Mechanisms of carbaryl resistance in the fall armyworm, Spodoptera frugiperda

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Title:
Mechanisms of carbaryl resistance in the fall armyworm, Spodoptera frugiperda
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Carbaryl resistance in the fall armyworm, Spodoptera frugiperda
Spodoptera frugiperda
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xv, 103 leaves : ill. ; 28 cm.
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McCord, Elzie
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Subjects / Keywords:
Fall armyworm -- Insecticide resistance   ( lcsh )
Insecticide resistance   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
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bibliography   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1985.
Bibliography:
Bibliography: leaves 92-101.
Statement of Responsibility:
by Elzie McCord Jr.
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Typescript.
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Vita.

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University of Florida
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Full Text











MECHANISMS OF CARBARYL RESISTANCE IN THE FALL ARMYWORM,
Spodoptera frugiperda










BY

ELZIE McCORD, JR.


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA


1985



























I Elzie McCord, Jr., dedicate this dissertation to:

o my family, Pinkie W., wife, Rogers Christopher and

Timothy Ryan, sons, for their continued support,

understanding and companionship, and

o Ms. Lue Vester Davis for being an ideal role model,

for forfeiting her one free hour during the school

day to teach a select few of us the slide rule, basic

and advanced algebra and trigonometry, for coercing

parents to impress upon their children the importance

of performing well in school and for inspiring my

career in the biological sciences.










ACKNOWLEDGEMENTS


Since it is nearly impossible to thank everyone who has contributed

to the success of my career, this research and the preparation of this

manuscript, I will attempt to mention those who have contributed the

most without diminishing the roles of those not named here.

I wish to thank the following people:

o My parents, Pearlence C. and Elzie, Sr., for their continued

encouragement and support in my pursuit of personal goals and for

guiding me in a direction that made goal selection possible,

o Dr. S. H. Kerr, for supporting me throughout my pre- and post-

graduate studies at the University of Florida,

o Dr. Simon S. J. Yu for allowing me the opportunity to study and

work with him under somewhat accelerated conditions; for providing

encouragement, constructive criticism, and assistance in performing

the complex biochemical processes for which he is highly regarded,

and for his belief in me and my abilities to accomplish the program

described herein,

o Drs. J. L. Nation, J. R. Strayer, D. L. Shankland, and R. B.

Shireman for quality educations instructions, for serving on my

supervisory committee and for reviewing this manuscript,

o Mr. and Mrs. Siegfried J. Schulze, Mr. Willie Foresto, Ms. Alice

Foresto and Mr. Lutz Schulze for being such wonderful neighbors who

took care of my family while I completed the research herein,

o Dr. J. R. Young, USDA, Agricultural Research Center, Tifton,

Georgia, for inspiring this research project and for providing me

with the resistant fall armyworm strain,










o Drs. A. B. Meade, K. S. Amuti, Der-I Wang, T. M. Priester, Ms.

Babirette Babineaux, Ms. C. N. Selz, Mr. Matthew McGirr and others

who have contributed moral support, ideas, constructive criticisms,

drawings, computer wizardry, etc.,

o The Agricultural Chemicals Department of the E. I. Du Pont de

Nemours & Company, Drs. Dale Wolf, K. A. Saegebarth, G. D. Hill, H.

M. Loux, J. W. Searcy, E. J. Soboczenski and others who assisted in

my leave of absence request and those who approved the granting of

that request,

o Mr. Philip N. Chaney and Mr. J. L. Jenkins for being the true

friends I always wanted and needed,

o Mrs. Jo Ann Ledford and Mrs. Glinda Burnett for their continued

friendship and assistance since 1973, and

o Last but not least, my wife Pinkie W. and my sons Rogers

Christopher and Timothy Ryan for enduring the hardships of daily

activities without me for 18 months, for tolerating my absence

while in Florida and while I was locked away in my home office.

















TABLE OF CONTENTS


ACKNOWLEDGEMENTS . .


LIST OF TABLES . .


LIST OF FIGURES . .


LIST OF ABBREVIATIONS .


ABSTRACT . .


INTRODUCTION . .


LITERATURE REVIEW . .


Status of Resistance .


Genetics of Resistance .


Resistance Mechanisms .


Reduced Penetration .


Altered Site Insensitivity


Increased Detoxication .


Cytochrome P-450 Monoo


Hydrolases .


A. Phosphotriest


B. Arylester Hyd


C. Carboxylester


Glutathione S-transfer


Epoxide Hydrolases


History of Carbaryl Resistance


Fall Armyworm Resistance to Carb


Page


. . iii


. . viii


. . x


ro. . xii


Hy. . xiv


. . 1


. . 4


. . 4


. . 6


.a . 9


. . 9


. . 11


. . 14


xygenases . 17


. . 21


er Hydrolysis 22


rolysis . 22


Hydrolysis . 22


ases . 24


. . 25


. . 26


aryl . 27










. . . 28


Insects . . . 28

R Strain . . ... 28

S Strain . . ... .28

Chemicals . . . 29

Bioassay . . .. 29

Protein Determinations . ... 29

Epoxidation Assay. .. . . 30

Microsomal Biphenyl Hydroxylase Assay . .. 34

Microsomal N-Demethylase Assay . .. 37

Cytochrome P-450 Measurements . .. 37

Glutathione S-Transferase Assay . .. 40

In vitro Carbaryl Metabolism Study. .. .. 43

Epoxide Hydrolase Assay . ... 44

Esterase Assays . . ... .48

Acetylcholinesterase Assay. . .. 48

Cuticular Penetration by Carbaryl. .... 51

Statistics . . . 54

RESULTS . . ... . 55

Bioassays . . . 55

Enzymatic Assays . .... .... 58

A. Aldrin Epoxidase . ... 58

B. Biphenyl Hydroxylase . .. 58

C. N-demethylase . ... 61

D. Cytochrome P-450. ..... .. 61


E. Glutathione S-transferase and Epoxide Hydrolase

F. Esterase . . .


S 61

67


MATERIALS AND METHODS










G. AChE Kinetics . ... 67

H. In vitro Carbaryl Metabolism . .. 67

Cuticular Penetration . ... 75

DISCUSSION . . .. . 82

LITERATURE CITED . . ... .. 92

BIOGRAPHICAL SKETCH ...................... 103














LIST OF TABLES


Table Page



1 The primary action of MFO systems on specific chemical
configurations found in xenobiotic molecules .. .19

2 Rf values of carbaryl and its metabolites on silica gel
G plates in a developmental solution of acetic acid:
ethyl acetate:benzene (1:10:33 by volume) ...... 45

3 Comparison of toxicological responses of R and S fall
armyworm larvae topically treated with 6 insecticides 56

4 Comparison of toxicological responses of R and S fall
armyworm larvae topically treated with carbaryl + PB .57

5 Aldrin epoxidase activities of midgut microsomes and
homogenates from various instars of R and S fall
armyworm larvae . .... .59

6 Microsomal biphenyl 4-hydroxylase activity in various
instars of R and S fall armyworm larvae ... 60

7 Microsomal N-demethylase activity from sixth-instar R
and S fall armyworm larvae . ... 62

8 Cytochrome P-450 activity from midgut microsomes of
sixth-instar R and S fall armyworm larvae ... 63

9 Glutathione S-aryltransferase activity of midgut soluble
enzyme fraction from sixth-instar R and S fall armyworm
larvae . . ... ...... 64

10 Microsomal epoxide hydrolase activity in sixth-instar R
and S fall armyworm larvae . ... 65

11 General and carboxylesterase activities from crude homo-
genates of sixth-instar R and S fall armyworm larvae 66

12 General and carboxylesterase activities from microsomes
of sixth-instar R and S fall armyworm larvae 68

13 Acetylcholinesterase activity from moth heads of 1 to 2
day old mixed population R and S fall armyworms 69


viii










14 In vitro metabolism of carbaryl by midgut homogenate
from R and S fall armyworm larvae . .. 74














LIST OF FIGURES


Figure Page

1 Metabolism of lipophilic foreign compounds ..... 16

2 The reaction of aldrin with midgut microsomes to produce
the epoxide product, dieldrin . .... 33

3 The reaction of biphenyl with microsomes to produce the
oxidative metabolite 4-hydroxybiphenyl ... 36

4 The reaction of p-Chloro-N-methyl aniline with microsomes
to produce the demethylated product p-Chloroaniline 39

5 The reaction of 3,4-dichloronitrobenzene with glutathione
-S-aryltransferase to produce the conjugated product S-(2-
Chloro-4-nitrophenyl) glutathione . .... 42
14
6 The reaction of [ C] styrene oxide with water and
microsomes to produce the water soluble product, styrene
glycol . . . 47

7 The reaction of a-naphthylacetate with esterases to form
a-naphthol and acetic acid . .... 50

8 The reactions of acetylthiocholine with acetylcholin-
esterase producing thiocholine which produces a yellow
color when combined, in reaction, with 5-dithiobis-2-
nitrobenzoic acid . . .. 52

9 Lineweaver-Burke plot for the reaction of R and S fall
armyworm moth head acetylcholinestera e with acety thio-
choline V = product formed (nmol min mg protein );
[ATC] = substrate concentration (mM) ... 71

10 Carbaryl inhibition of AChE from heads of R and S fall
armyworm adult moths. .. . 73
14
11 Percent of applied dose of [ C] carbaryl remaining on
the cuticle of sixth-instar R and S fall armyworm
larvae . . ... .77
14
12 Percent of applied [ C] carbaryl extracted from homoge-
nate of sixth-instar R and S fall armyworm larvae 79
14
13 Percent of applied [ C] carbaryl recovered from excreta
of sixth-instar R and S fall armyworm larvae 81










14 Aldrin epoxidase activities of midgut microsomes from
various instars of R and S fall armyworm larvae .. 85

15 Microsomal biphenyl 4-hydroxylase activities from various
instars of R and S fall armyworm larvae ... 87

16 Metabolic pathways of carbaryl . ... .90














LIST OF ABBREVIATIONS


AChE Acetylcholinesterase enzyme

ATC Acetylthiocholine

BHC Benzene hexachloride (See HCH)

BSA Bouine serum albumin

CPB Colorado potato beetle

DBLS Diazoblue laurylsulfate

DCNB 1,2-dichloro-4-nitrobenzene

DDT p,p' dichloro-diphenyl trichloroethane

DEF S,S,S-tributyl phosphorotrithioate

DFP-ase Phosphotriester hydrolase

DMC bis-(p-chlorophenyl) methyl carbinol

DTNB 5,5-dithiobis-2-nitrobenzoic acid

FAW Fall armyworm

GSH Glutathione

HCH Hexachlorohexane (see BHC)

HC1 Hydrochloric acid

HPLC High performance liquid chromatography

IBP S-benzyl 0,0-disopropyl phosphorothioate

Kdr Knockdown resistance

K. Inhibition constant

K Binding affinity
m
MFO Microsomal mixed-function oxidase

a-NA a-naphthylacetate

B-NA B-naphthylacetate


xii










NADPH Nicotinamide adenine dinucleotide phosphate

O.D. Optical density

OP Organophosphate insecticide

p-NPA p-Nitrophenyl acetate

PB Piperonyl butoxide

PCA p-chloroaniline

PCMA p-chloro-N-methylaniline

PCMB p-chloromercuribenzoate

PDAB p-dimethylaminobenzaldehyde

PHMB p-hydroxymercuribenzoate

R Resistant insect strain

R-AChE Acetylcholinesterase enzyme from resistant strain

R-V Maximum reaction velocity of resistant strain
max
S Susceptible insect strain

S-AChE Acetylcholinesterase enzyme from susceptible strain

S-V Maximum reaction velocity of susceptible strain
max

TLC Thin layer chromatography

TOCP Tri-creosyl phosphate

TPP Triphenyl phosphate

USDA United States Department of Agriculture

V Maximum reaction velocity
max
WHO World Health Organization


xiii














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of
Doctor of Philosophy



MECHANISMS OF CARBARYL RESISTANCE IN THE FALL ARMYWORM,
Spodoptera frugiperda

By

Elzie McCord, Jr.

