Expressed sequence tags from larval gut of the European corn borer (Ostrinia nubilalis): Exploring candidate genes potentially involved in Bacillus thuringiensis toxicity and resistance
- Chitvan Khajuria1,
- Yu Cheng Zhu2,
- Ming-Shun Chen1, 3,
- Lawrent L Buschman1,
- Randall A Higgins^1,
- Jianxiu Yao1,
- Andre LB Crespo4,
- Blair D Siegfried4,
- Subbaratnam Muthukrishnan5 and
- Kun Yan Zhu1Email author
© Khajuria et al; licensee BioMed Central Ltd. 2009
Received: 27 February 2009
Accepted: 29 June 2009
Published: 29 June 2009
Lepidoptera represents more than 160,000 insect species which include some of the most devastating pests of crops, forests, and stored products. However, the genomic information on lepidopteran insects is very limited. Only a few studies have focused on developing expressed sequence tag (EST) libraries from the guts of lepidopteran larvae. Knowledge of the genes that are expressed in the insect gut are crucial for understanding basic physiology of food digestion, their interactions with Bacillus thuringiensis (Bt) toxins, and for discovering new targets for novel toxins for use in pest management. This study analyzed the ESTs generated from the larval gut of the European corn borer (ECB, Ostrinia nubilalis), one of the most destructive pests of corn in North America and the western world. Our goals were to establish an ECB larval gut-specific EST database as a genomic resource for future research and to explore candidate genes potentially involved in insect-Bt interactions and Bt resistance in ECB.
We constructed two cDNA libraries from the guts of the fifth-instar larvae of ECB and sequenced a total of 15,000 ESTs from these libraries. A total of 12,519 ESTs (83.4%) appeared to be high quality with an average length of 656 bp. These ESTs represented 2,895 unique sequences, including 1,738 singletons and 1,157 contigs. Among the unique sequences, 62.7% encoded putative proteins that shared significant sequence similarities (E-value ≤ 10-3)with the sequences available in GenBank. Our EST analysis revealed 52 candidate genes that potentially have roles in Bt toxicity and resistance. These genes encode 18 trypsin-like proteases, 18 chymotrypsin-like proteases, 13 aminopeptidases, 2 alkaline phosphatases and 1 cadherin-like protein. Comparisons of expression profiles of 41 selected candidate genes between Cry1Ab-susceptible and resistant strains of ECB by RT-PCR showed apparently decreased expressions in 2 trypsin-like and 2 chymotrypsin-like protease genes, and 1 aminopeptidase genes in the resistant strain as compared with the susceptible strain. In contrast, the expression of 3 trypsin- like and 3 chymotrypsin-like protease genes, 2 aminopeptidase genes, and 2 alkaline phosphatase genes were increased in the resistant strain. Such differential expressions of the candidate genes may suggest their involvement in Cry1Ab resistance. Indeed, certain trypsin-like and chymotrypsin-like proteases have previously been found to activate or degrade Bt protoxins and toxins, whereas several aminopeptidases, cadherin-like proteins and alkaline phosphatases have been demonstrated to serve as Bt receptor proteins in other insect species.
We developed a relatively large EST database consisting of 12,519 high-quality sequences from a total of 15,000 cDNAs from the larval gut of ECB. To our knowledge, this database represents the largest gut-specific EST database from a lepidopteran pest. Our work provides a foundation for future research to develop an ECB gut-specific DNA microarray which can be used to analyze the global changes of gene expression in response to Bt protoxins/toxins and the genetic difference(s) between Bt- resistant and susceptible strains. Furthermore, we identified 52 candidate genes that may potentially be involved in Bt toxicity and resistance. Differential expressions of 15 out of the 41 selected candidate genes examined by RT-PCR, including 5 genes with apparently decreased expression and 10 with increased expression in Cry1Ab-resistant strain, may help us conclusively identify the candidate genes involved in Bt resistance and provide us with new insights into the mechanism of Cry1Ab resistance in ECB.
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 m ori) [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.
