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The Society for Integrative and Comparative Biology
Drosophila: Sentinels of Environmental Toxicants1
1 Department of Entomology, 400 Aronoff Building, Ohio State University, Columbus, Ohio 43210
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 SYNOPSIS INTRODUCTIONINSECTICIDE RESISTANCENONTARGET ORGANISMS: DROSOPHILAFUTURE DIRECTIONSReferences |
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Synthetic insecticides have been used intensively for the past 50 years in many parts of the world. Insect populations, both target and nontarget, have responded by evolving resistance. One of the nontarget insects is Drosophila melanogaster, which is well-suited for genetic analysis and has been particularly well-studied in both laboratory and field populations. Resistance to several insecticides, including two for which significant resistance in field populations has not been found, has been generated in susceptible laboratory strains following mutagenesis, allowing comprehensive study of the resistance genes. Field populations of D. melanogaster have evolved resistance to many, but not all, insecticides in use today. Both the genetic and biochemical mechanisms that underlie resistance in this insect are similar to those in other insects. Therefore, D. melanogaster can be a sentinel organism for long-term release of toxicants into the environment. While it remains useful for genetic analysis of resistance, a better understanding of the movement and population structures of this insect will be a prerequisite for its sentinel utilization at specific locales.
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INTRODUCTION |
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SYNOPSISINTRODUCTIONINSECTICIDE RESISTANCENONTARGET ORGANISMS: DROSOPHILAFUTURE DIRECTIONSReferences |
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Synthetic agrochemicals have been increasingly used during the past 50 years. At present, the application of these toxic chemicals is routine to control insect and plant pests that are either detrimental to crop and fiber production or serve as pests/disease vectors to humans and domestic animals. The annual quantity of these toxicants released into the environment is huge (Zilberman et al., 1991
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The effect of these chemicals, particularly insecticides, has been studied for many decades. In most cases they have performed well to control the target organisms, a result that has undeniably contributed to the success of agriculture. However, nontarget organisms can be affected as well by toxicants, and I will focus this review on the response of one of these unintended animals, Drosophila melanogaster, to insecticides. Work has been presented at this symposium and published elsewhere (Sparling et al., 2001) examining detrimental effects of insecticides on nontarget species. In some instances the use of insecticides has been shown to be detrimental to invertebrates (Brown et al., 2000). Vertebrates can also be affected by chronic exposure to certain insecticides. For example, application of methoprene (Fig. 1), a juvenile hormone (JH) analog insecticide, to aqueous habitats of mosquito larvae has been blamed for morphological defects and increased mortality of certain amphibian species (La Clair et al., 1998) although other explanations have been forwarded (Henrick et al., 2002; Sessions et al., 1999). Therefore, these chemicals, even when properly used in the field, may be affecting nontarget species to a greater extent than had been predicted from laboratory studies early in their usage.
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An understanding of the impact on insecticides must begin with a consideration of the mode of action of an insecticide. Following entry into the insect, it must bind to a specific target protein, whose biological activity is consequently diminished. In almost all instances, the target protein is required for viability. A number of target molecules have been employed in the design of novel insecticides, and the most successful ones have been proteins critical for nerve function (Bloomquist, 1996
). Not surprisingly, since the target molecules vary, insecticide chemistries vary. An early favorite target was the vital enzyme acetylcholinesterase, required for hydrolysis of acetylcholine at neuromuscular junctions, and it has been exploited by agrochemical companies in a spectacular manner. Both the organophosphate (OP) and carbamate classes of insecticides bind to and inhibit this enzyme and consequently kill insects within minutes of exposure (O'Brien, 1976). |
INSECTICIDE RESISTANCE |
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SYNOPSISINTRODUCTIONINSECTICIDE RESISTANCENONTARGET ORGANISMS: DROSOPHILAFUTURE DIRECTIONSReferences |
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Insect populations are fighting back, however, by evolving resistance to insecticides, and few biologists are ignorant of the success that pest insects have demonstrated in this battle. In initial studies of resistance the focus was on the economic aspects: the cost to the grower and the design of strategies to circumvent resistance. While these practical aspects are undeniably important, the genetic and biochemical aspects of resistance evolution that have come to light are also important and, perhaps as a pleasant surprise, have made for fascinating biology. These include the biochemical basis of resistance, the molecular biology of the mutations that have led to the resistance, and the evolutionary and population aspects of the response (Roush and McKenzie, 1987
; Morton, 1993; Feyereisen, 1995; McKenzie, 1996; McKenzie and Batterham, 1998; Taylor and Feyereisen, 1996). Let me present an overview of our knowledge of these aspects before delving into that of D. melanogaster.
