ORGANOPHOSPHATE INHIBITION OF NEMATODE ESTERASES

AND INTERACTION WITH CHLORINATED HYDROCARBON

INSECTICIDES

By

DONALD S. CANNON

A Dissertation submitted to the

Graduate School-New Brunswick

Rutgers, The State University of New Jersey

in partial fulfillment of the requirements

for the degree of

Doctor of Philosophy

Graduate Program in Entomology

written under the direction of

Professor W.R. Jenkins

and approved by

W.R. Jenkins

Andrew Forgash

Dwight Taylor

New Brunswick, New Jersey

January 2009

ii

ABSTRACT OF THE DISSERTATION

Organophosphate Inhibition of Nematode Esterases and

Interaction with Chlorinated Hydrocarbon Insecticides

by Donald S. Cannon

Dissertation Director:

W.R. Jenkins

The discovery and subsequent development of a vast array of synthetic organic

pesticides has provided nematologists with a rich source of potential nematicides.

Certain types of insecticides, notably the organophosphates and more recently the

carbamates, have been of significant value in nematode control, whereas, the chlorinated

hydrocarbon insecticides are generally ineffective. The wide discrepancy in the chemical

structure-toxicity relationship between these two phytogenetically separate animals

serves to emphasize the need for greater understanding of nematode chemistry and

physiology. Development of a sophisticated nematicide technology requires greater

knowledge of nematode response to toxic and non-toxic chemicals.

In this study, investigations were carried out into nematode esterase inhibition by a

nematicidal organophosphate and the interactions with chlorinated hydrocarbon

insecticides. Phorate suppression of esterase activity and the interference by chlorinated

hydrocarbon insecticides were studied in whole homogenates of the free-living

nematodes Panagrellus redivivus and the plant parasitic species Ditylenchus dipsaci and

on their electrohporetically isolated enzymes.

iii

The NE enzymes of P. redivivus were somewhat more sensitive than cholinesterases

(ChE) to phorate with pI50 values (negative log of the molar concentration inhibiting

activity 50%) were 5.4 and 3.7 respectively. Greater resistance and more rapid recovery

from NE inhibition by the organophosphate was demonstrated in homogenates of D.

dipsaci. A comparison of the inhibition curves of both species indicates phorate to be

moderately toxic to NE activity in vitro, approximately one-tenth that of the standard

organophosphate paraoxon and slightly more inhibitory than the anti-ChE carbamate

eserine.

Seven of the eight esterases of P. redivivus and one of the three isolated from

homogenate of D. dipsaci were inhibited to varying degrees by phorate and the two

standard inhibitors. The pattern of relative degree of sensitivity among the esterases was

similar for the two organophosphates but differed with the carbamate eserine.

Homogenates pretreated with one of four chlorinated hydrocarbon insecticides,

chlordane, DDT, dieldrin, or lindane, reduced the antiesterase toxicity of 5 x 10-7M

concentration of phorate 26, 18, 20, and 12% (P. redivivus), respectively. Reduction of

inhibition by 10-3M concentration of chlordane increased inversely with phorate

concentration in homogenates of both species. A similar reduction, but to a higher

degree, was produced by pretreatment of homogenates with the microsomal stimulant

phenobarbitol. Both chlordane and phenobarbitol reduced esterase inhibition by the

carbamate nematicide aldicarb but had no effect on eserine toxicity. Electrophoretically

isolated esterases of P. redivivus were not protected from phorate inhibition by

pretreatment with chlordane.

iv

In an in vivo assay no reduction of phorate toxicity was found in P. redivivus cultured

in insecticide treated oatmeal or presoaked in an aqueous solution of the insecticide.

Chlorinated hydrocarbon insecticides appear to stimulate microsomal release of

aliesterases capable of hydrolyzing organophosphates in a manner similar to the

interaction phenomena occurring in rats and mice.

v

TABLE OF CONTENTS

Abstract ............................................................................................................................... ii

List of Tables .................................................................................................................... vii

List of Figures .................................................................................................................... ix

Introduction..........................................................................................................................1

Objectives ............................................................................................................................4

Literature Review.................................................................................................................5

Esterases.........................................................................................................................5

Physiological role...........................................................................................................5

Nematode esterases........................................................................................................6

Organophosphate inhibition...........................................................................................7

Methods and Materials.......................................................................................................11

Quantitative colorimetric analysis ...............................................................................12

Disc electrophoretic analysis .......................................................................................20

Chemical Materials ............................................................................................................25

Results and Discussion ......................................................................................................27

Factors influencing esterase activity............................................................................29

Inhibition of esterase activity.......................................................................................30

Esterase recovery from phorate inhibition...................................................................41

Inhibition studies on separated esterases .....................................................................42

Preparatory studies.......................................................................................................46

Inhibition of isolated esterases.....................................................................................46

Insecticide interaction with phorate .............................................................................49

vi

TABLE OF CONTENTS (continued)

Summary and Conclusions ................................................................................................61

Literature Cited ..................................................................................................................63

Curriculum Vita .................................................................................................................66

vii

LIST OF TABLES

Table 1. The wet, dry, and protein weights of Panagrellus redivivus and Ditylenchus dipsaci. Protein determinations are corrected for dilutions in homogenate preparation. Data are presented as the average of two measurements shown in parenthesis. .........................................................................................................28 Table 2. Esterase activity in homogenates of Panagrellus redivivus and Ditylenchus dipsaci measured as rate of substrate hydrolysis per mg of protein in thirty minutes .....................................................................................................................28 Table 3. Esterase activity in homogenates of Panagrellus redivivus and Ditylenchus dipsaci after storage at – 18oC for 0 to 14 days. ............................................33 Table 4. The influence of time on phorate inhibition of esterase activity in homogenages of Panagrellus redivivus. Substrate hydrolysis rates were measured after 0 – 30 minutes preassay incubation of a 5 x 10-4 M phorate treatment and untreated homogenates.......................................................................................................35 Table 5. Phorate, paraoxon, and eserine inhibition of non-specific esterase activyt in Panagrellus redivivus. Treated and control homogenates were preincubated 30 minutes at 28oC prior to assay of beta-naphthyl acetate hydrolysis. Each experiment was repeated three times. ...................................................37 Table 6. Phorate, paraoxon, and eserine inhibition of cholinesterase acticity in Panagrellus redivivus. Treated and control homogenates were preincubated 30 minutes at 28oC prior to assay of acetyl choline chloride hydrolysis. Each experiment was repeated 2 times. ......................................................................................39 Table 7. Phorate, paraoxon, and eserine inhibition of non-specific esterase activity in Ditylenchus dipsaci. Treated and control homogenates were preincubated 30 min at 28oC prior to assay of beta-naphthyl acetate hydrolysis. Each experiment was repeated 3 times. .............................................................................43 Table 8. The interaction of chlordane, DDT, dieldrin and lindane with the antiesterase activity of phorate. Homogenates of Panagrellus redivivus and Ditylenchus dipsaci were treated with 10-3 concentration of insecticide 15 minutes prior to 30 minutes incubation with 5 x 10-7M phorate. Controls consisted of insecticide and phorate treatments alone and untreated homogenate. ...............................51 Table 9. The influence of chlordane concentration and preincubation time on the reduction of phorate toxicity. Homogenates of Panagrellus redivivus were pretreated with various concentrations of chlordane and preincubated for various time periods prior to 30 minute inhibition treatment with 5 x 10-7M phorate. Phorate and phorate plus chlordane omitted controls were carried simultaneously through all treatment stages. ..............................................................................................52

viii

LIST OF TABLES (continued)

Table 10. Chlordane and phenobarbitol reduction of esterase inhibition relative to phorate concentration. Homogenates of Panagrellus redivivus and Ditylenchus dipsaci were preincubated for 5 minutes with 10-3M concentrations of the non-inhibitors prior to 30 minute treatment with phorate. Data presented are the average of the replicates shown in parenthesis. .................................................................54 Table 11. The influence of 15 minute homogenate pretreatment with 10-3M concentrations of chlordane or phenobarbitol on carbamate (aldicarb and eserine) inhibition of non-specific esterase activity in Panagrellus redivivus. ...............................56 Table 12. The influence of pretreatment with chlorinated hydrocarbon insecticides on subsequent response to phorate by intact Panagrellus redivivus. Nematodes held 24 hours in insecticide solutions (test A), or seven days in insecticide treated oatmeal culture (test B), were transferred to phorate solutions for 24 hours. .......................................................................................................................58

ix

LIST OF FIGURES

Figure 1. Standard curve for protein determination represented by egg albumin in the biuret assay method of Lowry (1958). One ml distilled water containing 1 to 10 mgs of the albumin were added to 7 ml of biuret reagent. After 30 minutes incubation at 37oC, the change in optical density at 555 mu was measured and plotted against mgs of albumin per ml...............................................................................14 Figure 2. The absorbance spectra for 8 ug of beta-naphthol after reaction with Fast blue B (curve A) according to the method of Gomori (1953) and the diasonium salt alone (curve B). Optical density values at each wave length were obtained against a water blank with a Beckman Model DB spectrophotometer. ..............16 Figure 3. Standard curve for beta-naphthol reaction with Fast blue B following the method of Gomori (953). The increase in optical density at various concentrations of buffered beta-naphthol was determined against a blank containing all reagents except the beta-naphthol. Both treatment and control were carried through all steps of the enzyme assay. Optical density was measured at 540 mu with a Beckman Model DB spectrophotometer....................................................18 Figure 4. Standard curve for residual acetycholine determination developed by the method of Augustinsson (19). One ml samples containing 1 to 4 u M of acetylcholine chloride were treated according to the procedure for enzyme assay. Absorbance was measured at 540 mu on a Bausch and Lom Spectornic 20. The spectrophotometer was adjusted to zero with a control sample containing buffer without ester carried through all assay steps......................................................................19 Figure 5. The relationship of temperature to non-specific esterase activity on homogenate of Panagrellus redivivus and Ditylenchus dipsaci. Substrate hydrolysis was measured colorimetrically after 30 minutes incubation with homogenate at 24, 30, 37 and 40oC. ..................................................................................31 Figure 6. The influenct of pH on non-specific esterase activity in homogenates of Panagrelus redivivus (a) and Ditylenchus dipsaci (b) cholinesterase activity (c) in the free-living species. Homogenate and substrate solutions adjusted to indicated pH (Nitrate-phosphate buffer to pH 6.5 and Trie-HCl to pH 9.0). ....................................32 Figure 7. The influence of substrate concentration on the esterase activity in homogenates of Panagrellus redivivus. The amount of substrate per ml was vried around the centration specified in the assay (x) where x = 70 ug beta naphthyl acetate per ml (dotted line) and 726 ug of acetylcholine chloride per ml (dashed line). ...................................................................................................................................34

