraying. Eight week old cabbage plants (cv. Flower of Spring) were sprayed (section 2.6) with the following solutions (jug per ml): 1. Safflower oil (2500) 2. Sunspray 6E oil (2500) 3. AYMB1 (4.5) 4. AVMB-, (4.5) + safflower oil (2500) 5. AVMB1 (4.5) + Sunspray 6E oil (2500) 6. Untreated control Treated plants (four per treatment) were kept under glass and bioassayed against third instar larvae (section 2.6) 1, 3 and 7 days after treatment. Forty larvae were tested per treatment.

3.1.7 Contact/ingestion toxicity of AVMB1 on Chinese cabbage and cotton against third instar larvae of S.littoralis and H.armigera.

1. S.1ittorali s Larvae were placed on Chinese cabbage (cv. Pe Tsai) leaves which had been sprayed (section 2.6) with 4.5, 9, 13.5 and 27 jig AVMB-, per ml, or on cotton leaves sprayed with 4.5, 9, 18, 27 and 36 pg AVMB-, per ml respectively. 45

2. H.armigera Larvae were placed on Chinese cabbage (cv. Pe Tsai) leaves which had been sprayed (section 2.6) with 0.54, 1.08, 2.16, 5.4 and 14.04 pg AVMB1 per ml respectively.

3.1.8 Contact/ ingestion toxicity of AVMB^ on cabbage and cotton against first instar larvae of S.1ittoralis, H.armigera and H.virescens.

1. $.1ittoralis. Larvae were placed on Chinese cabbage (cv. Pe Tsai) leaves which had been sprayed (section 2.6) with 0.27, 0.45, 0.72, 1.26 and 1.44 pg AVMB1 per ml; or on cotton leaves sprayed with 0.9, 1.2, 1.5 and 1.8 pg AVMB-, per ml respectively.

2. H.armigera. Larvae were placed on Chinese cabbage (cv. Pe Tsai) leaves which had been sprayed (section 2.6) with 0.18, 0.36, 0.54 and 0.72 pg AVMB-j per ml; or on cotton leaves sprayed with 0.09, 0.18, 0.263, 0.36 and 0.72 pg AVMB., per ml respecti vely.

3. H.virescens. Larvae were placed on cotton (cv. Delta Pine 61) leaves which had been sprayed (section 2.6) with 0.0216, 0.0432, 0.0648, 0.0864 and 0.1296 pg AVMB-, per ml respecti vely. 46

3.1.9 Assessment of feeding rate in third instar larvae of S.1ittoralis and H.armigera reared on cotton. Ten, preweighed third instar larvae of $.1ittoralis or H.armigera were placed onto a preweighed cotton leaf (cv. Delta Pine 61) within a covered plastic No.11 dish. After 24h, the larvae and the leaf (cleaned of detritus) were reweighed, each of the leaves used was also photocopied and cutouts of the photocopies were weighed and compared.

3.1.10 Effect of foliar residues of AVMBj| on the feeding of third instar larvae of S.1ittoralis. Larvae were placed on Chinese cabbage (cv. Pe Tsai) leaves which had been sprayed (section 2.6) with 1.5, 3.0, 4.5 and 9.0 jug AVMB1 per ml respectively. The effect on feeding was assessed as described in section 2.8a

3.1.11 Effect of topical application of AVMB^on feeding of third instar larvae of S.1ittoralis. Larvae were treated topically (section 2.4) with 3.1, 5.1 and 12.0 jug AVMB1 per ml respectively. The effect on feeding was assessed as described in section 2.8b

3.1.12 Distribution ("Choice") of third instar larvae of S.littoralis between untreated and AVMB^ -treated Chinese cabbage leaves. Chinese cabbage (cv. Pe Tsai) was sprayed (section 2.6) with 1.5, 3.0, 4.5 and 9.0 jjg AVMB1 per ml respecti vely. Leaf discs were cut and placed within a choice chamber (Section 2.8c) containing untreated leaf 4 7 discs. Ten larvae were placed into the chamber and their distribution assessed after 24 and 48h as described in section 2.8c 48

3.2 Results

3.2.1 Toxicity of topically applied AVMB1 and cypermethrin to different larval instars of S.littoralis reared on Chinese cabbage.

The LD5q value for AVMB1 against the third instar was greater than that found for the sixth instar. LD50 values could not be accurately estimated for AVMB1 against the fourth and fifth instars; no concentration of AVMB1 tested giving greater than 35% mortality (Table 3.1). However, on a ng per mg basis the fourth instar larvae would appear to be more sensitive to topically applied AVMB1 than fifth instar larvae. In contrast the LD5Q values for cypermethrin increased progressively from the third to the fifth instar (Table 3.2). Sixth instar larvae were more sensitive to cypermethrin than fifth instar larvae, although the difference was not significant (P>0.05).

3.2.2 Toxicity of topically applied AVMB^to third instar larvae of S.littoralis reared on various diets. There was some indication that larvae reared on cotton were more sensitive to AYMB-j than those larvae

reared on the other food sources. However, the LDC_ou values for the different batches of larvae were not found to be significantly (P>0.05) different as their 95% CL overlapped (Table 3.3). Table 3-1 Toxicity of topically applied AVMB ^ to larval Instars of Spodoptera littoralis reared on cabbage.

Instar 96h LD5Q (95%C.L.)ng/mg 96h LD95(95%C.L.)ng/mg Slope ± S.E.

Third 43.4 (32.8 - 65.6) 388.5 (175.4 - 2950) 1.73 ± 0.39

Fourth >45 * - -

Fifth >45 ** - -

Sixth 0.23 (0.20 - 0.26) 0.51 (0.43 - 0.67) 4.78 ± 0.63

* 35% Dead at16186 ppm.

** 10% Dead at 16186 ppm. Table 3 m2 Toxicity of topically applied cypermethrin to larval instars of S.littoralis reared on cabbage.

Instar 96h LD<.q (95%C.L.) ng/mg 96h LDq _(95%C.L.)ng/mg Relative Potency (95%C.L.)

Third 0.03 (0.01 - 0.05) 1.11(0.55 - 3.02) 1.0

Fourth 0.28 (0.16 - 0.49) 11.8(4.71 - 49.47) 0.09 (0.03 - 0.21)

Fifth 0.56 (0.34 - 0.98) 23.85 (9.35 -103.39) 0.05 (0.02- 0.10) Sixth 0.44 (0.25 - 0.73) 18.43 (8.11- 64.72) 0.06 (0.02- 0.13) cn O

The slope from joint probit line analysis (1.01 ± 0.12 is not significantly different (P> 0.05) from the single line slopes. (0.77 ± 0.26; 1.24 ± 0.34; 0.87 ± 0.21; 1.25 ± 0.25; for the third, fourth, fifth and sixth instars respectively). Table 3-3 Toxicity of topically applied AVMB ^ to third instar larvae of S.littoralis reared on various diets.

Treatment 96h LD5Q(95%C.L.)ng/mg 96h LD95(95%C.L.)ng/mg Relative Potency (diet) (95%C.L.)

Artificial 52.7 (28.0 - 128.6) 577.7 (202 - 7327.2) 1.0 diet

Cabbage 44.5 (25.3 - 90.7) 487.6 (187.4 - 5044.3) 1.2 (0.5 - 3.2)

Cotton 20.6(10.8 - 38.8) 226.4 ( 93.8 - 1843.0) 2.5 (1.1 - 7.7)

The value of the slope for joint probit line analysis = 1.58iO-3; value is not significantly (P> 0.05) different from the values obtained from probit analysis for single lines (1*3+0*3■ 1-7±0*4; 1-7+0-3; for artificial diet, cabbage and cotton respectively). 52

3.2.3 Toxicity of topically applied AVMB to third instar S.littoralis and H.armigera reared on artificial diet. There was no significant difference (P>0.05) between the LD5Q values for $.1ittoralis and H.armigera reared on artificial diet (Table 3.4).

3.2.4 Toxicity of injected AVMB1 to fifth and sixth instar S.littoralis larvae. The LD50 value obtained when AVMB., was injected into each larva was lower than when AVMB-, was applied topically (Table 3.5) although confidence limits were not calculated as the regression was not significant (P>0.05). The difference between the fifth and sixth injected values was also found to be less than the difference between the values obtained for topical application.

3.2.5 Contact/ ingestion toxicity of AVMB^ and cypermethrin on Chinese cabbage foliage (with and without the addition of oils) against first, third and fourth instar S.littoralis. AVMB-, (113M25) in distilled water with the addition of either Sunspray 6E or safflower oil was significantly (P<0.05) more toxic to first instar larvae of 5.1ittoralis when compared with AVMB1 alone. Sunspray 6E and safflower oil enhanced the toxicity of AVMB-, by 4.9 and 4.6 fold respectively (Table 3.6). Cypermethrin with Sunspray 6E or safflower oil was also found to be significantly (P<0.05) more toxic than cypermethrin alone against first instar larvae of Table 3*4 Toxicity of topically applied AVMB ^ to third instar larvae of S.littoralis and H.armigera reared on artificial diet.

Treatment 96h (95%C.L.)ng/mg 96h LDg5(95%C.L.)ng/mg Relative Potency (species) (95%C.L.)

S.littoralis 54.9 (37.6 - 95.7) 733.1 (295.4 - 4932.6) 1.0

H.armigera 32.3 (22.7 - 46.3) 431.7 (202.0 - 2114.3) 0.59 (0.3 - 0.97)

Ol CO The value of the slope for joint probit line analysis = 1.46-0*3; value is not significantly (P> 0.05) different from the values obtained from probit analysis for single lines (1*3±0*3; 1-7+0-4; for S*littoralis and H*armigera respectively* Table 3-5 Mortality of fifth and sixth instar S.littoralis following topical application or injection with AVMB ^•

Method of Instar 96h LD50(95%C.L.)ng/mg Slope ± S.E application

Topical application Fifth > 45 * -

Sixth 0.23 (0.20 - 0.26) 4.78 ± 0.63

Injection Fifth 1.91 ** 4.26 ± 4.72

Sixth 0.11 ** 1.07 ± 0.64

* See Table 3-1

** No C.L. given as regression not significant Table 3-6 Contact/ingestion toxicity of AVMB x on Chinese cabbage foliage (with and without the addition of oils) against first instar larvae of S.littoralis.

Treatment 96h LC5q (95%C.L. )jjg/ml 96h LC95(95%C.L. )jjg/ml Relative Potency (95%C.L.)

AVMB i 1.12(0.98 - 1.29) 4.02 (3.23 - 5.32) 1.0

AVMB i + 0.24 (0.19 - 0.29) 0.88(0.72 - 1.12) 4.57 (3.62 - 5.93) Safflower

AVMB x + 0.23 (0.18 - 0.28) 0.81 (0.64 - 1.09) 4.93 (3.88 - 6.33) Sunspray 6E

Ol 01

The value of the slope for joint probit line analysis = 2.97±0\2 value is not significantly (P> 0.05) different from the values obtained from probit analysis for single lines (3-3i0»6; 2-0±0-4; 3-1±0-4; for AVMB^ , AVMB-j* Safflower, AVMB-j + Sunspray 6E respectively) 56

5.1ittoralis. Safflower oil and Sunspray 6E oil enhanced the action of cypermethrin by 6.1 and 2.4 fold respectively (Table 3.7). AVMB-, in association with either Sunspray or safflower oil was significantly (P<0.05) more toxic towards third instar S.1ittoralis when compared with AYMB1 alone. Safflower and Sunspray 6E oil enhanced the action 2.6 and 7.5 fold respectively; Sunspray 6E oil being significantly (P<0.05) more effective than safflower oil (Table 3.8). Cypermethrin sprayed with either Sunspray 6E or safflower oil was significantly (P<0.05) more toxic than cypermethrin alone against third instar S.1ittoralis; safflower and Sunspray 6E oil plus cypermethrin being 2.7 and 1.9 fold more toxic when compared with cypermethrin alone respectively (Table 3.9). For both first and third instar larvae of S.1ittoralis, Sunspray 6E oil was more active than safflower oil when sprayed with AVMB1; the reverse being found for cypermethrin (Tables 3.6, 3,7, 3.8 and 3.9). An LC50 value of 34.18 ppm was given for fourth instar S. 1 ittoral is fed AYMB-j-treated cabbage leaves (Table 3.10).

3.2.6 Residual (Foliar) toxicity of AVMB1 on cabbage (cv. Flower of Spring) under glass against third instar larvae of Spodoptera littoralis at 1, 3 and 7 days after spraying. The residual toxicity of AVMB-j (applied at 4.5 ppm) against third instar larvae of S. 1ittoralis was difficult to assess as there was no endpoint mortality up Table 3*7 Contact/ingestion toxicity of clypermethrin on Chinese cabbage foliage (with and without the addition of oils) against first instar larvae of S.littoralis.

