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1982 Toxicity and acetylcholinesterase inhibition by carbofuran and terbufos insecticides on Diabrotica species (Insecta: Coleoptera: Chrysomelidae) Barbara Ann Solheim Iowa State University

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Recommended Citation Solheim, Barbara Ann, "Toxicity and acetylcholinesterase inhibition by carbofuran and terbufos insecticides on Diabrotica species (Insecta: Coleoptera: Chrysomelidae) " (1982). Retrospective Theses and Dissertations. 7542. https://lib.dr.iastate.edu/rtd/7542

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Universi^ Mcrdfilms Intemadonal 300 N. Zeeb Road Ann Arbor, Ml 48106

8224334

Solheim, Barbara Ann

TOXICITY AND ACETYLCHOLINESTERASE INHIBITION BY CARBOFURAN AND TERBUFOS INSECTICIDES ON DIABROTICA SPECIES (INSECTA:COLEOPTERA:CHRYSOMELIDAE)

Iowa State University PhlD. 1982

University Microfilms lnt0rnstiona.i 300 N.Zeeb Rœd. Ann Arbor. MI 48106

Toxicity and acetylcholinesterase inhibition by

carbofuran and terbufos insecticides on Diabrotica

species (Insecta:Coleoptera:Chrysomelidae)

by

Barbara Ann Solheim

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of the

Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Department: Entomology

Major: Entomology (Insecticide Toxicology)

Approved;

Signature was redacted for privacy.

In Charge of Major Work

Signature was redacted for privacy.

For the Major Department

Signature was redacted for privacy.

For the Graduate College

Iowa State University Ames, Iowa

1982 ii

TABLE OF CONTENTS

Page

INTRODUCTION 1

LITERATURE REVIEW 3

Corn Rootworm History and Control Measures 3

Acetylcholinesterase Characteristics and Inhibition 13

Carbofuran and Terbufos 28

MATERIALS AND METHODS 31

Com Rootworms 31

Bioassay Techniques 32

Acetylcholinesterase Assay Techniques 33

Protein Assay 35

RESULTS 36

Dosage-mortality Data 36

Acetylcholinesterase Characteristics 37

Inhibition Studies 53

DISCUSSION 67

REFERENCES CITED 75

ACKNOWLEDGMENTS 86

APPENDIX A: LD^^ VALUES OF INSECTICIDES ON DIABROTICA SPECIES 87

APPENDIX B: CHEMICAL STRUCTURES OF CARBOFURAN, TERBUFOS, AND

ANALOGUES 93

APPENDIX C: BIOASSAY PROCEDURES FOR DIABROTICA SPECIES 95

APPENDIX D: LD^^ VALUES OF CARBOFURAN, TERBUFOS, AND ANALOGUES

OF EACH ON DIABROTICA SPECIES 98 1

INTRODUCTION

In 1956, Sir Vincent E. Wigglesworth made the following statements.

"The question is whether the research structure is really sound. The farmer's needs are wholly practical: he wants improvements in applied entomology; and the agricultural entomologist, like every other scientist who serves some economic purpose, is always being urged to keep his feet firmly on the ground and to stick to down-to-earth problems. But the fact is that there is often very little useful research to do that is of immediate practical importance. At the present time such a variety of new insecticidal chemicals have come upon the market that there is a good deal to be done in the way of testing their use under field conditions. But work of this kind is soon accomplished; it took only a very few years before the usefulness of DDT had been gauged for most agricultural purposes. Whereas, the chemical and toxicological work which lies behind such practical tests is almost unlimited." (Wigglesworth, 1976)

"All these modem synthetic insecticides have been discovered empirically in the course of testing long series of chemicals. But once found it is evident that they must intervene in some very special way with the chemical metabolism of the insect. If we had sufficient knowledge of insect biochemistry, it should have been possible to predict the activity of these particular substances; and given sufficient knowledge we should be able to devise molecules which will disrupt the machine at whatever point we desire. Actually, it looks at the present time as though the boot will be on the other leg, and that it is the study of these insecticidal chemicals which will reveal how the physiology of the insect proceeds." (Wigglesworth, 1956)

The "boot is still on the other leg" 26 years later. Man, his goods, and crops are still protected from the ravages of insects by chemical means; yet many details of the control process are still not known.

At a recent conference on the neurobiological aspects of insecti­ cidal action, it was apparent that huge gaps remain in the fundamental knowledge of the structure and function of the insect nervous system

(Elliott, 1980). A major effort is needed to find out more about the 2 mode of action of currently used chemicals and the resistance factors that may develop.

Current pest management stratagems support the use of insecti­ cides only when necessary and at the lowest dosages needed tc control the pest. This dissertation supplies base-line data on the effects of the insecticides carbofuran and terbufos on the three species of com rootworms damaging com in the United States. Specifically, this work looks at the relationships of dosage-mortality, dosage-acetylcho- linesterase (AChE) inhibition, AChE inhibition-mortality, and the interaction of these three concepts. 3

LITERATURE REVIEW

Corn Rootworm History and Control Measures

Say (1824) on an expedition to the Rocky Mountains collected speci­ mens of a pale greenish beetle which he described and named Galleruca longicomis. The classification of these beetles was to remain in the genus Galleruca (Galeruca) into the 1850s (Melsheimer, 1853). The genus association was then changed to the current Diabrotica (LeConte,

1859; LeConte, 1865). In 1967, the species was divided into two sub­ species, Diabrotica longicomis longicomis (Say) and Diabrotica longi­ comis barberi Smith and Lawrence (Smith and Lawrence, 1967). D. barberi being the subspecies found as a pest on com in the Midwest. In

1880, Riley (1880) wrote of the damage to com roots in Missouri by a larva that had a "general resemblance to the known larvae of ]0. vittata".

It was suspected to be the immature stage of longicomis barberi, the northern com rootworm (NCR), "on account of the frequency with which this pretty, greenish species was found in com fields". The suspicion was later confirmed when adults were reared from larvae collected from the field. Suggested controls included rotation of crops, the destruction of ragweed, and the application of lime and ashes around the young com

(Riley, 1880).

LeConte (1868), while on a survey for the extension of the Union

Pacific Railway from Kansas to New Mexico, collected two specimens feeding on wild gourd- From these insects, the description of the westem com 4

rootworm (WCR), Diabrotica virgifera, was made. Krysan et al. (1980)

described two subspecies of virgifera, virgifera virgifera (WCR) and D. virgifera zeae (Mexican com rootworm). This new nomenclature for the WCR was officially accepted by the Entomological Society of

America in 1980 (Sutherland, 1980). The earliest published report of com damage by the WCR was in 1912 when Gillette (1912) wrote of sweet com in Loveland and Fort Collins, Colorado, that "was being killed by a little grub boring in the roots". From his observations, he concluded that the insect was monovoltine and that its characteristics were much like longicornis longicornis. As to a method to control the damage caused by the WCR, Gillette suggested "not to grow com after com where this insect has been at all common the preceding year".

Fabricius in 1775 is given credit for the first description of the southern com rootworm (SCR), also known as the 12-spotted cucumber beetle. He named this 12-spotted beetle Chrysomela duodecimpunctata

(Barber, 1947). Olivier (1808) classified it as Galeruca duodecim­ punctata. Hom (1886) reviewed the species described by Olivier and equated jG. duodecim-punctata with D. duodecim-punctata. In 1947, taxonomic procedure prompted Barber (1947) to write the following:

"Taxonomic trouble also involves our supposedly best known species, the 12-spotted cucumber beetle. An asparagus beetle, now in Crioceris, was named Chrysomela duodecimpunctata by Linnaeus, 1758, and when Fabricius, 1775, applied the same name to a different species it was a primary homonym. Source of the Fabrician type is unknown, as is also its identity with our pest. The Code forbids use of the homonym. So the paradoxical course of describing the form we have called Diabrotica duodecim­ punctata as if it were new seems necessary. It often demanded the attention of our good Chief, who built our Bureau, and it seems proper it should be named for one whom we so greatly loved." 5

Thus, Chrysomela duodecimpunctata F. 0. duodecimpunctata, auct.) became

2"- undecimpunctata howardi Barber; named for Dr. L. 0. Howard. The first substantiated report of SCR larvae damaging com is found in the annual report for 1887 of the entomologist of the National Department of

Agriculture; written by Webster (Carman, 1891a; Riley, 1891). He stated that damage differed from that of the NCR and the WCR in that the SCR did not appear to attack the fibrous roots or bury themselves in longitudinal channels excavated in the larger roots, rather, they burrowed directly into the plants at or near the upper whorl of roots which almost invariably resulted in the death of the plant (Carman, 1891a).

Corn rootworms have been cited as the most important insect pest of field corn in the Midwest (Chiang, 1978). Damage was first noted in the Colorado-New Mexico region. Expansion of this range was initially slow as the WCR was not commonly found in southern Nebraska and northern

Kansas until the late 1940s (Bryson et al., 1953). Movement from

Nebraska was north to South Dakota, east into Iowa, and southeast into

Missouri. The first beetles were found in northwestern Illinois in

1964 and within four years, 13 counties had economically damaging populations. The first WCR beetles were discovered in Indiana in 1968 and 1969 (Gould, 1971). The NCR and WCR are now found throughout the

Com Belt States (Meyer, 1979). The third species, the SCR, is not of economic importance on corn in the Midwest because it can not overwinter in this region. The SCR is the most damaging species in the southern growing area where continuous generations can occur.

Larval damage consists of root feeding and tunneling which may 6

totally destroy both the main and brace roots of the plant when larval

infestations are high. Root feeding can destroy the nutrient and water

uptake capabilities of the plant causing a loss of vigor in the plant

which results in smaller plants and lower yields. The destruction of

the main and brace roots also predisposes the plant to wind damage in

the form of "goose-necking" and lodging. These aberrations of normal

plant posture make the harvesting of the crop difficult. In the South,

the SCR also bores into the stalk just above the roots and may eat the

crown of a young plant, kill the bud, and thus destroy the plant. As

much as 25% of a com stand may be destroyed by this type of feeding

when the plants are six to 18 inches high (Pfadt, 1971).

Adult beetles feed on all plant parts above the ground and are

not considered serious pests unless large numbers appear in late-

planted com before pollination is complete. Their clipping of the

ear silks does not allow complete pollination to occur and sparsely

filled ears result.

In Iowa, NCRs and WCRs have a similar life cycle. Egg laying

starts about one week after the adult beetles emerge with peak

laying occurring about August 10 to 20. The eggs are laid in the

top few inches of soil. Normally, the beetles crawl into insect or worm holes or drought cracks at or near the base of com plants to lay their eggs. The egg stage is the overwintering stage. Eggs hatch in June. Larvae feed on com roots if they are available.

Feeding occurs for two to three weeks during June and July. At the end of the third stage, the larvae form pupation cells in the soil. Adults emerge from mid-July through September. Peak emergence occurs

in early August. Mating occurs shortly after emergence and the cycle starts again (Stockdale et al., 1979; Stockdale and DeWitt, 1980).

The SCR does not overwinter in Iowa as the eggs do not survive the low temperatures. Adults migrate into Iowa from southern states in late May and June. These adults may also lay eggs in com fields.

Not enough eggs are laid in any one field from these immigrant beetles to cause economic damage. However, they may add to the damage done by the other two species (Stockdale and DeWitt, 1980).

Extensive research has been conducted on the biology and economics of corn rootworms. Chiang (1973) has reviewed the bionomics of the

NCR and WCR. An excellent bibliography (Luckmann et al., 1974) and an update (Irwin, 1977) covering the literature through 1976 are available

This literature review will not be a repetition of that material; rather, it will be concerned with the more recent developments affecting com rootworm control practices and mode-of-action studies of anticho­ linesterase pesticides.

Good cultural practices have long been known to keep com rootworm populations below economically damaging levels. Crop rotation effec­ tively controls larval damage by the NCR and WCR (Riley, 1880; Gillette,

1912; Hill et al., 1948; Weekman, 1961; Chiang, 1973). Damage caused by the SCR may be reduced by timely planting, clean cultivation, destruction of weedy field margins, and crop rotation (Hill et al.,

1948).

