The genomic information on insects has increased tremendously during last several years. Whole genomes have been sequenced for several insect species, including the fruit fly (Drosophila melanogaster) , African malaria mosquito (Anopheles gambiae) , yellow fever mosquito (Aedes aegypti) , honey bee (Apis mellifera) , silkworm (Bombyx mori) [5, 6], red flour beetle (Tribolium castaneum) , and 11 other Drosophila species [8, 9]. Genome sequencing of other insect species, including pea aphid (Acyrthosiphon pisum), northern house mosquito (Culex pipiens), three species of parasitoid wasp (Nasonia sp.), Hessian fly (Mayetiola destructor), blood sucking bug (Rhodnius prolixus), and body louse (Pediculus humanus), are currently in progress [10–12]. The red flour beetle is the only agricultural insect pest whose whole genome sequence has become available to date.
Lepidoptera, the second most biodiverse group of insect species after Coleoptera, represents more than 160,000 species including many of the most devastating pests of crops, forests and stored products . The silkworm was the first lepidopteran insect to have its complete genome sequenced . However, genomic information for other lepidopterans, particularly agricultural pest species is limited but urgently needed due to their economic importance and biodiversity. Sequencing of the expressed sequence tags (ESTs) has been recognized as an economical approach to identify a large number of expressed genes that can be used in gene expression and other genomic studies [14–16]. Indeed, ESTs have been generated from several lepidopteran insects including the silkworm , spruce budworm (Choristoneura fumiferana) , cotton bollworm (Helicoverpa armigera) , diamondback moth (Plutella xylostella) , tobacco hawkmoth (Manduca sexta) [21, 22], and fall armyworm (Spodoptera frugiperda) [10, 23].
It has been long recognized that the insect gut is an important target for developing new strategies for insect pest management. Until now, however, only a few studies have focused on the development of gut-specific EST libraries of lepidopterans as a tool to identify candidate genes involved in the toxicity of insecticides and the development of insecticide resistance. Gut-specific EST libraries were reported for light brown apple moth (Epiphyas postvittana) (6,416 ESTs) , bertha armyworm (Mamestra configurata) (30 serine protease-related sequences) , and European corn borer (ECB, Ostrinia nubilalis) (1,745 ESTs) .
ECB is one of the most destructive pests of corn and can cause as much as $1 billion of economic loss annually in the United States alone [27, 28]. ECB also represents a complex of stalk borers, such as the southwestern corn borer (Diatraea grandiosella) and the sugarcane borer (Diatraea saccharalis). These stalk borers share similar ecosystem and create similar damage to corn plants. Although ECB has been successfully managed using transgenic Bt corn hybrids (plants that express insecticidal toxins of Bacillus thuringiensis or Bt), there are increasing concerns about the potential development of Bt resistance in ECB because of the widespread use of Bt corn [28, 29]. Indeed, several ECB colonies have developed resistance to Bt toxins under laboratory selection conditions [30, 31].
The main target for Bt toxins is the insect midgut, where Bt protoxins are activated by gut proteases to produce activated Bt toxins. The activated toxins then bind to specific receptor(s) to confer toxicity . This means that insect resistance to Bt toxins could be conferred by protease-mediated and receptor-mediated mechanisms [33–37]. Because Bt toxins and insect gut interactions are determined by many gene products in the insect gut, including many proteins/enzymes involved in Bt protoxin activation, toxin binding to receptors and toxin degradation, any change in these systems has the potential to affect a particular Bt's specificity and efficacy, and could lead to Bt resistance in insects.
Our goals are to develop a gut-specific EST database from ECB larvae and explore candidate genes that are potentially involved in insect-Bt interactions and Bt resistance. In this paper, we report the analysis and annotations of 15,000 ESTs derived from the gut of ECB larvae. We discuss the putative identities of the ESTs, their potential biological and molecular functions, and present comparative analyses of our ESTs with sequences from other insects. This work provides the opportunity for developing an ECB gut-specific microarray that can be used to study insect-Bt interactions and genetic basis of Bt resistance in ECB. Furthermore, we revealed 52 candidate genes that could be involved in Bt toxicity and resistance. Among the 41 selected candidate genes examined by RT-PCR, we found 5 genes with apparently decreased expressions and 10 with increased expressions in Cry1Ab-resistant strain of ECB as compared with the susceptible strain of ECB. Differential expressions of these genes in a Cry1Ab-resistant strain may suggest possible involvement of these genes in Cry1Ab resistance, and therefore provides us with new insights into the mechanism of Cry1Ab resistance in ECB. This study may serve as a model for studying Bt resistance mechanisms and for developing bio-pesticides for all closely related corn stalk borers.