SCOPE 49 - Methods to Assess Adverse Effects of Pesticides on Non-target Organisms

15.1 INTRODUCTION

What are the genetic changes that result from the use of pesticides? In populations of many pests and some non-target organisms exposed to lethal doses of insecticides, resistance has developed, so that rare genes conferring decreased susceptibility to a pesticide become common genes in the population, due to selection. In many cases, susceptibility becomes very rare after many years of selection against it, so that even in the absence of the pesticides, resistance often remains in the population for many generations. This phenomenon is genetic change on the population level. Although certain pesticides are known to be mutagenic, it is unknown whether the rare alleles for resistance in the field have been produced through mutation caused by any pesticide.

Resistance in pests is generally an adverse consequence of pesticide use, and it has resulted in the loss of many formerly efficacious pesticides; however, in some cases resistance can be viewed as having benefit. In non-target organisms, resistance can be beneficial, as in resistant predatory mites, which have been exploited for their biological control ability in the presence of pesticides in orchards. Herbicide resistance genes found in weeds can be genetically engineered into crops that were formerly intolerant, so that the range of crops for which a particular herbicide is useful can be expanded. Resistance is a major factor reducing DDT use, which has been beneficial to certain species of wildlife. The recognition and anticipation of resistance serves as a stimulation for the discovery of more diverse pesticide chemicals.

The serious threat is that the development of new pesticide chemicals and the discovery of new targets in the pests might not keep pace with the evolution of resistance. While there are approximately 2000 active pesticide ingredients, they act upon only about 25 physiological target sites, most of which have been relied upon for many years. Uncontrollable pests will be of great consequence in agriculture and in public health. The objectives of this chapter are to review the status of resistance, to discuss the ways in which it develops, and to consider methods for assessing the hazards of resistance in the laboratory and in the field.

Certain pesticides are mutagenic (Klopman et al., 1985); therefore, they could possibly cause change to the genetic code directly without lethality or population selection. These mutagenic changes may be heritable under certain circumstances, and they may be very difficult to detect. Mutagenicity of pesticides is of great concern, since the widespread use of pesticides in agriculture, in household pest control, and in public health leads to human and animal exposures on several levels, including residual amounts on agricultural products (for which legal tolerances have been established). The mutagenetic risk of pesticides is difficult to estimate, since mutagenic effects are often observed in the laboratory at concentrations much greater than those to which the public is exposed. The emphasis of this chapter is on resistance as a consequence of pesticide use. Mutagenicity is considered herein only as it relates to understanding the genesis of resistance genes.

15.2 STATUS OF PESTICIDE RESISTANCE

Resistance to pesticides has been established as a capability of a wide variety of pest species (Georghiou and Saito, 1983; Georghiou, 1986). Resistance has had the greatest impact on insecticides and acaricides (Brattsten et al., 1986), but it is gaining in importance as more critical situations are detected for fungicides (Georgopoulos, 1986; Köller and Scheinpflug, 1987), herbicides (LeBaron and Gressel 1982; LeBaron and McFarland, 1990) and rodenticides (MacNicoll, 1986).

15.2.1 INSECTICIDES AND ACARICIDES

Resistance in arthropods is already a serious practical problem with major impact on public health and agricultural pest control. Over 490 species of arthropods are resistant (Georghiou, 1986), and this includes most of the major pests of agriculture and public health. In the last decade, there has been an alarming increase in resistance to organophosphorus, carbamate, and pyrethroid insecticides, so that many pests have developed resistance to multiple major classes of insecticides. The registration and use of insecticides of greater intrinsic toxicity has been a consequence of, and not a solution to, this problem. Approximately 50 species have resistance to photostable pyrethroid insecticides, which were marketed just 10 years ago (Brown, 1982; Georghiou, 1986). The first practical problem in the field with full documentation and surveillance data was the devlopment of fenvalerate resistance in the diamondback moth on cole crops near Taipei (Liu et al., 1981, 1984).

Table 15.1. Arthropod pests in which resistance has led to serious difficulty in control in many areas (adapted from Voss, 1987) 

In 1987, the pesticide manufacturing industry noted that approximately 40 species of arthropods had developed resistance that caused serious practical difficulty for their control in many areas (Table 15.1). These were referred to as Category 1 species in regard to the seriousness of resistance; it was recommended that they be studied intensively (Voss, 1987). Additionally, 33 species were listed as Category 2 speciesthose in which resistance is of practical consequence in some, but not many, areas; careful monitoring of them was recommended, since the potential for more serious resistance exists. It is clear that only rapid advances in pesticide resistance management will prevent the increasingly serious impact of resistance in arthropods.

