encyclopedia of biodiversity || insecticide resistance

29

Insecticide ResistanceIan Denholm, Rothamsted Research, Harpenden, UKGreg Devine, Queensland Health, Cairns, QLD, Australia

r 2013 Elsevier Inc. All rights reserved.

GlossaryAcetylcholinesterase (AChE) The enzyme responsible for

breaking down the neurotransmitter acetylcholine (ACh) at

nerve synapses, thereby preventing hyperexcitation of

cholinergic pathways in the nervous system. The target of

organophosphate and carbamate insecticides.

Bioassay (biological assay) A laboratory test for

evaluating the response of organisms to a toxin and for

diagnosing the presence or absence of resistance.

Cross-resistance The ability of a single gene or

mechanism to confer resistance to more than one toxin.

Cytochrome P-450 monooxygenases A ubiquitous group

of enzymes involved in the NAPDH-mediated oxidation

and metabolism of a broad range of endogenous and

exogenous substrates.

GABA receptor Part of the inhibitory ion channel

complex gated by GABA (g-aminobutyric acid) in

Encyclopedia of Bi8

postsynaptic nerve membranes. The target site of

organochlorine insecticides and some other chemical

groups.

Glutathione S-transferases (GSTs) Enzymes that catalyze

the metabolism of a range of substrates following their

conjugation with the endogenous tripeptide glutathione.

Multiple resistance The occurrence of more than one

resistance mechanism in the same individual or pest

population.

Sodium channel A large transmembrane protein that

regulates the flow of sodium ions across axonal membranes

and mediates the rising phase of action potentials. The

target site of pyrethroid insecticides.

Synergist A chemical used at sublethal concentrations to

inhibit particular groups of detoxifying enzymes and

therefore to implicate these enzymes in resistance.

600

Total500

Introduction

The diversity of organisms and their genetic variation have

been forged by evolution. In evolution, undirected mutations

generate unstructured diversity, which is then structured by

selection. The time scales over which selection and adaptation

occur in eukaryotes are usually too large to observe in situ

(except in a few cases, such as industrial melanism in some

moths and ladybirds), but there is one microevolutionary

process for which many of the factors driving selection and

adaptation are well understood, a process that is being in-

creasingly studied and manipulated by a large number of

biologists. This is the phenomenon of pest resistance to in-

secticides, and its study can reveal much about how bio-

diversity originates, at the intraspecific level at least. Similar

processes underpin the evolution of herbicide resistance by

weeds, fungicide resistance by plant pathogens, and drug

resistance by disease-causing microbes.

01930 1940 1950 1960

Year1970 1980 1990 2000

PyrethroidCarbamate

Organophosphate

Cyclodiene

100

200Res

ista

nt s

peci

es

300

400

Figure 1 Increase in the number of arthropod species reported toresist insecticides over time, in total, and in response to the fourmost widely used classes of insecticide (courtesy GP Georghiou).

Extent of Resistance

Although a relatively recent phenomenon (resistance to the

first synthetic insecticide, dichlorodiphenyltrichloroethane or

DDT, was initially reported in the 1940s), insecticide resist-

ance is now very widespread. Available statistics (Figure 1)

show that reports of resistant arthropod species increased al-

most exponentially between 1950 and 1980, following the

successive introduction of different classes of synthetic in-

secticides. By 1990, more than 500 species were reported to

resist chemicals of at least one insecticide class, and many of

these resisted several classes simultaneously. Of the resistant

species reported in 1990, 88% were insects (class Insecta) and

12% were mites and ticks (class Arachnida, order Acarina).

Four insect orders – Coleoptera (beetles), Diptera (true flies),

Hemiptera (aphids, bugs, hoppers, and whiteflies), and Lepi-

doptera (moths) – accounted for 92% of the resistant insect

species; the remainder mostly comprised of cockroaches,

thrips, lice, and fleas. Additional resistant species have been

reported since 1990, but a change in survey methods makes it

difficult to extend the temporal trend evident in Figure 1.

Although almost all insecticide classes are now affected by

resistance, its extent varies greatly between species. In some

insects, resistance only extends to a few closely related com-

pounds in a single class; it may be very weak or restricted to a

small part of their geographical range. At the other extreme,

odiversity, Volume 4 http://dx.doi.org/10.1016/B978-0-12-384719-5.00104-0

MAC_ALT_TEXT Figure 1

(a) (b)

(c) (d)

Figure 2 Examples of arthropod pests that have developed resistance to insecticides, (a) The bollworm, Helicoverpa armigera, a major pest ofcotton and vegetables in the Old World; (b) the tobacco or cotton whitefly, Bemisia tabaci, which threatens a wide range of crops through direct-feeding damage and the transmission of virus disease; (c) the peach–potato aphid, Myzus persicae, a major pest of vegetable and ornamentalcrops in temperate countries; and (d) the two-spotted spider mite, Tetranychus urticae, a cosmopolitan pest of fruit, vegetable, andornamental crops.

Insecticide Resistance 299

some widespread pests such as the cotton bollworm (Heli-

coverpa armigera; Figure 2(a)), the tobacco whitefly (Bemisia

tabaci; Figure 2(b)), the peach–potato aphid (Myzus persicae;

Figure 2(c)), and the two-spotted spider mite (Tetranychus

urticae; Figure 2(d)) can resist most or all of the insecticides

available for their control. The most extensively used insecti-

cide classes – organochlorines, organophosphates, carba-

mates, and pyrethroids – have generally been the most

seriously compromised by resistance, and many principles

relating to the origin and evolution of resistance can be

demonstrated solely by reference to these fast-acting neuro-

toxins. In recent years, however, there has also been a worrying

increase in resistance to more novel insecticides, including

ones attacking the nervous system (e.g., neonicotinoids), de-

velopmental pathways (e.g., benzoylphenylureas), respiratory

processes (e.g., mitochondrial electron-transport inhibiting

(METI) acaricides), and digestive systems (e.g., Bacillus

thuringiensis (Bt) endotoxins).

