Veterinary Parasitology, 18 (1985) 155--166 155 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

MECHANISMS OF RESISTANCE TO CHEMICALS IN ARTHROPOD PARASITES OF VETERINARY IMPORTANCE

JAMES NOLAN

CSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag No. 3, Indooroopilly, Queensland 4068, Australia

ABSTRACT

Nolan, J., 1985. Mechanisms of resistance to chemicals in arthropod parasites of veterinary importance. Vet. Parasitol., 18: 155-166.

This review evaluates the significance, for ectoparasites, of the four resistance mechanisms that have emerged in arthropods as defence against chemicals used for their control. In particular the contrast of the relative importance of each mechanism in each of the major ectoparasitic pests of economic importance (sheep blowflies, mosquitoes and ticks) is discussed. Use is made of the experience gained in attempting to overcome these mechanisms to assess the consequences of resistance, and the chances for prolonging the useful l i f e of pesticides, by suppressing the mechanisms, as an alternative to seeking replacement compounds.

INTRODUCTION

During the last two decades considerable research effort has been directed

into concepts aimed at reducing the reliance on chemicals for the control of

ectoparasites on domestic animals. These have included the selection and use

of breeds of host animals which resist infestation by ectoparasites, and the

present interest being shown in the development of vaccines, which would

provide animals of al l breeds with at least partial immunity against such

infestations. Although these concepts wi l l hopefully form the basis for

ectoparasite control in the future, i t is l ikely that they wi l l constitute

only part of an integrated control strategy which wi l l s t i l l require a

chemical input for operative s tab i l i ty . Th is wi l l be particularly true in

cases where ectoparasites act as vectors of highly pathogenic organisms.

There is no doubt that the greatest threat to the input of successful

chemotherapy, as part of an overall strategy of ectoparasite control, is the

continuing emergence of new resistance mechanisms. These mechanisms, as well

as producing crises in parasite and disease contrel, ~hen they become

established in the population, also add considerably to the cost of finding

effective replacement chemicals.

In this review we shall consider how and to what extent such mechanisms

have affected the efficacy of chemicals used for the control of

0304-4017/85/$03.30 © 1985 Elsevier Science Publishers B.V.

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ectoparasites. Included are not only those pest species which are controlled

in order to maximise production from domestic animals, such as blowflies,

bit ing f l ies and ticks, but also those species which are known to transmit

diseases considered to be of veterinary importance, e.g. mosquitoes, the

established vector of equine encephalitis in America. Because of the

extensive l i terature, and many reviews, that have been published on the

chronology of resistance development in various species of ectoparasites, and

the degree and spectrum of these resistances, no attempt has been made to

cover al l facets of this subject. Rather pertinent examples have been

selected, from the many available, to i l lus t ra te the significance of various

mechanisms between and within species.

RESISTANCE MECHANISMS

Any chemical applied for the specific purpose of controlling an arthropod

species, by interfering with some biochemical or physiological syste~ to

produce a lethal effect, must pass through several obstacles before eventually

reaching its target as an active toxicant. Any change in the nature of these

barriers, or extent of their act iv i ty, can lower the effective concentration

of the toxic compound. Such a change may occur through a spontaneous chance

mutation, occurring either before or during the use of a certain pesticide,

producing i n i t i a l l y a few heterozygote individuals with this beneficial

characteristic. Selection, part icularly with lower than normal concentrations

of the toxicant, wi l l give individuals possessing this a l le le a survival

advantage, although a period of consolidation may be necessary before this

a l le le is combined with other characteristics to produce fitness in other

respects. As well, as a result of previous selection by a structurally

unrelated chemical, a resistance mechanism capable of affecting an entirely

new pesticide class may already exist in the population. I t wi l l be seen,

from later discussions, that such resistance al leles, once selected and

established in the population, persist and accumulate, even when selection

from the original chemical agent ceases. There is l i t t l e evidence to support

the theory, sometimes advanced, that the pesticide may act as a mutagen

causing alterations at the nucleotide level, which are expressed

phenotypically as a resistance mechanism.

