occurrence, genetics and management of insecticide resistance

174 Parasitology Today, vol. 9, no. 5, 1993

References I Wemsdorfer, W.H. ( 1991 ) Parasitology Today

7, 297 303 2 Spencer, H.C. (1985) Trans. R. Soc. Trop. Meal

Hyg. 79, 748 758 3 Bloland, P.B. et al.J. Infect. Dis. (in press) 4 Carme, B. et al. (I 991 ) J. Infect. Dis. 164,

437 438 5 Koella, J.C. et al. (1990) Trans. R Soc. Trop.

Med Hyg. 84, 662-665 6 Guiguemde, T.R. et al. (1991) Ann. Soc. Beige

M~d Trop. 7 I, 199-207 7 Hoffman, S.L. et al. (1984) Trans. R. Soc. Trop.

Med Hyg. 78, 175-- 178 8 Rooth, I. and Bj6rkman, A. (1992) Trans. R.

Sac. Trop. Med. Hyg. 86, 479482 9 Khoromana, C.O. et al. (1986) Am. J. Trop.

Med. Hyg. 35, 46547 I 10 Greenberg, A.E. et al. (1989) Bull. WHO 67,

189 196 II Franco, S.M. (1991) Malaria Treatment.

A Review of Experience in Four Countries PRICOR Center for Human Services, 7200 Wisconsin Ave, Suite 500, Bethesda, Maryland 20814, USA

12 Sauerbom, R., Nougtara, A. and Diesfeld, H. (I 989) Sac. Sci. Med. 29, I 163 I 174

13 Foster, S. (1991) Soc. Sci. Med. 32, 1201 1218 t4 Trope, J.F. et al. (1987) Trans. R Soc. Trop.

Med. Hyg. 81 Suppl. 2, 3442 15 Ezedinachi, E.N., Ejezie, G.C. and Emeribe,

A.O. (1991) Cen. Afr.j. Meal 37, 16 20 16 WHO (1992) Guidelines for the Diagnosis and

Treatment of Malaria in Africa, WHO Regional Office for Africa, Brazzaville

17 USAID and CDC (1992) ACSI/CCCD 199~ 1991 Bilingual Annual Report, Centers for Disease Control, Atlanta, GA, USA

18 WHO (1990) Practical Chemotherapy of Malaria, WHO

19 Sudre, P. et al. (1992) Int. J. Epidemiol. 2t, 146-154

20 Campbell, C.C. (1991) J. Infect. Dis 163, 1207 1211

21 Paluku, K.M. et al. (1988) Trans. R %c Trop. Meal Hyg. 82, 353 357

22 Breman, J.G. et al. (1987) Am. J. Trap. Med. Hyg. 36, 469473

23 Sexton, J.D. et al. (1988) Am. j Trop. Med. Hyg. 38, 237 243

24 Soro, B. et al. (I 989) Ann. Trop. Meal Parasitol. 83. 101 106

25 Brandling-Bennet~, A.D. eta/, (1988) Trans. R. Sac. Trap. Med. Hyg. 82, 833- 837

26 Schapira, A. et al. (1988) Trans. R. Soc. Trop. Meal Hyg. 82, 39-43

27 Molineaux, L. and Gramicoa, G. (1980) The Garki Project, pp 153-157, WHO

28 WHO (1992) Global Malaria Control Strategy, WHO Document CTD/MCM/92.3, WHO

29 Shepard, D.S. et al. (1991) Trop. Med. Para- sitol. 42, 199 203

30 Sauerborn, R. et al. (1991) Trap. Med. Para- sitol. 42, 219 223

31 Sudre, P, Breman, J.G. and Koplan, J.P. (I 991 ) Lancet i, 772

32 Et%ng, M.B. and Shepard, D.S. (1991) Trop. Med. Parasitol. 42, 214 218

33 Brewster, D.R., Kwiatkowski, ©. and White, N.J. (1990) Lancet ii, 1039 1043

34 Schapira, A. (1989) Health Policy and Planning 4,17 28

35 Brinkmann, U. and Brinkmann, A. (199 I) Trop. Med. Parasitol. 42, 204 213

36 Luxemburger, C. etal. (1991) Lancet ii, 1268

Allan Schapira is at the Malaria Unit, Division of Control of Tropical Diseases, World Health Organization, Geneva, Switzerland. Peter Beales is at the Training Unit, Division of Control of Tropical Diseases, World Health Organization, Geneva, Switzerland M. Eliza- beth Halloran is at the Division of Biostatistics, Emon/ University School of Public Health, Atlanta, GA, USA.