x

LIST OF FIGURES (continued) Figure 8. Dosage effect curves for the phorate (p), paraoxon (px), and esterine (e) inhibition of non-specific esterase activity and homogenates of Panagrellus redivivus. Each point represents the mean of three replicates (Table 5). .........................38 Figure 9. Dosage effect curves for the phorate (p), paraoxon (px), and the eserine (e) inhibition of cholinesterase activity in homogenates of Panagrellus redivivus. Each point represents the mean of three replicates (Table 6). ...........................................40 Figure 10. Dosage effect curve for the phorate (p), paraoxon (px), and eserine (e) inhibition of non-specific esterase activity in homogenates of Ditylenchus dipsacii. Each point represents the mean of three replicates (Table 7). ...........................44 Figure 11. The recovery of non-specific esterase activity in homogenates of Panagrellus redivivus from inhibition by 5 x 10-7M phorate. The change in percent activity relative to a non-phorate control was measured at 15 minute intervals following 30 minute preincubation with the organophosphate...........................45 Figure 12. Anionic proteins isolated on acrylamide gels electrophoretically at pH 9.3 from homogenates of Panagrellus redivivus (gels a, b, and c) and Ditylenchus dipsaci( gels d, e, and f). Bands were visualized by immersion of developed gels in 0.1% amido Schwartz or 1.0% beta-naphthyl acetate and Fast Blue RR for protein (gels a, b, d and e) and esterase (gels c and f) staining respectively. ....................47 Figure 13. Diagramatic representation of the relative position and concentrations of protein and esterases bands isolated electrophoretically at pH 9.3 from homogenates of Panagrellus redivivus and Ditylenchus dipsaci. .....................................48 Figure 14. The inhibitory effect of phorate (T), paraoxon (P), and esterine (E) on anionic esterases isolated electrophoretically at pH 9.3 from homogenates of Panagrellus redivivus (top) and Ditylenchus dipsaci (bottom). Code letter designationof the treatment are followed by the negtive log of the inhibitor molar concns. (U=untreated control). ..........................................................................................50 Figure 15. The percent reduction in phorate inhibition of the non-specific esterase enzymes of Panagrellus redivivus (dashed line) and Ditylenchus (dotted line) following pretreatment with 10-3M concentration of chlordane (•) and phenobarbitol (x)................................................................................................................55

1

INTRODUCTION

The discovery and subsequent development of a vast array of synthetic organic

pesticides has provided nematologists with a rich source of potential nematicides.

Certain types of insecticides, notably the organophosphates and more recently the

carbamates, have been of significant value in nematode control, whereas, the chlorinated

hydrocarbon insecticides are generally ineffective. The wide discrepancy in the chemical

structure-toxicity relationship between these two phytogenetically separate animals

serves to emphasize the need for greater understanding of nematode chemistry and

physiology. Development of a sophisticated nematicide technology requires greater

knowledge of nematode response to toxic and non-toxic chemicals.

Several investigations, suggest that efficacy of a pesticide is probably governed by

one or more factors. While the nematode is anatomically a less complex organism, it

exposes a far greater surface area relative to its volume than most higher animals. The

nature of it’s cuticle, including composition and thickness may be of critical importance

in regulating the nature and extent of nematicide penetration. Hollis (1962) has

demonstrated the entrance of coal tar dyes through natural body openings of 28 genera of

plant parasitic and free-living nematodes. Corporal invasion of a toxicant may not be

necessary if interference with the normal function of surface exposed sensory organs or

of the body wall occurs. At subcellular levels the nature of the site under attack and the

ability to detoxify or withstand the effect of a poison are presumably significant.

The toxicity of those organophosphate insecticides which are currently finding wide

application in nematode control is mainly attributed to inhibition of cholinesterase (ChE)

type enzymes in insects. In a recent review, O’Brien (1968) concludes that

2

acetylcholinesterase (Ache) inhibition is of singular significance in the mode of action of

organophosphates in insects due mainly to the neurological importance and susceptibility

of this enzyme. The organophosphate inactivation of 10 of 13 insect non- ChE type

esterases by Cook (1963) demonstrates the broad target range of this chemical group.

The physiological role of most esterases in higher animals is unknown. Since even less is

known about nematode esterases, the significance of inhibition of any single enzyme in

this group is at best speculative.

Organophosphate inhibition of various esterases in intact nematodes has been

demonstrated by Rhode (1960) and Lee (1964). The AchE enzymes of Ascaris

lumbricoides appear to differ in activity and specificity according to Knowles and Casida

(1966). Furthermore, phosphorothionate and phosphorodithioate insecticides were not

converted in vivo to the more toxic oxygen analogs as in higher animals, thereby

suggesting unusual intracellular properties in this animal parasite. Spurr and Harvey

(1967) found atypical esterase activity in homogenates of Panagrellus redivivus. It

should therefore be of significant value to clarify the mode of action of organophosphates

in nematodes. While the initial approach to such a problem would be the investigation of

toxicological effects at subcellular levels, the examination of the net effects of pesticide

combinations should also be considered.

The chlorinated hydrocarbon insecticides are widely employed in soil insect control

in agricultural soils. According to Frear (1963), they may persist in the soil for many

years at insecticidal concentrations. Consequently soil-inhabiting nematodes may be

exposed to these non-nematicidal chemicals for several generations. Rats and mice

injected with chlordane, aldrin, or lindane insecticides have been found resistant to

3

organophosphate poisoning (O’Brien, 1968). A similar insecticide effect in nematodes

could result in severe interference with the use of organophosphate nematicides. It is

proposed, accordingly, to study the nature of nematode esterase inhibition by phorate,

O,O-diethyl-S-[(ethyl=thio) methyl] phosphorodithioate, and subsequently the influence

of chlorinated hydrocarbon insecticides on phorate toxicity.

4

OBJECTIVES

Relatively little attention has been given to the biochemistry and physiology of soil

inhabiting nematodes. Conventional analytical methods, often require greater numbers of

nematodes than can be provided by available rearing procedures. Panagrellus redivivus

(Linne) can be produced in adequate quantities on simple media for esterase type enzyme

studies. Although this species does not inhabit the soil, it does appear similar to soil

dwelling nematodes in its’ basic physiology and biochemistry (Lee, 1965). The

phytoparasitic species, Ditylenchus dipsaci Kuhn (Chitwood, 1938) can be reared in

sufficient amounts in the laboratory to permit a limited number of assays.

A major portion of the following investigations was conducted on the free-living

species with selected studies on the plant parasite for comparative purposes. The

following list outlines the objective of this thesis:

1. The quantitative effect of phorate on esterase activity in homogenized nematode

(homogenates) was measured.

a. Inhibition of non-specific esterase activity in P. redivivus and D. dipsaci was

compared.

b. Cholinesterase and non-specific esterase inhibition in P. redivivus was contrasted.

2. Phorate inhibition of esterases isolated electrophorectically on acrylamide gels was

examined.

a. Variation in susceptibility among the separated esterases was determined.

b. A comparison was made of the inhibition of esterases isolated from the two

nematode species.

3. Chlorinated hydrocarbon interaction with phorate inhibition was determined.

5

LITERATURE REVIEW

Esterases

Definition of Terms

Until recently, enzyme nomenclature was poorly organized and consequently many of

these proteins presently carry several trivial names. Much of the confusion in esterase

terminology results from the great variety of ester substrates susceptible to hydrolysis by

these enzymes.

In 1962 an international commission proposed specific rules for enzyme classification

and nomenclature (Dixon and Webb, 1964). Esterases were assigned to the class

hydrolase and subdivided according to their substrate preference and degree of sensitivity

to certain inhibitors. In this investigation the use of crude homogenates and simple

substrates precludes the specific indentification of the esterases involved. Trivial

nomenclature adopted here is similar to that employed in preceding nematological studies

of these enzymes. The non-specific esterase group (NE) includes those capable of

hydrolyzing beta napthyl acetate and contain various types known as aliesterases,

aromesterases, lipases and others. Those esterases which attack the substrate

acetylcholine such as acetylcholinesterase and pseudo-cholinesterase are grouped under

the term cholinesterase (ChE). Both NE and ChE enzymes are referred to collectively as

esterases.

Physiological role

Esterases appear to be ubiquitous among plants and animals, having been

demonstrated in single cell organisms as well as in the most highly evolved (Dixon and

Webb, 1964). Although these enzymes have been known for many years, their exact

6

physiological role and metabolic significance remains vague in most cases. They have

been implicated directly or indirectly in intermediary metabolism, cellular digestion, and

regulatory systems. Of the two major esterase groups NE and ChE, the former includes

several enzymes with varying but wide substrate specificity. Cells contain numerous

aromatic and aliphatic neutral esters susceptible to attack by NE enzymes. The ChE

enzymes are so named for their ability to hydrolyse choline esters but, like the NE group,

readily cleave other ester substrates. Acetylcholinesterase (AchE) has been studied

extensively and its role discussed by O’Brien (1967). In the central nervous system,

AchE is believed to hydrolyse protein bound acetylcholine to choline. The release of

choline has been shown to be a critical stop in nerve impulse transmission. Substrate

specificity of AchE is relatively high in contrast to other ChE enzymes.

