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Insecticide resistance: from mechanisms to management*
The scale of the problem
Butterflies and moths
Tackling fly pests
A continuing need for insecticides
Mechanisms of resistance
Aphids
Mosquitoes
Studies in fruit flies
The genomics approach
Future management strategies
Changing industrial perspectives
Predicting resistance
Malaria control
Transgenic plants
Broader lessons
The need for integrated pest management
Socio-economic factors
Summary
All pests can develop resistance to the insecticides which are used to control them. Insecticide resistance causes losses in food and fibre crops and has an impact on public health, for example in malaria control. The insecticide acts as an environmental pressure, selecting for populations of insects that are most able to survive its application. The genes for resistance may then spread among the local insect population, and even around the world. The phenomenon is then an example of evolution in action, illustrating how selective forces, genetic variability, gene flow, migration and life history can interact to produce changes in gene frequency.
Insecticide resistance has undermined any control programme that fails to take account of this evolutionary process and, therefore, represents a considerable challenge to industry, farmers and the research community. Many tools for managing resistance have been developed, from new insecticide chemistries to transgenic plants bearing the genes for natural insecticides. However, single strategies all have the potential for failure and may exacerbate existing resistance problems. The best hope for saving future food and fibre crops, and safeguarding health therefore lies in adopting integrated pest management strategies, where many different tools are used to delay the emergence of resistance.
The lessons of insecticide resistance may also be applicable to other areas of science - in antibiotic resistance, for example, or for predicting how animals and plants may respond to the stress of climate change.
The scale of the problem
All chemical insecticides, to a greater or lesser extent, exert a selective evolutionary pressure upon the insect pests they are intended to control. Therefore, over a period of time, resistant strains of insects are certain to emerge. The time to resistance depends on a number of factors, including the frequency and nature of resistance genes, pest management strategies, and the relative fitness of the resistant strains relative to the wild type (which is still sensitive to the insecticide in question). Currently, around 500 species of insect pest are resistant to one or more common insecticides. This includes pests on important food and fibre crops, such as cotton, and public health pests such as mosquitoes which carry disease.
Butterflies and moths
Butterflies and moths (heliothine lepidoptera) are among the world's most important pests. The damage they inflict has been reviewed by Dr Alan McCaffery of the University of Reading, with particular reference to resistance to pyrethroid insecticides. These compounds affect the insect's nervous system, causing paralysis and death.
Cotton bollworm (Heliocoverpa armigera) is a major pest in the Old World where it attacks cotton, maize, groundnut, sorghum and vegetables. Cotton bollworm (Helicoverpa zea) is an important pest on maize, sorghum, cotton and tomato in the New World. A third important species is tobacco budworm (Heliothis virescens) which is a pest of tobacco, cotton, sunflower, soya and other species in the New World.
As far as resistance is concerned, H armigera is the source of most problems in the Old World, with H virescens being the major species showing resistance in the New World. Pyrethroid resistance in H armigera was first detected in the late 1970s in the US, in 1983 in Australia, and more recently in other countries, notably India, Pakistan and China. However, there has not been resistance in Israel, largely because a management strategy there has been able to mitigate the problem. There are serious resistance problems with tobacco budworm in the US, while H zea is still under control.
Many resistance problems with these species arise on cotton which is a high-value crop, spending a lot of time in the ground - a factor which contributes to selection pressure. The pests feed on the cotton boll and this causes decreased yields all around the world. Indeed, an unparalleled variety and quantity of insecticides are used on cotton with up to 20% of global insecticide use being devoted to the control of cotton pests.
Dr Mike Broadhurst of Zeneca Ag Products, California, presented an analysis of the extent of the H virescens resistance problem. The toxicity of four different insecticides, with three different modes of action, to lab and field strains of H virescens, was assessed in 1993 (see figure 1). While the lab strains were still susceptible, those collected in the field had developed considerable resistance. 1995 was one of the worst years in history for control of this pest in the US with some farmers in Alabama and Mississippi losing their entire crop. However, the next two seasons showed some improvement thanks to the introduction of new tools for control, such as BollgardØ, Monsanto's transgenic cotton which carries the gene for a Bacillus thuringiensis (Bt) toxin. The gene makes an insecticidal protein which kills pests by destroying their gut wall.