Occurrence, Genetics and Management of Insecticide Resistance

R.T. Roush

A lot has been learned about insecticide resistance in the past 40 years. The problem is more extensive and widespread than could have been predicted. In this review, Richard Roush discusses the genetics and management o f resistance to insecticides (especially as related to arthropod vectors o f human and animal diseases), with the objective o f suggesting future directions.

The spectacular successes of DDT (dichlorodiphenyltrichloroethane) in suppressing arthropod-borne diseases among allied troops during World War II convinced many biologists that pest control had moved into a new era. In their enthusiasm, some even made bold predictions that certain scourges, such as the house fly, could be elim- inated from the face of the earth. Nonetheless, there were at least a few skeptics who noted that these pesticides often had deleterious effects on beneficial organisms and speculated that their effectiveness might be short- lived due to the evolution of resistanceL In fact, there were already about a dozen cases of resistance to inorganic insecticides, one dating back to 1908 (Refs I-4). Not even the first

cases of resistance to DDT in the late 1940s and early 1950s did much to dampen the enthusiasm of some chemists and their entomological col- laborators. So many new insecticides were being discovered that it was assumed that we could simply stay ahead of resistance. Resistance was not really a problem, but a new business opportunity for those who could find compounds to overcome it.

Unfortunately, the history of insecticide resistance prior to 1950 gave little inkling of how extensive the problem would become. With the widespread introduction of synthetic organic insecticides, the number of species of insects, mites and ticks showing resistance to one or more pesticides mush- roomed to more than 500 (Ref. 4). Resistance has been documented to virtually every kind of widely used toxi- cant, including arsenicals, sulfur, sulfur compounds, nitrophenols, phosphine fumigants, formamidines, organotins, selenium, insect growth regulators and the insecticidal crystals produced by Bacillus thuringiensis <5. In many cases, populations were resistant to novel insecticides even before they were introduced, due to the phenomenon of

cross-resistance, where a single resistance mechanism causes resistance to several compounds, sometimes even to some with a different mode of action. (Parasitologists often distinguish side-resistance, the result of selection with another compound of similar mode of action, from cross-resistance, referring to different modes of action 6, but in this article I have used the broader meaning of cross-resistance as typically used in entomology.)

Roughly 56% of the species now showing resistance are pests of crops, 40% are parasites of man or animals, and just 4% are beneficial species (eg. predators) 4. Although resistance in any one species may often be quite localized such that no species should be assumed to be universally resistant, there are about 50 key pests for which resistance is now a chronic problem across most of their geo- graphic ranges. Perhaps most important are Anopheles mosquitoes (Fig. I), which, due to their role in the transmission of malaria, should rightfully be considered the most dangerous animals in the world. However, almost all major groups of disease vectors (including flies, ticks, fleas, lice, bed

© 1993. Elsevier Science Publishers Ltd. (UK~

Parasitology Today, vol. 9, no. 5, 1993 175

bugs and mites) are represented among species documented to show resistance 4.

The main purpose of this review is not to lament the past, but to discuss the genetics and management of resistance to insecticides. In many cases, it is somewhat easier (and no less important) to identify myths about resistance than it is to describe what is known with certainty. Much data have been gathered and a clear goal is to put this information into practice.

Genetics of Resistance

Resistance mechanisms: At least in principle, insecticide resistance may occur through either behavioral or more strictly physiological means 7,8. However, very little is known about the significance of behavioral resistance in the field 3. A commonly cited example is the avoidance of pesticide residues by exophilic ('preferring to stay outdoors') behavior in Anopheles mosquitoes in Africa 8, but these obser- vations were made before Anopheles gambiae was resolved as a complex of sibling species, some of which are exophilic independent of resistance 3.

In contrast, specific genes controlling major mechanisms of physiological resistance to insecticides are relatively well described, and have often been mapped to a specific chromosomal region in flies and mosquitoes 9. The most important classes of resistance mechanisms are increased metabolism and decreased sensitivity of the target site. Decreased penetration of insecticide into the insect appears to be a common mechanism, but usually confers less than fivefold resistance 10.

