Resistance to insecticides in heliothine Lepidoptera: a global view

Resistance to insecticides in heliothine

Lepidoptera: a global view

Alan R. McCa ¡ery

Zoology Division, School of Animal and Microbial Sciences, and Crop Protection Unit,The University of Reading,Whiteknights,

PO Box 228, Rea ding RG6 6AJ, UK (a.r.mcca¡ery@reading.ac.uk)

The status of resistance to organophosphate, carbamate, cyclodiene and pyrethroid insecticides in the

heliothine Lepidoptera is reviewed. In particular, res istance in the tobacco budworm, Heliothis virescens,

and the corn earworm, Helicoverpa zea, from the NewWorld, and the cotton b ollworm, Helicoverpa armige ra,

from the Old World, are considered in detail. Particular emphasis has been placed on resistance to the

most widely used of these insecticide groups, the py rethroids. In each case, the incidence and current

status of resistance are considered before a detailed view of the mechanisms of resistance is given. Contro-

versial issues regarding the nature of mechanisms of resistance to pyrethroid insecticides are discussed.

The implications for resistance management are considered.

Keywords: insecticide resistance; Heliothinae; Heliothis virescens; Helicoverpa armigera; mechanism s of

resistance

1. INTRODUCTION

Lepidopteran species in the genera Heliothis and Helicov-

erpa are grouped together in the Tri¢ne subfamily

Heliothinae of the family Noctuidae (Hardwick 1965;

Mitter et al. 1993). The biology and ecology of t he species

within this complex have recently been reviewed by Fitt

(1989), Zalucki (1991) and King (1994). It is signi¢cant

that within the group there exists a large number of

highly destructive crop pests against which an unparal-

leled variety and quantity of insecticides have been used.

The polyphagous nature of a number of these species,

their wide geographic range and their ability to adapt to

diverse cropping systems have contributed to this pest

status. Moreover, the ability of certain species within the

complex to develop resistance to insecticides has placed

the heliothine Lepidoptera among a handful of the

world's most signi¢cant crop pests.

The genus Helicoverpa (designated Heliothis for a period)

includes the Old World species Helicoverpa armigera, gener-

ally considered to b e the most important species within

this group. Commonly known as the cotton bollworm,

gram p odborer or American bollworm, H. ar migera occurs

in Africa, Asia, southern Europe and Australia and is a

major pest of cotton, maize, sorghum, pigeonpea,

chickpea, soyab ean, groundnut, sun£ower and a range of

vegetables. It, above all others in this genus, has

developed resistance to virtually all of the insecticides

that have been deployed against it in any quantity.

Helicoverpa punctigera is a pest of cotton, sun£ower, lucerne,

soyabean, chickpea and sa¥ower in Australia and is

commonly found alongside H. armigera. Interestingly, until

recently H. punctigera had not developed resistance to

insecticides (Gunning & Easton 1994; Gunning et al.

1994), and this may have been because the pool of

unsprayed insects is so vast that the treated proportion of

the total populat ion is only trivial (Forrester et al. 1993).

Any resistance genes would be swamped by susceptible

genes in the unsprayed refugia. Nevertheless, a ¢eld

population of H. punctigera from New South Wales was

recently shown to have developed resistance to a

pyrethroid (Gunning et al. 1997). The oligophagous

species Helicoverpa assulta feeds on tobacco and other

solanaceous plants and is found in Africa, Asia, parts of

Australasia and the South Paci¢c. It is normally

considered to be a minor pest and there is no evidence of

it having developed resistance anywhere with in its range

since it is not subject to any signi¢cant insecticide treat-

ment (Armes et al. 1996). In the New World this genus is

represented by the corn earworm, Helicoverpa zea, a key

pest of maize, sorghum, cotton, tomato, sun£ower and

soyabean. It too has developed res istance to a number of

the insecticide g roups used against it (Sparks 1981;

Wolfenbarger et al. 1981; Stadelbacher et al. 1990),

alt hough not al l the crops it attacks are sprayed and the

species remains reasonably amenable to control with

insecticides.

Within the genus Heliothis there are two species of note.

The New World representative, Heliothis virescens, is

distributed throughout the Americas, is commonly known

as the tobacco budworm and is a major pest of cotton,

tobacco, tomato, sun£ower and soyabean. Like H. armi-

gera above, it has developed resistance to all t he insecti-

cides that have been used against it in signi¢cant

quantities (Sparks 1981; Wolfenbarger et al. 1981; Sparks et

al. 1993). The polyphagous species, Heliothis peltigera, has a

broad distribution across central and southern Europe,

the Canary Islands, Asia Minor and India and it is a pest

of sa¥ower, tobacco, cotton, chickpea, fodder crops,

grapevines and various fruit trees. Resistance to

insecticides has not been reported in this species and this

is presumed to be due to lack of intense selection.

Phil. Trans. R. Soc. Lond. B (1998) 353, 1735^1750 1735 & 1998 The Royal Society

It is clear that within the Heliothinae there are two

very signi¢cant pest species that have been subjected to

intense selection with a range of insecticides and which

have developed signi¢cant levels of resistance to

insecticides: H. armigera and H. virescens. This review will

therefore concentrate on resistance in these species and

will attempt to compare and contrast the phenomenon,

particularly with respect to the mechanisms of

resistance. There are a large number of reviews that

document the historical development of resistance in

these species and it is not the intention of the present

author to rehearse all of this literature here. The reader

is directed to earlier works by Sparks (1981),

Wolfenbarger et al. (1981) and Sparks et al. (1993). Most

studies on resistance in heliothine insects in the past 15

years have concerned the pyrethroid insecticides and it

is for this reason that this review will place special

emphasis on this group, although other insecticide

groups will also be considered.

An understanding of the mechanisms underlying

resistance is central to an ability to continue to e¡ectively

use existing insecticide chemistry to which resistance has

already developed. A knowledge of the mechani sms of

resistance enables one to understand not only the cross-

resistance patterns within insecticide groups but also

those between them. Thus, the mechanisms of resistance

determine the use of `resistance-breaking' compounds

and areas of new insecticide chemistry. Such considera-

tions are crucial in res istance management. A detailed

knowledge of resistance mechanisms could also be consid-

ered as essential in the formulation of diagnostics for use

in resistance management although, as will be empha-

sized later, the ver y diversit y of response to selection in

these insects could make the practical use of such diag-

nosis e specially di¤cult. The design of expression systems

for use in insecticide discovery might also be usefully

in£uenced by such information. This review therefore

places considerable emphasis on the mechanisms of resis-

tance and compares them in heliothine populations

around the world.

2. RESISTANCE TO ORGANOPHOSPHATES

(a) Heliothis virescens

After the development of resistance to DDT and toxa-

phene, organophosphates (OPs), particularly methyl

parathion, were introduced into the USA to control

H. virescens and H. zea. Resistance developed in H. virescens

within a few years of OP introduction (Wolfenbarger &

Mc Garr 1970; Harris 1972), and had become widespread

throughout the southern states of the USA by 1980

(Sparks et al. 1993). Numerous reports have detailed the

progress of resistance to OPs including methyl parathion,

sulprofos and profenofos both within and between seasons

(e.g. Wolfenbarger 1981; Elzen et al. 1992; Kanga & Plapp

1992, 1995; Sparks et al. 1993; Graves et al. 1994; Kanga et

al. 1995; Martin et al. 1995, 1997). Outside the USA, a low

level of resistance to monocrotophos was noted in

Colombia (Ernst & Dittrich 1992).

(b) Helicoverpa zea

Resistance to methyl parathion was reported in some

states of the USA and Central America (Wolfenbarger et

al. 1981), although there is little supporting information

in the literature that resistance to OPs is a signi¢cant

problem in the control of t his species (Sparks et al.

1993).

H. armigera in Australia have generally been considered

to be relatively susceptible to OP insecticides. Gunning &

Easton (1993) found no evidence of resistance to methyl

parathion and today only low levels of resistance are

found to profenofos, chlorpyrifos and methyl parathion

(N. W. Forrester, personal communication). In c ontrast,

high levels of resistance to monocrotophos and low levels

of resistance to chlorpyrifos and profenofos have been

recorded in populations of H. armigera in Pakistan

(Ahmad et al. 1995), alt hough resistance to profenofos is

continuing to rise as growers opt to use OPs rather than

the pyrethroids, to which there is resistance. Low-to-

moderate resistance was found to quinalphos in Indian

and Pakistan i p opulations of H. armigera (Armes et al.

1996), but t here was no evidence of signi¢cant resistance

to monocrotophos. Since 1980, phoxim has been the most

widely used OP for the c ontrol of H. armigera in China. It

was highly e¡ective until 1990, when it failed to control

populations in North China. Bioassays with insects

collected from di¡erent geographical areas of China

during 1994 and 1995 showed resistance to phoxim to be

widespread (Wu et al. 1997). No resistance to monocroto -

phos was observed in 1992 and 1993 (Wu et al. 1995), but

higher levels were recorded in 1995 (Wu et al. 1996). No

resistance to OPs was detected in H. armigera in Thailand

(Ahmad & McCa¡ery 1988).