Biochemically, insects become resistant by either of two major mechanisms: (1) detoxification achieved by either enhanced metabolism of the chemical to a less toxic form or sequestration prior to its interaction with the target macromolecule. Enhanced metabolism is the more important mechanism, and a major detoxification mechanism is the cytochrome P450 enzyme-mediated pathway (Berge et al., 1998; Feyereisen, 1999; Scott, 1999), which is found throughout the animal kingdom. (2) Reduction in the level of toxicity of the chemical resulting from an alteration in the target molecule (caused by a mutation in the gene encoding the target macromolecule) and consequent poor insecticide: target interaction. This mechanism is termed target-site insensitivity. Since the altered macromolecule precludes optimal binding of insecticide, a greater quantity within the insect is required for toxicity. Resistant insects can achieve high resistance levels—in some instances several thousand-fold relative to susceptible ones (ffrench-Constant et al., 1990)—by either mechanism. There are also minor mechanisms of resistance, such as altered behavior of the insect toward insecticide exposure and reduced penetration of the insecticide into the insect, neither of which has been well-studied nor their importance documented.
A major problem in insect control is cross-resistance. This type of resistance can be pronounced for enhanced metabolism resistance, particularly P450 resistance. Insects (and other organisms) carry a family of P450 genes, some of which are inducible by the xenobiotic chemical itself or can be transcriptionally activated in a constitutive manner due to a genetic change (Feyereisen, 1999; Scott, 1999). Once induced by either method, the P450(s) usually acts to metabolize and detoxify the chemical, thus rendering the insect resistant. Cross-resistance occurs when the enhanced P450 enzyme has a broad spectrum of substrate specificity and can metabolism different insecticides, resulting in resistance to a variety of insecticides directed at different target macromolecules and having very different chemistries (Feyereisen, 1999; Scott, 1999; Daborn et al., 2002). Cross-resistance can also occur with target-site resistance if two or more different insecticides target the same macromolecule, and the altered macromolecule binds both insecticides with less affinity than does the susceptible macromolecule (ffrench-Constant, 1999). A well-studied example of this type of cross-resistance will be described for DDT and pyrethroid insecticides (Fig. 1).
Genetically, target-site resistance is better understood. Those target-site resistance genes that have been isolated and sequenced show a preponderance of point mutations (ffrench-Constant et al., 1993; Mutero et al., 1994; Williamson et al., 1996; Dong, 1997; Soderlund and Knipple, 2003). This is logical if one considers that only a small change in the target protein may be required for reduced insecticide binding (and tolerated for viability in the absence of insecticide). Similarly, point mutations are important in resistance shown by other organisms; for example, in plant resistance to herbicides. Four families of herbicides target the enzyme acetolactate synthase, and target-site resistance found in various weed biotypes results from point mutations at multiple locations in the encoding gene (Gutteri et al., 1996).
The importance of point mutations is well-illustrated in insect resistance to pyrethroid insecticides. These chemicals are widely and heavily used for insect control and, like DDT, owe their toxicity to interaction with a sodium channel protein in neuronal membranes (Soderlund and Bloomquist, 1989; Zlotkin, 1999). Binding disrupts the gating kinetics of action potentials and results in rapid paralysis (termed "knockdown") and subsequent death of the insect. Resistance to both of these effects has been recognized in a variety of insects (Soderlund and Knipple, 2003). Resistance was genetically found in house flies to be due to a target-site resistance gene (kdr), and when the sodium channel gene was cloned and sequenced in these insects, the allele in a variety of resistant pest insects could be evaluated following PCR amplification and sequencing. Resistance was found to be due to a point mutation in the sodium channel gene (Williamson et al., 1996). Despite the large size of this protein (>2,000 amino acids), only about six or so different point mutations have been identified in a variety of resistant insects, and one of these, a leucine-to-phenylalanine substitution at amino acid 1,014, is the predominant one in seven pest species belonging to several different orders (ffrench-Constant, 1999; Soderlund and Knipple, 2003). Some high pyrethroid resistance strains ("super-kdr") have an additional nearby point mutation that accounts for the enhanced resistance (Crow, 1954; Miyazaki et al., 1996; Williamson et al., 1996). Therefore, resistance to these insecticides (and cross-resistance to DDT) is conserved, geographically widespread, and presumably due to direct selection pressure from pyrethroids (or DDT) on the insect populations.