There are three distinct kinds of target-site resistances. The most common is to cyclodiene insecticides (eg. dieldrin, endrin) and lindane, which can be found in at least 291 arthropod species 4, and seems similar in all species that have been studied in any detail ~0,~ ~. The mechanism of resistance, at least in two species of Drosophila, is a single amino acid substi- tution in a GABA receptor gene ~2.

Because organophosphorous (eg. chlorpyrifos, diazinon, malathion) and carbamate (eg. carbaryl) insecticides suppress acetylcholinesterase, it may not be surprising that resistance has been found to be due to a less sensi- tive acetylcholinesterase in several species, including mosquitoes, cattle ticks and the house fly. Many of these altered acetylcholinesterases have been

extensively characterized at the level of substrate specificity and enzyme kineticsl0.t 113

The pyrethroids and DDT apparently act on the sodium channels of the nerve axon. Although the specific bio- chemical change that confers resistance is still unknown, it is clear that many strains of insects and ticks are resistant to these pesticides due to changes in this target. In the house fly, the gene conferring relative insensitivity has been called kdr (standing for knock down resistance). Resistances that are at least superficially similar to kdr, perhaps most appropriately called kdr-like, have been found in many other species I°,11.13

Increased detoxication of many types of insecticides can occur from a variety of esterases, microsomal monoxygenases (also known as mixed function oxidases), and glutathione-S- transferases ~°. Enhanced oxidative metabolism appears to be a major mechanism of resistance for all insecticide classes except the cyclodienes ~. DNA sequences for the cytochrome P-450s involved in this mechanism have been identified for at least a few insect strains, but it is not yet clear whether resistance is due to changes in the structural gene or changes in regulatory genes that alter the amount of protein produced ~0,,3.

Esterases (or hydrolases) are important in resistance to organophosphorus insecticides and occasionally pyreth- raids (eg. permethrln). Some of these esterases result from gene amplification, particularly in Culex mosquitoes. So much esterase is produced in some insects (34% of the total protein) that at least part of the resistance is due to the pesticides being bound to the esterase and so prevented from reach- ing the target H. Thus, some pesticides are effectively sequestered by the enzyme ~ i.

Glutathione transferases are most important in the metabolism of organophosphorus insecticides. DDT dehydrochlorinase (or DDT-ase), one mechanism for the detoxication of DDT in house flies, mosquitoes, and a few other species of Diptera ~0, is prob- ably a glutathione transferase. A resistant house fly strain with high glutathione transferase activity has been found to have amplified DNA sequences not found in a susceptible strain d ~.

In general, most mechanisms of resistance tend to be co-dominant to fully dominant in expression (ie. heterozygotes generally show levels of resistance more similar to the resistant

than susceptible parents), but kdr-like resistances are generally nearly fully recessive 3, ~ 0

Number of genes controlling resistance: Levels of resistance sufiqcient to cause control failures in the field often evolve through a single gene 3,9, but whether resistance is nearly always monogenic in the field is controversial, at least in part because many classical inheritance studies were ambiguous ~s due to the lack of genetic markers. On the other hand, largely because laboratory colonies are generally too small to carry rare genes of major effect, there is evidence that laboratory selection generally leads to polygenic resistance, thereby producing resistant strains very atypical of those from the field 3,9.

Prior to the introduction of a novel insecticide, resistance alleles for the compound are assumed to be rare. Although the frequencies of resistance alleles have never been successfully measured prior to selection, it is generally believed that they are on the order of 10 6 or less 16, but this still leaves considerable doubt about where resistance alleles originate. One hypothesis is that resistance alleles are always present in populations prior to selection due to recurrent mutation3. 9. A con- trasting hypothesis posits that mutation rates for resistance alleles are so low that they are usually absent in most, or all, unselected populations at any given time, and that significant resistance can usually evolve only after a resistance allele arrives from another population by insect dispersal ('migration', as defined in population genetics).

In the only published survey that examined the molecular genetics of insecticide resistance in widely distrib- uted populations, it was discovered that a single amplified esterase-resist- once allele in Culex mosquitoes prob- ably originated from a single mutation which had subsequently spread around the world t7. It would be premature to extrapolate broadly from this one case, but at least in this example, pest dispersal was apparently a more readily available means of providing this kind of resistance than was local mutation. In a sense, not only was resistance monogenic, it was monoallelic.