Treatment 96h LC5q (95%C.L. )jjg/ml 96h LC95(95%C.L. )jjg/ml Relative Potency (95%C.L.) •

Cypermethrin 0.214 (0.161 - 0.273) 1.12 (0.85 - 1.60) 1.0

Cypermethrin + 0.035 (0.026 - 0.046) 0.18 (0.13 - 0.29) 6.12 (4.09 - 8.79) Safflower

Cypermethrin + 0.088 (0.068 - 0.12) 0.46 (0.29 - 0.84) 2.43 (1.53 - 3.55) Sunspray 6E U! " s i

The value of the slope for joint probit line analysis = 2.89 ± 0*2 value not significantly (P> 0.05) different from the value obtained from probit analysis for single lines, ( 2-4±0-4; 2-3+0-4; 2-1±0-4; for Cypermethrin. (CYP),CYP+ Safflower, CYP+ Sunspray6E respectively). Table 3-8 Contact/ingestion toxicity of AVMB i on Chinese cabbage foliage (with and without the addition of oils) against third instar larvae of S.littoralis.

Treatment 96 LC5q (95%C.L. )jjg/ml 96h LCg5(95%C.L. )jjg/ml Relative Potency (95%C.L.)

AVMBi 16.29 (13.14 - 20.07) 64.29 (50.05 - 87.51) 1.0 .

AVMB! + 6.33 ( 5.24 - 7.70) 25.04 (19.23 - 34.84) 2.57 (1.92 - 3.41) Safflower

AVMBl + 2.16 ( 1.82 - 2.52) 8.54 ( 6.99 - 10.94) 7.55 (5.82 - 9.83) Sunspray 6E tn 00

The value of the slope for joint probit line analysis = 2.751 0*2 value not significantly (P> 0.05) different from the values obtained from probit analysis for single lines (3-510-5; 2-410-3; 2-7± 0-3; for AVMB-,, AVMB-| + Safflower, AVMB-,-*- Sunspray 6E respectively). Table 3-9 Contact/ingestion toxicity of cypermethrin on Chinese cabbage foliage (with and without the addition of oils) against third instar larvae of S.littoralis.

Treatment 96h LC50(95%C.L.)jjg/ml 96h LC95(95%C.L.)pg/ml Relative Potency (95%C.L.)

Cypermethrin 0.84 (0.71 - 0.98) 2.43 (2.05 - 2.98) 1.0

Cypermethrin + 0.31 (0.25 - 0.37) 0.89 (0.71 - 1.16) 2.73 (2.12 - 3.48) Safflower

Cypermethrin + 0.43 (0.39 - 0.49) 1.25 (1.04 - 1.58) 1.94 (1.58 - 2.35) Sunspray 6E

The value of the slope for joint probit line analysis = 3.58± 0-3 value not significantly different (P> 0.05) from the values obtained from probit analysis for single lines ( 4 0 + 0 -5 ; 3-2*0-3; 4-7±0-9; for Cypermethrin(CYP), CYP+Safflower, CYP+Sunspray 6E respectively). Table 3*10 Toxicity of AVMB i against fourth instar larvae of S.littoralis by contact/ingestion and topical application methods.

Method of 96h LC5q (95%C.L. )jjg/ml 96h LCg5 (95%C.L. )jjg/ml Slope ± S.E. application

Foliar 34.18 (27.53 -41.94) 113.6 (77.85 - 267.0) 3.15 ± 0.66

Topical >16,1.86

o> * See Table 3*1 o 61 to 9 days exposure, although the rate of development to the sixth instar was impaired. Addition of Sunspray 6E oil enhanced the toxicity of AVMB-| up to 3 days after treatment (DAT), and both Sunspray 6E and safflower oil appreciably reduced the number of treated larvae which had developed to the sixth larval stage after 9 days exposure to AVMB1 (Table 3.11).

3.2.7 Contact/ingestion toxicity of AVMB^ on Chinese cabbage and cotton against third instar larvae of S.littoralis and H.armigera. There was no significant (P>0.05) difference in the LC5q values obtained for third instar S.littoralis feeding on AVMB-j-sprayed cabbage or cotton leaves . The LC50 value for H.armigera feeding on AVMB-j-sprayed cotton leaves was found to be significantly (P<0.05) less than that found for S.1ittoralis on cotton or cabbage (Table 3.12).

3.2.8 Contact/ingestion toxicity of AVMB^ on Chinese cabbage and cotton leaves against first instar larvae of S.1ittoralis, H.armigera and H.virescens. There was a significant (P-40.05) difference in the LC50 values obtained for first instar H.armigera, S.1ittoralis and H.virescens reared on AYMB1-sprayed cabbage or cotton leaves (Table 3.13). For S.littoralis the foliar toxicity of AYMB-, on cabbage was significantly (P<0.05) greater than on cotton (Table 3.13). Whereas residues on cotton were significantly more toxic against H.armigera than residues on cabbage. For both crops, the Table 3-11 Residual (foliar) toxicity of AVMB on cabbage (c.v. Flower of Spring) against third instar larvae of S.littoralis under glass.

(1 )

% mortality (% survivors at sixth instar) Age of residue at start of bioassay (days)

Treatment pg/ml a.i. 1 3 7

Untreated 17 (85) --

Safflower (2500) 17 (79) - 20 (82)

Sunspray 6E (2500) 25 (67) - 20 (78)

AVMB l (4.5) 47 (48) 25 (30) 40 (58)

AVMB i (4.5) + 50 ( 5) 17 ( 3) 40 (33) Safflower (2500)

AVMB i (4.5) + 94 ( 0) 50 (35) 25 (17) Sunspray 6E (2500)

n = 40

(1) = Observations made 9 days after introduction of larvae onto treated foliage. Table 3-12 Contact/ingestion toxicity of AVMB ^ on Chinese cabbage and cotton against third instar larvae of S.littoralls and H.armigera.

Treatment Species 96h LC:50(95%C.L.)jjg/ml 96h LC 9 5 (95%C.L.)ng/ml Slope ± S.E (diet)

Cabbage S.littoralis 10.18 (8.19 - 12.10) 28.12 (21.40 - 47.59) 3.73 ± 0.68

Cotton S.littoralis 10.96 (9.30 - 12.68) 31.69 (25.75 - 42.71) 3.53 ± 0.39

Cotton- H.armigera 1.29 (0.89 - 1.71) 8.86 ( 5.65 - 19.44) 1.96 ± 0.31

0> co

Parallel line analysis was not carried out as the larvae were reared on different food plants and Table 3*3 shows that diet may affect an insects toxicological response to AVMB -. Table 3-13 Contact/ingestion toxicity of AVMB ^ against first instar larvae of S.littoralis, H.armigera and H.virescens.

Treatment Species 96h LC _n(95%C.L. )jjg/ml 96h LC Q_ (95%C.L. )jjg/ml Slope ± (S.E.) (diet) 95

Cabbage S.littoralis 0.89 (0.79 - 1.04) 2.59 (2.00 - 3.91) 3.581(0.46)

Cabbage H.armigera 0.26 (0.21 - 0.30) 0.63 (0.53 - 0.85) 4.291(0.65)

Cotton S.littoralis 1.23 (1.14 - 1.31) 2.0 (1.79 - 2.41) 7.77±(1.11)

Cotton H.armigera 0.15 (0.11 - 0.19) 0.69 (0.50 - 1.23) 2.45±(0.39)

Cotton H.virescens 0.033 (0.027 - 0.039) 0.106 (0.085 - 0.149) . 3.281(0.45)

Parallel line analysis was not carried out as the larvae were reared on different food plants and Table 3*3shows that diet may affect an insects toxicological response on AVMB .. 65

LC50 values showed there to be a significant (P<0.05) difference in the toxicity of AVMB1 against S.littoralis and H.armigera (Table 3.13); H.armigera being more

sensitive to AVMB1 than S.1i ttoral i s. H. vi rescens was

found to be significantly (P<0.05) more sensitive to AVMB1 residues on cotton than was H.armigera (LC5Q values of 0.033 and o.ts ppm respectively).

3.2.9 Assessment of feeding rate in third instar S.littoralis and H.armigera. The 24h weight gain by S.1ittoralis and H.armi gera (2.5 and 2.8 mg per larva respectively) was not significantly (P>0.05) different (Table 3.14). However, the significantly (P<0.05) greater weight of cotton leaf consummed by H.armi gera larvae indicated that this species had a slightly greater feeding rate (Table 3.14). The unit area of leaf eaten was not significantly (P>0.05)

different between S.1 ittoralis and H.armigera.

3.2.10 Effect of AVMB ^ on the feeding of third instar larvae of S.littoralis.

[a] Effect of foliar residues. The weight gain of larvae fed on AVMB-,-sprayed cabbage leaves was significantly (P<0.01) reduced compared with larvae reared on untreated plants (Table 3.15); AVMB.,

concentrations less than the LC50 value (Table 3.8) significantly (P<0.05) reducing weight gain compared with controls. Table 3-14 Assessment of feeding rate in third instar larvae of S.littoralis and H.armigera reared on cotton.

X + SE- Treatment Weight gain per Weight leaf eaten Area leaf eaten (species) larva in 24h (mg) per larva in 24h (mg) per larva (cm2)

S.littoralis 2.51 ± 0.25 10.28 ± 0.79 4.80 ± 0.48

H.armigera 2.83 ±0.2 13.98 ± 1.94 * 4.42 ± 0.25

o> O) h=4 (10 Insects per treatment) £ Significant at 5% level (t) Table 3*15 Effect of foliar residues of AVMB ^ on the feeding of third instar larvae of S.littoralis.

______XtS-E-______AVMB ^ treatment (jjg/ml)

Control 1.5 3.0 4.5 9.0

Mean weight of larvae (mg) 8.91 ± 0.26 8.65 ± 0.21 8.27 ± 0.23 8.55 ± 0.30 8.72 ± 0.30 ± S.E. (N =11)

•k k k k Mean increase in weight (mg) 16.11 ± 0.81 12.11 ± 0.73 6.02 ± 1.07 5.34 ± 0.77 1.94 ± 0.48 ± S.E. 24h post-treatment ______:______o> ■ Nl

* Significant difference between control and treatment weight gain (P<0.01) 68

[b] Effect of topically-applied AVMB1 The weight gain by third instar larvae fed on untreated leaves was significantly (P<0.05) reduced compared with untreated and EMK treated controls (Table 3.16). AVMBi concentrations less than the LD_rt value, for topically applied AYMB1 against this instar significantly (P<0.05) reduced larval weight gain compared with controls (Tables 3.1 and 3.16).

[c] Distribution of third instar larvae between AVMB1 sprayed and untreated cabbage leaf discs. No discrimination was made between AVMB1-sprayed and unsprayed leaf discs 24h after the larvae were placed in the choice chamber. After 48h, 65% mortality was recorded in those larvae present on the 9.0 ppm AVMB-| treated leaf discs compared with 30% mortality on untreated discs. The number present on the treated discs after 48h appeared to be greater than on untreated discs (Table 3.17). Table3*16 Effect of topically applied AVMB^on feeding of third instar larvae of S.littoralis.

______AVMB ^ Treatment ng/mg______

Untreated Control 7 11 26 control EMK

Mean weight of larvae (mg) 7.72 ± 0.29 7.17 ± 0.29 7.34 ± 0.31 7.20 ± 0.31 7.11 ± 0.32 ± S.E.

n 10 10 10 10 10

* G) Mean weight CO change (mg) 13.57 ± l.81a 10.64 ± 1.14a 4.90 ± 1.87b 2.42 ± 0.89b -0.12 ± 0.51° ± S.E. 24h post treatment

n 7 10 10 10 10

* Values sharing a common letter are not significantly different (P> 0.05) Table 3-17 Distribution of third instar larvae of S.littoralis between control and AVMB ^ - treated leaves.