Nematode esterases

The requirements for large amounts of tissue in earlier enzyme assay procedures has

restricted nematode esterase studies to the large animal-parasitic species. Recent

developments in nematode rearing methods and microanalytical techniques permit

investigation of the smaller free-living and plant-parasitic forms. A most extensive

exploration of these enzymes in Ascaris lumbricoides has been reported by Lee (1961)

who employed histochemical stains and specific inhibitors to indicate esterase types. NE

enzymes distribution was general, appearing in muscle cells, body and organ walls, and

throughout the nervous system. In contrast, ChE enzymes were restricted to the nervous

system and the region about the feeding mechanism. Studies of other animal-parasitic

species show similar distribution with the only major difference apparently related to

feeding habits. Thorson (1953) found large amounts of NE enzymes in the pharyngeal

7

glands and anterior lumen of the rat parasite which suggests function in extracorporeal

digestion. Esterase distribution in Dorylaimus keilini was compared by Lee (1962) with

the predatory Actinolaimus hintoni. High concentrations around the base of the jaws of

the predator is believed to cause paralysis and aid in predigestion of the prey. On the

other hand, the dorylaim nematode, which feeds on soluble cell contents, showed heavy

esterase straining in the intestine and only light staining about the stylet base.

Dissimilarity in esterase localization in Meloidogyne sp. was related by Bird (1965) to

sex and age. Esterases of the NS type were found only in the amphid region of larvae in

contrast to general distribution throughout the nervous system and sensory structures of

adults. Heavy staining of the amphids of mature females and around the spicule bases of

males suggests an association with sexual attraction. Acetylcholinesterase localization

was found by Rhode (1960) to be similar in five plant-parasitic nematodes representing

five genera. The presence of typical cholinergic nerve impulse transmission was

indicated by heavy staining of various parts of the nervous system and sensory structures.

Benton and Myers (1966) demonstrated a qualitative difference in esterase patterns of

two nematodes. Electrophoretic separation on acrylamide gels of proteins from

homogenate extracts showed four NE enzyme bands for P. redivivus which were distinct

from six bands produced with a Ditylenchus triformis extract. Esterases were partially

purified from P. redivivus homogenates by Spurr and Chancy (1966). Substrate

specificity tests indicated the presence of two ChE type enzymes and one esterase unable

to hydrolyze choline esters.

Organophosphate inhibition

Mode of action in insects

8

Inhibition of AchE in the central nervous system is generally accepted as the primary

toxic effect of insecticidal organophosphates (Casida, 1964; O’Brien, 1968). The

mechanism of toxicity is described as enzymatic hydrolysis of these insecticides in a

reaction analogous to the estrolytic cleavage of bound acetylcholine. The product of this

reaction is an inactive phosphorylated enzyme. Reaction rate and reversibility are

influenced by the nature of the groups attached to the phosphorus atom as well as other

physical and chemical properties. Studies with phosphorus labeled inhibitors such as

diisopropyl flourophosphate (DFP) indicated that the hydroxyl group of the seryl residue

in the enzyme is esterified with the phosphorous atom to produce an inactive DFP-AchE

complex. Weaker organophosphate insecticides react slowly to form a readily reversible

inhibitor-enzyme complex. Variation in insect ChE enzymes is believed to account for

differences in response to any one organophosphate among insect species; however, other

factors such as penetration, detoxification, and inhibition of NE enzymes could be

important. Among numerous other enzymes reported susceptible to inhibition by this

insecticide group are trypsin, aliesterase of rat liver, B-esterase and aliesterases of various

sera, and acetylesterases of plant. Since the esterase function in nematodes is even more

vague than in insects and, further, because of the wide range of substrates susceptible to

attack, the consequence of the inhibition of any particular enzyme must remain

speculative.

Inhibition of nematode esterases

Indirect evidence has accumulated to show that AchE inhibition by organophosphate

nematicides occurs in nematodes. The contorted spastic response, symptomatic of

organophosphate poisoning in higher animals, has been described by Rhode (1960) and

9

Cannon (1966) in nematodes exposed to phorate and zinophos. Detailed studies of the

effects of organophosphates on cholinesterases by Lee (1963) and Knowles and Casida

(1966) indicate dissimilarity with findings in higher animals. Neither Ascaris

lumbricoides nor Haemonchus contortus respond typically to known phosphate and

carbamate anticholinesterases. The former species failed to convert phorate to a highly

toxic oxygen analog, a metabolic phenomena common in insects, vertebrates, and plants.

Lack of greater toxicity in homogenate over topical treatments may also be related to the

absence of this oxidative mechanism. Non-specific esterases were generally less

sensitive to organophosphates than cholinesterases.

Phosphate and carbamate inhibition of esterases partially isolated by salt precipitation

of P. redivivus homogenates was studies by Spurr and Chancy (1967). Two ChE and one

NE enzyme were identified and inactivated by the organophosphate bidrin and the

carbamate aldicarb. The former was more toxic to homogenates than intact worms which

is in disagreement with the results obtained using Ascaris spp. by Knowles and Gasida

(1966).

Interaction with chlorinated hydrocarbon insecticides

The possibility of significant interaction between organophosphates and chlorinated

hydrocarbon insecticides has been considered by entomologists. In a review by Hewlett

(1960), certain combinations were more toxic to insects than expected. This reaction was

attributed to an interaction probably more fundamental than synergism. In contrast, Ball

(1954) has demonstrated reduction in parathion toxicity in rats pretreated with certain

chlorinated hydrocarbon insecticides. Subsequent investigation has established

interference with esterase inhibition by several potent organophosphates in mice and rats

10

pretreated with aldrin, chlordane, DDT, dieldrin or lindane. Although the mechanism in

vertebrates is not fully understood, it appears to involve microsomal stimulation with

concomitant increase in organophosphate degrading esterases. Welch and Coon (1963)

have induced parathion resistance in mice by oral administration of known microsomal

stimulants such as phenobarbitol. Qualitative changes in tissue esterases were also found.

11

METHODS AND MATERIALS

Tissue preparation

After consideration of numerous enzyme methods and tissue preprations, some

general recommendations of Bergemeyer (1965), Boyer et al. (1961), Dixon and Webb

(1964) and others were adopted. The use of whole homogenates in assays and

separations avoids major disadvantages commonly encountered in most protein

purification procedures, especially denaturation, and qualitative and quantitative loss of

enzymes, prosthetic groups, or cofactors. Simple substrates were preferred to produce

the greatest expression of non-specific esterase and cholinesterase activity. Since the

solubility and nature of nematode esterases is unknown, the use of specific substrates

may exclude the presence of enzymes important to inhibition studies. Homogenate

temperature was maintained at about 0o C during preparatory work and at at –18oC in

storage. Buffers were used in all assays and at low concentrations to avoid enzyme

injury.

D. dipsaci was cultured axenically on alfalfa callus tissue according to the method of

Krusberg (1961). Collection was facilitated by mixing media and tissue with an equal

volume of water in a Waring Blender at low speed for 30 seconds. Nematodes were

collected after passing through two layers of Kleenex tissue in a standard Baermann

funnel apparatus. The procedures of Benton and Myers (1966) were followed in culture

and collection of Panagrellus redivivus. Collection was followed by washing in 100 ml

of a 5 ppm aureomycin-1000 ppm streptomycin sulfate solution for 30 minutes on a

magnetic stirrer. The antimicrobial wash was removed by vacuum filtration on Whatman

no. 1 filter paper. Nematodes were immediately rinsed, while on the filtration apparatus,

12

with several hundred ml of 0.01M phosphate buffer at pH7.4. For all assays,

homogenates were prepared by disintegration in the above buffer on an M.S.E. ultrasonic

disintegrator at 18,000/20,000 c.p.s. for 20 minutes. Cholinesterase assays required

freshly prepared homogenates, however, the stability of the non-specific esterases

permitted use of samples stored at –18oC for several weeks. Two ml were added to 3 ml

screw cap vials and frozen.

Quantitative colorimetric analysis

Protein determination

The standard biuret method of Lowry (1958) was selected for quantitative

estimate of protein content. This colorimetric method is based on a coordination complex

formed by a copper atom and four nitrogen atoms, of two peptide chains and is

independent of protein character and other nitrogenous compounds. Protein

concentration of whole homogenates was estimated by comparison with a standard

crystalline egg albumin*. To 6 ml of biuret reagent* was added 2 to 10 mg of egg

albumin and the volume brought to a total of 8 ml with distilled water. After incubation

for 30 minutes in a 37oC water bath, particulate matter was removed by vacuum filtration

through a fine scintered glass funnel. The solutions were transferred to matched

colorimeter tubes and this color was measured at 555 mu on a Bausch and Lomb

Spectronic 20 photometer. A standard curve was prepared (Fig. 1) from the average

absorption values of at least two replicates. In subsequent tests with homogenates as

known amount of egg albumin was included as a control. *

* See reagent data list, page 36.

