Biochemical mechanisms responsible for stage-dependent propoxur tolerance in the German cockroach

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Biochemical mechanisms responsible for stage-dependent propoxur tolerance in the German cockroach
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Thesis (Ph. D.)--University of Florida, 1995.
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Includes bibliographical references (leaves 95-115).
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by Steven M. Valles.
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Vita.

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BIOCHEMICAL MECHANISMS RESPONSIBLE FOR
STAGE-DEPENDENT PROPOXUR TOLERANCE
IN THE GERMAN COCKROACH

















By

STEVEN M. VALLES


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

1995


UNIVERSITY OF FLORIDA LIBRARIES




































For Sara.















ACKNOWLEDGEMENTS


I would like to thank the following people who, in one

way or another, contributed to the completion of this work:



Keith Heberlein for clean labware.

Euripides Mena for rearing cockroaches.

Deanna Harrison for rearing cockroaches and allowing me

to often dictate her schedule.

Myrna Litchfield for guiding me through the graduate

school bureaucracy.

Chuck Strong for his friendship and teaching me cockroach

biology.

Sam Nguyen for her friendship, technical expertise, and

moral support.

Dr. Robert Koch for supplying the radiolabeled propoxur

and metabolites.

Dr. Jim Maruniak for autoradiography assistance and

laboratory space.

Dr. Peter Teal for helpful discussions.

Dr. John Capinera for advice and moral support.

Dr. Rachael Shireman for serving on my committee and for

helpful discussions.


iii









Dr. James Nation for serving on my committee and for

helpful discussions and advice on experimental methods.

Mr. and Mrs. James Gahan for providing the financial

support enabling me to pursue a doctorate in entomology.

Dr. Philip Koehler for providing the impetus for this

study, for his friendship, and for many opportunities.



Finally, I would like to express my profound gratitude to

Dr. Simon J. Yu for serving as my committee chairman. I would

also like to thank Simon for his guidance, patience, and

friendship during this often frustrating but fulfilling period

of my life.















TABLE OF CONTENTS



ACKNOWLEDGEMENTS . . iii

Abstract . . vii

INTRODUCTION . . 1

LITERATURE REVIEW . . 4
Biology . . 4
Pest Status . . 5
Insecticide Resistance . 6
Insecticide Resistance Mechanisms . 7
Decreased Penetration . 8
Target Site Insensitivity . 8
Enhanced Metabolism . 11
Insecticide Resistance Mechanisms in the German
Cockroach . . 14
Village Green Strain . 18

MATERIALS AND METHODS . . 31
Insects . . 31
Chemicals . . 31
Insecticide Bioassays . 32
Microsome Preparation . 33
Tissue Localization . 34
Enzyme Assays . . 34
Microsomal Oxidases . 35
Reductases . . 38
Glutathione S-Transferases . 40
Esterases . . 42
Glucosidase . . 44
Propoxur Metabolism . 45
Penetration Studies . 47
Enzyme Induction . . 48
Statistics . . 49

RESULTS . . . 50
Microsome Preparation . 50
Tissue Localization . 51
Developmental Expression . 52
Enzyme Induction . . 52
Insecticide Bioassays . 53
Enzyme Assays . . 54









Insecticide Penetration . 56


DISCUSSION . . 85

REFERENCE LIST . . 95

BIOGRAPHICAL SKETCH ................ 116















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

BIOCHEMICAL MECHANISMS RESPONSIBLE FOR STAGE-DEPENDENT
PROPOXUR TOLERANCE IN THE GERMAN COCKROACH

By

Steven M. Valles

December 1995


Chair: Simon J. Yu
Cochair: Philip G. Koehler
Major Department: Entomology and Nematology

Methods of microsome preparation were evaluated to

determine their influence on the activity of cytochrome P450

monooxygenases in adult males and final instar nymphs of the

German cockroach, Blattella germanica (L.). Aldrin epoxidase

activity was significantly improved when homogenization

occurred in a protected buffer and when alimentary canal

contents were removed prior to homogenization. Adult male

aldrin epoxidase activity was highest in midgut and Malpighian

tubules, while no single tissue dominated in methoxyresorufin

O-demethylase activity. In final instar nymphs, aldrin

epoxidase specific activity was 9- to 11-fold higher in midgut

than in fat body, while methoxyresorufin (MR) O-demethylase

specific activity was 17- to 22-fold higher in fat body than

in midgut.


vii









Final instar nymphs and adult males of the Village Green

strain of German cockroach were investigated to determine the

mechanisms responsible for conferring stage-dependent propoxur

tolerance. Propoxur susceptibility was dependent upon age

within the final instar. Final instar nymphs (1-day-old) were

as susceptible to topically applied propoxur as adult males

(7- to 14-day-old) while 7- and 12-day-old nymphs were up to

16-fold more tolerant. Piperonyl butoxide (PBO), a cytochrome

P450 monooxygenase inhibitor, almost completely eliminated the

tolerance of propoxur in male and female nymphs compared with

adult males. The rate of in vitro microsomal oxidation of

propoxur was three times higher in male and female final

instars than in adult males. Similar k, (bimolecular rate

constant) values for propoxur in nymphs and adult males

indicated that acetylcholinesterase insensitivity did not

contribute to the enhanced nymphal tolerance. Cuticular

penetration studies using [14C]propoxur revealed that there was

no marked difference in the rate of penetration of propoxur in

nymphs and adults. The results indicated that nymphal

tolerance toward propoxur was largely due to enhanced

microsomal oxidation.


viii














INTRODUCTION


The German cockroach, Blattella germanica (L.), is

considered a serious pest in the urban environment (Cornwell

1968). Large quantities of insecticides are applied annually

in an effort to control this insect. The combination of

isolated populations and intense insecticide pressure, which

often typify the habitat of the German cockroach, are ideal

conditions for the development of insecticide resistance

(Atkinson et al. 1991). Several biochemical mechanisms have

been reported to be responsible for conferring insecticide

resistance in this species, including increased detoxication

via oxidative and hydrolytic enzymes (Bull et al. 1989,

Siegfried and Scott 1991, Hemingway et al. 1993b, Prabhakaran

and Kamble 1994), nerve insensitivity (kdr-type) (Scott and

Matsumura 1981, Umeda et al. 1988, Bull and Patterson 1993),

and decreased cuticular penetration (Siegfried and Scott

1991).

Insecticide resistance is a serious threat to the

effective control of German cockroaches. Indeed, resistance

ratios as small as 5-fold are considered significant enough to

result in control problems (Cochran 1989, Scott 1991a).









2

However, Koehler et al. (1993) have recently demonstrated that

stage-dependent insecticide susceptibility may also preclude

effective control of the German cockroach. Specifically, they

showed that late stage nymphs (4th-6th instars) were up to 27-

fold more tolerant of bendiocarb, chlorpyrifos, and

cypermethrin than adult males. Adult males are typically used

in toxicological bioassays to determine the effectiveness of

insecticides and to estimate the resistance level of various

strains. However, since nymphs are more tolerant of

insecticides, resistance levels may be underestimated and

insecticide efficacy may be exaggerated when based on

toxicological evaluations using adult males. This stage-

dependent tolerance phenomenon is compounded by the fact that

nymphs have been estimated to comprise >80% of field

populations (Ross and Mullins 1995).

Cytochrome P450 monooxygenases are an important

detoxication system by which insects, such as the German

cockroach, may develop insecticide resistance or exhibit

insecticide tolerance. Methods used for the evaluation of

German cockroach cytochrome P450 monooxygenases have relied

upon microsomes derived from either whole insects (Hemingway

et al. 1993a) or thorax-abdomen combinations (Siegfried and

Scott 1992a, Valles and Yu 1995). Further, most of these

investigations have dealt with adult males, or rarely nymphs,

of unknown age. Since numerous factors, including tissue,

sex, developmental stage, age, and hormonal status have been









3

reported to profoundly affect the activity of insect

cytochrome P450 monooxygenases (Hodgson 1985), a thorough

investigation of these factors in the German cockroach is

warranted, especially since a paucity of information exists on

these enzymes in the German cockroach. Therefore, to

adequately evaluate the contribution of cytochrome P450

monooxygenases in stage-dependent insecticide tolerance and to

develop a better general understanding of these enzymes in the

German cockroach, methods of microsome preparation from adult

males and final instar nymphs were studied. In addition,

constitutive expression of cytochrome P450 monooxygenases,

tissue-specificity, the inducibility of these enzymes and the

effects of age on enzyme induction were also investigated.

Although stage- and gender-dependent differential

insecticide susceptibility have been documented in the German

cockroach by other investigators (Dewey 1942, Fales and

Bodenstein 1963), the mechanisms responsible for this stage-

dependent phenomenon have not been reported. Therefore, the

biochemical mechanisms responsible for conferring stage-

dependent insecticide susceptibility to the carbamate

insecticide, propoxur, in the Village Green strain of German

cockroach were investigated.















LITERATURE REVIEW


Biology


The German cockroach, Blattella germanica (L.)

(Dictyoptera: Blattellidae), has three life stages typical of

insects with incomplete metamorphosis: the egg, larva or

nymph, and adult. The entire life cycle is completed in about

100 days (Archbold et al. 1987). However, factors such as

temperature, nutritional status, and strain differences may

influence the time required to complete a life cycle (Kunkel

1966, Cooper and Schal 1992a, Cooper and Schal 1992b). Once

fertilized, eggs pass into the vestibulum where they are

individually covered by secretions from the collateral glands

(Roth 1968). As additional eggs pass into the vestibulum an

egg case or ootheca begins to protrude from the genital

chamber. The female carries the ootheca until hatch or drops

it shortly before hatch (Barson and Renn 1983a). Typically 30

to 40 eggs are contained within an ootheca (Willis et al.

1958).

The nymphal stage begins with egg hatch and ends with the

emergence of the adult. The number of molts required to reach

the adult stage varies, but the most frequently reported

number of molts is 6 (Ross and Mullins 1995). Nymphs comprise









5
a significant proportion of the age structure of German

cockroach populations. Owens and Bennett (1983) reported

that 85-90% of the populations in low income housing in

Indianapolis, Indiana, were nymphs.

Adults are about 16 mm in length and tan to light brown

in color. The pronotum is characterized by two longitudinal

stripes and, although fully winged, adults cannot fly. Males

are easily distinguished from females by their slender

tapering shape (Ebeling 1975).


Pest Status


The German cockroach has worldwide distribution and is

strictly a domiciliary pest (Patterson and Koehler 1992).