Results and discussion
Development and analysis of the ECB gut ESTs
Summary of the analysis of 15,000 ESTs from the guts of the European corn borer larvae
Number of clones sequenced
Chromatographs checked (EST number)
Sequence quality checked (EST number)
Average length (bp)
Number of contigs b
Number of singletons
Poor quality a
List of 20 largest contigs assembled from 15,000 ESTs from the guts of European corn borer larvae
Number of ESTs
Trypsin-like protease T25 precursor
Chymotrypsin-like serine protease
Trypsin-like serine protease
Trypsin-like serine protease
Trypsin-like serine protease
Ribosomal protein s13
Trypsin-like serine protease
Thymosin isoform 1
Trypsin-like serine protease
Pancreatic triacylglycerol lipase
Chymotrypsin-like serine protease
Identification of the ORF and putative secretary proteins
To identify the secretory proteins, putative protein sequences were examined to identify potential secretion signal peptide using SignalP software . A total of 439 (15.2%) putative proteins were predicted to contain signal peptides (Figure 2B). Among the putative secretory proteins, 298 sequences (67.9%) had matches with known proteins in the NR protein database, whereas 141 putative secretory proteins (32.1%) were unique, sharing no significant sequence similarity with any known protein. This information is valuable since secretory proteins are important components of biological processes in the gut [44, 45].
Comparative analyses of ECB gut ESTs
Because B. mori genome has not been fully annotated, we have also compared our ESTs with all available B. mori ESTs using BLASTN. Among the 2,895 contigs and singletons, 579 (20.0%) had hits with B. mori sequences at E-value < 10-3 (Figure 4A). The remaining 2,316 ESTs (80.0%) did not show a significant match with the B. mori sequences. Among the 579 unique ESTs which had hits in the database, 43 (7.4%) had matches with E-value less than E-150, 64 (11.1%) had E-values between E-150 and E-100, 156 (26.9%) had E-values between E-100 and E-50, 135 (23.3%) had E-values between E-50 and E-20, and 181 sequences (31.3%) had E-values between E-20 and E-5 (Figure 4B).
Identification of ESTs potentially relevant to the Bt toxicity and resistance
List of genes potentially involved in Bt toxicity and resistance as identified by EST analysis from the guts of the European corn borer larvae
European corn borer
Trypsin-like serine proteases
Chymorypsin-like serine proteases
Our EST analysis also revealed 13 ESTs putatively encoding aminopeptidases (E-value 1e-64 to 1e-116), 1 encoding a cadherin-like protein (E-value 1e-35), and 2 encoding alkaline phosphatases (E-value 1e-115 to 1e-131). Aminopeptidase N, cadherin-like proteins, and alkaline phosphatases have been found to serve as Bt toxin binding receptors in other insect species [57–59]. To verify the function of aminopeptidase N as a receptor for Bt Cry1Ac toxin in Spodoptera litura, RNAi technology was used to reduce the expression of aminopeptidase N. This resulted in a significant reduction in the susceptibility of the insect to Cry1Ac toxin . Gahan et al.  showed that in a resistant strain (YHD2) of Heliothis virescens, there was a disruption of a cadherin-superfamily gene by a retrotransposon-mediated insertion that resulted in high levels of resistance to the Bt toxin Cry1Ac. Fernandez et al.  also reported that a GPI (glycosylphosphatidyl-inositol)-anchored ALP (alkaline phosphatase) was an important receptor molecule involved in Cry11Aa interactions with midgut cells and toxicity to Ae. aegypti larvae. These studies demonstrate that aminopeptidases, cadherin-like proteins, and alkaline phosphatases can serve as Bt toxin receptors involved in Bt toxicity and resistance. Thus, identification of these candidate Bt receptor genes in this study will allow us to further examine whether receptor-mediated resistance is involved in Bt resistance in ECB.