Table 15.2. Resistance to 12 types of herbicides in 64 species of weeds (LeBaron and McFarland, 1990)

15.2.2 HERBICIDES

Herbicide resistance has been observed in the field and confirmed in 64 species of weeds (Table 15.2). Although the initial cases of herbicide resistance were considered as rather isolated curiosities, the problem is intensifying in that certain species are spreading, acquiring multiple resistances, and threatening major crops (LeBaron and McFarland, 1990). Triazine resistance has evolved in 53 species of weeds; however, it is clear that other classes of herbicides are inducing resistance at an increasing rate. Trifluralin resistance in cotton-field goosegrass (Eleusine indica), has spread in the US from South Carolina (Mudge et al., 1984) to several neighbouring states, and has shown cross-resistance to oryzalin and phosphoric amide herbicides (Vaughn et al., 1987). In Australia, the annual ryegrass Lolium rigidum has developed resistance to dinitroaniline, sulphonylurea, and aryloxyphenoxyproprionate herbicides, so that there are no herbicides registered that control multiple resistant populations of this type in wheat (Heap and Knight, 1982, 1986; Powles, et al., 1990). Black-grass (Alopecurus myosuroides) has shown resistance to chlortoluron, diclofopmethyl and other herbicides in the United Kingdom (Kemp et al., 1990).

15.2.3 FUNGICIDES AND BACTERICIDES

Fungicide resistance has been reported in at least 90 species of pathogens (Table 15.3). There are 59 species with benomyl resistance and 22 with resistance to other benzimidazole fungicides. Resistance to other single-site inhibitors is less common, and resistance to sterol biosynthesis inhibitors of the C-14 demethylation step has been slow to develop (Locke, 1986; Köller and Scheinpflug, 1987).

Benzimidazole resistance is of practical importance; however, it is fortunate that substitute chemicals are available for the majority of cases so that the problem is not as serious as the Category 1 arthropod resistance to insecticides. Multiple resistances may be of most immediate consequence in grey mould from Botrytis cinerea, powdery mildews caused by Erysiphe cichoracearum, E. graminis, or Sphaerotheca fuligenea, peach brown rot from Monilinia fructicola, and apple scab due to Venturia inaequalis. Resistance to copper was widespread in recent surveys for plasmid-borne resistance genes in certain strains of plant pathogenic bacteria (Cooksey, 1990).

15.2.4 RODENTICIDES

Rodenticide resistance has been limited to anticoagulants, mainly warfarin. Warfarin resistance in Rattus norvegicus is widespread in Europe and North America (Jackson and Ashton, 1986). Warfarin resistance is also common in the house mouse (Mus musculus) and to a lesser extent in the roof rat (Rattus rattus) in the US. Fortunately, second-generation halogenated anticoagulants, such as brodifacoum, are not subject to cross-resistance to warfarin. Cases of resistance to the newer halogenated derivatives have been reported; should this become as widespread as warfarin resistance, then rodent control would depend on a very limited supply of viable replacement chemicals.

Table 15.3. Pathogens having resistance to fungicides or bactericides applied in practice (Ogawa et al., 1983; Cooksey, 1990) 