3. MECHANISMS OF RESISTANCE TO

ORGANOPHOSPHATES

(a) Insensitive acetylcholinesterase: target-site

resistance to organophosphates

The enzyme acetylcholinesterase resides on the post-

synaptic membrane of cholinergic synapses and is respon-

sible for the breakdown of acetylcholine after stimulation

of nicotinic acetylcholine receptors on the postsynaptic

neuron. Both organophosphate and carbamate insecti-

cides prevent the breakdown of acetylcholine by inh ibiting

the activity of this enzyme. The increased residence time

of acetylcholine in the synapse causes repeated stimula-

tion of the postsynaptic neuron and hence neuronal

hyperactivity. Commonly, resistance to OPs involves the

selection of mutants that pos sess a form of the enzyme

insensitive to inhibition.

A large number of reports have shown that resistance

to OPs in H. virescens may be due, at least in part, to a

target-s ite resistance involving decreased sensitivity of

acetylcholinesterase to inhibition (Brown & Bryson 1992;

Kanga & Plapp 1995; Brown et al. 1996a; Harold & Ottea

1997). In resistant strains of H. virescens, Brown & Bryson

(1992) and Gilbert et al. (1996) demonstrated the presence

of acetylcholinesterase insensitive to inhibition by methyl

paraoxon and G. Zhao et al. (1996) demonstrated

acetylcholinesterase insensitive to paraoxon. Although

this mechanism may be c ommon in OP-resistant insects it

may not be universal within ¢eld populations of

H. virescens, as shown by Harold & Ottea (1997).

1736 A. R. McCa¡ery Resistance to insecticides in heliothine Lepidoptera

Phil. Trans. R. Soc. Lond. B (1998)

(b) Metabolic m echanisms of resistance to

organo phos phates

Metabolic resistance to organophosphate insecticides in

heliothine insects has been thought to be due to elevation

in the activity of number of detoxi¢cation systems. Mo st

frequently, resistance to these insecticides has b een corre-

lated with elevated esterase activity, especially when the

model substrate 1-naphthyl acetate (1-NA) is used; this

result suggests a strong a ssociation between these enzy mes

and OP resistance. Esterase synergists such as TBPT and

EPN were shown to be e¡ective against methyl parath ion

resistance in H. virescens (Payne & Brown 1984). Impor-

tantly, in this New World species, elevated esterase activ-

ities were shown to be responsible for resistance to OPs

such as methyl parathion, profenofos and azinphosmethyl

and for cross-resistance between carbamate, OP and pyre-

throid insecticides (Goh et al. 1995; G. Zhao et al. 1996).

Higher phosphotriester hydrolase activity was reported to

be involved in resistance to methyl parathion in H. virescens

from North Carolina (Konno et al. 1989). In a recent study,

high frequencies of profenofos resistance were recorded in

larvae of all of a number of ¢eld strains of H. virescens

collected from Loui siana in 1995 and were strongly corre-

lated with esterase activity (Harold & Ottea 1997).

Glutathione S-transferases have also been frequently

associated w ith resistance to OPs and thought to be

responsible for metabolism of these compounds (Whitten

& Bull 1978). Resistance to profenofos was show n to be

only moderately correlated with glutathione S-transferase

activity towards 1-chloro-2,4-dinitrobenzene (CDNB)

and had no correlation with glutathione S-transferase

activity to 1,2-dichloro-4-nitrobenzene (DCNB)

(Ibrahim & Ottea 1995; Harold & Ottea 1997). This

correlation of profenofos resistance wit h activity of GST

towards CDNB but not DCNB suggests that these GST

enzymes have di¡erent identities and therefore likely

contributions to profenofos resistance. No di¡erences in

GSTactivity were observed by Konno et al. (1989).

Metabolism of OP insecticide s by P450 monooxy-

genases was reported in a number of early studies

(W hitten & Bull 1974; Reed 1974; Brown 1981; Bull 1981).

Mart in et al. (1995) identi¢ed low to moderate levels of

resistance to profenofos and sulprofos in Louisiana,

Mississippi and Texas and later showed that profenofos

was synergized by PBO in populations of H. virescens from

Texas, Mississippi and Oklahoma (Martin et al. 1997).

Other research suggested that monooxygenases might not

be involved in the elimination of OPs (Gould & Hodgson

1980; Payne & Brown 1984; Konno et al. 1989). Most

recently, Harold & Ottea (1997) found no correlation

between profenofos resistance and P450 monooxygenase

activity towards the model substrate p-nitroanisole.

4. RESISTANCE TO CARBAMATES

(a) Heliothis virescens

Resistance to the oxime carbamates thiodicarb and

methomyl has been recorded a number of times in popu-

lations of H. virescens from Louisiana, Mississippi, Texas

and Arkansas (Spark s 1981; Elzen et al. 1992; Martin et al.

1992, 1995; Sparks et al. 1993; Kanga & Plapp 1995), and

also in populations from Mexico (Roush & Wolfenbarger

1985).

(b) Helicoverpa zea

There are no reports of signi¢cant resistance to carba-

mates in H. zea.

Thiodicarb and methomyl are the carbamates most

widely used against H. armige ra in Australia. Methomyl

resistance was noted in 1986 but the insect remained

susceptible to thio dicarb for a number of years more

(Gunning et al. 1992). Resistance to thiodicarb was

detected in New South Wales in 1993 and this gave cross-

resistance to methomyl (Gunning et al. 1996b). Since then

resistance to thiodicarb has increased and moderate

resistance to carbamates is now common (N. W. Forrester,

per sonal communication). In China, signi¢cant resistance

to methomyl was recorded in strains of H. armigera from

Shandong province (Wu et al. 1995, 1996). Low-level

resistance to thiodicarb was seen in H. armigera from

Pakistan (Ahmad et al. 1995). Substantial resistance to

methomyl was recorded in populations from cotton-

growing areas of Andhra Pradesh, India (Armes et al.

1992, 1996), with lower levels being more typical of other

locations including Nepal, Gujarat and Maharashtra.

5. MECHANISMS OF RESISTANCE TO CARBAMATES

(a) Insensitive acetylcholinesterase: target-site

resistance to carbamates

Target-site resistance to carbamates is similar to that

found with organophosphates (see ab ove). Acetylcholines-

terase insensitive to inhibition by propoxur and methomyl

was observed in a selected strain of H. v irescens (Brown &

Bryson 1992). More recently, insensitive acetylcholines-

terase was shown to be a major mechanism of resistance

to methomyl and carbaryl in strains of H. virescens and to

thiodicarb in a thiodicarb- and pyrethroid-resistant strain

(G. Zhao et al. 1996). In Australia the recently developed

resistance to thiodicarb in H. armigera has been shown to

be due to a form of acetylcholinesterase that is insensit ive

to bot h thiodicarb and methomyl.

(b) Metabolic resistance to carbamates

Both enhanced esterase and enhanced monooxygenase

activity have been found to be signi¢cant mechanisms of

resistance to carbamates. In one recent study, substan-

tially increased esterase activity was observed and

thought to be responsible for res istance in a thiodicarb -

resistant (and pyrethroid-resistant) strain of H. virescens

(Goh et al. 1995). Rose et al. (1995), using a similar strain,

found high levels of P450 monooxygenase activity as well

as increased esterase activity. The involvement of P450

monooxygenases was c onsidered likely by G. Zhao et al.

(1996), who showed signi¢cant synergism of thiodicarb

with PBO. They also inferred the involvement of

enhanced esterase activity in this resistance. Very recently,

PBO was shown to synergize the action of methomyl and

thiodicarb in a number of ¢eld strains although it

antagonized the action of thiodicarb in some strains

(Martin et al. 1997).

6. RESISTANCE TO CYCLODIENES

(a) Heliothis virescens

Resistance to endosulfan has been demonstrated in

strains of H. virescens f rom Louisiana, Mississippi, Texas

Resistance to insecticides in heliothine Lepidoptera A. R. Mc Ca¡ery 1737

Phil. Trans. R. Soc. Lond. B (1998)

and Arkansas (Elzen et al. 1992; Kanga et al. 1995; Martin

et al. 1995).

(b) Helicoverpa zea

Increased tolerance to endosulfan was found in ¢eld

populations of H. zea from Texas in 1994 (Kanga et al.

1996). There appear to be no other records of signi¢cant

resistance to endosulfan in this species.