Comparison of expression profiles between Cry1Ab-susceptible and resistant strains of ECB
Although RT-PCR is not quantitative, reproducible results of such differential expression patterns for these candidate genes in the Cry1Ab-susceptible and resistant strains of ECB may imply their potential roles in conferring or contributing to Cry1Ab resistance as well as genetic differences between the susceptible and resistant strains of ECB. Indeed, certain trypsin-like and chymotrypsin-like proteases have previously been found to activate or degrade Bt protoxins and toxins, whereas several aminopeptidases, cadherin-like proteins and alkaline phosphatases have been demonstrated to serve as Bt receptor proteins in other insect species. Thus, our results may help conclusively identify the candidate genes involved in Cry1Ab resistance and provide us with new insights into the mechanism of Cry1Ab resistance in ECB. Nevertheless, further research will be needed to confirm their involvements and to elucidate their roles in Cry1Ab resistance in ECB.
Our study resulted in a gut-specific EST database containing 12,519 high-quality ESTs from a total of 15,000 ESTs sequenced in an agriculturally important lepidopteran pest. To our knowledge, this database represents the largest gut-specific EST database from a lepidopteran pest. Our analysis using ORF predictor software showed that approximately 11.2% of the protein coding genes in our database may be specific to ECB as these sequences have an ORF of at least 450 bp but did not have significant matches with known sequences in NCBI database. We have also identified 52 candidate genes that are relevant to Bt toxicity and resistance. These genes encode trypsin-like proteases, chymotrypsin-like proteases, aminopeptidases, cadherin-like protein, and alkaline phosphatases. Furthermore, we showed differential expressions of 15 out of the 41 representative candidate genes that were examined by RT-PCR, including 5 genes with apparently decreased expressions and 10 with increased expressions in Cry1Ab-resistant strain. These results may help us further narrow down the candidate genes possibly involved in Cry1Ab resistance, and provide us with new insights into the mechanism of Bt resistance in general in ECB.
We are in the process of developing a microarray using our unique ESTs together with the ECB gut-specific sequences which are already available in the GenBank. The microarray technology will help us analyze the global change of gene expression in response to Bt protoxins/toxins. It will also allow us to analyze any genetic differences between Bt resistant and -susceptible strains of ECB. Our genomic information on ECB could also serve as a valuable resource for identifying critical/vulnerable genes from the gut of ECB that would make useful physiological targets for new toxins that could be developed for use in pest management.
Insects rearing and dissection
The KS-SC Bt-susceptible ECB colony was used for generating EST libraries. This colony originated from the egg masses collected from the cornfields near St. John, Kansas, in 1995. The colony has been reared since then on artificial diets in the laboratory at Kansas State University according to Huang et al. . The resistant ECB strain originated from a field collection of 126 diapausing larvae obtained from non-Bt hybrids in Kandiyohi Co., MN in 2001. The resistant strain was initiated from 14 larvae that survived exposure to a diagnostic Cry1Ab concentration used to identify potential changes in susceptibility to Cry1Ab [64, 65]. To minimize inbreeding or founder effects, the resistant insects were backcrossed twice with the susceptible strain which originated from the same collection. Because the resistance was incompletely recessive and involved multiple factors , the F1 progeny were randomly mated to obtain recombination of resistance factors in the F2 progeny to allow selection of resistant genotypes. The insects were then subjected to selection at a Cry1Ab concentration corresponding to two- to three-fold the LC50 for the F1 progeny (150 ng/cm2) . This selection event was designed to eliminate all the susceptible homozygotes and most of the heterozygotes. The resistant survivors from this selection event were then subjected to a second cycle of backcrossing, random mating, and selection. After six generations, the Cry1Ab concentration used in selections was gradually increased to achieve 750 ng/cm2 at generation F10, a concentration that kills virtually all F1 progeny. At generation F17, the resistance to Cry1Ab in the re-selected strain was in excess of 800-fold. The guts were dissected from fifth-instar larvae in DEPC (diethylpyrocarbonate)-treated distilled water and were stored in TRI reagent™ (Molecular Research, Inc., Cincinnati, OH) at -80°C until used.