Cockroaches adulterate food or food products with their feces

and defensive secretions (Ebeling 1975), physically transport

and often harbor pathogenic organisms (Roth and Willis 1960),

cause severe allergic responses (Brenner et al. 1991), and in

extremely heavy infestations have been reported to bite humans

and feed on food residues on the faces of sleeping humans

(Frings 1948, Roth and Willis 1957). In addition, Brenner

(1995) suggests that German cockroach infestations cause human

psychological stress. He proposes that the stigma associated

with infestations alters human behavior. For example, people

with infested houses do less entertaining, and avoid the

kitchen at night for fear of encountering a cockroach. Most

people associate cockroach infestations with poor sanitary









6
conditions and will go to excessive lengths to eradicate them

from their dwelling. It is no wonder that cockroaches are

considered the number one household pest in the United States

(Brenner 1995).


Insecticide Resistance

Insecticide resistance is the "development of an ability

in a strain of some organism to tolerate doses of a toxicant

which would prove lethal to the majority of individuals in a

normal population of the same species (Anonymous 1957) ."

Insecticide resistance is arguably the most important problem

currently facing entomologists. The number of arthropod

species that have developed resistance to insecticides exceeds

500 (Georgiou 1990) and is growing at a near exponential rate

(Scott 1991a). Public concern over the environmental impact

of insecticides, increasingly stringent government regulations

and exorbitant costs associated with insecticide development,

and insecticide resistance have significantly reduced the

number of effective insecticides available for insect control.

Therefore, it is imperative that measures are taken to prolong

the effectiveness of our dwindling insecticide resources.

Understanding the mechanisms and genetics of insecticide

resistance (and tolerance) will allow the development of

effective strategies to delay or prevent its occurrence. For

example, if the biochemical mechanism responsible for

resistance to a particular insecticide is known, it may be









7

possible to develop population monitoring techniques as part

of a resistance management program or devise ways to overcome

the resistance.

Insecticide resistance became a problem shortly after the

introduction of synthetic organic insecticides during World

War II. The first report of insecticide resistance in the

German cockroach was to chlordane (Grayson 1951). Since that

time, numerous investigations concerning the resistance

phenomenon in German cockroaches have been conducted (Table

1).


Insecticide Resistance Mechanisms


Insects exhibit three physiological insecticide

resistance mechanisms, including decreased cuticular

penetration, enhanced metabolism, and target-site

insensitivity. Insecticide sequestration and behavioral

modification are additional mechanisms reported in insects.

However, sequestration is considered rare (Devonshire and

Moores 1982) and the extent to which behavioral modification

contributes to resistance is difficult to quantify (Hostetler

and Brenner 1994). Although the expression of only one

mechanism is sufficient for resistance to occur, usually

several mechanisms work in concert to confer resistance (i.e.

multiple resistance mechanisms).










Decreased Penetration


To be effective, insecticides must reach their target

site in a lethal dose. Thus, delaying the rate of insecticide

penetration through the integument is an important mechanism

of resistance (Brattsten et al. 1986). Functionally, delayed

penetration reduces the rate of insecticide influx into the

animal which provides time for supplementary detoxication

mechanisms (e.g. enhanced metabolism, accelerated excretion)

to exert their effects (Matsumura 1985). Alone, this

mechanism confers only low levels of resistance (less than 3-

fold) (Plapp and Hoyer 1968), but, when present with other

resistance mechanisms may contribute significantly to

insecticide resistance. Decreased penetration is not specific

for any particular class of insecticide and, as a result,

provides protection against a variety of insecticides

(Oppenoorth 1985, Plapp and Hoyer 1968).


Target Site Insensitivity


Insecticide resistance may result from modification of

the toxicant's target site. Such modifications may result in

reduced sensitivity to the toxicant. The most extensively

studied example of target site insensitivity is altered

acetylcholinesterase, which confers resistance to

organophosphorus and/or carbamate insecticides (Fournier and

Mutero 1994). Acetylcholinesterase mutations reduce its

reaction rate with these insecticides, whereas the normal









9

function of the enzyme hydrolysiss of acetylcholine) is either

unaffected or decreased but still sufficient to be compatible

with survival (Oppenoorth 1985). Mutero et al. (1994)

reported that 5 point mutations in the acetylcholinesterase

gene (Ace) of Drosophila were responsible for resistance to

various organophosphorus and carbamate insecticides. They

showed that a combination of several point mutations within

the same protein conferred greater levels of resistance than

any single point mutation. They concluded that outbreeding

would favor this intracistronic recombination and promote

resistance development while inbreeding and parthenogenetic

reproduction would prevent it.

To date, altered acetylcholinesterase has not been

confirmed as a mechanism of resistance against

organophosphorus or carbamate insecticides in the German

cockroach. Siegfried and Scott (1992b) suggest that the

German cockroach lacks the genetic plasticity for modification

of acetylcholinesterase. However, the lack of altered

acetylcholinesterase as a mechanism of resistance in the

German cockroach may be the result of isolated populations

thus preventing intracistronic recombination of Ace genes

possessing specific point mutations (Mutero et al. 1994).

Another important target site modification resulting in

insecticide resistance is modification of the voltage-gated

sodium channels. Mutation of these sodium channels results in

resistance to pyrethroids (and DDT) and is known as knockdown









10
resistance (kdr). Knockdown resistance was first observed by

Busvine (1951) who noted that a housefly strain survived DDT

and pyrethrins treatment without being initially knocked down

as in normal housefly strains. Therefore, kdr is strictly a

housefly specific term. The terms kdr-like or kdr-type are

more appropriate to describe cases of resistance to DDT and

pyrethroid insecticides in other insects. Pyrethroid

insecticides and DDT modify the sodium current of nerve cells

by slowing down the inactivation of the sodium channel

(Narahashi and Lund 1980, Scott and Dong 1994). Therefore,

sodium channel modification may prevent or reduce insecticide

binding and prevent their toxic action.

Similar target site modifications resulting in

insecticide resistance in insects have been identified for

cyclodiene (ffrench-Constant et al. 1993a, ffrench-Constant et

al. 1993b) insecticides. ffrench-Constant et al. (1993a)

reported that a single point mutation (alanine to serine) in

the y-aminobutyric acid receptor of Drosophila was responsible

for conferring high levels of resistance to cyclodiene

insecticides. Further, target site insensitivity has been

reported against non-traditional insecticides such as juvenile

hormone analogs (Wilson and Turner 1992), and Bacillus

thuringiensis (Bt) toxins (Van Rie et al. 1992). Resistance

to Bt toxins has been shown to be the result of a change in

the receptor for these proteins.











Enhanced Metabolism


Increased metabolism of insecticides leading to the

formation of less toxic products (i.e. detoxication) is the

most common and arguably the most important mechanism of

insecticide resistance in insects (Oppenoorth 1985).

Insecticide detoxication occurs by way of oxidation,

reduction, hydration, conjugation, and hydrolysis of

xenobiotics. The primary goal of detoxication reactions is to

create more water soluble products from hydrophobic substrates

to facilitate excretion.

Xenobiotic metabolism occurs in three characteristic

phases. Phase I reactions result in the functionalization of

the xenobiotic. Specifically, functional groups are added or

uncovered providing a "handle" for subsequent reactions to

occur. Phase II reactions are biosynthetic; they catalyze the

conjugation of the xenobiotic with endogenous compounds such

as glutathione, phosphate, sulfate, or amino acids. This

conjugation tends to produce water soluble metabolites that

are more easily excreted. An added benefit of conjugation

with an endogenous compound is that in some cases the added

chemical group is recognized by specific carrier proteins

involved in facilitated diffusion or active transport

(deBethizy and Hayes 1989). Finally, phase III in the

detoxication process is transport of the metabolite out of the

cell. For example, recent evidence suggests that a distinct









12

type of ATP-dependent export pump transports a variety of

glutathione conjugates out of cells (Ishikawa 1992).

Although any number of enzymes may contribute to the

detoxication of xenobiotics, cytochrome P450 monooxygenases

(also known as microsomal oxidases or mixed-function

oxidases), glutathione S-transferases, and hydrolases play the

most significant role in this process. Cytochrome P450

monooxygenases oxidize their substrates by the introduction of

a single molecule of oxygen. The cytochrome P450

monooxygenase system is located in the endoplasmic reticulum

and is composed of NADPH-cytochrome P450 reductase and

cytochrome P450. In addition to these proteins, NADPH,

oxygen, a substrate, and phosphatidylcholine are required for

activity. Electron equivalents are passed from NADPH to

NADPH-cytochrome P450 reductase which in turn passes them on

to the cytochrome P450. Cytochrome b5 has also been shown to

participate in the catalytic process as a mediator of electron

flow from NADH to cytochrome P450 (Zhang and Scott 1994).

Another group of enzymes known to play an important role

in insecticide detoxication in insects are the glutathione S-

transferases. Glutathione S-transferases are a large group of

enzymes that catalyze the conjugation of organic molecules

possessing an electrophilic center with the tripeptide

glutathione (Clark 1989). These enzymes are composed of two

subunits (homodimers or heterodimers between 20-27kDa) and

each has an independent catalytic center. Catalysis is









13

initiated by abstraction of the glutathione thiol proton by a

basic amino acid residue (possibly histidine) near the active

site. This enhances the reactivity of the thiol group by

ionization. The enzyme strategically places the electrophilic

center of the substrate in proximity to the active thiolate

ion which facilitates catalysis (nucleophilic attack). Many

insecticides, especially in the organophosphorus class,

possess an electrophilic center that may serve as substrate

for glutathione S-transferases. Indeed, glutathione S-

transferases have been reported to be an important

detoxication mechanism against organophosphorus insecticides

(Lamoureux and Rusness 1987). After conjugation, glutamate

and glycine residues are cleaved from the conjugate leaving a

water soluble mercapturic acid derivative. In addition to

their catalytic activity, glutathione S-transferases may also

serve to sequester potentially toxic organic molecules as well

as endogenous compounds such as steroids (Wilkinson 1985).

Finally, various esterases or hydrolases are involved in

insecticide detoxication in insects. Most traditional

insecticides (organophosphates, carbamates, pyrethroids)

contain either a carboxyl ester, phosphoester, or carbamate

ester bond in their structure. The rate at which an insect

can hydrolyze these bonds to unmask these functional groups

can influence their toxicities. These enzymes often have

broad substrate specificity. They are intended to catalyze

the hydrolysis of endogenous compounds but often metabolize









14

xenobiotics that have similar structures to their endogenous

substrates. Esterases may be classified by how they interact

with organophosphates and by their substrate specificity. A-

esterases (= arylesterases) prefer carboxylesters with aryl

groups bonded to the carbonyl carbon and can use

organophosphate esters as substrates. B-esterases (=

aliphatic esterases) prefer esters with alkyl groups bonded to

the carbonyl carbon and are inhibited by organophosphate

esters. Finally, C-esterases prefer acetate esters and do not

interact with organophosphates. Many insects have low levels

of A-esterases compared with mammals which often explains the

selective toxicity of the organophosphorus insecticide

malathion to insects and birds (deBethizy and Hayes 1989).