13

Non-specific esterase assay

The enzymatic liberation of free napthol which can readily complex with a diazonium

salt such as Fast blue B will form a soluble azo dye. Quantitative values for this reaction

can be determined photometrically. The method of Gomori (1953) was adopted and Fast

Blue B salt∗ was selected from among the numerous diazonium salts suggested by that

author. Substrate solution was freshly prepared by the addition of one ml of beta-

naphthyl acetate* stock solution (0.03M in acetone to 100 ml of 0.02M phosphate buffer,

pH 7.15. One ml of dilute homogenate was added to 5ml of substrate solution in a

reaction tube and mixed for about three seconds on a Vortex Jr. mixer (Scientific

Industries Inc., Springfield, Mass.). The tubes were immediately transferred to a 37oC

water bath and incubation proceeded for 30 minutes. Enzymatic activity was stopped by

the addition of 1 ml of diazonium salt solution and agitation on the Vortex Jr. mixer. The

dye solution was freshly prepared during the incubation stop by dissolving 80 mg of Fast

blue B in 22 ml of 3.5% sodium lauryl sulfate solution and filtration to remove solids.

In accordance with the recommendation of Gomori (1953), spectral scans of the

dye solution and the diazo beta-naphthol complex were completed to locate the optimal

wavelength of measurement in the NE activity assay. The procedure was identical with

that for the enzyme assay with the substitution of beta napthol for the acetate and buffer

solution replaced homogenate. From the absorption curves shown in Figure 2, 540 mu

was chosen as the most suitable wavelength for measurement of the diazotonization

reaction.

∗ See reagent data list, page 36

14

Figure 1. Standard curve for protein determination represented by egg albumin in the biuret assay method of Lowry (1958). One ml distilled water containing 1 to 10 mgs of the albumin were added to 7 ml of biuret reagent. After 30 minutes incubation at 37oC, the change in optical density at 555 mu was measured and plotted against mgs of albumin per ml.

15

A standard curve was constructed at 540 mm by plotting the optical densities for

known concentrations of beta napthol substituted for the acetate and in the absence of

homogenate. The absorption was found to be linear with beta-napthol concentration as

shown in Figure 3. The dye solution was freshly prepared during incubation by

dissolving 80 mg of Fast blue B salt in 22 ml of 3.5% sodium lauryl sulfate* solution

followed by filtration to remove solids. Denatured enzyme controls were included in

each experiment and prepared by gentle boiling of aliquots of homogenate diluted for

assay. The boiled homogenate was assayed for NE activity exactly as described for

untreated homogenate.

Cholinesterase assay

The reaction product of acetycholine and hydroxylamine hydrochloride can be

converted stoichometrically to the brown colored acetylhydroxamide-ferric complex.

Color development can be measured photometrically. The reduction in color formation

due to enzymatic hydrolysis of acetylcholine is a common means of measuring

cholinesterase activity.

* See reagent data list, page 36.

16

Figure 2. The absorbance spectra for 8 ug of beta-naphthol after reaction with Fast blue B (curve A) according to the method of Gomori (1953) and the diazonium salt alone (curve B). Optical density values at each wave length were obtained against a water blank with a Beckman Model DB spectrophotometer.

17

Several distinct advantages of the cholinesterase assay procedure of Augustinsson

(1965) led to its adoption. Although the accuracy is somewhat less than non-colorimetric

methods, it is adaptable to a wide range of experimental use and is suitable for crude

homogenate studies. Furthermore, it has been extensively employed in organophosphate

inhibition studies on these enzymes. Reagent* solution preparation and test procedures

were followed in accordance with Augustinssons’ direction. One-tenth ml of

homogenate solution was mixed on the Vortex Jr. mixer for 2-3 seconds with one ml

buffered substrate solution. Reaction tubes were placed in a 37oC water bath. The

addition of the alkaline hydroxylamine solution stopped enzyme activity at exactly 30

minutes. After centrifugation for 10 minutes at 2800x g, the clear supernatant was

decanted into matched colorimeter tubes. Optical density was measured at 540 mu on a

Bausch and Lomb Spectronic 20 photometer adjusted to 100% transmission with distilled

water.

An acetylcholine standard curve was prepared by the addition of 0.2 to 1.0 ml

aliquots of substrate solution plus buffer to give a 1.1 ml total volume. All the incubation

and color development steps were followed as described above. Absorption was found

linear with micrograms of acetylcholine chloride as shown in Figure 4.

The influence of inhibitors on esterase activity

Inhibition studies were conducted on homogenates by preassay exposure followed by

the non-specific esterase or cholinesterase colorimetric test as previously described.

* See reagent data list, page 36.

18

Figure 3. Standard curve for beta-naphthol reaction with Fast blue B following the method of Gomori (1953). The increase in optical density at various concentrations of buffered beta-naphthol was determined against a blank containing all reagents except the beta-naphthol. Both treatment and control were carried through all steps of the enzyme assay. Optical density was measured at 540 mu with a Beckman Model DB spectrophotometer.

19

Figure 4. Standard curve for residual acetycholine determination developed by the method of Augustinsson (19). One ml samples containing 1 to 4 u M of acetylcholine chloride were treated according to the procedure for enzyme assay. Absorbance was measured at 540 mu on a Bausch and Lomb Spectronic 20. The spectrophotometer was adjusted to zero with a control sample containing buffer without ester carried through all assay steps.

20

Inhibitor stock solution (1.0M in acetone) were prepared from which subsequent

serial dilutions were made with buffer solution. Homegenates were incubated with an

equal volume of dilute inhibitor solutions for 30 minutes at 0-5oC. The mixtures were

thoroughly intermixed by agitating 2-3 seconds on a Vortex Jr. mixer. At the completion

of inhibitor treatment, substrate solution was added and enzyme activity was measured as

described.

Insecticide interactions with phorate

Aliquots of whole homogenate were incubated 15 minutes at 0-5oC with 10-3M

solutions of one of four chlorinated hydrocarbon insecticides. The insecticides were

prepared as 10-1 M acetone stock solutions and diluted to 10-3 M concentration with

0.0001 M phosphate buffer, pH 7.4. Following chlorinated hydrocarbon treatment,

homogenates were incubated with inhibitors as described in the preceding paragraph and

subsequently analyzed for NE activity. Controls consisted of insecticide treatment

without inhibitor and homogenate alone. Buffer was substituted for each omission and

controls accompanied interaction experiments through the entire procedure.

Disc electrophoretic analysis

Instrumentation

The equipment employed in the subsequent esterase studies is shown in Figure 5.

Essential components include a power supply and a vertical electrophoresis unit. The

Simpson model Buchler D.C. power supply unit *Buchler Instruments Co., Inc., Fort

Lee, N.J.) delivers maxima of 1000 volts and 250 milliamps. Electrophoretic separations

were conducted in the Buchler Analytical Polyacrylamide Vertical Gel Electrophoresis

Apparatus #3-750 otherwise known as the “Polyanalyst”. Twelve samples may be

21

separated simultaneously in separate glass gel tubes 75 mm x 5 mm I.D. x 8mm O.D.

Sample capacity per tube is in the range of 10 to 100 ul. Acrylamide gel formation was

facilitated by use of the Buchler gel polymerization rack #3-1762 and fluorescent light

assembly #3-1764.

Procedure for electrophoretic separations

Uniform and reproducible protein separations were achieved with the ammonic gel

system described in the Buchler instruction manual which accompanies the

“Polyanalyst”. This diphasic acrylamide system has an upper stacking gel at pH 6.7 and

a lower separation gel at pH 9.1. The buffer system includes an upper Tris-glycine buffer

at pH 8.91 and lower Tris-HCl buffer at pH 8.07 with a running pH of 9.3.

Three modifications in technique were found to greatly facilitate gel preparation. The

bottom of the gel tubes were capped by stretching a small section of Parafilm “M”

(American Can Co. Neenah, Wis.) over the lower opening. The Parafilm “M” which

replaced the rubber caps provided by Buchler, produced a more even bubble-free surface

and was more readily removed. The overlayering of water, a critical step in the

prepolymerization of polymer reagent-loaded tubes, was accomplished more rapidly with

the wicked (threaded) dropping tube described by Clarke (1963). Gel removal from the

separation tubes must be done quickly following electrophoresis. The standard technique

employs a hypodermic needle or Pasteur pipette which must be worked between the gel

and the tube wall while water is forced through the orifice. This method is time

consuming and frequently results in torn gels. The use of a 6-inch piece of flexible

bronze-wound banjo wire (#7000 Mapes Piano String Co., New York, N.Y.) permitted

rapid and simple removal without injury to the gel. The wire is inserted between the gel

22

and tube wall while held under water and spun between the fingers down and around the

gel.

Gels were used within one-half hour of the final polymerization step. A 100 ul

syringe fitted with a no. 23 needle was used to deposit 10 to 75 ul of whole homogenate

on the upper gel. Upper buffer was layered onto the homogenate with the wicked

dropping pipette. The high specific gravity of the homogenate eliminated the need for

the density-increasing sucrose solution. Six mm glass beads were placed atop each filled

tube inserted in the electrophoretic apparatus.

Buffer solutions, chilled to 8oC were added along with a 1.0 ml of 0.001 methyl green

tracking dye mixed into the upper buffer. The power supply unit was activated about 15

minutes prior to electrophoresis. Current was adjusted to deliver 1.25 ma per tube until

the tracking dye passed through the stacking gel. Separation was accomplished at 2.50

ma per tube. Voltage was slightly variable between runs probably due to minor

differences in gel resistance. This effect could not be overcome despite rigorous attempts

at uniformity in gel preparation and electrophoretic procedure. Voltage differences also

occurred where particulated-free homogenate extracts were used thereby eliminating the

possibility of homogenate interference. Voltage remained relatively constant within each

run at about 450 V, however, an increase of 100 to 120 V occurred just prior to

completion of the separation. Electrophoresis was continued 60-70 minutes until the

tracking dye front was 1.0 cm from the bottom of the gel. Gels were immediately

removed from the tubes, washed in cold 0.01 M phosphate buffer pH 7.4, and subjected

to protein or esterase analysis. Electrophoresis was conducted at 8-10oC by immersion of

the lower buffer tank in an ice bath.