Genetic changes that result in upregulated transcription of detoxification genes are more poorly understood, but include transposable genetic element-associated transcriptional up-regulation (Daborn et al., 2002) and gene amplification (Devonshire and Field, 1991). Examples of resistance resulting from either loss of the target-site gene or failure of transcription are uncommon, but one has been shown for laboratory-generated resistance to the JH insecticide methoprene (Wilson and Ashok, 1998).
Do these resistant alleles exist in the population prior to exposure to insecticide or do they arise de novo following selection pressure? The origin of resistance gene mutations is not clear and unfortunately is difficult to determine. Presumably, these mutations are pre-existing in populations in very low allele frequencies, but since insect populations are not genetically examined prior to the introduction of a novel insecticide, solid data is unavailable. Once the insect population contacts an insecticide, then presumably selection of individuals carrying a resistance allele leads to a much higher allele frequency and resistance in the population. Aiding the rapid evolution of resistance in a population is insect movement, either by migration or by human activity. It is possible that resistance gene mutations arise de novo as a stress response to contact with insecticide, but evidence for an adaptive elevated mutation rate has been reported primarily in microorganisms (Hastings and Rosenberg, 2002; Kivisaar, 2003).
In summary, we have in-depth knowledge of the biochemical mechanisms and their underlying genetic changes leading to insecticide resistance in a variety of insects.
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SYNOPSISINTRODUCTIONINSECTICIDE RESISTANCENONTARGET ORGANISMS: DROSOPHILAFUTURE DIRECTIONSReferences |
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Nontarget organisms are affected by insecticide usage. Have populations of these organisms also responded by evolved resistance? Examination of the cosmopolitan fly D. melanogaster indicates that resistance has evolved to many insecticides, yet these flies are rarely intentional targets. Examination of cultures of these flies established as isogenic strains from field populations has also established resistance as a recent, not an ancient, occurrence. Since this insect is genetically well-understood, any resistance detected can be analyzed with a degree of sophistication not readily available for other nontarget insects (Wilson, 1988
, 2001; Morton, 1993). Examples of genetic exploitation of two resistance genes from field populations and one from laboratory flies will be described.
Enhanced metabolism by Cyp6g1
Insecticide resistance was found in field populations of D. melanogaster soon after the beginning of widespread and heavy use of synthetic insecticides in the 1950s. Early work examining DDT resistance laid the basis for a genetic analysis of resistance and documented that this basis could be genetically understood (Crow, 1954). Through the use of a collection of genetically marked chromosomes, DDT resistance was chromosomally traced, and the results suggested a polygenic basis for DDT resistance (King and Somme, 1958). Polygenic resistance subsequently has proven to be common in field populations of insects, both target and nontarget.
DDT resistance has persisted in field populations of D. melanogaster long after the withdrawal of this insectide from use in most locales. Resistance was genetically mapped to a single gene, termed R(1)DDT, on the second chromosome, and resistance due to this gene was found for other insecticides (Merrell and Underhill, 1956; Shepanski et al., 1977; Cuany et al., 1990; Bride et al., 1997). Subsequent biochemical analysis showed R(1)DDT resistance to result from P450 overexpression, presumably resulting in enhanced metabolism of DDT, as well as other insecticides, within the fly (Daborn et al., 2001). Recently, the genetic basis of this resistance (and cross-resistance) in strains from worldwide populations showed overexpression of a single P450 gene, Cyp6g1, associated with the insertion of an Accord transposable genetic element in the promoter region of this gene (Daborn et al., 2002). A broad substrate specificity of CYP6G1 presumably accounts for cross-resistance to the wide range of insecticides.
Other DDT-resistant populations of D. melanogaster show overexpression of other P450 genes (Maitra et al., 1996), so populations have responded differently to the presence of this insecticide. Possibly, the presence of other xenobiotics have also been involved in the response of a particular population, altering the constellation of P450s that have responded to the array of chemicals. One study of the RDDTR strain showed not only upregulation of Cyp6a2 but also several point mutations in the gene, suggesting additional resistance due to qualitative changes in the P450 enzyme (Berge et al., 1998).
DDT resistance in D. melanogaster has also been shown to result from overexpression of glutathione transferase (GST). GSTs are a family of proteins that conjugate the thiol group of the tripeptide glutathione to a variety of organic molecules, facilitating their metabolism and subsequent excretion (Daniel, 1993). In D. melanogaster, 42 GST genes have been found to exist, and this family of detoxifying enzymes may prove to be as complex and versatile as P450s. Gst D1 clearly has been shown to have DDT dehydrochlorinase activity, which is overexpressed in the DDT-resistant PSU-R strain and at least contributes to the resistance seen in that strain (Tang and Tu, 1994). Gst 2 was suggested as the gene for OP resistance in Israeli vineyards (one of the few locales where D. melanogaster is economically important) based on gene mapping data (Ringo et al., 1995).