Insecticide Resistance Mechanisms in the German Cockroach


Although insecticide resistance mechanisms in the German

cockroach have been investigated by numerous authors (see

Table 1), only a handful of these reports identify the

specific mechanisms responsible for the resistance.

Resistance mechanisms in the German cockroach have been

investigated at various levels, including (1) investigations

that implicated the biochemical mechanisms) based on

synergist studies and the resistance spectrum (cross

resistance), (2) reports that compared various physiological

or behavioral differences between resistant and susceptible









15
German cockroach strains, and (3) complete elucidation of the

resistance mechanism.

Insecticide synergists have been used by some researchers

to provide indirect evidence about the resistance mechanisms

of several German cockroach strains (Cochran 1987a), (Scott et

al. 1990), (Cochran 1994c), and (Valles and Yu 1995).

Synergists are compounds that greatly enhance the toxicity of

an insecticide, while they are not toxic themselves (Matsumura

1985). Synergists act by inhibiting a given detoxication

pathway. Two of the most common insecticide synergists are

piperonyl butoxide (PBO) and S,S,S-tributyl

phosphorotrithioate (DEF) which inhibit cytochrome P450

monooxygenases and hydrolases, respectively. Based on

synergist bioassay data against resistant and susceptible

insect strains, it is possible to show that a given class of

detoxication enzymes is involved in the resistance.

Ku and Bishop (1967) were the first to identify the

specific insecticide resistance mechanisms in a strain of the

German cockroach. They evaluated in vivo carbaryl metabolism,

insecticide penetration rates, and excretion of metabolites in

a carbaryl-resistant and susceptible strain of German

cockroach. They concluded that the major mechanism

responsible for the resistance was enhanced hydrolytic

metabolism of carbaryl to 1-naphthol and more rapid

conjugation (glucose and sulfate) of 1-naphthol. Decreased









16

cuticular penetration and accelerated excretion were found to

be minor, but contributing factors in the resistance.

Similarly, enhanced metabolism and decreased penetration

were reported to be the major mechanisms of resistance to

propoxur in the Baygon-R strain of German cockroach (Siegfried

and Scott 1991). Siegfried and Scott (1991) found that in

vivo and in vitro oxidative and hydrolytic metabolism of

propoxur was significantly greater in the Baygon-R strain than

in a standard susceptible strain. The penetration of propoxur

was found to be significantly slower in the Baygon-R strain

and resistance was reduced 3.4-fold when the insecticide was

injected compared to topically applied. These results

implicated decreased cuticular penetration as a resistance

mechanism.

Enhanced metabolism was also demonstrated to be

responsible for resistance to malathion and carbaryl (Bull et

al. 1989), and chlorpyrifos (Siegfried et al. 1990). Several

reports have implicated enhanced insecticide metabolism as the

mechanism of resistance to chlorpyrifos, propoxur,

cypermethrin (Prabhakaran and Kamble 1993, 1994), cyfluthrin,

fenvalerate, cypermethrin, lambda-cyhalothrin (Hemingway et

al. 1993a), chlorpyrifos, propoxur (Hemingway et al. 1993b),

and permethrin (Anspaugh et al. 1994).

Prabhakaran and Kamble (1995) reported that an esterase

(E6) from two resistant strains of German cockroach was more

active than in a susceptible strain. Kinetic analyses









17
revealed no differences in K, values between the strains.

However, V.x was significantly higher in the resistant

strains. Inhibition studies with paraoxon, chlorpyrifos, and

propoxur did not suggest any structural differences in the E6

esterase between resistant and susceptible strains.

Therefore, they concluded that resistance in these strains was

due to the increased production of the E6 esterase. In

addition, they tentatively concluded that the role of E6 was

sequestration of toxic molecules rather than hydrolysis.

kdr-Type resistance is apparently a common mechanism of

resistance to pyrethroid, DDT, and other sodium channel

neurotoxins in German cockroaches (Umeda et al. 1988, Cochran

1989, Bull and Patterson 1993, Scott and Dong 1994). kdr-Type

resistance was first documented in the VPIDLS German cockroach

strain by Scott and Matsumura (1981). In the German

cockroach, kdr-type resistance is incompletely recessive,

monogenic, and is not sex linked (Scott and Dong 1995).

Additionally, the kdr locus is tightly linked or identical to

the para-homologous sodium channel locus in several insect

species, suggesting that this resistance is due to mutations

in the para-homologous sodium channel gene (Dong and Scott

1994).

Recently, a new resistance mechanism has been reported in

the German cockroach. Silverman and Bieman (1993) discovered

a strain that avoided hydramethylnon bait stations. They

concluded that the avoidance behavior was because of an









18
aversion to D-glucose in the bait. The avoidance behavior was

not learned, rather was inherited as an autosomal incompletely

dominant trait.


Village Green Strain

The Village Green strain of German cockroach is one of

the most extensively studied German cockroach strains.

Complete characterization of a strain is essential to

advancing our understanding of the insecticide resistance

phenomenon, as well as aiding in the development of methods to

prevent or delay the evolution of insecticide resistance. The

Village Green strain was colonized from an infested apartment

(Village Green complex) in Gainesville, Florida, in 1988

(Atkinson et al. 1991). This strain was reported to possess

some of the highest resistance ratios for pyrethroid

insecticides ever documented (up to 337-fold). Pyrethroids

with an alpha-cyano functional group were generally more toxic

than those lacking this moiety (Atkinson et al. 1991).

Synergist experiments conducted with piperonyl butoxide and

,SS,S-tributyl phosphorotrithioate resulted in partial

elimination of the resistance (Atkinson et al. 1991). It was

tentatively concluded from this study that the resistance

mechanism to pyrethroids involved enhanced metabolism and

target-site insensitivity (kdr-like). A similar conclusion

was also reached by Anspaugh et al. (1994) using the

pyrethroid insecticide permethrin. They reported that









19

cuticular penetration of [14C]permethrin was significantly

slower and that in vivo metabolism of [14C]permethrin was

significantly greater in the Village Green strain as compared

to a susceptible strain (Orlando). They concluded that

multiple mechanisms of insecticide resistance, including

decreased cuticular penetration, and enhanced metabolism were

responsible for permethrin resistance in the Village Green

strain. In contrast to these studies, Bull and Patterson

(1993) reported that the primary resistance mechanism of

permethrin resistance in the Village Green strain was a kdr-

like target-site insensitivity possibly augmented by decreased

cuticular penetration. They also concluded that although

metabolism was a very important defensive mechanism of both

strains, it was not considered a major contributing factor in

the resistance.

Hostetler et al. (1994) reported that there was no

energetic cost associated with the insecticide resistance of

Village Green cockroaches. Metabolic rates, based on oxygen

consumption, were similar for Village Green and Orlando

strains. Further, Hostetler and Brenner (1994) detected no

behavioral traits that contributed to insecticide resistance

in the Village Green cockroach. They concluded that

behavioral resistance was not a contributing factor in Village

Green resistance because both resistant and susceptible

strains exhibited similar levels of irritability toward

insecticides.









20

Koehler et al. (1993) investigated the tolerance level of

each developmental stage in the Village Green strain toward

chlorpyrifos, bendiocarb, and cypermethrin. They found that

late-stage nymphs (4th to 6th instars) were significantly more

tolerant (up to 27-fold) to a topically applied insecticide

challenge than adult males. Adult males are typically used to

assess insecticide resistance levels and to determine the

efficacy of insecticides. Since nymphs are more tolerant of

insecticide exposure, tolerance levels of German cockroach

populations may be significantly underestimated and

insecticide efficacy may be exaggerated when based solely on

toxicological data collected from adult males.

The Village Green strain was also used as a model insect

in the development of a technique capable of detecting

insecticide resistance in the field (Moss et al. 1991, Moss et

al. 1992). Moss et al. (1991, 1992) used cockroach traps

containing an insecticide in the glue matrix in an effort to

detect the resistance spectrum of a particular population of

German cockroach. With this information, pest control

operators could choose the most effective insecticide against

individual populations.

Finally, Valles and Koehler (1994) evaluated the

influence of carbon dioxide anesthesia on chlorpyrifos

toxicity in the Village Green German cockroach strain. They

concluded that CO2 used either once of limited duration (<15

min) or multiple times of short duration, not to exceed 4









21
total knockdowns, did not increase chlorpyrifos toxicity in

the German cockroach. However, if these guidelines were not

followed, CO2 could potentiate the toxicity of chlorpyrifos

and probably other insecticides as well.











Table 1. References of Research Conducted on Insecticide
Resistance in the German Cockroach, Blattella germanica (L.).


Insecticide Research Citation

Class Areab


DOC, PE,


Grayson (1951)


OC

OC

OC, OP, PYR


INH

PE

CONT


DOC


OC, OP


CONT, SS

CONT

INH

CONT


Cochran (1952)

Babers and Roan (1953)

Fisk and Isert (1953)

Grayson (1953)

Heal et al. (1953)

Munson (1954)

Grayson (1954)

Butts and Davidson (1955)

Laake (1955)

Grayson and Jarvis (1956)

Jarvis and Grayson (1957)


a OC, organochlorine; OP, organophosphorus; CB, carbamate;
PYR, pyrethroid.
b BEH, behavioral effects associated with resistance; CONT,
control of resistant cockroaches and insecticide evaluations;
DOC, documentation of resistance; DEC, loss of resistance;
INH, mode of inheritance of resistance; MECH, mechanism of
resistance; MON, resistance detection and monitoring methods;
PE, physiological effects other than resistance mechanisms;
SS, selection studies including cross resistance.


OC, OP

OC

OC, OP











Table 1--Continued.


Citation


Insecticide

Class

OC, OP

OP

OC

OC, OP

OC, OP

OC

OC

OC

OC

OC



OC, OP

OC

OC, OP, CB

OP

OC, OP, CB

OP


Research

Areab

PE

MECH

DOC

DOC

SS

PE

DOC

PE

INH

MON



SS

SS

DOC

DOC

DOC

PE


Alexander et al. (1958)

Krueger and O'Brien (1959)

Zwick (1959)

Burden et al. (1960)

Grayson (1960)

Agosin et al. (1961)

Webb (1961)

Perkins and Grayson (1961)

Cochran and Ross (1962)

Fales and Bodenstein

(1963)

Grayson (1963)

Cochran and Ross (1964)

Stapp (1964)

Grayson (1965)

Ishii and Sherman (1965)

Mansingh (1965)











Table 1--Continued.


Citation


Insecticide

Class

OC, OP

OP



OP, CB

CB

OC, PYR

OC, OP, CB

CB

OC

CB

OC

OC

CB

OP, CB, PYR

OC, OP, CB

OC


Research

Areab

DOC, PE

MECH, SS



SS

MECH, PE

MECH, PE

DOC

DOC, SS

PE

MECH

DOC, MECH

PE

MECH

CONT, PE

DOC

PE


Russell and Frishman (1965)

Vanden Heuvel and Cochran

(1965)

McDonald and Grayson (1966)

Ku and Bishop (1967)

Matsumura et al. (1967)

Bennett and Spink (1968)

McDonald and Cochran (1968)

Wright (1968)

El-Aziz et al. (1969)

Hooper (1969)

Patil et al. (1969)

Shrivastava et al. (1969)

Bennett and Wright (1970)

Johnson and Young (1970)

Telford and Matsumura

(1970)











Table 1--Continued.


