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Pyrethroid resistance is becoming widespread in Anopheles gambiae mosquitoes, coinciding with expanded use of insecticide-treated nets (ITNs) throughout Africa. To investigate whether nets in use are still protective, we conducted [...]

The evolution of insecticide resistance in mosquitoes is threatening the effectiveness and sustainability of malaria control programs in various parts of the world. Through their unique mode of action, entomopathogenic fungi provide promising alternatives to chemical control. However, potential interactions between fungal infection and insecticide resistance, such as cross-resistance, have not been investigated. We show that insecticide-resistant Anopheles mosquitoes remain susceptible to infection with the fungus Beauveria bassiana. Four different mosquito strains with high resistance levels against pyrethroids, organochlorines, or carbamates were equally susceptible to B. bassiana infection as their baseline counterparts, showing significantly reduced mosquito survival. Moreover, fungal infection reduced the expression of resistance to the key public health insecticides permethrin and dichlorodiphenyltrichloroethane. Mosquitoes preinfected with B. bassiana or Metarhizium anisopliae showed a significant increase in mortality after insecticide exposure compared with uninfected control mosquitoes. Our results show a high potential utility of fungal biopesticides for complementing existing vector control measures and provide products for use in resistance management strategies. biopesticide | DDT | pyrethoids | resistance management | vector control doi/ 10.1073/pnas.0908530106

Transposable elements are a major mutation source and powerful agents of adaptive change. Some transposable element insertions in genomes increase to a high frequency because of the selective advantage the mutant phenotype provides. Cyp6g1-mediated insecticide resistance in Drosophila melanogaster is due to the upregulation of the cytochrome P450 gene Cyp6g1, leading to the resistance to a variety of insecticide classes. The upregulation of Cyp6g1 is correlated with the presence of the long terminal repeat (LTR) of an Accord retrotransposon inserted 291bp upstream of the Cyp6g1 transcription start site. This resistant allele (DDT-R) is currently at a high frequency in D. melanogaster populations around the world. Here, we characterize the spatial expression of Cyp6g1 in insecticide-resistant and -susceptible strains. We show that the Accord LTR insertion is indeed the resistance-associated mutation and demonstrate that the Accord LTR carries regulatory sequences that increase the expression of Cyp6g1 in tissues important for detoxification, the midgut, Malpighian tubules, and the fat body. This study provides a significant example of how changes in tissue-specific gene expression caused by transposable-element insertions can contribute to adaptation.

Abstract: Apple grain aphid, Rhopalosiphum padi (Linnaeus), is an important wheat pest. In China, it has been reported that R. padi has developed high resistance to carbamate and organophosphate insecticides. [...]

Abstract: A P-glycoprotein-like protein (Ha-Pgp) was detected in a membrane preparation from the insecticide-resistant pest Helicoverpa armigera (Lepidoptera: Noctuidae) using C219 antibodies that are directed towards an epitope in the [...]

Neotropical poison frogs (Dendrobatidae) contain a variety of lipophilic alkaloids in their diffusely distributed cutaneous glands, including a major class of compounds known as pumiliotoxins. Pumiliotoxins are highly toxic and are believed to protect frogs against predators. Their potential activity against ectoparasites, however, has not been investigated. We tested female yellow fever mosquitoes (Aedes aegypti) for responses to 8-hydroxy-8-methyl -6-(2'-methylhexylidene)-1-azabicyclo[4.3.0]nonane, designated pumiliotoxin 251D [PTX (+)-251D], a skin alkaloid present in all genera of dendrobatids and in other anurans, and to its unnatural enantiomer, PTX (-)-251D. Both enantiomers of PTX 251D presented on silicone feeding membranes reduced landing and feeding by A. aegypti, but PTX (+)-251D did so at lower concentrations. PTX (+)-251D also induced toxicosis, shown when mosquitoes failed to fly off membranes. Similarly, mosquitoes confined with copper wires coated with PTX (+)-251D exhibited greater latencies to fly off the substrate and a higher incidence of leg autotomy than did those confined with the (-)-enantiomer. Our results on the contact toxicities of PTX 251D enantiomers parallel those reported for mice injected with them. The presentation of serial dilutions of PTX (+)-251D to A. aegypti revealed a minimum toxic concentration of 0.1 [micro]g/[cm.sup.2]. This value is substantially lower than that estimated for the cutaneous abundance of this compound in some frogs, an observation consistent the function of PTX 251D in anuran chemical defense against ectoparasitic arthropods. alkaloids | chemical defense | dendrobatid poison frogs | insecticidal activity | skin secretions