24 h 48h %Dead Treatment Total Number present Total Number present (ppm AVMB^) larvae (Number alive) larvae (Number alive) 24h 48h Control Treated Control Treated Control Treated Control Treated side side side side

Control vs 40 21 (21) 19 (19) 40 27 (26) 13 (12) 0 0 3.7 3.7 Control

Control vs 40 16 (16) 24 (24) 40 22 (20) 18 (16) 0 0 9.1 11.1 1.5

Control vs 40 14 (12) 26 (22) 34 19 (18) 15 ( 9) 14.3 15.4 5.3 40 3.0

Control vs 40 22 (21) 18 (18) 39 18 (18) 21 (13) 4.5 0 0 38.1 4.5

Control vs 39 15 (12) 24 (20) 33 10 ( 7) 23 ( 8) 20 16.7 30 65.2 9.0 71

3.3 Discussion

3.3.1 Symptoms of poisoning with AVMB1 In the present work, larval instars of

S.1 ittoralis showed signs of paralysis within two days of

AYMB1 -treatment; death occurring up to four days post-treatment. The onset of paralysis in adult male P.americana was found to occur in the metathoracic legs within six hours of injection with AVMB-| (Section 4.2.2). There have been several other reports on the symptoms of poisoning of AVM in insects. For example, Putter et £l_. (1981) observed that lepidopteran larvae were fairly rapidly paralysed by AYMB1 ; being unable to move their abdominal legs. While, Abro (1985) reported that fourth instar larvae of the diamondback moth Plutella

xylostel1 a showed marked signs of paralysis on the second

or third day of treatment with AVMB1 , usually about one day before they died. With houseflies, Botham and Nicholson (1985) reported that the onset of paralysis in Musca domestica treated with a LD5Q dose of AVMB-, (4ng) was in the metathoracic legs. Thus, AVM killsinsects and mites directly but rather less rapidly than many other neuroactive pesticides such as the pyrethroids. However, it is uncertain whether the relative speed of kill with AVM is due to their mode of action, either because of the lack of hyperactive symptoms compared with other compounds, or to a relatively slow accumulation of AVM at critical target sites (Wright, 1986). The first symptoms of AVM poisoning which are often 72 observed within a few hours of application, such as the. paralysis of the hind legs of lepidopteran larvae (Putter et al., 1981) mentioned previously may thus reflect an initial action at relatively exposed peripheral target sites (Wright, 1987). Some AVMB-j-treated S.littoralis larvae failed to pupate properly. The pupation of larval V.maculifrons was also reported to be inhibited when larvae were fed AVMB-j at a concentration of 10 ppm (Parrish and Roberts, 1984). Whilst, some larvae of H.vireseens, H.zea and S.frugiperda treated with AVMB-, were found to develop into larval-pupal intermediates (Bull, 1986) (See Section 1.2.2). The above intermediate forms may have been produced when partially paralysed larvae started to moult, but were unable to completely shed the last larval skin due to a paralysis of the abdominal legs. The latter is supported by the report of Jyothi et al_. ( 1986) that larvae of the castor semilooper, Achaea janata treated topically

with AVMB1 showed abnormalities in growth and development only at the time of eedysis. Treated larvae were totally or partly unable to moult their old larval exuviae and apolysed larvae were found moving within their intact exuviae and died after one or two days. When the cuticle of apolysed larvae was removed the new cuticle was found

underneath. This suggests that AYMB1 does not interfere with the physiological processes underlining metamorphosis, but by paralysing the larval appendages prevents the larvae from shedding the old skin, resulting in death. 73

3.3.2 Toxicity of AVMB1 to larval instars. 3.3.2.1 Spodoptera 1i ttorali s It has been reported by various authors (Aikins and Wright, 1985; Lund et aK , 1979; Abro, 1985) that a decrease in insecticide susceptibi1 iy occurs with increasing larval instar. It was W particular interest therefore that topically applied AYMB1 was found to be relatively effective against third and sixth instar $.1 ittoralis, but ineffective against the fourth and fifth instars. Whereas cypermethrin was effective against all instars tested (Tables 3.1 and 3.2). The sensitivity to topically applied AVMB1 and cypermethrin decreased from the third to the fifth instar. The sixth instar was found to be slightly more sensitive than the fifth instar to cypermethrin. In contrast, the sixth instar was more sensitive to AYMB1 than the third instar. The observed insensitivity of the fourth and fifth larval instars of 5.1ittoralis to topically applied AVMB1 compared with the third and sixth larval instars could have been due to a number of factors such as an increased level of detoxification, reduced penetration, or altered site, of action (oppenorth and welling, 1976). The involvement of a penetration factor was implied by the finding that injection of AVMB-, increased its relative toxicity against the fifth compared with the sixth instar of S.1 ittoralis. The above observations, together with the finding that third and fourth instar S.1ittoralis fed on AVMB1-treated cabbage leaves had similar 96h LD50 values (16.2 and 34.2 ppm respectively) could also indicate that AVMB1 exerted 74

its action more readily when consummed in the food. A similar conclusion was made by Hollingshaus and Little (1984) who reported that less than 3% of an acetone solution of the amidinohydrazone insecticide AC 217,300 (AMDRO) topically applied to fourth instar larvae of H.virescens penetrated the cuticle after 72h. Whereas larvae which ingested AMDRO had residues in their tissues which were 45 to 5.5% of the applied dose; the toxicity of the compound being increased 40 to 140 fold over topical application. Insects which had been treated topically with AMDRO were seen under the microscope to have a dispersion of fine yellow crystals on the cuticle which was more pronounced at higher doses. The latter suggested that the compound could not readily penetrate the epicuticular waxes. The mechanism by which the cuticle inhibits the penetration of insecticides is not fully understood, but it has been related to increased cuticular thickness (Weismann, 1947), increased lipid and protein content (Weismann, 1957; Vinson and Law, 1971) and to altered lipid composition (Pati1 and Guthrie, 1979a,b). Differences in tolerance to an insecticide have also been related to changes in the cuticle (Busvine, 1971). It was suggested by Klinger (1936) that the relative immunity of later insect stages was due to an increase in cuticular thickness which was often associated with a sclerotized exocuticle and sometimes with a reduction in the number of pore canals. The insensitivity of the fourth and fifth instars of 5 .1ittoralis to AVMB1 compared with the third instar 75

also may have been due to an increased storage of AYMB1 in the former. For example, it has been shown by Chapman

(1969) that later instars of lepidopteran larvae possessed more adipose tissue than the early instars. However, this does not explain why the sixth instar was more sensitive than the third instar to AVMB1. It is likely therefore that the increasing tolerance of the instars to AVMB1 up until the sixth instar was at least partly the result of reduced cuticular penetration. The fall in tolerance during the sixth instar may imply a reduction in the cuticle thickness, or a change in its chemistry. Recent work has also suggested that changes in pesticide metabolism appear to be involved as the synergist piperonyl butoxide (PB) a mixed-function oxidase inhibitor was found to enhance the relative toxicity of AVMB1 against fifth compared with sixth instar 5.1ittoralis (P.T.Christie, personal communication).

3.3.2.2. Comparative toxicity First instar larvae of S.eridania were found to be 4-times more sensitive to foliar residues of the pyrethroid fenvalerate when compared with AVMB.J, whereas H.virescens was 26-times more sensitive to AYMB1 than to fenvalerate. For both species the activity of fenvalerate but not AVMB-j could be increased by the addition of PB

(Anderson et ali_., 1986). In contrast, third instar H.virescens were found to be equally sensitive to topically-applied AVMB-, and permethrin, but AVMB^ was less toxic than permethrin 76

towards H.zea (Wolfenbarger et £]_•» 1985). Similarly, third instar larvae of H.virescens were found to be equally sensitive to topically-applied AVMB1 and fenvalerate (Anderson £t al_., 1986), whereas Spodoptera eridania was 990-fold more sensitive to fenvalerate than AVMB-j. Topical application of Sunspray oil or PB with AVMB1 potentiated the toxicity of AVMB1 towards S.eridania but not towards H. vi rescens. Application of PB or Sunspray oil alone did not increase mortality significantly (P>0.05) compared with controls. PB had no effect on the toxicity of AVMB1 towards third instar S.eridania when applied two hours prior to AVMB1 but did have an effect when applied with AYMB1. The latter suggested that PB enhanced penetration of AVMB-j (Anderson et a]_., 1986).

In the latter study, Anderson et (1986) suggested that any potentiation of AVMB1 and fenvalerate towards S.eri dani a by PB or Sunspray oil was a result of an increased cuticular penetration by these insecticides. In contrast H.vi rescens was not found to be especially resistant to penetration by insecticides as neither Sunspray oil or PB caused any potentiation of AVMB1. Anderson et (1986) commented that in comparison with fenvalerate, AYMB1 was not readily potentiated by PB-induced enzyme inhibition, but application with oils promoting cuticular penetration could significantly potentiate the activity of AVMB1 against S.eridania. An enhancement of the activity of topically applied AVMB^ by dimethyl sulphoxide or cottonseed oil has also been reported with the boll weevil, Anthonomus 77 grandi s (Wol f enbarge r al_., 1985).

3.3.3 Effect of larval diet on insecticide toxicity. Beach and Todd (1985) have reported differences in the toxicity of AVMB1 against soybean looper, Pseudoplusia includens reared on artificial diet compared with certain soybean varieties. However, in the present work, a comparison of the 96h LD50 values obtained for third instar $.1ittoralis reared on different diets indicated that larvae on cotton were not significantly more sensitive to topically applied AVMB1 than were larvae reared on Chinese cabbage or artificial diet. Similarly, no significant difference was noticed in the 96h LD5Q values obtained when third instar S.littoralis larvae reared on Chinese cabbage or cotton were fed AVMB1-treated cabbage or cotton leaves respectively. This suggested that AVMB-, applied in food or onto the cuticle could enter the larva, reach the target site and exert its action equally effectively irrespective of diet. However, different 96h LD5Q values were obtained for first instar S.li ttorali s and H.armi gera exposed to AVMB1-treated cabbage or cotton leaves. In all cases, the 96h LDg0 values obtained for cotton were significantly (P<0.05) less than those on cabbage, although the differences between the species were greater than between crops within a species. Whether this was due to different feeding rates, or to differences in physiology is not known. One possible reason why H.armi gera was more sensitive to foliar residues of AVMB1 on cotton than 78

S.1ittorali s was given by a comparison of the weight gain per third instar H.armigera and $.11ttoralis larva fed on untreated cotton leaves. The amount of cotton leaf eaten per larva in this case suggested that H.armigera may feed at a slightly faster rate which would result in a larger dose of AVMB1 being consummed. The foliar toxicity of AVMB1 residues towards larval P.xylostel1 a was similarly found to be affected by the cabbage cultivar tested (Abro, 1985). The cultivar on which larvae were reared also influenced their sensitivity to topically-applied AVMBn (Abro, 1985). In the present study, a 96h LC50= 0.033 ppm was recorded for first instar H.virescens reared on AVMB-| -treated cotton leaves. Whereas, Anderson et £]_• (1986) reported a 12Oh LC5Q of 0.006 ppm against this species. The five fold difference in toxicity may have been due to the larvae being reared on different food plants and/or to the differences in bioassay times. In fact, Anderson et al. (1986) compared 120h LC5Q values recorded for 5.1ittoralis and H.virescens although these species were reared on different food plants. The present study has shown that larval diet may sometimes influence an insect’s toxicological response, and therefore comparisons between species or larval instars of the same species should strictly only be made when larvae are reared on the same food plant.