Fitness costs of resistance: Resistance is generally assumed to have some costs in the absence of pesticide use, but the more critical question from a resistance-management standpoint is whether these costs are substantial enough to cause significant declines in the frequencies of resistance over a reasonably short time period 3,9. Even

176 Parasitology Today. vol. 9. no. 5. 1993

then, recent theoretical work suggests that fitness disadvantages are somewhat irrelevant to choosing specific resistance management tactics, as virtually all tactics are more effective when susceptibility has an advantage~8 20. Although relatively few studies have tested for fitness disadvantages under conditions as rigorous as the field, significant fitness disadvantages seem to be clearly associated only with a few specific resistance mechanisms. The most serious and consistent disadvantages seem to be due to esterases and glutathione transferases produced by gene amplification, cyclodiene insensitivity, and perhaps with kdr-like resistances to DDT and pyrethroids3.9.1621.22.

Resistance Management

Reduction in the number of treatments: There are a wide range of potential tactics for managing insecticide resistance t6, but the single most effective tactic to date has been to reduce the number of pesticide treatments made 16,18. Not only does this minimize the duration of selection, reducing the number of treatments often increases the fraction of a population that escapes exposure alto- gether. One of the most consistent dif- ferences between those cases where there are serious resistance problems and those where there are not is the presence of a large fraction of the population that escapes exposure in the latter. Reductions in pesticide use have often been achieved through 'Integrated Pest Management' (IPM) programs that seek to take a holistic approach towards regulating rather than eliminating pest populations, and use a combination of means where pesticides are intended as the last resort. Although pesticide users often complain that alternative controls are not cost effective, those who have suf- fered through serious resistance problems nearly always ultimately adopt alternatives, usually at the cost of lower efficiency and less flexibility. With respect control of disease vectors, there were well-established management programs before the introduction of insecticides, and many effective and creative tactics have been added in the past few years 23.

Avoiding high doses: it is widely thought that resistance often occurs primarily because low doses (appli- cation concentrations) of pesticides are used. This notion underlies recommen-

dations to manage resistance by high doses.

In theory, if the dose of insecticide delivered to the target is sufficiently high to kill heterozygotes (ie. those carrying RS alleles), the onset of resistance can be delayed since heterozygotes are the most common carriers of resistance while it is rare (ie. under Hardy-Weinberg conditions, the prob- ability of occurrence of heterozygotes will be 2pq and the resistant homozygotes p2 where p, the frequency of the resistance allele, is small and q is the frequency of the susceptible allele). Any resistant homozygotes that may exist are assumed to be most likely to mate with susceptible immigrants, thereby converting their offspring to heterozygotes susceptible to the dose used 16,t8,24. In practice, however, it is difficult to apply this strategy to exter- nal parasites and even more difficult to use it against pests of field or orchard crops. High-dose tactics suffer from environmental limitations on the doses needed, the difficulty of maintaining doses high enough to kill heterozygotes, the deleterious effects of pesticide residues on the inward migration of susceptible insects, and the difficulty of maintaining an untreated source for immigrants t6,18,24. High doses can be most effective only when resistance is still so uncommon that RR homozygotes are virtually too rare to exist in finite populations; there is very little advantage to a high-dose approach unless the resistance allele frequency is less than about 10 3. Unfortunately, the first reaction of most pesticide users when resistance appears is to increase the dose used. In addition, the dose applied must be sufficient to kill at least 95% of the heterozygotes to provide much benefit (Fig. 2). A serious and common problem for controlling the heterozygotes is uneven coverage and decay of pesticide residues, either of which can reduce mortality of the heterozygotes to less than 95% (Refs 18,24). Every pesticide residue must decay. If the rate of decay is slow com- pared to the recruitment of individuals into the population, there will inevitably be a residue that selects in favor of resistance, even if the initial concentration of pesticide is sufficient to kill all heterozygotes. The high-kill strategy also assumes that the dose necessary to kill resistant homozygotes can be known (perhaps by studies of strains that evolved resistance elsewhere) and that new alleles or altemative mechanisms will not appear to provide even higher levels of resistance 24.