Origins of Resistance

As insecticides are not normally considered mutagenic at field

application rates, it is assumed that resistance mutations occur

independently of insecticide exposure, and are as likely to

occur before an insecticide is introduced as they are during its

use in the field. Resistance is therefore a preadaptive phe-

nomenon reflecting the selection of individuals possessing

heritable genetic traits that promote their survival or repro-

duction in environments treated with insecticides. Estimates

of mutation rates are as imprecise as they are for other adap-

tive traits, but will depend on the mutational event involved


300 Insecticide Resistance

(see Mechanisms of Resistance). The role of enzyme induction

in resistance has not been demonstrated satisfactorily but is

likely to be only of secondary importance. Increased tolerance

due to environmental or biological factors such as diet, age, or

climate can sometimes be significant but is outside the scope

of this article.

Mechanisms of Resistance

Some of the most significant recent progress in understanding

resistance result from applying advances in molecular biology

to resistance research. Depending on the mechanism involved,

resistance can arise through structural alterations of genes

encoding target-site proteins or detoxifying enzymes, or

through processes affecting the expression of gene products

(e.g., gene amplification or altered transcription rates). Other

mechanisms that have been demonstrated or postulated in-

clude reduced penetration of insecticides through the insect

cuticle, enhanced excretion of insecticides, and behavioral

traits enabling pests to reduce or avoid exposure to a toxin.

However, the latter are generally considered to be relatively

minor in effect or to arise only under very specialized cir-

cumstances. Figure 3 is a schematic representation of some of the

resistance mechanisms discussed in the following two sections.

Increased Detoxification of Insecticides

The three major routes of detoxification implicated in resist-

ance are as follows: Enhanced metabolism of insecticides by

cytochrome P-450 monooxygenases can potentially confer

resistance to most chemical classes. Previously, much of the

evidence for this mechanism was indirect, based on the ability

of compounds such as piperonyl butoxide, which are known

inhibitors of monooxygenases, to reduce the magnitude of

Na+

ac

b

channel Modified AChE

Acetylcholine

Presynaptic neurone

Figure 3 Schematic diagram of a nerve synapse showing examples of inssodium channel confer knockdown resistance (kdr) to pyrethroids. (b) Modorganophosphates and remains available to breakdown acetylcholine molecuenzymes degrade or sequester insecticides before they reach their targets in

resistance when used as synergists in bioassays. However, re-

cent developments in insect genomics are enabling complex

families of genes such as those encoding monooxygenases to

be cloned and characterized and single enyzymes to be im-

plicated directly in resistance through crossing studies and

experiments on insecticide metabolism and binding.

Enhanced activity of glutathione S-transferases (GSTs) is

considered potentially important in resistance to some classes

of insecticide, including organophosphates. However, infor-

mation on the role of GSTs in resistance is still sketchy but

these enzymes, like monooxygenases, exist in numerous mo-

lecular forms with distinct properties, and are proving amen-

able to molecular screening and characterization.

Enhanced hydrolysis or sequestration by esterases (e.g.,

carboxylesterases) capable of binding to and cleaving car-

boxylester and phosphotriester bonds undoubtedly plays a

significant role in resistance to organophosphates and pyr-

ethroids. Of the three main types of detoxification mech-

anisms, this has historically been the best characterized

biochemically. Resistance due to increased esterase activity can

arise through either a qualitative change in an enzyme, im-

proving its hydrolytic capacity, or (as in mosquitoes and

aphids) a quantitative change in the titer of a particular

enzyme that already exists in susceptible insects.

Alterations to Insecticide Target Sites

Resolving target-site modifications that lead to resistance re-

quires some knowledge of the mode of action of insecticides

themselves. At present, this information is most advanced for

insecticides binding to enzymes or receptors in the nervous

system of arthropods. Three examples of target-site resistance

are well understood:

Organophosphates and carbamates exert their toxicity by

inhibiting the enzyme acetylcholinesterase (AChE), thereby

Esterases/oxidases/transferases

Carbamate/organophosphate molecules

Postsynaptic cell

Acetylcholine receptorsAChE

ecticide-resistance mechanisms. (a) Changes in the structure of theified acetylcholinesterase (AChE) is no longer bound byles after neurotransmission across the synapse. (c) Detoxifyingthe nervous system.



impairing the transmission of nerve impulses across cholinergic

synapses. Mutant forms of AChE showing reduced inhibition

by these insecticides have been demonstrated in several insect

and mite species. Biochemical and molecular analyses of in-

secticide-insensitive AChE have shown that pests may possess

several different mutant forms of this enzyme with contrasting

insensitivity profiles, thereby conferring distinct patterns of re-

sistance to these two large insecticide classes.

Pyrethroids act primarily by binding to and blocking the

voltage-gated sodium channel of nerve membranes. A resist-

ance mechanism causing insensitivity of this target site was first

identified in houseflies (Musca domestica) and termed knock-

down resistance (kdr). Kdr is attributable to structural modifi-

cations of the sodium channel protein and the genetic

mutations responsible have been identified. This mechanism

has been found in many pest species. As with insensitive AChE,

there can be different forms of kdr resistance (e.g., a more po-

tent ‘‘super-kdr’’ form in which a second mutation potentiates

the level of resistance conferred by the original one).

GABA receptors are targets for several insecticide classes,

including cyclodienes (a subclass of the organochlorines),

avermectins, and fipronils. The primary mechanism of resist-

ance to cyclodienes and fipronils involves modification of a

particular GABA receptor subunit, resulting in substantial

target-site insensitivity to these insecticides.