Resistance to endosul fan in H. armigera has recorded in

Australia since the early 1970s and a number of reports

have demonstrated the substantial and continuing nature

of this problem (Kay 1977; Forrester et al. 1993; Gunning

& Easton 1994). Current levels of resistance to endosulfan

in Australia are moderate. Relatively low levels of resis-

tance were characteristic of H. armigera in various regions

of India from 1988 to 1992 (McCa¡ery et al. 1989; Armes

et al. 1992). Rather higher levels of resistance to this

compound were found in later years by Armes et al.

(1996), who suggested that incipient resistance to endo-

sulfan was present in this species in India, Nepal and

Pakistan. Low resistance to endosulfan characterized

populations of H. armigera from Pakistan between 1991

and 1993, but thereafter resistance rose to peak frequen-

cies in 1995, falling back somewhat in later years (Ahmad

et al. 1995, 1998). Populations collected from Indonesia in

1987 and 1988 were reported to be resistant to pyrethoids

(McCa¡ery et al. 1991a).

7. MECHANISMS OF RESISTANCE TO CYCLODIENES

(a) Altered GABA receptor: target-site resistance to

cyclodienes

The GABA-gated chloride-ion channel receptor

complex is generally considered to be the target for cyclo-

diene insecticides such as endosulfan. These compounds

act as GABA antagonists and hence, because they

suppress the inhibitory transmitter action of GABA, their

action results in increase d postsynaptic neuronal activity.

Although no direct evidence has been obtained with

heliothines, target-site insensitivity to cyclodiene action

has been inferred in adult H. virescens on the basis of

highly correlated toxicities of dieldrin and endosulfan

(Kanga & Plapp 1995).

8. RESISTANCE TO PYRETHROID INSECTICIDES

(a) Introduction

The pyrethroid insecticides were introduced to replace

the resistance-prone and environmentally unsuitable

organochlorines (OCs), cyclodienes and organo-

phosphates (OPs) (Morton & Collins 1989). They clearly

had a number of distinct advantages over insecticides

used previously. They possessed an inherently high

activity and could be applied at extremely low doses for

the control of a huge range of public health and agricul-

tural pests. Their high activity meant that e¡ective foliar

pro¢les were maintained for considerable periods. They

were safe to mammals, had low environmental impact

and were immobile in the soil (Elliott 1989). The

pyrethroids were especially useful in cotton, where their

contact activity and good e¤cacy enabled the grower to

regain control of pest species that had become resistant to

previously used insecticides. The global demise of the

e¡ectiveness of pyrethroids has provoked a huge research

e¡ort directed at understanding the nature of this

resistance and hence alternative control strategies.

(b) Resistance to pyrethroid insecticides around the

world

(i) Heliothis virescens in the USA

Following the development of resistance to DDT,

methyl parathion and a growing number of other OPs

(Sparks et al. 1993), the pyrethroid insecticides were intro-

duced into the USA and became available for use on

cotton in 1978, quickly becoming the insecticides of

choice. A small number of studies had inferred a deg ree

of cross-resistance to pyrethroids in methyl parathion-

resistant strains of the tobacco budworm, although

analysis of these data revealed no signi¢cant trends.

Nevertheless, susceptibility to pyrethroids was correlated

with that to methyl parathion (Sparks et al. 1993), and

suggested that di¡erences in susceptibility were already

present in populations of the tobacco budworm in cotton.

Numerous studies have documented resistance to

pyrethroids in H. virescens in the USA and the reader is

directed to the comprehen sive review by Sparks et al.

(1993) for more details. Although signi¢cant changes in

susceptibility had been noted in the Imperial Valley of

California in the early 1980s (Twine & Reynolds 1980;

Mart inez-Carrillo & Reynolds 1983), these were not

considered to have led to any ¢eld failure. The ¢rst reports

of signi¢cant resistance appeared in 1985 in west Texas

(Plapp & Campanhola 1986) and these were quickly

followed by a range of similar ¢ndings throughout the

cotton-belt state s of the southern USA, including Alabama

(Mullins et al. 1991), Arkansas (Plapp et al. 1987, 1990),

Louisiana (Leonard et al. 1988; Plapp et al. 1990; Elzen et

al. 1992), Mississippi (Luttrell et al. 1987; Plapp et al. 1990;

Elzen et al. 1992; Ernst & Dittrich 1992), Oklahoma (Plapp

et al. 1990) and Texas (Plapp et al. 1987, 1990). In many

cases this resulted in considerable cross-resistance between

pyrethroids and this was thought to imply the presence of

a target-site mechanism of resistance (Martin et al. 1992;

Graves et al. 1993; Sparks et al. 1993). Because a complete

loss of pyrethroids was feared, resistance monitoring

programmes were instituted (Plapp et al. 1987), and

management plans organized in Texas and the mid-south

in an e¡ort to provide pyrethroid-free windows during the

cotton-growing season (Sparks et al. 1993). Interestingly,

the continued used of pyrethroids in the USA has led to

what appears to be a shift in the mechanisms of resistance

to pyrethroids, as detailed below.

(ii) Heliothis virescens in Mexico

H. virescens is a common pest of cotton in Mexico and

pyrethroids have been extensively used for its control since

the early 1980s. Monitoring for resistance to pyrethroids

has been conducted in agricultural regions of northwestern

Mexico since 1984, when resistance was ¢rst noted. High

levels of resistance were recorded in 1987 from populations

from theYaqui and Mexicali valleys and in t he 1988 season

from the Costa de Hermosillo and Region de Caborca

(Martinez-Carrillo 1991, 1995). These high levels of resis-

tance prompted the introduction, in 1989, of a strategy to

1738 A. R. McCa¡ery Resista nce to insecticides in heliothine Lepidopte ra

Phil. Trans. R. Soc. Lond. B (1998)

reduce pyrethroid selection pressure in theYaqui Valley. As

a result, pyrethroid resistance decreased in this area in

1988 and 198 9 and has remained stable since 1990

(Martinez-Carrillo 1995). In contrast, levels of resistance

in the northeast of the country are high and would be

expected to cause control problems.

(iii) Heliothis virescens in Colombia

Pyrethroids became available for use in cotton in the

late 1970s and early 1980s and were very extensively used,

to the exclusion of other products. Very substantial

resistance to cypermethrin in the tobacco budworm was

noted from 1985 and has been documented by Ernst &

Dittrich (1992) and con¢rmed by McCa¡ery (1994).

(iv) Helicoverpa zea in the Americas

Very extensive resistance to DDT was a feature of early

control of H. zea in the USA (see, for example, Graves et

al. 1963; Wolfenbarger et al. 1981; Sparks et al. 1993). The

¢rst substantial report of resistance to pyrethroids in

H. zea was that of Stadelbacher et al. (1990). Following

this, a number of other authors noted a loss of

susceptibility to pyrethroids in this species (Graves et al.

1993; Abd-Elghafar et al. 1993; Kanga et al. 1996; Bagwell

et al. 1997). Despite this loss of susceptibility, pyrethroid

insecticides presently remain e¡ective for the control of

H. zea in US cotton, even at low ¢eld application rates. In

one of the few studies on this species conducted outside

the USA, strains of H. zea from the Tiquisate area of

Guatemala and the Leon area of Nicaragua were found

to be very substantially resistant to cypermethrin (Ernst

& Dittrich 1992).

(v) Helicoverpa armigera in Australia

Before the introduction of pyrethroids in 1977 in

Australia, H. armigera had developed severe resistance to

DDT in the Ord River Valley (Wilson 1974), New South

Wales (Goodyer et al. 1975; Goodyer & Greenup 1980)

and Queensland (Kay 1977). Resistance to endosulfan

(Kay 1977; Kay et al. 1983; Gunning & Easton 1994), OPs

(Goodyer & Greenup 1980; Kay et al. 1983) and

carbamates ( Gunning et al. 1992) was also known to be

present. Resistance to pyrethroids ¢rst appeared in 1983

(Gunning et al. 1984), and immediately a resistance

management strategy was implemented, which restricted

the use of pyrethroids to a 42-day window during

January

February (from 1990 they were restricted to a

35-day window) (Forrester 1990; Forrester et al. 1993).

Endosulfan use was also limited. An e¡ective weekly

monitoring scheme based on survival of fourth-instar

larvae of H. armigera after treatment with a diagnostic

dose of fenvalerate was initiated and much data accumu-

lated on the e¡ects of selection and survival of resistant

individuals. Later monitoring also determined the l ikely

presence of a metabolic resistance based on enhanced

monooxygenase activity by treating larvae with both

fenvalerate and the metabolic inhibitor piperonyl butoxide

(PBO) (see below). Based on these results, PBO could be

added to the last of the three (maximum) sprays in the

pyrethroid window. This strategy undoubtedly held pyre-

throid resistance in check for a number of years although

there appeared to be a steady rise in the proportion of the

population that was resistant to pyrethroids (Forrester et

al. 1993). H. armigera in unsprayed refugia readily became

contaminated with resi stant individuals (Gunning &

Easton 1989; Forrester et al. 1993), and similar levels of

resistance were found in other crops, such as maize

(Glenn et al. 1994). Thi s gradual los s of pyrethroid e¤-

cacy together with the development of an immunodiag-

nostic to distinguish the eggs of H. armigera from those of

H. punctigera, the use of Bacillus thuringiensis (Bt) and other

insecticides and the advent of Bt-transgenic cotton led to

a complete reorganization of the strategy and a relaxation

on the use of the now less useful pyrethroids. The situa-

tion is continuing to deteriorate, with resistance to pyre-

throids increasing steadily (N. W. Forrester, personal

communication).