On the other hand, it is also a persistent myth that resistance can be managed by low doses. In general, models suggest that a disadvantage of the low dose approach is that one must allow a large proportion of the treated individuals to survive if resistance is to be significantly delayed, which is not always practically accept- able 2s. There seems to be no resistance-management program where low doses in themselves are an effective part of the strategy ~8.

Pesticide rotations, mosaics and mixtures: Given two or more insecticides that are thought not to share the potential for cross-resistance (ie. have unique modes of action and metabolism), one could use each indepen- dently until it loses its effectiveness (sequential introduction), alternate their use either over time (rotations) or space (mosaics), or use them in combination (mixtures). Simple genetic simulation models and experiments suggest that rotations across generations (or where generations overlap, at some long time interval such as an entire season) will never be worse, and may sometimes (where there are high fitness costs) be much better than mosaics or rotations within a single generation 18. This can be illustrated with selection experiments on mosquitoes (Fig. 3).

Successful resistance-management programs often follow insecticide- rotation schemes ~6,t8, but rotation by itself shows little theoretical advantage over simply using the first insecticide until resistance occurs, and then substi- tuting the next one t9,2°. Rotational programs are often successful for other reasons: rotation can reduce the use of a favored insecticide, extending its useful life for the most critical applications 16, and may encourage the use of typically unfavored pesticides at those times when they can be adequate.

Whether one should rotate or mix insecticides depends on the circum- stances. Simulation models have been used to explore the conditions under which mixtures are likely to have a significant advantage; the key feature for the success of mixtures has been called 'redundant killing', which can be explained as follows. If resistance alleles at each of two separate loci exist in the population at very low frequency, it is extremely unlikely that any one individual will carry both alleles. Thus, insecticide A can kill individuals that are heterozygous for insecticide B and vice versa. As long as there is a significant fraction of the population that

Parasitology Today. vol. 9. no. 5, 1993 177

escapes all pesticide exposure, genes carded by the few resistant survivors will be greatly 'diluted', delaying the increase in resistance 2S. Because each insecticide is applied at a concentration that would have killed susceptible individuals if used alone, the pesticides are 'redundant' in killing fully susceptible individuals.

While redundant killing is the strength of a mixture approach, the failure to achieve nearly complete redundant killing dramatically weakens the benefits of mixtures ~8. For example, if pesticide A has significantly longer residue persistence than pesticide B, B will always benefit from redundant killing, whereas A will only benefit while B lasts. Although equal persistence has long been recognized as important to the success of mixtures, equal persistence alone does not maximize their benefits. As with the high-dose approach, it is even more critical that both pesticides have no

residue decay (because residues are either very short or very long) with respect to recruitment of unexposed individuals. Unfortunately, even a 5-10% loss in redundant killing, as measured by increased survival of individuals susceptible to either pesticide used alone (not to mention that the resistances should be somewhat recessive and that the genes controlling them not chromosomally linked), can reduce the effectiveness of mixtures from a greater than 1000-fold delay in resistance to less than a fivefold delay. Although the effects of pesticide decay on redundant killing are especially important to the effectiveness of pesticide mixtures, it is also important that the initial frequencies of resistance are low rs.

On the other hand, it is di~cult to find conditions under which mixtures are much worse than rotations, ignor- ing the adverse effects of multiple pesticides on costs, environment, and

biological control from natural enemies. The latter are especially important considerations in the kind of broadcast sprays that are necessary for control of pests in fields and orchards 26, but might be less relevant in medical and veter- inary applications, where both cost and residue decay might be more pre- cisely controlled 27. Perhaps the most- favored current tactic for managing mosquitoes and the diseases they carry is the use of insecticide-treated bednets 2s, which have the particular advantage that essentially only the female mosquitoes are exposed, leaving males as a refuge for susceptibility 20. Especially as resistance to pyrethroid insecticides seems to remain relatively uncommon among mosquitoes 28 (see Fig. I), mixtures of pyrethroids with some other insecticide would seem to be a very effective method for delaying resistance to treated bednets 20.