Homology of Resistance Genes

Although there are several possible mechanisms, the options

for resisting insecticides can also be very limited, especially for

mechanisms based on decreased sensitivity of insecticide tar-

get sites. The target-site mechanism of cyclodiene resistance

involves the same amino acid substitution (alanine-302 to

serine) in GABA receptors of several species of diverse taxo-

nomic origin, including beetles, mosquitoes, whiteflies, and

aphids. Work on the two other principal target-site mech-

anisms – altered AChE and kdr – has proved more challenging

due to the occurrence of multiple resistance alleles at the same

loci. In the case of kdr, however, there is also evidence for the

same amino acid substitution (leucine-1014 to phenyl-ala-

nine) in the sodium channel protein conferring a ‘‘basal’’ kdr

phenotype in a wide range of species. This phenotype can be

enhanced (to ‘‘super-kdr’’ resistance) by further mutations that

also recur between species. Despite the structural complexity

of the receptors involved, these parallel mutations imply that

the opportunities for insects to modify them to avoid or re-

duce binding of insecticides, while retaining normal func-

tioning of the nervous system, are very limited indeed.

When susceptible individuals of the sheep blowfly (Lucilia

cuprina) were exposed to the mutagen ethyl methanesulfonate

(EMS) and their progeny screened for resistance to dieldrin

(a cyclodiene), surviving insects not only exhibited a GABA

receptor-based mechanism analogous to that found in nature

but also exhibited an identical alanine to serine amino acid

substitution in the receptor gene. Similarly, mutagenesis fol-

lowed by screening with diazinon (an organophosphate) led

to the recovery of a resistance mechanism showing identical

toxicological, biochemical, genetic, and molecular properties

to one that had previously evolved to diazinon under field

conditions. These findings reinforce the tight evolutionary

constraints on the number of viable resistance mutations, even

in the laboratory, where mutations conferring deleterious ef-

fects on overall fitness might be expected to survive better than

in the open field. Interestingly, they also highlight the po-

tential of using mutagenesis to predict likely resistance

mechanisms to novel insecticides in advance of them being

used commercially and to tailor resistance-management rec-

ommendations accordingly.

In other cases, different types of resistance to the same

toxin exist and can account for differences in the toxicological

and genetic basis of resistance between species or between

different geographical populations of the same species. In-

secticidal proteins from the soil bacterium Bt are becoming

increasingly important in pest management, especially in re-

lation to insect-tolerant transgenic crops (see The Special Case

of Transgenic Plants). To date, the only species to have evolved

Bt resistance on a large scale in the field is the diamondback

moth, Plutella xylostella. The majority of Bt-resistant popu-

lations examined have exhibited very similar characteristics,

including a very consistent pattern of cross-resistance to dif-

ferent Bt toxins and recessive inheritance. However, there are

also strains of P. xylostella in which the breadth and inheritance

of Bt resistance differ markedly from this ‘‘mode,’’ implying the

existence of distinct resistance genes and mechanisms.

How often do Resistance Genes Arise?

The recurrence of specific resistance mutations within and

between taxa begs another question of fundamental signifi-

cance to the origins of biodiversity: Have such mutations

arisen repeatedly within the same species, or appeared on only

a limited number of occasions and subsequently spread

through migration or human agency? This question is amen-

able to investigation by sequencing not only the resistance

genes themselves but also flanking regions and introns, which

would be expected to vary between alleles that have arisen

independently. In the mosquito, Culex pipiens, organo-

phosphate resistance is primarily conferred by allozymes at

two closely linked loci (esterases A and B) coding for insecti-

cide-detoxifying carboxylesterases. Overproduced allozymes

(resulting from amplification of A or B genes) tend to recur in

geographically disjunct areas. This situation could be ex-

plained by recurrent mutation generating each amplification

event de novo or by a nonrecurrent mutation that has sub-

sequently spread between populations. Restriction mapping

of DNA around the esterase genes points to the latter ex-

planation, with large-scale gene flow (even between contin-

ents) most likely attributable to passive migration of

mosquitoes on ships or airplanes. It is notable that the ap-

pearance of a new resistance allele in southern France ori-

ginated in the vicinity of the international airport and seaport

at Marseilles.

Organophosphate resistance in the aphid M. persicae is

also attributable to the amplification of a gene encoding an

insecticide-detoxifying carboxylesterase. Despite the often

widespread dispersion of these amplified genes in the aphid

genome, restriction analyses have indicated that all copies are

in the same immediate genetic background. This suggests that


amplification occurred only once, with the amplified DNA

subsequently being moved intact around the genome through

chromosomal rearrangements, or perhaps mediated by trans-

posable elements. Similarities in the position and structure of

these genes in aphids of diverse geographic origin reinforce

the likelihood of a single amplification event that has sub-

sequently become widely dispersed around the world.

However, there is also molecular evidence for some resist-

ance genes having several independent origins in the same

species (e.g., for target-site resistance to cyclodienes in the red

flour beetle, Tribolium castaneum). Results for mosquitoes and

aphids, nonetheless, highlight the potential for large-scale

inadvertent movement of resistant insects between countries

or even continents. For crop pests, these risks are particularly

acute due to the increasing international trade in edible and

ornamental plants, many of which have been treated with

insecticides at their point of origin. In such cases, growers at

the receiving end of the trade network face a dual threat: (1)

the establishment of new pest species or more aggressive

biotypes of existing ones and (2) the possibility that such

pests are already strongly resistant to compounds that might

otherwise be used to suppress or eradicate them.