Toxicity of Various Insecticides to Field Collected Heliothis virescens 3rd Instar Topical Bioassay:
Average % Mortality of 8 Populations - Louisiana, 1992
JB Graves et al 1993 Proceeding Beltwide Cotton Production
Conf pp 788-90
Tackling fly (homopteran) pests
The cotton whitefly, or Bemisia tabaci, is another widespread pest. Twenty years ago it emerged from relative obscurity in the tropics and subtropics and underwent a massive extension in its geographical range. It is now a key pest in the field on both food and fibre crops, and on vegetables and ornamentals under glass.
Whitefly has even attained notoriety in the press as a 'superbug'. Several factors have contributed to its success. The adults are especially fecund, laying between 50 and 400 eggs on the underside of a host leaf which then hatch into mobile crawlers. The pest has over 500 different hosts and is particularly well dispersed. It feeds on phloem, the tissue which carries nutrients throughout the plants. It is also a carrier of gemini viruses which are important plant pathogens, and so poses a double threat to the host.
Dr Ian Denholm of the Institute of Arable Crops Research in Rothamsted and colleagues have been studying the emergence of resistance in whitefly around the world. Development of resistance depends upon the crops surrounding those that are sprayed and when crops are planted relative to one another. For instance, in the desert areas of the south west US, resistance may be accelerated by treating vegetables planted before and after the sprayed cotton crop, or delayed by the presence of alfalfa which is untreated and present all year round. The alfalfa acts as an important 'refuge' for susceptible insects where they are not subject to the same selection pressure as the treated crop.
Another key issue is that greenhouse crops seem to accelerate resistance with species such as peppers and gerbera (for the cut flower trade) being intensively sprayed; often the first reports of resistance to various insecticides have been reported under such circumstances. There is also evidence of the movement of resistance genes around the world via the pot plant trade on, for example, poinsettias.
A continuing need for insecticides
Despite the problems they cause, insecticides do save crops according to Dr Broadhurst (see figure 2). Crop protection, along with irrigation, fertilisers, and breeding techniques, has played an important role in today's food and fibre supply. Pesticides also reduce storage losses and improve food quality, reduce soil erosion, as well as controlling public health pests such as mosquitoes.
The role of crop protection is likely to become even more important in the future. World population is projected to reach around 11 billion by 2025-2050 while the amount of land available to grow food and fibre crops will continue to decrease from 0.29 hectares per head today to, potentially, less than 50% of this area. Moreover, the population has become more sophisticated, demanding more quality and variety in foodstuffs.
Crop production and crop protection EC Oerke et al Elsevier 1994
Mechanisms of resistance
There are two broad mechanisms by which insect pests develop resistance to insecticides. They may produce large amounts of enzymes, such as esterases which either break down the insecticide molecule or bind to it so tightly that it cannot function (a process known as sequestration). The second mechanism involves mutation of the insecticide target site, such as the acetylcholinesterase enzyme in the nervous system. This effectively blocks the action of the insecticide. Both types of mechanism have been studied in various species of insect.
Aphids
Professor Alan Devonshire and colleagues at the Institute of Arable Crops Research in Rothamsted have identified both types of mechanism in the peach-potato aphid (Myzus persicae), an important pest worldwide which is resistant to a wide range of insecticides.
Some resistant strains of the aphid make excess amounts of esterase enzyme. In some cases, the overproduction is up to 70 times more than the wild type so that it makes up 1% of the aphid's total body protein. This over-production has been shown to involve amplification and increased expression of esterase genes.