Choice of insecticide: Within any class of insecticides, there are nearly always

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178 Parasitology Today, vol. 9, no. 5, 1993

some chemicals that more effectively select for resistance either because they provide higher levels of resistance or because they are more persistent ~8. For example, the case could be made that because lindane provides lower resistance levels than true cyclodienes (eg. dieldrin)10,29, there would be fewer problems with cyclodiene resistance in mosquitoes today had dieldrin (and its close allies) been banned from use in favor of lindane 21. Although under- utilized, improved pesticide choice seems to have significant potential for the management of insecticide resistance 18, and could perhaps be used by establishing the pattern of cross-resistance to new classes of compounds in the very first cases where resistance occurs to them in the field.

Resistance monitoring: Resistance monitoring has been most effectively applied in testing continued pesticide efficacy and, in a few cases, the progress of an established resistance management program 16. Resistance is difficult to detect in natural populations on the basis of traditional LCso methods; until resistance has reached appreciable proportions, there may be little change in these values 3°,31, Thus, most programs now use diagnostic bioassays, thereby enhancing the detec- tion of resistance. Discriminatory doses also allow data collection in terms of gene frequencies, which more closely tie resistance monitoring to resistance models. Nonetheless, largely because of the expense of collecting and testing a sufficient number of individuals to detect resistance at low frequencies, insecticide resistance monitoring prob- ably cannot be used routinely to trigger the implementation of resistance management tactics once some detectable frequency has been reached, because the minimum detectable frequency is likely to be so high that control failures are imminent, especially where diagnostic assays are poor 32. In species that have shown a propensity for resistance, a more practical approach is to assume that resistance will occur and adopt a preventative strategy 3°,3~.

Interactions with agricultural pesticide use: Finally, there is evidence that in- advertant exposure to agricultural pesticide use can select for resistance in species such as mosquitoes 33. Although the impact of this selection in any given case is likely to be controversial, it is important to consider the possible implications of agriculture on vector control. It may well be true in some cases that control tactics for agricultural and medical pests should

be integrated, and that IPM in agriculture may improve human health both by direct reductions of pesticide exposure as well as in reducing resistance problems in medically important pests.

Conclusions and Recommendations

Wherever a pesticide has a specific mode of action, resistance by a single major gene, which may control either decreased sensitivity at the target or increased degradation of the insecticide, seems likely. The first and most important step in managing resistance is to reduce the fraction of the pest population that is exposed and the number of generations treated through the use of alternative non-chemical controls. The fitness disadvantages of resistance genes in the absence of pesticide residues vary with resistance mechanism, and are often too low to be counted upon to prevent resistance, but may help to delay resistance where there is competition with completely unexposed individuals, emphasizing the importance of untreated 'refuges' for susceptible individuals to reproduce. Resistance may be effectively managed for insect vectors of disease (though perhaps not for crop pests) through the use of carefully deployed insecticide mixtures. High doses of single components are less likely to be effective. Resistance management programs using insecticide rotations may help to manage resistance, but theory suggests that their effectiveness is not due to rotation alone.

It is still not unusual to hear the attitude voiced in many quarters that insecticide resistance is not really a problem because one new chemistry or another has just been introduced, or is about to be. Unfortunately, since we humans tend to be crisis oriented, it is very difficult to motivate policy makers in both the public and private sector to do much about resistance before it has occurred. The paradox is that once resistance has occurred, the frequencies are so high that most of the best options to manage resistance (especially mixtures) are no longer as effective. Although resistance management programs can always benefit from more information, we know enough now to take sensible steps for the management of resistance. Our problem is not so much a lack of knowledge as a lack of will to act. Evolution seems not to work via a

crisis mentality; we can rest reasonably assured that insect populations are preparing their solutions to our insecticides while we dither over what to do next.

Acknowledgement For the sake of brevity, I have often cited reviews or the most recent articles rather than the original work. My apologies go to the primary authors, especially George Georghiou, G.S. Mani, Hugh Comins, and Bruce Tabashnik. I thank Chris Curtis for providing an advance copy of a manuscript.