Following World War II, research on chemical control of insect 8

pests rapidly expanded. Fulton (1946) applied DDT or benzene hexa-

chloride (BHC) during fertilization or the seeding operation to test

plots and found promising results for the practical protection of

young corn from the SCR. Hill et al. (1948) demonstrated economic

control of corn rootworm larvae using the gamma isomer of BHC sprayed

on the soil before plowing or as a side-dressing prior to the first

cultivation. Larval populations were reduced 96.4% by the preplowing

application method and 77.4% by side-dressing as compared to the check

plots. DDT applied in the same manner as BHC did not give effective

control. Com yields were not significantly increased by any of the treatments. Muma et al. (1949) conducted further field experiments testing BHC, toxaphene, or rotenone as soil insecticides. The results indicated that BHC applied at a rate of one to two pounds of the gamma isomer per acre would give control of com rootworm larvae for at least two seasons. These studies also showed a direct correlation between significantly increased yields and the soil fertility as expressed by available nitrogen.

Lindane, the y-isomer of BHC, was the first chlorinated hydro­ carbon recommended for corn rootworm control by Nebraska. Aldrin and chlordane were added to the recommendations in 1952 and heptachlor in 1954 (Ball, 1977).

During the 1950s, additional testing of insecticides provided more information on chemicals that effectively controlled com rootworms.

Cox and Lilly (1953) applied aldrin, BHC, chlordane, dieldrin, hepta­ chlor, and DDT by six different application methods. Larval counts 9

averaged 17.9 per hill in untreated checks. The average per hill was

reduced to 4.1 larvae or less with the use of one pound per acre aldrin,

dieldrin, or chlordane mixed as wettable powders into fertilizer.

Lodging was reduced from 24-8% to 1.8% or less and yields were increased

from 28.0 bushels per acre in checks to 36.0 bushels in treated plots-

Applying 0.75 pound per acre of BHC, dieldrin, chlordane, and aldrin

as a broadcast spray produced larval reductions of 97, 91, 78, and 75%,

respectively. Lilly (1954) found 96, 95, 93, and 75% larval reductions

when one pound per acre heptachlor, BHC, endrin, and aldrin, respec­

tively, were applied as broadcast sprays. Lodging was reduced from

79% in checks to 4% or less in treated areas; yields were increased from 49.2 bushels per acre to 100.5.

Burkhardt (1954) tested reduced dosages and incorporated the chemicals in starter fertilizer. A quarter pound per acre of BHC, heptachlor, and aldrin gave 90, 90, and 69% reduction in larval counts, respectively; while a half pound per acre of each insecticide gave reductions of 100, 90, and 56%, respectively. Chlordane at a half and one pound per acre reduced larval counts by 93 and 100%, respectively.

Lodging was reduced from 47% in the check to 9% or less in all treat­ ments. Apple (1957) evaluated reduced dosages of aldrin and heptachlor applied broadcast or in a starter-fertilizer mix. One half pound per acre aldrin and heptachlor reduced lodging from 22% in checks to 10% or less by broadcast application and from 30% in checks to 11.8% or less by incorporation with starter-fertilizer. Yields were increased from 6.9 to 55%. 10

The chlorinated hydrocarbon insecticides proved to be so effective

at controlling com rootwonn larvae that crop rotation fell into disuse

as the major means of population suppression and prophylactic chemical

use became the mean rather than the exception. Indiscriminate insecti­

cide use leads to a corresponding selection pressure on a population.

Those individuals susceptible to the toxicant are removed from the

population; leaving the more resistant genotypes to produce the next

generation. Prior to 1946, ten insect species were showing resistance

to inorganic and botanical insecticides. By 1951, 11 species were

showing specific resistance to the chlorinated hydrocarbon DDT (Brown,

1968). It should have come as no surprise when rootworm resistance

started appearing in 1959 (Weekman, 1961; Ball, 1977).

Before progressing further, I should like to address the definition

of resistance as it will be used in this dissertation. There seems to

be much disagreement today among scientists as to how much change in

LDgg values is necessary before we call a group of insects resistant

(Ball, 1981a). As was pointed out in a "Letter to the Editor" by

Roush (1980), "resistance" designates a genetic change, such as a

response to pesticide selection (Crow, 1957; Hoskins and Gordon, 1956).

"Tolerance" is a natural ability found in the absence of selection

pressure (Hoskins and Gordon, 1956). Therefore, any change in values found in a population which has been under selection pressure due to pesticide usage will in all probability be due to a "genetic change" in the genetic equilibrium of the population and should be termed resistance. 11

Ball and Weekman (1962) collected WCR adults from central and

eastern Nebraska. Bioassays indicated that 100 times as much aldrin

or heptachlor was needed to produce 50% mortality in the central

Nebraska adults as compared to the eastern population. The LD^g

value of diazinon was the same with both populations. Within the next

few years, resistance was also found in Illinois, Indiana, Iowa, Kansas,

Minnesota, Missouri, Ohio, and South Dakota (Ball and Weekman, 1963;

Ball, 1969; Ball, 1977; Bigger, 1963; Blair et al., 1963; Gould, 1971;

Hamilton, 1965).

As the resistant populations developed and spread, growers

switched to newer classes of insecticides, carbamate and organophos-

phorus compounds. Cognizant of the possibility of resistance developing

to these compounds. Ball (1968) started determining values of the

major insecticides in use each year. Ball (1968, 1973) published five-

and ten-year studies of the changes in of diazinon and phorate

tested on WCR adults collected from 18 sites in Nebraska. Ball (1969)

published a summary of the values of 29 compounds compiled over

the course of six years. Ball (1977) summarized the larval and adult control recommendations for 1948 through 1976 and the results of his bioassays for the years 1961 through 1976. Ball (1981b) updated this work to include the years 1977 through 1981.

Other researchers have determined values of various insecti­ cides on com rootworm adults and larvae; though none has looked at it from a yearly basis as Ball does (Chio et al., 1978; Gould et al.,

1970; Metcalf, 1979; Smith, 1977; Sutter, 1979; Waller, 1972). A 12

summary of values found in the literature for various insecticides

is given in Appendix A.

Adult population suppression as an alternative to soil insecticide usage in the spring has been suggested in recent years. Musick (1971) evaluated azinphosmethyl, carbaryl, and diazinon for control of NCR adults. At 21 days following treatment, com treated with carbaryl or azinphosmethyl had significantly fewer beetles than the check or the diazinon treatment. Pruess et al. (1974) aerially applied malathion in an ultralow volume (ULV) formulation to a 42 sq. km. (16 sq. mi.) area to control corn rootworm adults in 1968, 1969, and 1970. Adult WCR populations were reduced 39, 54, and 72%, respectively, during the following seasons and no economic larval damage occurred. These researchers felt that widespread adult suppression would not be eco­ nomically feasible, but that adult control in individual fields could be an acceptable alternative to soil insecticides applied for larval control. Mayo (1979) has also been comparing adult control in the fall to larval control in the spring.

Prior to 1982, Iowa recommendations were that when adult counts in com fields averaged one beetle per plant or 0.6 beetle per ear zone and 10% of the females checked were gravid, then the number of eggs they would potentially lay would be high enough to cause economic damage the next year (Stockdale et al., 1979; Stockdale and DeWitt, 1980).

Iowa recommendations now state that adult beetles can be monitored by a sequential sampling method to decide whether a rootworm insecticide is necessary the following year. A sequential sampling method has been developed to provide a quick, statistically sound decision (Foster et 13

al., 1982). If beetle counts reach the "treatment necessary" decision

level, growers then have the choice of crop rotation, soil insecticide application in the spring, or adult suppression through the use of

insecticides immediately.

Acetylcholinesterase Characteristics and Inhibition

Carbamate and organophosphorus insecticides work by interfering with the function of the nervous system. These insecticides are thought to inhibit neural function by acting on one or more of the following sites, (1) AChE, (2) axonal transmission, (3) the acetylcholine (ACh) receptor, (4) receptors of other kinds, (5) transmitter synthesis and release, (6) presynaptic vesicle formation, or (7) various electro- genic processes (Shankland et al., 1978). The accepted theory as to the mode of action of carbamate and organophosphorus insecticides in insects and other invertebrates is as follows (Chadwick, 1963; O'Brien et al., 1974):

1. AChE is vital to nervous function, probably by removing ACh,

which is the normal nerve impulse transmitter. ACh blocks

transmission when it is allowed to accumulate to excess.

2. Anticholinesterase agents inhibit the enzyme(s) responsible

for the rapid destruction of ACh released during normal

nervous activity. In the poisoned animal ACh accumulates,

nervous function is wholly blocked or at least greatly impaired,

and death ensues.

The insect central nervous system (CNS) consists of a brain situated 14

dorsally in the head and a ventral chain of ganglia from which nerves

extend to the sense organs and muscles. The ganglia are formed of

nerve cell bodies, perikarya, surrounding a central neuropile. From the

perikarya extend the axons, along which nerve impulses are conducted

(Chapman, 1971; Lane, 1974).

The passage of a nerve impulse along the axon of a nerve cell is

the result of changes in the permeability of the axonal membrane to

sodium and potassium ions. Before the passage of an impulse, the

electrical potential inside the membrane is negative with respect to

the potential outside the cell. The concentration of sodium and

potassium ions are low and high, respectively, relative to the concen­

trations of these ions outside the cell. A nerve impulse is described

as a wave of changing polarity along the axon with the polarity at the

"crest" of the wave being completely the reverse of that before the

occurrence of the impulse or, in other words, the impulse is essentially

an electrical process in which the current is carried by ions. The

wave is caused by (1) the "sodium gate" opening with sodium ions flowing

in and (2) the "potassium gate" opening with potassium ions flowing

out. When the impulse has passed, it is thought that a single sodium/

potassium exchange pump ejects sodium ions from the inside of the axon

while simultaneously taking in potassium ions until the initial state

of the cell is again reached (Corbett, 1974).

The central neuropilar region of the ganglia is the area where

synaptic contacts between nerve cells take place. Synapses between axon terminals and nerve cell bodies do not occur in insects because 15

the perikarya are completely surrounded by protective layers including

the fat body sheath, neural lamella, perineurium, and glial cells.

The gap between cells is called the synaptic cleft and is 10 to 20 nm wide. The transmission of a nerve impulse across a synapse is, in

most cases, of a chemical nature and, therefore, distinctly different than axonal conduction which is electrical (Corbett, 1974; Lane, 1974).

As in vertebrates, it is presumed that transmitter substances are held in synaptic vesicles (25 to 45 nm in diameter) in insects and are released from the presynaptic membrane upon arrival of the nerve action potential. The transmitter substance then combines with either its inactivating enzyme or the receptor site on the postsynaptic membrane which further propagates the impulse (Figure 1).

The following criteria should be met before a substance can be identified with confidence as a synaptic transmitter (Mordue et al.,

1980);

1. The substance should be identified on the synaptic junction.

2. An enzyme for its synthesis and another for its inactivation

should be found at the synaptic endings.

3. The substance should be released on stimulation.

4. The action of the substance should be inhibited by competitive

antagonists.

5. The physiological action should be mimicked by applying the

substance artificially.

It has not been rigorously proven that ACh is a true transmitter in insects (Callec, 1974). The most extensive work to date has been on Figure 1. Schematic diagram of a neural synapse in insects

(Shankland, 1976). Ac = acetate, Ch = choline,

ACh = acetylcholine, AChR = acetylcholine receptor,

AChE = acetylcholinesterase, and = synaptic

vesicles. 17

Synaptic Presynaptic ^left j Subsynaptic * Membrane

Q D 1 / a Dm * Axon Hillock

Presynaptic Terminal Dendritic Terminal 18

the Ag ganglion of Periplaneta americana. ACh is present in the CNS

along with high levels of AChE activity. The synthesis of ACh is

substantiated by the presence of cholineacetylase- ACh applied to _2 the intact Ag ganglion at concentrations as high as 10 M failed to

induce synaptic transmission. The apparent failure may be caused by

a perineurial cell layer, acting as the blood-brain barrier, that does not allow the ACh to diffuse to the nerve cell. When the ganglion is

desheathed, ACh induces transmission (Callec, 1974). AChE activity has also been demonstrated in the neuropile of Formica, Musca, and

Rodnius and in Schistocerca metathoracic ganglia (Lane, 1974; Booth and Metcalf, 1970).

