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Genome-wide microarray analysis (Affymetrix array) was used (i) to determine whether only one gene, the cytochrome P450 enzyme Cyp6g1, is differentially transcribed in dichlorodiphenyltrichloroethane (DDT)-resistant vs. -susceptible Drosophila; and (ii) to profile common genes differentially transcribed across a DDT-resistant field isolate [Rst(2)DDT Wisconsin ] and a laboratory DDT-selected population [Rst(2)DDT 91-R ]. Statistical analysis (ANOVA model) identified 158 probe sets that were differentially transcribed among Rst(2)DDT 91-R , Rst(2)DDT Wisconsin , and the DDT-susceptible genotype Canton-S (P < 0.01). The cytochrome P450 Cyp6a2 and the diazepam-binding inhibitor gene (Dbi) were over transcribed in the two DDT-resistant genotypes when compared to the wild-type Drosophila, and this difference was significant at the most stringent statistical level, a Bonferroni correction. The list of potential candidates differentially transcribed also includes 63 probe sets for which molecular function ontology annotation of the probe sets did not exist. A total of four genes (Cyp6a2, Dbi, Uhg1, and CG11176) were significantly different (P < 5.6 e ؊06 ) between Rst(2)DDT 91-R and Canton-S. Additionally, two probe sets encoding Cyp12d1 and Dbi were significantly different between Rst(2)DDT Wisconsin and Canton-S after a Bonferroni correction. Fifty-two probe sets, including those associated with pesticide detoxification, ion transport, signal transduction, RNA transcription, and lipid metabolism, were commonly expressed in both resistant lines but were differentially transcribed in Canton-S. Our results suggest that more than Cyp6g1 is overtranscribed in field and laboratory DDT-resistant genotypes, and the number of commonalities suggests that similar resistance mechanisms may exist between laboratory-and field-selected DDT-resistant fly lines.T he evolution of insecticide resistance is often, but not always, based on major effect alleles (1-4). It has been hypothesized that high selection pressure in the field will favor monogenic forms of pesticide resistance, and that selection for resistance in the laboratory will favor polygenic resistance (5-7). In early genetic studies in Drosophila, dichlorodiphenyltrichloroethane (DDT) resistance was mapped to multiple locations on chromosomes II and III (8-14). Subsequently, low-level DDT resistance was mapped to 64.5 Ϯ 2 centiMorgans on the second chromosome (15), a locus (loci) known as Rst(2)DDT.Recently, Daborn et al. (16) suggested that resistance to DDT in the field is monogenic and is due to the overexpression of a single P450 gene, Cyp6g1. Le Goff et al. (17) suggested that resistance in field isolates of both Drosophila melanogaster and Drosophila simulans is associated with overtranscription of Cyp6g1, whereas prolonged laboratory selection with DDT apparently coselects additional genes such as Cyp12d1 (18) To date, no genome-wide expression profile has been evaluated to investigate the extent to which gene transcription varies between genotypes that are resis...

Mutagenesis can be used as a means of predicting likely mechanisms of resistance to novel classes of insecticides. We used chemical mutagenesis in Drosophila to screen for mutants that had become resistant to imidacloprid, a neonicotinoid insecticide. Here we report the isolation of two new dominant imidacloprid-resistant mutants. By recombinational mapping we show that these map to the same location as Rst(2)DDT. Furthermore, we show that pre-existing Rst(2)DDT alleles in turn confer cross-resistance to imidacloprid. In order to localize the Rst(2)DDT gene more precisely, we mapped resistance to both DDT and imidacloprid with respect to P-element markers whose genomic location is known. By screening for recombinants between these P-elements and resistance we localized the gene between 48D5-6 and 48F3-6 on the polytene chromosome map. The genomic sequence in this interval shows a cluster of cytochrome P450 genes, one of which, Cyp6g1, is over-expressed in all resistant strains examined. We are now testing the hypothesis that resistance to both compounds is associated with over-expression of this P450 gene.