References I Metcalf, Ri. (1980) Annu. Rev Entamol. 25,

219 256 2 Georghiou, G.P. (1986)in Pesticide Resistance:

Strategies and Tactics for Management (National Academy Press, ed.), pp 14 43, National Academy Press

3 Roush, R,T. and Daly, J.C. (1990) in Pesticide Resistance in Arthrapods (Roush, R.T. and Tabashnik, B.E., eds), pp 97 152, Chapman & Hall

4 Georghiou, G.P. and Lagunes, A.T. ( 1991 ) The Occurrence of Resistance to Pesticides in Arthro- pods, Food and Agric@tural Organization

5 McGaughey, W.H. and Whalon, M.E. (1992) Science 258, 1451 1455

6 Roush, R.T. (1991) in Resistance of Parasites to Antiparasitic Drugs (Boray, J.C., Martin, P.J. and Roush, R.T., eds), pp 197 207, MSD Agvet, Division of Merck & Co.

7 Georghiou, G.P. (1972) Annu. Rev. Ecol. Syst. 3, 133 168

8 Lockwood, J.A., Sparks, T.C. and Story, R.N (1984) Bull. Entomol. Sac. Am. 30, 41 S t

9 Roush, R.T. and McKenzie, J.A. (1987) Annu. Rev. Entomol. 32, 361 380

I00ppenoor th , FJ. (1985) in Comprehensive Insect Physiology, Biochemistry and Pharma- cology (Vol. 12) (Kerkut, C.A. and Gilbert, L.I., eds), pp 73 I 773, Pergamon

I 5odedund, D.M. and Bloomquist, J.R. (1990) in Pesticide Resistance in Arthrapods (Roush, R.T. and Tabashnik, B.E., eds), pp 58 96, Chapman & Hall

2 ffrench-Constant, R.H. et al. Proc Natl Acad. Sci. USA 90 (in press)

3 ffrench-Constant, RH., Roush, R.T. and Carino, F.A. (1993) in Molecular Approaches to Fundamental and Applied Entomology (Oakeshott, J. and Whitten, bl,J., eds), pp I 37, Spnnger Verlag

14 Devonshire, A i . and Field, L.M. (1991) Annu. Rev. Entomol. 36, I 23

15 Tabashnik, B.E. (199 I) j. Econ. Entomol. 84, 703 712

16 Roush, R.T. and Tabashnik, B.E., eds (1990) Pesticide Resistance in Arthropods, Chapman & Hall

17 Raymond, M. et al. (1991) Nature 350, 151 153

18 Roush, R.T. (1989) Pesticide Sci. 26, 423 44 I 19 Curtis, C.F. (1987) in Combatting Resistance to

Xenobiotics (Ford, M.G. et al., eds), pp 150 161, Ellis Horwood

20 Curtis, C.F., Hill, N. and Kasim, S.H. (1993) Biol. J. Linn. Sac. 48, 3 18

2/ Rowland, M. (1991) Med Vet. Entomol. 5, 207 222

22 McKenzie, J.A. (1990) Aust. J. Zool. 38, 493 50 I

23 Curtis, C.F. (ed.) (}990) Appropnate Tech- nology in Vector Control, CRC Press

24 Tabashnik, B,E. and Croft, B.A. (1982)

Parasitology Today, vol. 9, no. 5, 1993 179

Environ. Entomot. 11, II 37-II 44 25 Curtis, C.F. (1985) Bull. Entomol. Res. 75,

259-265 26 Tabashnik, B.E. (1989)J. Econ. Entomol. 82,

1263-1269 27 $utherst, R.W. and Comins, H.N (1979) Bull.

Entomol. Res. 69, 5 t 9 537 28 Malcolm, C.A. (1988) Parasitology Today 4,

SI3 SI5

29 Rawlings, P. et al. (1981) Bull. WHO 59, 631 640

30 Roush, R.T. and Miller, G.L (1986) J. Econ. Entomol. 79, 293-298

3t ffrench-Constant, R.H. and Roush, R.T. (1990) in Pesticide Resistance in Arthropods (Roush, R.T. and Tabashnik, B.E., eds), pp 4 38, Chapman & Hall

32 Halliday, W.R. and Burnham, K.P. (1990)

J. Econ. Entomot. 83, I 151 I 159 33 Georghiou, G.P. (1990) in Pesticide Resistance

in Arthropods (Roush, R.T. and Tabashnik, B.E., eds), pp 183 202, Chapman & Hall

Richard Roush is at the Department of Ento- mology, Cornstock Hall, Cornell University, Ithaca, NY 14853, USA.