(vi) Helicoverpa armigera in New Zealand

A programme to monitor resistance to fenvalerate in

H. armigera was initiated in 1991 in tomato, maize and

lucerne crops in New Zealand. A sign i¢cant trend of

declining mortality from 1992 to 1994 was seen and this

suggests an increase in the frequency of resistance to the

pyrethroids (Cameron et al. 1995; Suckling 1996).

Management strategies have been devised to counter this

problem (Suckling 1996).

(vii) Helicoverpa armigera inThailand

Wangboonkong (1981) ¢rst reported inadequate control

of H. armigera in Thailand soon after the introduction of

pyrethroids, but it was not known whether resistance was

the cause. Signi¢cant resistance to pyrethroid s was found

in populations of H. armigera from the Tak Fa area of

Nakonsawan in Thailand in 1985 (Ahmad & McCa¡ery

1988). These insects were also resistant to DDT and

carbaryl. Pyrethroid resistance was again noted in Thai

populations of the insect by Ernst & Dittrich (1992).

(viii) Helicover pa armigera in Indon esia

After the introduction of pyrethroids in the 1980s, resis-

tance to was found in populations of H. armigera collected

from the cotton-growing areas of South Sulawesi, Indonesia,

in 1987 and early 1988 (McCa¡ery et al. 1991a). These

populations were also resistant to endosulfan and DDT.

(ix) Helicoverpa armigera in China

Almost all groups of conventional in secticides have been

used to control H. armigera in China. DDT resistance was

¢rst detected in H. armigera in Henan province (Anon 1974),

and subsequently in Jiangsu and Hebie provinces (Zhu et

al. 1982), together wit h resistance to carbaryl. Pyrethroids

such as fenvalerate and deltamethrin have been widely

used since 1983 with others such as cyhalothrin, cyperme-

thrin, esfenvalerate, fenpropathrin and cy£uthrin being

used from the mid- to late-1980s.There were no substantial

changes in susceptibility until around 1989, but in the

following years resistance to pyrethroids was widely

detected in a number of areas including Jiangsu, Henan

and Shandong provinces (Tan et al. 1987; Shen et al. 1991,

1992, 1993; Wu et al. 1996, 1997b). The development of this

resistance led to calls for a resistance management strategy

to restrict pyrethroid use, to promote greater emphas is on

the use of alternations with other insecticides and to

promote the use of biological control (Shen et al. 1992).

Although level s of resistance to pyrethroids are still high,

Resistance to insecticides in heliothine Lepidoptera A. R. Mc Ca¡ery 1739

Phil. Trans. R. Soc. Lond. B (1998)

recent lower populations have alleviated the problem to

some degree (Y.Wu, personal communication).

(x) Helicoverpa armigera in Central Asia

High levels of resistance to pyrethroids (as well as to

OCs and OPs) have been found in H. armigera from Tajik-

stan and Azerbaijan (Sukhoruchenko 1996). In a similar

study, resistance to pyrethroids was found to be present in

populations of H. armigera from Russia (Leonova &

Slynko 1996).

(xi) Helicoverpa armigera in India

Pyrethroid insecticides were ¢rst used in India in 19 80

for the control of a number of p ests, including H. armi-

gera. In 1987 resistance to pyrethroids was ¢rst noted in

India in Andhra Pradesh (Dhingra et al. 1988;

Mc Ca¡ery et al. 1988, 1989; Phokela et al. 1989) in popu-

lations that were also resistant to DDT and slightly resis-

tant to endosulfan (McCa¡ery et al. 1989). Numerous

other studies con¢rmed the high incidence of pyrethroid

resistance, especially in the cotton- and pulse-growing

regions of central and southern India, and also

con¢rmed its gradual spread to other regions of the

country (see, for example, Phokela et al. 1990; Mehrohtra

& Phokela 1992; Armes et al. 1992, 1996; Sekhar et al.

1996; Jadhav & A rmes 1996). Pyrethroid resistance has

recently been found in the Punjab close to populations

over the border in Pakistan, leading Armes et al. (19 96)

to the conclusion that pyrethroid resistance is ubiquitous

in H. armigera in the Indian subcontinent. Resistance to

pyrethroids is frequently accompanied by resistance to

endosulfan, to OPs such as qu inalphos and monocroto-

phos, and to the oxime carbamate methomyl (Armes et

al. 1992, 1996).

(xii) Helicoverpa armigera in Pakistan

As a result of pyrethroid use since the early 1980s,

moderate to high levels of resistance to pyrethroids were

found in p opulations of H. armigera collected from

various regions of Pakistan from 1991 onwards (Ahmad

et al. 1995). Thes e insects were also resistant to the OP

monocrotophos, showed moderate resistance to endo-

sulfan and had low-level resistance to the OPs chlorpyr-

ifos and profenofos and the carbamate thiodicarb.

Interestingly, in a subsequent study these authors showed

variations in resistance to pyrethroids depending on

their structure. Although resistance varied from location

to location, the general t rend was for moderate to high

resistance to chemicals like cypermethrin, a low-to-

moderate resistance to compounds like deltamethrin and

comparatively low resistance to others like lambda-cyha-

lot hrin (Ahmad et al. 1997). With t he loss of e¤cacy of

the pyrethroids farmers have begun to use other non-

pyrethroid compounds, with the result that levels of

pyrethroid resistance were lower in 1997 than in

previous years (Ahmad 1998).

(xiii) Helicoverpa armigera in Africa

In the Ivory Coast pyrethroids have been applied for

15 years to control H. armigera and other bollworms. These

pyrethroids were always mixed or rotated with organopho-

sphate insecticides in an e¡ort to prevent or delay resis-

tance in bollworms (A laux et al. 1997). Ernst & Dittrich

(1992), in a comparative survey of resistance in heliothines

around the world, could ¢nd no evidence for resistance to

pyrethroids in the Ivory Coast. Vassal et al. (1997)

con¢rmed that before 1992 there was no change in resis-

tance to pyrethroids but in subsequent years susceptibility

decreased and by 1995 and 1996 signi¢cant resistance was

recorded. This is the ¢ rst documented evidence for resis-

tance to pyrethroids in bollworms inWest Africa. No resis-

tance to pyrethroids was found in populations of H.

armigera from Chad, although some changes in tolerance

were believed to be occurring (Martin & Renou1995).

(xiv) Helicoverpa armigera inTurkey

Resistance to synthetic pyrethroids was found in popu-

lations of H. armigera in 1984, after their initial use around

1980 (Anon 1986). Similar ¢ndings were reported by

Ernst & Dittrich (1992).

(xv) Helicoverpa armigera in Israel

Since 1987 a strictly observed insecticide resistance

management strategy has been in place in cotton ¢elds in

Israel. This is designed to maintain susceptibility to a

range of insecticides, including pyrethroids, in H. armigera

and other cotton pests. Monitor ing studies show that,

de spite slight £uctuations during the season, susceptibility

to cypermethrin did not alter during the period 1987^

1991 (Horowitz et al. 1993); control continued to be

ach ieved despite a very marked decline in the number of

sprays applied (Horowitz et al. 1995).

(xvi) Helicoverpa punctigera in Australia

A population of H. punctigera collected from New South

Wales in 1994 was shown to be resistant to fenvalerate

(Gunning et al. 1997). This is the ¢rst report of signi¢cant

resistance in this species.

9. MECHANISMS OF RESISTANCE TO PYRETHROIDS

(a) Nerve insensitivity: target-site resistance to

pyrethroids

The principal site of action of DDT and pyrethroid s is

the voltage-gated sodium channel of nerve cells (Soder-

lund & Bloomquist 1989; Narahashi 1992; Bloomquist

1996). These insecticides alter the gating kinetics of the

sodium channel so that the open time of the channels is

increased after the passage of t he dep olar izing pulse of an

action potential. This inhibition of sodium-channel inacti-

vation leads to the development of prolonged sodium

currents and accounts for the prolonged depolarizing

after-potential. This action causes the repetitive ¢ring of

neurons that is typically found in pyrethroid-poisoned

insects. Pyrethroids also cause membrane depolarization

due to the prolonged opening of sodium channels. Type II

pyrethroids, which c ontain a cyano group at the 

position, are generally more potent in this respect than

type I pyrethroids, which lack this -cyano group. Thus

sensor y neurons are stimulated as a result of membrane

depolarization. Membrane depolarization at nerve term-

inals causes massive release of neurotransmitter, resulting

in severe disruption of synaptic transmiss ion.