It is generally agreed that ACh is a major, but not sole chemical transmitter of impulses across the synaptic junction. Other molecules such as L-glutamate, dopamine, norepinephrine, glycine, 5-hydroxytrypta- mine, histamine, and even adenosine triphosphate may act as excitatory neurotransmitters in some cases (Bullock, 1977; Chadwick, 1963).

There is some evidence that y-aminobutyric acid (GABA) is the inhibitory transmitter (Lane, 1974).

From the brain and other ganglia, peripheral nerves emanate to various parts of the insect. Peripheral nerve axons terminate on muscle at what is called a neuromuscular junction. Synaptic vesicles are also present in the terminal ends of axons; presumably, to store transmitter substances until the arrival of an impulse triggers the release of the chemical into the synaptic cleft. The excitatory neurotransmitter in insect neuromuscular junctions is not ACh since the junctions have been 19 shown to be noncholinergic and AChE is also not present (Lane, 1974).

It appears that L-glutamate is the major excitatory transmitter in both fast and slow axons. There is some evidence, however, that other compounds may play a role in neuromuscular transmission; such as octopamine, found as a modulator compound in a neurone in the locust

(Evans and O'Shea, 1977). As in the CNS, GABA appears to be the inhib­ itory compound.

It is generally accepted that the reaction of organophosphorus and carbamate compounds with AChE is the same process as that which occurs when the enzyme catalyzes the hydrolysis of its substrate (Eq. 1).

EH + ACh = EHACh ^ EA ^ * EH + AOH (1) ChH H^O

EH = AChE

ACh = ACh, (CHg)^^H^CH^OC(0)CH^

ChH = choline, (CH^)^&H^CH^OH

EA = acetylated enzyme, E0C(0)CH^

AOH = acetic acid, CH^C(0)OH

Equations 2-4 show the process in generalized form.

EH + AB EHAB (2) ^-1

^2 EHAB — EA + BH (3)

EA + HgO ^ EH + AOH (4) 20

EH is the free enzyme, AB is the ester (substrate or an ester of an

organophosphorus or carbamic acid), EHAB is the Michaelis complex

which breaks down to give BH and EA as first products, and AOH and

EH as the second products (Aldridge and Reiner, 1972). The acylated

enzyme (EA) could be acetylated AChE formed during the hydrolysis of

ACh or a phosphorylated or carbamylated esterase produced by inter­

action with organophosphorus or carbamate compounds. The return of

enzyme activity is due to hydrolysis of the acylated enzyme (Eq. 4).

Procedures have been developed to determine the presence and

activity of AChE. All methods are based on a determination of the

rate of hydrolysis of ACh or of a similar ester that is hydrolyzed by

AChE. There are two prerequisites for a satisfactory procedure. The

rate of the reaction measured must be proportional to the amount of

the enzyme present, i.e., a linear relationship must exist between

enzyme concentration and activity. Also, the enzyme measured under

the conditions of the assay must be AChE (Witter, 1963).

The earliest method used in determining AChE activity was based

on the ACh remaining after incubation with the enzyme. A number of

methods have been developed to determine the remaining ACh. The

Hestrin method is a colorimetric determination of the amount of iron

(III) hydroxamate formed after ACh in alkali reacts with hydroxylamine

to form acetohydroxamic acid which is then reacted with ferric

chloride (Witter, 1963). The Schonemann method reacts ACh with alkaline hydroperoxide to produce a peracid which can then oxidize various amines to produce colored oxidation products which can be 21

detected colorimetrically (Witter, 1963). A gas chromatographic

method measures demethylated choline esters (Cranmer and Peoples, 1973).

These methods are considered inferior because they do not directly measure the rate of disappearance of the substrate (Holmstedt, 1971).

A number of methods are based on the formation of acid from the hydrolysis of acetyl- or butyrylcholine. The Warburg manometric technique measures the change in CO^ volume that occurs when the acid from hydrolysis of substrate reacts with bicarbonate ion to form carbonic acid which disassociates to CO^ and HgO. This method is one of the most accurate procedures available. It is limited in that the useable pH range is 7.2 to 7.7 and substrate concentrations below 0.4 mM cannot be studied as insufficient CO^ is liberated (Witter, 1963;

Holmstedt, 1971). The Michel method measures the change in pH units of the hydrolysis of ACh by AChE over an interval of time. This method lacks the accuracy and sensitivity of some of the other methods because the acid production is not measured directly; rather, the pH, which is a logarithmic function of the acid concentration, is measured. A number of techniques have also evolved that measure the change in pH by the change of color of an acid-base indicator. The Acholest method utilizes the color change of an indicator. A slip of filter paper is wet with a mixture of choline ester and bromothymol blue and dried.

The enzyme source is added to the paper and the activity is measured as the time needed for the reaction to change the color of the test paper to the color of the control paper. The pH-stat method is a titrimetric determination of the acid released during hydrolysis. 22

The automated pH-stat has the advantage that the rate of reaction can

be continuously recorded (Nabb and Whitfield, 1967).

Radiometric assays have used substrates labeled in the acyl moiety.

After hydrolysis, either the radioactive acid or the unhydrolyzed sub­

strate is counted after they have been separated by volatilization,

extraction, precipitation followed by centrifugation, or ultrafiltration

(Winteringham and Disney, 1964a; Winteringham and Disney, 1964b; Disney,

1966; Lewis and Eldefrawi, 1974).

The last group of methods to be discussed is based on the hydroly­ sis of thiocholine esters. Two iodometric procedures have been developed to determine the amount of thiocholine produced according to equations

5 and 6.

2(CH2)2&CH2CH2SH-I + (CH^)(CH^)^-21 + 2HI (5)

lOg" + 51 + 6H'^ —31^ + SH^O (6)

A number of procedures follow the hydrolysis of thiocholine esters colorimetrically. The decoloration of a blue 2,6-dichlorophenolindo- phenol solution (Dye) with thiocholine (Eq. 7) is an example of this type (Witter, 1963).

2(CH2)2&CH2CH2SH'I + Dye—(CH^)^&H2CH2SSCH2CH2&(CS^) ^ ' 21" +

Dye&g (7) 23

Another colorimetric method is based on the oxidation of thiocholine

by sodium nitroprusside to produce a yellow-orange nitroprusside

(Witter, 1963). The best known of these colorimetric methods is the

Ellman method (Ellman et al., 1961). This method is based on the

reaction of thiocholine with dithiobisnitrobenzoic acid (DTNB) to

produce a yellow color (Eq. 8 and 9).

H^O + (CH2)2&CH2CH2SC(0)CH2 (^3)2^^2^28" + CH2C(0)0" +

2H''" (8)

(^2)3^^2^25" + RSSR—^(CH3)3$CH2CH2SSR + RS~ (9)

R = —i)-B02

coo"

The resultant yellow color comes from the anion, 5-thio-2-nitrobenzoic acid denoted as RS in the above scheme. This compound has an absorp­ tion maximum at 412 nm. In contrast to other colorimetric methods, the Ellman method is based on the measurement of the reaction product instead of the remaining unhydrolyzed substrate. It also has the advantages of simplicity, high precision, pH constancy, flexibility, short incubation period, and continuous increase in color density as a function of incubation time. It has been used with an auto analyzer system to give continual ^ vivo analyses on the AChE activity in the blood of laboratory animals (Bajgar et al., 1977).

Reviews giving more detail on the procedures, usefulness, and 24

limitations of these methods have been published by Augustinsson (1971),

Holmstedt (1971), Smyth (1977), and Witter (1963).

All of the above methods were developed for use with mammalian

systems and some have been adapted for use in insect studies. The house

fly has been used as a source for insect AChE because the fly is easy

to rear, has a short reproductive cycle, and the collection of AChE-rich

fly heads is very simple (Moorefield, 1957). Investigators of house fly AChE have utilized the radiometric method (Winteringham and Disney,

1964b), the Warburg mancmetric method (Asperen, 1959; Dauterman et al.,

1962; Metcalf and March, 1950), titrimetric methods (Abd-Elroaf et al.,

1977; Tripathi et al., 1973; Chadwick et al., 1953), and the Ellman colorimetric method (Tripathi and O'Brien, 1973a; Tripathi and O'Brien,

1973b; Tripathi, 1976; Eldefrawi et al., 1970; Booth and Lee, 1971;

Hellenbrand, 1967:; Lee et al., 1978). Chiu et al. (1972) developed a procedure which can be used to determine kinetic data from electro- phoretic polyacrylamide gels (O'Brien et al., 1974). Research on AChE of other insect species, mites, and ticks has almost exclusively utilized the Ellman colorimetric method (Abdel-Aal, 1977; Abdel-Aal et al., 1979; Call et al., 1977; Callaham et al., 1975; Callaham et al.,

1977; Joiner et al., 1973; Knowles and Arurkar, 1969; Nolan and Schnit- zerling, 1976; Smissaert, 1976; Stone et al., 1976; Voss, 1980; Zahavi et al., 1972).

The Michaelis constant, K , is the concentration of substrate or m inhibitor (AB) required to ensure half of the maximum rate of production of both products or of the maximum rate of regeneration of free enzyme. 25

It is defined by equation 10 (Aldridge and Reiner, 1972) where k., k ,, 1 —1 k^, and k^ are rate constants as shown in equations 2-4.

k_i + kg k \ = —k— (10)

Published data for the associated with mammalian AChE ranged from

120 to 790 fiM. for ACh as substrate and 140 to 500 /zM for ASCh (Aldridge

and Reiner, 1972). Kinetic data.obtained from a number of insect species are shown in Table 1. A number of authors have studied the effects of insecticides on AChE from both resistant and susceptible strains of insects and ticks (Nolan and Schnitzerling, 1976; O'Brien et al., 1974; Roulston and Nolan, 1974; Tripathi and O'Brien, 1973b; Voss,

1980). The k^ (bimolecular rate constant of inhibition) and k^ values

have been determined for various insecticides in house flies (Lee et al-,

1978; O'Brien et al., 1974; Tripathi, 1976; Tripathi and O'Brien, 1973b;

Tripathi et al., 1973; Voss, 1980), honey bee (Abdel-Aal, 1977;

Abdel-Aal et al., 1979), cotton leafworm (Abdel-Aal, 1977;

Voss, 1980), red spider mites (Smissaert, 1964), and cattle tick

(Lee and Batham, 1966; Stone et al-, 1976). AChE inhibition by insecti­ cides (% inhibition or values) has been determined for house fly

(Lee et al., 1978; Metcalf and March, 1950; O'Brien et al., 1974;

Tripathi and O'Brien, 1973a), honey bee (Abd-Elroaf et al., 1977;

Abdel-Aal et al., 1979; Guilbault et al., 1970; Metcalf and March, 1950), bean leaf beetle (Sadar et al., 1970), boll weevil (Guilbault et al-,

1970; Joiner et al-, 1973), white fringe beetle (Sadar et al-, 1970), 26

Table 1. Optimum substrate concentration, K^, and substrate inhibition

determined for AChE from various insect species.

Insect Optimum K Inhibition Reference Substrate(M) (H0) by Substrate

ACh as Substrate

-2 House fly 1 x 10 Yes Metcalf and March, 1950 -4 7 X 10 Yes Winteringham and Disney, 1964b -3 Face fly 3 X 10 Yes Knowles and Arurkar, 1969

-2 Honey bee 1 X 10 Yes Metcalf and March, 1950 -3 5 X 10 Yes Guilbault et al., 1970 -3 Boll weevil 1 x 10 Yes Guilbault et al., 1970

ASCh as Substrate

House fly 28.8 Booth and Lee, 1971 Isozyme I 183 none in the Tripathi and O'Brien, head . II 687 range 2 to Tripathi and O'Brien, III 435 0.1 mM Tripathi et al., 1973 IV 770 ( V 158 thorax 1 VI 657 (vii 386

Cricket 167 Booth and Lee, 1971

Green peach 60 none to Smissaert, 1976 aphid 0.5 mM

Spirea aphid saturated _ 67 none to Manulis et al., 1981 at 1 X 10 1 mM

spirea aphid (Manulis et al., 1981), and cotton leafworm (Zaazou et al.,

1973).