In summary, the many studies examining resistance to DDT and other insecticides in field populations show enhanced resistance mechanisms primarily due to elevated transcription of P450 and GST detoxification genes. Cyp6g1 is a major responding gene, but clearly other P450 and GST genes are involved in some populations, showing a heterogeneous response to xenobiotics.
Target-site resistance
Populations of D. melanogaster can also evolve target-site resistance to insecticides. The apparent ease with which this has occurred is well illustrated by resistance to cyclodiene insectides. This class of insecticides was widely used but has been withdrawn in recent years. Populations of D. melanogaster were collected initially in upstate New York in apple orchards, and they showed a high allele frequency (as high as 10%) of a resistance gene termed Resistance to dieldrin (Rdl), a major cyclodiene (Fig. 1) insecticide (ffrench-Constant et al., 1990; ffrench-Constant and Roush, 1991). These mutant flies exhibited phenotypic characteristics consistent with those expected from a lesion in the central nervous system, not with enhanced metabolism of dieldrin. Rdl was cloned by positional cloning methodology and identified as a subunit in the gamma amino butyric acid (GABA) receptor (ffrench-Constant et al., 1991). Since the action of cyclodiene insecticides was known to involve GABA receptors, this result identified the resistance as target-site and also represented a novel approach to cloning a GABA receptor gene by utilizing a target-site resistant mutant to identify the gene.
When Rdl was sequenced and compared with the susceptible allele, a single point mutation replacing alanine302 with a serine in the critical chloride ion channel pore was found (ffrench-Constant et al., 1993). This mutation was identical in resistant worldwide populations of D. melanogaster. Significantly, ffrench-Constant and his group PCR amplified Rdl from a variety of dieldrin-resistant insects from other insect orders and found the identical amino acid mutation in nearly all of the species (ffrench-Constant, 1994). The degree of gene homology among the insects was not surprising for a conserved protein such as a GABA receptor, but the fact that this one amino acid change is conserved is remarkable. How many other mutations in Rdl can result in a phenotype of both resistance and sufficiently high fitness to enable these mutants to compete successfully with wild-type flies in field populations (as has Rdl) remains an interesting question. Creating other Rdl alleles in the laboratory and testing each for resistance is relatively easy, but measuring their competitive ability in the field would be more difficult.
Therefore, a variety of insects have utilized target-site insensitivity as a resistance mechanism for this class of insecticide. It is tempting to conclude that since Rdl-mediated resistance results in very high (>1,000-fold) resistance, insect populations bearing Rdl are afforded high protection from cyclodiene extermination; consequently, the driving force for spread (or maintenance once established) of a P450-based resistance allele is small when Rdl is in high allele frequency. A "selective sweep" by a high-resistance gene is proposed to account for monogenic resistance found in insect pest populations (Roush and McKenzie, 1987). Other possible explanations are that the cyclodiene insecticides are not as readily detoxified by P450 or GSH or that the presence of both Rdl and Cyp mutations in flies has a severe fitness disadvantage.
Laboratory selection
Laboratory populations of D. melanogaster have long been the subjects of selection experiments for a variety of traits. Usually, selection has been on fly strains that exhibit variability for the trait under selection. These selection regimes have ranged from bristle number to resistance to chemicals, and the flies usually respond to varying degrees in 6–12 generations (Lee and Parsons, 1968). Insecticide resistance alleles are usually not found in laboratory strains because most of these strains were derived from flies collected from field populations prior to the widespread use of synthetic organic insecticides. Therefore, studies that select for lines with increased resistance have utilized recently collected field populations carrying pre-existing resistance alleles in low allele frequency. These selection experiments are almost guaranteed to meet with success, and in some cases have provided useful strains in which the genetic component(s) could be analyzed (Merrell and Underhill, 1956). Many resultant strains become polygenic for the resistance, resulting from the genetic accumulation of several resistance mutations, often minor, whose products physiologically sum to higher resistance level (Dapkus and Merrill, 1977). Analyzing these minor resistance mutations has not been easy, and the value of the strains for understanding the contributions of minor resistance genes has yet to be realized.