August, 1985

Chairman: Dr. S. J. Yu
Major Department: Entomology and Nematology


Mechanisms of resistance to carbaryl were investigated in larvae of

the fall armyworm, Spodoptera frugiperda (J. E. Smith). Piperonyl

butoxide greatly reduced the resistance ratio from > 90-fold to 6-fold

suggesting the involvement of microsomal cytochrome P-450-dependent

monooxygenases. In vitro metabolic studies revealed that oxidative

metabolism of carbaryl by midgut microsomes was 5 times more active in

the resistant strain compared to the susceptible strain. In addition,

activities of midgut microsomal epoxidase and hydroxylase were signifi-

cantly higher during the larval stage in the resistant strain than in

the susceptible strain.
14
Cuticular penetration studies using [ 14C] carbaryl showed that 60%

of the applied radioactivity remained on the cuticle of resistant larvae

while 32% remained on susceptible larvae 24 hr after topical treatment.

There was no difference in the amount of radioactivity found internally

in the two strains. Susceptible larvae, however, excreted 4 times more


xiv










radioactivity than resistant larvae. It is concluded that enhanced

oxidative metabolism of carbaryl plays an important role in the carbaryl

resistance. Slower penetration of carbaryl in the resistant armyworm

may be a minor factor contributing to resistance.














INTRODUCTION


Resistance has been defined as "the developed ability in a strain

of insect to tolerate doses of toxicants which would prove lethal to the

majority of individuals in a normal population of the same species"

(Anonymous 1957). It is preadaptive in nature, representing a selection

of genes already present in the population. As susceptible individuals

are killed from the selected population, resistant individuals breed and

pass resistance genes to their progeny. The continued use of the same

or similar insecticides increases the selection pressure on the popula-

tion and causes resistance expression in the majority of the individuals

in that population. Georghiou and Mellon (1983) reported that a total

of 428 insect and acarina species were resistant to one or more

insecticide classes including those commonly used today. These insecti-

cide classes include DDT-analogues, cyclodiene/BHC, organophosphates

(OP's), carbamates, insect growth regulators, pyrethrins and the newer

synthetic pyrethroids (Priester 1979; Wolfenbarger et al. 1981; Sparks

1980; Bull 1981; Brown 1981).

Insect resistance to insecticides can be divided into two types,

behavioral resistance and physiological resistance. Recent evidence

shows that both types of resistance often coexist in resistant individ-

uals (Lockwood et al. 1984). Behavioral resistance is mostly stimulus

dependent, requiring sensory stimulation to achieve avoidance. Insects

with behavioral resistance are more sensitive and are able to respond to

lower concentrations of insecticides than are susceptible insects.










There are three main types of physiological resistance, namely, in-

creased detoxication, reduced penetration, and target site insensitivity.

Increased insecticide metabolism by specific detoxication enzymes

was found to confer carbamate, organophosphate and/or chlorinated

hydrocarbon resistance in numerous species of insects (Hughes 1982; Yu

and Terriere 1979; Motoyama et al. 1980; Kuhr 1970; Kao et al. 1984;

Devonshire and Moores 1982; Clark et al. 1984; Rose and Sparks 1984;

Plapp 1970; Wool et al. 1982).

Reduced penetration as a resistance mechanism was reported in

several insect species (Eldefrawi and Hoskins 1961; Ku and Bishop 1967;

Hanna and Atallah 1971; Ahmad et al. 1980; Ariaratnam and Georghiou

1975; Patil and Guthrie 1979; Sinchaisri et al. 1978).

Target site insensitivity, including insensitive acetylcholines-

terase as a resistance mechanism was reported in several insect species

(Roulston et al. 1968 and 1969; Iwata and Hama 1972; Hama and Iwata 1971

and 1978; Devonshire 1975; Yamaoto et al. 1977; DeVries and Georghiou

1981a and 1981b; Yeoh et al. 1981; Devonshire and Moores 1984).

Carbaryl (1-Naphthyl-N-methylcarbamate), a reversible cholinester-

ase inhibitor, is an agricultural pesticide used in the control of over

150 major pests (Mount and Oehme 1981). Carbaryl is safe to mammals,

having an acute oral LD50 greater than 500 mg/kg body weight in rats

(Mount and Oehme 1981; Terriere 1982). It is short lived in the

environment. However, its high toxicity to honey bees has restricted

its use on some highly pollinator dependent crop plants, and has limited

its time of application on others. The apparent success of carbaryl

since its introduction in 1956 has been due to its reliability of

control, safety to humans and wildlife, and the array of insects con-

trolled.










The fall armyworm, Spodoptera frugiperda (J. E. Smith), is a vora-

cious phytophagous insect pest of the southeastern U.S. and the tropics

(Luginbill 1928; Vickery 1929). The fall armyworm (FAW) damages many

crop plants by feeding on leaves and fruit, often consuming the entire

leaf, except the mid-rib, or producing holes in the leaves (Vickery

1929) as a result of sporadic feeding.

Young and McMillian (1979) reported that FAW had become resistant

to carbaryl insecticide but remained susceptible to a related carbamate

insecticide, methomyl. FAW resistance to the organophosphates, tri-

chlorfon, diazinon, methyl parathion and parathion was reported by Bass

(1978).

It is important to study the mechanisms of resistance in order to

better understand how to slow down or lessen the severity of widespread

insect resistance to insecticides. The research reported here was

designed to investigate resistance mechanisms in a field collected

resistant strain of FAW.

Specific objectives were to determine the following:

1. The susceptibility of the field collected strain to related

carbamates, organophosphorous and synthetic pyrethroid insecticides as

compared to a susceptible laboratory strain.

2. The activities of various detoxication enzymes in the resistant

and susceptible strains.

3. The differences in the rate of cuticular penetration of car-

baryl in both strains.














LITERATURE REVIEW


Status of Resistance

Insect resistance to insecticides has been known since the early

1900s. Melander reported San Jose scale resistance to lime-sulfur in

1914 and Quayle reported resistance in the California red scale to

cyanide in 1916 (O'Brien 1967; Forgash 1984). Since these early

reports, resistance has been reported in organisms other than insects

such as bacteria, sporozoa and mammals (Georghiou and Mellon 1983).

Nowhere has the impact of organisms expressing resistance been as great

as with insects. Georghiou and Mellon (1983) reported, conservatively,

428 known insect and acarina species world-wide that have developed

resistance. Georghiou (1980) emphasized that the number of resistant

insect species is not as staggering as the number of chemicals that many

insect strains can now tolerate and the increased geographical distribu-

tion of resistant insect populations.

The wide distribution of resistant species suggests a common

phenomenon called cross-resistance which allows one organism to become

resistant to insecticides of the same and different classes due to the

same resistant mechanism (Oppenoorth and Welling 1976). Priester (1979)

reported cross-resistance in Culex quinquefasciatus Say to synthetic

pyrethroids with implication of prior DDT exposure. Scott et al. (1983)

reported cross-resistance in six predatory mite strains to permethrin

that also had previous exposure to DDT, azinphosmethyl, parathion and

carbaryl.










Resistance that is related to previous exposure suggests a genetic

change that influences massive physiological and biochemical changes in

an organism. Plapp (1984, p. 194) states that "it is becoming apparent

that changes at only a few loci are responsible for resistance to many

insecticides. That is, the genetic basis for resistance is relatively

simple. This is why cross-resistance to insecticides is such a severe

problem. Selection for resistance to a specific chemical often confers

resistance not only to the selecting agent, but sometimes to all insec-

ticides having the same mode of action and other times to virtually all

chemicals metabolized by one or more of the major detoxification enzyme

systems".

Wolfenbarger et al. (1981) reported geographical locations of

resistant Heliothis zea (Boddie), H. virescens (F.), H. armigera (Hubner)

and H. puntigera (Wallengren) in Mexico, Central America, South America,

Australia, Africa and Asia. Wolfenbarger's survey included countries or

continents where each species was indigenous. Sparks (1981) emphasized

the severity and importance of resistant Heliothis zea (Boddie) and H.

virescens (F.) in North America, concluding that these species are two

of the most serious agricultural pests. Bull (1981) noted that H.

virescens (F.) had become resistant to many of the older chlorinated

hydrocarbon and organophosphorus insecticides and apparently has some

cross tolerance to certain of the new synthetic pyrethroids and organo-

phosphorus insecticides recently developed for its control.

Graham-Bryce (1983) concluded that increases in resistance to

conventional pesticides require investigation of novel chemical ap-

proaches to crop protection. He suggested the investigation of










unexploited target sites, the modification of chemical properties of

pesticides to increase mobility and availability, the exploration of

novel formulations, and the investigation of chemical compounds that

suppress chemically mediated processes rather than functioning by direct

toxic action. The approaches suggested by Graham-Bryce (1983) would

serve to slow down resistance, produce selective compounds, reduce

mammalian toxicity and afford control comparable to more toxic, environ-

mentally persistent compounds now in use.

Genetics of Resistance

The World Health Organization's (WHO) definition of resistance

denotes resistance as a property of a population and not the result of

alterations within individual insects (Oppenoorth and Welling 1976). It

is the individual insect that possesses the preadaptive ability to

withstand higher than normal toxic doses of pesticides. Resistance is

assumed to be preadaptive arising through recurrent mutation of existing

alleles (Sawicki and Denholm 1984). Mutations of genes can be monogenic

or polygenic, and those terms are synonymous with mono- and multifac-

torial, respectively, meaning resistance is under the control of one or

several genes. It is not known which mutation will occur under which

insecticidal pressure for a given insecticide. However, Oppenoorth and

Welling (1976) predict monogenic resistance will occur if a single gene

can confer high resistance in an organism. Polygenic resistance is less

likely to occur, but may occur in organisms exposed to the selecting

agent over long periods of time.

Genetics offer a valuable tool in analysis of resistance (Oppen-

oorth and Welling 1976). Genetics can aid the separation of different

resistance mechanisms that occur simultaneously in a strain. Also

continuous environmental selection can provide researchers with rare










mutants that without genetic analysis would not be detected (Oppenoorth

1965).