Pathogen	Fungicide or bactericide
Alternaria spp. (2)	Polyoxin B
Aspergillus nidulans	Benomyl, calcium succinate
Botrytis spp. (2)	Benomyl, carbendazim, dichloran, iprodione,
	polyoxin B, quinomethionate, quintozene,
	tecnazene, thiophanate methyl
Ceratocystis ulmi	Benomyl
Cercospora spp. (4)	Benomyl, fentin acetate, fentin hydroxide
Cercosporella herpotrichoides	Benomyl, carbendazim
Cercosporidium personatum	Benomyl
Cladosporium spp. (2)	Benomyl
Cochiobolus carbonum	Cadmium succinate, thiram
Colletotrichum spp. (3)	Benomyl, carbendazim, thiabendazole, thiophanate methyl
Didymella ligulicola	Benomyl
Diplodia spp.	Diphenyl
Erwinia amylovora	Streptomycin
Erysiphe spp. (2)	Benomyl, ethirimol, thiophanate methyl, hydroxypyrimidine, SBI
Erythronium spp.	Benomyl
Fulvia fulva	Benomyl
Fusarium spp. (6)	Benomyl, tecnazene, thiabendazole, thiophanate methyl, thiram
Fusicalidium effusum	Benomyl
Gilbertelia persicaria	Dichloran
Hypomyces solani	Quintozene
Monilinia spp. (3)	Benomyl, iprodione, polyram
Mycospharella fragaries	Benomyl
Neurospara crassa	Benomyl
Oidiopsis taurica	Benomyl
Oidium begoniae	Benomyl
Penicillium spp. (6)	2-Aminobutane, anilazine, benomyl, diphenyl, 2-phenylphenol,
	thiabendazole, thiorhanate methyl
Phytophthora infestans	Metalaxyl
Plasmopara viticola	Phenylamides
Pseudocercosporella herpotrichoides	Benomyl
Pseudomonas spp. (4)	Copper, streptomycin
Pseudoperonospora cubensis	Metalaxyl
Puccinia spp. (2)	Dichlone, oxycarboxin
Pyrenophora spp. (2)	Methoxyethylmercury, organomercury
Pyricularia oryzae	S-Benzyl-O,O-diisopropropyl phosphorothioate, blasticidin-S, cadmium
	succinate, edifenphos, isoprothiolane, kasugamycin
Rhizoctonia solani	Benomyl, quintozene, thiram
Rhizopus stolonifer	Dichloran
Sclerotinia spp. (2)	Anilazine, benomyl, cadmium, succinate, thiram
Sclerotium spp. (2)	Benomyl, dichloran, quintozene
Septoria spp. (4)	Benomyl, carbendazim, edifenphos, thiophanate methyl
Sphaerotheca spp. (3)	Benomyl, dimethirimol, dinocap, pyrazophos, quinomethionate, thiophanate methyl
Sporobolomyces roseus	Carbendazim
Tilletia foetida	Hexachlorobenzene
Uncinula necator	Benomyl
Ustilago spp. (2)	Benomyl, carboxamide
Venturia spp. (3)	Benomyl, dodine, thiophanate methyl
Verticillium spp. (4)	Benomyl
Xanthomonas spp. (3)	Copper, streptomycin

Thus, it appears that resistance to insecticides and acaricides is the most urgent problem; but this same status could apply to herbicides, fungicides, and rodenticides in the next decade. The greatest threat is the growing number of pest species with multiple resistances, whereas the number of new chemicals appears very limited. The challenge is to find practical ways to manage pesticides so that resistance is prevented before many more species become even more difficult to control.

15.3 DEVELOPMENT OF RESISTANT POPULATIONS 

Resistance is a practical problem in population genetics. It is usually considered to be a consequence of rapid Darwinian selection brought about by the lethality of the pesticide to the normal susceptible organism, resulting in the concentration of rare, innately tolerant individuals, which then pass on to their progeny the genes responsible for a resistant population. Its consequences can be found on many levels, from the loss of a farm crop to resistant fungi, weeds, or arthropods, to the environmental hazard due to increased use of pesticides to gain some control of a resistant pest, to the decline of human health due to resistant vectors of disease or resistant rodents.

The potential hazard for resistance to a pesticide is difficult to predict from experimental evidence, due to the complex array of operational factors that can influence the degree of selection in the field, and the general lack of knowledge of pest genetics; however, several general principles have been learned through experience. Resistance generally develops most rapidly against those pesticides that are persistent in the field, so that one application exerts selection pressure on an entire generation or several consecutive generations of the pest. Examples of this phenomenon are the organochlorine insecticides, such as DDT and dieldrin, to which resistance had become widespread within ten years of their introduction.

Pesticides that are ephemeral due to photolability or reactivity are slower to induce resistance. Photolabile natural pyrethrins have produced only a few cases of low-level resistance due to their use alone (Brown, 1982). Organophosphorus and carbamate pesticides, most of which are reactive with nucleophiles and only moderately persistent, were used in enormous quantities for thirty years before resistance began seriously to diminish their effectiveness (Georghiou and Saito, 1983). Resistance is more likely to develop against pesticides with a specific mode of action (i.e., a single target), and less likely against general poisons with multiple targets and actions in the pest (Köller and Scheinpflug, 1987). Resistance generally develops in proportion to the intensity of selection pressure against the pest population. The resistance problem is aggravated by reliance upon broad-spectrum pesticides, which, when sprayed to control one problem pest, also apply selection pressure for resistance in other species that are present at levels not requiring concurrent control. This type of mismanagement was recognized as a major factor in multiple antibiotic resistance, so that an important countermeasure was replacement, whenever possible, of broad-spectrum antibiotics (Skurray et al., 1988).

Resistance develops as a response of the target population to the selection to which it is subjected by pesticide. It requires that one or more gene alleles that confer reduced pesticide susceptibility be present in the population at some low level, or that such an allele arise through spontaneous or induced mutation. For plant pathogens, three general cases are recognized: unavailability of appropriate mutant; one-step pattern of development due to one major gene; and multistep pattern due to accumulation of several genes (Georgopoulos and Skylakakis, 1986).