Resistance to dimethylcarbamates, an important class of aphicides, develops through modification of the acetylcholinesterase target. This mechanism can occur in combination with the esterase mechanism - giving the resistant strain two ways of blocking the insecticide. Field studies are revealing just where in the UK this target modification mechanism has emerged in the last three years; this knowledge is important because it tells us where use of this class of insecticide should be avoided so that the resistance problem is not exacerbated.
Mosquitoes
Professor Janet Hemingway and colleagues at the University of Wales, and Dr Michel Raymond and his team at the University of Montpellier in France, have been studying resistance mechanisms in Culex mosquitoes which cause tropical diseases such as filariasis (in which parasitic worms invade the lymphatic system causing swelling of surrounding tissue). The mosquitoes act as an intermediate host for the nematode worms which are the infective agent in filariasis. As with aphids, the amplification of esterase genes is important in the resistance of mosquitoes to a wide range of insecticides. According to Professor Hemingway, either esterases with enhanced binding to insecticide molecules are preferentially amplified or such genes are pulled out of an amplified pool by natural selection. Either way, there is more to the resistance mechanism than amplification in aphids for example, differential regulation of the different esterases also seems to be important - with those that are already amplified perhaps also having enhanced expression.
Dr Raymond has studied the evolution and spread of Culex resistance genes in the Montpellier area over the last 23 years; the two resistance mechanisms are overproduced esterase and altered sensitivity to the acetylcholinesterase target. Methods of mosquito control in the region have changed - from using DDT in the early years through to organophosphates and then, more recently, Bacillus sphaericus, a method of biological control. The Montpellier team has looked at how the cost to the insect of specific resistance genes, along with environmental pressure exerted by control methods, have affected the frequency distribution of particular resistance genes over time. While population studies suggest that mutation events conferring resistance have only arisen a few times, mutants have migrated extensively so that a combination of the genes A2 and B2, for instance, which causes resistance through the esterase mechanism, now has a worldwide distribution.
Studies in fruit flies (Drosophila)
Dr Jean-Baptiste Bergˇ and his team at the Institut National de la Recherche Agronomique in Antibes, France, have studied a further mechanism by which resistant insects break down insecticides. The cytochrome P450 enzyme system is present in many organisms including humans. It metabolises xenobiotics (substances not found in nature) such as insecticides by oxidising them. In Drosophila, resistance can be overcome by inhibiting P450. Evidence from genetic and molecular biological studies suggests that overexpression of the P450 gene is associated with resistance. However, this is not the full story as resistance is not always lost if overexpression disappears. Comparison of the P450 genes in both susceptible and resistant Drosophila shows that three different point mutations of the gene in the latter give rise to an enzyme with higher specific metabolic activity towards insecticides. Therefore P450-based insecticide resistance is based on both overexpression and mutation to a more active form.
The genomics approach
Genomics - the comparative study of the structure and function of entire genomes - is proving to be a powerful tool for elucidation of pesticide resistance mechanisms. Dr David Heckel of Clemson University, South Carolina, has been working on a genetic linkage map of tobacco budworm. A complete physical map of this pest is also being constructed. This will facilitate the complete analysis of the multigene mechanisms which can be so important in insecticide resistance. The map has revealed sites on the genome which are close to genes conferring resistance to organophosphates, carbamates and pyrethroid classes of insecticide. The approach is now being extended to cotton bollworm using a whole genome scan, and so testing every possible chromosome for linkage to resistance. Genomics is also being used to find genes for resistance to the Bt toxin, to discover strategies for combating resistance to Monsanto's transgenic cotton. Comparisons between the genomes of the silk moth, the fruit fly and tobacco budworm should be particularly fruitful and may even speed up the search for insecticidal targets for important pests.
Future management strategies
The future will undoubtedly see a move towards integrated pest management, bringing in a better balance of biological control agents, transgenics and chemicals. The use of multiple tools should decrease the selection pressure towards any one component.