To use genetics as a tool, researchers have developed various

cytogenic techniques whereby marker genes can be located on chromosomes,

and these chromosomes mapped to determine specific location of alleles

on those chromosomes. Priester (1979) used genetic crosses of Culex

quinquefasciatus Say to study inheritance of pyrethroid resistance to

isomers of permethrin. Farnham (1973) isolated four genetic resistance

factors from the house fly, Musca domestic (L.), to natural pyrethrins

and resmethrin. Priester (1979) and Farnham (1973) used bioassay

technique to determine the presence or absence of expected resistance

genes acquired during crossings.

Farnham (1973) found that the resistance genes carried no markers.

He replaced the marked autosomes of a quadruple susceptible strain with

unmarked resistance genes in an attempt to associate visible phenotypic

characters with resistant characteristics. By crossing and back-cross-

ing progeny from both fly strains, he developed four strains which were

visibly distinct and which conferred resistance factors specific for

penetration, kdr (knockdown resistance), natural pyrethrin resistance

and resistance to synergized pyrethrins. These genes were located on

chromosomes 3, 3, 5, and 2, respectively.

Predecessors of the above techniques were performed soon after the

discovery of organic insecticide resistance. Lovell and Kearns (1959)

selected house flies, Musca domestic (L.), with DDT and DMC (bis-(p-

chlorophenyl) methyl carbinol). The amount of DDT-ase present in the

fly strain selected with DDT alone was much less than in those selected

with DDT and DMC. Subsequent back crosses provided initial clues that










DDT resistance may be governed by a single partially dominant gene which

behaved according to simple Mendelian principles.

Georghiou et al. (1961) and Georghiou (1962) selected laboratory

house flies with various carbamates and tried to reverse resistance with

piperonyl butoxide (PB). They concluded that some unknown factor in the

fly was insensitive to PB because resistance could not be eliminated

entirely. They also recognized that factors other than those inhibited

by PB played a major role in carbamate resistance in highly resistant

fly strains.

Plapp and Hoyer (1968a), investigating resistance in the mosquito

Culex tarsalis Coquillet and the house fly, found that a kdr gene for

DDT resistance also conferred resistance to DDT analogues and pyrethrins

+ PB. By crossing groups of individuals in both species and using

discriminating insecticidal doses to isolate the desirable genotypes,

Plapp and Hoyer relocated unmarked genes in individuals with phenotypic

marker. No metabolic differences were found in the Resistant (R) or

Susceptible (S) strain of the mosquito or house fly that could explain

the high degree of resistance found to DDT and pyrethrin. This experi-

ment showed that resistance could occur without the presence of high

levels of detoxication enzymes and pointed toward some insensitive

resistance mechanism.

Plapp and Casida (1969) reported that genes on autosome 2 and 5 in

two house fly strains, controlled the tissue level of NADPH (reduced

nicotinamide adenine dinucleotide phosphate). NADPH levels were

controlled genetically to confer resistance to chlorinated hydrocarbon,

pyrethroid, organophosphate, and methyl carbamate insecticides.










Plapp (1970) used back crosses of two carbamate resistant house fly

strains to demonstrate resistance inheritance. By isolating heterozygo-

tes with Isolan and carbaryl, Plapp distinguished resistant flies

phenotypically and chemically. Isolated genes were located on chromo-

some 2; however, genes on chromosome 3 and 5 contributed insignificantly.

Resistance Mechanisms

The mechanisms of pesticide resistance are classified into two

categories; behavioral and physiological. Behavioral resistance is

defined as those actions that have evolved as the result of pesticide

selection which aid the organism in avoiding toxicosis (Lockwood et al.

1984). Insects that are behaviorally resistant usually avoid pesticide

residues and treated surfaces either by direct stimulation or host

and/or habitat selection.

Physiological resistance is categorized as follows: I. Physical or

restricted cuticular penetration, II. Increased enzymatic detoxication,

and III. Altered site or reduced sensitivity of a physiological endoge-

nous target (Busvine 1971; Devonshire 1973; Plapp 1976; Oppenoorth and

Welling 1976; Oppenoorth 1984; Hodgson and Motoyama 1984).

Reduced Penetration

Early researchers investigating the rate at which insecticides

penetrated the cuticle of various insects (Eldefrawi and Hoskins 1961;

Plapp and Hoyer 1968b; Ku and Bishop 1967; Camp and Arthur 1967; Hanna

and Atallah 1971; Ahmad et al. 1980) correlated that rate with the rate

of internal metabolism. Resistance attributed to the rate of penetra-

tion produced a comparable rate of metabolism except in those species

that were deemed highly resistant (Ku and Bishop 1967). Busvine (1971)










reported on work of other researchers who tried to explain resistance by

this route. Several researchers measured cuticle thickness in R and

S insect strains while others measured the protein and lipid content in

the cuticle of R and S insect strains (Oppenoorth and Welling 1976).

Patil and Guthrie (1979) altered the lipid composition of house fly

cuticle by feeding artificial diets with and without DL-carnitine and

2-dimethylaminoethanol. House flies with abnormally high cuticular

phospholipids did not always show a decrease in insecticide absorption.

Strain and insecticide differences showed trends toward reduced penetra-

tion, thus, partially supporting the theory that a gene for penetration

resistance can alter the cuticular composition to slow the rate of

insecticide moving into organisms. Generally, the slower penetration

rate allows the usually slow metabolic detoxication process to protect

the organism from toxicosis.

Busvine (1971) also cited one case where excessive peritrophic

membrane development accounted for the rapid excretion of DDT in a

mosquito strain. Ariaratnam and Georghiou (1975) reported slight, but

not statistically significant differences in rates of metabolism in R

and S strains of Anopheles albimanus Wiedemann to carbaryl. They

concluded that high resistance in this mosquito strain was yet uniden-

tified but alluded to reduced penetration as the probable cause.

DeVries and Georghiou (1981b) found decreased cuticular penetration as

one of the resistance mechanisms in a permethrin selected strain of

house fly. Devonshire (1973) showed that the gene for house fly pene-

tration resistance was located on chromosome 3.

Sinchaisri et al. (1978) reported cuticular penetration as a

possible mechanism of resistance in Leucania separate Walker to methyl










parathion, fenithrothion, diazinon, and phenthoate because each chemical

showed variable rates of penetration. They concluded that penetrability

can be influenced by solubility, lipophilicity and hydrophilicity of a

compound, thus accounting for the variability in penetration rates in

this insect strain. Oppenoorth and Welling (1976) also agreed that the

effectiveness of the penetration gene is dependent on the nature of the

insecticide and its avenue of administration.

Altered Site Insensitivity

Altered site insensitivity varies among organisms and between

pesticides. Altered site insensitivity can take the form of

o Less sensitive AChE to inhibition by carbamate and OP Compounds

(Oppenoorth 1984).

o Kdr (knockdown resistance), where the immediate immobility of an

organism treated with DDT or pyrethroids does not occur. This

phenomenon was first observed in the house fly (Oppenoorth and

Welling 1976) and has subsequently been found in the cattle tick

(Busvine 1971).

o Target site change. Evidence of HCH and dieldrin cyclodieness)

resistance in several mosquito, house fly and bed bug strains

suggests target site change because no differences in metabolism

or cuticular penetration was found between R and S strains

(Oppenoorth 1965; Oppenoorth and Welling 1976).

The nervous system is an integral part of an organism thus making

it a suitable target for alteration, inhibition or direct poisoning.

The nervous system of both vertebrates and invertebrates is the most

exploited target site for natural poisons and the majority of organic










insecticides carbamatess, organophosphates and chlorinated hydrocarbons)

(Shankland 1976).

There is a multitude of papers describing the function of this

chemically mediated cholinergic system. Also, recent reviews employing

electrophysiological techniques for measuring electrical impulses and

the effects of substrates on axonal sodium channels have been published

(Shankland 1976; Edwards 1980; Laufer et al. 1984). In view of the

above published works, description and operations of the nervous system

will not be described here.

It is general knowledge that carbamate and organophosphorus

insecticides exert their toxic action on the nervous system by

inhibiting acetylcholinesterase (AChE) (Oppenoorth and Welling 1976;

Hodgson and Motoyama 1984). Kinetic studies have shown that AChE of

some R species is less sensitive to inhibition than their S counterparts

(Hodgson and Motoyama 1984; Plapp 1976; Oppenoorth 1984) indicating an

alteration or site change (Oppenoorth and Welling 1976; Busvine 1971).

Site changes or alterations can occur quantitatively or qualita-

tively, i.e., more sites of action or less sensitive sites (Oppenoorth

1984). Site alterations have only been found in AChE. The first

evidence of altered AChE was found in the red spider mite, Tetranychus

urticae Koch, by Smissaret in 1964 (Plapp 1976; Busvine 1971; Oppenoorth

and Welling 1976; Oppenoorth 1984). Other mite strains showing altered

AChE had slight changes in an imidazole residue relative to the serine

hydroxyl necessary for acetylcholine hydrolysis (Plapp 1976).









Roulston et al. (1969) showed that the R-AChE of a Biarra strain of

cattle tick, Boophilus microplus (Canestrini), was less sensitive to

inhibition by organophosphate and carbamate insecticides than a was

susceptible strain. R-AChE of the Biarra tick strain also showed 60%

less activity toward acetylthiocholine than did the susceptible strain

suggesting that their enzymes were different. Hama and Iwata (1971 and

1978) and Yamamoto et al. (1977) found that a strain of green rice

leafhopper, Nephotettix cinctipes Uhler, was resistant to organophos-

phates and selected carbamates by insensitive AChE. Hama and Iwata

concluded that insensitive AChE was controlled genetically by an incom-

pletely dominant autosome.

Devonshire and Moores (1984) showed that differences in R-AChE from

house flies were unusual in having a greater affinity for acetylthio-

choline converse to previous works where R-AChE showed less affinity for

ATC. They concluded that AChE should be partially protected from

inhibitors by substrates present in the synapse, even if the enzyme was

not also intrinsically insensitive to inhibition.

Biochemical differences in R and S AChE of the house fly was

described by Devonshire (1975). The R and S enzymes showed no differ-

ences electrophoretically when applied on the surface of polyacrylamide

gels with a surfactant. In the absence of the surfactant, R-AChE

produced two distinct electrophoretic bands indicating heterogeny or

isozymic forms but acted as one enzyme in vitro. R-AChE showed slower

organophosphate inhibition than the S-AChE in this house fly strain.

Altered AChE has been predominantly found in mosquitoes, house

flies, planthoppers, ticks and several mite strains (Voss 1980). Voss

(1980) found that a related armyworm species, Spodoptera littoralis










Boisduval, was resistant by this mechanism. These findings indicate

that lepidopterous larvae that are exposed to heavy selection pressures

from various insecticidal classes possess the capability of altered AChE

resistance.

Oppenoorth et al. (1977) found house fly R-AChE in combination with

other metabolic detoxication mechanisms providing resistance to paraoxon

and tetrachlorvinphos. DeVries and Georghiou (1981a, 1981b) found that

decreased nerve sensitivity to permethrin combined with reduced

cuticular penetration provided resistance in another house fly strain.