In the presence of the pesticide, the rarer resistant genotype has a tremendous fitness advantage, because the susceptible genotype will be killed. When the pesticide is removed, this artificial fitness advantage is usually lost, but few resistance genes have been systematically studied to determine whether they inflict any fitness deficit in the absence of the pesticide that would contribute to reversion to susceptibility in the population. This gap is crucial to an understanding of the development of resistance and must be considered to design counter-strategies (Keiding, 1986; Roush and McKenzie, 1987).

It is also possible, but not proven or generally considered, that pesticides that are mutagenic in the target pest might induce resistance through the mutation of a normal gene into one that confers resistance, assuming the individual in which the mutation occurs is not killed by exposure to the pesticide. In one experiment in Drosophila, a chemical mutagen was used to produce a resistance gene, which was then subjected to selection pressure using a non-mutagenic pesticide, methoprene (Wilson and Fabian, 1986).

It is more likely that resistance in the field begins with the genetic diversity that is often present in pests; it has also been the basis of their evolution and survival of the many natural toxicants to which they have been exposed through the ages. In this regard, mutation leading to genetic diversity becomes an advantage. The mutagenicity of a variety of naturally occurring substances has recently been reviewed (Ames, 1983; Ames et al., 1987). Whereas genetic diversity and selection have led to an evolved natural resistance to toxicants in some species, the contribution of mutation induced by natural substances to observed genetic diversity is unclear. Mutagenicity is an individual consequence of exposure to certain pesticides, but it may or may not result in heritable change; resistance is a consequence of the selection of a rare allele in a population group, and it may or may not be due to an induced mutation.

Once resistance arises in one location, it can spread outward fastest if the surrounding areas remain treated, since resistant progeny will replace the susceptible ones at the interface of the genotypes. In the US, cyclodiene resistance in corn rootworm spread from one county in Nebraska to all the surrounding states over several years (Brown, 1971). Cypermethrin resistance in the tobacco budworm was detected in 1987 in Texas, and its spread will be closely monitored through a network of surveillance (Simonet et al., 1988). DDT and organophosphorus resistances in Heliothis spp. were also found first in the Midsouth (Sparks, 1981).

Resistance alleles can spread through mating, through natural migration of the pest, or even through the assistance of man, as in the case of resistant strains of stored-grain beetles which apparently escape fumigation efforts in commercial grain shipping and are often detected first at seaports of entry. Resistance to benomyl fungicide was spread across Michigan golf-course greens on the spikes of golf shoes as golfers moved from one course to another. Triazine herbicide resistance apparently spread most rapidly along railroad rights of way.

A much better understanding of the dynamics of resistance in the field is needed. This effect can be achieved only by observing the population genetics of resistance. The lack of techniques for estimating frequencies of expression of individual alleles has been the limiting factor in studying this phenomenon.

Compounds exist that can produce pest resistance to other pesticides within the same chemical class and to other classes of pesticides. This response is due to the breadth of action of the selected resistance mechanism in the pest, such as development of a less sensitive target or production of an enzyme catalysing the same form for detoxication of several compounds. Pyrethroids and DDT are common victims of reduced target sensitivity, probably in the sodium ion channel of nerve (Lund and Narahashi, 1983), and can be in the same cross-resistance group when the insect of interest has this mechanism, as noted in cattle ticks (Nolan et al., 1977), house flies (Plapp and Hoyer, 1968; DeVries and Georghiou, 1980), horn flies (Byford et al., 1985), tobacco budworms (Payne, 1982) and three mosquito species (Chadwick et al., 1984; Omer et al., 1980; Priester and Georghiou, 1980). It must be recognized that the unnecessary choice of a relatively dispensable compound might jeopardize a very valuable, economical, biodegradable, or selective compound by inducing cross-resistance. An example of this mismanagement is the use of persistent pyrethroid insecticides in situations where natural pyrethrins belonging to the same resistance group are more desirable, and would otherwise have practically no chance of falling to resistance (Keiding, 1986).

Cross-resistance has been distinguished from multiple resistance, in which a pest accumulates several mechanisms each giving resistance to one or more compounds (Metcalf, 1971). Certain species have accumulated multiple resistance to each of the pesticides used against them (Table 15.1). Some species seem to have an array of mechanisms to meet any challenge. Chemical control of certain populations of many of these pests cannot be achieved at the level of previous standards. On the other hand, a few important species, such as the boll weevil and the European corn borer, seemed to lack certain resistance mechanisms, so that they retained susceptibility to azinphosmethyl and DDT, respectively (Brown, 1971).