(i) Indirect evidence for nerve insensitivity

Evidence for the involvement of a kdr-like mechanism of

resistance in helioth ine insects has not been easy to

obtain. There is much indirect evidence that implies the

1740 A. R. McCa¡ery Resista nce to insecticides in heliothine Lepidopte ra

Phil. Trans. R. Soc. Lond. B (1998)

involvement of a target-site mechanism of resistance to

DDT and pyrethroids, although such evidence is never

wholly reliable. The most frequently used criterion has

been the lack of synergizable resistance. Weekly estimates

of the survival of third-instar H. armigera treated with a

discriminating dose of fenvalerate both with and without

PBO have been used in Australia to provide an estimate of

the percentage of non-synergizable resistance (Forrester et

al. 1993). It is inferred that this residual resistance is due to

other mechanisms, including a target-site resistance.

Similar ¢ndings were presented by Armes et al. (1996)

working with Indian populations of H. armigera. In a study

in Maharashtra State in India, it was suggested that the

residual non-metabolic resistance remaining after syner-

gism with both the monooxygenase synergist piperonyl

butoxide (PBO) and the esterase synergist S,S,S,-tributyl

phosphorotrithioate (DEF) was likely to be due to target-

site insensitivity (Kranthi et al. 19 97), and this is being

veri¢ed now. Given that the principal mechanisms of

resistance in these insects are considered to be enhanced

monooxygenase activity and/ or esterase activity and a

target-s ite resistance of the kdr type, this approach might

appear reasonable. Nevertheless, a s indicated below, PBO

may not be a reliable synergist for monooxygenases and it

may indeed synergize other forms of metabolic resistance

as suggested by Gunning et al. (1996a). Moreover, non-

synergizable penetration resistance also contributes to this

residual resistance. Given such considerations the premise

that non-synergizable resistance represents target-site

resistance is at best equivocal.

The presence of cross-resistance between DDT and

pyrethroids is frequently used as evidence for the involve-

ment of resistance at the target site. H. armigera from

Thailand were shown to possess high levels of resistance

to DDT and cypermethrin; this observation implies a

common mechanism. Lack of synergism by the DDT-

dehydrochlorinase synergist FDMC reinforced this view

(Ahmad & McCa¡ery 1991); as discussed below, the

insects were indeed shown to pos sess a nerve insensitivity

(Ahmad et al. 1989). Recent studies with strains of

H. armigera selected from ¢eld c ollections from Jiangsu

province in China have shown highly signi¢cant, non-

synergizable cross-resistance between DDT and fenvale-

rate (J. Tan & A. R. McCa¡ery, unpublished results).

This res istance has subsequently been shown to be due to

nerve insensitivity and its molecular basis is being studied

presently (see below). The inabilit y to identify metabolites

of pyrethroids in biochemical studies of pyrethroid

metabolism has also been use d to imply that resistance

may be due to target-site resistance, although it is clear

that the common involvement of both metabolic and non-

metabolic mechanisms in the same individuals in resistant

strains makes such an approach di¤cult.

Finally, because individuals with target-site resistance

might the oretically be able to withstand higher internal

concentrations of insecticide than their susceptible

counterpart s, it has been thought t hat the presence of

high titres in insects that survive such treatments indicates

the presence of this mechanism. The nervous system of

resistant third-instar larvae of the PEG 87 strain of

H. virescens was shown to contain up to tenfold greater

concentrations of cis-cypermethrin than those of

susceptible larvae of the BRC strain (Wilkinson &

Mc Ca¡ery 1991). In addition, the behavioural responses

of these intoxicated insects suggested that comparable

symptoms of intoxication occurred at higher concentra-

tions in larvae of the resistant strain than in lar vae of the

susceptible strain. This was again taken to imply a

decreased interaction of the pyrethroid wit h its target site.

At best, such evidence is tenuous. Such considerations

form the basi s of behavioural assays for ner ve

insensitivity, typi¢ed by the hot-needle assay developed

by Bloomquist & Miller (1985) and a locomotory assay

developed by Gunning (1996).

(ii) Direct evide nce for nerve insensitivity

There now exists a large body of direct ev idence that a

form of nerve insensitivity contributes substantially to

many cases of resistance to DDT and pyrethroids in

heliothine insects. Nicholson & Miller (1985) ¢rst demon-

strated this neurophysiological ly in a resistant strain of H.

virescens collected from cotton-growing areas of southern

California. A similar technique was used to demonstrate

nerve insensitivity in a pyrethroid- and DDT-resistant

Thai strain of H. armigera (Ahmad et al. 1989). At the

onset of pyrethroid resistance in Australia in 1983 a

strong super-kdr-like mechanism was demonstrated by

using a simple single-dose neurophysiological technique

(Gunning et al. 1991), but in a survey during the period

from 1997 to 1990 no evidence was found for the presence

of this super-kdr-like mechanism. Instead, another distinct

kdr-type mechanism with l ittle or no tox icological signi¢-

cance was found. By means of a cumulative dose

response neurophysiological assay for spontaneous

neuronal activity, nerve insensitivity to cypermethin was

demonstrated in resistant laboratory strains of H. virescens

(Gladwell et al. 1990) and in ¢eld strains collected from

various parts of the US cotton belt (McCa¡ery et al. 1995;

Ottea et al. 1995). Since monitoring of pyrethroid

resistance in the USA has been based upon the adult vial

test (Plapp et al. 1987), it is signi¢cant that nerve insensi-

tivity in adult stages of resi stant strains of H. virescens was

correlated with that in larval stages (Holloway &

Mc Ca¡ery 1996). Modi¢cations of this technique have

also been used to demonstrate high levels of nerve insensi-

tivity to pyrethroids and DDT in H. armigera from

Andhra Pradesh state in India (West & McCa¡ery 1992)

and from various parts of China (Y. Zhao et al. 1996;

Mc Ca¡ery et al. 1997; Ru et al. 1997; Zhang et al. 1997),

and H. zea from the USA (Holloway et al. 1997).

(iii) Molecular basis of nerve insensitivity resistance to pyrethroids

Although pyrethroids may interact with a number of

sites within the nervous system and although a range of

e¡ects may be produced by these interactions, the principal

site of action is considered to be the voltage-gated sodium

channel. For thi s reason e¡orts to determ ine the mol-

ecular basis of resistance to pyrethroids in heliothine

insects have centred on changes in sodium channels and

have followed similar pioneering studies on house £ies

and cockroaches. Experiments conducted by Church &

Knowles (1992) on binding to neural membranes of

saxitoxin, a high-a¤nity neurotoxin binding to site 1 on

the sodium channel, suggest that there is no di¡erence in

the number of sodium channels between pyrethroid-resis-

tant and -susceptible strains of H. virescens. Further work

Resistance to insecticides in heliothine Lepidoptera A. R. McCa¡ery 1741

Phil. Trans. R. Soc. Lond. B (1998)

has shown that binding of batrachotoxin, a sodium-

channel neurotoxin, is enhanced by pyrethroid binding;

by means of this assay these authors have provided

evidence t hat t he a¤nity for pyrethroids on the sodium

channels is considerably reduced in resi stant H. virescens

compared with susceptible counterparts (Church &

Knowles 1993). Taken together these studies imply that

reduced a¤nity of binding is resp onsible for resistance to

pyrethroids at the sodium channel in this species.

The evidence reviewe d above suggested that resistance

to pyrethroids and DDT might be expected to result from

the selection of genetic mutants with altered sodium

channels. Molecular genetic studies on sodium channels

would clearly be essential to understand the basis of t his

resistance. By using degenerate oligonucleotide pr imers

based on conserved amino-acid sequences in so dium

channels of Drosophila melanogaster, para-homologous

sodium-channel genes were isolated from a range of

insects including H. virescens (Doyle & Knipple 1991). The

polymerase chain reaction was used to amplify sequences

from genomic DNA from the PEG87 strain of H. virescens

by using degenerate pr imers homologous to the fourth

transmembrane domain of the -subunit locus para of

D. melanogaster (Taylor et al. 1993). One genomic clone

encoding a putative sodium channel in H. virescens was

obtained and designated hscp (Heliothis sodium channel para

homologue). In a subsequent experimental analysis,

markers for hscp were found to be linked to resi stance

phenotypes and this provided the ¢rst molecular genetic

evidence for such a link in any heliothine.