AChE inhibition and axonal sodium channel interference by pesti-

cides can selectively produce organisms that are resistant. Also

important are the new techniques available for determining effects on

these insect systems by extrapolations from giant axons of crayfish or

squids.

Increased Detoxication

A compound which is biologically active by virtue of interactions

with biochemical systems such as enzymes and membranes will be vulnerable

to attack by other enzymes in the same cells and tissues (Terriere

1982). "Attack" denotes metabolism of the compound. Metabolism gener-

ally results in detoxication and subsequent elimination of the metabo-

lized compound from the organism's system. The original function of the

MFO system is assumed to be that of metabolizing toxic allelochemicals

(Dowd et al. 1983) and to a lesser extent, juvenile hormones (Yu and

Terriere 1975) followed by juvenile hormone analogues (Yu and Terriere

1978). A typical metabolic scheme indicative of most lipophilic insec-

ticides is shown in Figure 1. This scheme was derived from the many

studies of insecticide metabolism in various organisms.





































Figure 1. Metabolism of lipophilic foreign compounds.


















4 HYDROPHILIC


Drugs Primary
Insecticides
Other Foreign
Compounds











Oxidation
Reduction
Hydrolysis
Group Trans-
fer


Primary Secondary Secondary
Products Products














Conjugation
with Sugars,
Amino Acids,
Sulfates,
Phosphates,
etc.



EXCRETION


Figure 1. Metabolism of lipophilic foreign compounds.


LIPOPHILIC










Most of the more active insecticides are non-polar, lipophilic, fat

soluble compounds which readily penetrate insect cuticle and gut walls.

Non-polar compounds are usually insoluble in water; therefore, they are

difficult to excrete without some biochemical modifications. However,

some insects have developed the ability of rapidly excreting intact

unchanged toxic molecules (Devonshire 1973; Matthews 1980; Ivie et al.

1983). Insects that possess this ability are considered highly resis-

tant by virtue of rapid elimination.

Metabolism of lipophilic compounds may follow primary and/or

secondary pathways, (Fig. 1) (Wilkinson and Brattsten 1972). Primary

metabolism of lipophilic compounds takes the form of oxidation, reduc-

tion, group transfer, or hydrolysis. Some primary products are bio-

transformed into hydrophilic, water soluble products and are readily

excreted. Those primary products that are not readily excretable are

biotransformed into secondary products which are conjugated either with

sugars, amino acids, phosphates, sulfates, glutathione or other endogen-

ous conjugative compounds and excreted (Wilkinson 1983; Terriere 1982;

Hollingworth 1976).

Cytochrome P-450 Mono-oxygenases

The most important oxidase enzymes are found in the endoplasmic

reticulum membranes of cells. Cells which contain the most abundant

oxidase enzymes are species specific. That is to say some organisms

show higher oxidative activity from preparations of the midgut (Krieger

and Wilkinson 1969; Yu and Ing 1984), fatbodies (Kuhr 1971; Price and

Kuhr 1969; Brattsten et al. 1980), and less activity in preparations

from malpighian tubules, fore- and hindgut, and the whole body (Krieger

and Wilkinson 1969; Yu 1982b).










Fragmented endoplasmic reticulum membranes are called microsomes

and are the results of tissue grinding or homogenation. The oxidase

enzymes associated with microsomes are termed microsomal oxidases (Yu

1983a), mixed-function oxidases (MFO), or cytochrome P-450-dependent

mono-oxygenases.

The MFO system accomplishes its functions by inserting one atom of

molecular oxygen into a xenobiotic and combining the other oxygen atom

with hydrogens from NADPH to form water (H20). Wilkinson (1983)

depicted a generalized reaction for this procedure:

+ +
RH + 02 + NADPH + H -------> ROH + H20 + NADP


RH represents the lipophilic toxicant.

ROH represents the hydrophilic metabolite.


In the above reaction, electrons flow from NADPH + H and a flavo-

protein, cytochrome P-450 reductase (Terriere 1982) to an enzyme known

as cytochrome P-450. Cytochrome P-450 binds to the xenobiotic (RH) and

to oxygen (02) resulting in the splitting of molecular oxygen, inserting

one atom in the xenobiotic (ROH) and combining the other with hydrogens

from NADPH + H to form water.

MFO actions on xenobiotics including insecticides are listed in

Table 1, which was derived from Terriere (1982) and Yu (1982, personal

communications).

The diversity of compounds attacked by MFO is shown to some extent

in Table 1. The wide tissue distribution of MFO systems in insects

demonstrates the ubiquitous nature of this important enzyme system.

Increases in the rate of deactivation (detoxication) of toxic molecules

can demonstrate the evolution of a resistance mechanism, particularly if





19



Table 1. The primary action of MFO systems on specific chemical
configurations found in xenobiotic molecules.


Reaction Chemical Reaction Consequence*
Type Configuration Products


Epoxidation


Sulfoxidation






Phosphorothioate
Oxidation




N-Dealkylation





O-Dealkylation


Hydroxylation


-C-S-C-




S
II
>P-



CH3
/3
-N





-O-CH3


-C-H


0

-C---C-


-C-S-C-
II
0


0
II
>P-



H
/-N
-N
\


-C-OH


-C-OH


Activation


Activation






Activation





Deactivation


Deactivation


Deactivation


* Activation means the metabolite is more toxic than the parent compound;
deactivation means the metabolite is less toxic than the parent
compound.










that rate is high enough to protect the organism from toxicosis.

Indeed, this phenomenon occurs widely in the insect world. Insecticides

detoxified by increased oxidation include DDT, carbamates, organophos-

phates and pyrethroids (Devonshire 1973).

Increased MFO deactivation of diazinon and diazoxon in a resistant

house fly strain compared to a susceptible strain was demonstrated by

Yang et al. (1971). Kuhr (1971) found increased fatbody MFO responsible

for resistance in a cabbage looper strain to carbaryl. Feyereisen

(1983) found high oxidative metabolism in a resistant house fly strain

when measuring NADPH:cytochrome C reductase, cytochrome P-450 and aldrin

and heptachlor epoxidase systems.

Multiple forms of cytochrome P-450 have been credited for the

ability of insects to metabolize almost any foreign compound (Wilkinson

1983). Yu and Terriere (1979) found different forms of cytochrome P-450

in resistant and susceptible house fly strains. The resistant strain

showed absorbance maxima lower than that found in the susceptible strain

which resembled the high spin hemoprotein type cytochrome found in

mammals. Terriere et al. (1975) used temperature, pH, ionic buffer

strength and spectral data to determine microsomal oxidase differences

in several R and S house fly strains. They found that a WHO standard

reference strain showed abnormalities in the oxidase enzyme system and

concluded that this strain may not be suitable as a reference strain.

Also, this work suggested the presence of multiple forms of cytochrome

P-450 as described by Yu and Terriere (1979).

Moldenke et al. (1984) isolated two forms of cytochrome P-450 from

a house fly strain with different absorbance maxima and aldrin epoxidase

activities. 0-demethylase activity was detectable in one cytochrome

P-450 fraction and not the other.









The MFO system is even more flexible in the metabolism of various

chemical compounds. The induction of MFO systems provides this flexi-

bility (Brattsten et al. 1977). Yu et al. (1979) and Berry et al.

(1980) showed that peppermint plant leaves induced microsomal oxidases

and cytochrome P-450 in the variegated cutworm. Brattsten et al. (1980)

showed that epoxidation, N-demethylation and cytochrome P-450 reductase

could be induced with phenobarbital either in midgut or fat body prep-

arations from the southern armyworm. Yu and Ing (1984) demonstrated

that another oxidase, fall armyworm microsomal hydroxylase, was induced

by allelochemicals, drugs and host plants. Wood et al. (1981) and

Farnsworth et al. (1981) showed that certain host plants could increase

the tolerance in the fall armyworm and cabbage and alfalfa loopers

when fed host plants that induced microsomal oxidases.

MFO induction appears to be age dependent. Yu (1982b, 1983a)

showed that MFO in young fall armyworm larvae were less inducible than

in older larvae. MFO induction also appears to be host plant and insect

specific. Brattsten et al. (1984) showed that certain monoterpenes

isolated from carrots induced MFO in the southern armyworm.

The significance of induction to the survival of an organism is yet

unclear (Busvine 1971; Oppenoorth and Welling 1976; Wilkinson 1983);

however, Perry et al. (1971) viewed chemical induction as a possible

enhancement to the development of insect resistance.

Hydrolases

Hydrolases are those enzymes that catalyze the cleavage of mole-

cules with water thus producing an acid and a leaving group, usually an

alcohol or an amide. These include the esterases, phosphotases and

amidases (Terriere 1982). Each group contains several different kinds










of hydrolases. Oppenoorth and Welling (1976) and Dauterman (1976)

emphasized the importance of hydrolase attack on ester groups of many

insecticides such as organophosphates, carbamates and pyrethroids but

stated that the effects on organophosphates are most important in

resistance.

A. Phosphotriester Hydrolysis

Phosphotriester hydrolase has been named DFP-ase, paraoxonase,

A-esterase, phosphorylphosphatase, aryl esterase, phosphatase, etc.

(Dauterman 1983). This enzyme or enzyme complex catalyzes the hydro-

lysis of organophosphate insecticides to produce phosphorus containing

molecules that are poor cholinesterase inhibitors and are generally

water soluble (Dauterman 1976, 1983).

B. Arylester Hydrolysis

Arylester hydrolases are implicated in the detoxication of aryl

esters of organophosphorus compounds such as parathion or paraoxon

(Dauterman 1976). Ahmad and Forgash (1976) described arylester hydro-

lases as 1) preferentially reacting with phenolic esters, 2) being

inhibited by PCMB (parachloromercuribenzoate), 3) being activated by

Ca2+, and 4) readily hydrolyzing organophosphate compounds.

C. Carboxylester Hydrolysis

Carboxylesterases are known to catalyze the hydrolysis of aliphatic

and aromatic carboxyl esters (Dauterman 1976; Ahmad and Forgash 1976) in

many insecticides and is responsible for resistance. The hydrolysis of

malathion by carboxylesterases produces malathion acid(s) and an

alcohol(s). Zettler (1974) found that the carboxylesterase titre in a

malathion resistant Indian meal moth strain was greater than in that of

a susceptible strain. He also concluded that this strain of Indian meal









moth was resistant only to malathion and not other organophosphate

compounds. Devonshire and Moores (1982) characterized carboxylesterase

from the peach-potato aphid and found that the enzyme had broad sub-

strate specificity thus contributing to organophosphate, carbamate and

possibly pyrethroid resistance.

Motoyama et al. (1980) described a house fly strain that had multi-

ple resistant mechanisms responsible for organophosphorus resistance.

They concluded that a carboxylesterase from the nuclei, the mitochondria

and the microsomal fraction was predominantly responsible for malathion

resistance in this fly strain. Kao et al. (1984) selected two suscep-

tible house fly strains with malathion and found that carboxylesterase

activities and LD50 values were significantly increased after treating

only three generations. Carboxylesterases were credited for rapid

development of resistance to malathion in this house fly strain.