15.4 MECHANISMS OF RESISTANCE

15.4.1 BEHAVIOURAL AND PHYSIOLOGICAL MECHANISMS 

Resistance usually results from genetically controlled biochemical or physiological changes from the normal type that alter pesticide pharmacokinetics or pharmacodynamics (Oppenoorth and Welling, 1976; Plapp, 1976; Soderlund and Bloomquist, 1990). Documented cases of behavioural avoidance of the pesticide are less common, but they can lead to serious consequences (Lockwood et al., 1984). Several cases of behavioural resistance selection are known, and they include both stimulus-independent phenomena, such as exophily of mosquitoes, and stimulus-dependent cases, generally known as increased irritability. The genetic bases of behavioural resistance have not been described.

Less methoprene uptake was the result of behavioural change selection in Culex pipiens (Brown and Brown, 1980). These larvae became quiesent when disturbed and exhibited significantly reduced movement and feeding behaviour for 3 h after disturbance. This selected behaviour was independent of methoprene stimulation. Lack of feeding behaviour was shown to reduce uptake by about 25 per cent, so that this mechanism alone did not confer all of the 100-fold increase in methoprene resistance in this strain. These larvae also produced 11 per cent more of an oxidative metabolite (Brown and Hooper, 1979); however, considering the wide range of juvenile hormone mimic chemistry to which there was full cross-resistance in this otherwise highly insecticide-susceptible strain (Brown et al., 1978), it is likely that behaviour and metabolism were combined with reduced sensitivity of an undefined molecular target to produce such a high level of resistance. Pyrethroid-resistant horn flies likewise displayed the behavioural mechanism combined with target insensitivity (Sparks et al., 1985). Horn flies with an 85-fold increase in pyrethroid resistance were highly irritated by cypermethrin and delta-methrin (but not by DDT), and they were 50 times more cross-resistant to directly applied DDT. This latter effect may have been due to nerve insensitivity combined with low level metabolic resistance indicated by synergistic activity.

Physiological mechanisms can provide resistance without behavioural avoidance of the pesticide. A pesticide must penetrate the pest and reach the target, where a molecular lesion is initiated, leading to death. Pesticide pharmacokinetics include the processes of penetration, distribution and biotransformation of the pesticide; pesticide pharmacodynamics, by contrast, describe the interactions with the target site. Resistance has been associated with pharmacokinetics, pharmacodynamics, and combinations of these sets of processes.

One of the pharmacokinetic changes possible is pesticide detoxication, often increased in resistant strains due to increased amounts or increased efficiency of detoxication enzymes: less pesticide reaches the target site. There are many types of detoxicative reactions, so that one species might have several different mechanisms of detoxication resistance.

Targets of toxicity (often enzymes, receptor proteins, or ion channels) can be altered qualitatively (e.g., to have less affinity for the pesticide), so that a greater concentration must reach the target to cause death.

Resistance can be conferred by a single mechanism, as has been observed for many genes in mosquitoes and house flies (Brown, 1971; Tsukamoto, 1983). Clear segregation of resistance was demonstrated in progeny of appropriate backcrosses. In these cases, it is easy to determine the degree of dominance of the resistance gene allele and to identify doses that discriminate among genotypes; however, it would be a gross simplification to consider resistance as usually monofactorial.

In some strains combinations of mechanisms are present, as in the Learn, New York, strain of house flies having pyrethroid resistance conferred by genes on three chromosomes (Scott and Georghiou, 1986). Development of strains in which single mechanisms are combined has demonstrated that multiple mechanisms can interact synergistically as demonstrated by the addition of the des detoxication gene and pen permeability gene in diazinon-resistant house flies (Sawicki, 1970). This condition was confirmed in other strains of house flies, in which the permeability gene was named tin because it gave resistance to organotin compounds (Hoyer and Plapp, 1968).

Using genetic markers and the natural recombination that occurs in backcrosses, the relative importance of the various genes can be estimated. However, for most major agricultural pests, practically nothing is known of genetic linkage, which makes such evaluation nearly impossible. Even when one factor is strong enough that segregation of resistance can be observed, there may be secondary factors involved that complicate the interpretation of results (Payne et al., 1988).

Changes in protein quality or quantity that cause resistance are inherited as alleles that vary from the normal type. Although few cases of resistance have been studied at the molecular genetic level, conferral of resistance through mutation of a single codon in a structural gene has been shown to result in an insensitive target protein, which is changed by only one amino acid substitution (Hirschberg and McIntosh, 1983). For the vast majority of resistance cases, very little is known of the inheritance of the trait. For many insecticides, the trait of resistance has been mapped genetically in the house fly and in several mosquitoes; however the structural and regulatory genes for the proteins likely to be involved as biochemical mechanisms have not been mapped. Work in this area has often been complicated by the multiplicity of isozymes occurring for the various detoxifying enzymes (Agosin, 1982; Motoyama et al., 1983).