3.3.4. Action of oil enhancers. An enhanced foliar activity of both AYMB1 and cypermethrin when applied with Sunspray 6E or safflower oil 79 was found for both first and third instar $.1ittoralis, possibly due to increased penetration. Similarly, Abro (1985) found that fourth instar larvae of the Diamondback moth, Plutella xylostella (Thailand strain) had an increased sensitivity to topically applied AVMB1 when mixed with the above oils. A faster rate of penetration would result in an increased toxicity of the insecticide due to there being less time for detoxification processes to occur. The potentiation of both AYMB1 and cypermethrin by Sunspray 6E and safflower oil was probably a result of an increase in the rate of penetration of the insecticide into the insect. Sun and Johnson (1972) have described increased toxicity due to an enhanced cuticular penetration as 'quasi-synergi sm*. Potentiation of the activity of AVMB1 by Sunspray oil was also reported by Anderson et aK (1986) for S.eridania and H.virescens; larvae acquiring the insecticide-oil residue on feeding and by contact with the leaf surface. In this case, the oil may have enhanced the rate of penetration of AVMB1 into the insect, but may also have had a stabilising effect on the pesticide. The latter effect was supported by the finding in the present study that Sunspray 6E oil enhanced the residual activity of AVMB1 (4.5 pg per ml) on cabbage (cv. Flower of Spring) against third instar larvae of S.1ittoralis. In addition, the residual activity of AVMB1 against T.urticae, both under glass and outdoors was reported to be enhanced when sprayed with safflower or Sunspray 6E oil (Wright et al., 1985bV 80

3.3.5 Larval weight gain. Low doses of an insecticide may cause a reduction in feeding due to one or more of the following actions : antifeedant activity, where the compound reduces the palitability of the food (Watanabe and Fukami, 1977); repellancy, defined as the active avoidance of a volatile substance (Hirata and Sogawa, 1976); and anorexia or appetite loss (Beeman and Matsumura, 1978). Foliar residues of AVMB1 were found to significantly reduce the weight gain of third instar $.1ittoralis; the reduction being more or less dose dependent. A reduction in feeding and weight gain in insects due to sublethal doses of insecticide have been reported previously by a number of other authors (Tan, 1981; Kumar and Chapman, 1984; Corbitt etaK, 1985). The weight gain by third instar 5.1ittoralis was significantly reduced compared with controls when larvae were fed on AVMB1-sprayed cabbage leaves. A similar observation was made by Abro (1985) working on fourth instar P.xylostella. In both cases, concentrations of AYMB1 below the 96h LD50 value caused a significant reduction in weight gain. That AVMB1 has no repellant action towards third instar S.1ittoralis larvae was shown in the choice experiment using leaf discs, where larvae appeared to feed equally well on untreated or AVMB1-treated leaf discs. The relatively high mortality observed on the untreated side of the choice chamber (30%) was possibly due in part to the larvae receiving a lethal dose of AVMB1 before passing onto the control side. Similar observations were made using AVMB1 81

-treated or untreated food with nymphs of the German cockroach Blatella germanica (Cochran, 1985). The above observations suggest that the effect of AVMB-j on larval weight gain was at least in part due to an anorectic rather than a repellant action. This was further supported by the finding that the weight gain by third instar S.littoralis larvae receiving lpl of AVMB1 by topical application was significantly less than controls; levels greater than 7 ng/mg insect causing a loss in weight to occur. In the latter experiment, AVMB1 was applied to the dorsal posterior region of each larvae to ensure that chemoreceptors located on the mouthparts were unaffected thereby reducing the possibility that AVMB1 has an antifeedant action on the mouthpart chemoreceptors. A reduction in food intake by Soybean looper larvae, Pseudoplusia includens treated topically with AYMB1 has also been reported by Beach and Todd (1985), and by Pienkowski and Mehring (1983) working on second and fourth instar Hypera postica. One possible basis for an anorectic action of a pesticide was suggested by Ishaaya £t aK (1974) who found that the main digestive enzymes present in the midgut of S.1ittoralis were inhibited by the compound AC-24,055. This inhibition was thought to be indirect, the compound inhibiting a biochemical system affecting the production of

these enzymes which would lead to the insect being unable to

digest its food.

Finally, Putter et jaK (1981) have put forward the view that AVMB-j reduced the food intake of lepidopteran 82

larvae by impairing their locomotory activity. Certainly, in the present study, larvae which gained the least weight were often paralysed. While Schuster and Everett (1983) observed a reduced level of stippling by female L.trifoli on AVMB^treated leaves which may have been due to a paralysis of the ovipositor musculature. The above findings would seem to indicate that AVMB1 interferes with one or more physiological/behavioural processes; low levels of AVMB-, partially inhibiting the insect, higher levels completely inhibiting the insect and leading to death. It is therefore possible that the effect of AVMB-j in reducing food intake and weight gain is a subtle interaction of anorectic, antifeedant and impaired locomotory processes. 83

UPTAKE OF [3H]AVMB1 BY Periplaneta americana TISSUES,

4.1 Materials and Methods.

4.1.1 Localisation of total radioactivity in Periplaneta americana tissues. Adult, male Periplaneta americana were lightly anaesthetized with carbon dioxide and injected through the posterior ventral abdomen with 2pl of an aqueous solution of [3H]AVMB1 (18 ppm, 11103 dpm/2pl). Injection was carried out as described in Section 3.1.4. When symptoms of poisoning were observed (lethargy, paralysis of appendages, movement only on prodding) in the majority of adults (24h) the animals were dissected. The testes and ventral nerve cord up to and including the metathoracic ganglion were taken together with samples of fat body and metathoracic muscle. Each tissue (5 insects per replicate, 3 replicates) was placed into individual pre-weighed glass scintillation vials and the vials reweighed. To each vial was then added 0.4 ml of methyl benzethonium hydroxide (1.0 M in methanol) to bring about dissolution of the tissues. The vials were stored at room temperature. Once digestion was complete (7-10 days), 10 ml of cocktail T scintillant (BDH Ltd) were added to each vial and the vials were left for several days in the dark until spontaneous chemiluminescent reactions had abated. The vials were then counted in a Beckman-250 liquid 84 scintillation counter for 100 min or until a 2sigma error value of 0.2% was recorded. Disintegrations per minute (dpm) were then calculated from counts per minute (cpm) using a standard curve for [3H] in cocktail T and the dpm values converted into nM equivalents of [3H]AVMB1 per mg wet weight tissue. As an internal standard, a 10ul of [3H]AVMB1 (158410 dpm) was added to a known weight of nerve or muscle tissue in a vial (n=2), the tissue was allowed to dry before digestion and counting procedures were carried out as above. Tissues were taken from untreated adults, digestion and counting procedures were carried out as above to determine the control tissue dpm/mg.

4.1.2 Uptake of radioactivity by P.americana nerve and muscle tissues with time. Adult, male P.americana were treated as in Section 4.1.1. At 6, 18, 24, and 48h post-injection, four cockroaches were dissected. The ventral nerve cord up to and including the metathoracic ganglion was taken together with samples of muscle from the metathoracic region, samples were pooled, digested and radioactivity counted as in Section 4.1.1. The control dpm/mg was determined from tissues taken from untreated insects. 85

4.1.3 Isolation of radioactivity from P.americana nerve and muscle tissues. Adult, male P.americana were treated as in Section 4.1.1, left for 24h and then dissected. Tissue samples were taken as in Section 4.1.2.

4.1.3a Extraction of [3H]AVMB.|. The radioactive fraction containing [3H]AVMB1 was extracted from nerve and muscle tissues using the method of Burg et a]. (1979). Known weights of tissue were shaken for 2 min in 10 ml of a 100:1 (v/v) acetone/hydrochloric acid (1M) solution. Five ml of chloroform were added to each sample which was shaken for a further 2 min. The samples were then centrifuged (1500g) the supernatent decanted off and the pellet discarded. Two ml of distilled water was added to the supernatent and the above procedures repeated, resulting in the formation of an upper aqueous and a lower chloroform layer. The upper aqueous layer was pipetted off, its volume determined and a 20iil aliquot taken for scintillation counting (Section 4.1.1). The lower solvent layer was evaporated to dryness under nitrogen and the residue taken up in 1.0 ml of chloroform and three seperate 20ul aliquots counted (Section 4.1.1). The extraction efficiency of the above procedure was evaluated using the following internal standards:

(1) A 20jj1 aliquot of [3H]AVMB1 (113282 dpm) was added to a vial (n=3), the extraction procedures were carried out as before and the % recovery determined.

(2) A IOjjI aliquot of [3H]AVMB1 (158410 dpm) was added to 86 a known weight of tissue in a vial. The tissue was allowed to dry before extraction procedures were carried out and the mean % recovery determined (n=2). Untreated adults were also dissected and nerve and muscle samples taken. Extraction procedures and counting were carried out as previously described.

4.1.3b Thin layer chromatography (TLC) of [3H]AVMB1.

Twenty jjI aliquots of the lower solvent layer from the tissue, internal standard, and control extracts

(Section 4.1.3a) together with 5jj1 aliquots of 180ppm [3H]AVMB1 (302217 dpm) and lOpl of unlabelled AYMB-, (90pg) were spotted seperately onto silica gel (0.2mm) aluminium TLC plates (Merck) which had been pre-heated at 100°C for 24h. The plates were allowed to dry completely at room temperature then placed within a solvent tank containing ethyl-acetate, dichioromethane, methanol, and chloroform in a ratio of 9:2:1:9. After the solvent had ascended a distance of 15 cm (ca. 45 min) each plate was removed from the tank and allowed to dry. The plates were cut into sections and those sections with unlabelled standards sprayed with 1% (w/v) ceric sulphate in 10% (v/v) sulphuric acid and heated to 100°C in an oven to visualise the AVMB-j . Once the position of AVMB1 (Rf=0.33) was determined, the silica layer at corresponding positions on unsprayed sections (referred to as band 3) were scraped off, placed into pre-counted vials containing 10 ml of Cocktail T scintillant and counted (Section 4.1.1). Sections from other regions of the TLC plate (bands 1-2 + 4-8) were also taken for counting. 87

4.1.3c Bioassay of silica gel samples. In addition, some fractions from TLC plates were scraped into seperate vials and 1 ml of 1% (v/v) acetone in Triton-X 100 (100pg/ml) was added to each vial to extract AVMB-i or its metabolites. The extracts were then bioassayed against the second stage juvenile (J2) of the plant parasitic nematode Meloigdogyne incognita to determine the biological activity present in the sample; the activity (undulations per minute) of the nematodes in the extracts being compared with controls (Wright, et al., 1984). 88

4.2 Results

4.2.1 Distribution of radioactivity in P.americana

tissues. The level of radioactivity present in selected tissues from adult, male P.americana is given in Table 4.1. Little difference was found between tissues with the ventral nerve cord for example containing the equivalent of 33 nM AVMB-, per mg wet weight tissue (assuming all radioactivity was present as AVMB1). When nerve and muscle tissues from [3H]AVMB1 -treated adult cockroaches showing a range of symptoms were compared, it was found that the degree of poisoning appeared to be reflected in the level of radioactivity present in the tissues. Thus, adults which were paralysed and unable to walk had significantly (P<0.05) higher levels of radioactivity in their ventral nerve cords than partially paralysed adults able to walk. While all paralysed insects had significantly (P<0.05) greater levels of radioactivity (nerve and muscle) compared with treated but unaffected insects (Table 4.2). Internal standards showed that 93 and 91% respectively of radioactivity added to nerve and muscle tissue was recovered on digestion and counting (Table 4.3).

4.2.2. Uptake of radioactivity by P.americana nerve and muscle tissues with time. The level of radioactivity present in the ventral nerve cord appeared to decrease with time, whereas the 89

Table 4.1 Localisation of radioactivity in selected body tissues of Periplaneta americana 24h after injection of [3H]AVMB1:*=

Tissue * Dpm/mg nM equivalents Estimated^ AVMBj/mg XtSE nM AYMB-j X +S-E-

Ventral nerve cord 15.0 38.2 12.0 30.6 2 5. 1± 1.8 12.4 31.6 (33. 5±2.4)

Muscle 11.7 29.8 11.4 29.9 8.1±0.51 9.5 24.2 (27.9U.9)

Fat body 14.3 36.4 16.0 40.7 13.5 34.4 (37.2+1.9)

Testes 8.3 21.1 9.9 25.2 13.5 34.4 (26.9-3.9)

=t= Insects injected with 36ng [3H]AVMB1 (18ppm; 23969 dpm/2pl) * n=3, 5 insects per replicate + See Table4-5( section 4.2.3b) Table 4.2 Levels of radioactivity in nerve cord and muscle of [3H]AVMB1-treated adult, male Periplaneta americana (24h post-injection)(l)

X±SE * + Symptoms Tissue X dpm/mg wet nM equivalents Estimated (n) weight tissue of AVMB-j nM AVMB-j

Unaffected Nerve cord 2.4 ±1.9* 6.114.8 4.6 i 3.6 (4) Muscle 2.3 - 0.8a 5.9± 2.0 1.7 i 0.6 Paralysed- Nerve cord 11.1 ± 1.58 28.3 t 3.8 21.1 ± 2.8 walk (8) Muscle 11.8±1.0b 30.012.5 8.7 ± 0.7 Paralysed- Nerve cord 17.5 ±1.4C 44.61 3.6 33.4 12.7 no walk (16) Muscle 12.5 ± 1.4b 31.81 3.6 9.2 1 1.0

0) Insects injected with 36 ng [3H]AVMB1 (11103 dpm/2/il)

* Values sharing a common letter not significantly different at 5% level (t) * Estimated nM AVMB-j assumming 75 and 29% respectively of dpm/mg in nerve cord and muscle due to AVMB-j (Section 4.2.3b). 91

Table 4.3 Level of radioactivity recovered from nerve and muscle tissues of adult, male Periplaneta americana.

X dpm/mg Treatment Initial Recovered % Recovery (ti ssue) (i) Nerve cord 11147.4 10406.3 93.3 Muscle 4888.5 4473.4 91.5

(D lOpl [3H]AVMB1 (158410.5 dpm) added to known weight of tissue In vial. n=2 92

amount in muscle remained fairly constant (Table 4.4). The number of individuals affected and their level of paralysis increased with time (Table 4.4).