Hemingway and Georghiou (1984) found a mosquito strain resistant to

organophosphorus insecticides by increased levels of esterase enzymes.

They were able to reverse resistance below the susceptible level by

treating the larvae with known esterase inhibitors, IBP (S-benzyl

0,O-diisopropyl phosphorothioate), DEF (S,S,S-tributyl phosphorotrithi-

oate) and TPP (triphenyl phosphate), thus partially confirming the

resistance mechanism.

Recent studies of synthetic pyrethroid resistance have shown that

hydrolases are responsible for flucythrinate, decamethrin, and fenva-

lerate resistance in an Egyptian cotton leafworm strain (Riskallah

1983). Resistance to another synthetic pyrethroid, permethrin, was

found in a predatory mite strain by Scott et al. (1983). Several mite

strains were investigated that had a prior exposure to DDT, azinphos-










methyl, carbaryl, and permethrin. In all cases, resistance was due to

either a kdr type resistance or to increased ester hydrolysis. Hydro-

lase activity is generally measured with one of these commonly used

substrates, a-naphthyl acetate (a-NA), B-naphthyl acetate (B-NA) and/or

p-nitrophenyl acetate (p-NPA). Comparison of hydrolase activities of

susceptible and resistant insects is a good measure of hydrolase resis-

tance.

Glutathione S-transferases

Glutathione S-transferases are enzymes that catalyze the conjuga-

tion of glutathione (GSH) with many foreign compounds (Chasseaud 1973).

Chasseaud (1973) and Dauterman (1983) explained the two main roles of

GSH S-transferase as the conjugation of potentially harmful electro-

philes with the nucleophile, GSH, thus protecting cell nucleophilic

centers which occur in proteins and nucleic acids. Secondly, GSH

provides an avenue for excretion of the potentially harmful electrophile

through the formation of anionic, water-soluble products. GSH S-trans-

ferases catalyzes two type of reactions, the conjugations of GSH with

epoxides and unsaturated compounds and the substitution of GSH with

alkyl and aryl halides (Dauterman 1983).

There are many such transferases as described by Ahmad and Forgash

(1976). These authors listed all known transferases requiring GSH in

the metabolism of insecticides. GSH S-transferases act directly on the

insecticide without the need for hydroxylation by MFO.

Usui and Fukami (1977) found two transferases from cockroach fat

bodies active on diazinon and three transferases active on methyl

parathion. Wool et al. (1982) correlated high GSH S-transferase levels

with resistance to malathion in a flour beetle strain. Motoyama et al.

(1980) determined that resistance in a house fly strain was in part due










to elevated levels of GSH S-transferase. Oppenoorth et al. (1977) found

GSH S-transferase levels in a resistant house fly strain 9 to 120-fold

more than a susceptible strain to methyl parathion, parathion, methyl

paraoxon and paraoxon.

GSH S-transferases are known to be induced by allelochemicals (Yu

1982a) and insecticides. Xanthotoxin, an allelochemical from parsnip,

induced GSH S-transferase by 39-fold in an insecticide resistant and

susceptible strain of fall armyworm (Yu 1984). Permethrin, a synthetic

pyrethroid, induced GSH S-transferase 296% of the control when fed to

groups of adult honey bees for two days (Yu et al. 1984). Hayaoka and

Dauterman (1982) induced GSH S-transferases in a strain of house fly

with phenobarbital and several chlorinated hydrocarbon insecticides.

House fly pretreatment with phenobarbital afforded some protection from

toxicosis by several organophosphorus insecticides, thus further empha-

sizing the importance of GSH S-transferases in insecticide detoxication.

Epoxide Hydrolases

Epoxide hydrolases are enzymes that hydrate epoxides of certain

arene, alkene and cyclodiene compounds to trans-diols by the inclusion

of water in the molecules (Dauterman 1976; Oesch et al. 1971). Enzyma-

tic hydration of epoxides is recognized as an important metabolic

reaction in protecting organisms from potentially hazardous labile

epoxides which are considered carcinogens (Yu 1982, personal communica-

tion; Dauterman 1976). I have not found literature articles where

epoxide hydrases contribute significantly to insect resistance; however,

their presence is unquestionably important in the detoxication of

cyclodienes such as dieldrin, enzymatically altered compounds such as

heptachlor epoxide, and other more stable deleterious epoxides.










History of Carbaryl Resistance

Carbaryl was introduced to the commercial market in 1956 to control

a variety of insect pest species including those that were highly

resistant to DDT (Harding and Dyar 1970). The first reported cases of

resistance to this compound were against the light brown apple moth in

1963 in New Zealand and in 1966 against the tobacco budworm in the U.S.

(Mount and Oehme 1981).

Since carbaryl controlled important agronomic and urban insects, it

was no surprise when Ku and Bishop (1967) reported that carbaryl resis-

tance in a cockroach strain was due to three resistance mechanisms. The

primary mechanism was reduced cuticular penetration while increased

excretion and metabolism contributed significantly to the elevation of

resistance in this strain.

Roulston et al. (1968, 1969) reported insensitive AChE in a Biarra

strain of cattle tick while Schuntner et al. (1972) found increased

metabolism responsible for resistance in a Mackay strain of cattle tick.

Increased oxidative metabolism was found to be responsible for

carbaryl resistance in a resistant cabbage looper strain (Kuhr 1971).

Atallah (1971) selected several strains of Egyptian cotton leafworms

with carbaryl for 15 generations and found a 30-fold increase in resis-

tance. Biochemical identification of the resistance mechanism proved to

be increased metabolism and restricted cuticular penetration (Hanna and

Atallah 1971). Atallah (1971) simultaneously selected individuals of

the same leafworm strain used for carbaryl selection with DDT. He found

that DDT resistance developed much more slowly than that of carbaryl.

DDT resistance was 24-fold after 26 generations. This work indicated

multifactorial resistance to carbaryl while DDT resistance was probably

due to a single mechanism.









Hama and Iwata (1971) found insensitive AChE responsible for

carbamate resistance, including carbaryl, in a resistant strain of green

rice leafhopper. Hama and Iwata (1978) described the heritability of

resistance in this leafhopper strain as being controlled by an incom-

pletely dominant autosomal gene.

Wolfenbarger et al. (1981) reported increases in carbaryl LD50

values from the American bollworm, Heliothis armigera, from 1969 to 1973

in Thailand as 94 ug/g to 310 ug/g, respectively. Increases of this

magnitude over this period indicate the tremendous insecticidal selec-

tion pressure applied to this insect.

Rose and Brindley (1985) showed that a carbaryl resistant Colorado

potato beetle strain from New Jersey was resistant due to an increase in

oxidative metabolism. Potato beetles in the northeastern U.S. are

subjected to tremendous insecticide selection pressures because they

have developed resistance to many of the highly toxic persistent insec-

ticides including chlorinated hydrocarbons, organophosphates, and carba-

mates.

Fall Armyworm Resistance to Carbaryl

The fall armyworm is a highly mobile phytophagous pest of many

grasses, corn, oats, rye, cotton, garden vegetables, and other succulent

plants (Quaintance 1897). This species migrates from the tropics,

Florida and Gulf coast states (Luginbill 1928; Vickery 1929) as far

north as Canada (Snow and Copeland 1969; Combs and Valerio 1980). In

1979, using diet spray bioassay techniques, Young (1979) found that the

fall armyworm was resistant to carbaryl. Many researchers believe that

wide-spread resistance in this species could prove devastating to

farmers from the tropics to Canada and west to southern California.














MATERIALS AND METHODS


Insects

R Strain

The carbaryl resistant strain of fall armyworm was collected near

Tifton, Georgia, by Dr. J. R. Young. Larvae were reared on a meridic

diet (Burton 1969). Environmental conditions were 27 2 degrees C with

50 5% relative humidity and 16:8 light:dark photoperiod. Moths were

held in a separate environmental chamber whose atmospheric conditions

were 26 2 degrees C, 50 70% relative humidity, and 16:8 light:dark

photoperiod.

S Strain

Eggs of the carbaryl susceptible strain were obtained twice weekly

from the United States Department of Agriculture (USDA), Gainesville,

Florida. Larvae were reared under the same conditions as the R strain;

however, old sixth instar larvae were discarded.

The rearing procedures utilized have been previously described by

Young (personal communication) and Shorey and Hale (1965). Modifica-

tions to each were made to accommodate current laboratory conditions.

Moths of the R strain were housed in one-gallon cardboard ice-cream

containers. The lid was removed and fitted with an absorbent paper

towel. The moths were fed a 10% sucrose solution saturated on sterile

cotton in a 4 oz squat cup. Eggs were removed thrice weekly by anesthe-

tizing the moths with 12 second bursts of CO2. The moths were










transferred to a clean container and provided clean towelling and fresh

sucrose solution (10%).

Eggs on paper towelling were sterilized in a 10% formaldehyde

solution, rinsed in tap water and allowed to dry. The paper towelling

was glued to tab lids of 16 oz plastic cups, each containing about

inch of the artificial diet.

Chemicals
14
14C] carbaryl was purchased from the California Bionuclear Cor-
14
portion, Sun Valley, CA, and [8- 14C] styrene oxide was purchased from

the Amersham Corporation. All insecticides and chemical reagents were

of the highest purity available commercially. Carbaryl metabolites were

a gift from The Union Carbide Corporation.

Bioassay

The bioassay methods used were as described by Mullins and Pieters

(1982). Twenty 4th instar Spodoptera frugiperda larvae (22 3 mg in

weight) were placed into a four inch glass petri dish. An ISCO Model M

microapplicator was used to treat the larvae topically on the dorsal

prothorax with 1 ul of insecticide diluted with acetone. Controls were

treated with 1 ul of acetone only. After treatment, the larvae were

transferred individually to glass scintillation vials, each containing

about 1 gram of artificial diet. Mortality was recorded 24 and 48 hours

post-treatment with the end point being a completely moribund condition

unresponsive to prodding. Only 48 hour data were used in probit

analysis. All insecticides were tested at a minimum of five dosages, on

at least four different days. Probit analyses were made by a computer

program.

Protein Determinations

The protein content of each preparation, midgut microsomal suspen-

sion or crude homogenate was measured by the method of Bradford (1976).










A protein reagent was made by adding precisely the ingredients described

by Bradford:

a). Coomassie Brilliant Blue G-250 dye (100 mg)

b). Ethanol-95% (50 ml)

c). Phosphoric acid-85% (100 ml)

This solution was brought to a final volume of 1 liter, stirred, filter-

ed twice and used for all assays. A standard curve was made with

multiple determinations of known quantities of bovine serum albumin

(BSA), Fraction V.

A typical mixture included 0.1 ml of 10 ug BSA protein pipetted

into a test tube and 3.0 ml protein reagent added. This mixture was

shaken and incubated at room temperature for a minimum of 2 minutes. A

blank was prepared with 0.1 ml warm 0.1 M sodium phosphate buffer, pH

7.5, plus 3.0 ml protein reagent and handled as above.