Lufenuron is an insect growth regulator insecticide mainly used for the control of the cat flea. To understand mechanisms of resistance to lufenuron, we have characterized lufenuron resistance in a natural population of Drosophila melanogaster. In this study we have used precise genetic mapping to identify a mechanism of lufenuron resistance: the overexpression of the cytochrome P450 gene Cyp12a4. Cyp12a4 is predicted to encode a mitochondrial cytochrome P450 enzyme. Expression of Cyp12a4 in D. melanogaster third-instar larvae was detected in the midgut and Malpighian tubules of both lufenuron-resistant and wild-type strains. The level of Cyp12a4 expression in the midgut is higher in the lufenuron-resistant strain than in wild-type strains. Driving the expression of Cyp12a4 in the midgut and Malpighian tubules by using the GAL4/UAS gene expression system results in lufenuron resistance, but it does not result in resistance to three other insecticide classes. Transgenic expression of Cyp12a4 in a ubiquitous expression pattern results in late embryonic lethality, suggesting that high-level ectopic expression of Cyp12a4 is detrimental to development. cytochrome P450 | insecticide resistance | insect growth regulator | genetic mopping

Metabolic pathways play an important role in insecticide resistance, but the full spectra of the genes involved in resistance has not been established. We constructed a microarray containing unique fragments from 230 Anopheles gambiae genes putatively involved in insecticide metabolism [cytochrome P450s (P450s), GSTs, and carboxylesterases and redox genes, partners of the P450 oxidative metabolic complex, and various controls]. We used this detox chip to monitor the expression of the detoxifying genes in insecticide resistant and susceptible An. gambiae laboratory strains. Five genes were strongly up-regulated in the dichlorodiphenyltrichloroethane-resistant strain ZAN/U. These genes included the GST GSTE2, which has previously been implicated in dichlorodiphenyltrichloroethane resistance, two P450s, and two peroxidase genes. GSTE2 was also elevated in the pyrethroid-resistant RSP strain. In addition, the P450 CYP325A3, belonging to a class not previously associated with insecticide resistance, was expressed at statistically higher levels in this strain. The applications of this detox chip and its potential contribution to malaria vector insecticide resistance management programs are discussed. mosquito | cytochrome P450 | GST | carboxylesterase

Commercial varieties of canola (Brassica napus L. and B. rapa L.) are susceptible to infestations by the root maggots Delia radicum (L.) and Delia floralis (Fallen) (Diptera: Anthomyiidae) in western Canada. Although cultural strategies can ameliorate crop damage from root maggot infestations, these methods are not sufficiently effective to prevent substantial economic losses. In this paper we report the development of germplasm for resistance to root maggot infestations and the introgression of genes from a resistant relative (Sinapis alba L.) to susceptible B. napus. The effectiveness of the conferred resistance to root maggot damage was validated by comparing different genotypes for pest damage and yield loss with and without insecticide applications. Yield of B. napus plants, susceptible to root maggot infestations, increased when insecticide was applied (by up to 24%), but no significant yield differences were observed among resistant intergeneric hybrids that were treated or not treated with insecticide. One hundred and thirty-five restriction fragment length polymorphisms (RFLPs) were used to construct a B. napus genetic linkage map and to identify quantitative trait loci (QTLs) associated with resistance to root maggot damage. Two QTLs, RM-G8 and RM-G4, were found to be associated with resistance to root maggot damage. Together, these two QTLs explain 54.6% of the total variation observed., IN WESTERN CANADA, infestations of root maggots are responsible for substantial economic losses in the production of canola (Liu and Butts, 1982; Griffiths, 1986, 1991; Dosdall, 1998). Primary plant damage [...]

Aflatoxin is a carcinogenic and toxic compound produced by the fungus Aspergillus flavus (Link:fr) that can be found at detrimentally high concentrations in maize (Zea mays L.) grain. Screening has led to the discovery of sources of resistance to aflatoxin accummulation in maize, but associated poor agronomic characteristics and complex inheritance have limited transfer of resistance to elite inbreds. A set of 210 [F.sub.2:3] families derived from a cross between inbred lines Mp313E (resistant) and B73 (susceptible) was evaluated in replicated trials in four environments for resistance to aflatoxin accumulation. Families were also genotyped using simple sequence repeal (SSR) markers to develop a genetic map for quantitative trait loci (QTL) analysis. Composite interval mapping (CIM) was used to identify 2, 3, 5, and 3 QTL within the tests at Stoneville (2000) and Mississippi Stale (2000, 2001, 2002), respectively. The QTL were primarily additive in nature, with Mp313E contributing to reduced aflatoxin concentration in all but one case. Two QTL regions were significant in at least three environments. The afl3 locus, represented by marker bnlg371, was located on chromosome two and accounted for 7 to 18% of variation in aflatoxin levels depending on environment. The afl5 locus, represented by marker bnlg2291, was located on chromosome four, with explained variance ranging from 8 to 18%. This QTL has been noted in earlier studies whereas afl3 is new. Identified QTL confirm important regions influencing aflatoxin accumulation previously identified and present new ones of equal effect., AFLATOXINS ARE TOXIC secondary metabolites produced by the fungal pathogen Aspergillus flavus. These toxins have carcinogenic, immunosuppressive, and hepatotoxic properties in animals (Castegnaro and McGregor, 1998). Aflatoxin B1 is the [...]