(a)

(b)

Figure 4 Examples of resistance diagnostics for the peach–potatoaphid, Myzus persicae; (a) a bioassay in which insecticide dropletsare applied topically to individual aphids; and (b) a biochemicalimmunoassay to determine levels of an insecticide-detoxifyingesterase in individual aphids. The top row shows results for threeinsects from each of four resistance categories, with resistanceincreasing from left to right. The remaining insects were drawnrandomly from a field sample.

Cross-Resistance and Multiple Resistance

Arthropods seldom resist just one toxin. Most commonly, they

exhibit differing levels of resistance to a range of related and

unrelated insecticides. In its strictest sense, the term ‘‘cross-

resistance’’ refers to the ability of a single mechanism to confer

resistance to several insecticides simultaneously. A more

complex situation is that of ‘‘multiple resistance,’’ reflecting

the coexistence of two or more resistance mechanisms, each

with their own specific cross-resistance characteristics. Dis-

entangling cross-resistance from multiple resistance is a chal-

lenging aspect of resistance research. However, knowledge of

the mechanisms involved is often essential in order to develop

resistance-management recommendations based, for example,

on the alternation of insecticides to avoid continuous selec-

tion for the same resistance gene or mechanism.

Unfortunately, cross-resistance patterns are inherently dif-

ficult to predict in advance, because mechanisms based on

both increased detoxification and altered target sites can differ

substantially in their specificity. The most commonly en-

countered patterns of cross-resistance tend to be limited to

compounds within the same chemical class (equivalent to the

term ‘‘side resistance’’ as used by parasitologists). However,

even these patterns can be very idiosyncratic. For example,

organophosphate resistance based on increased detoxification

or target-site alteration can be broad-ranging across this group

or highly specific to a few chemicals with particular structural

similarities. The breadth of target-site resistance to pyrethroids

also depends on the resistance allele present. The most fre-

quent mutation affects almost all compounds in this class to a

similar extent (approximately 10-fold resistance), whereas re-

sistance due to other mutations is highly dependent on the

alcohol moiety of pyrethroid molecules, ranging from ap-

proximately 10-fold to virtual immunity. Cross-resistance be-

tween insecticide classes is even harder to anticipate, especially

for broad-spectrum detoxification systems whose specificity

depends not only on insecticides having the same mode of

action but also on the occurrence of common structural fea-

tures that bind with detoxifying enzymes.

Empirical approaches for distinguishing between cross-re-

sistance and multiple resistance include (1) repeated back-

crossing of resistant populations to fully susceptible ones to

establish whether resistance to one chemical cosegregates

consistently with resistance to another and (2) reciprocal se-

lection experiments whereby populations selected for resist-

ance to one chemical are examined for a correlated change in

response to another. If available, biochemical or molecular

diagnostics for specific resistance genes can assist considerably

with tracking the outcome of genetic crosses or with assigning

cross-resistance patterns to particular mechanisms.

Diagnosis of Resistance

A large number of laboratory bioassay methods (e.g., Figure 4(a))

have been developed for detecting and characterizing resistance.



Most of these are limited to defining phenotypes and provide

little or no information on the underlying genes or mech-

anisms. Thus, although bioassays remain the mainstay of most

large-scale resistance monitoring programs, much attention

has been paid to develop more incisive techniques that not

only offer greater precision and throughput but also diagnose

the type of mechanism(s) present and, whenever possible, the

genotypes of resistant insects. A variety of approaches are

being adopted for this purpose, including electrophoretic or

immunological detection of resistance-causing enzymes, kin-

etic and end-point assays for quantifying the activity of en-

zymes or their inhibition by insecticides, and DNA-based

diagnostics for mutant resistance alleles. The sensitivity that

these techniques can provide is exemplified well by work on

the aphid M. persicae, which in northern Europe possesses at

least three coexisting resistance mechanisms: (1) an over-

produced carboxylesterase conferring resistance to organo-

phosphates, (2) an altered AChE conferring resistance to

certain carbamates, and (3) target-site (kdr) resistance to

pyrethroids. These mechanisms collectively provide strong

resistance to virtually all available aphicides. Fortunately, it is

now possible to diagnose all three in individual aphids using

an immunoassay for the overproduced esterase (Figure 4(a)),

a kinetic microplate assay for the mutant AChE, and a DNA-

based diagnostic for the kdr allele. The combined use of these

techniques against field populations provides up-to-date in-

formation on the incidence of the mechanisms and is used to

alert growers to potential control problems.

Selection of Resistance Genes

The rate at which resistance genes are selected reflects the

combined influence of numerous biotic and abiotic factors.

Resistance offers several advantages for research to resolve

these factors and their interactions. First, the selecting agent

(exposure to insecticides) is well understood; it can usually be

carefully documented using treatment histories or manipu-

lated to investigate its effect on selection rates. Second, the

selective advantages conferred by resistance genes are often

very large, leading to substantial changes in genetic com-

position over a measurable time frame. Third, most of the

major mechanisms of insecticide resistance, unlike those for

many stress-related adaptations, are controlled by single genes

of major effect (monogenic) rather than many genes, each of

small effect (polygenic). This renders resistance more readily

amenable to analysis within the theoretical framework of

ecological genetics. Finally, the frequent availability of bio-

assays for quantifying the frequency of resistance phenotypes,

or even in vitro assays for specific genotypes, enables accurate

documentation of responses to selection applied in popu-

lation cages in the laboratory or under open field conditions.

Factors determining the selection (and hence risk) of re-

sistance to insecticides can, for convenience, be classified into

genetic or ecological ones relating to the intrinsic properties of

pests and resistance mechanisms and operational ones re-

lating to the chemical itself and how it is applied. Some of the

most important factors apparent from the large body of ex-

perimental and theoretical research on resistance selection are

summarized in the following three sections.