Citation


Insecticide

Classa

OC

OC

OC

PYR

OP

OC, OP, CB, PYR

OP

OP, CB

OC, OP, CB, PYR

OC, OP, CB

OC, OP, CB

OP, CB

CB

OC

OC, PYR


Research

Areab

MECH, PE

MECH

PE

INH

INH

MECH, SS

INH

DOC, SS

SS

DOC

DOC, SS

DOC

MON

MECH, PE

MECH


Wang and Matsumura (1970)

Matsumura (1971)

Khan and Matsumura (1972)

Cochran (1973a)

Cochran (1973b)

Collins (1973)

Cochran (1975)

Collins (1975)

Collins (1976)

Batth (1977)

Barson and McCheyne (1978)

Rust and Reierson (1978)

Barson and McCheyne (1979)

Ghiasuddin et al. (1981)

Scott and Matsumura (1981)











Table 1--Continued.


Citation


Insecticide

Class

OC, OP, CB

OC, OP, CB, PYR

PYR

CB

OC



CB

OC, OP, CB

OC, PYR

CB, PYR

PYR

CB

OP

OP, PYR

OP, CB, PYR

PYR


Research

Areab

DOC

DOC, SS

MECH, PE

BEH

MECH



BEH

DOC

PE, SS

DOC, MECH

SS

PE

MON

CONT

DOC

MECH


Nelson and Wood (1982)

Barson and Renn (1983b)

Scott and Matsumura (1983)

Brett and Ross (1985)

Rashatwar and Matsumura

(1985)

Brett and Ross (1986)

Koehler and Patterson (1986)

Scott et al. (1986)

Cochran (1987a)

Cochran (1987b)

Harmon and Ross (1987)

Milio et al. (1987)

Koehler and Patterson (1988)

Schal (1988)

Umeda et al. (1988)











Table 1--Continued.


Citation


Insecticide

Class

OP, CB

OP, CB, PYR

CB

OP, CB, PYR

ABAMECTIN

HYDROPRENE

OP, CB, PYR

OP, CB

OP

PYR

PYR

OC, PYR



OP

HYDRAMETHYLNON

OP, CB, PYR


Research

Areab

MECH

DOC, MON

PE

DOC

CONT

PE

DOC, MECH

MECH, PE

MECH

DOC, MECH

SS

INH,

MECH, PE

CONT

DOC

MON


Bull et al. (1989)

Cochran (1989)

Kramer et al. (1989)

Wadleigh et al. (1989)

Cochran (1990)

Kramer et al. (1990)

Scott et al. (1990)

Siegfried and Scott (1990)

Siegfried et al. (1990)

Atkinson et al. (1991)

Cochran (1991)

Dong and Scott (1991)



Koehler and Patterson (1991a)

Koehler and Patterson (1991b)

Moss et al. (1991)











Table 1--Continued.


Citation


Insecticide

Class

PYR

ABAMECTIN,

HYDRAMETHYLNON

CB

PYR

PYR



OP, CB, PYR

PYR

OP, PYR

SULFLURAMID,

HYDRAMETHYLNON

OP, CB

OP, CB

PYR

PYR

PYR


Research

Areab


PE

DOC



MECH

PE

MECH, PE



MON

BEH

BEH

DOC



MECH, PE

MECH

MON

MECH

DOC


Ross (1991)

Scott (1991)



Siegfried and Scott (1991)

Wadleigh et al. (1991)

Charalambous and Matsumura

(1992)

Moss et al. (1992)

Ross (1992)

Ross and Cochran (1992)

Schal (1992)



Siegfried and Scott (1992a)

Siegfried and Scott (1992b)

Zhai and Robinson (1992)

Bull and Patterson (1993)

Cochran (1993)











Table 1--Continued.

Insecticide

Classa

PYR

PYR

OP, CB

OP, CB, PYR

OP, CB, PYR



PYR




OP, CB, PYR

GLUCOSE

PYR

ABAMECTIN

CB

PYR

OP, CB, PYR

PYR

OC, PYR


Citation


Research

Areab

MECH, PE

DOC, MECH

DOC, MECH

DOC, PE

MECH



BEH

PE

CONT, DOC

MECH

MECH

SS

INH

DOC, MECH

DOC

INH

PE


Dong et al. (1993)

Hemingway et al. (1993a)

Hemingway et al. (1993b)

Koehler et al. (1993)

Prabhakaran and Kamble

(1993)

Ross (1993)

Ross and Crouch (1993)

Rust et al. (1993)

Silverman and Bieman (1993)

Anspaugh et al. (1994)

Cochran (1994a)

Cochran (1994b)

Cochran (1994c)

Cochran (1994d)

Cochran (1994e)

Dong and Scott (1994)











Table 1--Continued.


Insecticide

Class"

PYR






OP, CB, PYR



OC, PYR

OP

OP, CB



PYR

BORIC ACID

OP, CB, PYR


Research

Areab

PE

MECH



MECH



MECH, PE

PE

MECH



INH

PE

DOC, MECH


Citation


Hostetler et al. (1994)

Hostetler and Brenner

(1994)

Prabhakaran and Kamble

(1994)

Scott and Dong (1994)

Valles and Koehler (1994)

Prabhakaran and Kamble

(1995)

Ross and Cochran (1995)

Cochran (1995)

Valles and Yu (1995)















MATERIALS AND METHODS


Insects


The multi-insecticide-resistant Village Green German

cockroach strain was used in all tests. The cockroaches were

obtained from the USDA, Gainesville, Florida and reared at 260

C, 55% relative humidity, with a 12:12 light:dark photoperiod

as described by Koehler and Patterson (1986). To acquire

final instar nymphs of specific age, newly-molted (white)

final instars were removed from a colony rearing tub every 3

hours throughout a 12 hour period using a gentle vacuum to

prevent damaging the fragile nymphs. Newly-molted adult males

were collected on a similar time regime using featherweight

forceps. The cockroaches were subsequently placed into 4

liter glass containers with 2 cotton-stoppered 20 ml

scintillation vials of water, 2 rolled cardboard harborages

(15 x 5 cm width), and an unlimited supply of Purina rat

chow. Nymphs were sexed immediately prior to use by the

method of Ross and Cochran (1960).


Chemicals


[14C]Propoxur with radiocarbon in the ring position

(specific activity, 23.5 mCi mmol"') was generously supplied









32
by Dr. R.G. Koch, Miles, Inc., Stilwell, KS. [14C]Propoxur

was purified on Redi/Plt Sil Gel G thin-layer chromatography

plates (Fisher, Pittsburgh, PA), using hexane:ethyl

acetate:ethanol (35:6:5 by volume). [1C]Propoxur was eluted

from the silica gel with acetone. Propoxur metabolite

standards, N-hydroxymethyl propoxur, o-isopropoxyphenyl

carbamate, o-hydroxy propoxur (o-depropyl propoxur), and 2-

isopropoxyphenol were also supplied by Miles, Inc. All other

chemicals were of analytical quality and procured from

commercial suppliers.


Insecticide Bioassays


Last instar male and female nymphs (1, 7, and 12 days

after ecdysis) and adult males (7- to 14-days-old) were

anesthetized with CO2 as described by Valles and Koehler

(1994) and topically treated with the appropriate insecticide

dissolved in 1 Al acetone. The insecticide solution was

applied to the first abdominal sternite in 5 concentrations

causing >0% and <100% mortality. Three replications

containing 10 cockroaches per concentration were conducted.

When synergism was studied, piperonyl butoxide (100

pg/cockroach) was applied to the first abdominal sternite 2

hours prior to insecticide application. Mortality (no

response to probing) was recorded 24 hours after treatment.











Microsome Preparation


Male or female final instar nymphs or adult males of

appropriate age were decapitated, then cut with scissors

latitudinall cross section) to remove the last 2 to 3

abdominal segments. The entire alimentary canal was gently

removed from the cockroach through the newly-cut posterior

opening. The alimentary canal was placed into ice-cold 1.15%

KCl and the contents of the foregut, midgut, and hindgut were

removed by gently teasing open the tissues longitudinally with

fine forceps. The tissues were recombined and homogenized in

a protected buffer (0.1 M sodium phosphate, pH 7.5, containing

10% glycerol, 0.1 mM dithiothreitol, 1 mM

ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl

fluoride, and 1 mM l-phenyl-2-thiourea) or 0.1 M sodium

phosphate buffer, pH 7.5, using a teflon pestle and glass

mortar. The homogenate was filtered through two layers of

cheesecloth, then centrifuged at 10,000gmax for 15 min. The

supernatant was filtered through glass wool and further

centrifuged at 105,000g9ax for 1 h. The resulting pellet

microsomess) was suspended in 0.1 M sodium phosphate buffer,

pH 7.5.

To further investigate the midgut inhibitor, aldrin

epoxidase activity was measured throughout development of

final instar nymphs using the currently accepted method for

microsome preparation (Siegfried and Scott 1991). Final

instar (0, 1, 3, 5, 7, 9, and 11 days after ecdysis) male and









34
female nymphs (whole thorax and abdomen) were homogenized in

0.1 M sodium phosphate buffer, pH 7.5, centrifuged, and

resuspended as described above. The microsomes were used as

the enzyme source.


Tissue Localization


When evaluating tissue specificity the alimentary canal

was dissected from sixty to eighty 7-day old male and female

final instar nymphs and adult males as described above.

Foregut, midgut, and hindgut contents were again removed prior

to homogenization. After removal of the alimentary canal, a

longitudinal incision was made in the ventral abdomen and

thorax of the remaining carcass creating an opening from which

fat body was removed. Cleaned foregut, midgut, and hindgut,

and Malpighian tubules, fat body, and the remaining tissues of

the carcass were placed into separate 10 ml beakers with 1.15%

KC1. Tissues were removed, homogenized separately in

protected buffer, centrifuged, and the resulting microsomal

pellets were resuspended as described above.

The above procedures were all performed at 0-40 C.

Protein determinations were made by the method of Bradford

(1976) using bovine serum albumin as the standard.


Enzyme Assays

All enzyme assays were performed within linear ranges for

protein level and time. These experiments were accomplished









35
by varying the protein content or incubation time for a

particular enzyme assay and plotting the results. All enzyme

assays were then performed at protein levels and incubation

times within the linear portion of the respective curve.

Additionally, all enzyme assays were performed using fresh

tissue preparations.