Throughout the 20th century, scientists developed a variety of chemical compounds to kill insects and other menaces of agriculture and public health. Yet, in many, cases, the target insects outmaneuvered the scientists by developing resistance to insecticides--in much the same way as some bacteria can tolerate antibiotics. Insecticide resistance research has involved scientists from a range of disciplines and a variety of institutional contexts that have often guided research strategy. For example, entomologists working at agricultural stations and universities concentrated on insect physiology and evolutionary genetics, while industrial chemists continued the development of novel compounds capable of killing resistant pests. Collaboration between the two groups beginning in the 1940s did not provide a solution to the resistance, but did lead to a strategic shift from pest control to pest management that continues to the present.

The inheritance of deltamethrin resistance in the codling moth (Lepidoptera: Tortricidae) has been studied with attention to the number of genes involved. The hypothesis that there is a polygenic response to deltamethrin selection in the resistant strain is supported. The resistance seen in southeastern France appears to come from an adaptive sequential selection process though the sequential addition of resistance genes.

Two forest pests appear to become partially resistant to the insect toxins produced by Bacillus thuringiensis. Both Thaumetopoea pityocampa and Lymantria monacha lose binding sites for the Cry1Ab toxin during larval development.

The antagonistic roles of selection and migration involved in local adaptation were examined with regard to allelic frequencies at two resistance loci in the mosquito Culex pipiens. Their frequencies were found to follow an annual cycle of large amplitude and the results provide a firm base on which insecticide treatment strategies could be developed that would give better control of the evolution of resistance genes and the growth of populations of mosquitos.

Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton boll-worm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000-10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.

Insecticidal proteins from the soil bacterium Bacillus thuringiensis (Bt) are becoming a cornerstone of ecologically sound pest management. However, if pests quickly adapt, the benefits of environmentally benign Bt toxins in sprays and genetically engineered crops will be short-lived. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to Bt in open-field populations. Here we report that populations from Hawaii and Pennsylvania share a genetic locus at which a recessive mutation associated with reduced toxin binding confers extremely high resistance to four Bt toxins. In contrast, resistance in a population from the Philippines shows multilocus control, a narrower spectrum, and for some Bt toxins, inheritance that is not recessive and not associated with reduced binding. The observed variation in the genetic and biochemical basis of resistance to Bt, which is unlike patterns documented for some synthetic insecticides, profoundly affects the choice of strategies for combating resistance.

Cry proteins produced by Bacillus thuringiensis are selective biodegradable insecticides used increasingly in bacterial insecticides and transgenic plants as alternatives to synthetic chemical insecticides. However, the potential for development of resistance and cross-resistance in target insect populations to Cry proteins used alone or in combination threatens the more widespread use of this novel pest control technology. Here we show that high levels of resistance to CryIV proteins in larvae of the mosquito, Culex quinquefasciatus, can be suppressed or reduced markedly by combining these proteins with sublethal quantities of CytA, a cytolytic endotoxin of B. thuringiensis. Resistance at the [LC.sub.95] level of 127-fold for a combination of three CryIV toxins (CryIVA, B, and D), resulting from 60 generations of continuous selection, was completely suppressed by combining sporulated powders of CytA in a 1:3 ratio with sporulated powders of a CryIVA, CryIVB, and CryIVD strain. Combining the CytA strain with a CryIVA and CryIVB strain also completely suppressed mosquito resistance of 217-fold to the latter toxins at the [LC.sub.95] level, whereas combination of CytA with CryIVD reduced resistance in a CryIVD-selected mosquito strain from greater than 1,000-fold to less than 8-fold. The CytA/CryIV model provides a potential molecular genetic strategy for engineering resistance management for Cry proteins directly into bacterial insecticides and transgenic plants.