Genetic Influences

For resistance to evolve, resistance genes must confer a se-

lective advantage over their susceptible counterparts. One of

the primary challenges for describing resistance is therefore to

estimate the relative fitness of different genotypes under ex-

posure to insecticides. There are different ways of achieving

this, the most direct being to release individuals of known

susceptible and resistance genotypes into insecticide-treated

environments and to monitor their survival. This has been

done for a variety of pest species and has identified many,

often subtle, influences on how resistance genes are expressed

in the field. The dominance of resistance genes, which exerts a

major influence on selection rates, is a case in point. In la-

boratory bioassays evaluating the relative survival of suscep-

tible homozygotes (SS), heterozygotes (RS), and resistance

homozygotes (RR) over several insecticide concentrations,

dominance can be measured precisely, with RS individuals

usually responding in an intermediate manner. In the field,

dominance is a changeable phenomenon, depending on the

concentration of insecticide applied and its uniformity over

space and time. Even when the initial concentration is suf-

ficient to kill RS individuals (rendering resistance effectively

recessive), the weathering or decay of residues may result in

this genotype showing increased survival and resistance be-

coming functionally dominant in expression. When resistance

genes are still rare, and hence mainly present in heterozygous

condition, this can have a profound effect in accelerating the

selection of resistance genes to economically damaging

frequencies.

The diverse mating systems of insects also influence the

rate at which resistance evolves. Although most research has

focused on outcrossing diploid species (typified by members

of the Lepidoptera, Coleoptera, and Diptera), systems based

on haplodiploidy and parthenogenesis also occur among key

agricultural pests. In haplodiploid systems, males are pro-

duced uniparentally from unfertilized, haploid eggs, and fe-

males are produced biparentally from fertilized, diploid eggs.

The primary consequence of this, exemplified by whiteflies

(Figure 3(b)), spider mites (Figure 3(c)), and phytophagous

thrips, is that resistance genes are exposed to selection from

the outset in haploid, hemizygous males, irrespective of in-

trinsic dominance or recessiveness. Whether a resistance gene

is dominant, semidominant, or recessive, resistance can de-

velop at a similar rate under haplodiploidy, whereas re-

cessiveness can cause significant delays (initially at least) in

diploid populations.

Most species of aphid undergo periods of parthenogenesis,

promoting the selection of clones with the highest levels of

resistance or the most damaging combination of resistance

mechanisms. However, in holocyclic populations (ones that

alternate between sexual and asexual reproduction), this effect

is at least partially countered by genetic recombination and

the subsequent reassortment of mechanisms during sexual

reproduction. In fully anholocyclic (asexual) populations,

such as those of M. persicae in northern Europe, the influence

of parthenogenesis is much more severe and has led to strong

and persistent associations between resistance mechanisms

within clonal lineages exposed to a succession of different

selecting agents.


Ecological Influences

Several aspects of pest ecology, including the dynamics,

phenology, and dispersal capabilities of pest organisms,

act as primary determinants of resistance development.

However, their influence on selection rates can be un-

predictable without a sound knowledge of how they in-

teract with patterns of insecticide use. As an example,

movement of pests between untreated and treated parts of

their range may delay the evolution of resistance, due to

the diluting effect of susceptible immigrants. Conversely,

large-scale movement can also accelerate the spread of

resistance by transferring resistance alleles between

localities.

For highly polyphagous crop pests, interactions between

pest ecology and insecticide treatments play a particularly

critical role in determining selection pressures. Key factors

to be considered are the seasonality and relative abundance

of treated and untreated plant hosts and patterns of migration

between hosts at different times of the year. A good example

relates to the two major bollworm species (Lepidoptera:

Noctuidae) attacking cotton in Australia. Only H. armigera

(Figure 3(a)) has developed a strong resistance; H. punctigera,

despite being an equally important cotton pest, has remained

susceptible to all insecticide classes. The most likely explan-

ation is that H. punctigera occurs in greater abundance

on a larger range of unsprayed hosts than H. armigera,

thereby precluding a significant increase in resistance on

treated crops.

However, polyphagy can sometimes be deceptive. In the

cotton/vegetable/melon production systems of the south-

western USA, the highly polyphagous whitefly B. tabaci has

become a devastating pest and a primary target of insecticide

sprays. The consequences for resistance have varied sub-

stantially on a regional basis. Resistance problems have proved

much more severe and persistent on cotton in south-central

Arizona than in the extreme southwest of the state and the

adjacent Imperial Valley of California. This appears attrib-

utable to the higher proportion of unsprayed hosts, especially

alfalfa, acting as a buffer to resistance in the latter areas and

preventing any directional increase in the severity of resistance

over successive seasons. In south-central Arizona, these un-

treated refuges are much less abundant during the time that

cotton is treated with insecticides. Thus, a large proportion of

the local whitefly population is forced through a selection

‘‘bottleneck’’ on cotton and exposed to intense selection for

resistance, despite an abundance of alternative hosts at other

times of the year.

Enclosed environments, such as greenhouses and glass-

houses, which restrict migration and escape from insecticide

exposure under climatic regimes favoring rapid and continu-

ous population growth, provide ideal ecological conditions

for selecting resistance genes. Very low or zero damage toler-

ance thresholds for high-value ornamental or vegetable pro-

duce accentuate the problem by promoting overfrequent

spraying and hence intensify selection for resistance. Over the

years, these environments have proved potent sources of novel

resistance mechanisms for a diverse range of control agents

and have presented a particular challenge to attempts at

resistance management.

Operational Influences

Although closely linked to the aspects of pest genetics and

ecology, operational factors are best distinguished as ones

which, in principle at least, are at human’s discretion and can

be manipulated to influence selection rates. Factors exerting a

major influence in this respect include the rate, method, and

frequency of applications, their biological persistence, and

whether insecticides are used singly or as mixtures of active

ingredients.