Most researchers working with insect AChE have treated it as a single kind of enzyme without attempting to separate it into isozymes. 27

O'Brien et al. (1974) have demonstrated that at least seven isozymes of AChE are present in the house fly, four in the head and three in the thorax. Using an electrophoretic method, they were able to establish substrate kinetics for the isozymes and found that the for the four head or three thoracic isozymes varied approximately 4.2-fold (Table 1).

In vitro studies on the isolated isozymes revealed significant, though not large, variation in their sensitivities to inhibition by four organophosphorus compounds. The largest range of sensitivity of AChE to inhibition by a specific chemical was in the case of malaoxon where -5 -1 -1 the bimolecular rate constant, k^, ranged from 3.42 x 10 M min -5 -1 -1 to 7.85 X 10 M min . This indicates a 2.3-fold difference in rate between the least and most sensitive isozyme (Tripathi et al., 1973).

O'Brien et al. (1974) also conducted in vivo time-course studies of the inhibition of each isozyme. There was a marked difference in response from the various isozymes. Some were partially inhibited and returned to near normal in a short period of time, whereas, others were almost totally inhibited and did not recover to any great extent.

Later, analysis of the isozymes of a population of house flies resistant to the organophosphorus insecticide stirofos (Rabon®) showed that the binding site for ACh was little changed while the binding site for the inhibitor was quite altered (O'Brien et al., 1974).

This led these authors to the following hypotheses:

1. The old concept of the existence of the same two sites for

binding of substrate or inhibitor to the enzyme is no longer

accurate. 28

2. One site, the esteratic site which probably contains a serine

hydroxyl, is most likely the same for acetylation by ACh or

carbamylation and phosphorylation by insecticides.

3. The second site which is now known as the anionic site is

probably the part that binds the quaternary nitrogen of ACh

to the enzyme surface but probably does not play an active

role in the binding of insecticides as very few organophosphates

or carbamates have ionic groups in them.

4. It would appear likely that more binding sites exist in the

enzyme and would encompass such things as hydrophobic sites,

indophenyl sites, or charge-transfer sites.

In other words, the catalytic or esteratic site appears to be surrounded by a variety of potential binding sites. This idea really is not surprising because AChE, like any enzyme, is a polypeptide. It is well- known that the coiling of a polypeptide caused by its secondary and tertiary structure will bring into close proximity to the catalytic site a whole host of different amino acids. Amino acids, depending on the characteristic of the substituent group attached to it, are defined as having neutral, acidic, or basic properties. These properties of amino acids give rise to the variety of binding sites.

Carbofuran and Terbufos

Carbofuran is an N-methylcarbamate insecticide/nematocide. The scientific name is 2,3-dihydro—2,2-dimethyl—7-benzofuranyl-N-methyl- 29

carbamate. The FMC Corporation has manufactured it commercially under

the trade name Furadair since 1969. Carbofuran controls soil nematodes,

soil insects, and foliar insects both by contact and systemic means.

FMC claims control of the following com pests with the use of

carbofuran: com rootworms, wireworms, seedcom maggot, fall armyworm,

European com borer, stalk borer, armyworm, southern com borer, and

nematodes (FMC Corporation, 1975). The two most widely used formulations

of carbofuran are Furadan 5-G and Furadan 10-G. These are dense, sand- core granules containing 5% and 10% carbofuran by weight. Biomagnifi- cation of carbofuran and its metabolites does not occur. The major degradation pathway is through hydrolysis at the carbamate linkage.

The products of hydrolysis are carbon dioxide, methylamine, and the corresponding 7-phenol compound. The half-life in soil is decreased with increasing pH, increasing temperature, increasing moisture content, increasing clay content, and decreasing organic matter (FMC Corporation,

1975). The half-life in most surface waters (pH range of 7.0 to 8.5) varies from 40 days to less than two days (FMC Corporation, 1975).

Chio and Metcalf (1979) have published their findings of the major metabolites of carbofuran found in the NCR, WCR, and SCR.

Terbufos is a phosphorodithioate insecticide. The scientific name is S-[[(1,1-dimethylethyl)thio]methyl]0,0-diethylphosphorodithioate.

The American Cyanamid Company manufactures it commercially under the trade name Counter . Terbufos is labeled for the control or reduction of the following pests in com: com rootworms, seedcom maggot, wireworm, symphytans, nematodes, adult southem com billbugs, and 30

white grubs (American Cyanamid Company, technical information label).

Terbufos is sold as Counter 15-G. This formulation is made on 24/48

mesh size attapulgite-type carrier containing 15% terbufos by weight

(American Cyanamid Company, technical information flyers). Terbufos will undergo hydrolysis in basic solution. Applied to the soil, terbufos has a half-life of four to 24 days (Ahmad et al., 1979;

Chapman and Harris, 1980; Laveglia and Dahm, 1975). The degradation product, terbufos sulfoxide, reaches a maximum soil concentration after two to nine weeks. Another degradation product, terbufos sulfone, does not appear until one week after application (Chapman and Harris,

1980; Laveglia and Dahm, 1975). Chio and Metcalf (1979) have determined the major metabolites of terbufos in NCR, WCR, and SCR. 31

MATERIALS AND METHODS

Com Rootworms

Three species of com rootworms were utilized in various parts of

this work. They were the NCR, Diabrotica longicomis longicomis Smith

and Lawrence; the SCR, undecimpunctata howardi Barber; and the WCR,

2" virgifera virgifera LeConte. Larvae and adults of laboratory-reared

SCR were obtained from a population maintained at Iowa State University,

Ames, lA. Some SCR adults were collected on Curcurbita texana from the

Plant Introduction Station, Ames, lA. Larvae and adults of the WCR

were either collected from fields in central Iowa or from lab-reared

F1 generation rootworms. All NCR larvae and adults were collected

from central Iowa.

Laboratory rearing procedures were similar to those described by

Branson et al. (1975) and Jackson and Davis (1978). Field-collected

larvae were maintained on flats of germinated com for at least 24 hours

prior to testing. All field-collected adults were held for at least

24 hours prior to testing. Lab-reared adults were used two weeks

after emergence. Field-collected SCR adults in 1980 were initially

fed C^. texana fruit and gradually weaned to the normal diet of the lab colony. All other field-collected adults were maintained on a diet of young corn ears and water. 32

Bioassay Techniques

Two major com rootworm insecticides and their analogues were

utilized in this study. The chemical structures of carbofuran,

terbufos, and analogues may be found in Appendix B. Carbofuran,

3-hydroxycarbofuran, 3-ketocarbofuran, 7-phenolcarbofuran, 3-hydroxy-

7-phenolcarbofuran, and 3-keto-7-phenolcarbofuran were obtained from

FMC Corporation, Middleport, NY. Terbufos, terbufos sulfoxide,

terbufos sulfone, terbufoxon, terbufoxon sulfoxide, and terbufoxon

sulfone were obtained from American Cyanamid Company, Princeton, NJ.

These chemicals were all analytical standards. Thin layer chromatog­

raphy was utilized to confirm the purity of the compounds. Glass-

distilled acetone was used as the carrier solvent in all bioassays.

The procedures followed for both adults and larvae were

adapted from the standard method for detection of resistance in

Diabrotica (Anonymous, 1972). Specific steps followed for larvae

or adults can be found in Appendix C. A microvacuum device set at the lowest suction necessary to hold the insect was used to handle the insect during the application of chemicals. A Hamilton foot-operated

microapplicator and a calibrated syringe were used to deliver the insecticide. The syringe was calibrated by filling it with a radio­ labeled DDT solution, delivering an aliquot of solution to a scintillation vial, and counting the delivered amount in a Packard scintillation counter. The average of 10 aliquots was used as the volume delivered per application. 33

Mortality readings were taken after 48 hours. Adults were considered

dead if they could not regain their footing after the petri dish holding

them was shaken. Larvae were considered dead if they were unable to

move away from tactile stimulus.

Thirty insects (10 insects per replicate, three replicates) were

tested for each dose except in the following cases, WCR larvae, 1978,

15 larvae total; WCR larvae, 1979, 10 larvae total; NCR larvae, 1979,

10 larvae total; NCR adults, 1979, 20 adults total- Five or six dosages

and an acetone control were used to determine values except in

the following cases, WCR larvae, 1978, three doses with carbofuran; WCR

larvae, 1979, four doses with carbofuran; NCR adults, 1979, four doses

with carbofuran. A Statistical Analysis System (SAS) Probit computer

program (Helwig and Council, 1979) based on Finney (1971a) was used to

analyze the data except in the following cases, WCR adults, 1979,

carbofuran and terbufos; NCR adults, 1979, terbufos; lab-reared SCR

adults, 1980, carbofuran and terbufos, where the data were hand

calculated using the probit procedure described by Finney (1971b).

Acetylcholinesterase Assay Techniques

The Ellman colorimetric method (Ellman et al., 1961) was utilized

in gathering data concerning AChE characteristics, dosage-inhibition,

and inhibition-mortality. DTNB was from Aldrich Chemical Company,

Milwaukee, WI. Acetylthiocholine iodide (ASCh) was from Scientific

Products, McGaw Park, IL. All other chemicals were reagent grade. 34

The water used in these experiments was double-distilled, deionized

water. The insecticides used were all analytical standards dissolved

in glass distilled acetone.

The initial method utilized was published by Call et al. (1977).

This procedure used 5% wt/vol (50 mg/ml) whole insect homogenates in

0.1 M phosphate buffer at pH 7.5 as the enzyme source, ^81 [M ASCh as

substrate, and 310 (M DTNB as color agent.

Whole insect homogenates were prepared in cold 0.1 M phosphate

buffer, pK 7.5, in a glass-Teflon tissue homogenizer. Initial homo-

genate concentration was 5% wt/vol but was later changed to 2.5% wt/vol

(25 mg/ml) for both adults and larvae. The homogenate was filtered through four layers of cheesecloth to remove gross particles and then centrifuged at 4® C for 30 minutes at 12,000 x The resultant supernatant was used as the enzyme source.

AChE activity was determined by the method of Ellman et al. (1961).

Assays were performed at 28° C. Standard assay conditions consisted

of 3.12 ml 0.1 M phosphate buffer (pH 7.5), 310 filî DTNB, 25 ASCh,

and 0.1 ml of 2.5% wt/vol homogenate. If conditions varied from

the standard, then exact concentrations are given in the figure legends. Rate in moles ASCh hydrolyzed per liter homogenate per minute (M/min) is found by dividing by the extinction coeffi­ cient (1.36 X 10^/M). Equation 11 describes the relationship between change in absorbance per minute and moles ASCh hydrolyzed per minute per gram insect tissue. 35

R = — (11) 1.36 X 10 V /V^(C ) o t o

where R = rate in moles of ASCh hydrolyzed per minute per gram of

tissue,

AA = change in absorbance per minute,

= volume of homogenate used,

= total volume in cuvette,

= original concentration of homogenate (mg/ml).

Data from experiments were fitted by a weighted least-squares

method written in the OMNITAB computer language (Siano et al., 1975).

Protein Assay

Protein concentration was determined with the use of the Bio-Rad

Protein Assay Kit from Bio-Rad Laboratories, Richmond, CA. This assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. A linear response is seen as protein concentration increases. 36

RESULTS

Dosage-mortality Data

There has been much debate concerning whether resistance may be

developing to certain currently used rootworm insecticides. The object

of this section of my research was to monitor the values of

carbofuran and terbufos insecticides on field populations of NCRs and

vJCRs in central Iowa over a period of three years to fill in the

lack of data from Iowa and to compare these results with those of

studies in neighboring states. Additionally, LD^g values were deter­

mined for field-collected and laboratory strains of SCR.