Microsomal Oxidases


Epoxidase activity was measured by the epoxidation of

aldrin to dieldrin as described by Yu (1982b). The microsomal

fraction was suspended in 0.1 M sodium phosphate buffer, pH

7.5. Incubate mixtures in a 5 ml final volume contained 0.1-

0.5 mg protein; 0.1 M sodium phosphate buffer, pH 7.5; an

NADPH-generating system comprised of 1.8 Aimol NADP, 18 pmol

glucose-6-phosphate, and 1 unit of glucose-6-phosphate

dehydrogenase; and 250 nmol aldrin in 0.1 ml of ethylene

glycol monomethyl ether. Incubations were conducted in a

water bath with shaking at 300 C in an atmosphere of air for

15 minutes. The reaction was terminated by the addition of 10

ml n-hexane. The product, dieldrin, was extracted by shaking

for 1 hour followed by centrifugation (500g) for 3 minutes.

An aliquot of the supernatant was removed and dried over

anhydrous Na2SO4. The sample was analyzed by gas

chromatography on a Varian Model 3740 gas chromatograph

equipped with an electron capture detector. The column was









36
1.22 m X 2 mm i.d. glass, packed with a 1:1 mixture of 5% DC

11 and 5% QF 1 on 100-120 mesh high performance Chromosorb W.

Microsomal O-dealkylase activities were measured by the

O-demethylation of methoxyresorufin and the O-deethylation of

ethoxyresorufin to resorufin (Mayer et al. 1977) as modified

by Yu (1991). The 2.015 ml reaction mixture contained 0.1 M

sodium phosphate buffer, pH 7.5; 0.05-0.1 mg microsomal

protein suspended in 0.1 M sodium phosphate buffer, pH 7.5; 5

Al of 0.5 mM methoxyresorufin or ethoxyresorufin in ethylene

glycol monomethyl ether; and 10 Al of 0.2 mM NADPH. The

buffer and protein were incubated at 300 C in a water bath for

3 minutes. The reaction was initiated by the addition of

substrate followed immediately by NADPH. Specific activity

was determined spectrofluorophotometrically on a Shimadzu

Model RF5000U spectrofluorophotometer by the formation of the

product, resorufin, and was based on the initial reaction

rate. The excitation and emission wavelength settings and

their respective slit settings were 560 nm (1.5 nm) and 580 nm

(3.0 nm).

N-Demethylase activity was based on the N-demethylation

of p-chloro-N-methylaniline to p-chloroaniline (Kupfer and

Bruggeman 1966). The 5 ml reaction mixture contained 0.25-

0.75 mg microsomal protein; 0.1 M sodium phosphate buffer, pH

7.5; an NADPH-generating system (as described in the aldrin

epoxidase assay); 3 Amol of p-chloro-N-methylaniline in 0.1

ml of ethylene glycol monomethyl ether. The reaction mixture









37

was incubated in a water bath with shaking at 340 C in an

atmosphere of air for 20 minutes. Complete reaction mixtures

containing equivalent microsomal protein which had been

denatured by boiling for 15 minutes were used as reference

blanks. The reaction was terminated by the addition of 2 ml

of 6% p-dimethylaminobenzaldehyde in 3 N sulfuric acid. The

samples were centrifuged at 10,000 g for 15 min in a Beckman

J-21 preparative centrifuge. Specific activity was determined

by measuring the product, p-chloroaniline contained in the

supernatant, spectrophotometrically on a Beckman Model 5260

uv/vis spectrophotometer equipped with a scattered

transmission accessory at 445 nm against the corresponding

boiled tissue reference.

Microsomal sulfoxidase activity was determined with

phorate as substrate (Yu 1985, Szeto and Brown 1982). The 5

ml reaction mixture contained 0.2-0.5 mg microsomal protein;

0.1 M sodium phosphate buffer, pH 7.5; an NADPH-generating

system (as described in the aldrin epoxidase assay); and 100

jg phorate in 0.1 ml of ethylene glycol monomethyl ether.

Incubations were conducted in a water bath with shaking at 300

C in an atmosphere of air for 15 minutes. The reaction was

terminated by the addition of 10 ml ethyl acetate. The

product, phorate sulfoxide, was extracted with the ethyl

acetate by shaking for 1 hour followed by centrifugation

(500g) for 3 minutes. A 1 ml aliquot of the supernatant was

removed and dried over anhydrous Na2SO4. The sample was









38
analyzed by gas chromatography on a Varian Model 3740 gas

chromatograph equipped with a thermionic specific detector.

The column was 1.22 m X 2 mm i.d. glass, packed with 2% OV-101

on 80-100 mesh Ultra-Bond 20M.

Total cytochrome P450 content was determined by the

method of Omura and Sato (1964). Microsomes were suspended in

0.07 M sodium phosphate buffer in 30% glycerol. The sample

cuvette was saturated with CO (CO gently bubbled into the

cuvette for 1 minute), followed by the addition of

approximately 0.3 mg Na2S204 to both reference and sample

cuvettes. The mixtures were stirred with a glass rod for 20

seconds, then immediately scanned from 500 to 400 nm. Spectra

were recorded until no further increase in cytochrome P450

content (Amax 448-450) occurred on a Beckman Model 5260 uv/vis

spectrophotometer equipped with a scattered transmission

accessory. Change in absorbance between 490 nm and

approximately 450 nm was determined. The molar extinction

coefficient of 91 mM"' cm'1 was used to calculate the specific

content of cytochrome P450 in the sample (expressed as nmol

cytochrome P450/mg protein).


Reductases


Quinone reductase was measured using juglone as substrate

by the method of Yu (1987). Cockroach microsomes were

suspended in sodium phosphate buffer, pH 6.0. The 4.6 ml

reaction mixture containing suspension buffer and 0.3-0.5 mg









39

microsomal protein was incubated in a 30 C water bath for 3

minutes. After incubation, 0.4 ml of 1.25 mM NADPH was added.

The mixture was shaken well for 30 seconds and 2.5 ml was

placed into a sample and reference cuvette. Five microliters

of 50 mM juglone in ethylene glycol monomethyl ether was then

added to the sample cuvette and stirred thoroughly. The rate

of NADPH oxidation was recorded at 340 nm on a Beckman Model

5260 uv/vis spectrophotometer equipped with a scattered

transmission accessory. Juglone reductase activity was

expressed as nmol NADPH oxidized per min per mg protein.

Cytochrome c reductase activity was determined by the

reduction of cytochrome c (Masters et al. 1967). The 6 ml

reaction mixture contained 0.05-0.3 mg microsomal protein and

0.1 M sodium phosphate buffer, pH 7.5, in a total volume of

3.6 ml; 0.3 ml of 50 AM/ cytochrome c; and 2.1 ml deionized

water. The mixture was shaken for 20 seconds, then 2.5 ml was

pipetted into sample and reference cuvettes. Fifty

microliters of sodium phosphate buffer, pH 7.5, was added to

the reference cuvette and 50 il of NADPH generating system (as

described in the aldrin epoxidase assay) was added to the

sample cuvette to start the reaction. The initial increase in

absorbance at 550 nm was recorded on a Beckman Model 5260

uv/vis spectrophotometer equipped with a scattered

transmission accessory. Specific activity was determined with

the molar extinction coefficient, 21.01 mM-1 cm-1.









40

Total cytochrome b5 content was determined by the method

of Omura and Sato (1964). Microsomes were suspended in 0.07

M sodium phosphate buffer in 30% glycerol. Approximately 0.3

mg of Na2S204 was added to the sample cuvette and the mixture

was stirred with a glass rod for 20 seconds, then immediately

scanned from 500 to 400 nm. Spectra were recorded until no

further increases in cytochrome b5 content (A max 424)

occurred on a Beckman Model 5260 uv/vis spectrophotometer

equipped with a scattered transmission accessory. Change in

absorbance between 424 nm and 409 nm was determined. The

molar extinction coefficient of 185 mMn1 cm'1 was used to

calculate the specific content of cytochrome b5 and was

expressed as nmol cytochrome b5 per mg protein.


Glutathione S-Transferases


Glutathione S-transferase (GST) activity was measured

with 3,4-dichloronitrobenzene (DCNB), 1-chloro-4-nitrobenzene

(CDNB), and p-nitrophenyl acetate (pNPA) as substrates.

Enzyme preparation was accomplished as described in the

preparation of microsomes section using mixed-sex final instar

nymphs and adult males of unknown age. The 105,000 g soluble

fraction supernatantt) was used as the enzyme source.

Homogenization and centrifugation took place in 0.1 M Tris-HCl

buffer, pH 9.0, for DCNB; 0.1 M sodium phosphate buffer, pH

6.5, for CDNB; and 0.1 M sodium phosphate buffer, pH 7.0, for

pNPA conjugation. The method of Booth et al. (1961) as









41

modified by Yu (1982a) was used to measure DCNB conjugation.

The 3 ml reaction mixture contained 1 ml of 15 mM glutathione

and 0.2-0.8 mg protein in 0.1 M Tris-HCl buffer, pH 9.0, in 2

ml. The mixture was incubated at 370 C for 3 min, then 0.02

ml of 150 mM DCNB in ethylene glycol monomethyl ether was

added and mixed. The change in absorbance at 344 nm was

measured on a Beckman UV 5200 series split-beam

spectrophotometer against a reference containing DCNB and

glutathione. Enzyme activity was expressed as nmol DCNB

conjugated per min per mg protein using the molar extinction

coefficient of 10 mM-1 cm'" for the product S-(2-chloro-4-

nitrophenyl)glutathione.

For CDNB conjugation the method of Habig et al. (1974) as

modified by Yu (1984) was used. The 3 ml reaction mixture

contained 1 ml of 15 mM glutathione and 0.004-0.016 mg protein

in 0.1 M sodium phosphate buffer, pH 6.5, in 2 ml. The

mixture was incubated at 250 C for 3 min, then 0.02 ml of 150

mM CDNB in ethylene glycol monomethyl ether was added and

mixed. The change in absorbance at 340 nm was measured on a

Beckman UV 5200 series split-beam spectrophotometer against a

reference containing CDNB and glutathione and the enzyme

activity was expressed as nmol CDNB conjugated per min per mg

protein using the molar extinction coefficient of 9.6 mMI cmn1

for the product S-(2,4-dinitrophenyl)glutathione.

pNPA conjugation was measured by the method of Keen and

Jakoby (1978). The reaction mixture contained 0.1 ml of 30 mM









42

glutathione, 0.05-0.2 mg protein in 0.1 M sodium phosphate

buffer, pH 7.0, in 2 ml, and 0.9 ml deionized water. The

mixture was incubated at 250 C for 3 min, then 0.02 ml of 30

mM pNPA in ethylene glycol monomethyl ether was added and

mixed. The change in absorbance at 400 nm was measured on a

Beckman UV 5200 series split-beam spectrophotometer against a

reference containing pNPA and glutathione. Enzyme activity

was expressed as nmol pNPA conjugated per min per mg protein

using the molar extinction coefficient of 8.79 WmM' cm"' for

the product S-(p-nitrophenyl)glutathione.