Equating operational factors with selection is often dif-

ficult, because without a detailed knowledge of the resistance

mechanisms present it is impossible to test many of the as-

sumptions on which genetic models of resistance are based.

Anticipating the selection pressure imposed by a particular

application dose of insecticide is a case in point. If resistance

alleles are present, the only entirely nonselecting doses will be

ones sufficiently high to overpower all individuals, regardless

of their genetic composition, or ones sufficiently low to kill no

insects at all. The latter is obviously a trivial option. Prospects

of achieving the former depend critically on the potency and

dominance of resistance genes present. A pragmatic solution

to this dilemma is to set application doses as far above the

tolerance range of SS individuals as economic and environ-

mental constraints permit, in the hope that at least RS geno-

types will be effectively controlled. Even this approach can

backfire badly if resistance turns out to be more common

than suspected (resulting in the presence of RR homozygotes)

or resistance alleles exhibit an unexpectedly high degree of

dominance. Unless a high proportion of insects escape ex-

posure altogether, the consequence could then be to select

very rapidly and effectively for homozygous resistant

populations.

In practice, concerns over optimizing dose rates to avoid

resistance are secondary to ones regarding the application

process itself. Delivery systems or habitats promoting uneven

or inadequate coverage will generally be more prone to se-

lection for resistance, because pests are more likely to en-

counter exposure conditions under which selection is most

intense. This was elegantly demonstrated through experiments

assessing the relative survival of endosulfan-susceptible and -

resistant phenotypes of the coffee berry borer (Hypothemus

hampei) in coffee plantations treated with this chemical in

New Caledonia. The practice of spraying plantations from

roadsides with vehicle-mounted mistblowers generated gra-

dients in the concentration of endosulfan that resulted in

different selection pressures in different parts of each field.

Similarly, underdosing with the fumigant phosphine in in-

adequately sealed grainstores has been implicated as a primary

cause of resistance to this chemical in a range of stored

product pests.

The timing of insecticide applications relative to the life

cycle of a pest can also be an important determinant of re-

sistance. A good example relates to the selection of pyrethroid

resistance in the cotton bollworm, H. armigera, in Australia.

On cotton foliage freshly treated with the recommended field

dose, pyrethroids killed larvae up to 3–4 days old irrespective

of whether they were resistant or not by laboratory criteria.

As the sensitivity of larvae of all genotypes to pyrethroids was

found to decline with increasing larval size, the greatest


discrimination between susceptible and resistant phenotypes

occurred only when larvae achieved a threshold age. Targeting

of insecticides against newly hatched larvae, as is generally

advocated for bollworm control, not only increases the like-

lihood of contacting larvae at the most exposed stage in their

development but also offers the greatest prospect of retarding

resistance by overpowering its expression.

In practice, persistent insecticides are often essential to

ensure an acceptable period of control, especially when con-

tending with disease vectors or continued invasion of crop

pests from alternative host plants. However, persistent appli-

cations can accentuate resistance development by exposing a

larger number of individuals to the selecting agent. Another

problem is that residues of persistent insecticides decay or

become diluted through plant growth, so that resistant insects

may survive more effectively than they did at the time of ap-

plication. Empirical studies with a range of pests including

mosquitoes, bollworms, and blowflies have demonstrated that

aged deposits discriminate more readily between genotypes or

phenotypes than ones freshly applied.

In theory, the coapplication of two or more unrelated

chemicals as insecticide mixtures offers substantial benefits for

delaying the selection of resistance. The underlying principle is

one of ‘‘redundant killing,’’ whereby any individual already

resistant to one insecticide is killed by simultaneous exposure

to another, and vice versa. However, achieving this objective

requires not only that each type of resistance is still rare but

also that the ingredients confer mutual protection throughout

the effective life of an application. Failure to ensure that they

exhibit similar biological persistence may lead to one com-

pound exerting greater selection pressure than the other,

thereby accelerating the selection of doubly resistant pheno-

types. Two potentially conflicting challenges of choosing ideal

mixture partners are therefore (1) to ensure maximum simi-

larity in efficacy and persistence against the target pest(s) and

(2) to ensure maximum dissimilarity in chemical structure

and mode of action to minimize the likelihood of cross-

resistance. Difficulties with identifying candidate molecules

that meet all these criteria have greatly limited the use of

mixtures for combating resistance to conventional insecticides,

although they have considerable appeal for sustaining the ef-

fectiveness of insect-tolerant transgenic crops (see The Special

Case of Transgenic Plants).

Fitness Costs Associated with Resistance

Despite the advantages they confer under exposure to in-

secticides, it is often assumed that resistance genes also confer

physiological costs that could lead to counterselection when

insecticides are not applied. Some of the best examples of such

fitness costs come from studies conducted under harsh or

stressful environmental conditions, when even slight differ-

ences in relative fitness are likely to have major consequences

for the survival of genotypes. For example, resistant strains of

bollworms, blowflies, and aphids have all been demonstrated

to overwinter less successfully than their susceptible counter-

parts. Possible explanations for these fitness differentials in-

clude the reduced viability of certain life stages, a slower

reproductive rate rendering resistant insects more vulnerable

to adverse climatic conditions or to predation, or a reduced

ability to respond to environmental cues promoting survival.

In the aphid M. persicae, resistant individuals are less inclined

to move from senescing to younger leaves and are therefore

more vulnerable to isolation and starvation after leaf abscis-

sion. Many resistant aphids also appear less able to avoid at-

tack by parasitoids and predators – a more subtle side-effect

with interesting implications for combining chemical and

biological control strategies.

Fitness costs associated with resistance can be difficult to

demonstrate experimentally, since deleterious effects may only

be expressed under particular environmental conditions or be

conferred by other genes closely linked to the resistance locus.