4.2.3 Isolation of radioactivity from nerve and muscle tissues of P.americana. 4.2.3a Extraction of radioactivity from nerve and muscle tissues of P.americana. The aqueous component (containing polar metabolites of AVMB1) of the nerve and muscle extracts was found to have 311 dpm/mg and 55 dpm/mg of [3H] respectively compared with 21 dpm/mg and 4 dpm/mg respectively in the chloroform fraction (containing AVMB1 and non.-polar metabolites) (Table 4.5). In the internal standard extracts, the radioactivity appeared to be largely confined to the chloroform fraction (Table 4.6).

4.2.3b TLC of chloroform extracts from nerve and muscle tissue.

Visualisation of unlabelled AVMBi (90pg in IOjjI) on a TLC plate revealed that AVMBi was located in band 3 (Rf=0.33±0.01, n = 14). TLC of the [3H]AVMB1 stock solution

(0.18 pg in IOjjI aqueous formulation) revealed that 91% of the activity was located in band 3 (Rf=0.31) (Table 4.7) and that there was a loss of only S% activity compared to direct measurement by vial. Internal standards (Table 4.8) revealed that there was relatively little loss of radioactivity during the extraction and seperation procedures. 93

Since there was little loss of activity from internal standards, the % activity in TLC band 3 of the extracts was used to estimate the dpm/mg (nM equivalents) due to AYMB-j . The % of the total dpm/mg found in band 3 of nerve and muscle extracts was 75 and 29% respectively (Table 4.5). Thus, if all activity in band 3 was due to AVMB-, and not to other non-polar metabolites the maximum dpm per mg (nM equivalents) due to AVMB-j was 16 (40) and 1 dpm/mg (3) for nerve and muscle extracts respectively (based on data in Table 4.5).

4.2.3c Bioassay of extracts from TLC plate bands. Bioassay of the extracts from bands on the TLC plate against M. incognita gave rather di ssapoi nti ng results. It was found that the AVMB-J fraction was active. However, all the other bands appeared to contain extracts which considerably reduced nematode activity compared with controls (Appendix 4.1). Chemical studies beyond the scope of this thesis are required to determine the identity of the other substances. Table 4.4 Uptake of radioactivity by P.americana tissues with relation to time.

Time after X dpm/mg _Estimated (,) Degree of injection (nM AVMB1 equi valents ) X±SE nM AVMB1 paralysi s (h) Nerve cord Muscle Nerve Muscle (number affected out of 8)

6 6.1 (15.5) 5.0 (12.7) 13.211.5 3.9-4.3 PW (8) 7.7 (19.6) 5.6 (14.3) CO - F t

12 4.3 (10.9) 4.8 (12.2) 8.510.2 3.3±0.1 PW (8) 4.6 (11.7) 4.2 (10.7)

24 3.0 (7.6) 2.4 (6.1) 5.7 - 2.9+1.2 [PW (4) 5.6 (14.3) P (4)]

48 1.8 (4.6) 2.5 (6.4) 5.211.7 4.012.2 [PW (2) 3.6 (9.2) 8.4 (21.4) P (6)] n= 2, 4 insects per replicate PW= Paralysed able to walk P = Paralysed unable to walk (I) See Section 4*2-3b Table 4.5 Level of radioactivity in chloroform and aqueous phases taken from nerve and muscle extracts of AVMB^injected adult, male Periplaneta americana after 24hl')

T reatment Extract Dpm/mg Dpm/mg X Dpm/mg X % Activity (tissue) i njected recovered recovered (nM) band 3 TLC

Nerve cord Chioroform 970.4 30.6 21.1 (40) 75 750.6 11.6 Nerve cord Aqueou s 970.4 486.7 310.7 750.6 134.8 (0 cn

Muscle Chioroform 224.7 4.1 4.1 (3.5) 29 240.8 5.3 Muscle Aqueou s 224.7 75.4 55.2 240.8 35.1

(i) Insects injected with 36 ng [3H]AVMB-, (3483.6 dpm/2»jl) * Estimated nM AVMB1 assumming 75 and 29% respectively of dpm/mg in nerve cord and muscle due to AVMB1 . N=2, (8 insects per replicate) Table 4.6 Level of radioactivity in chloroform and aqueous phases from internal standard nerve and muscle extracts of adult, male P.americana.

Treatment Extract X Dpm/mg X Dpm/mg % Dpm/mg % of Total (tissue) Initial recovered of initial activity recovered A vs C

Nerve cord * Chioroform 9130 7819 86 - 7293 5806 80 - Aqueou s ND Muscle * Chioroform 1559 1301 83 1440 1287 89 - Aqueou s ND

Nerve cord ** Chioroform - 111216 - 99.7

Aqueou s - 347 - 0.3

Muscle ** Chioroform - 51798 - 99.5

Aqueous - 250 - 0.5

* = IOjjI [3H]AVMB1 (18ppm, 158410.5 dpm) added to tissue in vial. Tissue allowed to dry prior to extraction (n=2). ** = Tissue bathed for 30 min in [3H]AVMB1 (180ppm) prior to rinsing and extraction (n=l) ND= Not determined. 97

Table 4.7 Level of radioactivity by vial and TLC methods of determination.

Treatment X total X dpm % dpm % recovery 5jj1 [ 3H]AVMB1 dpm band 3 band 3 (TLC vs vial) 180ppm

Vi al 302217 -- - TLC 277957 253947 91 92 n=3

Table 4.8 Level of radioactivity in chloroform phases following evaporation in vial overnight (A)

or to point of dryness (B) •

Vial TLC Treatment X dpm X dpm % X dpm X dpm % dpm ini ti al recovered recovery bands band 3 band 3 1-8

A 112382 110403 98 102962 87011 84 B 112382 113875 100 107591 96972 90

A= ca. 16h evaporation. B= ca. 4h evaporation. n= 3 98

4.3 Discussion In the present study, injection of [3H]AVMB1 into adult, male P.americana resulted in the uptake of radioactivity by all tissues investigated including the fat body which would be expected to act as a storage reservoir for non-polar toxicants. It was found that paralysed insects had higher levels of radioactivity compared with treated but unaffected insects, pointing to a relationship between the level of radioactivity and the stage of paralysis reached. The latter indicates that radioactivity can accumulate in the nerve cord during the normal time course of poisoning.

The localisation of radioactivity within the ventral nerve cord may point to an action of AVMB-j at GABA-mediated receptors in the nerve cord of P.americana. since Lummis and Sattelle (1985) showed that AVMBi (lpM) enhanced the binding of C3H}GABA in nerve cord extracts of P,americana, whereas binding was inhibited by low concentrations of AVMB-j (10nM) . Similarly, Abalis and Eldefrawi (1986) reported the high affinity binding of C3H3muscimol to a putative GABA receptor in the brain of the honey bee, Apis mellifera to be inhibited by AVMB-j (IC5o=10pM) and by GABA agonists. The latter suggests the action of AVM on ligand binding is concentration dependent.

In a study on the post-treatment fate of AVMB-j on three noctuid species Bull (1986) reported that the substantial differences in susceptibility to AVM were probably due to differences in the affinity of AVM for, and its interaction with GABA receptors. Differences in rate of cuticular penetration suggested by Anderson et al.(1986), and/ or rate of metabolism were not sufficient to explain the differential susceptiblity towards AVM (Bull, 1986). In the present study, the level of radioactivity within the 99

ventral nerve cord was not found to remain constant but to reach a peak and then decrease with time over a 48h period. However, the level of AVMB1 found was lower than in the localisation or extraction studies. The reason for the latter is not known. The level of [3H] within aqueous samples from [3H]AVMB1-injected insects was found to be greater than the level found in aqueous extracts of internal standards (Tables 4.5 and 4.6). The latter indicates that some degradation of the [3H]AYMB1 occurs within the insects but little occurred during the isolation procedure. The level of radioactivity in the aqueous fraction of nerve cord and muscle extracts was 15 and 11 times greater respectively than found in the chloroform fraction (Table 4.5). The latter points to there being a high level of metabolism in the 24h following injection with [3H]AVMBi . However, the role of metabolism in the toxicity of AVMB1 against insects is not known. That some of the radioactivity identified in distribution and uptake studies was attached to an AYMB1 -like molecule was shown by AVMB1 isolation procedures; the % radioactivity located in TLC band 3 (band 3 = Rf 0.29 to 0.37) of nerve and muscle extracts being 75 and 29% respectively. This suggested that 75 and 29% of the total radioactivity present in the ventral nerve cord and muscle respectively 24h after injection was the parent compound AVMB1 . The maximum dpm/mg (nM equivalents AVMB1) within nerve and muscle tissues was estimated to be 16 (40) and 1

(3) respectively. The identity of the other substances is not known from the present data though their activity against M .incognita suggests they are similar to AVM. Chemical analysis, requiring greater amounts of these substances is needed to determine their structure. 100

ACTIONS OF AYMB1 ON A VENTRAL NERVE CORD PREPARATION OF Perl pianeta americana

5.1 Materials and Methods.

5.1.1 In vitro effects of AVMB on spontaneous and evoked activities recorded extracel1ularly from the ventral

nerve cord of Periplaneta americana. Adult, male Peri pianeta americana were anaesthetized with carbon dioxide and their wings, legs (up to the femur) and antennae removed prior to being pinned into a Petri dish (5cm dia) partially filled with wax. Insectswere held in position by pins positioned around the edges of the abdomen and through the femur of the metathoracic legs. A 'window' was excised in the dorsal abdomen and the alimentary canal, testis and fat body removed so as to expose the ventral nerve cord. The body cavity was kept moist at all times with cockroach saline (Appendix 5-1). In some experiments, the sixth abdominal ganglion was partially desheathed using fine pins to facilitate faster penetration of the ganglion by test solutions. A glass microcapi11 ary tube filled with saline and fitted with a silver wire passing from a Grass SD9 stimulator was positioned over the cut end of a cercus. A suction electrode mounted on a micromanipulator (Prior, England) was positioned over the ventral nerve cord near to the sixth abdominal ganglion; suction being applied so that the nerve cord was firmly attached to the 101 electrode. The preparation was housed within a Faraday cage; all electrical equipment being exterior to the cage to minimise electrical interference. Nerve activity recorded by the electrode was passed to a CFP preamplifier 8121 (Gain set at 1000X). Two types of activity were monitored : [1] Spontaneous activity travelling along ascending and descending pathways in the ventral nerve cord. This was recorded through a spike integrator, the level of activity recorded being adjusted by means of the integrator. The resulting frequency data was displayed on a

Tele-equipment oscilloscope (type D1010) and on a Venture RE

Sll.20 Potentiometric chart recorder at 600 mm/h.

[2] Evoked activity in the form of compound action potentials produced when an electrical stimulus was applied to the cercus. The resulting action potentials were displayed on an Enertec Schlumberger 5072 storage osci11oscope.

The preparation was left after the dissection procedure until : [a] The spontaneous activity was regular and non-pertubating. [b] The action potentials were evoked by a stimulus of similar voltage and were of similar amplitude compared with previous recordings when tested at 10 min i ntervals. Once both criteria were satisfied, a saline wash was carried out. If there was no change in spontaneous

activity and in the voltage required to evoke a compound 102

action potential, the saline was exchanged for one containing AYMB1 (O.Ol-l.OpM). Recordings were carried out as previously described until the level of spontaneous activity was consistently different from that observed in saline alone. The saline containing AYMB1 was then exchanged for one containing picrotoxin (100 pM) and AVMB1 (1:1); readings being carried out as before.

5.1.2 Recording of spontaneous and evoked activities from the ventral nerve cord of P.americana injected with AVMB invivo. ----- 1------Adult, male P.americana were injected through the posterior, ventral abdomen with 2jj1 of an 18ppm solution of AVMBi. After 24h, dissection procedures, recordings and measurements of spontaneous and evoked activities were carried out as in section 5.1.1. Recordings were made over a 20 min period.

5.1.3 Relationship between spontaneous and evoked

activities present in the ventral nerve cord of

adult male P.americana pre-injected with [3H]AVMB1 and the level of radioactivity within nerve and

muscle tissues 24 and 96h after injection. Adult, male P.americana were injected with 2pl of an aqueous solution of [3H]AVMB1 (18ppm, 11058 dpm/2pl) through the posterior, ventral abdomen. Recordings of spontaneous and evoked activities were carried out as in section 5.1.1 over a 20 min period. The ventral nerve cord and a sample of metathoracic muscle were then taken and the 103 level of radioactivity present determined as in section 4.1.1. A record of the toxic symptoms shown by each insect was also made• 104

5.2 Results

5.2.1 In vitro preparations

5.2.1.1 Controls. The mean spontaneous and evoked activities for each 10 min period are shown in Table 5.1 and Fig. 5.1. Spontaneous activity fluctuated over the experimental time course, although the average spontaneous activity remained essentially unchanged. The evoked activity appeared to remain stable for a desheathed preparation but increased for a sheathed preparation. However, difficulty was found in recording evoked activity over the required experimental period of 160-180 min ( See Table 5.1 ) , and only one set of data for sheathed and desheathed preparations was obtained (Table 5.1).