The European corn borer, Ostrinia nubilalis (Hubner), is a devastating insect pest in the corn (Zea mays L.) growing regions of North America and Europe. Field evaluations in the USA and Belgium showed that transgenic corn events expressing Cry9c, an insecticidal crystal protein from Bacillus thuringiensis subsp. tolworthi, very effectively control both generations of the European corn borer. Second to fourth instar larvae fed with leaf material of event CBH351 expressing the Cry9C protein all died within 4 d. Cry9C events, expressing high levels of the insecticidal protein, showed minimal stalk tunneling after heavy artificial infestations. Event CBH351 tested in plots containing only Cry9C transgenic plants had 0.14- and 0.09-cm tunneling per stalk compared with more than 30- and 23-cm tunneling per stalk for the negative controls, in the Belgium and Iowa field trial, respectively. In plots containing 30% non-transgenic plants the event CBH351, showed only 1.45-cm tunneling per stalk. Leaf, tassel, and pith tissue contained 39.0, 17.4, and 84.8 [micro] g Cry9C protein [mg.sup.-1] soluble protein, respectively, in analyses conducted at harvest of the Belgium trial. The implications of Cry9C use for resistance management strategies are discussed., The european corn borer is a devastating insect pest in the corn growing regions of North America and Europe (Beck, 1987; Hudon et al., 1987). In the Corn Belt and [...]

Microbiological research shows that insecticidal toxins produced by the microorganism Bacillus thuringiensis subspp. kurstaki and tenebrionis retain their toxicity when bound to montmorillonite and kaolinite homoionic clay in the presence of reduced nitrogen. The insecticidal toxins exhibited toxic activity for up to forty days following their exposure to nonsterile soil. Thus, toxins may build up in soils following harvest.

A mutation proximal to a voltage-sensitive sodium channel gene caused the kdr insecticide resistance tract, observed in the house fly Musca domestica. Insecticides such as pyrethroids and 1,1, 1-trichloro-2,2-bis (p-chlorophenyl) ethane (DDT), which cause rapid paralysis when sprayed on insects, are ineffective in house flies that possess the kdr insecticide resistance trait.

Crop yield and quality could be affected in transgenic crop plants carrying foreign genes which confer resistance to plant pests. Scientists at Monsanto Company produced nine transgenic cotton (Gossypium hirsutum L.) lines, each containing one of the two Bacillus thuringiensis var. kurstaki (Berliner) constructs, cryIA(b) or cryIA(c), that encode a protein toxic to lepidopteran insects. We undertook this study to determine whether these transgenic lines exhibit any undesirable agronomic or fiber properties. At Maricopa, AZ, we grew five, four, and three transgenic lines in field plots in 1990, 1991, and 1992, respectively; the parental 'Coker 312' and the locally adapted, nectariless, high fiber-strength 'Md 51 ne' were included each year. Somaclonal variation, insertion of the insect-control protein genes, and the expression of the insecticidal protein caused no general reduction in lint yield, primary or secondary yield components, or fiber properties. The transgenic lines varied significantly in every trait measured. Mean lint yields over the 3 yr were as follows: transgenic lines, 135.4 + or - 4.5 g m to the -2; Coker 312, 115.1 + or - 7.7 g m to the -2, MD 51 ne, 141.2 + or - 11.4 g m to the -2. Six of the nine transgenic lines yielded significantly more lint (13-30%) than did Coker 312. Lint yields of the same six lines (10% less to 7% more) were not significantly different than that of MD 51 ne. Compared with Coker 312, four of nine transgenic lines had longer fiber, eight had stronger fiber, two had higher fiber elongation, and two did not differ significantly in micronaire. Compared with MD 51 ne, five of the nine transgenic lines did not differ significantly in fiber length, two did not differ in strength, three did not differ in elongation, and six did not differ in micronaire. These transgenic lines are valuable sources of germplasm, not only for insect resistance but also for agronomic and fiber properties.

Two headwater streams draining catchments 53 and 54 (C53 and C54, respectively) at the Coweeta Hydrologic Laboratory in western North Carolina were treated with insecticide in 1980 (C53) and 1986-1988 (C54). During recovery periods in both streams, Lepidostoma spp. were abundant, early colonizers. Densities of Lepidostoma in litterbags and benthic samples collected from recovering streams were substantially higher than in untreated streams (pretreatment and reference streams). During treatment years, leaf-litter processing rates were severely reduced relative to pretreatment and reference streams. In contrast, litter processing rates in C53 and C54 during recovery were faster than those in untreated streams (pretreatment and reference). Rhododendron is one of the most refractory leaves commonly found in Coweeta streams; however, percent increase of rhododendron processing rates from treatment periods to recovery was greater than that of more labile red maple. Laboratory feeding experiments were performed in order to examine and quantify use of rhododendron and red maple litter by Lepidostoma larvae. In the laboratory, Lepidostoma consumed significantly more rhododendron (1.062 mg AFDM/mg AFDM body wt/day) than red maple (0.479 mg AFDM/mg AFDM body wt/day) (P = 0.001). Results suggest that changes in the taxonomic composition of stream macroinvertebrate communities due to disturbance may have significant effects on ecosystem processes for at least 2 yr after termination of disturbance. Lepidostoma colonize rapidly following disturbance, enhancing the restoration of vital ecosystem processes such as litter decomposition in Coweeta streams.