15.4.2 PROTEIN PRODUCTS INVOLVED IN RESISTANCE

Enzymes catalysing pesticide biotransformation have been implicated in pesticide resistance. Detoxication of many insecticides is catalysed by monooxygenase, glutathione S-transferase, or carboxylester hydrolase. Detoxication is a common mechanism for resistance in insects and plants, but it is very rarely a factor in resistance to fungicides. Decreased activation has been detected as a possible mechanism of resistance to the fungicides pyrazophos (De Waard and van Nistelrooy, 1980) and triadimenol (Kalamarkis et al., 1986) and to the insecticide methyl parathion (Konno et al., 1989).

Target proteins with reduced sensitivity to pesticides include -tubulin in fungi resistant to benzimidazole fungicides, leaf plastid thylakoids in weeds resistant to triazine herbicides, and acetylcholinesterase in insects resistant to organophosphate and carbamate insecticides.

Detailed biochemical analysis of proteins from resistant and susceptible strains has been accomplished rarely. In the case of a carboxylester hydrolase, E4, from the aphid Myzus persicae, it was found that resistance was due to a much greater quantity of a protein with the same biochemical characteristics as that found in the susceptible strain (Devonshire and Moores, 1982). This protein has carboxylester hydrolase activity; however, its action is largely due to its very high titre and the noncatalytic reaction with, or the sequestering of, organophosphorus, carbamate, and pyrethroid insecticides. It is unusual that this appears to be the only resistance mechanism available to this aphid, while most insects have many possible mechanisms.

15.4.3 GENETIC MECHANISMS CONFERRING RESISTANCE 

Increased detoxication enzyme activity can be the result of genetic changes at any of several levels. The quantity of protein with unchanged structure can be increased by: gene duplication or amplification, in which a pest acquires multiple copies of the same gene; increased gene transcription, which may result from a mutation of a regulatory sequence; or increased translation due to mutation in the processing rate of mRNA. Altered quality of protein (e.g., changed catalytic centre activity) can result either from changes in the structural gene coding protein sequence or from processing or regulatory gene changes. Similar alterations are possible for target proteins, which might have their affinity for the pesticide or their number reduced to provide a lower sensitivity.

Amplification of the carboxylester hydrolase gene responsible for 250 times the normal number of gene copies has been found to be a genetic mechanism of resistance to organophosphurus insecticides in mosquitoes (Mouches et al., 1986). Biochemically, this protein appears to resemble the E4 sequestering protein with carboxylester hydrolase activity in aphids which is also increased through gene amplification (Field et al., 1988); however, in mosquitoes, other mechanisms are also present, such as target insensitivity (Raymond et al., 1985).

The genetic basis has not been identified for a large number of species that have devloped increased detoxication rates through distinctly catalytic processes, such as those mediated by the microsomal monooxygenases. A report of the first cloning of the cytochrome P₄₅₀ gene from an insect species suggest that resistant house flies have more constitutive transcription of this monooxygenase than their susceptible counterparts (Feyereisen et al., 1989). The sequence of the gene has been reported only for the resistant strain. The gene sequence for coding the haeme binding site of the house fly is similar to that of known, primarily mammalian, genetic codes; however, the overall sequence is sufficiently dissimilar from that of other species to be designated a new gene family for this species.

Resistance to the herbicide glyphosate can be genetically engineered into crops either by a gene for increased detoxication or by a gene for target site insensitivity. Experimental resistance to sulphonylurea herbicides is easily conferred by many nucleotide substitutions for several bases in the gene for the target enzyme acetolactate synthase (Yadav et al., 1986).

Perhaps the best studied gene encodes the target protein for triazine herbicides. In this case, a single amino acid substitution in the active site produces resistance that is maternally inherited, since the gene is a constituent of the chloroplast DNA and not the nuclear genome (Hirschberg and McIntosh, 1983).

Fungicide resistance to benzamidazoles has recently been studied at the level of the gene sequence for the target protein, which in this case is ,-tubulin (Fujimura et al., 1990). In Neurospora crassa, as occurs in strain F914, benomyl resistance can be accompanied by hypersusceptibility to the N-phenylcarbamate fungicide, diethylfencarb. It was hypothesized that this negatively correlated cross-resistance would always result from benomyl selection, so that benomyl susceptibility would be restored by subsequent selection with diethylfencarb; however, that hypothesis is less likely to be correct, since other strains such as B511 are resistant to benomyl but are no more susceptible to diethylfencarb than wild-type strains. Single amino acid substitutions in -tubulin that serve as a basis of these resistances are as follows: amino acid 198 is changed from glutamic acid to glycine in strain F914, making this strain benomyl-resistant but simultaneously very susceptible to diethylfencarb; if, instead, there is substitution of tyrosine for phenylalanine at position 167, then benomyl resistance is produced without the increased susceptibility to diethylfencarb.