However, when high resistance cannot be found in field populations, laboratory strains can be utilized to study resistance. In this approach mutagenesis is used to create resistance mutations, which can then be selected with the toxin of interest. One of the first demonstrations of the usefulness of this approach was shown for 
-amanitin resistance in work utilizing ethyl methane sulfonate (EMS) as a chemical mutagen that primarily results in point mutations in DNA. Resistance alleles were recovered following -amanitin selection, and they were subsequently identified as mutations in the RNA polymerase II locus (Greenleaf et al., 1979). Since this protein is the target molecule for -amanitin, these mutants represent target-site resistance and provided a mechanism for identifying this gene before the availability of the sequenced D. melanogaster genome.
An advantage of laboratory mutagenesis studies is that knowledge gained from studies of the resistance gene(s) for a particular insecticide can then be used to presage resistance in field populations of pest insects if usage of that insecticide becomes widespread and, ideally, aid in the design of control measures. For example, work generating D. melanogaster resistant to cyromazine (Adcock et al., 1993) should enable knowledge transfer to pest populations of other fly species.
We followed a mutagenesis approach to examine the genetic basis for resistance to JH insecticides. These compounds mimic JH and, when present at an inappropriate stage in development, disrupt metamorphosis and lead to death of the insect (Restifo and Wilson, 1998; Zhou and Riddiford, 2002). Although resistance (as cross-resistance) to the JH insecticide methoprene was shown in a pest insect soon after its introduction in the early 1970s (Cerf and Georghiou, 1974), the genetic basis was unknown. Low-level resistance was seen in some D. melanogaster field strains (Wilson and Thurston, 1988), but identifying the genetic basis for this resistance by conventional mapping is difficult and was not attempted.
Instead, we carried out EMS mutagenesis of susceptible laboratory strains and selected progeny on a toxic level of methoprene incorporated into the diet (Wilson and Fabian, 1986). Two strains were established from this initial effort and remained resistant upon retesting. Genetic analysis showed the resistance of both to map to a gene that was termed Methoprene-tolerant (Met). Met was readily mapped by recombination to 33 on the X-chromosome, a region that fortunately was well-represented by deficiency chromosomes. These chromosomes are useful not only for corroborating the map position determined by genetic recombination but also for establishing the consequences of a null on that chromosome. The ability to rapidly map resistant genes by both recombination and deficiency chromosomal analysis attests to the advantage that these insects possess for resistance studies.
Resistance of Met flies was also demonstrated to other JH analog insecticides and to JH III, the naturally occurring JH in these flies. However, cross-resistance to other classes of insecticides proved to be absent to very low (Wilson and Fabian, 1986). Therefore, Met results in resistance to the JH insecticides, but it is not a "general" insecticide-resistance gene.
Biochemically, resistance mechanisms of reduced cuticular penetration and enhanced metabolism were ruled out by direct experimentation (Shemshedini and Wilson, 1990). Target-site resistance was evaluated by examining binding of radiolabelled JH III to larval fat body, a JH target tissue in these flies. When intracellular binding to Met and susceptible Met+ fly extracts were measured and compared by Scatchard binding analysis, binding affinity of hormone to Met flies was about 10-fold poorer than to Met+ flies (Shemshedini and Wilson, 1990). Therefore, Met results in resistance by a target site insensitivity mechanism.
Met was cloned by transposon-tagging using P-transposable genetic elements (Ashok et al., 1998). DNA sequencing identified Met to be a member of the bHLH-PAS transcription factor family. Our current interpretation is that MET is a JH receptor protein component which, when mutated, binds JH III or methoprene with much less affinity. Since either of these compounds is toxic to certain insects when present at the onset of metamorphosis, poorer methoprene binding in Met flies results in resistance to the toxic effects. Over the last few years we have selected 14 Met alleles in our screening protocol (Wilson and Fabian, 1987) for methoprene resistance using either 
-irradiation or EMS (Wilson and Fabian, 1986; Ashok et al., 1998). DNA sequence analysis of these alleles has revealed the lesions to be primarily point mutations at various locations in the coding region of the gene, although they are not confined to the three bHLH or PAS gene domains (T.G.W., unpublished data). So, the variety of laboratory-generated Met alleles conferring resistance presents a picture that differs from the situation with field strains of Rdl having a highly conserved mutation (ffrench-Constant et al., 2000). Site-directed mutagenesis of Met and Rdl to investigate other regions of each gene for resistance should provide a picture of changes that can be made in each gene that will result in resistance to the respective insecticides. Overall, it is likely that each resistance gene will be unique: some (Met) may have several mutations resulting in resistance, but others (Rdl) may have far fewer.