Table 2 shows the average values for carbofuran and terbufos

obtained on the three species of corn rootworms over the years of

this study. Table 3 shows the values obtained for carbofuran,

terbufos, and five analogues of each on WCR adults and 3-hydroxycarbo-

furan on SCR larvae. Detailed tables of yearly bioassays are in

Appendix D.

The yearly carbofuran LD^^ values within species and stage remained similar over the testing period. There was an approximately 8.7x

difference in the average susceptibility between larvae and adults of

the WCR. There was an approximately 5.9x difference in susceptibility

between larvae and adults of the NCR. The lab-reared SCR larvae are approximately 17.8x less susceptible to carbofuran than the lab-reared adults. The SCRs were less susceptible to carbofuran than the NCRs and WCRs. 37

Table 2. Average values for carbofuran and terbufos on western,

northern, and southern corn rootworms.

Carbofuran Terbufos

Insect Stage Ug/insect ug/g ug/insect Wg/g

WCR Larval 0.12 12.68 0.01 1.08

Adult 0.02 1.46 0.08 4.74

NCR Larval 0.10 9.20

Adult 0.01 1.56 0.07 7.34

SCR Larval 0.21 23.14 0.04 3.70

Adult 0.04 1.30 0.12 4.30

The terbufos values within species and stage remained similar

over the testing period with the exception of the values found for NCR adults. These values decreased over the three year period. The average

LD^Q values for terbufos on NCR adults were calculated using a value of

0.01 /xg/insect and 1.00 ptg/g as the 1981 values. There is a reversal of the trend of stage susceptibility seen in the values with carbofuran.

With terbufos, the WCR adults are about 4.4x less susceptible than the larvae. Lab-reared SCR adults are 1.2x less susceptible than the larvae.

Acetylcholinesterase Characteristics

Studies on enzyme systems of insects under the selection pressure of insecticides are of interest not only to physiologists, biochemists. 38

Table 3. values of carbofuran, terbufos, and five analogues of

each on field-collected WCR adults and 3-hydroxycarbofuran

on SCR larvae.

^50 ^50 Compound Insect Jig/insect Aig/g

Carbofuran WCR 0..03 2.20

3-hydroxycarbofuran WCR 0,.14 10.45

3-ketocarbofuran WCR 0..11 9.69

7-phenolcarbofuran WCR >8..76 > 768.07

3-hydroxy-7-phenolcarbofuran WCR >8.,58 > 660.78

3-keto-7-phenolcarbofuran WCR >8..58 > 657.82

Terbufos WCR 0.06 4.52

Terbufos sulfoxide WCR 0.04 3.11

Terbufos suifone WCR 0.05 3.87

Terbufoxon WCR 0.05 3.81

Terbufoxon sulfoxide WCR 0.08 5.15

Terbufoxon suifone WCR 0.09 5.95

Carbofuran SCR 0.25 25.89

3-hvdroxvcarbofuran SCR 1.12 109.18 39

and geneticists, but should also be of great interest to economic

entomologists monitoring field populations of insects for an expected

or real development of resistance.

Only one paper has been published concerning AChE activity in

com rootworms (Call et al., 1977). These authors did not look at the

effect of substrate concentration on AChE activity. Consequently,

their work was carried out at a substrate concentration far above the

K of rootworm AChE and at a level where substrate inhibition occurred. m Over 50% inhibition of AChE by the substrate ASCh occurs at the concen­ tration of 481 ptM that they used. I investigated effects on AChE activity of such variants as homogenate concentration, ASCh concentra­ tion, DTNB concentration, and larval weight.

For the first few months of enzyme analysis, I followed the procedure published by Call et al. (1977). This procedure used 0.3 ml of 5% wt/vol homogenate. Since substrate hydrolysis rates were fast, I decided to reduce the amount of homogenate used to 0.1 ml. This reduction was desirable because it reduced the total amount of homogenate needed per experiment and it also allowed for a more accurate addition of homogenate (one addition of 0.1 ml with a capillary pipet versus three such additions or one 0.3 ml addition with a graduated pipet). The rate of ASCh hydrolysis at this lower homogenate concentration was not linear. Since I hadn't noticed this nonlinearity previously, I decided to look at the linearity of the rate of ASCh hydrolysis at various concentrations of homogenate. The initial rates of hydrolysis were linear at all concentrations tested except with the addition of 0.1 ml 40

of 5% wt/vol homogenate (50 mg/ml). I have no explanation as to why

the nonlinearity occurred only when the 5% wt/vol homogenate was added

to the reaction, but nonlinear hydrolysis did occur every time that

amount of homogenate was used. Since linear hydrolysis did occur

when 0.1 ml of 2.5% wt/vol homogenate was used, I made that the

standard amount of homogenate added to the reaction mixture.

Figure 2 is a plot of initial rate of ASCh hydrolysis

versus homogenate concentration (% wt/vol). The best fitting line was

drawn to the nonlinear hydrolysis response with the 5% wt/vol homogenate

in order to estimate the initial rate at that homogenate concentration.

A linear relationship exists between initial rate and increasing homo­

genate concentration.

Mammalian AChE is subject to substrate inhibition by both ACh and

ASCh (Aldridge and Reiner, 1972). There are reports of insect AChE

being inhibited by ACh but not to ASCh (Table 1). Call et al. (1977) did not examine the effect of substrate concentration on com rootworm

AChE, therefore, I looked at substrate effects on rootworm AChE. Figures

3, 4, 5, and 6 show the Lineweaver-Burk (double reciprocal) plots determined for various ASCh concentrations with homogenates of third- stage SCR larvae, third-stage WCR larvae, field-collected WCR adults, and two-week-old, F1 WCR adults, respectively. Table 4 summarizes the

K , V , K , and concentration of ASCh at which inhibition is first m max ss seen for the insects tested.

The K is the substrate concentration required to inhibit the ss enzyme 50% (Aldridge and Reiner, 1972). Published values of in Figure 2. Plot of change in absorbance versus

homogenate concentration (% wt/vol). Assays

contained a total volume of 3.22 ml, 449 ASCh,

311 juM DTNB, and were carried out at pH 7.5 in

0.1 M phosphate buffer. Data were computer fitted 2 with an R value of 0.999. 42

5 10 15 Homogenate concentration (%wt/vol) Figure 3. Lineweaver-Burk plot of the initial velocity of the

rate of ASCh hydrolysis versus concentration of ASCh

for AChE homogenate from third-stage SCR larvae.

Assays contained a total volume of 3.12 ml, 310 pM

DTNB, 25 /iM ASCh, 0.5 ml 2.5% wt/vol homogenate, and

were carried out at pH 7.5 in 0.1 M phosphate buffer.

V = 4.097 X 10 ^ mole/l-min and K = 9.28 uM ASCh. max m Velocity (V) is expressed in moles ASCh hydrolyzed/l-min

and ASCh concentration is expressed in moles/1.

The average % difference = 0.89. I I so 100 150 200 (1/ASCh) X 10'® Figure 4. Lineweaver-Burk plot of the initial velocity of the

rate of ASCh hydrolysis versus concentration of ASCh

for AChE homogenate from third-stage, Fl WCR larvae.

Assays contained a total volume of 3.145 or 3.17 ml, 0.025

or 0.050 ml 2.5% wt/vol homogenate, respectively, 310 /iM

DTNB, 25 /iM ASCh, and were carried out at pH 7.5 in 0.1 M

phosphate buffer. At 0.025 ml homogenate, V = max

3.336 X 10 ^ mole/l-min and K m = 24.27 r-uM ASCh. At 0.050 ml homogenate, = 5.827 x 10 ^ mole/l-min

and = 15.80 /iM ASCh. Velocity (V) is expressed in

moles ASCh hydrolyzed/l-min and ASCh concentration is

expressed in moles/1. The average % difference = 3.07 at

0.025 ml and 6.04 at 0.050 ml. 20

16

<0 12 'O 0.050 ml >8 N,

100 150 200 (1/ASCh) X 10"3 Figure 5. Lineweaver-Burk plot of the Initial velocity of the

rate of ASCh hydrolysis versus concentration of ASCh

for AChE homogenate from field-collected WCR adults.

Assays contained a total volume of 3.22 ml, 310 piM DTNB,

25 /iM ASCh, 0.1 ml 2.5% wt/vol homogenate, and were

carried out at pH 7.5 in 0.1 M phosphate buffer.

V = 5.121 X 10 ^ mole/l-min and K = 24.80 uM ASCh. max m Velocity (V) is expressed in moles ASCh hydrolyzed/l-min

and ASCh concentration is expressed in moles/1.

The average % difference = 1.27. 12

50 100 150 200 (1/ASCh) X 10^ Figure 6. Lineweaver-Burk plot of the initial velocity of the

rate of ASCh hydrolysis versus concentration of ASCh

for AChE homogenate from two-week-old, F1 WCR adults.

Assays contained a total volume of 3.22 ml, 310 fiM

DTNB, 25 fxM ASCh , 0.1 ml 2.5% wt/vol homogenate, and

were carried out at pH 7.5 in 0.1 M phosphate buffer.

V = 2.212 X 10 ^ mole/l-min and K = 21.20 uM ASCh. max m Velocity (V) is expressed in moles ASCh hydrolyzed/l-min

and ASCh concentration expressed In moles/1.

The average % difference = 4.00. 30

24

18

12

50 100 ISO 200 (1/ASCh) X 10^ 51

Table 4. K , V , K , and concentration of ASCh at which inhibition m max ss is first seen for whole insect homogenates of third-stage SCR

and F1 WCR larvae and field-collected (FC) and F1 generation

WCR adults.

Amount homo K Inhibiting K ss used m conc. ASCh V * Insect Stage (ml) (uM) max (mM) (im)

SCR larval 0.100 9.28 1.023 369 25

WCR larval 0.025 24.27 1.679

larval 0.050 15.80 1.478 120

FC WCR adult 0.100 24.80 0.660 496 93

F1 WCR adult 0.100 21-20 0.285 794 52

^max terms of /mole ASCh hydrolyzed/min/g insect tissue.

mammalian systems range from 11 to 22 mM for ACh and 3.3 mM for ASCh.

ACh was inhibitory to four species of insects (Table 1) but I could not find reports of ASCh inhibition in insects. The K values for ASCh ss inhibition in corn rootworms ranged from 369 to 794 /iM. The ASCh concentration used in the procedure published by Call et al. (1977) was

481 fiK. My studies indicate that inhibition by ASCh to SCR larval AChE occurs at concentrations greater than 25 ^M. This finding led to the reduction of ASCh used in each assay from 481 /xM to 25 /xM.

There are reports in the literature of DTNB (10 pM to 3,000 /iM) acting as an inhibitor of AChE in the green peach aphid (Zahavi et al.,

1972; Smissaert, 1976) and the spirea aphid (Manulis et al., 1981). 52

To determine if DTNB had an inhibitory effect on rootworm AChE, concen­ trations of DTNB from 31 to 1,242 were added to the standard assay mix and the reaction followed for five minutes. The activity at the five concentrations used is given in Table 5. No inhibition by DTNB to

AChE from third-stage SCR larvae occurred at the concentrations studied, therefore, no change of DTNB concentration (310 pM) was made from that used by Call et al. (1977).

Table 5. Third-stage SCR larval AChE activity at various concentrations

of DTNB.

DTNB ASCh hydrolyzed (txM) (umole/min/g body wt)

31 0.69

155 0.64

311 0.74

621 0.59

1242 0.62

The standard bioassay method for detection of insecticide resistance in Diabrotica species suggests the use of larvae with weights in the range 7 to 11 mg (Anonymous, 1972; Sutter, 1979). Assuming that inhibition of AChE is the major cause of death, I questioned whether or not the ratio of AChE activity to unit of body weight would remain constant over this range of larval weights. The AChE activity was determined at different body weights (stages) of SCR 53

larvae. The larval stage of each weight of larvae used was determined

by measuring the width of the head capsule according to the following

(Branson et al., 1975):

first-stage 0.25 - 0.30 mm

second-stage 0.39 - 0.42 mm

third-stage 0.58 - 0.64 mm

Homogenates were prepared as described in the materials and methods

section and assays were run. Protein determinations were also made

at this time. Figures 7 and 8 show the relationship between;

activity and weight (stage) or protein concentration. These

data show that as larval size increases, the amount of AChE

activity increases proportionally. When AChE activity is

compared on a per gram of body weight or per /zg protein basis, the

activity remains constant over the larval weights studied except in the

first-stage and early second-stage. The first-stage has approximately

2x the AChE activity on a per gram basis than the later stages.