Essentially four types of resistance mechanisms have been established:

a) Avoidance of contact with the pesticide, thus lowering the deposition of

toxicant.

b) Lower than normal rate of penetration of pesticide through the cuticle.

c) Increased detoxication of the pesticide, or a toxic metabolite where a

propesticide such as an organophosphorus (OP) compound is used.

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d) A change in sensi t iv i ty of the target s i te , as in an alteration to acetyl-

cholinesterase (ACHE) the target of OP tox ic i ty or to the nerve membrane,

the target of DDT and pyrethroid tox ic i ty .

Avoidance

This mechanism involves a behavioural characteristic of the arthropod pest

which enables i t to escape contact with a pesticide, or at least to avoid the

degree of contact ~hich would prove lethal to i t . The mechanism is of minor

importance in re la t ion to arthropods of veterinary signi f icance, although i t

has been recorded in mosquitoes (Brown and Pal, 1971) e.g. an Anopheles

albimanus strain which was more i r r i t a b l e , in the presence of DDT deposits,

than two other strains with which i t was compared.

Penetration

A lower rate of penetration of the toxicant through the cuticle has been

well known for many years as an important resistance mechanism in the house

f ly (Husca domestica). However, i t remains a re lat ively rare phenomenon in

ectoparasites. Apperson and Georghiou (1975) showed that less parathion was

absorbed in an organophosphorus (OP) resistant strain of Culex tarsal is . In

addition, McDonald and Wood (1979) suggested that reduced absorption of DOT

may have been part of the explanation for the lower ODT and DOE content found

internally in larvae of the two most resistant strains of Aedes aegypti they

examined. In the catt le t ick, Boophilus microplus, a species which has shown

outstanding ab i l i t y to develop and accumulate a variety of resistance

mechanisms, lowered rates of penetration of a pesticide have only recently

been documented and then only in a laboratory selected resistant strain

(Schnitzerling et a l . , 1982b). A lower rate of penetration of both cis and

trans isomers of cypemethrin was demonstrated in the Malchi pyrethroid

resistant strain compared with susceptible Yeerongpilly t icks.

Often this mechanism, when considered in isolat ion, appears to be of l i t t l e

significance because of the small differences involved between strains. In

the Culex case, the difference in penetration rates was 1.3-fold and in

Boophilus a maximum of 2-fold. However, as considered by O'Brien (1967), when

he coined the term "opportunity factor", the mechanism can have a "multiplying

ef fect" , when acting in association with other mechanisms, such as

detoxication or insensi t iv i ty . This effect is expressed by slowing the rate

of accumulation of the toxicant internal ly in an arthropod, to the extent

where the presence of an enhanced t i t e r of a detoxifying enzyme, or a more

e f f i c ien t mutant form can reduce the supply of toxic chemical to a sub-lethal

level.

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Detoxication

This mechanism has assumed particular importance in ectoparasitic Diptera

while i t is only of secondary significance, so far, in Acarina. Increased

metabolism of the chlorinated hydrocarbon DDT was identif ied as a contributing

factor to resistance in Aedes aegypti by several workers. Kimura and Brown

(1964) suggested that dehydrochlorination of DDT, as measured by DDE

production, was the principal resistance mechanism in certain American strains

of this species. McDonald and Wood subsequently (1979) found increased

metabolism of DDT to DDE to be an important factor in the resistance of f ive

strains of Aedes aegypti. Other work carried out by Rathor and Wood (1981)

indicates dehydrochlorination is a mechanism of greater significance in adult

Aedes than in larvae of this species. One must conclude from the results of

many studies conducted into DDT-resistance in Aedes aegypti that

dehydrochlorination is a major resistance mechanism in this species, although

often acting in conjunction with other non-metabolic resistance mechanisms.

In the only example of DDT resistance in acarines, where the mechanism was

investigated in detai l , Schnitzerling et al. (1970) could find no evidence

that increased production of DDE, or of any other metabolite, contributed to

this resistance.