15.5 METHODS TO ASSESS THE PROBABILITY OF RESISTANCE

Experimental selection for resistance in the laboratory provides some knowledge of the potential development of resistance to a given pesticide by a pest (Brown and Payne, 1988). The rapid and high-level development of resistance may indicate the presence of genes for resistance in this species; however, because of the genetically limited sample available in the laboratory, failure to develop resistance cannot be interpreted as a lack of its potential for expression. Many laboratory experiments on numerous compounds have demonstrated resistance, a phenomenon later observed in the wild (Brown and Payne, 1988). Since the mechanisms of resistance found in experimental laboratory strains are not known to be identical to those found in the field, the full significance of experiments remains unclear.

Another approach is selection of strains that are also exposed to mutagen. This exposure produces resistant strains whose mechanism(s) of resistance can be examined. However, the possibility of false-negative results exists and is complicated by the fact that induced mutations might not have occurred otherwise.

A refinement of this approach is to produce specific mutant genes through molecular site-directed mutagenesis and then to insert these genes into the organism of interest to determine whether resistance can be conferred. In the genetic engineering of crops, researchers have been successful with this approach by inserting the acetolactase synthase gene in the gene target of sulphonylurea herbicides (Falco et al., 1985). Resistance expressed in plants engineered in the laboratory, while not as yet observed elsewhere, suggests that it might be inducible in the field also.

Although this approach appears to be a very powerful and scientifically defined method of experimentally inducing resistance by anticipated mechanisms, it is less capable of uncovering unforeseen mechanisms than the cruder methods of experimental selection of the organism discussed previously. 

15.5.1 CONVENTIONAL PESTICIDES

Resistance to DDT and its analogues, to cyclodiene, and to organophosphate, carbamate, and pyrethroid insecticides has been induced in over 143 experiments, in which insects were exposed to doses killing from 50 to 90 per cent of the colony for each of 1060 generations (Brown and Payne, 1988). DDT, dieldrin, and permethrin generally induced resistance rapidly and to very high levels, while organophosphorus and carbamite insecticides produced a slower response to less intense resistance. In the broadest sense, this relationship was the same as that observed in the field, where DDT resistance was actually found prior to most laboratory studies, and organophosphate resistance had only limited occurrence for nearly 20 years.

15.5.2 JUVENILE HORMONE MIMICRY, Bacillus thuringiensis TOXIN, AND OTHER NOVEL PESTICIDES

Mimicry of juvenile hormone is not an exception to the phenomenon of resistance, as demonstrated in a completely susceptible wild strain of the northern house mosquito, Culex pipiens, which increased methoprene resistance 100-fold in 34 generations of selection and developed lesser levels of resistance to triprene and the chitin synthesis inhibitor diflubenzuron (Brown et al., 1978). In a confirmatory selection of a new collection of larvae from the same site two years later, high methoprene resistance was developed in just nine generations. Hydroprene produced selective resistance in the confused flour beetle, Tribolium confusum, whereas the milkweed bug, Oncopeltus fasciatus, developed a fourfold increase in tolerance to kinoprene (Brown et al., 1978). Methoprene resistance has also been developed in Drosophila melanogaster by selection following mutagen treatment of males (Wilson and Fabian, 1986). While there are no reports of resistance in agriculture to this class of novel insecticides, they are not used as extensively as conventional pesticides.

Bacillus thuringiensis toxin added to larval diet produced a 14-fold increase in resistance in 27 generations of selection of house fly larvae (Harvey and Howell, 1965). From that point, resistance was not increased in 23 additional generations of selection, but it was relatively stable in the absence of selection for 20 generations. This toxin has attracted renewed interest with successful expression of its recombinant gene in plants (Vaeck et al., 1987). Protection of crop plants from lepidopterous and coleopterous pests is the primary goal of this research. Recently, two lepidopterous pests of stored products responded to selection with this toxin mixed into a wheat-based larval diet. The Indianmeal moth, Plodia interpunctella, developed a 310-fold increase in resistance in 35 generations, while the almond moth, Cadra cautella, exhibited a 7.8-fold increase in resistance in 23 generations (McGaughey and Beeman, 1988). Resistance was generated very rapidly in several Indianmeal moth colonies, even though the selecting dose was adjusted only once per experiment, and many generations were selected at less than the median lethal dose. No reports exist on the selection of insect pests with the genetically engineered toxin in plants or soil microbes.