The most convincing examples are ones in which costs have

been found consistently in resistant populations of diverse

geographic origin or have persisted after several generations of

back-crossing to susceptible insects in order to exclude linkage

effects. The potential for fitness drawbacks to be overcome by

a process of coadaptation, i.e., the integration of resistance

genes with other ‘‘modifier’’ loci that ameliorate fitness costs,

has also proved challenging to demonstrate. In a few cases,

however, repeated back-crossing of resistant insects to sus-

ceptible ones has led to seemingly fit resistance phenotypes

acquiring a fitness penalty, apparently due to the uncoupling

of resistance genes from modifier loci.

Combating Insecticide Resistance

In most studies of evolution, the primary challenge is to

identify selective forces and to interpret their effects on the

genetic composition of individuals and populations. With

insecticide resistance, it is also necessary to intervene in the

evolutionary process and find ways of reducing its deleterious

impact on pest management. Failure to do so in the past has

had many severe consequences, including the economic fail-

ure of cropping systems, the resurgence of insect-transmitted

pathogens, and damage to the environment by way of in-

creased insecticide applications.

The concept of insecticide-resistance management (IRM)

aims to address these concerns through the development of

control strategies for overcoming resistance to currently used

compounds, or preventing its appearance in the first place.

Although drawing extensively on the theoretical and empirical

framework that evolutionary biology provides, IRM strategies

must also contend with several practical, economic, and pol-

itical constraints on the choice of possible management tactics

and the precision with which they can be applied. The most

important of these are as follows:

1. The properties of any resistance genes present will often be

unknown, and knowledge of pest ecology may still be

rudimentary.

2. It is often necessary to contend with a whole pest complex

rather than just a single pest species.

3. There will often be a very limited number of insecticides

available for use in management strategies.

4. For highly mobile pests at least, countermeasures may

need to be standardized and synchronized over large

areas, sometimes whole countries.

5. Resistance is a dynamic phenomenon; i.e., any mech-

anisms already known to exist may change over time.


Continued monitoring is vital to determine whether

management recommendations remain valid or need to

be revised in light of changing circumstances or new

knowledge gained.

6. To promote compliance with management strategies, the

countermeasure adopted should be as unambiguous,

rational, and simple as possible.

A strategy first implemented on Australian cotton in 1983 to

contend with the bollworm, H. armigera, illustrates many

features of large-scale attempts at resistance management. It

was introduced in response to unexpected, but still localized,

outbreaks of pyrethroid resistance in H. armigera and was

based primarily on the concept of insecticide rotation. The

threat of pyrethroid resistance was countered by restricting

these chemicals to a maximum of three sprays within a pre-

scribed time period coincident with peak bollworm damage.

Farmers were required to use alternative insecticide classes at

other stages of the cropping season, in order to diversify the

selection pressures being applied.

Compliance with this strategy was excellent, and initially it

had the desired effect of preventing a systematic increase in the

frequency of pyrethroid-resistant phenotypes. Additional rec-

ommendations resulting from work on the ecological genetics

of pyrethroid resistance, including the targeting of insecticides

against newly hatched larvae (when even resistant insects can be

killed) and the plowing-in of cotton stubble to destroy resistant

pupae overwintering in the soil, undoubtedly contributed to

this success. Unfortunately, the restrictions placed on pyrethroid

use were inadequate to prevent a gradual, long-term buildup of

pyrethroid resistance. As a result, pyrethroids are no longer

considered reliable control agents for H. armigera, although they

remain highly effective against a coexisting species, H. puncti-

gera. The strategy has therefore been revised extensively to place

greater emphasis on distinguishing between the two Helicoverpa

species and on the strategic use of nonpyrethroids against H.

armigera. Transgenic cotton plants expressing Bt toxins have

since become the mainstay for controlling H. armigera in Aus-

tralia, and tactics for deploying these without selecting rapidly

for Bt resistance were implemented at the outset.

Another strategy incorporating a wide range of chemical and

nonchemical countermeasures was introduced on Israeli cotton

in 1987. This had the primary objective of conserving the ef-

fectiveness of insecticides against the whitefly, B. tabaci. Under

recommendations coordinated by the Israeli Cotton Board,

important new whitefly insecticides were restricted to a single

application per season within an alternation strategy optimized

to contend with the entire cotton pest complex and to exploit

biological control agents to the greatest extent possible. One

major achievement of this strategy has been a dramatic re-

duction in the number of insecticide applications against the

whole range of cotton pests, but especially against B. tabaci.

Sprays against whiteflies now average less than two per growing

season compared with more than 14 per season in 1986. Most

importantly of all, the strategy has generated an ideal en-

vironment for releasing additional new insecticides onto cotton

and for managing them effectively from the outset.

Given the threats posed by resistance to pest control,

resistance management has become an increasing pre-

occupation among crop-protection scientists, and also

pesticide manufacturers and bodies that regulate the release

and use of insecticides. Major agrochemical companies work

jointly to publicize resistance through the global Insecticide

Resistance Action Committee (IRAC). In the European Union,

resistance risk assessment is an integral part of approving new

products and maximizing their effective lifespan.

Resistance in Nonpest Species

Compared with its prevalence in arthropod pests, insecticide

resistance is still relatively rare among nonpest species in-

cluding beneficial organisms. However, it has been well

documented in a few species of hymenopteran parasitoids and

predatory mites, some of which are being exploited in inte-

grated pest management (IPM) systems. Its rarity among

beneficial organisms is probably due in part to difficulties in

locating hosts and prey (and hence surviving) under exposure

to insecticides. The likelihood of resistance developing in

beneficial arthropods may be increased if the insects they de-

pend on as prey are already resistant, although this requires

further research. It is also likely that, in comparison with

herbivorous species, the enzyme systems of predators and

parasites are less well adapted to detoxify xenobiotics.