This work has shown that not only can resistance genes be readily analyzed in flies, but that an important physiology such as the GABA or JH receptor can be identified and studied as well. Both studies illustrate the utility of D. melanogaster for rapid and in-depth (both genetic and molecular) study of the resistance gene.
Chronological appearance of resistance
Examination of D. melanogaster strains collected at various times from field populations during the past century and maintained under laboratory conditions in world-wide collections has shown a correlation of resistance with the widespread usage of certain insecticides. The resistant D. melanogaster strains brought into the laboratory during the past several decades show remarkable stability of resistance in the absence of insecticidal selection pressure. For example, WC2 has been husbanded in the laboratory since its collection in 1991 and has maintained high resistance (Wilson and Cain, 1997; Daborn et al., 2002). Therefore, even after years of laboratory passage without selection pressure, resistance is stable and reflects the level of the original population at the time of capture. There is little evidence that resistance is "lost" during laboratory culture (unless by accidental introduction of a susceptible allele into the culture). Since resistance usually results from one or more gene mutations, this stability of the resistance phenotype is not surprising and in fact is expected.
The chronological appearance of resistance in field strains has been shown with resistance to lufenuron, a benzoylphenyl urea insect growth regulator insecticide, in D. melanogaster strains collected in the early 1990s at two widely separate locales in the United States. Since lufenuron at that time had experienced negligible field applications, any resistance seen would be cross-resistance. Resistance in field populations was found to be common to a level of 100-fold over laboratory strains that were collected more than 60 years ago (Wilson and Cryan, 1996; Wilson and Cain, 1997). These strains included some collected at sites near agricultural insecticide usage while others were collected distant (>30 km) from these sites; there was no obvious correlation with resistance and proximity to high insecticide usage. However, not all of the strains were resistant; about 15–20% of the strains showed susceptibility levels near that of laboratory strains. One of the most resistant of the strains examined was WC2, which was subsequently shown to owe its resistance to elevated Cyp6g1 gene expression described earlier (Daborn et al., 2002).
Examination of strains with well-documented dates of laboratory husbandry showed that resistance both to lufenuron and to the carbamate insecticide propoxur correlated with usage of insecticides during the past three decades and were especially high in strains established since 1990 (Wilson and Cain, 1997). Therefore, resistance in field populations is a relatively recent phenomenon. We favor the hypothesis that these populations are responding to some agent in the environment, probably an insecticide, that has become prevalent during the past several decades.
Cross-resistance
A major impediment to understanding the relationship of a resistant D. melanogaster population to the presence of a xenobiotic in its environment is cross-resistance. The initial interpretation of resistance evolution stated that an insect population responds by evolving resistance to the one found in its environment. As new insecticides were developed and deployed, it soon became clear that frequently a population was cross-resistant to the new insecticide soon after it was applied in the field, long before resistance could evolve via selection to that insecticide in the population. Resistant insects are cross-resistant to a novel insecticide either because the two chemicals target the same macromolecule (target-site resistance) or because a metabolic detoxification mechanism recognizes both chemicals (enhanced metabolism). An example of the former has resulted from the targeting of acetylcholinesterase by both OP and carbamate insecticides, and the latter by P450 enzymes substrate nonspecificity (Le Goff et al., 2003).
Cross-resistance makes it difficult to ascribe resistance to a particular chemical in the environment as the response of the population to that chemical since the resistance may be a response to an earlier chemical, not the one in question. If this knowledge is desired, then resistance of a population predicted to be in contact with the insecticide must be determined in laboratory studies. Knowledge of the target macromolecule helps to predict cross-resistance, as evident with acetylcholinesterase-targeting insecticides. This knowledge is sometimes difficult to obtain; for example, the molecular target for cyromazine is still unknown (Bel et al., 2000). Therefore, conclusions about a population responding to a new insecticide with resistance must be drawn with care, since resistance due to cross-resistance must be ruled out.
Resistance is widespread but not ubiquitous
D. melanogaster populations that have evolved resistance to different classes of insecticides are widespread. In addition to the examples of resistance to DDT, cyclodienes, lufenuron, and carbamate insecticides mentioned above, there exists much work examining resistance to OPs. These chemicals continue to see enormous usage worldwide, and resistance to them, both metabolic and target-site, is widespread in D. melanogaster. The metabolic resistance is likely P450-mediated in many instances. However, resistance resulting from changes in the target macromolecule acetylcholinesterase is common and well-understood. A single F368 to T change was found when the resistant Ace allele was sequenced (Fournier et al., 1992), and examination of other resistant populations showed four additional point mutations that result in OP or carbamate resistance (Fournier et al., 1993; Mutero et al., 1994). In some strains, the point mutations "accumulated," presumably via recombination, in Ace to result in very high resistance (Mutero et al., 1994).