5.2.1.2 Sheathed versus desheathed preparations. Application of AVMB-j (lpM) to sheathed preparations resulted in a gradual decline in spontaneous activity. The time required to reduce the level of spontaneous activity consistently below that of the mean control activity was about 117 min (Table 5.2). The action of AVMB-| on desheathed preparations was found to be dose dependent. AYMB-j at lpM took 38 min to reduce the level of spontaneous activity, whereas O.OlpM AVMB1 took 91 min (Table 5.2; Figs 5.2 and 5.3, Appendix5*2) Picrotoxin (100pm) was found to consistently reverse the action of AVMB1 (O.OlpM and l.OpM) on 105

Table 5.1 Sheathed and desheathed control ventral nerve cord spontaneous and evoked activites in Perlplaneta americana.

X ±SE (n) Sheathed Desheathed Time Spontaneous Stimulus Spontaneou s Stimulus (min) activity voltage acti vi ty voltage Hz (4) V (1) Hz (3) V (1)

0 21.92:3.3 0.30 22.1* 7.0 0.12 10 28.6*8.9 0.29 17.9*10.9 0.12 20 26.419.7 0.33 11.6* 2.6 0.12 30 14.9*3.5 0.31 23.9* 4.6 0.13 40 18.6*6.7 0.42 13.4* 1.6 0.11 50 23.5* 6.9 0.39 15.0* 4.2 0.12 60 20.9*5.7 0.34 18.6— 3.9 0.12 70 25.4*5.0 0.36 10.2* 1.0 0.12 80 26.8*4.9 0.35 22.6* 8.2 0.12 90 20.9*2.4 0.39 15.4* 5.1 0.12 100 23.5*5.3 0.41 23.4* 7.2 0.12 110 22.4*1.9 0.43 10.4* 0.6 0.12 120 16.3*2.5 0.52 18.8* 2.6 0.12 130 19.2*4.2 0.54 24.7* 6.9 0.12 140 22.2*9.2 0.54 12.2* 2.9 0.12 150 19.9*4.0 0.56 13.3* 5.6 160 24.8*8.6 0.40 29.1 170 20.5*2.9 — 25.5 180 22.6*6.6 — fig- 5.1 Control sheathed and desheathed ventral nerve cord spontaneous ($) and evoked (E) activities in Periplaneta americana. Sheathed ------Desheathed ------106 107

Table 5.2 Effect of AVMB1 on spontaneous activity within sheathed and desheathed ventral nerve cord preparations of Periplaneta americana.

AYMB1 treatment (jjM) n X miniSE to reduce spontaneous activity below control activity

Sheathed (1.0) 2* 117.5 —17.5 Desheathed (1.0) 3 38.3 + 6.0 Desheathed (0.01) 4 91.2 + 22.0

* In one other preparation there was no action of AVMB-j on spontaneous acti vi ty within time constraints of experiment. Fig-5-2 Effect of AVMB1 (l.QjuM) on evoked (a) and spontaneous (b) activities within a desheathed ventral nerve cord preparation of Periplaneta americana.

A B c D Treatment

A 0-1% v/v bMSO SALINE (a) B AVMB1 10>JM C Plcrotoxin 100 pM + AVMBi 1-0 JjM (l-l) V D 0-1% v/v DMSO SALINE 108

(*>)

H 2 Fig-5-3 Effect of AVMB] (O.QluM) on evoked (a) and spontaneous (b) activities within a desheathed ventral nerve cord preparation of Periplaneta americana.

A B c Treatment

A 0-1% v/v DMSO Saline 0 2 (a) - -. B AVMB1 0-01 jjM V C Picrotoxin lOOpM + 00 AVMB1 0-01 jjM (1:1) 109 110

desheathed preparations (eg Fig. 5.2), but had no effect on sheathed preparations. In the two cases tested, washing of the desheathed preparation with 0.1% (v/v) DMSO saline reversed the action of picrotoxin (Fig. 5.2).

5.2.1.3 Effect of AVMB-) on evoked activity. Readings of evoked activity were attempted in all preparations. However, in many cases difficulty was encountered (see Section 5.2.1.1) probably due to damage caused to the nerves during dissection procedures. In some preparations readings could be made initially but it became progressively more difficult to do so, possibly due to the suction electrode changing its position on the nerve during the experiment. The results obtained were variable, some indicated that AYMB1 acted to increase the stimulus voltage required to evoke an action potential whilst in other recordings AVMB1 had no effect.

5.2.2 In vivo preparations.

5.2.2.1 Effect of AVMBt on spontaneous and evoked activities 24h after injection. Injection of 2jul aqueous solution of AVMB1 (18ppm) into adult male P.americana resulted in some insects displaying signs of poisoning after 24h whilst others appeared to be apparently unaffected. Recordings were made of the level of spontaneous activity present in the ventral nerve cord of [a] control insects; [b] treated insects that appeared unaffected; [c] treated insects which were partially paralysed but still 111

able to walk; and [d] treated Insects that were unable to walk. These revealed that paralysed insects (c and d) had a significantly (P<0.05) reduced level of spontaneous activity compared with controls (Table 5.3). However, treated insects which were able to walk (c) had a mean spontaneous activity which was significantly (P<0.05) greater than more paralysed insects (d). Those insects which were treated but apparently unaffected (b) had a level of spontaneous activity similar to controls (Table 5.3). In contrast, AVMB1 had a less marked effect on evoked activity (Table 5.3); although the stimulus voltage required to evoke an action potential was significantly (P<0.05) greater in insects showing increasing signs of paralysis (c and d). Treatment with picrotoxin was not carried out in the above experiments as j_n vitro studies described earlier had indicated that picrotoxin had no action on sheathed preparations during the experimental period.

5.2.2.2 Level of radioactivity present in nerve and muscle 24 and 96h after injection of [3H]AVMB1in relation to spontaneous and evoked activities in the ventral nerve cord. As in the previous experiment (Table 5.3), the level of spontaneous activity was found to be significantly (P<0.05) reduced in treated-paralysed insects (c/d) compared with controls (a). Whereas the treated but unaffected insect (b) had spontaneous activity greater than controls (Table 5.4). After 96h all treated insects were 112

Table 5.3 Effect of AVMB1 on spontaneous and evoked activities within the ventral nerve cord of adult, male P.americana 24h post-injection.

X tSE *

Treatment (n) Spontaneous Stimulus acti vity voltage Hz V a. Control (8) 34.4± 3.7* 0.22± 0.023 o o b. Unaffected (4) 37.3 ± 2.83 0.251 • b c. Paralysed-walk (9) 12.li 0.9b 0.281 0.02 b d. Paralysed-no walk (18) 6.6 t l.lc 0.30 i 0.02

* Values sharing a common letter not significantly different at 5% level (t) 113

Table 5.4 Level of radioactivity present In nerve and muscle tissues 24 and 96h after injection with [3H]AVMB1 in relation to spontaneous and evoked activities.

Xt SE * Treatment Spontaneous Stimulus Dpm/mg (nM AVMB^) (n) acti vi ty voltage Hz V Nerve cord Muscle

24h

a. Control 34.4*3.7* 0.22+0.02* (8) b. Unaffected 42.8 0.24 8 (15) 3 (2) (1) c. Paralysed- 12.9±0.9b 0.27±0.02b lO+l3 (19) 11±1* (8) walk (8) b b d. Paralysed- 7.9±0.9C 0.28+0.02b 15±1 (29) 8+1 (6) no walk (8) 96h d b d. Paralysed- 3.8i0.9 0.31+0.02 9+1* (17) 11±1* (8) no walk (11)

* Values sharing a common letter not significantly different at 5% level (t) * Estimated nM AVMB1 assumming 75 and 29% respectively of dpm/mg in nerve cord and muscle due to AVMB-. (Section 4.2.3b). Insects injected with 36ng [3H]AVMB1 (18ppm; 11058 dpm/2ul) 114

paralysed (d) and the level of spontaneous activity was significantly (P<0.05) reduced compared with treated insects at 24h post-treatment. The voltage required to evoke an action potential was also found to be slightly but significantly (P<0.05) greater in treated insects. Fully paralysed insects (d) at 96h required a significantly {P<0.05) greater stimulus voltage to evoke an action potential than fully paralysed insects (d) at 24h (Table 5.4). The level of radioactivity present in nerve cord and muscle tissues taken from treated-paralysed insects (c/d) after 24h was greater than in tissues from the treated but unaffected insect (b) (Table 5.4). There was found to be a significant (P<0.05) difference in the level of radioactivity found in tissues taken from the two types of paralysed insects (c/d). Paralysed insects unable to walk (d) had levels of radioactivity in their nerve cords significantly (P<0.05) greater than those paralysed insects able to walk but the reverse was the case for muscle. The levels of radioactivity present in tissues of treated-paralysed insects (d) after 96h were found to be similar to the levels found in paralysed insects (c) after 24h (Table 5.4). It was found in Section 4.2.3b that 75 and 29% respectively of the radioactivity present in nerve and muscle extract was due to AVMB1. Thus, the level of AVMB1 present in nerve cords of treated insects can be seen to increase along with the level of paralysis (Table 5.4) 24h post-treatment- 115

5.3 Piscussion

Within the sixth abdominal ganglion of Periplaneta americana the giant interneurones passing anteriorly along the ventral nerve cord receive an excitatory, cholinergic synaptic input from cereal nerve 10 (Satelle, 1980; 1985). The giant interneurones also appear to be associated with pre- and post-synaptic inhibitory mechanisms probably utilising GABA as the transmitter (Hue and Callec, 1983). The action of GABA is to induce a chloride ion conductance increase (Hue et a K , 1981) and has been found to have an inhibitory effect on the giant interneurones (Kerkut et al., 1969a,b; Pitman and Kerkut, 1970; Callec, 1974). The amplitudes of the EPSPs evoked by cereal nerve stimulation are also reduced by GABA (Hue et al., 1981).

In the present study, it was found that in in vivo preparations the voltage required to evoke an action potential increased as symptoms of poisoning with AVMB^ became more apparent. The latter is consistent with AVMB-, mimicking the action of GABA on the giant interneurones and cereal nerves by increasing membrane conductance which would reduce the amplitudes of the action potentials evoked on stimulating the cercus. As a result the stimulus voltage would need to be increased to evoke an action potential of amplitude similar to before.