Esterases


General esterase and carboxylesterase activities were

measured as described by Van Asperen (1962) using a-naphthyl

acetate (a-NA) as substrate. Ten adult males or mixed-sex

final instar nymphs were decapitated, then homogenized in 20

ml of 0.04 M sodium phosphate buffer, pH 7.0. The homogenate

was filtered through 2 layers of cheesecloth, then centrifuged

at 1000 g for 15 min. The supernatant was used as the enzyme

source. For general esterase measurement, the 5.88 ml

reaction mixture contained 50 pi of a-NA, 0.04 M sodium

phosphate buffer, pH 7.0, and 0.005-0.05 mg protein in 1 ml

buffer. The reaction mixture was incubated in a water bath

with shaking for 30 min at 270 C. The reaction was terminated

by the addition of 1 ml of diazoblue and sodium lauryl sulfate

mixture (2 parts 1% diazoblue, 5 parts 5% sodium lauryl









43

sulfate). To measure carboxylesterase activity 0.005-0.03 mg

protein was used and, eserine (10'4 M final concentration) and

p-hydroxymercury benzoate (10'4 M final concentration) were

added to the reaction mixture to eliminate cholinesterase and

arylesterase activities, respectively. The product of both

reactions, a-naphthol, was quantified spectrophotometrically

at 600 nm on a Beckman UV 5200 series split-beam

spectrophotometer by reference to a standard curve prepared

from known amounts of a-naphthol. Complete reaction mixtures

containing equivalent microsomal protein which had been

denatured by boiling for 15 minutes were used as reference

blanks.

Acetylcholinesterase activity was measured with

acetylthiocholine as substrate as described by Ellman et al.

(1961). Fifty adult males or mixed-sex final instar nymphs

were decapitated and the heads homogenized in 10 ml of 0.1 M

sodium phosphate buffer, pH 8.0. The homogenate was filtered

through 2 layers of cheesecloth, then centrifuged at 1000 g

for 15 min. The supernatant was used as the enzyme source.

The 3.2 ml reaction mixture contained 0.1 M sodium phosphate

buffer, pH 8.0, 0.1 ml of 0.01 M dithiobisnitrobenzoic acid

(DTNB), 0.1 ml of 0.03 M acetylthiocholine iodide (ATC), and

0.1-0.75 mg protein. A complete reaction mixture, less ATC,

was used as reference. The initial reaction rate was followed

spectrophotometrically on a Beckman Model 5200 uv/vis

spectrophotometer at 412 nm. Specific activity was calculated









44

using the extinction coefficient 13.6 mM'1 cm'1 for 5-thio-2-

nitrobenzoic acid. The bimolecular rate constant (ki) for the

inhibition of acetylcholinesterase by propoxur was determined

by the method of Aldridge (1950).

Permethrin esterase was determined using trans-permethrin

as substrate by the method of Yu (1990). The 5 ml reaction

mixture contained 0.5-1.75 mg protein (105,000 g soluble

fraction prepared in 0.1 M Tris-HCl buffer, pH 8.3, as

described in the preparation of microsomes section); 400 fg

trans-permethrin; 2.5 mg bovine serum albumin (BSA); and 0.1

M Tris-HCl buffer, pH 8.3. The reaction mixture was incubated

in a water bath with shaking for 15 min at 30 C. The

reaction was terminated by the addition of 10 ml of ethyl

acetate. The product, 3-phenoxybenzyl alcohol, was extracted

in the ethyl acetate by gentle shaking for 1 hour followed by

centrifugation for 3 minutes. An aliquot of the supernatant

was removed and dried over anhydrous Na2SO4. The sample was

analyzed by high-performance liquid chromatography on a

Beckman Series 340 high-performance liquid chromatograph

operated at 254 nm while using an Ultrasphere-Si column (25 cm

X 4.6 mm i.d.) eluted with 1 ml/min of 5% isopropyl alcohol in

n-hexane.


Glucosidase


fl-Glucosidase activity was measured with helicin as

substrate as described by Yu (1989). The 5 ml reaction









45
mixture contained 0.75-2.75 mg protein (105,000 g soluble

fraction prepared in 0.1 M sodium phosphate buffer, pH 7.0, as

described in the preparation of microsomes section); 0.1 M

sodium phosphate buffer, pH 7.0; and 17.5 pmol helicin. A

complete reaction mixture containing boiled tissue was used as

a reference blank. The reaction mixture was incubated in a

water bath with shaking at 300 C in an atmosphere of air for

1 hr. The reaction was terminated by immersing the incubation

tube in boiling water for 10 min. The samples were then

immediately deproteinized by adding 1 ml each of 0.3 M barium

hydroxide and 0.3 M zinc sulfate to the reaction mixture. The

mixture was then centrifuged at 10,000 g for 15 min to obtain

a clear supernatant fraction. The hydrolysis product,

glucose, was determined from the supernatant fraction by the

glucose oxidase-peroxidase reaction (Sigma Diagnostics, 1984).

To this end, 0.5 ml of the supernatant was mixed with 5 ml of

the combined enzyme-color reaction solution and incubated at

room temperature for 30 min. At the end of the incubation

period, absorbance was measured at 450 nm. ?-Glucosidase

activity was expressed as nmol glucose formed per min per mg

protein.


Propoxur Metabolism


The oxidative metabolism of propoxur was studied in vitro

with microsomal enzymes from adult males (7- to 14-days-old)

and male and female final instar nymphs (7 days after









46

ecdysis). Microsomes were prepared as described in the

preparation of microsomes section. To evaluate metabolites

produced by the microsomal oxidation of propoxur, the 5 ml

incubation mixture contained 2.5 mg microsomal protein

suspended in 0.1 M sodium phosphate buffer, pH 7.5; 0.05 ml

methyl Cellosolve containing 0.48 pg [14C]propoxur (120,000

dpm) and 10 jg cold propoxur; and an NADPH generating system

comprised of 1.8 nAmol NADP, 18 unol glucose-6-phosphate, and

1 unit glucose-6-phosphate dehydrogenase. Duplicate

incubations were carried out at 300C in a water bath with

shaking for 2 hours. The reaction was terminated by the

addition of 5 ml chloroform. Propoxur and its metabolites

were extracted in the chloroform by shaking for 1 hour. After

centrifugation for 5 minutes at 500g, the chloroform phase was

removed and the extraction process repeated. For metabolite

identification, an aliquot of the combined extracts was

evaporated to approximately 100 Al and spotted onto a 5 x 20

cm TLC plate (Redi/Pit Sil Gel GF, Fisher Scientific,

Pittsburgh, PA) along with a mixture of known propoxur

metabolites (N-hydroxymethyl propoxur, o-isopropoxyphenyl

carbamate, o-hydroxy propoxur, and 2-isopropoxyphenol). The

plates were developed once in benzene and twice in

hexane:ethyl acetate: ethanol (35:6:5) as described by

Shrivastava et al. (1969). After drying overnight, the plates

were exposed to X-ray film for 10 days. Metabolite

determinations were made by comparing the positions of unknown









47

autoradiographic bands with known metabolites under UV light.

When propoxur metabolism was quantified, a 5 ml aliquot of the

combined extracts was evaporated to approximately 150 Al and

streaked onto a 5 x 20 cm TLC plate (Redi/Plt Sil Gel G). The

plates were developed as described above and scanned for

radioactivity in a Packard Model 7220/21 radiochromatogram

scanner. The zone of radioactivity corresponding to the major

oxidative metabolites (N-hydroxymethyl propoxur, o-hydroxy

propoxur, and unknowns C and D) was scraped from the plates

and counted in a Tracor Analytic Data 300 liquid scintillation

counter. All sample peaks produced were identified with known

unlabelled propoxur and its metabolites. In all instances

boiled microsomal tissue was used as a blank to correct for

nonenzymatic activity.


Penetration Studies


Cuticular penetration of propoxur was measured by a

method slightly modified from Argentine et al. (1994).

Sublethal doses of [14C]propoxur for final instar male and

female nymphs and adult males (15,000 dpm, 0.04 Ag) were

applied to the first abdominal sternite in 1 Al acetone. The

cockroaches were dried briefly with gentle fanning, then

placed individually into glass scintillation vials for varying

periods of time (0, 1, 2, and 6 hours). After the specified

period of time, 2 cockroaches were placed together into 5 ml

of acetone and swirled gently for 15 seconds. This external









48

extraction method was repeated and the acetone extracts

combined. The amount of unpenetrated [1C]ipropoxur was

determined from aliquots of acetone washings using liquid

scintillation counting.

Internalized radioactivity was determined by homogenizing

the solvent-rinsed cockroaches with a motor-driven teflon

pestle and glass mortar in 5 ml of acetone then centrifuged

for 10 minutes at 500g. This extraction was repeated three

times and aliquots were taken to quantify internalized

radioactivity.

Radioactivity remaining in the holding vials (excreta)

was determined by adding scintillation cocktail directly to

the vials and counting. Four replicates for each time

interval were conducted.


Enzyme Induction


When enzyme induction was studied, food and water were

mixed with sodium phenobarbital. A rat chow pellet was

crushed to a fine powder in a mortar and pestle and the

appropriate quantity of sodium phenobarbital dissolved in

deionized water (1 ml volume per 2.5 g rat chow) was then

mixed thoroughly with the rat chow. The mixture was poured

into a 6 cm section of PVC pipe (1.5 cm diameter) and

compressed with a glass rod. The newly-formed pellet was

allowed to dry at room temperature overnight before use.









49

Sodium phenobarbital dissolved in deionized water was provided

in 25 ml scintillation vials.

To determine the dose that caused maximal enzyme

induction, varying concentrations of sodium phenobarbital in

food and water were fed to newly-molted adult males for 3

days. Microsomes were then prepared as described above and

methoxyresorufin (MR) O-demethylase activity was determined.

After identifying the maximal inducing sodium phenobarbital

concentration, the developmental profile study was conducted.

Phenobarbital (0.2% in food and water) was fed to male and

female nymphs and adult males throughout their development.

No alternative food or water source was provided to the

cockroaches. Cockroaches fed unadulterated rat chow and water

as controls were run in tandem with sodium phenobarbital-

treated cockroaches. Microsomes were prepared as described

above.


Statistics


Data obtained from different treatments were analyzed by

Student's t test or by analysis of variance (ANOVA) followed

by Fisher's least-significant-difference (LSD) test when

appropriate. Insecticide bioassay data were analyzed by

probit analysis (Finney 1971). Significant differences

between LD50 values were determined by nonoverlapping fiducial

limits.