In respect of OP compounds, detoxication is of considerable significance

again in mosquitoes, is the only apparent resistance mechanism elucidated in

blow f l ies (Hughes and Devonshire, 1982) but again is of relat ively minor

importance in ticks. With this group of compounds, detoxication of either the

parent compound, or of i ts oxon, a toxic metabolite, occurs by hydrolysis,

dealkylation or oxidation. Increased detoxication of malathion in Cu!ex

tarsalis (Matsumura and Brown, 1961), fenthion in Culex pipiens fatigans

(Stone and Brown, 1969), parathion in Culex tarsalis (Apperson and Georghiou,

1975) and chlorpyrifos in Culex pipiens quinquefasc!atus (Georghiou et a l . ,

1975) appears to be the sole reason for resistance for al l the compounds

studied, with the exception of the parathion study where penetration was also

implicated.

In their work with the Australian sheep blow f ly (Lucil ia cupr!na), another

ectoparasite of considerable significance, Hughes and Devonshire (1982) found

that in the diazinon resistant Q strain, resistance was due mainly to an

enhanced ab i l i ty in vivo of this strain to detoxify the toxic oxon derivative

of the thionate parent compound. Although a mixed function oxidase system was

implicated as a secondary resistance mechanism, responsible for enhanced

degradation of the phosphorothionate.

In the catt le tick detoxication appeared for the f i r s t time as a mechanism

of resistance against the OP class of tickicides and then only after

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insensit iv i ty had featured strongly in the resistance of this pest to this

particular class. Roulston et al. (1969) showed that in the Mackay strain of

the catt le tick detoxication represented i n i t i a l l y the sole mechanism.

Subsequently in the further evolution of this strain, AChE insensit ivi ty was

to develop as well and provide, along with detoxication, a jo int resistance

mechanism. A similar situation was found to exist later in the Gracemere and

Mt Alford strains of ticks where detoxication was found to supplement

Ridgelands and Biarra type insensitive AChE respectively. Subsequently in the

Bajool and Tully strains, which emerged in 1972, increased detoxication, of

coumaphos and chlorpyrifos respectively, was established as a factor in

resistance while a year later the Ingham strain was shown to have an increased

capability to detoxify both of these compounds.

Synthetic pyrethroids have in many areas replaced OP pesticides for

mosquito control and amidines for tick control. Although cross-resistance

from a previous mechanism selected during widespread use of DDT is the major

cause of resistance to pyrethroids, there have been several cases recorded

where detoxication of pyrethroids has been found. Based on evidence from the

use of piperonyl butoxide as a synergist, Prasittisuk and Busvine (1977)

suggested detoxication by microsomal oxidases contributed to resistance in a

Guyana strain of Aedes aegypti. Similarly, Chadwick et al. (1977), using

again a synergist technique, found evidence for detoxication of bioresmethrin

as a resistance mechanism in Aedes aegypti from Bangkok. In a laboratory

selected strain, the pyrethroid-resistant (Malchi) Boophilus microplus,

detoxication of permethrin and cypermethrin isomers, has been proven to be an

important potential resistance mechanism over and above nerve insensit ivi ty or

kdr type cross resistance selected by previous use of DDT (Schnitzerling et

a l . , 1982). Further work demonstrated that a carboxyesterase was responsible

for this increased hydrolysis of the pyrethroids (de Jersey et a l . , 1985).

Insensit ivi ty

The outstanding difference between resistance mechanisms in the

ectoparasitic Diptera and Acarina is the significance of detoxication in the

former group compared with alterations in sensit ivity of the target site, the

predominant mechanism with al l chemical classes for the lat ter .

Certainly the ab i l i ty to tolerate higher internal levels of DDT has been

documented in mosquito larvae (Brown and Pal, 1971; McDonald and Wood,

1979). I t has been assumed rather than proven, in the absence of evidence of

other mechanisms, that this target site insensit ivi ty is similar to that

documented as "kdr" resistance in houseflies. In certain mosquito strains

resistant to DDT, this insensit ivi ty has occurred in association with

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increased metabolism of DDT to DDE as a j o i n t cause (Brealey et a l . , 1984), in

others i t apparently has been the sole mechanism (Plapp et a l . , 1965a). A

s im i l a r assumption was made by Schni tzer l ing et a l . (1970) who, through

detox icat ion studies in v ivo, e l iminated the p o s s i b i l i t y of advantageous

metabolism of DDT as the basis for resistance in the ca t t l e t i ck .