The induction of plant defenses by insect feeding is regulated via multiple signaling cascades. One of them, ethylene signaling, increases susceptibility of Arabidopsis to the generalist herbivore Egyptian cotton worm (Spodoptera littoralis; Lepidoptera: Noctuidae). The hookless1 mutation, which affects a downstream component of ethylene signaling, conferred resistance to Egyptian cotton worm as compared with wild-type plants. Likewise, ein2, a mutant in a central component of the ethylene signaling pathway, caused enhanced resistance to Egyptian cotton worm that was similar in magnitude to hookless1. Moreover, pretreatment of plants with ethephon (2-chloroethanephosphonic acid), a chemical that releases ethylene, elevated plant susceptibility to Egyptian cotton worm. By contrast, these mutations in the ethylene-signaling pathway had no detectable effects on diamondback moth (Plutella xylostella) feeding. It is surprising that this is not due to nonactivation of defense signaling, because diamondback moth does induce genes that relate to wound-response pathways. Of these wound-related genes, jasmonic acid regulates a novel ␤-glucosidase 1 (BGL1), whereas ethylene controls a putative calcium-binding elongation factor hand protein. These results suggest that a specialist insect herbivore triggers general wound-response pathways in Arabidopsis but, unlike a generalist herbivore, does not react to ethylene-mediated physiological changes.Resistance or tolerance of plants to insect herbivores and pathogens is mediated via constitutive or induced defense mechanisms (Mauricio et al., 1997; Buell, 1998). Inducible defenses play a major role in conferring disease resistance against plant pathogens (Maleck and Dietrich, 1999), and their effects on phytophagous insects can include increased toxicity, delay of larval development, or increased attack by insect parasitoids (Baldwin and Preston, 1999). Inducible defenses are thought to compromise plant fitness less, and maybe more durable, than constitutive defense mechanisms (Agrawal, 1998).During their evolution, specialist herbivores have explored new ecological niches and adapted to novel plant chemical defenses (Ehrlich and Raven, 1964). It is therefore not surprising that specialist herbivores are frequently attracted to secondary metabolites from their hosts. For instance, glucosinolates and their hydrolysis products are feeding and oviposition attractants for crucifer specialists (Gupta and Thorsteinson, 1960; Hicks, 1974), but deterrents for nonadapted insects (McCloskey and Isman, 1993). Specialist herbivores frequently detoxify or sequester plant defense compounds. The latter form of adaptation can even result in protection against parasitoids and predators. Differences in metabolism of plant toxins may be one reason why some induced defenses protect against generalist, but not specialist insect herbivores (Agrawal, 1999).Several signaling pathways, including jasmonic acid (JA), salicylic acid (SA), ethylene, and perhaps hydrogen peroxide (H 2 O 2 ; Reymond and Farm...

Indirect evidence previously suggested that Arabidopsis (Arabidopsis thaliana) vegetative storage protein (VSP) could play a role in defense against herbivorous insects. To test this hypothesis, other AtVSP-like sequences in Arabidopsis were identified through a Basic Local Alignment Search Tool search, and their transcriptional profiles were investigated. In response to methyl jasmonate application or phosphate starvation, AtVSP and AtVSP-like genes exhibited differential expression patterns, suggesting distinct roles played by each member. Arabidopsis VSP2 (AtVSP2), a gene induced by wounding, methyl jasmonate, insect feeding, and phosphate deprivation, was selected for bacterial expression and functional characterization. The recombinant protein exhibited a divalent cation-dependent phosphatase activity in the acid pH range. When incorporated into the diets of three coleopteran and dipteran insects that have acidic gut lumen, recombinant AtVSP2 significantly delayed development of the insects and increased their mortality. To further determine the biochemical basis of the anti-insect activity of the protein, the nucleophilic aspartic acid-119 residue at the conserved DXDXT signature motif was substituted by glutamic acid via site-directed mutagenesis. This single-amino acid alteration did not compromise the protein's secondary or tertiary structure, but resulted in complete loss of its acid phosphatase activity as well as its anti-insect activity. Collectively, we conclude that AtVSP2 is an anti-insect protein and that its defense function is correlated with its acid phosphatase activity.