While the reader may be drawing the conclusion that high resistance, either novel or cross-resistance, is a foregone conclusion with a particular insecticide, this is not the case. Resistance to methoprene was minimal in field strains examined (Wilson and Thurston, 1988), and resistance to cyromazine was found to be virtually nonexistent, even in the strains highly resistant to lufenuron and propoxur (Wilson, 1997). Therefore, Cyp6g1 upregulation in field strains does not result in cross-resistance to these two chemicals, presumably because they are not substrates for the P450 enzyme.
Resistance to metal ions
While I have focused on insecticide resistance, Drosophila species have been shown resistance to other toxicants, including phytotoxins in their natural diet and certain metal ions whose presence has increased in the environment. The former has been well-studied by Fogelman (Fogleman, 2000) and offers insight into the evolution of certain Drosophila species. The latter offers another example of a response to toxicant release by human activity, and I will remark on this work. The best-studied is resistance to copper in wine-producing regions of Europe, where CuSO4 has long been used for control of plant pathogens in vineyards, and the grapes attract large populations of D. melanogaster. In laboratory trials cultures from these populations show resistance to toxic levels of copper incorporated into the diet (Maroni et al., 1987). The resistant flies have been biochemically examined and found to have elevated levels of metallothionein, a small cysteine-rich protein that sequesters heavy metal ions to facilitate detoxification. Genetically, one of the metallothionein genes, Mtn, was found to be duplicated in certain resistant strains, leading to higher MTN levels and resistance. The occurrence of Mtn duplications was higher in strains from Europe and North America and none of the strains examined from tropical Africa showed the duplication, suggesting that the resistance resulted from environmental metal ion contamination selection (Maroni et al., 1987). Gene duplication in response to environmental changes is not common in D. melanogaster, and this response offers an opportunity to understand the mechanism of gene duplication.
Resistance in D. simulans
Several studies have examined resistance in D. simulans, a sympatric sibling species that is very similar to D. melanogaster, having separated evolutionarily only 2–3 million years ago. Their geographical ranges overlap, and both species occur at common locales where insecticide usage can be heavy. Yet, the two species have responded differently at one locale near Tampa, Florida in the U.S. D. simulans was shown to be as much as 100-fold more resistant to the OP malathion than D. melanogaster (Windelspecht et al., 1995). An examination of why D. simulans, but not D. melanogaster, is responding so strongly may provide insight into how nontarget species evolve resistance.
However, D. simulans has shown a common mechanism to D. melanogaster in other populations. A strain taken in Brazil showed resistance due to upregulation of the Cyp6g1 ortholog (Le Goff et al., 2003). Whether an Accord element is associated with this upregulation as in world-wide D. melanogaster strains remains to be seen.
Fitness
Mutations in certain genes, such as those involved in longevity or reproduction, can lower the fitness of an organism. Since the target molecules for insecticides are nearly always vital genes, one might predict that mutations in these genes that confer resistance would lower the fitness of the insects. In general, however, resistant strains of D. melanogaster are remarkably fit. This seems evident by the persistence of the Rdl allele in U.S. populations for many years following the withdrawal of cyclodienes from widespread usage. In the absence of a chemical(s) maintaining the resistance allele frequency (by killing flies carrying the susceptible allele), this observation suggests that the point mutation in the GABA receptor has little or no effect on the normal functioning of the GABA receptor protein as it relates to the fitness of flies carrying it. Similarly, the previously described R(1)DDT allele found world-wide appears to have little or no effect on the fitness of the flies carrying it. When husbanded in the laboratory, strains carrying this allele show survival and reproductive traits similar to those of wild-type flies (T.G.W., unpublished data). Certainly, mutations in resistance genes that result in resistance but lower fitness are unlikely to persist in field populations unless they remain under strong selection pressure. This was shown after examination of a collection of D. melanogaster sodium channel para mutants that were resistant to DDT. Sequence analysis of each allele showed the lesion in some to be analogous with those found for other insects in field populations, but other lesion sites to be novel (Pittendrigh et al., 1997). Perhaps this result together with the paucity of Rdl mutations found in field populations of pest insects is an indication that field populations can tolerate very few amino acid changes in either the sodium channel or GABA receptor protein and still retain reasonable fitness.