Inhibition Studies

When mammalian AChE is incubated with an organophosphorus compound, the activity of the enzyme will decrease in a time-dependent fashion.

The decreased activity cannot be restored by quick dilution, quick dialysis, or by addition of substrate. These properties are indicative of covalent bond formation between enzyme and inhibitor rather than formation of a reversible complex as seen when AChE reacts with ACh. Figure 7. Plot of change in absorbance /min/g body

weight (7a) and change of absorbance

insect (7b) versus weight (stage) of SCR larvae. Assays

contained a total volume of 3.22 ml, 310 DTNB,

25 ASCh, 0.1 ml 2.5% wt/vol homogenate, and

were carried out at pH 7.5 in 0.1 M phosphate buffer.

Lines were computer fitted. Calculated equations for

fit were:

A0D^12/min/g body weight = 2.868 - 0.004(weight),

= 0.021;

(AOD^^^/min/insect) x 10^ = 0.070 + 0.275(weight),

= 0.996.

Data for first-stage, early second-stage, and prepupae

were not included in the calculations. 55

(7b) N / S » X î î bo .s \ \ s c * (7a) s S II

6 9 12 15 18 21 WEIGHT(mg) 12 2 3 3 3 PP 3 STAGE Figure 8. Plot of change in absorbance /mxiL/ng protein (8a)

and pig protein/g body weight (8b) versus weight (stage)

of SCR larvae. Assays contained a total volume of

3.22 ml, 310 /xM DTNB, 25 fM ASCh, 0.1 ml 2.5% wt/vol

homogenate, and were carried out at pH 7.5 in 0.1 M

phosphate buffer. Lines were computer fitted.

Calculated equations for fit were:

AOD^^2/min/;fg protein = 0.000180 - 0.00000248(weight),

= 0.971;

fig protein/g body weight = 17,180 + 175(weight),

R^ = 0.350.

Data for first-stage and prepupae were not included

in the calculations of the first equation; datum for

prepupae was not included in the calculations of the

second equation. 57

X O S % ® —.5

< •

3 6 9 12 15 18 21 WEIGHT (mg) 12 2 3 3 3 PP 3 STAGE 58

The half-life of the acetylated enzyme formed by the reaction with

-6 ACh is 2.3 X 10 minute or less, whereas the half-life of a dimethyl-

phosphorylated AChE is about 90 minutes (Aldridge and Reiner, 1972).

Since these data were from mammalian sources, I wondered what

would occur when carbofuran or terbufos was added to rootworm AChE.

To 2.5% wt/vol homogenates of third-stage SCR larvae, I added approxi­

mately the concentrations of carbofuran (0.60 /xg/ml) or terbufos

(0.093 pig/ml) and assayed for activity over time. Figures 9 and 10

are plots of the results. When carbofuran was added, the activity was

inhibited 82.5% of the control within five minutes, fell steadily to

92.0% inhibited after two hours, and was 94.1% inhibited at the end of

six hours. There was no inhibition when terbufos was added. This

is not entirely surprising as terbufoxon, not terbufos, is the

form that inhibits AChE. What is surprising is that I saw no

conversion of terbufos to terbufoxon during the seven hours of

this study.

In order to compare what happened ^ vitro to ^ vivo, I applied

approximately the 1981 concentration of carbofuran (0.029 ptg/insect)

or terbufos (0.05 pg/insect) to the ventral abdomen of field-collected

WCR adults. Groups of 30 insects were held in glass petri dishes under

the same conditions used during bioassays. At the end of an appropriate

time interval (1, 2, 4, 8, 12, 24, or 48 hours), insects in each group were separated into live and dead categories. Live insects were those that showed any response to tactile stimulation. Each category of insects was rinsed in acetone, homogenized, and the AChE activity assayed. Figure 9. Plot of percent remaining activity versus time when

the concentration of carbofuran is added to SCR

larval homogenates. Assays contained a total volume

of 3.22 ml, 310 /iM DTNB, 25 /xM ASCh, 0.1 ml 2.5% wt/vol

homogenate, and were carried out at pK 7.5 in 0.1 M

phosphate buffer. % Remaining activity

o> w M Figure 10. Plot of percent remaining activity versus time

when the LD^g concentration of terbufos is added

to SCR larval homogenates. Assays contained a

total volume of 3.22 ml, 310 fiM DTNB, 25 fzM ASCh,

0.1 ml 2.5% wt/vol homogenates, and were carried out

at pH 7.5 in 0.1 M phosphate buffer. % Remaining activity M M K> <^00 O h) O O O O O 63

Table 6 and Figure 11 give the results of this study. The 24-hour values for terbufos in Table 6 are not plotted in Figure 11 and the

48-hour values are not given because of interference in the assay.

The initial rates of ASCh hydrolysis with both the live and dead categories were negative at 48 hours. The cause may be the binding of thiol metabolites of terbufos to the anion, 5-thio-2-nitrobenzoic acid

(RS in equation 9 on page 23). When thiocholine reacts with DTNB, the anion (RS ) is produced. The anion absorbs at 412 nm and the increase in the production of anion is what is measured colorimetrically.

A solution of DTNB has a yellow color and an absorbance at 412 nm.

This indicates that some anion is free in solution. If the anion is binding with thiol metabolites of terbufos faster than ACh is being hydrolyzed, then the amount of anion in solution would be decreasing and would account for the negative initial rates observed. The initial rate of ASCh hydrolysis at 24 hours is lower in the live category than expected for recovering insects, therefore, I suspect that thiol meta­ bolites are interfering at 24 hours, too. 64

Table 6. In vivo AChE activity of field-collected WCR adults treated

with the LD^g concentration of carbofuran or terbufos.

Time mole (x 10 ASCh hydrolyzed/min/g (hr) Carbofuran Terbufos

Control 2.42 3.03

1 1.16 2.16

2 1.46 1.76

4 - alive 1.38 1.86

- dead 1.27

8 - alive 1.61 2.54

- dead 1.76 0.97

12 - alive 1.61

- dead 1.53

24 - alive 1.82 2.24

— dead 1.34 0.81

48 - alive 2.48

— dead 1.38 Figure 11. Plot of percent remaining AChE activity versus

time from WCR adults treated in vivo with the 50 concentration of terbufos or carbofuran. Assays

contained a total volume of 3.22 ml, 310 ftM DTNB,

25 /iM ASCh, 0.1 ml 2.5% homogenate, and were

carried out at pH 7.5 in 0.1 M phosphate buffer.

Terbufos - • = alive/dead through four hours;

• = dead and •= alive at eight hours.

Carbofuran - # = alive/dead through two hours;

#= dead and 0= alive at four hours

and after. 66

100

>. 40

80

60

40

12 24 36 48 Hours 67

DISCUSSION

Mullins and Pieters (1982) studied the relationship of values

{ligJz body weight) for methyl parathion and permethrin over a range of

tobacco budworm larval weights. They found that the LD^g values based

on p#/g body weight were not constant over the weight classes they

studied (20 to 60 mg/larva, second- to third-stage larvae). Robertson

et al. (1981) also studied weight as a variable in the response of an

insect to an insecticide. Robertson et al. (1981) used sixth-stage

western spruce budworm larvae in a range of weights (15 to 120 mg/larva)

and treated them with either mexacarbate, pyrethrins, or DDT. They also

found that response was not a simple proportional function of weight.

Assuming that the cause of death in com rootworm larvae is the

inhibition of AChE, my findings of similar ID^g values based on ^g/insect,

and inferring from the above findings in tobacco and western spruce

budworm larvae, I would have expected that the amount of activity of

AChE per SCR larva would remain fairly constant over a range of weights

within a stage. This is not what I found. My study showed that a plot

of AChE activity per SCR larva versus weight (Figure 7, 7a) has an

increasing slope. This indicates that as the larva increases in size,

the amount of AChE also increases proportionally. The amount of

protein per gram of insect (Figure 8, 8b), the AChE activity per micro­

gram protein (Figure 8, 8a), and the AChE activity per gram body weight (Figure 7, 7b) remain fairly constant through the second and third stages of SCR larvae. I infer from these results that the larger the larvae, the larger the LD^^ values will be unless there 68

are target sites other than AChE that are not weight related. Therefore,

expressing values in ^g toxicant/g body weight would be the more

accurate form to use when comparing larval LD^o This inference

is in direct opposition to that found by Mullins and Pieters (1982) and

Robertson et al. (1981). I would like to see a study where the relation­

ships between larval weight, AChE activity, and value were examined over a range of weights within a population of insects. A study of

this type would clarify on which basis (pig/g body weight or /ig/insect)

LD^g values of different sized larvae should be compared. Also, a study of this sort would indicate whether target sites other than AChE (that are not weight related) have any influence on the response of larvae to an insecticide.

Toxicity trends that were discernible when compared on a pg/insect basis are not so clear-cut when expressed as pg/g. One of these is the similarity of the degree of susceptibility between the same stages of

NCR and WCR. Table 2 (average values over the three years of the study) shows that on a fxg/insect basis, the SCR adults and larvae were less susceptible to carbofuran and terbufos than their respective stages in NCRs and WCRs- This is not so clearly seen when examined on a pg/g basis. In fact, the terbufos value for SCR adults is lower than that for NCR adults.

The degree of susceptibility to an insecticide of one stage compared to a later stage is not consistent between carbofuran and terbufos.

For all three species of com rootworms, the third-stage larvae are less susceptible to carbofuran than the adults. The reverse 69

is true with terbufos and WCR and SCR- Toxicity studies on carbofuran and terbufos conducted in South Dakota confirm that all the above trends occur in other populations of WCRs and SCRs (Sutter, 1982 and personal communication). Hamilton (1966) found that aldrin-resistant WCR larvae were less susceptible to aldrin than the adults. Since the trend of stage susceptibility is not consistent between chemical classes, both adults and larvae of a species should be assayed when monitoring for developing resistance to insecticides.

The agreement of the values in my study with those from surrounding states is very good. For carbofuran on WCR adults, my average value over the three years was 0.02 pig/insect. Metcalf (in preparation) found an average value of 0.03 /xg/insect for WCR adults treated with carbofuran in 1980. My values (}JLg/g) are approximately two times greater than those reported by Ball (1981b) for 1979-1981.

These differences in values are reduced if one takes into account the fact that Ball's values are determined by two hour assays, whereas, my values are based on 48 hour studies, the differences in techniques used, and the statistical deviations of each value. For terbufos on WCR adults, my average value over the three years was 0.08 ^g/insect. This is the same value found by Metcalf at the University of Illinois (in prepa­ ration) for WCR adults in 1980. Comparing my terbufos data to those of

Ball (1981b), my values are 0 to 1.5 times -greater than Ball's 1979-1981

LD^Q values. I infer from these results that no "pockets" of resistance are developing to either of these chemicals

Sutter (1982) has determined LC^^ values for carbofuran and 70

terbufos in soil of third-stage SCR and WCR larvae- Using his

values and the average values (;

the ratio of LC^Q/LD^Q equals 4.67 in both species for carbofuran and

2.00 and 4.00 in SCR and WCR larvae, respectively, for terbufos (Table

7). It would be of great interest to see how these ratios vary with

different soil types.

Table 7. LC^g, and LC^q/LD^q values for carbofuran and terbufos

on third-stage WCR and SCR larvae.