The contrast of resistance mechanisms between f l i e s and mosquitoes on one

hand, and t icks on the other, became p a r t i c u l a r l y apparent in the era of OP

resistance in these species. Whereas in mosquitoes only one s t ra in of

Anopheles albimanus (Ayad and Georghiou, 1975} has been shown to possess a

mutant acety lchol inesterase with decreased s e n s i t i v i t y to cer ta in OP

i nh i b i t o r s and one repor t , (Schuntner and Roulston, 1968) pointed to the

presence of AChE of decreased s e n s i t i v i t y in the thorax but not the head of

OP-resistant L u c i l i a , th is mechanism predominates in OP resistance in t i cks .

AChE i n s e n s i t i v i t y in t icks was documented f i r s t by Lee and Batham in 1966.

Subsequently a to ta l of nine OP res is tan t s t ra ins of Boophilus emerged in

Aust ra l ia between 1963 and 1973 and decreased s e n s i t i v i t y of ACHE, as

expressed to the oxons of the i nh ib i t o r s coumaphos, diazinon and ch lo rpy r i fos ,

was present in a l l .

A considerable amount of research has been conducted into the biochemistry

of the insens i t ive AChE resistance mechanism. Much information has been

accumulated on the st ructure a c t i v i t y re la t ionsh ips between the various

aberrant forms of the enzyme and a series of i nh ib i t o r s and substrateso The

ra t iona le for th is e f f o r t was for the c l a s s i f i c a t i o n of s t ra ins , to pred ic t

sa t is fac to ry a l te rna t i ve chemicals for t i ck control from wi th in the an t i -

chol inesterase group, and to determine whether a rat ional attack could be made

on the resistance mechanism through st ructura l manipulation of the compounds

af fected. The work culminated eventual ly in the e luc idat ion of the changes in

the enzyme (ACHE) i n h i b i t o r (OP) react ion which form the basis for the

mechanism (Schni tzer l ing et a l . , 1982a; Nolan and Schni tzer l ing, 1985).

F ina l ly in re la t i on to i n s e n s i t i v i t y as a resistance mechanism one must

include the recent ly developed amidine resistance in the ca t t l e t i c k . The

mechanism has not been explained but the lack of any evidence of increased

detoxication of amitraz, chloromethiuron or cymiazole, the broad spectrum of

affect and the high level of resistance factors for al l amidines leads one to

infer that a change in sensi t iv i ty of the target is most l i ke ly .

CONSEQUENCES AND COUNTERMEASURES OF RESISTANCE MECHANISMS

The consequence of a newly emerged resistance mechanism may appear to be

obvious as being a control fa i lure for the compound, which selected the

mechanism, against the species involved. However wide variations exist in the

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seriousness of the mechanism in terms of loss of efficacy and the spectrum of

effect between and within chemical groups and between species. I t is worth

considering these points in relation to their significance for the various

chemical control agents such as arsenic, the chlorinated hydrocarbons and

cyclodienes, the anti-cholinesterase OP's and carbamates, the amidines and the

contemporary synthetic pyrethroids. Although the era of many of these

compounds has passed, their resistance mechanisms, and the effects of them,

s t i l l persist in the pest populations. Th i s consideration in particular

allows a review also of the suggestions and strategies that have been put

forward to suppress various mechanisms. With ticks and blowflies the

consequences of resistance have had greater impact in the f ie ld because,

chemical control of these veterinary ectoparasites is practised widely to

al leviate the damage inf l ic ted by the pests per se. With mosquitoes, the

organism or parasite carried is usually the target of control programs.

Arsenic resistance, the f i r s t type to be recognized in ticks (Du Toit,

1941), is a simple case of a specific mechanism which so far has had no affect

on subsequent classes of tickicides - a result probably arising from the

relat ively simple chemistry of the compound and its unique mode of action,

which is most l ike ly inhibit ion of the vital function of the sulphydryl

group. Although the biochemistry of the mechanism is s t i l l in doubt no ef for t

was devoted in attempting to combat the mechanism or to eradicate the

strain. Fortunately the chlorinated hydrocarbon DDT was developed meanwhile

and was available as an alternative.

