MECHANISMS OF CARBARYL RESISTANCE IN THE FALL ARMYWORM,
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
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.
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
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
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
B. Arylester Hyd
History of Carbaryl Resistance
Fall Armyworm Resistance to Carb
. . 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 . . .
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
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
14 In vitro metabolism of carbaryl by midgut homogenate
from R and S fall armyworm larvae . .. 74
LIST OF FIGURES
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
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
11 Percent of applied dose of [ C] carbaryl remaining on
the cuticle of sixth-instar R and S fall armyworm
larvae . . ... .77
12 Percent of applied [ C] carbaryl extracted from homoge-
nate of sixth-instar R and S fall armyworm larvae 79
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
BHC Benzene hexachloride (See HCH)
BSA Bouine serum albumin
CPB Colorado potato beetle
DBLS Diazoblue laurylsulfate
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
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
MFO Microsomal mixed-function oxidase
NADPH Nicotinamide adenine dinucleotide phosphate
O.D. Optical density
OP Organophosphate insecticide
p-NPA p-Nitrophenyl acetate
PB Piperonyl butoxide
R Resistant insect strain
R-AChE Acetylcholinesterase enzyme from resistant strain
R-V Maximum reaction velocity of resistant strain
S Susceptible insect strain
S-AChE Acetylcholinesterase enzyme from susceptible strain
S-V Maximum reaction velocity of susceptible strain
TLC Thin layer chromatography
TOCP Tri-creosyl phosphate
TPP Triphenyl phosphate
USDA United States Department of Agriculture
V Maximum reaction velocity
WHO World Health Organization
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,
Elzie McCord, Jr.
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.
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
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.
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-
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
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.
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
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
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
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
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
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.
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).
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
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
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)
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
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
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.
Primary Secondary Secondary
Figure 1. Metabolism of lipophilic foreign compounds.
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;
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
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
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
Table 1. The primary action of MFO systems on specific chemical
configurations found in xenobiotic molecules.
Reaction Chemical Reaction Consequence*
Type Configuration Products
* Activation means the metabolite is more toxic than the parent compound;
deactivation means the metabolite is less toxic than the parent
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 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
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
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-
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 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-
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
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
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.
14C] carbaryl was purchased from the California Bionuclear Cor-
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.
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
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
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
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
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
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).
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
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
milligram of protein using an extinction coefficient of 10 mM cm for
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
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.
(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
Screw cap tubes were used to hold the incubation mixture which
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).
Methylol-carbaryl (N-hydroxymethyl) 0.29
acetate, and the product in the ethyl acetate was quantified by liquid
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
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
All incubations were duplicated and each experiment was repeated
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
0 0 1
Cl) b "
^ 2 e
Z O .
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
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
Ten milliliters of Scinti-Verse I scintillation cocktail were added
and radioactivity was counted in a Tracor Analytic Data 300 liquid
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.
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
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/
/Synergist R S R/S
Carbaryl 10343b/ 115 90
Butoxide 400.43 67 5.98
a/ Each observation consisted of at least two different tests.
b/ outer 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.
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
(pmol dieldrin min mg protein )
Instar R S
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
6the/ 237.33 1.25 171.39 1.38
Larvae used in all assays were age synchronized.
Mean SE of at least three experiments, each assayed
Value significantly different (< 0.05) from S strain.
1 day old
2 day old.
TABLE 6. Microsomal biphenyl 4-hydroxylase activity in various instars
of R and S fall armyworm larvae.a
(pmol min mg protein )
Instar R S
4th 663.76 2.25b,d/ 177.28 2.75
5th 959.23 29.15d/ 548.27 57.90
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
/ 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 <
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
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
TABLE 7. Microsomal N-demethyl se activity from sixth-instar R and S
fall armyworm larvae
pmol min pmol min
Strain mg protein midgut
R 849.38 59.42b/ 39.96 2.78
S 883.35 141.62 129.70 25.24c
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.
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
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
Strain (nmol DCNB conjugated min-I mg protein )
R 22.73 0.45b/
S 24.12 0.30
a/ Larvae used in all assays were age synchronized.
Mean SE of at least three experiments, each assayed in duplicate.
Microsomal epoxide hydrolase activity in sixth instar R and
S fall armyworm larvae.
Strain (nmol min mg protein )
R 33.92 0.69b/
S 30.63 0.77
SLarvae used in all assays were age synchronized.
Mean SE of at least three experiments, each assayed in duplicate.
4-1 4 1
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
ATC. Although the maximum reaction velocity (V ) is different (R-V
-1 -1 -1 -1
= 0.345 nmol min- my protein ; S-V = 0.208 nmol min ng protein )
toward the hydrolysis of ATC, their substrate binding affinities, Km,
are the same (K = 38.46). Attempts to obtain an inhibition constant
(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
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
General and Carboxylesterase activities from microsomes of
sixth instar R and S fall armyworm larvae.
(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
/ 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.
Acetylcholinesterase activity from moth heads of 1 to 2 day
old mixed population R and S fall armyworms
Strain (nmol ATC metabolized min-I mg protein )
R 339.05 4.93b/
S 253.21 16.73
/ Larvae used in all assays were age synchronized.
Mean SE of at least three experiments, each assayed in duplicate.
Q) 4J 4
C > J aI
O *H *
r) c) 0
0 -H O
13 0 0
) *-4l r
d U .-
(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.
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.
X [i4]-C CARBARYL RECOVERY (OPM)
SI I i i
i v .
(WdO) AU30A33 1AUV9IVO3 0- -[PT] X
i~ ~ I I I I I I I I
1' t i I I i i i .
Si Si k [r X !
(WdO) Al13A03:U 7:AUVB:V:O 3-"T] X
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
NI3108d OW/NIW/NIU073IO 70oWd