23

Protein and esterase staining with histochemical reagents.

Protein and esterase bands were assayed by the method of Benton and Myers (1966).

Immediately following electrophoretic separation, buffer-washed gels were transferred to

a petri dish filled with 0.1% Amido Schwartz black* in 7% acetic acid solution at 28oC.

After 60 minutes, treated gels were destained for 24 hours in several changes of 7%

acetic acid. Fast blue B and beta-naphthyl acetate were substituted for the dye and

substrate, which were employed in the esterase assay of Benton and Myers (1966). Gels

were incubated at 8oC in a solution containing 25 mgs of dye, 1.0 ml of .03 M substrate,

and sufficient 0.01 M phosphate buffer at pH 7.4 to bring the total volume to 50 ml.

Optimal band development was achieved in 15-20 minutes. Incubation beyond 20

minutes caused loss of resolution through diffusion of hydrolysed substrate into adjacent

bands. Enzymatic activity was stopped by immersion of the incubating gels in 7% acetic

acid fixative.

Inhibition of isolated esterases

The repressive effect of phorate, eserine, and paraoxon on the NE activity of

separated esterases was studied in situ on the freshly developed gels. The gels were

immersed in buffered inhibitor solution for 30 minutes at 8oC. Treated gels were rinsed

briefly in 8oC buffer prior to staining with Fast blue B. Interference of insecticide with

phorate inhibition was determined by preincuabting freshly developed gels for 15

minutes in buffered 10-3 M chlordane solution at 8oC prior to treatment with inhibitors.

In addition, whole homogenate was mixed with an equal volume of 2 x 10-3 M buffered

chlordane solution at 0-5oC for 25 hours prior to electrophoresis. The developed gels

were subsequently treated with inhibitor and assayed for NE activity. * See reagent data list, page 36.

24

Phorate-chlordane interaction in intact nematodes

Several thousand P. redivivus cultured in oatmeal media were collected by the

Baermann funnel technique and rinsed with several hundred ml of deionized water as

described earlier in the tissue preparation section. Populations of mixed age were rinsed

once with, and then transferred to, a 10-3 M chlordane solution (buffered at pH 7.4 with

0.001 M phosphate) for one to twenty-four hours at 25oC. One-half ml of buffered

phorate solution was added to an equal volume of chlordane solution containing

approximately 100 nematodes in a one-ounce vial. The vials were 20 mm in diameter

and, consequently, the one ml of solution layers in a thin film (1 to 2 mm depth) would

presumably permit adequate respiratory gas exchange. The vials were loosely capped

with aluminum foil and held at 25oC for the duration of the assay. A control treatment

where nematodes were pretreated with buffer solution was included for comparison. The

influence of the organophosphate on undulatory movement was measured and recorded.

Five ml of buffered chlorinated hydrocarbon insecticide solution and an equal volume

of buffer solution containing several thousand P. redivivus was thoroughly mixed into ten

grams of oatmeal (“Mothers Quick Oatmeal”, Quaker Oats Co., Minneapolis, Minn.)

containing a few mgs of yeast (“Fleischmann’s Active Dry Yeast”, Standard Brands, Inc.,

New York, N.Y.) in one ounce glass vials. The vials were lightly capped with aluminum

foil and held at 25oC. After seven days, nematodes were extracted from the culture

media by the Baermann funnel technique and washed as previously described. The

nematodes were then transferred to buffered phorate solutions and tested for resistance to

toxicant. A control treatment where the insecticide was omitted from the culture media

was included for comparison.

25

CHEMICAL MATERIALS

Special reagents:

Acetyl choline chloride – Nutritional Biochemicals Corp.

Amido Schwartz – Matheson, Coleman, and Bell

Beta-napthol – Eastman Kodak

Beta-napthyl acetate – Eastman Kodak

Beta-naphthyl butyrate – Eastman Kodak

5-Bronoindoxyl acetate – Nutritional Biochemical Corp.

Brom thymol blue – Aldrich Chemical

Egg albumin (crystallized 3x) – Fisher Scientific

Eserine sulfate (pure) – Fisher Scientific

Fast blue B – Dajac Laboratories

Fast blue RR – Dajac Laboratories

Phenobarbitol, sodium (pure) – donated by American Cyanamid Co.

Insecticides:

Aldicarb – 99% 2-methyl-2-(methylthis) propionaldehyde o-methylcarbamoyl oxime.

Union Carbide, N.Y.

Chlordane – (reference grade) 60% 1,2,4,5,6,7,8,8,-octachloro-2,3,3a,4,7,7a hexa hydro

4,7-methanoindene and 40% insecticidally active related compounds. City Chem.

Corp., N.Y.

DDT - p,p1-99% 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane (p, p1-isomer). City

Chem. Corp., N.Y.

26

Dieldrin – 99% recrystallized 1,2,3,4,10,10, hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-

octahydro-1,4-endo-exo-5,8-dimethanonaphthalene. Shell Chem. Co., N.Y.

Lindane – 99% 1,2,3,4,5,6-hexachlorocyclohexane (gamma isomer). City Chem. Corp.,

N.Y.

Paraoxon – 95% O,O-diethyl O-p-nitrophenyl phosphate. American Cyanamid Co.,

Princeton, N.J.

Phorate – 98% O,O-diethyl S-(ethylthis)ethyl phosphorodithioate. American Cyanamid

Co., Princeton, NJ.

27

RESULTS AND DISCUSSION

Homogenate analysis

The wet weight of intact nematodes was measured immediately prior to

homogenization and compared with the weight of homogenate samples oven-dried 20

hours at 75oC. Wet and dry weight values for each nematode species are given in Table 1

along with biuret analysis for protein content.

Esterase activity in freshly prepared homogenates of each nematode species is

represented in Table 2. Serial dilutions were made with 0.001 M phosphate buffer and

assayed immediately for NE activity. The homogenate dilution which produced

photometer readings in the 60 to 80 O.D. unit range in the NE assay was chosen for

subsequent enzyme studies. Dilutions of 1:1000 and 1:500 in the homogenates of

Panagrellus redivivus and Ditylenchus dipsaci, respectively, gave absorption values

consistently within the 60 to 80 O.D. unit range. Aromatic esterase activity in P.

redivivus was investigated by the substitution of beta-naphthyl benzoate for beta-naphthyl

acetate in the Gomori (1953) method. ChE studies were conducted on undiluted

homogenate assayed within one hour of preparation. Homogenate solutions boiled 15

minutes failed to show activity in either the NE or ChE assay.

28

Table 1. The wet, dry, and protein weights of Panagrellus redivivus and Ditylenchus dipsaci. Protein determinations are corrected for dilutions in homogenate preparation. Data are presented as the average of two measurements shown in parenthesis.

mgs per ml

P. redivivus D. dipsaci

Wet weight* 324.0 (310.5-337.5) 413.3 (410.0-416.7)

Dry weight 72.3 (69.8-74.8) 67.1 (65.0-69.2)

Protein** 26.4 (25.2-27.5) 17.6 (16.1-19.1)

* Wet weight determinations made on live worms which were subsequently homogenized. Aliquots of homogenate were removed for dry weight and protein determinations. ** Protein content was measured colorimetrically by the biuret method of Lowry (1958). Table 2. Esterase activity in homogenates of Panagrellus redivivus and Ditylenchus dipsaci measured as rate of substrate hydrolysis per mg of protein in thirty minutes. homogenate* Substrate hydrolysis** dilution B-napthyl B-napthyl Acetyl choline Acetate benzoate chloride P. redivivus none 2.8 uM 1:500 3.0 ug 1:1000 19.7 ug D. dipsaci 1:200 2.6 ug 1:500 7.4 ug * Dilutions made with 0.001 M phosphate buffer (pH7.4). ** Homogenate boiled 10 t0 15 minutes and included in each esterase assay produced optical density readings of less than 2 0.D. units which appear to be due to the slight colloidal appearance of boiled solutions.

29

The stability of esterase activity in homogenates stored at –18oC for up to fourteen

days is presented in Table 3. The NE enzymes of P. redivivus appeared reasonably stable

after two weeks, however, the ChE enzyme activity declined rapidly between 24 and 48

hours. For tests involving NE activity, 3 ml samples of freshly prepared homogenate

were stored in 3 ml screw-cap glass vials and frozen immediately. Once thawed, a

sample was used and the excess discarded. Studies on NE enzymes were limited to

homogenate frozen no more than eight days. The rapid loss of ChE activity necessitated

the use of fresh tissue preparations in inhibition studies with these enzymes.

Factors influencing esterase activity

Variables which could significantly affect enzyme activity were investigated.

Temperature response in the NE assay was relatively linear between 20oC and 40oC as

shown in Figure 5. The 37oC temperature specified in the Gomore (1953) assay appeared

to be suitable for measuring esterase activity. The influence of pH on NE activity was

determined by substituting 0.001M citrate-phosphate buffers from pH 5.5 to 7.5 and

0.001M phosphate buffers in the pH 8.0 to 9.0 range. Both homogenate and substrate

dilutions were freshly prepared with their respective pH buffer and absorption was

measured against a blank containing substrate and buffer adjusted to the specified pH.

Homogenates of both nematode species exhibited maximum NE activity in the pH range

of 6.5 to 7.5 as shown in Figure 6. The pH response of ChE type enzymes of P. redivivus

was also included because of the abnormal pH curve found by Spurr and Chancy (1966).

Citrate-phosphate and phosphate buffers were substituted for the homogenate and

substrate buffer specified in the Augustinsson (1965) assay. The failure to show a high

activity peak above pH 8.0, normally indicative of acetylcholinesterase activity, may

30

indicate a deviation from the pH curve of ChE type esterases in higher animals, according

to Dixon and Webb (1964). The effect of substrate concentration on esterase activity in

homogenates of P. redivivus is shown in Figure 7. The rate of hydrolysis increased non-

linear from one-half to twice the substrate concentration specified in both esterase assay

methods. The preceding studies provide reasonable assurance that with respect to

temperature, pH and substrate concentration, the Gomori (1953) and Augustinsson (1965)

methods are suitable for the investigation of esterases in nematode homogenates.