Modification of Arthropod Vector Competence via Symbiotic Bacteria

C.B. Beard, S.L. O'Neill, R.B. Tesh, F.F. Richards and S. Aksoy

Some of the world's most devastating diseases are transmitted by arthropod vec tars. Attempts to control these arthropods are currently being challenged by the widespread appearance of insecticide resistance. It is therefore desirable to develop alternative strategies to comp- lement existing methods of vector control. In this review, Charles Beard, Scott O'Neill, Robert Tesh, Frank Richards and Serap Aksoy present an approach for introducing foreign genes into insects in order to confer refractoriness to vector populations, ie. the inability to transmit disease-causing agents. This approach aims to express foreign anti-parasitic or anti-viral gene products in symbiotic bacteria harbored by insects. The potential use of naturally occumng symbiont- based mechanisms in the spread of such refractory phenotypes is also discussed.

Attempts to eradicate disease-transmit- ting insects by chemical means are being frustrated by the appearance of resistant strains ~. The common occurrence of insecticide resistance, coupled with environmental safety issues and the high cost associated with heavy insecticide usage 2 is generating interest in alternative methods for reducing vector-spread diseases. Genetic/recom- binant DNA technology is being inves- tigated as a means to insert disease resistance genes into plant genomes 3 or to reduce or modify the competence of insect vectors to transmit pathogenic agents 4, s

Insects that are susceptible or refractory to infection or to transmission of infectious microorganisms occur naturally in vector populations. Thus, if the genes encoding these refractory phenotypes could be identified 6 and spread, or if susceptibility genes could be altered, disease transmission would © 1993. Elsevier Science Publishers Ltd, (UK)

be affected. However, in many cases, naturally occurring refractoriness may have a complex genetic basis and therefore a simpler approach may be to introduce foreign genes expressing anti-parasitic or anti-viral products into insects 4. The goal is to interfere with differentiation, replication or transmission of infective agents in the insect.

In order to achieve this goal, a reliable method for introducing and expressing genes (transformation) in insects other than Drosophila is needed. In Drosophila, direct germ-line transformation can be accomplished routinely by the use of native transposable elements, ie. the P and hobo elements 7,8. The P element has the added advantage of being able to spread through Drosophila populations by virtue of the hybrid dysgenesis phenomenon it gen- erates 9,~°. In theory, any gene that could be linked to a P element would spread with that element into a naive population. Although P-element mediated germ-line transformation works efficiently in members of the genus Drosophila, attempts to use P elements as transformation vectors in other insect species have met with only limited success s,~o. It is possible that other transposable elements with wider host ranges will be found, but it still remains to be seen whether these would induce a mechanism similar to P-element mediated hybrid dysgenesis, which would enable them to spread into populations.

Here, we review another method for expressing foreign genes in arthropods by using permanently associated symbiotic bacteria. It is possible to exploit these naturally occurring bac- terial symbionts as vehicles for the introduction of foreign genes into insects instead of direct genome trans-

formation ~ u2. The desirable genes can be introduced, expressed and the products secreted in these simple bac- terial systems with greater ease than they can in complex eukaryotic insect tissues. These modified symbiotic bacteria can then be re-introduced into insect populations to replace their native counterparts.

Association of Symbionts with Insect Tissues

Arthropods that are dependent on restricted diets, such as blood or plant juice, commonly carry symbiotic microorganisms ~3 that are thought to provide nutritional supplements for their hosts (Box I). These bacteria supply nutrients such as amino acids, vitamins, nucleic acid bases and heme, and/or synthesize proteins needed by their nutritionally restricted host ~4. These symbionts are either localized extra- cellularly (eg. symbionts of Rhodnius prolixus in the midgut lumen ~S) or intracellularly [eg. 'Rickettsia-like organisms' (RLOs) found in the midgut epithelial cells or large bacteroids associated with midgut mycetomes of the tsetse fly~6,~7]. There are also organisms in the gonadal tissues of many arthropods (eg. Wolbachia-like bacteria) that are not mutualists. These bacteria form stable associations with their hosts by enhancing their own transmission through the expression of cyto- plasmic incompatibility ~8 or sex ratio distortion 19.

Symbiotic bacteria have, in many instances, co-evolved with their hosts to form highly specialized associations, thereby allowing their hosts to exploit their environment more fully and simultaneously anchoring the symbiont

occurrence, genetics and management of insecticide resistance

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