Changing industrial perspectives
Attitudes within the chemical industry are changing. Consideration of the mechanisms of resistance now have a big impact on the research and development of new chemicals, according to Dr Broadhurst. Up to 20% of promising candidates might be rejected at an early stage because of the likelihood of developing cross-resistance and therefore shortening the lifetime (and financial returns from) the chemical. For instance, Zeneca recently abandoned a potential insecticide in the new aryl heterocycle class because cross-resistance to the cyclodiene insecticides, widespread among public health pests, appeared likely.
There is also increased cooperation between companies in an effort to anticipate resistance problems. Recently, four companies operating in Europe consulted prior to the launch of a new class of acaricide (mite killer) which each planned to market. There were impending problems of cross-resistance in which resistance to one compound would most likely confer resistance to one or more of the others. To avert this, the companies agreed to warning labels being distributed with the products which advise farmers on how best to use the chemicals to delay the emergence of cross-resistance.
Predicting resistance
Too often, management of insecticide resistance has been left until the problem becomes apparent. Professor John McKenzie of the University of Melbourne, Australia, has investigated whether the emergence of resistance can be predicted in advance so that management decisions can be taken earlier when they are more likely to be effective. He has modelled the interaction of Lucilia cuprina, the Australian sheep blowfly, with the various insecticides which have been used against it in the field.
Dieldrin, introduced in 1955, was the first insecticide used against L cuprina. Resistance set in only two years later and a second chemical, diazinon, was introduced in 1957. Diazinon resistance was rather slower to develop. A third compound, cyromazine, has been in use for 20 years, but resistance has not developed.
Professor McKenzie's studies involved mutagenesis to generate single gene variants resistant to dieldrin and diazinon; the genetic and molecular basis of resistance was identical to that seen in the field. Furthermore, analysis of cyromazine mutants suggests an explanation why this insecticide does not induce resistance. If cyromazine, and this information relating to it, had been available in the 1950s, it would have been used in preference to dieldrin and diazinon on the basis of their relative likelihood for selection for resistance in their target pest.
Malaria control
There are 300-500 million cases of malaria a year worldwide leading to 1.2-1.6 million deaths, 80-90% of which are in Africa. This makes the vector of this disease, the mosquito Anopheles gambiae, the world's most dangerous animal, according to Professor Chris Curtis of the London School of Hygiene and Tropical Medicine. However, pyrethroid-impregnated bed nets have been shown to be an effective means of control with protection lasting 6-8 months. They stop the mosquito from entering if there are holes in the net, they generally decrease the density of mosquitoes in the surrounding community by acting as traps and the insecticide has an irritant effect which may drive the mosquitoes away.
Studies by the Medical Research Council in the Gambia show a 63% decrease in child mortality from all causes where treated bed nets are used. Professor Curtis has investigated whether use of the nets could lead to pyrethroid resistance. Studies in China and Tanzania suggest that there is no increase in 'knockdown' time of mosquitoes with length of time the net has been in use, although other data from Kenya shows that there may be a resistance problem. Further studies show that lower doses of pyrethroid are less likely than higher ones to select resistance genes probably because they are less irritating to the mosquito so it stays on the net for longer and is so more likely to be killed. A further strategy for blocking the spread of the resistance genes, is to give an insect growth regulator alongside the pyrethroid to reduce the fecundity of adult female mosquitoes. So far this appears to lead to reduction in the number of eggs laid but not to complete sterility.
Transgenic plants
For several years now, the bacterium Bacillus thuringiensis (Bt) has been used as a method of biological pest control. This natural pathogen produces an insecticidal toxin which leaves humans and beneficial insects unaffected. The toxins form a family of proteins and 135 different Bt toxin genes have been sequenced to date. Of these, only a few will be toxic to any one pest species. The toxins are produced as crystalline inclusions during sporulation of the bacteria. On ingestion, they dissolve in the midgut of a susceptible insect and then bind to sites on the gut membrane. Once bound they cause pore formation and then bursting of cells which kills the insect.