Inhibition of esterase activity

The toxicity of the nematocidal organophosphate phorate to the esterases of the two

nematode species was studied and compared with standard esterase inhibitors, paraoxon

and the cholinesterase-specific carbamate eserine.

Prior to the investigation of esterase inhibition it was necessary to determine the

influence of time on the rate of interaction between enzymes and inhibitor. Phorate

suppression of esterase activity in homogenate solutions of P. redivivus after

preincubation periods up to 30 minutes is presented in Table 4. Maximum reduction of

NE activity was reached between fifteen and twenty minutes. No further decrease in ChE

activity occurred after fifteen minutes exposure to the toxicant. A thirty minute

preincubation period was therefore judged adequate to provide maximum esterase

inhibition by phorate in P. redivivus homogenates and presumed satisfactory for similar

studies with D. dipsaci. Enzyme inhibition (I) is expressed as the percent decrease in

micrograms of substrate hydrolyzed by the treatment (T) as compared with the untreated

control (u) and can be represented by the formula: %I = (U-T)/T x 100 where U and T

are given in indicated units of substrate hydrolyzed.

31

Figure 5. The relationship of temperature to non-specific esterase activity on homogenate of Panagrellus redivivus and Ditylenchus dipsaci. Substrate hydrolysis was measured colorimetrically after 30 minutes incubation with homogenate at 24, 30, 37 and 40oC.

32

Figure 6. The influence of pH on non-specific esterase activity in homogenates of Panagrellus redivivus (a) and Ditylenchus dipsaci (b) cholinesterase activity (c) in the free-living species. Homogenate and substrate solutions adjusted to in indicated pH (Nitrate-phosphate buffer to pH 6.5 and Trie-HCI to pH 9.0).

33

Table 3. Esterase activity in homogenates* of Panagrellus redivivus and Ditylenchus dipsaci after storage at –18oC for 0 to 14 days. Substrate hydrolysis / minute / mg protein Acetlycholine chloride (uM) beta-naphthyl acetate (ug) Days 0 1 2 0 1 5 7 14 1:1000* P. redivivus 2.3 1.5 1.1 17.4 18.2 17.9 17 1:400* D. dipsaci 5.1 5.3 4.7 *Homogenate dilutions were made with 0.001 M phosphate buffer, pH 7.4

34

Figure 7. The influence of substrate concentration on the esterase activity in homogenates of Panagrellus redivivus. The amount of substrate per ml was varied around the centration specified in the assay (x) where x = 70 ug beta naphthyl acetate per ml (dotted line) and 726 ug of acetylcholine chloride per ml (dashed line).

35

Table 4. The influence of time on phorate inhibition of esterase activity in homogenates of Panagrellus redivivus. Substrate hydrolysis rates were measured after 0 to 30 minutes preassay incubation of a 5 x 10-4 M phorate treatment and untreated homogenates. Substrate % inhibition* with time 0 10 20 30 minutes beta-naphthyl acetate 40 69 70 70 acetylcholine chloride 35 43 40 41 * Inhibition is expressed as the percent differences in substrate hydrolysis between treatment and control, each preincubated 30 min at 28oC.

36

Quantitative analysis of the anti-esterase activity of the organophosphates and eserine

is presented in Tables 5, 6, and 7 and Figures 8, 9, and 10. The relative toxicity of the

three inhibitors against the NE enzymes in both nematode species is similar to findings

with other animals (Dixon and Webb, 1964). The pI50 values (negative log of inhibitor

molar concentration which reduces enzyme activity by 50%) for paraoxon, phorate, and

eserine treatment of P. redivivus estimated from Figure 8 are 6.3, 5.4, and 3.2,

respectively. The low toxicity of the carbamate is indicative of its selectivity towards the

ChE enzymes which, according to Sternberg and Hewitt (1962), hydrolyze beta-naphthyl

acetate slowly. Both organophosphates are inhibited significantly at low concentrations

with paraoxon about ten times more toxic. The wide concentration range of the

inhibition curves is probably the net effect due to differences in degree and nature of

inhibition, concentration, and substrate specificity of the various NE type enzymes

hydrolyzing beta-naphthyl acetate (Dixon and Webb 1964). Inhibition of ChE enzymes

occurred over a more narrow concentration range as shown in Table 6 and Figure 9

which is probably indicative of the greater selectivity of the acetylcholine chloride

substrate. Approximate pI50 values of 5.8, 3.9, and 3.7 for paraoxon, eserine, and phorate

respectively demonstrates a greater toxicity to these enzymes than the NE type by the

carbamate but not the organophosphates. Insect Ch enzymes are completely inhibited by

most organophosphate and carbamate insecticides and eserine within a 10-6M to 10-10M

concentration range (O’Brien, 1968). The ChE of P. redivivus appears significantly less

sensitive to both eserine and phorate in comparison with insects. This may be a genuine

resistance as suggested in histochemical studies by Lee (1963) where 10-6M eserine had

no effect on ChE activity in Haemonchus contortus. Phosphates such as paraoxon

37

Table 5. Phorate, paraoxon, and eserine inhibition of non-specific esterase activity* in Panagrellus redivivus. Treated and control homogenates were preincubated 30 minutes at 28oC prior to assay of beta-naphthyl acetate hydrolysis. Each experiment was repeated three times.

% inhibition pI** phorate paraoxon eserine 1 2 3 Mean 1 2 3 Mean 1 2 3 Mean 2.5 95 91 90 93 100 100 99 100 60 57 38 58 3.5 75 82 83 79 86 90 91 89 47 46 43 45 4.5 60 60 67 62 74 78 81 77 36 20 33 29 5.5 48 45 44 46 70 68 64 66 20 19 16 18 6.5 27 33 31 30 51 51 45 48 18 8 11 12 7.5 24 12 23 20 42 40 34 38 7 0 4 5 * Phorate, paraoxon, and eserine at 5 x10-4 M concn inhibited beta-naphthyl butyrate hydrolysis 46%, 58%, and 0% respectively. ** pI = negative log of the molar concn.

38

Figure 8. Dosage effect curves for the phorate (p), paraoxon (px), and esterine (e) inhibition of non-specific esterase activity in homogenates of Panagrellus redivivus. Each point represents the mean of three replicates (Table 5).

39

Table 6. Phorate, paraoxon, and eserine inhibition of cholinesterase activity in Panagrellus redivivus. Treated and control homogenates were preincubated 30 minutes at 28oC prior to assay of acetyl choline chloride hydrolysis. Each experiment was repeated 2 times. % inhibition pC* phorate paraoxon eserine

1 2 Mean 1 2 Mean 1 2 Mean 2.5 80 78 79 93 95 94 3.0 64 70 67 89 89 89 3.5 55 50 52 69 74 72 4.0 41 44 43 46 51 49 4.5 31 36 34 20 27 24 5.0 90 93 92 5.5 75 80 77 6.0 41 42 42 6.5 28 21 25 *pC = negative log of molar concn.

40

Figure 9. Dosage effect curves for the phorate (p), paraoxon (px), and the eserine (e) inhibition of cholinesterase activity in homogenates of Panagrellus redivivus. Each point represents the mean of three replicates (Table 6).

41

exhibited high anti-ChE in vivo against Ascaris lumbricoides, however, Knowles and

Casida (1966) found phorate and other phosphorodithioates relatively nontoxic when

injected in intact, or added to homogenized worms. The apparent resistance of the ChE

enzymes to certain organophosphates was attributed to the absence of metabolic

activation in A. lumbricoides. The in vivo oxidation of phosphorodithioates to more

potent ChE inhibitors by higher animals has been established (O’Brien, 1966), however,

Van Asparan (1962) reported the loss of this activation potential by homogenization of

insect tissue. It is therefore not possible to explain the great difference in anti-ChE

activity between paraoxon and phorate as relavent to activation of the latter by P.

redivivus. Van Asparan’s findings also raise the possibility of significantly greater

phorate toxicity to this nematode in vivo. The NE enzymes of D. dipsaci were more

resistant in a similar degree to all three toxicants (Table 7 and Figure 10). A comparison

of the inhibition curves in Figures 8 and 10 of the two species shows phorate significantly

less toxic than paraoxon and more toxic than eserine. The close parallel in nature and

displacement of the curves suggests no great qualitative difference in esterase response or

in organophosphate activation. If this were established the disparity in degree of

sensitivity could result from either a broad quantitative difference in estrolytic activity or

subcellular protective mechanism such as detoxification favoring the plant-parasitic

species.

Esterase recovery from phorate inhibition

The recovery potential of NE enzymes from phorate inactivation was demonstrated

by measuring the change with time of substrate hydrolysis. Homogenate treated with 5 x

10-5M phorate was compared with an untreated control, each preincubated for 30 min.

42

then assayed with substrate solution for 15 to 60 minutes. Percent activity as shown in

Figure 11 indicates the change in the rate of hydrolysis of beta naphthyl acetate in the

treated sample at 15 minute intervals. The relatively rapid recovery from phorate

inhibition shown here is a characteristic of the weaker organophosphates according to

Casida (1964). The metabolic activation phenomenon discussed earlier could lead to one

of several oxygen analogs of phorate which have been found in insects to form more

stable enzyme-inhibitor complexes. The inactivated enzyme ionizes at a slower rate

depending upon the number and location of oxidized molecular sites. D. dipsaci

esterases exhibit a somewhat higher rate of recovery and this may provide some

explanation of their relatively greater resistance than the NE enzymes of the free living

species.