The propensity for beneficial insects to evolve resistance

obviously depends on the degree of selectable variation within

their populations. Although laboratory selection has been

used to enhance low levels of pesticide tolerance found in field

populations, there is a concern that such selection applied at

artificially low doses will promote polygenic traits that could

fragment and dissipate if released into natural populations.

However, when substantial resistance has evolved naturally in

the field, its mechanisms have tended to be similar to ones

found in pest species. Organophosphate-resistant strains of

the green lacewing, Chrysopa scelestes, have been shown to

exhibit increased activity of acetylcholinesterase (AChE)

compared with susceptible insects. A carboxylesterase enzyme,

very similar in amino acid sequence to that conferring or-

ganophosphate resistance in the aphid, M. persicae, has been

cloned and sequenced from a malathion-resistant strain of the

parasitic wasp Anisopteromalus calandrae.

As the development of resistance is dependent on the

ecology of systems in which it appears, interactions between

beneficial and pest species will greatly affect the epidemiology

and dynamics of resistance in both. For example, some para-

sitoids of stored-grain beetles are resistant to insecticides, and

it is thought that this adaptation has been encouraged by the

fact that their parasitic larvae are sheltered from insecticides by

the grain kernels inhabited by their hosts. This is thought to

protect a substantial part of the insect life cycle from insecti-

cide selection and ensures that relatively small shifts in in-

secticide tolerance by the parasitoid afford significant

protection against the decreased insecticide doses that do

penetrate their defenses.

Natural enemies may contribute to retarding resistance in

pest species by exerting sufficient control to decrease the

number of insecticide treatments required. Conversely, there

are ways in which natural enemies could promote the adap-

tation of pests to insecticides. For example, the selection

pressure for resistance would be increased if weaker,


sublethally affected individuals were more easily preyed upon

or parasitized than their fully resistant and therefore un-

affected counterparts.

The Special Case of Transgenic Plants

A major development in crop protection with an important

bearing on resistance is the release of crop plants genetically

engineered to express genes for insecticidal toxins derived

from the microbe Bacillus thuringiensis (Bt). Bt cotton and corn

is already being grown commercially on a large scale in the USA,

Canada, Australia, Mexico, South America, China, India, and

South Africa. In 2006, the total area worldwide planted with Bt

crops was estimated to exceed 28 million ha. Existing toxin genes

in Bt cotton and corn are active specifically against certain key

lepidopteran pests (especially bollworms and corn borers);

another engineered into potatoes provides protection against the

Colorado beetle, Leptinotarsa decemlineata.

Aside from their commercial prospects, insect-tolerant

transgenic crops offer numerous potential benefits to agri-

culture. The incorporation of Bt genes into crops offers con-

stitutive expression of toxins in plant tissues throughout a

growing season. This could reduce dramatically the use of

conventional broad-spectrum insecticides against insect pests

as well as remove the dependence of pest control on extrinsic

factors, including climate and the efficiency of traditional

application methods. Estimates differ, but one study con-

cluded that conventional insecticide use between 1996 and

2005 fell by more than 35 million kilograms of active in-

gredient, roughly equivalent to the amount applied per year to

arable crops in the European Union. However, the high and

persistent level of expression also introduces a considerable

risk of pests adapting rapidly to resist genetically engineered

toxins. To date, there have been no examples of large-scale

control failures due to resistance selected directly by com-

mercial transgenic crops, but resistance to conventional Bt

sprays (selected in either the laboratory or the field) has been

reported in more than a dozen species of insect. Research on

the causes and inheritance of such resistance is providing

valuable insights into the threats facing Bt plants and the

efficacy of possible countermeasures.

Tactics proposed for sustaining the effectiveness of Bt plants

have many parallels with ones considered for managing re-

sistance to conventional insecticides. However, they are more

limited in scope due to the long persistence and constitutive

expression of engineered toxins and the limited diversity of

transgenes currently available. Indeed, for existing ‘‘single-gene’’

plants, the only prudent and readily implementable tactic is to

ensure that substantial numbers of pests survive in non-

transgenic ‘‘refuges,’’ composed either of the crop itself or of

alternative host plants. The stacking (pyramiding) of two or

more genes in the same cultivar, or possibly mixtures of culti-

vars expressing different single toxins, are potentially more

durable options for resistance management. Whatever measures

are adopted, it is essential that Bt plants (and their successors

expressing other transgenes) are exploited as components of

multitactic strategies rather than as a panacea for existing

pest-management problems, including those arising from the

development of resistance to conventional insecticides.

Concluding Remarks

Over the past 25 years, few areas of entomology have ad-

vanced as rapidly or received such widespread attention as that

of insecticide resistance. Research on this topic has provided

invaluable insights into the origin and nature of adaptations,

and these are in turn proving of much broader significance for

understanding genetic responses to manmade change in the

environment. In many respects the continuing battle against

resistance is analogous to an evolutionary ‘‘arms race,’’ in this

case pitting human ingenuity in discovering new toxins

against the adaptive capacity of pest species. Debates as to who

will eventually win this race are of secondary importance to

the realization that for many species the race is probably un-

necessary. A wider adoption of resistance-management prac-

tices, especially through greater exploitation of nonchemical

measures, would assist with reducing both the economic im-

pact of resistance and any deleterious effects of pest manage-

ment on biodiversity in general.

See also: Agricultural Invasions. Agriculture, Industrialized. Diversity,Molecular Level. Ecological Genetics. Ecotoxicology. Genetic Diversity.Indigenous Strategies Used to Domesticate Plants in Brazilian Amazon.Pesticides, Uses and Effects of. Population Genetics

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