RESULTS


Microsome Preparation


Table 2 summarizes the results obtained from studies of

the effects of gut contents and buffer type on aldrin

epoxidase activity. The presence of gut contents, regardless

of buffer type, had a significant effect on enzyme activity in

both male and female final instar nymphs. Removal of gut

contents prior to tissue homogenization (in protected buffer)

increased enzyme activity 2.2-fold in final instar male nymphs

and 7.5-fold in female final instar nymphs. Conversely,

aldrin epoxidase activity in adult males was unaffected by the

presence of gut contents when microsomes were prepared in a

protected buffer. Enzyme activity was significantly reduced

in all stages when the unprotected buffer was used as the

homogenization medium in the presence of gut contents.

Aldrin epoxidase activity was lowest at the middle of the

instar (days 7-9) and highest at the time of ecdysis when

microsome preparation was accomplished using the whole body

(Figs. 1 and 2).











Tissue Localization


Tables 3, 4, and 5 summarize tissue-specific aldrin

epoxidase and MR O-demethylase activities for adult males, and

final instar female and male nymphs. Final instar nymphs were

analyzed for activity 7 days after ecdysis because intra-

instar enzyme activities were shown to be greatest at this

time (see Fig. 3). When expressed on a per mg protein basis,

adult male aldrin epoxidase activity was highest in the midgut

and Malpighian tubules. In comparison, MR O-demethylase

activity exhibited a great deal of statistical overlap among

the tissues, with no single tissue dominant. However, when

expressed on an insect equivalent basis, aldrin epoxidase

activity was highest in the midgut, and MR O-demethylase

activity was highest in the remaining tissues of the carcass.

Final instar 7-day old male and female nymphs exhibited

similar tissue-specific patterns of aldrin epoxidase and MR 0-

demethylase activities (Tables 4 and 5). On a per mg protein

and insect equivalent basis, aldrin epoxidase activity was

highest in the midgut and MR O-demethylase activity was

highest in the fat body in male and female final instar

nymphs. Aldrin epoxidase specific activity was 9- to 11-fold

higher in midgut than in the fat body, while MR O-demethylase

specific activity was 17- to 22-fold higher in the fat body

than in the midgut.











Developmental Expression


The final instar of the Village Green German cockroach

strain typically lasted 12 to 14 days under the rearing

conditions described previously. Therefore, cytochrome P450

monooxygenases were measured on alternate days of the final

instar, with the last determination made on day 11. Male and

female nymphs exhibited a similar pattern and level of

activity throughout the final instar (Fig. 3). Therefore,

only data for male nymphs are presented. Enzyme activities

were lowest at the time of ecdysis and highest near the middle

of the stadium. Over the course of the final instar, aldrin

epoxidase increased 4-fold, while cytochrome c reductase, MR

O-demethylase, and ER O-deethylase activities increased about

2-fold. Similar increases were observed in adult males for

aldrin epoxidase and cytochrome c reductase, but MR 0-

demethylase and ER O-deethylase activities only increased 1.3-

to 1.6-fold. Adult enzyme activities peaked 5-7 days after

eclosion.


Enzyme Induction


Maximal induction occurred when 0.2% phenobarbital was

present in the food and water. Concentrations greater than

0.8% decreased feeding and resulted in highly variable enzyme

activity. No mortality was observed in 0.2% phenobarbital-fed

nymphs. However, approximately 15% mortality occurred by day

21, and >80% by day 28 in adult males. Figure 4 illustrates









53

the net increase in aldrin epoxidase, cytochrome c reductase,

MR O-demethylase, and ER O-deethylase activities in response

to 0.2% phenobarbital in food and water. Sodium phenobarbital

caused maximal induction of aldrin epoxidase activity in

nymphs and adults in 3 days (Fig. 4). Maximal induction was

not achieved for MR O-demethylation, ER O-deethylation, and

cytochrome c reduction in the time course study as these

enzyme activities continued to increase throughout

development. Based on net increase in specific activity,

adults were less inducible than nymphs. However, based on the

level of induction (i.e. percentage of control activity) no

differences were noted at corresponding time intervals between

nymphs and adults. Aldrin epoxidase was induced 2.8- and 2.6-

fold over control values at 3 days in nymphs and adults,

respectively.

Dietary phenobarbital (0.2%) decreased the toxic

efficiency of propoxur, but not cypermethrin (Table 6).

Phenobarbital-treated adults were 3.8-fold more tolerant of

propoxur than control cockroaches.


Insecticide Bioassays


Results of propoxur and propoxur/PBO bioassays against

adult males and final instar males and females are summarized

in Tables 7 and 8. Adult male (7-14-day-old) LD50 values were

not significantly different, based on overlapping 95%

confidence intervals, from 1-day-old male and female final









54

instar nymphs. However, 7-and 12-day-old male and female

final instars were between 9- and 16-fold more tolerant of

propoxur than adult males. Final instar male and female (1-

and 7-day-old) LD50 values were not significantly different

from one another, while 12-day-old males were 1.7-fold more

tolerant of propoxur than 12-day-old females. No significant

differences in LD50 values were observed between 7- and 12-day-

old nymphs regardless of sex.

Similar results were observed with PBO treatment (Table

8). There was no significant difference in LD5s values between

adult males and 1-day-old nymphs (both sexes). PBO reduced

the tolerance level in 7-day-old male and female final instars

from 15- to 2-fold and from 10- to 3-fold, respectively, as

compared with adult males. Similarly, the tolerance level was

reduced from 16- to 2-fold in 12-day-old final instar males as

compared with adult males. Propoxur tolerance was completely

eliminated by PBO pretreatment in 12-day-old final instar

females as compared with adult males.


Enzyme Assays


Various cytochrome P450 monooxygenase activities in adult

males and 7-day-old male and female final instar nymphs are

shown in Tables 9 and 10. No significant differences were

observed in aldrin epoxidase and PCMA N-demethylase activities

between adult males and nymphs. Phorate sulfoxidase activity

was significantly higher in male and female nymphs as compared









55

with adult males when expressed on a per insect equivalent

basis. However, no differences were noted in phorate

sulfoxidase activity on a per mg protein basis. MR 0-

demethylase and ER O-deethylase activities were significantly

higher in final instar nymphs as compared with adult males.

Cytochrome c reductase activity was significantly greater in

final instar females that adult males (per insect equivalent);

however, when based on a per mg protein basis no differences

were noted.

No significant differences in glutathione S-transferase

activity were observed on protein basis regardless of

substrate (Table 11). However, glutathione S-transferase

activities toward PNPA and CDNB were significantly higher in

final instar nymphs than adult males on a per insect basis

(Table 12).

a-NA esterase, a-NA carboxylesterase, and permethrin

esterase activities were similar for both developmental stages

(Tables 11 and 12). Helicin glucosidase activity on a protein

basis was similar in nymphs and adults, but significantly

greater in nymphs on an insect basis.

Incubation of [14C]propoxur with adult and nymph

microsomes fortified with NADPH resulted in the formation of

7 and 8 metabolites, respectively (Fig. 5). With the

exception of unknown A, no qualitative differences in

metabolite formation were observed between nymphal and adult

stages. Adult males either did not produce unknown A or did









56

produce it in undetectable quantity. Since autoradiograms

from male and female final instar nymphs were identical, only

results from male nymphs are presented in Fig. 5. Figure 6

illustrates the metabolic pathway for propoxur. The major

metabolites produced were N-hydroxymethyl propoxur, o-hydroxy

propoxur, and unknowns C and D. Two minor metabolites,

unknown A (nymphs) and o-isopropoxyphenyl carbamate (nymphs

and adults) were also detected. 2-Isopropoxyphenol and

unknown B were produced with inactivated (boiled) microsomal

preparations, active microsomal preparations without added

NADPH, and a complete incubation mixture without microsomes

indicating that they were non-enzymatic products. Microsomal

oxidase activity (based on the total production of o-hydroxy

propoxur, N-hydroxymethyl propoxur, and unknowns C and D) was

significantly higher (2.7-fold, based on per mg protein) in

male and female final instars than in adult males (Table 13).

The bimolecular rate constant for inhibition of

acetylcholinesterase by propoxur was similar for adult males

and final instar male and female nymphs (Table 14).

Acetylcholinesterase activity and I50 values were also similar

between the stages.


Insecticide Penetration


["C]Propoxur recovered from external rinses of male

adults and male nymphs was similar at all times tested (Table

15). However, radioactivity recovered from female nymphal









57

cuticle at 2 hours was significantly higher than for the adult

male. Radioactivity from internal extracts of male and female

nymphs was significantly less than that of adult males at 2

and 6 hours after insecticide application. Conversely,

significantly more radioactivity was recovered from excreta at

1, 2, and 6 hours, and 1 and 2 hours after application in male

and female nymphs, respectively, as compared with adult males.













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Table 13. In Vitro Oxidative Metabolism of Propoxur by
Microsomes Prepared from Village Green Adult Male and Final
Instar Male and Female German Cockroaches.
Propoxur metabolized!

Stage Sex Age (d) nmol 2hr"1 mg nmol 2hr"1

protein"1 insect'-

Adult d 7-14 1.88 0.09 0.68 0.03

Nymph & 7 5.16 0.49* 2.21 0.09"

Nymph 9 7 5.13 0.31' 2.10 0.14'

a Mean SE of two experiments, each with duplicate
determinations. Metabolism was based upon the total
production of N-hydroxymethyl propoxur, o-hydroxy propoxur,
and unknowns C and D.
* Value significantly different (P < 0.001) from the adult
male.












Table 14. Acetylcholinesterase Activity and its Inhibition
by Propoxur in Village Green Adult Males and Final Instar
Nymphs.
Specific

activity

Stage Sex (nmol min"1 mg I50 (1O'TM) ki (M-min'1)

protein'1)b

Adult d 79.95 8.38 3.73 0.15 186,317 7,253

Nymph & 71.83 4.99 2.89 0.35 243,413 29,479

Nymph 9 58.84 8.38 3.32 0.36 211,520 22,652

a Insect heads were used as the enzyme source.
b Mean SE of two experiments, each with duplicate
determinations.












Table 15. Absorption of [14C]Propoxur (48 ng/cockroach) in
Adult Males (7-14-day-old) and Final Instar Male and Female
Nymphs (7-day-old) of the Village Green German Cockroach.
Percentage of total applied dose SE

Extract

category and Adult male Nymph male Nymph female

time (h)


External

rinse

0

1

2

6

Internal

extract

0

1

2

6


88.5 3.74

26.44 3.71

8.86 1.52

3.16 1.03






3.87 1.10

27.34 1.79

40.85 1.85

32.09 2.82


98.35 3.58

22.84 2.83

7.40 1.10

4.54 0.42






2.82 0.39

28.12 4.07

24.09 1.61*

22.23 3.33*


94.43 8.10

22.39 1.44

14.15 0.90*

3.60 0.66






1.95 0.78

26.99 2.42

28.88 3.76*

22.94 2.12*













Table 15- -Continued



Excreta


26.03 2.41

26.93 0.78

24.03 1.35


37.08 1.64*

47.72 2.76

41.45 2.04*


39.16 0.99*

41.01 5.16'

31.94 4.05


by Students t-


* Significantly different from the adult male
test, P < 0.05; n = 4.