Inhibition studies on separated esterases

A more qualitative investigation of the NE inhibition was undertaken to further

clarify the nature and significance of phorate anti-esterase toxicity in nematodes. Collins

(1964) demonstrated the use of acrylamide gel electrophoresis to study organophosphate

inhibition of separated insect esterases. Nematode esterases have been successfully

isolated by disc acrylamide gel electrophoresis and identified with histochemical reagents

by Benton and Myers (1966).

43

Table 7. Phorate, paraoxon, and eserine inhibition of non-specific esterase activity in Ditylenchus dipsaci. Treated and control homogenates were preincubated 30 min at 28oC prior to assay of beta-naphthyl acetate hydrolysis. Each experiment was repeated 3 times. % inhibition pC** phorate paraoxon eserine

1 2 3 Mean 1 2 3 Mean 1 2 3 Mean 2.5 79 76 75 77 88 85 84 86 49 45 45 46 3.5 69 65 63 66 76 70 71 72 38 35 34 36 4.5 57 55 49 54 62 58 59 60 29 27 25 27 5.5 41 41 43 42 51 49 46 49 18 9 7 11 6.5 24 26 29 26 43 34 35 37 0 5 2 3 7.5 23 15 24 21 31 29 25 28 0 0 0 0 * Phorate, paraoxon, and eserine at 5 x 10-4M concn inhibited beta-naphthyl butyrate hydrolysis 13%, 24%, and 0% respectively. ** pC = negative log of the molar concn.

44

Figure 10. Dosage effect curve for the phorate (p), paraoxon (px), and eserine (e) inhibition of non-specific esterase activity in homogenates of Ditylenchus dipsacii. Each point represents the mean of three replicates (Table 7).

45

Figure 11. The recovery of non-specific esterase activity in homogenates of Panagrellus redivivus from inhibition by 5 x 10-7M phorate. The change in percent activity relative to a non-phorate control was measured at 15 minute intervals following 30 minute preincubation with the organophosphate.

46

Preparatory studies

A preliminary examination of the effect of homogenate volume on electrophoretic

separation was necessary to demonstrate the maximum number of bands. Below 20

microliters both protein and esterase bands from homogenates of P. revivivus were weak

or absent while volumes in excess of 60 ul resulted in heavy staining and obliteration of

many bands. Optimal amounts of homogenate per electrophoretic tube were 50 ul for

protein and 75 ul for esterase tests with D. dipsaci and 20 and 40 ul respectively for

protein and enzyme studies with P. redivivus. A comparison of the protein-stained gels

in the photo, Figure 12 and diagrammatically represented in Figure 13, failed to establish

a definite correlation in electrophoretic mobility between protein and esterase bands for

either species. Cook (1963) also failed to match esterase and protein bands. Esterases

may occur in concentrations so low as to produce a weak protein stain reaction or the

difference in colorimetric methods may permit migration or diffusion. The possibility of

slight variations between gels developed simultaneously was eliminated when the halves

of a split gel stained for protein and esterase failed to show band correlation.

Inhibition of isolated esterases

The variation in sensitivity of the separated NE bands to phorate and the standard

inhibitors paraoxon and eserine is shown in the photo, Figure 14. Band Pl of P. redivivus

was totally resistant to all 3 inhibitors while the remaining esterases of both species

exhibited varying degrees of loss of activity. No qualitative difference is apparent

between the organophosphate treatments, however, the similarity in band intensity of 10-

4M phorate and 10-6 paraoxon treatments suggests close to 100 fold greater sensitivity of

the P. redivivus enzymes to the latter. The quantitative assay (Table 5) gave a phorate to

47

Figure 12. Anionic proteins isolated on acrylamide gels electrophoretically at pH 9.3 from homogenates of Panagrellus redivivus (gels a, b, and c) and Ditylenchus dipsaci (gels d, e, and f). Bands were visualized by immersion of developed gels in 0.1% amido Schwartz or 1.0% beta-naphthyl acetate and Fast Blue RR for protein (gels a, b,d, and e) and esterase (gels c and f) staining respectively.

48

Figure 13. Diagramatic representation of the relative position and concentrations of protein and esterases bands isolated electrophoretically at pH 9.3 from homogenates of Panagrellus redivivus and Ditylenchus dipsaci.

49

paraoxon toxicity ratio of about 1:20 based on estimated LD50 values which implies

suppression of phorate toxicity in homogenates. Direct comparisons of the results from

quantitative and qualitative studies are questionable, since in addition to the differences

in methods and materials the latter assay is limited to an unknown portion of the total NE

enzyme group. NE sensitivity towards the carbamate is demonstrated by reduced stain

intensity in bands P2 through P8. Comparison of the 10-6M eserine and 10-4M phorate

treatments demonstrates greater resistance of band P8 and higher sensitivity of P4 to the

carbamate. The toxicity of phorate, relative to eserine appears lower against

electrophoretically isolated NE enzymes, although this observation must be subjected to

the qualifications notes in the comparison with paraoxon.

Insecticide interaction with phorate

The reduction in the antiesterase activity of phorate following homogenate treatment

with four insecticides is demonstrated in Table 8. None of the control treatments which

included one percent acetone and each of the insecticides alone altered NE hydrolysis of

beta naphthyl acetate as measured colorimetrically by the method of Gomori (1953). The

cyclodiene chlordane induced the greatest suppression of phorate toxicity with

homogenates of both species and was therefore selected for a more definitive analysis of

the interaction phenomena in nematodes.

Ball and his coworkers (1954) found a four day delay following injection of

chlorinated hydrocarbon insecticides necessary to produce suppression of parathion

poisoning in rats. Neither extended preincubation time, nor reduced chlordane

concentration increased the NE resistance to 5 x 10-7M phorate as indicated by the data in

50

Figure 14. The inhibitory effect of phorate (T), paraoxon (P), and esterine (E) on anionic esterases isolated electrophoretically at pH 9.3 from homogenates of Panagrellus redivivus (top) and Ditylenchus dipsaci (bottom). Code letter designation of the treatment are followed by the negative log of the inhibitor molar concns (U=untreated control).

51

Table 8. The interaction of chlordane, DDT, dieldrin, and lindane with the antiesterase activity of phorate. Homogenates of Panagrellus redivivus and Ditylenchus dipsaci were treated with 10-3 concentration of insecticide 15 minutes prior to 30 minutes incubation with 5 x 10-7M phorate. Controls consisted of insecticide and phorate treatments alone and untreated homogenate. % inhibition Insecticide trial 1 2 3 4 avg. % reduction** Pretreatment* None 34 31 34 37 34 Chlordane 23 22 25 29 25 26 DDT 25 26 29 31 28 18 Dieldrin 24 26 29 31 27 20 Lindane 32 25 29 33 30 12 * Insecticides alone had no influence on NE activity. ** % reduction in % inhibition by insecticide pretreatment over control (homogenate preincubated with buffer solution alone prior to phorate treatment).

52

Table 9. The influence of chlordane concentration and preincubation time on the reduction of phorate toxicity. Homogenates of Panagrellus redivivus were pretreated with various concentrations of chlordane and preincubated for various time periods prior to 30 minute inhibition treatment with 5 x 10-7M phorate. Phorate and phorate plus chlordane omitted controls were carried simultaneously through all treatment stages. Chlordane % inhibition by phorate after indicated Concentration preincubation period* Time 0 15 min 30 min 3 hrs 24 hrs 96 hrs 0 29 34 31 28 24 ** 10-3M 23 19 25 20 16 10-4M 22 10-5M 19 10-6M 20 * Preincubation treatments beyond 30 minutes were held at 3-5oC in lightly capped test tubes. ** Only 37% of original NE activity remained after 96 hours.

53

Table 10. The interaction of 10-3M chlordane with phorate at varied concentrations is

shown in Table 9 and expressed as percent reduction of NE inhibition by

organophosphate. Sternberg and Hewitt (1962) suggest that at lower concentrations

enzymatic hydrolysis of organophosphates may reduce inhibition. The increased

resistance of chlordane treatments to lower phorate concentrations may be analogous to

increase in organophospholytic esterases. Triolo and Coon (1966) found an increase in

certain aliesterases paralleling an increase in microsomal activity and a decline in

parathion toxicity in rats treated with the cyclodiene insecticide aldrin. Pretreatment of

these animals with the microsome stimulant Phenobarbital produced similar effects with

other organophosphate produces similar effects with other organophosphate insecticides

and eserine. O’Brien (1968) concluded that certain chlorinated hydrocarbon insecticides

stimulate the release from microsomes of organophospholytic aliesterases in rats and

mice. Reduction of phorate anti-esterase activity in homogenates of P. redivivus

preincubated with phenobarbitol is shown in Table 10 and presented graphically in Figure

15.

In addition to organophosphates, many carbamate insecticides appear susceptible to

aliesterase hydrolysis in insects and higher animals. Temik inhibition of the esterases of

P. redivivus has been demonstrated by Spurr and Chancy (1966). The data illustrating

the influence of chlordane pretreatment of NE inhibition by Temik and eserine is

presented in Table 11. The chlordane induced resistance to the carbamate insecticide

gives further evidence of increased microsomal release of hydrolytic enzymes in

homogenates of nematodes. The lack of interaction with eserine is in agreement with the

earlier findings of Ball et al (1954). Dissimilar results with other chlorinated

54

Table 10. Chlordane and phenobarbitol reduction of esterase inhibition relative to phorate concentration. Homogenates of Panagrellus redivivus and Ditylenchus dipsaci were preincubated for 15 minutes with 10-3M concentrations of the non-inhibitors prior to 30 minute treatment with phorate. Data presented are the average of the replicates shown in parenthesis.