A similar situation in relation to lack of affect on other pesticides

appeared to apply when DDT resistance emerged in ticks (Legg et al . , 1955)

and in 1947 in mosquitoes (Brown and Pal, 1971). In ticks i t was established

that the mechanism did not affect the organochlorine alternative BHC (Stone,

1957) or the imminent release of OP's as tickicides. However, an observation

made at that time by Whitehead (1959) of cross-resistance between DDT and

pyrethrum was to have a significant effect on the use of synthetic pyrethroids

for tick control some twenty years later. The only resistance management

strategy attempted with ticks was to double the DDT use concentration from

0.5% ~/v p.p. isomer to 1.0%. The loss in efficacy generally would have been

tolerated and use of DDT continued for tick control, i f i t were not for the

release of the OP tickicides and the ban on DDT because of meat residue

problems. DDT resistance was never considered to be a problem in the control

of sheep blowfly or buffalo f ly .

In mosquitoes, probably because of the usefulness of DDT, the lack of

alternatives with low mammalian toxic i ty, and the eminence of detoxication as

a resistance mechanism, ways of combatting the mechanism were studied and

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ident i f ied. The analog deutero-DDT was found to be highly toxic to DDT-

resistant Aedes ae~ypti (P i l la i et a l . , 1963) apparently due to the

speci f ic i ty of the detoxifying enzyme DDT-dehydrochlorinase (DDT-ase) in this

species. However the expense in producing analogs of this type precluded

their commercial use. Chlorfenethol was f i r s t established as a synergist for

DDT-resistance in houseflies on the basis of being an effective DDT-ase

inh ib i to r (March, 1952). However the use of this compound, and alternatives

such as DMC, have largely been relegated as aids of research workers

investigating the mechanism of DDT resistance. A similar situation applies to

the use of mixed function oxidase inhibi tors, such as sesamex and piperonyl

butoxide, as microsomal oxidation has been of minor significance in DDT-

resistant mosquitoes.

BHC resistance in ticks appeared rapidly after i ts introduction (Whitnall,

1952). Probably the most signif icant consequence of this mechanism, s t i l l

unidentif ied in any species, was the to l l i t took on other compounds under

development as pesticides at the time. Cross-resistance meant that toxaphene

and dieldr in were rendered useless for t ick control.

The most complex patterns of resistance effects and mechanisms yet

encountered in ectoparasite control, between and within species occurred in

the era of OP use. This was also the era when considerable research e f for t

was expended into overcoming the mechanisms and documenting their spectrum of

effect. I t consequently provides us with some of the best examples not only

of the consequences of resistance but of the chances of successfully

combatting i t .

OP resistance in t icks, which f i r s t emerged in 1963 and reached i ts climax

with the advent of the Mt Alford strain in 1970, the highest and broadest-

based resistance recorded, provides a good example of the importance of

thorough characterisation of resistance mechanisms in understanding the

spectrum of their effect and in maximising the use of one chemical class.

Each of the nine resistant strains characterised had an incomplete spectrum of

effect on OP's and carbamates with the result that control could s t i l l be

achieved by selection of other compounds from ~4ithin the anti-cholinesterase

group. This research led to the continued effective use of the OP's and

carbamates for t ick control for many years after resistance was f i r s t

reported. This was achieved in some instances by a progression to other OP's

or carbamates and in other cases through increased concentration of the

compound affected, (Nolan and Roulston, 1979). A further strategy, described

in this review, of eliminating the resistance al le le from the population, by

the use of treatment with the compound to which resistance had developed

proved to be a feasible alternative.

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Although detoxication of OP's and carbamates developed some importance as

a supplementary mechanism to insensit ivi ty in ticks, l i t t l e was done in

attempting to restore act iv i ty by inhibit ing the detoxication process.

Schuntner et al. (1974), demonstrated the potential for synergism of carbaryl

with piperonyl butoxide but presumably formulation and cost prevented any

commercial acceptance of this finding.