Gregory et £l_. ( 1987) reported that application of picrotoxin to a desheathed sixth abdominal ganglion preparation of P.americana gave rise to excitatory effects in the ventral nerve cord. In the present study, 116

application of picrotoxin to AVMB1-treated in vitro preparations resulted in a reversal of AYMB1 action. The latter suggests that picrotoxin reversed AVMB1 action allowing the cereal nerve-interneurone synapse to function. In vitro experiments on the ventral nerve cord of adult male Peri pianeta americana have revealed that the level of spontaneous activity is reduced in AVMB-j-treated insects compared with controls. The effect of the pesticide appeared to be both concentration and time dependent. Partial desheathment of the sixth abdominal ganglion was found to reduce the time required for the pesticide to exert its effect. Application of picrotoxin was found to reduce the effect of AVMB1 on desheathed but not sheathed preparations; the latter probably being due to the failure of the relatively hydrophilic picrotoxin molecule to pass through the nerve sheath. In vivo experiments on [ 3H]AVMB-| - i njected adult male P.americana revealed that at 24h post-treatment the level of spontaneous activity within the ventral nerve cord was reduced in those insects which were partially (category c), or fully paralysed (category d); the latter having a significantly (P<0.05) lower spontaneous activity. Both groups were also shown to possess significantly (P<0.05) greater amounts of radioactivity compared with controls or treated but unaffected insects. As in section 4.2.1 there were significantly (P<0.05) greater levels of radioactivity within the ventral nerve cords of paralysed insects which were unable to walk compared with those paralysed insects which were able to walk at 24h post-treatment. This suggests that there is, initially at least a direct 117

relationship between the level of AVMB1 (plus any toxic metabolites) within the nerve cord and the latters spontaneous neural activity. However, significantly (P<0.05) less radioactivity was found in the nerve cords of cockroaches examined at 96h post-treatment despite the fact that all treated insects were fully paralysed. The latter finding is in agreement with earlier experiments (Section 4.2.2) which showed that the level of radioactivity in the nerve cord fell gradually over a 48h period. On the assumption that the proportion of radi oacti vi ty which is present in the nerve cord as AVMB-, is reasonably constant, these findings suggest that AYMB-j initially acts to bring about a decline in the insects activity by paralysing the appendages. One possibility is that AYMB1 causes an irreversible toxic lesion which remains when the level of radioactivity becomes reduced due to metaboli sm. The j_n vitro study also showed there was a transient increase in spontaneous activity in some preparations. Similarly, in the in vivo study, some AVMB1 treated but unaffected insects were found to possess a level of spontaneous activity significantly (P<0.05) greater than controls. Tanaka and Matsumura (1985) working on leg muscles of adult P.americana have also reported a transient increase in excitation elicited by the insecticide gamma-BHC before blockage with AVMBi occurred. While a similar transient increase in activity was observed in lobster muscle (Mel 1 i n et , 1983) and in an embryonic neuronal culture from P.americana where 10% of cells responding to DHAVMB1 depolarised prior to 118

hyp.erpolarising (Lees and Beadle, 1986). In the present study, both the j_n v1 tro and 1 n vlvo evidence suggested that AVMB1 had an effect on the central nervous system of P.americana. The finding that spontaneous activity was reduced but not extirpated in ventral nerve cords of fully paralysed insects (category d) at 24 and 96h post-treatment (Table 5.4) may point to AVMB1 also having an action peripherally. The fact that the onset of paralysis in P.americana was first observed in the metathoracic appendages some six hours after injection and thence in the other legs, antennae and mouthparts (Section 4.2.2) would suggest an action on the musculature, either directly or via neuromuscular junctions. The paralysing, action of AYMB1 on insect appendages has also been reported by Putter et aK (1981) and Botham and Nicholson (1985) as discussed in Section 3.3.1.

Tanaka and Matsumura ( 1985) found that IOjjM AVMB-, caused the coxal leg muscle of P.americana to fail to respond to stimuli within 30 min of application. However, the magnitude of contraction was not affected. This suggested that AVMB1 blocked the transmission of stimuli passing from the CNS to the muscle by opening chloride channels in the postsynaptic muscle membrane which would lead to hyperpolarisation of the post-synaptic region. This was in agreement with the proposal by several authors (Fritz et a]., 1979; KassetaK, 1980; Me 11 i n et al ., 1983) that the action of AVM was on postsynaptic chloride ion channels. The observation that picrotoxin reversed the action of AVMB1 in the present study and previous studies 119

(Fritz et aK , 1979; Mellin et , 1983) may indicate that AVMB1 acts via a GABA mediated chloride ion channel. However, picrotoxin is known to block other Cl"channels and also K+channels in some arthropod systems (Cul1-Candy, 1976; Marder and Paupardin-Tritsch, 1978) and a direct action of AVMB-j on chloride channels as proposed by Tanaka and Matsumura (1985) cannot be discounted. The last workers found that AYMB1 did not increase binding of C3H]dihydropicrotoxinin to brain and muscle membranes of P.americana and suggested that the action of AVMB1 was not mediated via the GABA receptor-ionophore complex to which the picrotoxinin receptor is closely associated. DHAVMB1g has been reported to have a reversible action on the GABA receptor chloride ion channel and an irreversible effect on the chloride ion channel of extrajunctional glutamate H receptors present on locust leg muscle (Duce and Scott, 1985a,b). The latter channel was suggested to have a greater selectivity for Cl ions, than the one(s) activated by GABA. However, it is possible that DHAVMB1a may have activated Cl'channels with different ion filtering properties or altered the selectivity filter in some way to induce an irreversible permeability to anions (Scott and Duce, 1986). The latter may explain why in the present study paralysed insects at 96h post-treatment remained paralysed even though the level of radioactivity in the ventral nerve cord had fallen. The level of radioactivity found in metathoracic muscle taken from fully paralysed P.americana 96h post-treatment with [3H]AVMB1 was significantly (P<0.05) greater than found in fully paralysed insects after 24h but 1 20

similar to levels found in paralysed insects (c) able to walk. While the presence of AVMB-j in the muscle may contribute to the paralysing action of AVMB1 in P.americana, it is not known from the present study whether AVMB1 had an action directly on the muscle as found by Duce and Scott (1985a,b). The level of radioactivity in muscle samples taken from treated-paralysed insects was greater than from the treated-unaffected insect so a direct action on extrasynaptic chloride ion channels cannot be ruled out. It is also possible that the AYMB1 was bound to the post-synaptic region of the neuromuscular junction, since Fritz et aK (1979) and Mellin et al_. (1983) proposed that AVM had an action at this site. An estimate of AVMB1 present in the ventral nerve cord of P.americana based on TLC (Section 4.2.3b) showed that there were 19 and 29 nM in paralysed insects able to walk (c) and paralysed insects unable to walk (d) respectively. These concentrations were in most cases similar, or less than those used in neurophysiological studies by other authors (Fritz et al_., 1979 (1.2 juM - 12 juM); Mellin et al ., 1983 (6 juM); Duce and Scott, 1985a,b

(90 pM - 1.2 juM); Bokisch and Walker, 1986 (0.01 /jM - 1.0 JLlM) . The above estimates for AYMB1 are for the overall concentration in the nerve cord and this may obviously vary both between and across ganglia. However, that the level of AVMB-j found in the ventral nerve cord of paralysed P.americana was similar to doses having an action on nerve preparations in this, and other studies suggests the estimated levels of AVMB1 found can reasonably be related to the symptoms exhibited. It has been shown in the nematode Ascaris suum that AVMB1a (6jum) blocked transmission between interneurones and excitatory motorneurones in the ventral nerve cord reducing depolarization of muscle cells (Kass et al., 1980; 1984). AVMB1a was also shown to block transmission between inhibitory motorneurons and muscle cells thereby reducing hyperpolarization of the latter. The former blockage was reduced by picrotoxin whereas the latter was not. Since the inhibitory motoneurones receive their sole input from axons of the excitatory motoneuron, control of the latter is central to the regulation of locomotion (Johnson and Stretton, 1980) and the principle action of AVM would appear to be within the ventral nerve cord in this species (Kass et aK , 1980; 1984). More recently, Martin et £]_. (1987) also found that DHAVMB 1a

(0.1 jjM) acted as a GABA antagonist at an extrasynaptic GABA receptor present on somatic cells of A.suum by reducing Cl"ion conductance. The concentration used by Martin (1987) is a more realistic dose when compared with doses found to have a biological activity against nematodes (Wright, 1986) and insects. 122

6.0 General Discussion and Conclusions

6.1 Toxicity of AVMB-, against lepidopteran larval instars. The present work has shown that abamectin (AVMB1) is active against larval instars of $.1ittoralis. The third and sixth instar larval stages were found to be sensitive to topically applied AVMB1 whereas the fourth and fifth larval instars were relatively insensitive. All larval instars of S.1ittoralis tested other than the sixth instar were more susceptible to the pyrethroid cypermethrin than to AVMB1. However, injection of AVMB1 into fifth and sixth instar S.littoralis resulted in a greater reduction in the LD50 value in the former compared with the latter instar suggesting that a penetration factor was involved in the relative sensitivity of S.littoralis larval instars to AVMB1 The sensitivity of the first and third larval instars of S.li ttorali s to foliar residues of AVMB-j was greater than to topically applied AVMB1. The fourth instar was more susceptible to AVMB1 residues than to topically applied AVMB1 suggesting that penetration of AVMB1 was inhibited by the cuticle. That foliar residues were more toxic than topically applied AVMB1 suggests that the insecticide acts as a stomach poison. The latter, together with an increased toxicity of AVMB1 by the use of oils indicates that AVMB-, might find a use in integrated control programmes in conjunction with predators and parasites of S.li ttorali s, such as Bracon brevicornis and Microgaster rufiventris. Certainly, AVMB1 has been shown to be more toxic towards 123

phytophagous than predatory mites (Grafton-Cardwell and Hoy, 1983). First Instar larvae feeding on AVMB1-treated foliage were the most sensitive Instar tested and control measures should be primarily directed at this Instar. In the field, first Instar larvae of $.1IttoralIs feed on the underside of leaves and AYMB1 has the advantage of possessing translamlnar activity (Green et , 1985; Wright et aK , 1985a') in contrast to the pyrethroids. Larval diet was shown to influence the susceptibility of first instar S.1ittoralis and H.armigera to foliar residues of AVMB-,. The latter suggests that a knowledge of an insects nutritional requirements could be used to reduce the level of pesticide required to be applied to certain crops. The present work has shown that AVMB1 reduces larval growth probably by an anorectic effect. Lower rates of AYMB-i were required to reduce feeding compared with the dose required to kill 50% of a population. A consequence of reduced feeding is that the development time of the larval instars is prolonged though ultimately the larva dies. During this extended period the larva is also more likely to be predated. It was found that both He!iothis species tested were more sensitive to AVMB-j when compared with 5.1ittoralis. However, $.1ittoralis was found to be far more susceptible to AVM than some other Spodoptera species (Bull, 1986; Anderson et aK , 1986) and the above work suggests that this pesticide may have some potential for control of S.1ittoralis. 124

6.2 Radiochemical and Neurophysiological studies. Radiochemical studies (Section4-2-l) with [3H]AVMB1 -injected Peri pianeta americana showed that all tissues examined contained radioactivity. Uptake studies indicated that the level of radioactivity within the ventral nerve cord declined and at 48h post treatment contained 3 dpm/mg compared with 7 dpm/mg at 6h although the number of affected insects increased with time. The latter would be expected, however, as AVMB1 is generally accepted to cause an irreversible lesion in insects (Wright, 1986). Separation of radi oact i vi ty from AVMB-|-injected adult, male P.americana into polar and non-polar phases showed that some metabolism of the AVMB-, occurred within the insect. The chloroform phase (containing AVMB1 and non-polar metabolites) of the extract was active against the nematode M.incognita. The majority of the activity within the tissue extracts co-chromatographed with standard AYMB1 and [3H]AVMB1 suggesting that the active moiety was AYMB-j. The level of [3H]AVMB1 within the nerve cord and metathoracic muscle was estimated by TLC to be 75 and 29% respectively of the total dpm/mg tissue taken from the insects (Section 4.2.3b). This gave estimated values for AVMB1 of 40 and 3 nM in nerve and muscle extracts respectively (Table 4.5). From the data in sections 4.2.1 and 4.2.2 (Tables 4.1, 4.2 and 4.5), estimated levels of AVMB1 extracted from nerve and muscle tissues of treated-paralysed insects after 24h were found to be of the same order of magnitude as in the above experiment (5-40 and 2-9 nM for ventral nerve cord and muscle respectively). 125

For unknown reasons, rather less radioactivity was detected in the nerve cord in the time-uptake study (Table 4.3). Neurophysiological studies on the action of AVMB-j on spontaneous and evoked neural activities within the ventral nerve cord of J_n vitro preparations of P.americana indicated that AVMB1 had a dose dependent action; desheathed preparations being affected more rapidly than sheathed preparations. However, j_n vi tro preparations were found to be unstable over the time required for AVMB1 to have an action, and in the case of the desheathed preparation this was possibly due in part to desheathing procedures. The action of AVMB1 was therefore investigated using j_n vivo preparations. It was found that AVMB1 reduced the level of spontaneous activity, but increased the stimulus voltage required to generate an action potential compared with controls. The jji vi vo preparations were far more stable over the thirty minute period required for each preparati on. The action of AVMB1 on spontaneous and evoked activities was found to be related to the degree of paralysis exhibited by the insect, and to the level of radioactivity taken up by the ventral nerve cord and possibly metathoracic muscle. At 24h post [3H]AVMB1 -treatment, insects displaying signs of paralysis were either partially (still able to walk) or fully (unable to walk) paralysed. Both types of paralysed insects had significant (P<0.05) levels of radioactivity within their ventral nerve cords. The radioactivity within the nerve cord of fully paralysed insects was significantly (P<0.05) greater than found in partially paralysed insects whereas 126

the level of spontaneous activity was significantly (P<0.05) reduced in the former. The latter suggests that the stage of paralysis reached is a reflection of the initial dose of AVMB1 accumulated. Further studies are required over shorter time intervals, to examine this relationship in more detail. At 96h post-treatment all insects were fully paralysed and unable to walk and the spontaneous activity was significantly (P<0.05) reduced compared with controls or fully paralysed insects at 24h. However, the level of radioactivity within the ventral nerve cord was lower than at 24h post-treatment. This suggested that a toxic dose could accumulate within the normal time course of poisoning. The GABA antagonist picrotoxin was found to reverse the effect of AVMB1 in desheathed preparations. This, together with the reduction in spontaneous activity and increased stimulus voltage required to elicit an action potential suggests that AYMB1 is a GABA mimetic. The site of AVM action has been proposed to be at GABA-sensitive (Fritz e_t 1979; Mellin £t aK, 1983) and GABA-insensitive (Duce and Scott, 1985a,b; Bokisch and Walker, 1986) chloride ion channels, although the relative importance of each is not known. The observation that paralysed insects gave no movements even on prodding but possessed nerve cords with a reduced but not extirpated level of spontaneous activity suggested that transmission between motor neurones and muscle might be inhibited by AVMB1 in P.americana. The latter is supported by various authors (Fritz et al ., 1979; 1 27