An exciting development over the last few years has been the engineering of the Bt toxin genes into many crop plants so they can produce their own natural insecticide. Currently 9 million acres of Bt-expressing transgenic cotton, corn and potatoes have been planted in the US, and there have been field trials of 15 more crops engineered with Bt, with the potential for many more.
Detecting and understanding resistance to Bt in the field will be crucial to optimal deployment of these transgenic plants. Professor Bruce Tabashnik of the University of Arizona detected resistance to Bt in the diamond-back moth (Plutella xylostella) in Hawaii as long ago as 1986. This moth feeds on crucifer species and is a worldwide pest causing damage whose estimated cost totals $1 billion per annum. More recently Professor Tabashnik and colleagues have identified and studied Bt resistance in three separate moth populations from Hawaii, Pennsylvania and the Phillipines. Genetic experiments suggest that the mechanism of resistance in the Hawaii and Pennsylvania populations involves reduced binding of the toxin to the mid gut. Resistance in the Phillipines population of diamond-back moth involves a different gene, at the same genetic locus, as reduced binding to the toxin was not seen and the resistant species had a narrower spectrum of resistance (that is they were resistant to fewer toxins in the Bt family than the other two populations).
Two different strategies of managing resistance in transgenic crops were outlined by Dr Rick Roush of the University of Adelaide. The first involves creating a 'refuge' area of non-transgenic plants within a transgenic crop. The refuge, as the name suggests, is a place where susceptible insects can survive (these would be killed by the transgenic plant). If they subsequently mate with a resistant insect, the offspring will be heterozygotes (insects with one resistance gene paired with one susceptible gene) which, because the mechanism is genetically recessive, will be killed off by the toxin in the transgenic plant thereby delaying the propagation of the resistance gene.
Using high doses of insecticide should maximise kill of heterozygotes and this strategy was popular among theorists in the 1970s. But to be really effective, the dose was too high to be acceptable on either environmental or cost grounds. Another drawback was that uniform coverage of the crop was not assured. The high dose strategy could be revived though with transgenic plants if it is possible to maintain a high level of expression of the toxin gene. The other strategy is pyramiding which involves creating trangenic plants with genes for two different toxins. Insects resistant to one will be killed by the other, and vice versa. This provides a double hit strategy for seeing off heterozygotes and discouraging the spread of resistance genes. It also parallels the successful use of combination drug therapy in leprosy, TB and HIV/AIDS.
There are also toxins other than Bt which could be engineered into crops, giving growers a wider repertoire of genes to exploit in the future and therefore improve prospects for delaying resistance. Entomophagous (insect-eating) nematodes are an established method of biological pest control. The two main groups are Steinernema and Heterorhabditis. The latter is infected by a luminescent bacterium Photorhabdus luminescens. When the nematode invades an insect, the bacteria are released into its body cavity where they have a killing role whose mode of action is not fully understood. The dead caterpillars are said to emit an eerie glow from the billions of luminscent bacteria within their bodies. The bacteria can also kill directly if they are ingested by a range of insects including butterflies and moths, beetles, wasps and cockroaches.
Dr Richard ffrench-Constant of the Department of Entomology, University of Wisconsin-Madison, has isolated toxins from Photorhabdus luminescens and cloned the corresponding genes. At least one of these toxins is as potent as Bt toxins. Furthermore, it is orally active against economically important pests such the southern corn rootworm which feeds on corn in the US. Further research could result in the development of transgenic plants bearing the gene for this toxin. Only a few of the 200 or so species of Photorhabdus luminescens carry toxin genes but similar species are being investigated for toxin production.
Broader lessons
Although scientific and technical understanding of insecticide resistance has advanced dramatically over the last five years, there is still a need for a broader appreciation of the issues. The key issue that needs addressing is that resistance is an evolutionary response to stress and can only be delayed rather than controlled. This may best be done by adopting a variety of management tactics rather than relying upon only one method.