A desk top Turner Model (330) single beam spectrophotometer was

used to measure optical densities (O.D.) at 595 nm. Each protein

concentration was replicated 3 times and run on at least 3 different

days. The average O.D. was plotted on graph paper against micrograms of

BSA protein to establish the standard curve.

To determine unknown protein quantities, 0.03 ml protein solution

and 0.07 ml of 0.1 M sodium phosphate buffer, pH 7.5, were added to a

standard test tube. A volume of 3.0 ml protein reagent was added, and

the tube shaken and incubated at room temperature for a minimum of 2

minutes. Optical densities were measured at 595 nm and compared to the

standard curve.

Epoxidation Assay

Aldrin epoxidation was assayed (Fig. 2) by the method of Yu et al.

(1979) and Yu and Terriere (1979). Aldrin epoxidation was assayed with










two types of enzyme preparations, crude homogenate and microsomal

fraction. Crude homogenates were uncentrifuged homogenates of fall

armyworm midguts. They were obtained by dissecting larval midguts,

removing the food containing peritrophic membrane and placing the

cleaned guts into ice-cold 1.15% KC1 solution. The clean guts were then

transferred to an ice-cold glass homogenizer tube into which 20.0 ml

ice-cold 0.1 M sodium phosphate buffer, pH 7.5, were added. The guts

were homogenized for about 30 seconds with a motor-driven teflon tissue

grinder. Homogenized guts were filtered through double layer cheese-

cloth and used as the enzyme source. Microsomal isolation followed the

above steps except the homogenate was centrifuged in a Beckman L5-50E

ultracentrifuge at 10,000g max at 0 to 4 degrees C for 15 minutes. The

pellet containing mitochondria and cell debris was discarded and the

supernatant filtered through glass wool. The supernatant was recentri-

fuged at 105,000g max for 65 minutes. The resulting microsomal pellet

was resuspended in ice-cold 0.1 M sodium phosphate buffer, pH 7.5, to

obtain a protein concentration 1.0 mg/ml and used immediately as the

enzyme source. A typical 5 ml incubation mixture contained 0.1 M sodium

phosphate buffer, pH 7.5, an NADPH generating system (1.8 umoles of

NADP; 18 umoles of glucose-6-phosphate; 1.0 unit of glucose-6-phosphate

dehydrogenase); 250 nmoles of aldrin in 0.1 ml methyl Cellosolve; and

2.0 ml of microsomal suspension (1 mg protein). Mixtures were incubated

in a water bath while being shaken at 30 degrees C in an atmosphere of

air for 15 minutes. Each incubation was duplicated and accompanied by a

blank or control which did not contain microsomes. After 15 minutes,

each reaction was stopped by adding 10 ml hexane and placing the incuba-

tion tube on ice. The epoxidation product, dieldrin, was extracted


























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from the mixture by slowly shaking it for one hour. Dieldrin formation

was analyzed on a Varian Model 3740 gas chromatograph equipped with an

electron capture detector. The column was 4 ft. X 2 mm i.d. glass,

packed with a 1:1 mixture of 5% DC 11 and 5% QF 1 on 100 to 120 mesh

high performance Chromosorb W (Yu and Terriere 1974; Yu 1982).

Microsomal Biphenyl Hydroxylase Assay

Microsomal biphenyl hydroxylation (Fig. 3) is a mixed-function

oxidase system that plays a major role in the oxidative metabolism of

foreign substances in insects (Yu and Ing 1984). The activity of this

enzyme system in the fall armyworm was determined by the method of Yu

and Ing (1984) which used biphenyl as substrate.

Microsomes were isolated from 25 cleaned guts by homogenizing the

guts in 20 ml ice-cold 0.1 M sodium phosphate buffer, pH 7.5, and

centrifuging as above. The resulting microsomal pellet was resuspended

in ice-cold 0.1 M sodium phosphate buffer, pH 7.5, and used immediately

as the enzyme source. A typical 5.0 ml incubation mixture contained 0.3

ml of an NADPH generating system as mentioned above; 2.6 ml of a 0.1 M

sodium phosphate buffer, pH 7.5; 2.5 mg biphenyl in 0.1 ml methyl

Cellosolve, and 2.0 ml of microsomal suspension (1 mg protein).

Mixtures were incubated in duplicate in a water bath while being shaken

at 30 degrees C in an atmosphere of air for 30 minutes.

The reactions were stopped by adding 5.0 ml ethyl acetate and

placing incubation tubes on ice. The hydroxylated product, 4-hydroxybi-

phenyl, was extracted twice with 5.0 ml of ethyl acetate, each time,

dried over anhydrous sodium sulfate and analyzed by high performance

liquid chromatography (HPLC). Analyses were performed on a Beckman

Series 340 HPLC at 254 nm. The column was an Ultrasphere-Si measuring



























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25 cm X 4.6 mm i.d. Isopropanol (5%) in hexane was used to elute the

column at a flow rate of 0.75 ml/minutes. Enzymes were denatured by

heat and tested as above to determine non-enzymatic product formation.

Microsomal N-Demethylase Assay

Microsomal N-demethylation of p-Chloro-N-methylaniline (PCMA) (Fig.

4) was carried out by the method of Kupfer and Bruggeman (1966).

Standard curves were obtained by measuring, spectrophotometrically,

known concentrations of p-Chloroaniline (PCA) in an aqueous solution at

445 nm. All assays, whether standard curve determinations or enzyme

activity determinations, consisted of a comparable blank, i.e., the

absence of PCA or the use of heat denatured protein. A Beckman Model

5260 spectrophotometer was used for all N-demethylation assays.

Microsomes were prepared as mentioned earlier and suspended in 0.1

M sodium phosphate buffer, pH 7.5. A 5.0 ml incubation mixture contain-

ed 0.3 ml NADPH generating system (1.8 umoles of NADP; 18 umoles of

glucose-6-phosphate, and 0.5 unit of glucose-6-phosphate dehydrogenase);

0.1 ml PCMA (30 umoles in aqueous HC1); 2.6 ml of 0.1 M sodium phosphate

buffer, pH 7.5; and 2.0 ml microsomal preparation (0.5 to 1 mg protein/

ml). The incubation mixture was shaken at 34 degrees C for 20 minutes.

The reaction was stopped with 2.0 ml of a 6% aqueous p-dimethylamino-

benzldehyde and centrifuged for 15 minutes at 10,000 RPM in a refrige-

rated Beckman Model JA-21 centrifuge. The incubation tubes were allowed

to reach ambient temperature before being analyzed spectrophotometrically

at 445 nm on a Beckman Model 5260 spectrophotometer. Each incubation

was duplicated and each experiment was repeated three times.

Cytochrome P-450 Measurement

Cytochrome P-450, a carbon monoxide-binding pigment of endoplasmic

reticulum, was determined by the method of Omura and Sato (1964).












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Midgut microsomes from 20 cleaned guts were homogenized in 20 ml of

ice-cold 0.1 M sodium phosphate buffer, pH 7.5, and centrifuged as

above. The resulting microsomal pellet was resuspended in ice-cold 0.07

M sodium phosphate buffer, pH 7.5, containing 30% glycerol and used

immediately as the enzyme source.

Baseline scans of the microsomal suspension alone were run on a

Beckman Model 5260 uv/vis spectrophotometer equipped with a scattered

transmission accessory at 300 to 500 nm. After recording the baseline,

the sample cuvette was removed and carbon monoxide (CO) was gently

bubbled through the preparation for 1 minute. This sample was reduced

with a few milligrams of sodium dithionite (Na2S204), stirred with a

glass rod and again scanned from 300 to 500 nm. Scanning was continued

until a maximum spectrum was obtained. This assay was duplicated and

run on at least 3 different days on both insect strains (Yu 1982b).

Glutathione S-Transferase Assay

Glutathione S-transferases (Fig. 5) are enzymes that catalyze the

conjugation of glutathione (GSH) with many foreign compounds (Chausseaud

1973). Conjugation products are usually water soluble, readily excret-

able substances and their formation generally results in a decrease in

xenobiotic toxicity (Yang 1976).

Glutathione S-transferase activity was measured by the method of Yu

(1982a). Midgut soluble enzyme fractions were used in lieu of the

resuspended microsomal pellet. Twenty cleaned guts were homogenized in

20 ml of ice-cold 0.1 M Tris-HC1 buffer, pH 9.0, and filtered through

double layered cheesecloth. The homogenate was centrifuged at 10,000g

max for 15 minutes. The resulting supernatant was filtered through

glass wool and recentrifuged at 105,000g max for 65 minutes. Prior to

decanting the supernatant from the centrifuge tube, all lipids were


















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removed from the supernatant surface with a medicine dropper and

discarded. The supernatant was then gently poured into a large test

tube so as not to disturb the microsomal pellet and kept on ice for

immediate use. A typical 3.0 ml reaction mixture contained 1.0 ml of 15

mM glutathione and 2.0 ml soluble fraction (2.0 ml 0.1 M Tris-HC1

buffer, pH 9.0, served as blank) was first incubated for 3 minutes at 37

degrees C, after which 0.02 ml 150 mM 1,2-dichloro-4-nitrobenzene (DCNB)

was added and mixed. The change in absorbance at 340 nm for 5.0 minutes

was measured with a Beckman Model 5260 uv/vis spectrophotometer. The

enzyme activity was expressed as nmoles DCNB conjugated per minute per
-1 -1
milligram of protein using an extinction coefficient of 10 mM cm for

S-(2-chloro-4-nitrophenyl) glutathione.

In vitro Carbaryl Metabolism Study

Carbaryl was metabolized in vitro by modifications of the methods

of Kuhr and Davis (1975), Kuhr and Hessney (1977), and Yu and Terriere

(1978). Midgut homogenate was prepared, as described earlier, from 2

day-old sixth instar larvae to obtain 4-5 mg protein/ml.

Midgut homogenates of R and S larvae were incubated with carbaryl

in an atmosphere of air for 2 hours at 30 degrees C. The 5.0 ml incuba-

tion mixture contained 0.3 ml of an NADPH generating system, as mention-

ed above; 0.58 ml of 0.1 M sodium phosphate buffer, pH 7.5; 0.437 ug
14
14C] carbaryl (100,000 dpm); 10 ug cold carbaryl; 10 mg bovine serum

albumin; 0.05 ml methyl Cellosolve; and 4.0 ml of midgut crude homoge-

nates. The NADPH generating system was omitted from some incubations in

order to study nonoxidative metabolism of carbaryl. The incubation

mixture was stopped with 5.0 ml chloroform, and carbaryl and its metabo-

lites were extracted by the solvent. The same extraction was repeated

again and the combined extracts were then dried over anhydrous sodium










sulfate. Two milliliter aliquots of chloroform were concentrated under

a stream of air to 0.2 ml and spotted on silica gel G thin layer

chromotographic (TLC) plates (0.25 mm). The TLC plates were developed

in a solution of acetic acid:ethyl acetate:benzene (1:10:33 by volume)

and scanned for radioactivity in a Packard Model 7220/21 radiochromato-

gram scanner. Individual spots were identified by Rfs of previously

chromatographed standard metabolites (see Table 2). Each peak was

scraped from the plates and counted in a Tracor Analytic Data 300 liquid

scintillation counter. The oxidative metabolites were combined due to

poor separation and resolution near the TLC plates' origin.