When novel pesticides are employed in selection experiments, the results can be very dependent on the methods of application and timing of the dose because of the greater lability of many of these compounds in relation to conventional pesticides, and because dose effects differ according to the stage of physiological development of the pest. This situation could result in very uneven selection within a generation so that some individuals are not actually selected. High doses timed to correspond to the most susceptible stage(s) or the use of slow-release formulations may overcome this technical problem (Brown and Payne, 1988). Similarly, consistency in the expression of toxin dose should be determined in the design of selection experiments in genetically engineered plants.

15.6 MOLECULAR TECHNIQUES TO DETECT RESISTANCE 

The new prospect for studying resistance before use requires advances in finding DNA cloning vectors for pests. To be fully exploited, the basic genetics of pest species should be studied by several approaches, including the development of genetic linkage maps. New technology of restriction fragment-length polymorphism mapping recently applied to the human genome can be used to make rapid progress in many pest species for which genetic information is usually absent.

Understanding of population genetics in resistance development is fragmented and incomplete. The major problem is that surveillance data are generally obtained for susceptibility of the population by measuring mortality due to the pesticide; from these data, the proportion of resistance is estimated. Since there are often several genetically distinct mechanisms of resistance to one pesticide, direct detection of individual mechanisms becomes necessary. From the perspective of the population geneticist, the allele frequency must be determined (Roush and McKenzie, 1987). This requires that both homozygous and heterozygous individuals be detected with substantial accuracy and that tests be capable of discriminating between multiple resistance mechanisms in an individual. Breakthroughs in this form of surveillance have been achieved with the aphid Myzus persicae, in which an immunosorbent assay for an active enzyme has been used, and in several mosquito species, in which exposure to the insecticide was followed by multiple assays for several mechanisms in aliquots from each individual.

In Haiti, microtitre plate assays for carboxylester hydrolase were found to be very sensitive in detecting resistant individuals of Anopheles albimanus, so that the initial development of fenitrothion resistance was documented in the field and diagnosed as this mechanism (Brogdon et al., 1988). In Guatemala, the additional, independent mechanism of less sensitive target acetylcholinesterase was also found when both mechanisms were assayed in each of 1100 mosquitoes (Brogdon et al., 1988). These studies demonstrate the potential for determining the frequencies of several different resistance genes in field populations with economical and practical field kits.

15.7 ECOLOGICAL CONSEQUENCES OF RESISTANCE

A major consequence of resistance is that it often leads to increased frequency of pesticide application. This increased application rate can be damaging to biological control and integrated pest management strategies.

In some situations, mixtures that are synergistic in the resistant pest have been marketed, so that some control is regained; however, the environmental consequences of these mixtures is usually unknown. Indeed, even the basic toxicology of such mixtures has seldom been investigated.

Genetic engineering of resistance in beneficial species of insects, or even in domestic animals, may be the logical extension of accomplishments with crops when adequate cloning vectors are identified. Inserting resistance genes uncommon in pests into beneficial species may pose a great risk; for instance, a resistance gene engineered through a transposon vector into a parasitic insect could lead to the unintended movement of that gene into the pest.

Genes for hydrolases that cause enhanced degradation of pesticides have been isolated and cloned for soil-inhabiting microorganisms. A catalytic hydrolase mechanism (in contrast to a sequestering type) is very rare for organophosphorothioate and carbamate insecticides in resistant insects; in fact, the lack of hydrolysis is probably a major factor in the insecticidal activity of these major insecticidal classes. The accidental introduction of this mechanism could lead to serious resistance problems in pests and must be avoided.

Progress at improving beneficial organisms will depend on an improved understanding of the biochemistry of herbivory versus predation. It appears that herbivores are more powerful in monooxygenase-catalysed detoxication of xenobiotics than are predatory arthropods (Mullin et al., 1982). Generalist herbivores have at least seven times more trans-epoxide hydrolase than cis-epoxide hydrolase, which is useful for the detoxication of epoxides sometimes generated through oxidation reactions. specialized herbivores and predators have much lower ratios (Mullin and Croft, 1984).

15.8 MUTAGENICITY OF PESTICIDES

Certain pesticides are known mutagens in bacteria and yeast assays (Klopman et al., 1985). In addition, it is known that certain pesticides are promutagens which are metabolized to mutagens (Casida and Ruzo, 1986). Since insects and plants are capable of activating promutagens (Zijlstra et al., 1987; Veleminsky and Gichner, 1988), it is possible that both mutagenic and promutagenic pesticides could produce mutations for resistance in alleles.

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