Sequence comparisons between resistant and suscep-

tible genotypes of house £y have revealed the presence of

a single leucine- to-phenylalanine mutation (L1014F) in

transmembrane segment 6 of domain II associated with

kdr resistance, and an additional methionine-to-threonine

mutation (M918T) associated with super-kdr resistance

(Williamson et al. 1996). Park & Taylor (1997) examined

H. virescens in a similar manner and revealed the existence

of a leucine-to-histidine change (L1029H) associated

with resistance to pyrethroids and located at a position

homologous to that in kdr strains of the house £y. No

mutation homologous to that found in super-kdr £ies was

found in H. virescens (Park & Taylor 1997). Interestingly,

the resistant PEG87 strain of this insect was not found to

carry this mutation; this observation leads to the sugges-

tion that more than one sodium-channel mutation may

be contributing to pyrethroid resistance in ¢eld popula-

tions of H. virescens. This contrasts with the situation in

Musca domestica, Blattella germanica, the diamondback

mot h, Plutella xylostella, and the peach

potato aphid,

Myzus persicae, in which the leucine-to-phenylalanine

substitution is always consistently present in resistant

genotypes (Martinez-Torres et al. 1997). More recently,

Park et al. (1997) reported a valine-to-methionine

(V421M) substitution in transmembrane segment 6 of

domain I (IS6) of the hscp locus of ind ividuals of the homo-

zygous resistant strain used by Taylor et al. (1993) for

linkage analysis. More recently still, Head et al. (1998)

made sequence comparisons between resistant and suscep-

tible strains of both H. virescens and H. armigera and showed

consistent aspartic acid-to-valine (D1561V) and glutamic

acid-to-glycine (E1565G) substitutions in the cytoplasmic

linker region between domains III and IV (III^IV) of the

para-homologous sodium-channel sequence of neurophy-

siologically resistant insects of both species; this region is

involved in channel inactivation. A further mutation in t he

IIS5^IIS 6 linker region was again consistently found in

both resistant H. armigera and resistant H. virescens (Head

1998). All these ¢ndings emphasize the likelihood that a

number of possible mutations can confer resistance at the

sodium channel, although the function of these mutations

clearly remains to be ascertained. The development of

diagnostic technologies based on mutations that unequivo-

cally indicate the kdr-like nerve insensitivity resistance to

pyrethroids and DDT is a clear aim of such studies. The

successful deployment of technology of this type would

provide a degree of precision and re¢nement that has so far

been lacking in the monitoring of resistance-gene

frequency in heliothine pests.

(b) Metabolic mechanisms of resistance to pyrethroids

Studies on the metabolism of pyrethroids in heliothine

insects have been characterized by a degree of contra-

diction, which has centred on the relative roles of the

principal systems of enzymic detoxication: ox idation by

the m icrosomal P 450-dependent monooxygenases (or

mixed-function oxidases) and hydrolysis by esterases.

Glutat hione S-t ransferases do not appear to be involved in

resistance to pyrethroids. The traditional use of synergists

to give preliminary indications of the type of metabolism

involved in resistance has been critically questioned in

relation to resistance to pyrethroids in v iew of ¢ndings,

discus sed below, which suggest t hat speci¢c synergists are

no longer (or possibly never were) e¡ective at suppressing

the enzyme systems with which they have traditionally

been associated. Other studies suggest that some synergists

inhibit enzy me systems alternat ive to those with which

they are normally as sociated. The use of model substrates

is also an area of some uncertainty: the isozymes respon-

sible for detoxication of speci¢c insecticides may not

necessarily be those involved in model substrate metabo-

lism. The latest and most comprehensive view of this ¢eld

would suggest that both oxidative and hydrolytic activity

is involved in resistance to pyrethroids in heliothines and

that, indeed, t he se species seem likely to be able to use

both types of metabolism in response to appropriate

selection. Such a view has considerable implications for

resistance management.

(i) Metabolic resistance in Heliothis virescens

Initial studies on metabolism of pyrethroids in

H. virescens suggested that monooxygenases were involved

in tolerance to trans-permethrin (Bigley & Plapp 1978). In

¢eld strains of this insect collected from the Imperial

Valley in California, enhanced metabolism of trans-perme-

thrin was a shown to be a mechanism of res istance

(Nicholson & Miller 1985) and it was thought that this was

likely to be due to oxidative hydroxylation. Dowd et al.

(1987) brought insect s from this location into the labora-

tory and selected them with £ucythrinate. In contrast to

the ¢ndings above, they demonstrated both a qualitative

and a quantitative enhancement in the ability of larvae to

hydrolyse pyrethroids compared with a susceptible strain.

The PEG87 strain of H. virescens has been used exten-

sively in research on resistance to pyrethroids in this

species and was derived from the US83 strain, itself

1742 A. R. McCa¡ery Resistance to insecticides in heliothine Lepidoptera

Phil. Trans. R. Soc. Lond. B (1998)

assembled from a ser ies of 19 collections across the US

cotton belt where control with pyrethroids had become

increasingly di¤cult. Despite e¡ectively being a labora-

tory strain, it was considered to possess the mechanisms

of resistance most likely to be representative of those in

the ¢eld. By using this strain it was shown that resistance

to trans-cypermethrin and cis-cypermethrin was largely

due to a PBO-synergizable monooxygenase, which

resulted in hydroxylation of the pyrethroid in the 4'; and

2'; positions on the phenoxybenzyl moiety (Lee et al.

1989: L ittle et al. 1989; Clarke et al. 1990; McCa¡ery et al.

1991c) and later elimination of conjugated metabolites.

Further studies on th is mechani sm showed that the resis-

tant strain possessed a s ixfold greater quantity of total

cytochrome P450 and a fourfold greater quantity of

cytochrome P450 reductase than did the comparable

susceptible strain (Clarke et al. 1990). Activity was shown

to be NADPH-dependent and PBO-suppress ible. Signi¢-

cantly, it was shown in these studies that the major

hydroxy-metabolites were likely to be better substrates for

hydrolysis than the parent compound. This ¢nding was

considered to explain the PBO-suppressible, NADPH-

dependent appearance of acid metabolites, although

carboxylesterase action was considered to play a minor

role in the direct hydroxylation of the pyrethroid (Clarke

et al. 1990). Using the PEG87 strain of H. virescens Abd-

Elghafar et al. (1994) presented si milar evidence for oxida-

tive metabolism of fenvalerate.

Despite the early demonstration of enhanced metabo-

lism in the ¢eld strain from Cal ifornia noted above

(Nicholson & Miller 1985; Dowd et al. 1987), metabolic

resistance was considered to be rare or absent in ¢eld popu-

lations of H. virescens for many years. Accordingly, a

number of studies showed that pyrethroid resistance was

not synergized by PBO or DEF (McCa¡ery et al. 1991b;

Clower et al. 1992), and it was cons idered that the majority

of the resistance was likely to be due to target-site

resistance of the kdr type. With continued use of

pyrethroids, evidence for PBO-synergi zable resistance

began to appear in the early 1990s in various US cotton-

belt states including Louisiana, Mississippi, Texas and

Oklahoma (Graves et al. 1991; McCa¡ery & Holloway

1992; Elzen et al. 1993; Kirby et al. 1994; Martin et al. 1994,

1997; G. Zhao et al. 1996), suggesting a widespread and

growing resistance problem based on enhanced monooxy-

genase activity as had been found with H. armigera. The

existence of enhanced resistance to cypermethrin through

selection with the oxime carbamate thiodicarb, and the

existence of PBO synergism of these insecticides, was

strongly indicative of the involvement of oxidative metabo-

lism (G. Zhao et al. 1996).

A number of other ¢ndings suggest that this might not

be wholly representative of the status of this mechanism.

Mart in et al. (1997) showed that application of PBO

delayed penetration of pyrethroids and suggested that

PBO could in£uence toxicity in other ways. Some strains

of H. virescens believed to possess enhanced mono-

oxygenase activity were shown to be entirely unresponsive

to the action of PBO and instead were synergized by

propynyl ethers such as TCPB (Brown et al. 1996b) (see

below). Nevertheless, many later biochemical studies

demonstrated the importance of oxidative attack in

resistance to pyrethroids in H. virescens (Ottea et al. 1995;

Ibrahim & Ottea 1995; G. Zhao et al. 1996). In a recent

study with the metab olically blocke d pyrethroid

fen£uthrin, a number of other structurally modi¢ed

pyrethroids and several synergists Shan et al. (1997)

con¢rmed that P450 monooxygenases were associated

with pyrethroid resistance in this species. Using a

pyrethroid- (and thiodicarb-) resistant strain of

H. virescens strain originally collected from ¢elds in

Louisiana where ¢eld failures with cypermethrin and

thiodicarb had been recorded, Rose et al. (1995)

examined monooxygenase, esterase and glutathione

S-transferase activity. Up to 4.4-fold higher quant ities of

cytochrome P450 were found in the gut, fat body and

carcass of the resistant strain than in those of the suscep-

tible strain and it was thought likely that these increased

P450 levels represented the sum of several P45 0 isozymes,

each of which may possess speci¢c yet overlapping

substrate speci¢cities. Esterases and transferases were

thought to be less important in conferring resistance in

this strain, although transferases may be important in the

production of conjugates, which form the bulk of excreted

metabolites in monooxygenase-resistant H. virescens (Little

et al. 1989). Interestingly, Rose et al. (1995) obtained

incomplete synergism with PBO in this strain and o¡ered

the suggestion that isozymes involved in pyrethroid

resistance might be made unresponsive to synergists by

selection pressure with PBO or other insecticides, as

appears to be the case in other insects. Incomplete

synergism with PBO was also obtained in another

H. virescen s strain derived from ¢eld collections in

Louisiana (Shan et al. 1997) although it could be

completely synergized by the propynyl ether TCPB. The

e¡ectiveness of TCPB as a monooxygenase synergist for

pyrethroid resistance in H. virescens was ¢rst shown by

Brown et al. (1996b), who concluded that di¡erent classes

of P450 monooxygenases were involved in resistance-

associated metabolism of pyrethroids. Such a ¢nding casts

considerable doubt on the validity of previous synergism

studies using PBO; the absence of PBO synergism should

perhaps not be taken as an indication of the absence of

enhanced oxidative metabolism.