Epoxide Hydrolase Assay

Epoxide hydrolase (Fig. 6) was assayed by the method of Yu et al.
14
(1984) using [ C] styrene oxide as substrate. Microsomes were prepared

as previously described and suspended in 0.5 M Tris-HCl buffer, pH 9.0,

to make a final concentration of 0.4 mg protein/ml. Heat denatured

enzyme was used as the control to correct for any non-enzymatic glycol

formation.

Screw cap tubes were used to hold the incubation mixture which
14
contained 0.6 ug (100,000 dpm) [ C] styrene oxide, 8.0 ug cold styrene

oxide in 7.0 ul of acetonitrile, and 0.5 ml microsomal suspension. This

mixture was incubated in a shaking water bath at 37 degrees C for 5

minutes. The reactions were stopped by the addition of 10 ml petroleum

ether and the unreacted styrene oxide was extracted by the solvent. The

petroleum ether was readily decanted by freezing the aqueous phase in a

dry ice-acetone mixture. The same extraction was repeated again after

the aqueous phase was thawed. The aqueous solution which contained the

polar product, [8-14C] styrene glycol, was then shaken with 2 ml ethyl










TABLE 2. R values of carbaryl and its metabolites on silica gel G
plates in a developmental solution of acetic acid:ethyl
acetate:benzene (1:10:33 by volume).


Compound Rf


a-naphthol 0.79

Carbaryl 0.64

5-hydroxy-carbaryl 0.49

4-hydroxy-carbaryl 0.40

Methylol-carbaryl (N-hydroxymethyl) 0.29
















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acetate, and the product in the ethyl acetate was quantified by liquid

scintillation counting.

Esterase Assays

Esterases were assayed by the method of van Asperen (1962) (Fig. 7)

using a-naphthylacetate (a-NA) as substrate. Both midgut microsomes and

crude homogenates were used to perform this assay. A typical 6.0 ml

incubation mixture contained 4.95 ml of 0.04 M sodium phosphate buffer,

pH 7.0; 0.05 ml of a 0.03 M a-NA in acetone, and 1.0 ml of midgut

homogenate or microsomal preparation. To assay for carboxylesterase

activity, eserine (10-4 M) and p-hydroxymercuribenzoate (PHMB) (10-4 M)

were added to the incubation mixture to inhibit cholinesterase and

arylesterases, respectively.

This mixture was incubated for 30 minutes at 27 degrees C and the

reaction was stopped by placing each incubation tube on ice and intro-

ducing 1.0 ml of diazoblue laurylsulfate solution (DBLS). A red color

developed and quickly changed to a dark blue color. The absorbance of

the reaction product, naphthol-diazoblue, was measured at 600 nm on a

Beckman Model 5260 spectrophotometer against a blank containing no

enzyme. Optical densities of the reaction products were compared to

known quantities of naphthol reacted with DBLS and plotted as a standard

curve.

All incubations were duplicated and each experiment was repeated

twice.

Acetylcholinesterase Assay

Acetylcholinesterase (AChE) (Fig. 8) was assayed by the method of

Ellman et al. (1961) using acetylthiocholine (ATC) as substrate.

Initially fall armyworm adult heads, whole larvae and larval heads were
























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assayed for AChE activity. Whole larvae larval heads showed low acti-

vity and were not used. Adult heads contained the highest activity and

were subsequently used in this assay.

Two-day old adults of both R and S strains were frozen and their

heads removed with forceps. The heads were homogenized for 30 seconds

in ice-cold 0.1 M sodium phosphate buffer, pH 8.0, in a glass homoge-

nizer with a teflon pestle attached to a motorized grinder, for 30

seconds. The homogenate was filtered through doubled layered cheese-

cloth and used as the enzyme source.

A typical 3.2 ml incubation mixture contained 2.75 ml of 0.1 M

sodium phosphate buffer, pH 8.0; 0.1 ml of 0.01 M 5,5-dithiobis-2-nitro-

benzoic acid (DTNB), 0.1 ml ice-cold 0.075 M acetylthiocholine (ATC);

and 0.2 ml enzyme. The reaction was initiated by adding 0.2 ml enzyme

to the incubation mixture. The blank contained all of the above rea-

gents excluding the enzyme. The buffer was increased in the blank to

compensate for the lack of enzyme. The yellow colored reaction product

formation was measured for 5 minutes at 412 nm against the blank which

showed some non-enzymatic ATC hydrolysis.

Cuticular Penetration by Carbaryl

Cuticular penetration of carbaryl was measured by a method modified

from Ku and Bishop (1967). The cuticular penetration was assayed by
14
topically applying [ 14C] carbaryl to the dorso-prothorax of fall army-

worm larvae. The treated larvae were rinsed with acetone at different

time intervals after treatment. The excreta was extracted directly from

scintillation vials with Scinti-Verse I scintillation cocktail. Carbaryl

that penetrated larval cuticle was extracted by homogenizing whole

larvae at various time intervals and washing the remaining carcasses and










utensils with aliquots of acetone. Washes were collected, concentrated,

and radioactivity measured.

In a typical experiment, duplicate groups of 4 larvae each of

2-day-old sixth instar were placed into a glass petri dish. Each larva

was treated topically with 1.0 ul acetone containing 0.048 ug [14C]

carbaryl and 0.952 ug cold carbaryl. Those larvae that were assayed at

zero hour were rinsed immediately in three 5.0 ml aliquots of acetone in

3 separate scintillation vials. Those larvae requiring rinses at other

time intervals were treated and placed into separate pre-labelled vials.

Larvae were removed individually from each duplicate vial set at the

designated time interval and rinsed as above. After being rinsed, each

larva was cut into four pieces and placed into a glass homogenizer.

After all larvae had been rinsed, the larval pieces (two larval equiva-

lents) were homogenized in 7.0 ml acetone. The pestle was rinsed with

another 5.0 ml acetone and the acetone was filtered into a 250 ml

Erlenmeyer flask. The filter paper (Whatman #1) and the precipitate

were rinsed twice into the same flask. The combined extracts were

concentrated to approximately 5.0 ml under a constant stream of air.

The Erlenmeyer flasks were rinsed 2 times with acetone, and the combined

rinses were concentrated and added to the original extract in the

scintillation vial.

Ten milliliters of Scinti-Verse I scintillation cocktail were added

and radioactivity was counted in a Tracor Analytic Data 300 liquid

scintillation counter.

Statistics

Computerized t- and F-tests were used to compute the significance

of the difference between means and to determine population normality,

respectively. All in vitro data, except carbaryl metabolism, were

analyzed by these methods.














RESULTS


Bioassays

All FAW bioassays were by the methods of Mullins and Pieters

(1982). Carbaryl, parathion, methomyl, diazinon, cypermethrin, and

permethrin were bioassayed initially to determine FAW susceptibility to

different insecticidal classes and to determine cross-resistance if any.

The LD50 values (ug/g larva) for each insecticide are shown in Table 3

along with the relative resistance ratios in descending order of suscep-

tibility. The resistant strain showed > 90X resistance to carbaryl but

remained susceptible to a related carbamate, methomyl (4.7X). The

resistant strain also showed tolerance to the organophosphates, para-

thion (5.8X), and diazinon (2.9X). The R and S fall armyworm strains

showed no tolerance or cross-resistance to the synthetic pyrethroids,

cypermethrin and permethrin.

In Table 4, the toxicological responses are shown for larvae

treated with PB, a known microsomal oxidase inhibitor (Yu, 1982b).

Resistance was reduced to 6X in the R strain when PB was topically

applied in conjunction with carbaryl at a 5:1 ratio. These results

suggest that microsomal oxidases are involved in carbaryl resistance.

The synergized resistance level of the R strain did not approach that of

the susceptible strain, further suggesting that other factors are pos-

sibly involved in carbaryl resistance. Also, the LD50 of the suscep-

tible strain was reduced by PB, suggesting that microsomal oxidases play

an important role in the detoxication of carbaryl in this insect strain.










TABLE 3. Comparison of toxicological responses of R and S fall armyworm
larvae topically treated with 6 insecticides.


LD50 (ug/g Larva)a/


Insecticide R S R/S


Carbaryl 10343b/ 115 90

Parathion 14.38 2.46 5.85

Methomyl 4.17 0.98 4.74

Diazinon 19.18 6.50 2.95

Cypermethrin 0.12 0.08 1.50

Permethrin 0.23 0.17 1.35


a/ Each observation consisted of at
/Computer estimate


least two different tests.









TABLE 4. Comparison of toxicological responses of R and S fall armyworm
larvae topically treated with carbaryl + PB.


LD50 (ug/g Larva)a/

Insecticide
/Synergist R S R/S


Carbaryl 10343b/ 115 90

Carbaryl +
Piperonyl
Butoxide 400.43 67 5.98


a/ Each observation consisted of at least two different tests.
b/ outer estimate
Computer estimate










These in vivo results may prove helpful in the discussion of results

found in in vitro detoxication assays. DEF, (S,S,S,tributyl phosphoro-

trithioate), a known esterase inhibitor, was too toxic when applied

alone for esterase comparisons. TOCP (tri-O-creosyl phosphate), another

known esterase inhibitor, had no effect on the LD50 levels in either

strain. These findings indicate that esterases do not play a role in

carbaryl resistance in this FAW strain.

Enzymatic Assays

A. Aldrin Epoxidase

The specific activities of aldrin epoxidase enzyme of various

larval instars are shown in Table 5. Larvae younger than fourth-instar

were not studied because of the difficulty in dissecting the midgut.

The R strain showed significantly higher epoxidase activity than the S

strain across all instars tested. These results support those of Yu

(1984) in that the R strain of fall armyworm possesses a higher level of

microsomal epoxidase activity than does the S strain.

B. Biphenyl Hydroxylase

The results summarized in Table 6 show that the biphenyl 4-hydroxy-

lase activity was significantly higher in R larvae. Larvae younger than

4th instar were not examined because of the difficulty in dissecting

midguts. The activity of biphenyl 4-hydroxylase enzyme increased in

both strains from 4th to 2 day-old sixth instar larvae. Three day-old

sixth instar individuals showed a slight decrease in activity thus

confirming results from Yu (1984) that the activity of important detoxi-

cation enzymes reached maximum capacity in the second day of the final

instar. High activity begins to decline with the onset of pupation.

Four day old sixth instar larvae were not observed feeding, and were










TABLE 5. Aldrin epoxidase activities of midgut microsomes and homoge-
nates from various instars of R and S fall armyworm larvae.a


Specific Activity
(pmol dieldrin min mg protein )

Larval
Instar R S


Microsomes

5thb/ 483.23 1.58c,d/ 237.94 1.41

5the/ 709.35 1.22d/ 445.67 3.17

6the/ 694.52 3.83d/ 475.67 3.37

Crude Homogenates

6the/ 237.33 1.25 171.39 1.38


Larvae used in all assays were age synchronized.
Newly molted
Mean SE of at least three experiments, each assayed
Value significantly different (< 0.05) from S strain.
1 day old
2 day old.


in duplicate.