"so" ^^50 ^S0/^°50 Insecticide Insect (ug/g soil) (Wg/insect)

Carbofuran WCR 0.56 0.12 4.67

SCR 0.98 0.21 4.67

Terbufos WCR 0.04 0.01 4.00

SCR 0.08 0.04 2.00

^Sutter (1982).

The degree of susceptibility between the adult stage of two

different families in the order Coleoptera is not the same for carbo­ furan and terbufos. The toxicity pattern of carbofuran > 3-keto- carbofuran > 3-hydroxycarbofuran is the same for Pterostichus chalcites (Hsin et al., 1979) and WCR although the values are 71

much greater for 2- chalcites than for WCRs (Table 8). Differences

in analogue toxicities between species are much less with the

terbufos series than the carbofuran series; possibly indicating

some physiological or morphological differences between the two species.

Table 8. Comparison of values of analogues of carbofuran and

terbufos insecticides on WCR beetles and Pterostichus chalcites

beetles.

Compound Pterostichus chalcites^ Diabrotica virgifera

Carbofuran 1.4 2.00

3-hydroxycarbofuran 126 10.45

3-ketocarbofuran 72 9.69

Terbufos 3.6 4.52

Terbufos sulfoxide 3.6 3.11

Terbufos sulfone 4.3 3.87

Terbufoxon 3.7 3.81

Terbufoxon sulfoxide 4.1 5.15

Terbufoxon sulfone 4.3 5.95

values from Hsin et al. (1979). 72

In looking at the characteristics of rootworm AChE, I determined that DTNB in the range 31 to 1,242 ;xM did not inhibit the enzyme.

The plot of initial rates of ASCh hydrolysis versus homogenate concen­ tration was linear. The rate of ASCh hydrolysis was linear at all homogenate concentrations studied except 5% wt/vol. The cause of this nonlinear rate of hydrolysis is not known. The K for SCR larvae in is 9.3 fiM. and approximately 21 ptM for both WCR larvae and adults. AChE showed characteristic substrate inhibition patterns when the concentration of ASCh was greater than 25 (xVL for SCR larvae and 50 fxM. for WCRs. The

values ranged from 369 for SCR larvae to 794 iM for F1 WCR adults.

When Manulis et al. (1981) incubated the LC^^ concentration of aldicarb ^ vitro for 15 minutes with spirea aphid AChE, they saw approximately 90% inhibition of enzyme activity. They felt that such a large amount of inhibition at the end of the 15 minute incubation period indicated that almost no decarbamylation occurred during this time.

This is analogous to the ^ vitro inhibition I saw with the concen­ tration of carbofuran added to third-stage SCR larval homogenate. AChE activity was severely inhibited (82%) within the first five minutes.

Inhibition increased steadily to 94% inhibition occurring at the end of six hours. There was no recovery of the enzyme in the in vitro study-

Spirea aphids fed a diet containing the LC^^ concentration of aldicarb had AChE that was inhibited 50% at the end of 24 hours (Manulis et al.,

1981). The relatively low ^ vivo inhibition as compared to ^ vitro inhibition was thought to be due to decarbamylation of aldicarb occurring in the intact aphid that did not occur in the homogenate. In vivo, in WCR 73

adults, the concentration of carbofuran caused 52% inhibition of

AChE at one hour post application. Moderate AChE recovery occurred

(38% inhibition) over the next 11 hours. Recovery of AChE activity in

the surviving 50% was complete by 48 hours (0% inhibition).

Manulis et al. (1981) saw no inhibition of spirea aphid AChE in

vitro when the LC^^ concentration of dimethoate was incubated with whole

insect homogenate. When I incubated the concentration of terbufos with third-stage SCR larval homogenates, the AChE activity was slightly greater than that of the control for the first three hours (101-108% of control). For the rest of the experiment, the AChE activity of the terbu­ fos treated homogenate was 98-100% that of the control. This indicates that no conversion of terbufos to terbufoxon occurred vitro•

Manulis et al. (1981) report 70% in vivo inhibition of AChE from spirea aphid which have fed for 24 hours on a diet treated with the LC^^ concentration of dimethoate. Tripathi and O'Brien (1973a) report that maximum vivo inhibition of house fly isozymes occurs within 20 to

80 minutes after treatment with the concentration of four organo- phosphorus insecticides. Maximum inhibition of head isozymes was 82% for malaoxon, 55% for paraoxon, 72% for diazinon, and 61% for dichlorvos.

All four compounds produced a maximum of 99% inhibition of house fly thoracic isozymes. Lee et al. (1978) applying slightly less than the

concentration of racemic fonofos to house flies found an in vivo 50 inhibition of 62% after one hour and a maximum of 89% after six hours.

The maximum ±a vivo AChE inhibition in surviving WCR adults treated with the LD^Q concentration of terbufos was 42% at two hours post treatment. 74

This maximum inhibition is much less than that reported for other organo- phosphorus compounds on house flies and spirea aphids. It could be due to slower penetration of terbufos through WCR cuticle and/or a faster system for metabolizing terbufos. Chio and Hetcalf (1979) have shown that at eight hours, only 34% of applied terbufos is present in WCR in the form of terbufos, terbufos sulfone, terbufos sulfoxide, terbufoxon, and terbufoxon sulfoxide; only 14% is present at 24 hours; and only

6% at 48 hours.

The interaction of dosage-mortality, dosage-AChE inhibition, and

AChE inhibition-mortality appears to be complex. More detailed experi­ mentation needs to be done before an attempt should be made to explain these interactions. A repetition of the procedures I used for determining

LD^Q values, in vivo inhibition, and in vitro inhibition conducted on the same stage within one or two generations of a population and over a range of insecticide concentrations would give a clearer "picture" of what is occurring. 75

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ACKNOWLEDGMENTS

"You will meet with several observations and experiments which disappoint your expectation, either not at all succeeding constantly, or at least varying much from what you expected." Robert Boyle, 1673

I give my thanks to Dr. Paul A. Dahm for his support of this

research and the hours he spent "tightening up" this dissertation.

My thanks also go to Dr. Jon J. Tollefson for the use of his insect

rearing equipment and labs. Special thanks to Alan Henn for his

extra WCR larvae when equipment failures "cooked" mine repeatedly.

Thanks to Dr. Herbert J. Fromm for the use of his labs and equipment;

without which, the enzyme work would not have been as easily done.

To the Fromm research group, Leif, Nancy, Sandy, and Mark, my thanks for adopting me and putting up with "Have lab - will travel". 87

APPENDIX A: LD^q VALUES OF INSECTICIDES ON DIABROTICA SPECIES 88

Table 9. LD^g values of insecticides on adult NCRs.

Insecticide (ftg/insect) (ug/ g) Reference

Chlorpyrifos 0. 049 7.00 Chio et al., 1978

Diazinon 0..03 (Missouri) Ball and: Weekman, 1963 0.,19 (Nebraska) Ball and. Weekman, 1963 0.01 (Iowa) Hamiltoni, 1965 0.02 (S. Dakota) Hamilton, 1965 0.076(Illinois) 10.81 Chio et al., 1978

Fensulfothion 0.010 1.44 Chio et al., 1978

Fonofos 0.058 8.25 Chio et al., 1978

Malathion 0.156 22.18 Chio et al., 1978

Phorate 0.080 11.38 Chio et al., 1978

Terbufos 0.026 3.71 Chio et al., 1978

Carbaryl 0.008 1.14 Chio et al., 1978 0.037 3.78 Metcalf, 1979

Carbofuran 0.0013 0.186 Chio et al., 1978 0.0131 1.34 Metcalf, 1979

Aldrin 0..79-1 .55 (lowa) Ball and Weekman, 1963 16.85 (Minnesota)— Ball and Weekman, 1963 1.77 (Missouri)— Ball and Weekman, 1963 0.77 (Nebraska)— Ball and Weekman, 1963 (Ohio)36.3-2395.0 Blair• et al., 1963 3..70-9 .87 (Iowa) Hamilton, 1965 0,.07-0 1.16 (Indiana) Gould et al., 1970 0,.07-29.2 (Indiana) Gould, 1971 (Illinois)>1000 Chio et al., 1978 89

Table 10. values of insecticides on adult SCRs.

^°50 ^50 Insecticide (A^g/insect) (ng/'g) Reference

Chlorpyrifos 0.101 4.81 Chio et al., 1978 0.188 9.02 Metcalf, 1979

Diazinon 0.743 Smith, 1977 0.43 20.47 Chio et al., 1978

Fensulf o thion 0.211 10.05 Chio et al., 1978

Foncfos 1.489 Smith, 1977 0.372 17.71 Chio et al., 1978 1.015 48.72 Metcalf, 1979

Malathion 7.36 350.41 Chio et al., 1978

Parathion 0.141 Smith, 1977

Phorate 0.481 Smith, 1977 0.19 9.26 Chio et al., 1978

Terbufos 0.11 5.24 Chio et al., 1978 0.195 9.36 Metcalf, 1979

Carbaryl 0.11 5.24 Chio et al., 1978 0.581 27.89 Metcalf, 1979

Carbofuran 0.0738 Smith, 1977 0.0046 0.23 Chio et al., 1978 0.086 4.15 Metcalf, 1979

Aldrin 0.365 Bigger, 1963 6.831 Smith, 1977

Landrin 1.031 Smith, 1977

Bux 0.736 35.32 Metcalf, 1979 90

Table 11. ED^g values of insecticides and analogues on larval SCRs

(Waller, 1972).

Chemical (^g/g)

Phorate 3.5

Phorate sulfoxide 3.1

Phorate suifone 20.5

Phoratoxon 2.1

Phoratoxon sulfoxide 3.3

Phoratoxon sulfone 13.0

Terbufos 1.6

Terbufos sulfoxide 2.0

Terbufos sulfone 2.3

Terbufoxon 1.0

Terbufoxon sulfoxide 1.3

Terbufoxon sulfone 0.8 91

Table 12. LD^g values of insecticides on adult WCRs.

Insecticide (ng/insect) (%%/%) Reference

Chlorpyrifos 0.048 4.87 Chio et al., 1978 C 060 6.12 Metcalf, 1979

Diazinon 0.04 (Kansas) 3.65 Bail and Weekman, 1963 0.06 (Missouri) Bail and Weekman, 1963 0.06-0.37(Nebraska) Bail and Weekman, 1963 0.05 (Iowa) Hamilton, 1965 0.01-0.03(5. Dakota) Hamilton, 1965 0.082(Illinois) Chio et al., 1978

Fensulfothion 0.0107 1.09 Chio et al., 1978

Fonofos 0.087 8.83 Chio et al., 1978 0.227 23.15 Metcalf, 1979

Malathion 0.116 11.78 Chio et al., 1978

Phorate 0.049 4.97 Chio et al., 1978

Terbufos 6.88-21.17 Bail, 1977 0.039 3.96 Chio et al., 1978 0.046 4.68 Metcalf, 1979

Carbaryl 0.017 1.73 Chio et al., 1978 0.073 7.49 Metcalf, 1979

Carbofuran 0.70-1.98 Bail, 1977 0.0009 0.091 Chio et al., 1978 0.0135 1.38 Metcalf, 1979

Thimet 15.65-68.42 Bail, 1977

Bux 3.52-16.13 Bail, 1977 0.042 4.34 Metcalf, 1977

Aldrin 0.04-73.87 3.8-4,476.9 Bail and Weekman, 1963 0.068-51.0 Bigger, 1963 0.02-15.50 Hamilton, 1965 120.00 Gould, 1971 1,000 Chio et al., 1978 237.5-1.399.1 Bail, 1977 92

Table 13. values of insecticides on WCR larvae (Sutter, 1982).