The doubts about the e¤cacy of PBO and the involve-

ment of monooxygenases are compounded by renewed

interest in the role of esterases in pyrethroid resistance in

¢eld strains of H. virescens. Graves et al. (1991) had initially

found evidence for synergism of pyrethroids with DEF,

inferring the involvement of esterases and con¢rming

earlier observations (Dowd et al. 1987). Martin et al. (1997),

however, showed antagonism of the esterase synergist TPP

to cypermethrin action. The larval stages of a strain of H.

virescens originally obtained from the ¢eld in Louisiana,

where control with cypermethrin and thiodicarb had failed,

were examined for esterases associated with resistance to

these two compounds (Goh et al. 1995). Esterase activity

against the model substrate 1-naphthyl acetate (1-NA) was

elevated in whole-body homogenates of resistant insects

compared with those of susceptible insects. Increased

esterase activity was attributed to three esterases, A1, B1and

C1, which were puri¢ed and compared by means of immu-

noblotting techniques.The most signi¢cant of these, esterase

A1, was considered to share common epitopes with the resis-

tance-associated esterase of other insects, although its role in

insecticide resistance in the tobacco budworm was not

Resistance to insecticides in heliothine Lepidoptera A. R. McCa¡ery 1743

Phil. Trans. R. Soc. Lond. B (1998)

entirely clear. In a very recent study, G. Shan and J. A.

Ottea (personal communication) have shown that metabo-

lism of cypermethrin in H. virescens larvae occurs by both

ox idative and hydrolytic pathways but that the hydrolytic

route appears to be the major resistance mechanism. The

production of metabolites of hydrolysis in laboratory and

¢eld strains, as well as observations that suggest that both

cypermethrin and1-NA inhibit esterases in a concentration-

dependent manner, provide further evidence that esterases

are the major metabolic mechanisms of resistance to pyre-

throids. Moreover, inhibition experiments with PBO and

paraoxon and studies with 1-NA all suggest that the mono-

oxygenase inhibitor also inhibits esterases. Such studies

clearly concur with the ¢ndings of Gunning et al. (1996a)

working with H. armigera (see below) and cast yet further

doubt on the validity of using PBO as a synergist. Urgent re-

evaluation of the action and usefulness of these synergists is

required.

(ii) Metabolic resistance in H. armigera

Australia too has been a focus of cons iderable debate

regarding the relative roles played by estera se- and

cytochrome P450-mediated pyrethroid metabolism. In

1983, with t he onset of resistance to synthetic pyrethroids

in H. armigera in Australia (Gunning et al. 1984), three

mechanisms of resistance were thought to be involved.

Both a strong nerve insensitivity (super-kdr) and a pene-

tration resistance (Pen) were believed to be present,

together with a th ird factor overcome by PBO (Pbo)

(Gunning et al. 1991). Between 1987 and 1990 these insects

were again examined to determine which mechanisms

were present. Both t he Pen and Pbo mechanisms had

increased in importance, although they conferred only a

low-order resistance of around 20-fold (Gunning et al.

1991). The ability of PBO to c ompletely suppress resis-

tance to pyrethroids in strains of the insects homozygous

for a metabolic detoxication mechanism was presumed to

be evidence of the involvement of P450-mediated meta-

bolic resistance (Forrester et al. 1993). Moreover, the rela-

tive metabolic resistance-suppressing activity of a range of

65 synergists including TCPB provided further strong

evidence of the involvement of P450-mediated metabo-

lism. Field populations of the insects were regularly tested

with a discriminating dose of fenvalerate both with and

without PBO.The evidence from this monitoring suggested

that the PBO-suppressible resistance component was

always predominant. As discussed earlier, the non-syner-

gizable component was assumed to represent other

mechanisms, in particular nerve insensitivity. Further

convincing evidence that the great majority of the resis-

tance to pyrethroids seen Australian H. armigera was due to

enhanced oxidation came from an important exami nation

of the structure

activity relations of a large range of pyre-

throid analogues with varying acid and alcohol structures

and a range of substitutions. Alterations in the alcohol

moiety of the pyrethroid structure could overcome most, if

not all, resistance. The nature of these changes in coun-

tering resistance provided strong evidence that the resis-

tance was due to oxidative metabolism. All of the se

¢ndings would seem to provide overwhelming, if somewhat

indirect, ev idence that resistance to pyrethroids in H. armi-

gera in Australia was based on enhanced P450-mediated

metabolism. Similar evidence has been put forward for

enhanced monooxygena se activity being a mechanism of

resistance to pyrethroids in H. armigera from India

(Phokela & Mehrohtra 1989; Kranthi et al. 1997) and

China (Wu et al.1997a).

This conventional view was challenged by Kennaugh et

al. (1993) using a strain of H. armigera derived from ¢eld

collections and subsequently backcrossed and selected.

Although the resistant strain was 19- to 33-fold resistant

to fenvalerate and this resistance could be eliminated

with PBO, these authors could ¢nd no increased levels of

P450 in the midguts of the resistant strain compared with

those of the susceptible strain. Further, there was no

evidence for increased permethrin detoxication in the

resistant strain. Signi¢cantly, PBO increased the rates of

metabolism in both susceptible and resistant strains.

Evidence was obtained which suggested the involvement

of a cytochrome P450 in the process of penetration of the

insecticide through the insect cuticle. The action of PBO

would thus be to inhibit a P450-dependent penetration

resistance (Kennaugh et al. 1993). These ¢ndings were

corrob orated by Gunning et al. (1995), who examined

esfenvalerate metabolism in a resistant strain of H. armi-

gera, in which the res istance was suppressed by PBO and

which lacked any nerve insensitivity. It was show n that

esfenvalerate metaboli sm was only slightly enhanced in

this resistant strain and that PBO did not in hibit this

metabolism. The authors concluded t hat reduced penetra-

tion appeared to be an important mechanism of esfenva-

lerate resistance in this strain.

An important study was then published, which

suggested that pyrethroid-resistant H. armigera in

Australia have enhanced esterase activity that is due to

increased production of enzymes (Gunning et al. 1996a).

The most resistant individuals were shown to have an

approximately 50-fold increase in esterase activity

compared with susceptible populations. Moreover,

resistant strains were shown to have additional esterases

not detectable in susceptible populations and increased

esterase hydrolysis of 1-NA was correlated with the

esfenvalerate resistance factor. Furthermore, evidence

was obtained which suggested that the esterase had a

poor catalytic activity towards the pyrethroids and that

esterases were also acting a s insecticide-sequestering

agents. It was concluded that detoxi¢cation by hydrolysis

together with sequestration would give H. armigera the

ability to detoxify signi¢cant quantities of fenvalerate,

consistent with the large resistance factors involved.

Together these ¢ndings imply that detoxication via

monooxygenases is no longer, or was never, a signi¢cant

mechanism of resistance to pyrethroids in H. armigera, a

situation that is paralleled in H. virescens in t he USA. To

further emphasize this revised view of metabolic resis-

tance to pyrethroids, Gunning et al. (1996b) have shown

that PBO can suppress esterase-mediated metabolism.

This crucial observation de¢es the conventional assump-

tion that PBO un iquely suppresses metab olic resistance

mediated by cytochrome P450 and is again mirrored in

recent studies in the USA on H. virescens (J. A. Ottea,

per sonal communication). More recently Gunning et al.

(1997) showed that fenvalerate toxicity in H. punctigera

was synergized by the esterase inhibitors DEF and

profenofos and that the resistant insects had increased

esterase activity to 1-NA.

1744 A. R. McCa¡ery Resistance to insecticides in heliothine Lepidoptera

Phil. Trans. R. Soc. Lond. B (1998)

(iii) Molecular studies on metabolic resistance in heliothines

The con£icting nature of these ¢ndings both in

Australia and in the USA emphasizes that a de¢nitive

role in metabolic resistance for speci¢c P450s or esterases

is likely to come only from expression studies using genes

cloned from pyrethroid-resistant strains.