TABLE 6. Microsomal biphenyl 4-hydroxylase activity in various instars
of R and S fall armyworm larvae.a


Specific Activity
(pmol min mg protein )

Larval
Instar R S


4th 663.76 2.25b,d/ 177.28 2.75

5th 959.23 29.15d/ 548.27 57.90

6th

1 Day 768.89 62.80d/ 386.26 16.14

2 Day 1045.28 62.70d/ 634.08 11.91

3 Day 730.37 28.78c/ 529.75 33.06


a/
/ Larvae used in all assays were age synchronized.
SMean SE of at least three experiments, each assayed in duplicate.
/Value significantly different ( 0.05) from S strain.
Value significantly different (p < 0.01 from S strain.










found to have clear guts and to be preparing cells in the artificial

diet for pupation.

The activities of microsomal biphenyl 4-hydroxylase of all R larval

instars in Table 6 were greater than in the S strain by the following

factors: 4th (3.74X), 5th (1.75X), one day-old 6th (2.09X), two day-old

6th (1.65X), three day-old 6th (1.38X). The activities of all larval

instars in the R strain on a per mg protein basis were statistically

different from the S strain at a probability of P < 0.01 except for

three day old 6th instar which showed a significance probability of P <

0.05.

C. N-demethylase

The activity of microsomal N-demethylase of two- day-old sixth

instar R and S fall armyworm larvae is summarized in Table 7. There are

no differences in activity on a per mg protein basis; however, the S

strain is significantly (P < 0.01) more active on a per midgut basis.

The maximum difference (3.2-fold) was observed in the sixth instar of R

and S larvae.

D. Cytochrome P-450

The results summarized in Table 8 show that there was no signifi-

cant difference in the Cytochrome P-450 content between R and S strains,

although the R strain appeared to be consistently higher than the S

strain.

E. Glutathione S-transferase and Epoxide Hydrolase

From Table 9, it can be seen that there was no significant differ-

ences in the glutathione S-aryltransferase activity between the R and S

strains. This is also true for the epoxide hydrolase activity (Table

10).










TABLE 7. Microsomal N-demethyl se activity from sixth-instar R and S
fall armyworm larvae


N-demethylase


-1 -1
pmol min pmol min
-1 1
Strain mg protein midgut


R 849.38 59.42b/ 39.96 2.78

S 883.35 141.62 129.70 25.24c


a/
b/ Larvae used in all assays were age synchronized.
Mean SE of at least three experiments, each assayed in duplicate.
/ Value significantly different (P < 0.01) from R strain.





63



TABLE 8. Cytochrome P-450 activity from midgua microsomes of sixth-
instar R and S fall armyworm larvae


pmol P-450 pmol P-450
Strain mg protein midgut


R 269.70 12.40b/ 17.97 0.10

S 235.30 6.60 11.53 0.31

a/
SLarvae used in all assays were age synchronized.
Mean SE of at least three experiments, each assayed in duplicate.










TABLE 9. Glutathione S-aryltransferase activity of midgut soluble
enzyme fraction from sixth instar R and S fall armyworm
larvae.


Specific activity


Strain (nmol DCNB conjugated min-I mg protein )


R 22.73 0.45b/

S 24.12 0.30


a/
a/ Larvae used in all assays were age synchronized.
Mean SE of at least three experiments, each assayed in duplicate.










TABLE 10.


Microsomal epoxide hydrolase activity in sixth instar R and
S fall armyworm larvae.


Epoxide Hydrolase
Strain (nmol min mg protein )


R 33.92 0.69b/
S 30.63 0.77

a/
SLarvae used in all assays were age synchronized.
Mean SE of at least three experiments, each assayed in duplicate.



















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F. Esterase

The activities of general esterases and carboxylyesterase in the R

and S strains are summarized in Table 11. The results show that there

was no significant difference in the general esterase and carboxylester-

ase activities between the R and S strains when midgut crude homogenates

were used as the enzyme source. However, the activity of general

esterases from midgut microsomes are significantly higher in the S

strain compared to the R strain (Table 12).

G. AChE Kinetics

AChE activity was not significantly different bewteen the R and S

strains (Table 13). Studies of AChE enzyme kinetics (Fig. 9) show that

the K values from both the R and S strains are not different toward
m
ATC. Although the maximum reaction velocity (V ) is different (R-V
max max
-1 -1 -1 -1
= 0.345 nmol min- my protein ; S-V = 0.208 nmol min ng protein )
max
toward the hydrolysis of ATC, their substrate binding affinities, Km,

are the same (K = 38.46). Attempts to obtain an inhibition constant
m
(K.) failed because carbaryl is a poor yet reversible inhibitor of

cholinesterase (Mount and Oehme 1981). Carbamates bind less tightly to

cholinesterase as compared to most organophosphorous insecticides (Mount

and Oehme 1981). At 107 M to 10- M concentrations, the inhibition

rate showed a flat, nonlinear response after 15 minutes of incubation

with carbaryl against moth head homogenate. A linear increase in
-4 -3
carbaryl inhibition from 10 to 10- M concentrations is shown in Fig.

10, thus verifying that high carbaryl molar concentrations are required

to inhibit AChE.

H. In vitro Carbaryl Metabolism

In vitro carbaryl metabolism studies showed that the R strain

produced 5.4X more carbaryl oxidative metabolites than did the S strain










TABLE 12.


General and Carboxylesterase activities from microsomes of
sixth instar R and S fall armyworm larvae.


Specific activity

(nmol a-naphthol min-i mg protein )


Strain General esterase Carboxylesterase


R 286.20 11.15a/ 196.23 0.32

S 477.57 6.88c/ 261.73 2.23

a/
/ Larvae used in all assays were age synchronized.
SMean SE of at least three experiments, each assayed in duplicate.
SValue significantly different (P < 0.05) from R strain.










TABLE 13.


Acetylcholinesterase activity from moth heads of 1 to 2 day
old mixed population R and S fall armyworms


Specific activity

Strain (nmol ATC metabolized min-I mg protein )


R 339.05 4.93b/

S 253.21 16.73

a/
/ Larvae used in all assays were age synchronized.
Mean SE of at least three experiments, each assayed in duplicate.
































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(Table 14). A 1.76X decrease in oxidative metabolites was seen when

NADPH was not used in an incubation mixture. This suggests that oxida-

tion depends on the NADPH cofactor for maximum reaction rate. The

addition of the esterase inhibitors, DEF (10-4 M) and TOCP (10-4 M), to

the incubation mixture reduced the esterase activity in the R strain by

99.5% and 100%, respectively. However, these inhibitors caused 65% and

72% reduction in esterase activity in the S strain suggesting that the S

strain has a different form of esterase than the R strain. Similarly,

microsomal oxidases which oxidized carbaryl were more susceptible to Pb

inhibition in the R strain (92%) than in the S strain (59%).

The most significant data in the in vitro metabolism of carbaryl

are the production of oxidative metabolites. The R strain metabolizes

more carbaryl per unit time than does the S strain, thus confirming that

oxidative metabolism plays a major role in resistance in this strain.

Cuticular Penetration
14
The rate of disappearance of [ 14C] carbaryl from the exterior

cuticle of sixth instar fall armyworm larvae is shown in Figure 11.

After 24 hours, there remains almost 2X more carbaryl on the exterior

cuticle of R larvae than of S strain. Figure 12 shows that the amount

of carbaryl found internally in both R and S larvae is about the same at

13 and 14%, respectively, of the controls (immediate wash-off) (Fig.

12). Data in Figure 13 show that the S strain excretes more than 2X

more carbaryl than the R strain in 24 hours, however. This suggests

that more carbaryl enters S larvae and more is excreted either as

carbaryl or as carbaryl metabolites while 60% of the applied carbaryl

remains on the cuticle of R larvae.


























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DISCUSSION


The results of this study show that the fall armyworm is resistant

to carbaryl and confirms the findings of Young and McMillian (1979) and

Wood et al. (1981). Young and McMillian (1979) also noted that R-FAW

larvae were resistant to carbaryl biochemically and behaviorally.

According to them, R-FAW larvae avoided carbaryl treated surfaces as

compared to a carbaryl susceptible strain. Lockwood et al. (1984) cited

this type of resistance as stimulus-dependent, i.e. requiring sensory

stimulation to exhibit avoidance in this case, to avoid carbaryl resi-

dues. To further demonstrate the complexity of resistance in FAW, Wood

et al. (1981) showed that FAW larvae that fed on previously carbaryl

treated corn and signalgrass were resistant to carbaryl while those that

fed on bermudagrass and millet were susceptible. These data agree with

those of Yu (1984) where he found that midgut microsomes of a carbaryl

resistant strain of FAW were highly induced by the allelochemicals,

indole 3-carbinol and flavone.

In vivo data (Table 4) show that resistance could not be eliminated

entirely by topical treatments of PB-carbaryl. These findings suggest

that microsomal oxidases play a major role in resistance; however, there

are other factors involved in resistance in this strain. Rose and

Brindley (1985) showed that the Colorado potato beetle (CPB) was highly

resistant to carbaryl. The topical treatment of these beetles with

PB-carbaryl did not eliminate the resistance completely. They concluded

that monooxygenases and other resistance mechanisms may be involved in









CPB resistance to carbaryl and carbofuran. This work agrees with my

findings in FAW.

Results of in vitro assays show that the activities of aldrin

epoxidase (Fig. 14) and biphenyl 4-hydroxylase (Fig. 15) are signifi-

cantly higher in R-FAW larvae compared to S-FAW over all instars tested.

Higher aldrin epoxidase and biphenyl 4-hydroxylase activities in R-FAW

larvae were also observed by Yu (1984) and Yu and Ing (1984), respec-

tively. These data further support in vivo findings that MFO enzymes

play a major role in resistance in this strain.

In vitro metabolism of carbaryl in the R strain showed a 5-fold

increase in oxidative metabolite production over the S strain (Table

14). The fact that differential inhibitions of carbaryl oxidation by PB

were observed between R and S strains suggests that the MFO enzymes from

the R strain were qualitatively different from the S strain. Kuhr

(1971) and Kuhr and Davis (1975) identified carbaryl metabolites pro-

duced by midgut homogenates of R and S cabbage looper and European corn

borer strains. They found that the oxidative metabolite, hydroxymethyl

carbaryl, was the major metabolite produced in vivo and in vitro.

Shrivastava et al. (1969) suggested that hydroxylation of substituted-

aryl methylcarbamate toxicants contributed significantly to the develop-

ment of resistance in a house fly strain. These findings are in agree-

ment with my observations from the fall armyworm.

In the present study, carbaryl metabolites produced in vitro were

not identified; however, those carbaryl metabolites that were found by

other researchers (Price and Kuhr 1969; Camp and Arthur 1967; Andrawes

and Dorough 1967; Kuhr 1970) were chromatographed by TLC and Rfs were

recorded (Table 2). These Rfs were used to isolate carbaryl radiocarbons



















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