Since resistant insects created in the laboratory can be nursed with care, they are exempted from the survival challenges of those in field populations. Consequently, they may have fitness deficits that result from mutations that would not be recovered from field strains. Using mutagenesis techniques, it is possible to create strains that have high resistance but suffer from fitness deficits. The Met27 allele was recovered following -ray mutagenesis, and homozygotes show high resistance to JH insecticides. A molecular analysis of Met27 showed that the mutation in the 5' end of the gene results in a total failure of gene expression. Met27 is thus a null allele of Met, and examination of the phenotype revealed a strong depression in oogenesis (Wilson and Ashok, 1998). Thus, null alleles of Met arising in field populations would be unlikely to persist in more than trivial allele frequency.
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SYNOPSISINTRODUCTIONINSECTICIDE RESISTANCENONTARGET ORGANISMS: DROSOPHILAFUTURE DIRECTIONSReferences |
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D. melanogaster populations have responsed to environmental factors, presumably insecticides, by evolving resistance. This response is reasonably well understood both biochemically and genetically. The advantages that this insect possesses for both genetic and biochemical analysis has contributed to the success of these analyses to no small extent, and they will continue to be important for further studies, such as microarray analyses of resistance (Daborn et al., 2002
).
As a sentinel for toxic compounds in the environment, how useful is this insect? Other organisms, both aquatic and terrestrial, have been shown to act as environmental sentinels of ecosystem health (Beeby, 2001). Most of these organisms have a limited range of movement and in fact some, such as mollusks, are sessile (Irato et al., 2003), allowing a relatively straightforward association with levels of toxins in the locale. D. melanogaster would represent a long-term response through resistance evolution and population change; thus, this insect might better reflect continued exposure to a toxin.
To better understand this response and possibly utilize its sentinel value, I believe that work has to focus on two areas that currently hinder our interpretations of resistance evolution in this insect.
Migration
Gene flow in this species is not well-understood, especially over small (<10 km) distances. Migration studies, especially on small insects such as D. melanogaster, are difficult to carry out. Moreover, migration distances are likely to vary with respect to geography, climate, and the impact of human activities, such as movements of foodstuffs, that may include D. melanogaster as hitchhikers. However, without migration estimates, measurement of the response of D. melanogaster especially at a local level to an environmental toxin is likely to harbor much guesswork.
It is clear from the spread of the Cyp6g1 resistance allele that D. melanogaster migration, either independent of or resulting from human activity, is occurring worldwide. It has been suggested that CYP6G1 enzyme has a broad substrate specificity, enabling selection from a variety of xenobiotics, depending on the locale, thus maintaining the allele at a relatively high frequency (Daborn et al., 2002). However, although widespread, this resistance allele is by no means fixed in global terms (Daborn et al., 2002). Resistance resulting from genes such as Cyp6g1 that harbor high cross-resistance will be less useful for interpreting a response to specific environmental chemicals than will target-site resistance loci that are specific for the chemical in question.
Maintenance of resistant alleles
A better understanding of the forces maintaining resistance alleles in a population is needed. When cultures from field populations are husbanded in the laboratory and found to be resistant to one or more insecticides, there is seldom a problem maintaining these cultures using minimal husbandry procedures. Even in the apparent absence of insecticide, field populations often maintain high resistance allele frequencies, as demonstrated for the Rdl allele. Are other xenobiotics acting to maintain selection pressure on these populations and maintaining the resistant allele frequency, or is the fitness of resistant flies so high that the allele persists in a population even in the absence of selection pressure? These questions are also difficult to answer, but studies to measure the relative fitness of flies carrying a resistant allele can be undertaken by competition studies of these flies in competition with susceptible ones. These studies can either be carried out in laboratory cultures or preferably in a field environment.
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ACKNOWLEDGMENTS |
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Work in my laboratory on juvenile hormone is funded by the National Science Foundation (IBN 0322136) and the National Institutes of Health (AI052290 [GenBank] ).
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FOOTNOTES |
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1 From the Symposium EcoPhysiology and Conservation: The Contribution of Endocrinology and Immunology presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 5–9 January 2004, at New Orleans, Louisiana.
2 E-mail: wilson.1457{at}osu.edu
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SYNOPSISINTRODUCTIONINSECTICIDE RESISTANCENONTARGET ORGANISMS: DROSOPHILAFUTURE DIRECTIONSReferences |
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