Mel 1i n et a K , 1983; Tanaka and Matsumura, 1985) who suggested that AVM has a direct action on chloride ion channels present at neuromuscular junctions. The report of Duce and Scott (1985 a,b) that DHAVMB1a has an action on extrajunctional glutamate H receptor chloride ion channels present on locust muscle also points to AYMB1 having a non-synaptic action. However, the role of extrajunctional AVM sites and the relative importance of synaptic and non-synaptic sites is unclear. In the present work, the estimated levels of AVMB1 in muscle taken from treated-paralysed insects were less than found in the ventral nerve cord. However, a non-synaptic action of AVMB1 cannot be discounted since the levels in muscle were similar to those found to have an action on locust muscle (Duce and Scott, 1985). In conclusion AVMBn is active by contact and as a stomach poison against lepidopteran larval instars with lethal and sub-lethal effects. In the American cockroach, P .americana, the mode of action of AVMB1 would appear to involve GABA-mediated synapses, both centrally and peripherally. The level of AVMB-, found in the ventral nerve cord of paralysed insects was similar to the levels of AVM used in this, and other studies (Fritz et al_.f 1979; Duce and Scott, 1985). The latter suggests there is a relationship between the stage of paralysis reached and the level of AVMB1 within the ventral nerve cord of P.americana. REFERENCES

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Appendix 2.1 Artificial diet for Spodoptera littoral Is, Hellothis armlgera and Hellothis vIreseens

Larval diet.

[a] Haricot bean seed (finely ground) 300.0 g [b] Ascorbic acid 4.7 g [c] Methyl-4-hydroxybenzoate 3.0 g

1 O. 1—1 1— Sorbic acid 1.5 g [e] Tetracycl1ne 1.5 g [f] Linseed oil 12.0 ml

1 1

_ 1— 1 IQ Vitamin stock solution (see below) 10.0 ml [h] Water 500.0 ml [i] Yeast 48.0 g

1 _ l—1 1 C_i. Agar 17.25 g 1 1—1 _ 1 Water (for yeast+agar) 810.0 ml

Adult diet.

Sucrose 50.0 9 Methyl-4-hydroxybenzoate 1.0 9 Vitamin stock solution (see below) 10.0 ml Water 500.0 ml 137

Vitamin stock solution (kept at 5°C).

Ingredients:

Nicotinic acid 1.528 9 Calcium pantothenate (B3) 1.528 9 Rib of1avi ne (B2) 0.764 9 Aneurine hydrochloride (Bl) 0.382 9 Pyridoxine hydrochloride (B6) 0.382 9 Folic acid (Be) 0.382 9 D-bi oti n (H) 0.305 9 Cyanocobalami ne (B12) 0.003 9 Water 500.0 ml

Preparation of larval diet.

Water [h] was added with stirring to ingredients a-g. The water (k) for the yeast/agar was brought to boiling, yeast and agar were then added while stirring and the mixture combined with ingredients a-h. The diet was poured into a plastic tray and placed in a laminar flow cabinet until set. The diet was then covered with silver foil and stored at 5°C. 138

Preparation of adult diet.

The ingredients were added to a 500 ml flask and mixed with the water. The diet was kept at 5°C and dispensed into pots containing cotton wool as required.

t 139

Appendix 3*1

------—Toxicity of------topically — -- -*»applied------—— AVMB — 1 1 ------——------to larval instars of— Spodoptera littoralis reared on cabbage (see Table 3.1). Instar Concentration n Number (ug/ml) respondi ng Thi rd Control 80 14 392.0 80 32 549.0 84 38 1079.0 40 20 2139.0 40 33 Fourth Control 40 0 4586.0 40 8 16186.0 40 14 F i f th Control 40 0 16186.0 40 4 Sixth Control 40 2 153.0 40 6 200.0 40 13 392.0 60 43 549.0 40 39 Toxicity of topically applied cypermethrin to 1arval instars of S.littoralis reared on cabbage (see Table 3.2). Instar Concentrati on n Number (jLig/ml) respondi ng Third Control 40 8 0.5 40 26 2.0 40 29 5.0 40 34 10.0 40 36 Fourth Control 45 6 5.0 40 13 10.0 40 16 25.0 40 22 48.0 45 32 F i f th Control 63 14 25.0 54 20 48.0 59 25 90.0 59 36 181.0 45 22 1023.0 40 32 Sixth Control 70 11 90.0 62 17 452.0 37 22 1023.0 55 38 2018.0 50 39 140

Toxicity of topically applied AVMB„ to thi rd instar larvae of S.littoralis reared on various diets (see Table 3.3). Treatment Concentration n Number (diet) (jjg/ml) respond!* ng

Artificial diet Xlarval weight=10*7mg Control 40 3 186.0 40 4 394.0 40 16 831.0 40 19 1871.0 40 22

Cabbage X larval weiglit=10-4mg Control 80 14 392.0 80 32 549.0 84 38 1079.0 40 20 2139.0 40 33 Cotton X larval weight=5 -2 mg Control 40 7 186.0 40 25 394.0 40 32 831.0 40 33 1871.0 40 39 Toxicity of topically applied AVMB to third instar larvae of S.1ittoralis and H.armigera reared on artificial diet (see Table 3.4). Treatment Concentration n Number (Species) (jug/ml ) respond!* ng

S . 1 i ttoral i S X larval weignt=10-7 mg ControI 40 3 186.0 40 4 394.0 40 16 831.0 40 19 1871.0 40 22

H.armigera x larval weights! 0-9 mg Control 39 2 392.0 40 11 549.0 40 19 831.0 44 27 1871.0 39 28 141

Mortal 1ty of f ifth and sixth instar S.1i ttorali s fol1 owing topical application or injection with AVMB„(see Table 3.5). T reatment Concentration n Number (ng/ml) responding Topical application Fifth instar Control 40 0 16186.0 40 4 Sixth instar Control 40 2 153.0 40 6 200.0 40 13 392.0 60 43 549.0 40 39 I nj ecti on Fifth instar Control 45 17 186.0 24 12 392.0 30 14 549.0 30 18 Sixth instar Control 35 11 80.0 33 20 186.0 30 23 394.0 30 24

Contact/i ngesti on toxicity of AVMB„ on Chi nese cabbage foliage (with and without the addition of oils ) against first instar larvae of S.littoralis (see Table 3.6). Treatment Concentration n Number (jLig/ml) respondi ng AVMB1 Control 80 2 0.45 40 3 0.7 40 14 0.9 40 15 1.25 80 47 1.5 40 26 AVMB-, + Safflower Control 40 2 0.15 40 18 0.45 40 29 0.7 40 36 0.9 40 37 AYMB1 + Sunflower 6E Control 41 0 0.072 40 3 0.18 40 14 0.36 40 17 0.81 40 37 142

Contact/ingestion toxicity of cypermethrin on Chinese cabbage foliage (with and without the addition of oils) against first instar larvae of S.littoralis (see Table 3.7). Treatment Concentration n Number (jig/ml) respondi ng Cypermethrin Control 41 0 0.15 40 15 0.45 40 28 0.7 40 35 0.9 40 39 Cypermethrln + Saf fl ower Control 40 3 0.09 41 7 0.015 40 12 0.045 40 21 0.15 40 39 Cypermethrln + Sunspray 6E Control 40 0 0.009 40 • 1 0.015 40 2 0.045 40 8 0.09 40 21 Contact/1 ngestlon toxicity of AVMB1 on Chinese cabbage foliage (with and without the addition of oils) against third Instar larvae of S.littoralls (see Table 3.8). Treatment Concentration n Number (pg/ml) respondi ng AVMB.1 Control 40 0 9.0 40 •7 18.0 40 20 36.0 40 37. 72.0 40 39 AVMB + Saf fl ower 1 Control 60 4 1.5 60 10 3.0 60 13 4.5 60 21 18.0 60 55 AVNB.1 + Sunspray 6E Control 40 0 0.9 40 9 1.8 40 15 3.0 40 27 4.5 40 31 6.0 40 36 9.0 40 37 143

Contact/inge.stion toxicity of cypermethrin on Chinese cabbage foliage (with and without the addition of oils) against third instar larvae of S.littoralis (see Table 3.9). Treatment Concentration n Number (ug/ml) respondi ng Cypermethri n Control 40 2 0.5 40 9 1.0 40 24 1.5 40 32 2.0 40 38 2.5 40 39 Cypermethrin + Saff1ower Control 40 3 0.09 40 4 0.15 40 7 0.3 40 17 0.5 40 35 Cypermethrin + Sunspray 6E Control 80 0 0.15 80 8 0.3 80 22 0.45 40 14 0.75 40 34 0.9 40 35 Toxicity of AVMB^ against fourth instar larvae of i S.littoralis by contact/i ngesti on and topical application methods (see Table 3.10). Treatment Concentration n Number (pg/ml) respondi ng Foliar Control 31 0 18.0 30 4 27.0 30 15 36.0 35 24 72.0 35 28 Topical Application Control 40 0 4586.0 40 4 16186.0 40 14 144

Contact/1ngestion toxicity of AVMB^ on Chinese cabbage and cotton against third instar larvae of S.littoralis and H.armigera (see Tab!e 3.12).

Species Concentration n Number (diet) (ng/ml) respondi ng

S. 1i ttorali s (Cabbage) X larval weights'! 0-4 mg Control 40 6 4.5 40 7 9.0 40 21 13.5 40 31 27.0 .40 37 S.1i ttorali s ( Cotton ) X larval weight=5*2 mg Control 40 0 4.5 40 2 9.0 40 16 18.0 40 35 27.0 40 36 36.0 40 37 H.armigera (Cabbage) X larval weights*-* mg Control 40 6 0.54 40 12 1.08 40 . 24 2.16 40 27 5.4 40 37 14.04 40 39 Contact/ingestion toxicity of AVMB^ against first Instar larvae of S. 1ittoralis, H.armigera and H.virescens (see Table 3.13). Species (diet) Concentration n Number (jug/ml) respondi ng S.littoralis (Cabbage) Control 40 0 0.27 40 2 0.45 40 4 0.72 40 15 1.26 40 28 1.44 40 31 H.armigera (Cabbage) Control 48 7 0.18 48 19 0.36 48 33 0.54 48 46 0.72 48 47 S.littoralis (Cotton) Control 40 0 0.9 40 5 1.2 40 20 1.5 40 31 1.8 40 35 145

conti nued.... H.armigera (Cotton) Control 40 4 0.09 40 12 0.18 40 28 0.263 40 31 0.54 40 36 0.72 40 . 38 H.virescens (Cotton) Control 40 0 0 .0216 40 11 0 .0432 40 23 0 .0648 40 36 0 .0864 40 37 0 .1296 40 38 146

Appendix4-1 Bioactivity of TLC plate silica dust samples against J2 M.incognita .

Treatment (1) X undulations per minute (tissue) f ------* band on TLC pi ate 1 2 3 4 5 6 7 8 00 o o o CO CVJ • Nerve cord 0.6 0.4 0.8 - • 1.6 . o 00 • • o Muscle 1.0 1.6 0.4 1.2 0.4 0.6 ro . Control ±S.E. 3.4± o ro

- Nematodes lost in silica dust * Band 1 = Origin

(1) Insects injected with 2pl 18ppm [3H]AVMBi 147

Appendix 5«1 Cockroach saline (Yarom et aj, 1982).

NaCl (214mM) 12.51 gms. KC1 (3.ImM) 0.23 gms. CaCl2 (7.0mm) 1.03 gms. Tris buffer (1. Omm) 0.12 gms. The Ingredients were made up to 1 litre with distilled water and buffered to pH 7.2-7.4 with HC1. 148

Appendi x 5’2 Effect of AVMB^ 0.01 jj M on spontaneous activity within a desheathed (ventra1 nerve cord preparation of P.americana