Ist-stage 2nd-stage Insecticide (ppm in soil) (ppm in soil)

Counter 0.03 0.04

Lorsban 0.06 0.26

Thimet 0.08 0.08

Bux 0.08 0.26

Dyfonate 0.12 0.25

Amaze 0.15 0.29

Mocap 0.16 0.41

Furadan 0.29 0.56 93

APPENDIX B: CHEMICAL STRUCTURES OF CARBOFURAN, TERBUFOS,

AND ANALOGUES 94

OH CH. .CH, CH: 'CH: OfNHCH. OgNHCH,

Carbofuran 3-hydroxycarbofuran 3-ketocarbofuran

OH .CH .CH, .CH, CH: CH: CH: OH OH OH 7-phenolcarbofuran 3-hydroxy-7-phenolcarbofuran 3-keto-7-phenolcarbofuran

S 0 EtO H EtO^jl EtO Il il ^PSCEgSCCCH^)^ PSCB^SCCCHg)^ PSCH_SC(CH_)_ Z|| J J EtO EtO EtO'

Terbufos Terbufos sulfoxide Terbufos sulfone

0 0 0 EtO. H EtO. S EtO Il II ^PSCH,SC(CH ) •PSCH SC(CH ) PSCH^SCCCH^)^ EtO^ EtO" ^ EtO 0 Terbufoxon Terbufoxon sulfoxide Terbufoxon sulfone 95

APPENDIX C: BIOASSAY PROCEDURES FOR DIABROTICA SPECIES 96

General Larval Bioassay Procedure for Diabrotica Species

1. Third-stage larvae weighing seven to 11 mg/larva should be used for bioassay purposes. If field-collected, then larvae should be held on com pans for 24 hours to eliminate those larvae that received damage during collection.

2. A minimum of 50 larvae should be weighed to determine the average weight per larva.

3. A minimum of three replicates, at 10 larvae per replicate, should be used for each dose (i.e., three petri dishes should be used with 10 treated larvae in each dish).

4. The insecticide should be dissolved in acetone or, if not soluble in acetone, a suitable solvent should be utilized.

5. A minimum of five dosages, preferably seven or eight, plus a solvent control should be chosen in a suitable dosage range.

6. One fil of test solution should be applied to the ventral abdominal region of each larva and allowed to air dry.

7. Treated larvae should be held in 100 x 15-mm glass petri dishes that have the bottom of the dish covered with moist filter paper and contain germinated com for food.

8. Treated larvae should be held at 25 ± 2® C and a 12h:12h photoperiod for 48 hours. Dishes should be checked frequently to ensure that the filter paper remains moist.

9. Larvae are counted as dead if unable to burrow into soil or show an inability to move away from tactile stimulus.

10. If the average control mortality exceeds 10%, discard data, and repeat test. 97

General Adult Bioassay Procedure for Diabrotica Species

1. Laboratory-reared adults should be treated at two weeks after emergence. Field-collected adults should be treated within 24 hours after collection.

2. A minimum of 50 adults should be weighed to determine the average weight per adult.

3. A minimum of three replicates, at 10 adults per replicate, should be used for each dose (i.e., three petri dishes should be used with 10 treated adults in each dish).

4. The insecticide should be dissolved in acetone or, if not soluble in acetone, a suitable solvent should be utilized.

5. A minimum of five dosages, preferably seven or eight, plus a solvent control should be chosen in a suitable dosage range.

6. One 111 of test solution should be applied to the ventral abdominal region of CO^ anesthetized adults.

7. Treated adults should be held in 100 x 15-mm glass petri dishes that have the bottom of the dish covered with dry filter paper and contain pieces of young corn ears for food (lettuce and dry diet may be substituted for laboratory-reared insects).

8. Treated adults should be held at 25 ± 2° C, 30 ± 10% relative humidity, and a 12h:12h photoperiod for 48 hours.

9. Adults are considered dead when they cannot regain or maintain their footing after the petri dish holding them is shaken.

10. If the average control mortality exceeds 10%, discard data and repeat test. 98

APPENDIX D: LD^q VALUES OF CARBOFURAN, TERBUFOS, AND ANALOGUES

OF EACH ON DIABROTICA SPECIES Table 14. LD^g values of carbofuran on larvae and adults of the WCR,

NCR, and SCR.

Weight Insect Stage (mg) Origin^ Date^

WCR Larval 8.4 FC 7-15-78 10.2 FC 7-18-79 10.9 Fl LR 10-27-80 8.2 Fl LR 8—19—81

WCR Adult 19.1 FC *8-17-79 17.2 FC 9— 8—80 12.0 FC 8— 5—81 16.1 FC 9- 5-81

NCR Larval 10.6 FC 7-18-79

NCR Adult 8.6 FC 8-16-79 10.8 FC 8—27—80 10.6 FC 8—16—81

SCR Larval 9.1 F36 LR 5-17-78 9.1 F36 LR 6—30—80 8.8 F36 LR 2-24-81 9.7 F36 LR. 2-25-81

SCR Adult 29.2 F36 LR *10-23-80 26.2 F36 LR 3- 5-81 28.9 F36 LR 3-10-81 18.6 FC 8—26—80

^C = field-collected, F1 LR = laboratory-reared Fl generation from field-collected adults, F36 LR = laboratory-reared, inbred SCR colony over 36 generations old.

^ates marked with * indicate manual calculation of probit analysis. Probit Equation ^50 95% Fiducial 95% Fiducial Probit = A + B(dose) ng/insect /zg/g Range

= p 3.505 + 10.074(d) 0.15 0.08 - 0.20 17.66 9.76 - 23.63 p 4.315 + 8.249(d) 0.08 0.03 - 0.25 8.14 2.95 - 24.12 = p 3.254 + 12.017(d) 0.15 0.11 - 0.19 13.19 10.13 - 16.96 = p 4.031 + 10.019(d) 0.10 0.08 - 0.12 11.75 9.24 - 14.35 p 7.656 + 1.527(log d) 0.02 0.01 — 0.03 0.95 0.66 _ 1.37 p — 3.810 + 73.282(d) 0.02 0.01 - 0.02 0.94 0.71 - 1.24 = p 3.682 + 49.838(d) 0.03 0.02 - 0.03 2.20 1.77 - 2.67 = p 3.562 + 50.498(d) 0.03 0.01 - 0.09 1.77 0.74 - 5.36

p 3.581 + 14.554(d) 0.10 0.04 - 0.17 9.20 4.10 - 16.46 p 3.619 + 130.870(d) 0.01 0.01 — 0.02 1.23 0.77 — 1.99 = p 4.394 + 26.712(d) 0.02 0.01 - 0.03 2.10 1.18 - 3.06 = p 3.710 + 90.383(d) 0.01 0.01 - 0.02 1.35 1.06 - 1.62 p = 4.053 + 3.804(d) 0.25 0.17 _ 0.63 27.36 18.92 _ 69.21 = p 3.901 + 7.192(d) 0.15 0.11 - 0.22 16.72 12.52 - 24.19 = p 3.319 + 8.436(d) 0.20 0.17 - 0.23 22.61 19.80 - 26.21 = p 3.611 + 5.525(d) 0.25 0.21 — 0.34 25.89 21.79 - 34.71

= p 8.794 + 2.323(log d) 0.02 0.02 — 0.03 0.80 0.59 — 1.07 = p 4.354 + 18.275(d) 0.04 0.02 - 0.05 1.35 0.59 - 1.92 = p 4.130 + 17.314(d) 0.05 0.04 - 0.06 1.74 1.22 - 2.18 = p 3.764 + 20.213(d) 0.06 0.03 - 0.12 3.29 1.47 - 6.60 Table 15. values of terbufos on larvae and adults of the WCR, NCR,

and SCR.

Weight Insect Stage (mg) Origin^ Date^

WCR Larval 10.2 FC 7-18-79 9.2 F1 LR 8—21—81

WCR Adult 19.1 FC *8-17-79 17.2 FC 9— 8—80 13.9 FC 7—30—81 16.5 FC 9- 5-81

NCR Adult 8.6 FC *8—16—79 10.8 FC 8—27—80 10.6 FC 8—14—81

SCR Larval 9.9 F36 LR 3-13-81

SCR Adult 27.7 F36 LR *10—21—80 18.6 FC 8—26—80

^FC = field-collected, F1 LR = laboratory-reared F1 generation from field-collected adults, F36 LR = laboratory-reared, inbred SCR colony over 36 generations old.

^Dates marked with * indicate manual calculation of probit analysis. 102

Probit Equation 95% Fiducial 95% Fiducial Probit = A + B(dose) ug/insect fxg/g

= p 2.245 + 262.317(d) 0.01 0.00 - 0.01 1.03 -0.15 - 1.44 = p 3.371 + 158.694(d) 0.01 0.01 - 0-01 1.12 0.95 - 1.28 p 7.155 + 2-277(log d) 0.11 0.08 — 0.15 5.93 4.41 - 7.79 = p 3.608 + 16.846(d) 0.08 0.07 - 0.12 4.81 3.86 - 6.77 = p 4.250 + 11.945(d) 0.06 0.03 - 0.09 4.52 2.44 - 6.39 = p 2.384 + 43.004(d) 0.06 0.05 - 0.11 3.69 3.00 - 6.58 p 7.155 + 2.277(log d) 0.11 0.08 — 0.15 13.16 9.80 -17.29 = p 3.856 + 13.449(d) 0.08 0.07 - 0.10 7.87 6.61 - 9.48 = p 5.021 + 12.623(d) 0.00 —0.08 - 0.02 -0.15 -7.77 - 2.09

= p 1.231 + 103.150(d) 0.04 0.03 - 0.04 3.70 3.38 - 4.10 p 8.168 + 3.430(d) 0.12 0.10 — 0.15 4.30 3.62 - 5.53 = p 4.502 + 4.605 0.11 0.04 - 0.16 5.83 2.42 - 8.83 Table 16. LD^g values of carbofuran, terbufos, and five analogues of

each on WCR adults and 3-hydroxycarbofuran on SCR larvae.

Weight Chemical Insect Stage (mg) Origin^ Date

Carbofuran WCR Adult 12.0 FC 8— 5—81

3-hydroxycarbofuran 13.0 8-13-81

3-ketocarbofuran 11.5 8-13-81 1 1 00 00 7-phenolcarbofuran 11.4 Ui

3-hydroxy-7-phenolcarbofuran 13.0 8— 5—81

3-keto-7-phenolcarbofuran 13.0 8— 5—81

Terbufos WCR Adult 13.9 FC 7-30-81

Terbufos sulfoxide 11.6 7—30—81

Terbufos sulfone 13.2 7-30-81

Terbufoxon 13.9 7-31-81

Terbufoxon sulfoxide 15.8 7-31-81

Terbufoxon sulfone 14.5 7-31-81

Carbofuran SCR Larval 9.7 F36 LR 2-25-81

3-hydroxycarbofuran 10.3 3— 9—81

3-hydroxycarbofuran 10.3 3-11-81

^C = field-collected, F36 LR = laboratory-reared, inbred SCR colony over 36 generations old- 104

Probit Equation 95% Fiducial 95% Fiducial Probit = A + B(dose) #g/insect Range ^g/g Range

p = 3.682 + 49.838(d) 0.03 0.02 - 0.03 2.20 1.77 - 2.67

p = 4.227 + 5.689(d) 0.14 0.09 - 0.17 10.45 6.79 - 13.21

p = 4.710 + 2.612(d) 0.11 -0.08 - 0.22 9.69 -7.41 - 19. 15

>8.76 >768.07

>8.58 >660.78

>8.58 >657.82

P = 4.250 + 11.945(d) 0.06 0.03 - 0.09 4.52 2.44 - 6.39

P = 4.427 + 15.866(d) 0.04 0.00 - 0.06 3.11 -0.23 - 4.90

P = 4.105 + 17.494(d) 0.05 0.04 - 0.06 3.87 2.77 - 4.86

P = 2.875 + 40.148(d) 0.05 0.04 - 0.06 3.81 3.19 - 4.40

P = 4.364 + 7.807(d) 0.08 0.03 - 0.11 5.15 1.84 - 7.17

P = 3.505 + 17.337(d) 0.09 0.07 - 0.10 5-95 4.63 - 7.15

P = 3.611 + 5.525(d) 0.25 0.21 - 0.34 25.89 21.79 - 34.71

P = 4.107 + 0.700(d) 1.28 0.98 - 1.57 123.79 94.70 - 152.73

P = 4.239 + 0.782(d) 0.97 0.59 - 1.25 94.56 56.90 - 121.17