The extensive AChE biochemical studies achieved the aim of elucidating the

mechanism of insensit iv i ty. Essentially i t was discovered that AChE in ticks

occurs as two components distinguishable on the basis of inhibi tor

sensit ivi ty. One of these components, as a result of a series of mutations,

has given rise to altered AChE which in i ts several forms is the basis of the

different levels of sensit ivity to OP's associated with the different

strains. The cause of the decreased sensit ivity is largely due to change

which decreases the a f f in i ty of the enzyme for the inhibitor. However the

complexity of these fidings in relation to the differences between the strains

thwarted the ultimate practical aim of developing concepts of design of

chemicals to overcome resistance. Partly also the ticks themselves defeated

the aim by adding detoxication as a resistance mechanism, while the work was

in progress.

I t is worthy of note in relation to OP resistance in ticks that on present

evidence, as noted by Nolan and Schnitzerling (1985), one cannot with

confidence assume that a similar successive series of strains, with similar

• resistance patterns, wi l l emerge in other countries where OP resistance has

not progressed or degenerated to the same extent i t has in Australia.

Certainly where the pattern is repeated the experience wi l l form a valuable

guide for the selection of alternatives.

In the sheep blowfly Lucilia cuprina detoxication of OP's as noted above

has emerged as the predominant resistance mechanism and unfortunately in

contrast to ticks, exerts a wide spectrum of effect within the OP group

(Shanahan and Hart, 1966). In both this species and in the mosquito, the two

alternatives in such a case, synergism or seeking replacement compounds have

been given considerable attention. Hughes (i982) showed the potential of DEF

as a synergist for diazinon in sheep blowfly control and the carboxylesterase

inhibitors EPN (Matsumura and Brown, 1961) tri-phenyl phosphate and IBP (S-

benzyl O,O-diisopropyl phosphorothionate)(Hemingway and Georghiou, 1984) have

all been shown to be effective synergists for malathion against resistant

strains of mosquitoes. Despite these results synergism has had l i t t l e effect

in providing an answer for OP resistance in f l ies or mosquitoes.

The only other attempt at a chemical counter-attack in OP resistance was

undertaken ~ Plapp et al. (1965b) who examined the potential of alkyl and

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carboxymethyl analogs of malathion as substitute compounds for the control of

malathion resistant Culex tarsal is . Certainly the isopropylalkyl analogs

showed promise but were not u t i l i sed. The l ike ly reason being the problems

often associated with innate mammalian tox ic i ty of higher alkyl OP analogs.

Amidines have been, and are, almost exclusively used as t ickic ides. The

emergence of high-level resistance to this group is a re lat ively recent

occurrence. The early history of use provides examples of low level

resistance which did not necessitate a hasty change to a new class. Amidine

resistance was known in Australia in f ie ld samples from 1977 onwards, was

d i f f i c u l t to define in laboratory tests, and only became obvious in the

treatment of infested catt le when such treatments were carried out below

normal recommended concentration. No action was taken and the widespread use

of the class was preserved for another three years, unti l ult imately severe

resistance ensued with a wide spectrum of effect on al l compounds of ,this

structure.

L i t t l e can be said of the consequences of pyrethroid resistance mechanisms

affecting ectoparasite control. Fortunately, at this stage, the only

signif icant mechanism is that resulting from previous DDT use. I t is not

surprising, in retrospect, that the "kdr" mechanism, preserved from the era of

DDT use, has had such an effect on the use of these highly active compounds

for the control of t icks, buffalo f l ies and mosquitoes. The successful

synergism of the pyrethroids with OP compounds has alleviated the problem for

certain pyrethroids in respect of t ick control. However i t must be remembered

that this synergism, through inhibi t ion of carboxyesterases, constitutes an

example of increasing the act iv i ty of the pyrethroid generally for al l strains

rather than defeating a detoxication resistance mechanism.

I t is apparent, from the foregoing discussion, that detailed understanding

of resistance mechanisms has not enabled us to restore the act iv i ty of a

chemical, where efficacy has been reduced to an unsatisfactory level by an

arthropod's defence mechanism. I t has however provided us with a rational

basis for the selection of suitable alternative compounds. The information

has also been of considerable assistance to the chemist and toxicologist

seeking guidelines in the search for new classes of compounds for future use

in ectoparasite control.

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