A study tested the hypothesis, suggested by researchers in 1992, that the insertion of a long terminal repeat of a transposable element 17.6 into the 3' untranslated area of a P450 gene causes vulnerability to the insecticide DDT in Drosophila melanogaster. The results suggest rejection of this hypothesis because the global screening of lines showed that the presence or absence of a long terminal repeat was not correlated to the resistance in 31 strains of D. melanogaster and Drosophila simulans.

The alteration in the metabolic enzymes capable of degrading pesticides or changes in the functionality of insecticide targets due to transposon insertion is discussed. Transposon alteration alters gene product function and thus causes resistance.

The emergence of insecticide resistance in the mosquito poses a serious threat to the efficacy of many malaria control programs. We have searched the Anopheles gambiae genome for members of the three major enzyme families--the carboxylesterases, glutathione transferases, and cytochrome P450s--that are primarily responsible for metabolic resistance to insecticides. A comparative genomic analysis with Drosophila melanogaster reveals that a considerable expansion of these supergene families has occurred in the mosquito. Low gene orthology and little chromosomal synteny paradoxically contrast the easily identified orthologous groups of genes presumably seeded by common ancestors. In A. gambiae, the independent expansion of paralogous genes is mainly a consequence of the formation of clusters among locally duplicated genes. These expansions may reflect the functional diversification of supergene families consistent with major differences in the life history and ecology of these organisms. These data provide a basis for identifying the resistance-associated enzymes within these families. This will enable the resistance status of mosquitoes, flies, and possibly other holometabolous insects to be monitored. The analyses also provide the means for identifying previously unknown molecules involved in fundamental biological processes such as development., Insecticides form a central component of most malaria control programs [see accompanying paper by Hemingway et al. in this issue (1)]. Mechanisms of resistance, such as reduced penetration, increased sequestration, [...]

The evolution of resistance in pests such as the European corn borer will imperil transgenic maize varieties that express insecticidal crystal proteins of Bacillus thuringiensis. Patchworks of treated and untreated fields can delay the evolution of pesticide resistance, but the untreated refuge fields are likely to sustain heavy damage. A strategy that exploits corn borer preferences and movements can eliminate this problem. Computer simulation indicates that this approach can delay the evolution of resistance and reduce insect damage in the untreated fields of a patchwork planting regime., Maize, cotton, potatoes, and tobacco have now been genetically engineered to express crystal protein (cry) genes from Bacillus thuringiensis. Maize varieties nearing commercialization are toxic to many Lepidoptera, including Ostrinia [...]

Mechanism of Insect Resistance to the Microbial Insecticide Bacillus thuringiensis PROSPECTS FOR THE FUTURE OF MICROBIAL insecticides are bright. In addition to conventional applications, recent advances in plant transformation with [...]

Strains of Wolbachia bacteria sharing the same genetic background but differing in their resistance alleles and infection status are compared, to discriminate between costs of resistance in Culex pipiens mosquitoes due to resistance genes from those associated with elevated Wolbachia densities. It is found that Wolbachia are capable of modifying the cost of resistance and that the cost of Wolbachia infections increases with their density.

The acquisition of insecticide resistance genes for large areas, combined with the absence or rarity of sexual recombination, led to the low genetic variability in the Myzus persicae in a wide geographical scale. The study suggests that human-induced selective pressures probably shape the genetic structure of Myzus persicae populations living on oilseed rape.

Adaptations conferring resistance to xenobiotics (antibiotics, insecticides, herbicides, etc.) are often costly to the organism's fitness in the absence of the selecting agent. Tests show parasitism as a source of selection capable of modifying the costs of organophosphate insecticide resistance in mosquitoes.

Several life history trait that could influence the fitness of the insects are compared between insecticide-susceptible and malathion-specific resistant populations of the red flour beetle (Tribolium castaneum). Results suggested that egg fertility and egg-to-adult development time did not differ susceptible and resistant populations.