The complete coding sequence and parts of the 3' and 5'

non-coding regions of a mRNA coding for a cytochrome

P450 from H. armigera was obtained (Wang & Hobbs 1995).

The sequence is most similar to member of the CYP6

family and has been designated CYP6B2. The cDNA

hybridizes to two major mRNAs, the larger of which i s

inducible by permethrin, although t he levels of induction

are generally low. These same authors have demonstrated

much higher quantities of the larger mRNA in individual,

pyrethroid-resistant lar vae collected directly from the

¢eld; this result implies the involvement of this P450 in

resistance. In a separate study, RT

PCR was used to clone

P450 gene fragments from the RNA of a pyrethroid-resis-

tant strain of H. armigera (Pittendrigh et al. 1997). By this

method eight new P450 genes were isolated, seven from

the CYP4 family and one CYP9. One of these genes,

CYP4G8, was twofold overexpressed in the resistant strain.

Although no di¡erence in express ion was noted in resistant

strains, CYP9A3 appeared to be a homologue of the puta-

tively resistance-associated CYP9A1 of H. v irescens (Rose et

al. 1997) (see below). Further, the authors found non-

detectable levels of expression of the CYP6B2 isolated by

Wang & Hobbs (1995) and reportedly overexpressed in

resistant strains. In H. virescens Rose et al. (1997) isolated a

P450 gene designated CYP9A1, the ¢rst member of family

9, from a pyrethroid-resistant strain.These studies indicate

that bot h qualitative and quantitative strain-to-strain

variations in P450 expression levels are important and

that recombinant expression will be necessary in order to

precisely de¢ne t he substrate speci¢cities and pyrethroid-

metabolizing abilities of individual P450s. The ability to

de¢ne the character istics of the detoxi¢cation systems of

resistant strains of insects would lead to a signi¢cant re¢ne-

ment in cross-resistance studies. On the basis of substrate

speci¢city it should prove possible not only to objectively

select insecticides between chemical groups but also to

select more e¤cacious analogues from within groups.

10. DISCUSSION

As summarized in this review, H. ar migera, H. virescens,

and to some extent H. zea, have developed substantial and

often uncontrollable levels of resi stance to virtually all the

neurotoxic in secticides that have been directed against

them. Ecological and physiological aspects of the biology

of these insects have made possible the emergence of pest

species, which have often proved di¤cult to control.

Continued selection with insecticides has allowed the

survival of resistant populations, which have generally

proved exceedingly di¤cult or impossible to control.

Despite th is there are examples of cropping systems in

which resistance is absent or in which resistance is a

minor problem. The resistance-management strategy

initiated in cotton in Israel to control a range of p ests has

left H. armigera there very largely susceptible to all insecti-

cides, despite, or more probably because of, the use of a

very small number of applications (Horowitz et al. 1995).

In other areas such as the Ivory Coast, low or restricted

use of insecticides has allowed a great many years of

resistance-free pest control and only now are levels of

resistance beginning to rise.

Species such as H. punctigera, which have, by virtue of

their biology and ecology, been considered capable of

escaping the development of res istance, have now been

shown to do so (see, for example, Gunning et al. 1994,

1997). It is essential that resistance-management strategies

are formulated in ways that do not enhance the resistance

status of such species. In the USA , where pyret hroids

have been very widely used, it is generally accepted that

the key feature that ha s prevented development of

resistance in the maize earworm, H. zea, i s its wider range

of unsprayed hosts: H. zea attacks maize and soyabeans

whereas H. virescens does not attack maize and prefers

cotton to soyab eans. Nevertheless, H. zea has recent ly

developed signi¢cant resistance to these compounds and

consideration must be given to the implications of this.

The most highly imitated resistance management

strategy for heliothines is that which was set up for the

control of pyrethroid- and endosulfan-resistant H. armige ra

in cotton (and other crops) in Australia (Forrester et al.

1993).The obvious success of this highly acclaimed scheme

was that control was maintained for well over ten years

with insecticides to which resistance had already devel-

oped. This was achieved largely through strict observance

of restrictions in use.The gradual loss of the pyrethroids to

resistance and the advent of new insecticides, Bt and Bt-

transgenic cotton has allowed a relaxation of the pyre-

throid use strategy and control is now based on a broad

range of chemical, biological and cultural methodologies.

Similar types of strategy have been initiated el sewhere

with varying degrees of success, as noted above.

The development of pyrethroid resistance in heliothine

species in various countries around the world cont inues

unceasingly, even in countries with management

strategies, although the rate of loss of e¤cacy is generally

slower in control led situations. That pyrethroid-resistant

insects can be found in unsprayed refugia in Australia

(Forrester et al. 1993), and pyrethroid-susceptible insects

are absent or exceedingly hard to ¢nd anywhere in

countries such as Pakistan and India, does not bode well

for much further use of these compounds unless new

chemistry is deployed or severe restrictions on use are

instigated. Such actions have complex economic and

political implications. Moreover, questions regarding the

¢tness costs of these resistances need to be addressed

urgently since there are important considerations for

insecticide-resistance management. Although new tech-

nology and new chemistry will enable the select ion

pressure from older insecticides to b e relaxed, it is likely

that the use of conventional insect icide chemistry will

continue for many years.

This review has placed considerable emphasis on the

mechanisms of resistance to the insecticide groups

considered. This is because patterns of cross -resistance

within and between insecticide groups are entirely

dependent on the biochemical and molecular nature of

the resistance mechanism. Even within the c onventional

insecticide groups there are many areas of susceptibility

to existing chemistry, which can be exploited for control.

In a previous brief review this author considered that

Resistance to insecticides in heliothine Lepidoptera A. R. McCa¡ery 1745

Phil. Trans. R. Soc. Lond. B (1998)

target-s ite resi stance to pyrethroids had developed in

many heliothines before the later emergence of metab olic

resistance (McCa¡ery 1994). Although this may still

hold true to some degree, the ability of these species to

diversify their mechanisms of resistance under selection

pressure is noteworthy. The current debate over the

nature of metabolic resistance in heliothines is confused

by technical controversy over the use of some of the

basic tools, such as synergists and model substrates, and

these arguments have been considered in detail in this

review. Likewise, the inconsistencies found in pyrethroid

target-s ite mutations are at variance with those found in

other insects. It is the view of this author that the

heliothines are especially £exible in the use of a variety

of modi¢cations in all of their resistance mechanisms.

Thus, even when a similar system of enhanced enzyme

activity is involved in resistance to the same group of

compounds, such as monooxygenases, di¡erent P450

forms seem likely to be found in individual populations

of the same species.

It could be argued that an ability to diagnose the precise

nature of the mechanisms of resistance would be a key

component of the management of resistance in the

heliothines. However, as emphasized in t his review, the

very diverse nature of the modi¢cations found so far makes

this enormously less easy than might otherwise be so and

possibly renders such an approach not practicable. It might

be considered that this diversity would allow the use of

resistance-breaking molecules within existing conven-

tional insecticide groups.The existence of such compounds

has been illustrated for both H. armigera (Forrester et al.

1993; J. Tan and A. R. McCa¡ery, unpublished obser-

vations) and H. virescens (Shan et al. 1997), but the usefulness

of such an approach again depends entirely on an ability to

correctly diagnose subtle changes in resistance mechanisms

in ¢eld populations. As highlighted above, this might

prove di¤cult in reality and it is disappointing that such

approaches have not yet resulted in commercially viable

products. A knowledge of the mechanisms of resistance

exist ing in these insects is clearly of value in devising new

insecticides to control them. The advent of new areas of

insecticide chemistry such as Bacillus thuringiensis, pyrroles,

phenyl pyrazoles, spinosad, nicotinyls and insect growth

regulators (IGRs) should make control of heliothines

(Tabashnik et al., this issue) considerably more e¡ective

and release selection on existing resistance mechanisms.

Nevertheless, incipient resistance to some of these materials

in a number of arti¢cial laboratory strains of Heliothinae is

surely a stimulus to e¡ective use of new materials and an

ever-watchful study of the development and nature of

possible resistances to them.

In conclusion, it is perhaps signi¢cant that, to date, the

most success ful resistance-management programme that

has been developed for t he se insects has been instituted in

cotton in Israel. Key features of this programme have been

a dramatic reduction in the number of sprays directed at a

number of pests, including H. armigera , together with the

considered use of a range of integrated pest management

(IPM) techniques, It would appear that, despite our

rapidly increasing knowledge of the biochemical and mole-

cular nature of this problem, the most e¡ective means of

managing resistance to insecticides in t he Heliothinae

remains a strict control of insecticide use.

I am very grateful to Dr Neil Forrester, Dr James Ottea and Dr

Yidong Wu for information on the current resistance situation in

their countries and for permission to quote unpublished infor-

mation and from papers currently in press.

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