% reduction in phorate inhibition* chlordane phenobarbitol phorate pI P. redivivus D. dipsaci P. redivivus 2.5 0 2(0,2) 0 3.5 1.0 (0,1) 0 12.0 (5, 19) 4.5 6.5 (4,9) 8.0 (3, 13) 32.0 (27, 37) 5.5 9.5 (7, 12) 14.5 (10, 17) 34.5 (31, 38) 6.5 24.0 (20, 28) 21.0 (20, 22) 39.5 (36, 43) 7.5 43.5 (42, 45) 32.5 (29, 36) 74.0 (62, 86) * Percent reduction in % inhibition by insecticide pretreatment over control (homogenate preincubated with buffer solution alone prior to phorate treatment). Neither chlordane nor phenobarbitol alone affected esterase activity.

55

Figure 15. The percent reduction in phorate inhibition of the non-specific esterase enzymes of Panagrellus redivivus (dashed line) and Ditylenchus dipsaci (dotted line) following pretreatment with 10-3M concentration of chlordane (•) and phenobarbitol (x).

56

Table 11. The influence of 15 minute homogenate pretreatment with 10-3M concentrations of chlordane or phenobarbitol on carbamate (aldicarb and eserine) inhibition of non-specific esterase activity in Panagrellus redivivus.

% inhibition following indicated pretreatment

none chlordane phenobarbitol

Inhibitor pI 1 2 1 2 1 2 Aldicarb 2.5 77 69 79 69 38 60 3.5 43 33 38 27 0 2 4.5 12 19 5 4 0 0 eserine 3.5 52 48 53 4.5 21 20 24 5.5 11 15 10

57

hydrocarbon and antiesterase combinations have led Triolo and Coon (1966) to suggest

variability in response among species or in pesticide combinations.

Chlordane treatment of homogenate prior to electrophoresis or of gels after separation

had no influence on NE sensitivity to 5 x 10-4M and 5 x 10-5M phorate. Direct visual

comparison with control gels which were carried simultaneously through all the treatment

steps without chlordane showed no difference in number, position, or band intensity in

response to phorate. The lack of evidence of interaction in the absence of homogenate

constituents is in agreement with the proposed mechanism of microsomal stimulation.

Efforts to demonstrate chlordane interference with phorate toxicity in intact

nematodes were unsuccessful. The response of mixed population of the free-living

species to phorate following treatment with chlordane is shown in Table 12. The

distorted body conformation and spastic locomotory response, characteristic of

organophosphate poisoning, was found to be a better criterion of toxicity since

immobility failed to occur after twenty-four hours in 5 x 10-3M phorate solution. No

change in locomotor response to phorate was observed after twenty-four hours exposure

in chlordane solution or seven day culture on chlordane treated oatmeal. Failure to

demonstrate chlordane-induced resistance to organophosphate poisoning in intact

nematodes cannot be accepted as evidence of non-interaction without reservations. It has

not been established that normal absorption or intake by nematodes will occur in

acqueous solutions. Myers (1967) has described significant morphological and internal

salt changes in P. redivivus in response to variation in the toxicity of external

environment which probably produces a physiologically atypical nematode. Yeast cells,

58

Table 12. The influence of pretreatment with chlorinated hydrocarbon insecticides on subsequent response to phorate by intact Panagrellus redivivus. Nematodes held 24 hours in insecticide solutions (test A), or seven days in insecticide treated oatmeal culture (test B), were transferred to phorate solutions for 24 hours.

Estimated % abnormal motility* Insecticide test A test B Treatment 0 25 50 ppm phorate 0 25 50 ppm phorate none 5 60 90 5 50 95 10-4M chlordane 10 60 95 5 60 95 10-4M DDT 5 70 95 5 60 100 10-4M dieldrin 5 70 90 5 60 95

* Complete loss of movement failed to occur in 24 hours. Abnormal serpentine motility was employed as evidence of phorate poisoning. Approximately 100 nematodes from each of two treatment replicates were exposed to thin films of phorate solution for 24 hours at 28°C.

59

required in the P. redivivus oatmeal culture method of culture, may have interfered with

chlordane absorption by the nematode or chemical stability.

Profound consequences are implied in the use of chlorinated hydrocarbon insecticides

where plant parasitic nematodes threaten crop production unless it can be shown that the

type of interaction demonstrated here does not occur in intact nematodes in the soil.

Chlordane, DDT, dieldrin, and lindane commonly employed in soil insect control

programs, may persist in the soil for many years and have no significant nematicidal

value. Many of the new nematicides are in the organophosphate and carbamate class and

interference in their effectiveness by soil insecticides may lead to failure heretofore

attributed to other factors such as resistance, soil conditions, or method of application.

Although resistance to nematicides has not been proven, it is likely that such a

phenomenon can occur in nematodes. A protective effect by chlorinated hydrocarbon

insecticides could conceivably accelerate resistance to organophosphates and carbamates.

Selection of candidate nematicides or the design of analogs highly resistant to aliesterase

hydrolysis may become necessary.

Undoubtedly there are numerous overlapping positive and negative effects as well as

interactions between agricultural chemicals employed in crop protection. Dieldrin has

been implicated by Chen et al (1962) in the selective destruction of soil organisms

leading to large increases in plant parasitic nematode populations. Van Gundy (1965)

found four saprophagous nematode species susceptible to carbamate herbicide while five

plant parasites were unaffected.

Although Roa and Prasted (1968) relate the higher susceptibility of rhabditids to

organophosphates to greater cuticular permeability, Haig (1966) found this difference

60

disappeared when D. dipsaci L4 larvae were exposed to these nematicides in the presence

of fungi. According to Alexander (1965), soil microorganisms may readily absorb,

chemically modify, or succumb to many of our conventional pesticides. The reduction in

non-plant parasitic nematodes may therefore be a consequence of the destruction of the

food supply or ingestion of contaminated microorganisms. Furthermore, microbial

activation or destruction of nematicides may be of greater importance than heretofore

realized. There are numerous other inadequately explored interrelationships between

nematodes and the soil flora and fauna with respect to predation, parasitism, nematode-

host orientation, and the soil environment in general. The development and utilization of

nematicidal chemicals therefore will require, in addition to elucidation of direct effects on

the nematode, an understanding of the interrelationships involving other organisms and

pesticides. Perhaps the increased coordination and investigation by agricultural workers

from various disciplines in the currently developing integrated control concept will

accelerate the development of chemical control of nematodes.

61

SUMMARY AND CONCLUSIONS

Investigations were carried out into nematode esterase inhibition by a nematicidal

organophosphate and interaction with chlorinated hydrocarbon insecticides. Phorate

suppression of esterase activity and the interference by chlorindated hydrocarbon

insecticides were studies in whole homogenates of the free-living nematodes Panagrellus

redivivus and the plant parasitic species Ditylenchus dipsaci and on their

electrohporetically isolated enzymes.

Beta-naphthyl acetate hydrolysis was employed as a measure of NE activity and

quantitated spectrophotometrically by the color reaction of beta naphthol with the diazo

dye Fast Blue B. The NE enzymes of P. redivivus were somewhat more sensitive than

cholinesterases (ChE) to phorate with pI50 values (negative log of the molar concentration

inhibiting activity 50%) were 5.4 and 3.7 respectively. Greater resistance and more rapid

recovery from NE inhibition by the organophosphate was demonstrated in homogenates

of D. dipsaci. A comparison of the inhibition curves of both species indicates phorate to

be moderately toxic to NE activity in vitro, approximately one-tenth that of the standard

organophosphate paraoxon and slightly more inhibitory than the anti-ChE carbamate

eserine.

Anionic proteins were separated from homogenates by disc acrylamide

electrophoresis. Isolated NE bands were identified by immersing gels in a buffered

solution of beta-naphthyl acetate and a diaz dye until optimal resolution was achieved.

Seven of the eight esterases of P. redivivus and one of the three isolated from

homogenate of D. dipsaci were inhibited to varying degrees by phorate and the two

62

standard inhibitors. The pattern of relative degree of sensitivity among the esterases was

similar for the two organophosphates but differed with the carbamate eserine.

Homogenates pretreated with one of four chlorinated hydrocarbon insecticides,

chlordane, DDT, dieldrin, or lindane, reduced the antiesterase toxicity of 5 x 10-7M

concentration of phorate 26, 18, 20, and 12% (P. redivivus), respectively. Reduction of

inhibition by 10-3M concentration of chlordane increased inversely with phorate

concentration in homogenates of both species. A similar reduction, but to a higher

degree, was produced by pretreatment of homogenates with the microsomal stimulant

phenobarbitol. Both chlordane and phenobarbitol reduced esterase inhibition by the

carbamate nematicide aldicarb but had no effect on eserine toxicity. Electrophoretically

isolated esterases of P. redivivus were not protected from phorate inhibition by

pretreatment with chlordane.

In an in vivo assay no reduction of phorate toxicity was found in P. redivivus cultured

in insecticide treated oatmeal or presoaked in an aqueous solution of the insecticide.

Chlorinated hydrocarbon insecticides appear to stimulate microsomal release of

aliesterases capable of hydrolyzing organophosphates in a manner similar to the

interaction phenomena occurring in rats and mice.

63

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66

Curriculum Vitae Donald S. Cannon

EDUCATION Doctoral Studies in Plant Nematology, 1967-1969 Rutgers University, New Brunswick, NJ

Masters of Science in Plant Nematology, 1966 Rutgers University, New Brunswick, NJ

Bachelor of Science in Entomology, 1953 University of Maine, Orono, ME Undergraduate Courses in Forestry Management, 1949 Colorado A&M, Fort Collins, CO

EMPLOYMENT American Cyanamid, Stamford, CT 1953 – 1961 Princeton, NJ 1961 – 1969 Research Scientist, Entomology and Nematology Union Chemical Company, Hope, ME 1969 – 1974 Vice President, Research and Development