The need for integrated pest management
Professor Marjorie Hoy of the University of Florida pointed out that all single strategies for pest management - from insecticides to transgenics - cause problems such as toxic residues and secondary pest outbreaks, over time.
There is an urgent need to return to the principles of integrated pest management (IPM) as advocated by Stern in 1959, namely that pesticides should only be used as a last resort when natural mortality factors cannot stop the pest population from reaching the economic injury level.
There are several elements to IPM including pest monitoring, effective agronomic practices and the integration of chemical and biological control methods. The latter include encouraging natural predators, mating disruption, sterile insect release and developing host plant resistance.
More co-operation between companies, regulators, scientists and growers is needed, and the adoption of specific practices such as changes in labelling of insecticides could also help delay the emergence of resistance.
Socio-economic factors
Sir Robert May FRS, Chief Scientific Adviser and Head of the Office of Science and Technology as well as one of the world's leading theorists in population ecology and disease epidemiology, spoke of the socio-economic factors inherent in delaying the onset of insecticide resistance.
Susceptibility to insecticides itself should be seen as a non-renewable resource. Strategies which are used to delay the onset of resistance, such as reducing insecticide applications, involve loss of immediate benefit in terms of reduced yields. But the short term loss could be more than offset by the long term gain of delaying resistance which makes control tools useful over a longer period. Much depends on the rate at which different interest groups 'discount the future' - a poorly understood aspect of natural resources which generates genuine tensions between, say, a chemical company wishing to push a product, and those more directly concerned with producing food. There are also important public perception issues to be addressed in the area of transgenic plants.
Dr Susan Aldridge
ABSW
October 1998
Contacts
Dr Alan McCaffery
School of Animal and Microbial Sciences, University of Reading
Tel 0118 931 8464 Fax 0118 931 0180
Dr Mike Broadhurst
Zeneca Ag Products, California, USA
Tel 001 510 231 1399 Fax 001 510 231 1369
Dr Ian Denholm
Institute of Arable Crops Research, Rothamsted
Tel 01582 763133 Fax 01582 762595
Professor Alan Devonshire
Institute of Arable Crops Research, Rothamsted
Tel 01582 763133 Fax 01582 762595
Professor Janet Hemingway
Department of Pure and Applied Biology, University of Wales
Tel 01222 874261 Fax 01222 874305
Dr Michel Raymond
University of Montpellier, France
Tel 00 33 4 67 14 46 15 Fax 00 33 4 67 14 36 22
Dr Jean-Baptiste Bergˇ
Institut National de la Recherche Agronomique, France
Tel 00 33 4 93 67 89 37 Fax 00 33 4 93 67 89 55
Dr David Heckel
Department of Biological Sciences, Clemson University, USA
Tel 001 864 656 3585 Fax 001 864 656 0435
Professor John McKenzie
Department of Genetics, University of Melbourne, Australia
Tel 00 61 3 9344 6246 Fax 00 61 3 9344 5139
Professor Chris Curtis
London School of Hygiene and Tropical Medicine
Tel 0171 927 2339 Fax 0171 436 5389
Professor Bruce Tabashnik
Department of Entomology, University of Arizona, USA
Tel 001 520 621 1141 Fax 001 520 621 1151
Dr Rick Roush
Department of Crop Protection and Centre for Weed Management Systems, Waite Institute, University of Adelaide, Australia
Tel 00 61 8 8303 6591 Fax 00 61 8 8379 4095
Dr Richard ffrench-Constant
Laboratory of Toxicology, University of Wisconsin-Madison, USA
Tel 001 608 263 7924 Fax 001 608 262 3322
Professor Marjorie Hoy
Department of Entomology and Nematology, University of Florida, USA
Tel 001 352 392 1901 ext. 153 Fax 001 352 392 0190
Sir Robert May FRS
Chief Scientific Adviser and Head of the Office of Science and Technology
Tel 0171 270 1234 Fax 0171 271 2003
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