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DISCUSSION


A components) of the Village Green German cockroach gut

contents, released upon homogenization, was found to be

inhibitory to cytochrome P450 monooxygenase activity.

Disproportionate inhibition of aldrin epoxidase activity

between nymphs and adults was observed (Table 2), indicating

that there may be quantitative and/or qualitative differences

in the inhibitor between nymphs and adults. Significant

improvements in aldrin epoxidase activity of male and female

nymphs (Table 2) were made when gut contents were removed

prior to homogenization and when enzyme protectants (10%

glycerol, 0.1 mMDTT, 1 mM EDTA, 1 mM PMSF, and 1 mM PTU) were

added to the homogenization buffer. When homogenization

occurred in protected buffer, aldrin epoxidase activity was

not affected in adult males whether gut contents were present

or absent (Table 2). However, aldrin epoxidase activity was

reduced 2.2-fold in male nymphs and 7.5-fold in female nymphs

under identical conditions.

Differences in the degree of inhibition between adult

males and final instar nymphs appear to be related to food

consumption. Since nymphs are an active feeding stage, the

85









86

inhibitor may be a digestive enzyme(s) or some other entity

released into the gut in response to food consumption.

Inhibition of aldrin epoxidase activity and the amount of food

consumed were directly correlated. Valles et al. (1995)

reported that all instars of the German cockroach

(insecticide-susceptible and -resistant strains) exhibit a

characteristic pattern of food consumption characterized by

minimal feeding near the time of ecdysis and maximal feeding

near the middle of the instar. Possibly, low aldrin epoxidase

activity near the middle of the instar is the result of

digestive enzymes released in response to feeding. These

enzymes could be degrading cytochrome P450 monooxygenases

during the microsome preparation process which takes

approximately 3 hours to complete. Indeed, digestive

proteases have been a common source of cytochrome P450

monooxygenase inhibitors in many insects, including the

southern armyworm (Krieger and Wilkinson 1970, Orrenius et al.

1971), caddisfly larvae (Krieger and Lee 1973), migratory

locust (Chakraborty et al. 1967), and the house cricket

(Brattsten and Wilkinson 1973). Preliminary characterization

of the gut content inhibitor indicated that it was heat

labile, causing a rapid decrease in microsomal oxidase

activity concomitant with an increase in cytochrome P420

content. Gut contents did not affect cytochrome c reductase

activity (Valles and Yu, unpublished results).









87

Based on these results, it is imperative that gut

contents be removed from the alimentary canal prior to

microsome preparation in German cockroaches. As a result of

disproportionate inhibition between nymphs and adults, gut

cleaning is a crucial step when comparing cytochrome P450

monooxygenases between these life stages.

Aldrin epoxidase and MR O-demethylase activities were

tissue- and stage-specific. Aldrin epoxidase activity was

highest in the midgut of final instar nymphs when expressed on

a per mg protein basis and was highest in the midgut and

Malpighian tubules of the adult male. Adult males exhibited

considerable overlap among the tissues in MR O-demethylase

activity; no specific tissue was dominant. On the other hand,

MR O-demethylase activity of final instar male and female

nymphs was highest in fat bodies when expressed on a per mg

protein and per insect equivalent basis. The higher specific

activities of these tissues midgutt, Malpighian tubules, and

fat bodies) indicate that they possess important detoxication

capacities. Plausibly, each tissue plays a different role in

the detoxication process depending upon the type of toxicant

and how the toxicant is encountered. For example, ingested

xenobiotics may be principally detoxified by enzymes in the

alimentary canal while those encountered by cuticular contact

may be primarily detoxified by the enzymes of the fat body.

Tissue-specific differences in cytochrome P450

monooxygenase activities have been reported in other insect









88

species. For example, Feyereisen and Farnsworth (1985)

similarly reported that aldrin epoxidase activity was highest

in midgut, whereas MR O-demethylase activity was highest in

fat bodies in final instars of the cockroach, Diploptera

punctata. Similar tissue-specific relationships were also

observed in final instar Manduca sexta larvae (Tate et al.

1982), the southern armyworm, Prodenia [Spodoptera] eridania

(Krieger and Wilkinson 1969, Brattsten et al. 1980), and the

fall armyworm, Spodoptera frugiperda (Yu 1995). Conversely,

Lee and Scott (1992) reported that aldrin epoxidase and MR 0-

demethylase activities of the housefly were highest in the

proximal intestine and Malpighian tubules.

Activity profile studies showed that cytochrome P450

monooxygenase activities are lowest at the time of ecdysis and

highest at the intermolt period in the final instar. This

developmental pattern was reported in other insect species,

including the Diploptera punctata (Feyereisen and Farnsworth

1985), Manduca sexta (Tate et al. 1982), Acheta domesticus

(Benke and Wilkinson 1970), and Gromphadorhina portentosa

(Benke et al. 1972).

Nymph and adult male cytochrome P450 monooxygenases of

the Village Green strain were inducible by dietary

phenobarbital. Phenobarbital also induced microsomal oxidases

in the Madagascar cockroach (Gil et al. 1974). Moreover,

moderate induction of benzo(a)pyrene hydroxylase by dieldrin

and DDT was demonstrated in the German cockroach (Khan and









89

Matsumura 1972). I was perplexed to discover that

phenobarbital-induced adults were more tolerant of propoxur

but not cypermethrin even though both insecticides are known

to be metabolized by microsomal oxidases (Shono et al. 1979).

Yu (1982b) demonstrated that induction of microsomal oxidases

by corn leaves served to protect fall armyworm larvae from

carbamate, organophosphate, and pyrethroid insecticides.

Apparently, phenobarbital selectively induced certain

cytochrome P450 isozymes which degraded propoxur but had no

activity toward cypermethrin in the cockroaches. In contrast,

Khan and Matsumura (1972) found that pretreatment of German

cockroaches with dieldrin, while making a susceptible strain

more sensitive, made a dieldrin-resistant strain and their

hybrids more tolerant of carbaryl and diazinon. These results

suggest that induction of microsomal oxidases may result in

decreased German cockroach susceptibility to insecticides.

Therefore, this area of research warrants further

investigation.

Nymphal age profoundly influenced tolerance toward the

carbamate insecticide, propoxur. The final instar of the

Village Green strain of German cockroach typically lasted

between 12 and 14 days. To date, all insects examined exhibit

a characteristic pattern of detoxication enzyme expression

within an instar; enzyme activity is lowest near the time of

ecdysis and highest near the middle of the stadium (Benke et

al. 1972, Feyereisen and Farnsworth 1985). Therefore, we









90

chose to evaluate nymphal tolerance to propoxur at the

beginning (1 day), middle (7 days), and end (12 days) of the

final instar. While 1-day-old final instar nymphs were as

susceptible to propoxur as adult males, 7- and 12-day-old

nymphs were up to 16-fold more tolerant. Generally, male and

female nymphs were equally susceptible to propoxur. However,

12-day-old male nymphs were slightly more tolerant (1.7-fold)

of propoxur than females. A similar relationship between

adults and final instar nymphs was reported in the migratory

locust (Onyeocha and Fuzeau-Braesch 1991). Final instar

nymphs were significantly more tolerant to methomyl and

permethrin than adults.

PBO, a well known cytochrome P450 monooxygenase

inhibitor, effectively reduced the nymphal tolerance to

propoxur. PBO reduced the tolerance level in 7-day-old male

and female final instars from 15- to 2-fold and from 10- to 3-

fold, respectively, as compared with adult males. Similarly,

propoxur tolerance was substantially reduced in 12-day-old

final instar males as compared with adult males and was

completely eliminated in 12-day-old final instar female

nymphs. These results strongly suggest that detoxication,

catalyzed by cytochrome P450 monooxygenases, is largely

responsible for the enhanced nymphal tolerance to propoxur.

This notion is supported by the fact that in vitro

propoxur metabolism by microsomal monooxygenases was 2.7-fold

greater (based on per mg protein) in 7-day-old male and female









91

nymphs than in adult males. Despite the nearly 3-fold

difference in propoxur metabolic rate between nymphs and

adults, the same number and type of metabolites were produced

by both stages with the exception of unknown A in adult males.

This suggests that either a qualitative difference in

monooxygenase expression occurs between nymphs and adults or

that adults produce unknown A in quantities too small to be

detected. The major metabolites produced, N-hydroxymethyl

propoxur and o-hydroxy propoxur, were also reported to be the

principal metabolites in a different German cockroach strain

(Siegfied and Scott 1991) and the spruce budworm, Christoneura

fumiferana (Shrivastava et al. 1969). Conversely, the major

metabolite in housefly was 5-hydroxy propoxur (Shrivastava et

al. 1969).

In addition to a higher propoxur metabolic rate,

cytochrome P450 content, and MR O-demethylation and ER 0-

deethylation activities were significantly higher in final

instar male and female nymphs compared with adult males.

Stage-dependent insecticide tolerance is often correlated with

enhanced detoxication enzyme activity. For example, Yu (1983)

reported that 6th instar fall armyworm larvae were

significantly more tolerant of methomyl, diazinon, and

permethrin than 5th instars. The enhanced tolerance was

associated with higher cytochrome P450 monooxygenase activity.

A similar relationship was reported for Spodoptera littoralis

where 5th instars were nearly 500-fold more tolerant of









92
topically applied abamectin than 6th instars (Christie and

Wright 1991). Enhanced oxidative detoxication and reduced

penetration were determined to be responsible for the

differential susceptibility.

Incomplete reduction of nymphal tolerance by PBO suggests

that additional mechanisms are responsible for enhanced

nymphal tolerance to propoxur. No significant differences in

general esterase, carboxylesterase, permethrin esterase, and

helicin fl-glucosidase activities were observed between final

instar nymphs and adult males of the Village Green German

cockroach strain. Additionally, there were no differences in

glutathione S-transferase activities (toward 3,4-

dichloronitrobenzene, 1-chloro-2,4-dinitrobenzene, and p-

nitrophenyl acetate) between these stages. These data suggest

that esterases and glutathione S-transferases probably do not

play a significant role in the observed stage-dependent

tolerance to propoxur.

Apparently decreased insecticide cuticular penetration

does not play a role in the nymphal tolerance to propoxur.

External cuticular rinses of adults and nymphs yielded similar

quantities of radioactivity at all times evaluated. However,

the amount of radioactivity recovered from internal extracts

was significantly higher in adult males as compared with

nymphs. Further, higher quantities of radioactivity were

recovered from the excreta of male and female nymphs as

compared with adult males. These data coincide with the in




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