cDNA library construction and sequencing
Total RNA was isolated from the whole guts of ECB larvae using TRI reagent™. The plasmid library was constructed using Creator SMART™ cDNA library construction kit from Clontech (Palo Alto, CA) following the manufacturer's protocols with one modification; instead of using the original phage vector, PCR fragments were cloned directly into a pPCR-XL-TOPO plasmid using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA). The λ-library was constructed using ZAP-cDNA synthesis kit and ZAP-cDNA Gigapack III gold cloning kit (Stratagene, La Jolla, CA) according to the manufacturer's protocols. Briefly, double stranded cDNA was synthesized from poly(A) RNA, size-fractionated through a Sepharose CL-2B gel filtration column, and ligated into λ Uni-ZAP XR vector. The ligated DNA was packaged with the Gigapack III gold packaging extract and the library was plated on LB/agar plates. Recombinant plasmid within the lambda Uni-ZAP XR vector was in vivo excised using the ExAssist helper phage and recircularized to generate subclones in the pBluescript SK phagemid vector. To sequence the clones, M13R and M13F primers were used for 5' and 3' sequencing, respectively. Plasmid DNA was isolated using Qiagen Bio Robot 3000 and sequenced using an ABI 3700 DNA analyzer.
EST analyses and annotations
The DNA sequences were preprocessed by using the online software EGassembler . Specifically, sequence cleaning process was employed to trim the vector and adaptor sequences from the ESTs. RepeatMasker process was used to mask the interspersed repeats and low complexity regions of the sequences by using Drosophila Repbase repeat library. The sequences were further masked by using vector masking against NCBI's vector library and organelle masking against mitochondrial library. The preprocessed ESTs were then assembled by using Sequencher software (Gene Codes Corp., Ann Arbor, MI). The ORF regions of the assembled ESTs were identified by using the ORF predictor software  and secretory proteins were identified by looking for signal peptide sequence using SignalP software . Gene ontology (GO) annotation was derived using Blast2GO software http://www.blast2go.de/.
Comparative analysis of ESTs
The ECB unique ESTs were comparatively analyzed for their sequence similarities against other organisms. The organism associated with the EST showing the highest BLAST score in GenBank databases was selected. The ECB gut ESTs were also compared with sequences from the silkworm and ECB that are currently available in the database by using BLASTN with a cutoff E-value of 10-3.
Expression profiling by RT-PCR
Forty-one out of the 52 candidate genes were selected for comparing their apparent gene expression profiles between the Cry1Ab-susceptible and resistant strains of ECB by using RT-PCR. These genes were selected solely based on their representations among different gene groups from our EST analysis. After total RNA was isolated from four midguts dissected from one-day-old fifth-instar larvae of each strain (Cry1Ab-susceptible and resistant strains) of ECB by using TRI reagentTM (Sigma, St. Louis, MO), it was treated with TURBO™ DNase (Ambion, Austin, TX)to remove any genomic DNA contaminations. Three micrograms of total RNA was used for synthesis of first strand cDNA using SuperScript® III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). cDNA prepared from total RNA was used as a template for RT-PCR. A minimum of two biological replications was used for all the PCR primer pairs. For all trypsin-like (except for ECB-30_C08) and chymotrypsin-like serine protease, alkaline phosphatase, and RPS3 genes, 25 PCR cycles were used whereas for aminopeptidase and cadherin-like protein, 27 PCR cycles were used. For one trypsin-like serine protease gene (ECB-30_C08), however, 33 PCR cycles were used as the expression of this gene using fewer cycles was not visible on agarose gels. Each PCR was performed for above mentioned number of cycles, each consisting of 94°C for 30s, 55°C for 60s, and 72°C for 60s. The sequences of forward and reverse PCR primers, and expected size of PCR product for each of 41 candidate genes are provided in Additional file 1.
The authors thank Yoonseong Park for his helpful comments on an earlier draft of this manuscript, Lisa Tan for maintaining the European corn borer colonies, Xiang Liu for his technical assistance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by U.S. Department of Agriculture or Kansas State University. This study was supported in part by the Kansas Agricultural Experiment Station and the Arthropod Genomics Center funded by K-State Targeted Excellence program at Kansas State University. This paper is contribution No 09-105-J from the Kansas Agricultural Experiment Station. The Ostrinia nubilalis voucher specimens (voucher No. 079) are located in the Kansas State University Museum of Entomological and Prairie Arthropod Research, Manhattan, Kansas.
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