Expressed sequence tags from the midgut of Epiphyas postvittana (Walker) (Lepidoptera: Torticidae). Insect Mol Biol

Horticulture and Food Research Institute, Palmerston North, New Zealand.
Insect Molecular Biology (Impact Factor: 2.59). 01/2008; 16(6):675-90. DOI: 10.1111/j.1365-2583.2007.00763.x
Source: PubMed


The midgut is a key tissue in insect science. Physiological roles include digestion and peritrophic membrane function, as well as being an important target for insecticides. We used an expressed sequence tag (EST) approach to identify candidate genes and gene families involved in these processes in the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae). Two cDNA libraries were constructed from dissected midgut of third to fifth instar larvae. Clustering analysis of 6416 expressed sequence tags produced 1178 tentative unique genes comprising 725 tentative contigs and 453 singletons. The sequences show similar codon usage to sequences from other lepidopterans, a Kozak consensus sequence similar to Drosophila and single nucleotide polymorphisms (SNPs) were detected at a frequency of 1.35/kb. The identity of the most common Interpro families correlates well with major known functions of the midgut. Phylogenetic analysis was conducted on representative sequences from selected multigene families. Gene families include a broad range of digestive proteases, lipases and carbohydrases that appear to have degradative capacity against the major food components found in leaves, the diet of these larvae; and carboxylesterases, glutathione-S-transferases and cytochrome P450 monooxygenases, potentially involved in xenobiotic degradation. Two of the larger multigene families, serine proteases and lipases, expressed a high proportion of genes that are likely to be catalytically inactive.

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Available from: Ross Crowhurst, Mar 15, 2014
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    • "The occurrence of multiple isoforms may provide adaptive advantages for insects feeding on plants containing inhibitors. Therefore, it is not surprising that during the past decades, a large number of protease encoding genes were identified to be present in the digestive system of many different insects (Bown et al., 1997; Gatehouse et al., 1997; Ge et al., 2012; Marshall et al., 2008; Oliveira-Neto et al., 2004; Pedra et al., 2003; Prabhakar et al., 2007; Simpson et al., 2007). "
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    ABSTRACT: While technological advancements have recently led to a steep increase in genomic and transcriptomic data, and large numbers of protease sequences are being discovered in diverse insect species, little information is available about the expression of digestive enzymes in Orthoptera. Here we describe the identification of Locusta migratoria serine protease transcripts (cDNAs) involved in digestion, which might serve as possible targets for pest control management. A total of 5 putative trypsin and 15 putative chymotrypsin gene sequences were characterized. Phylogenetic analysis revealed that these are distributed among 3 evolutionary conserved clusters. In addition, we have determined the relative gene expression levels of representative members in the gut under different feeding conditions. This study demonstrated that the transcript levels for all measured serine proteases were strongly reduced after starvation. On the other hand, larvae of L. migratoria displayed compensatory effects to the presence of Soybean Bowman Birk (SBBI) and Soybean Trypsin (SBTI) inhibitors in their diet by differential upregulation of multiple proteases. A rapid initial upregulation was observed for all tested serine protease transcripts, while only for members belonging to class I, the transcript levels remained elevated after prolonged exposure. In full agreement with these results, we also observed an increase in proteolytic activity in midgut secretions of locusts that were accustomed to the presence of protease inhibitors in their diet, while no change in sensitivity to these inhibitors was observed. Taken together, this paper is the first comprehensive study on dietary dependent transcript levels of proteolytic enzymes in Orthoptera. Our data suggest that compensatory response mechanisms to protease inhibitor ingestion may have appeared early in insect evolution.
    Insect biochemistry and molecular biology 05/2014; DOI:10.1016/j.ibmb.2014.03.002 · 3.45 Impact Factor
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    • "Moreover, Mir1-CP was found to synergize activity of a Bacillus thuringiensis toxin (Mohan et al., 2008) whose mode of action includes direct attack of the MG by forming ion pores through binding to cadherins (Gómez et al., 2002; Vachon et al., 2012). Most of the differentially expressed genes (N ¼ 133 for Q- value 0.05) have homologs present in the PM or MG tissue of other lepidopterans including H. armigera (Campbell et al., 2008; Pauchet et al., 2008,2010), Mamestra configurata Walker (Noctuidae ) (Toprak et al., 2008,2010b), Epiphyas postvittana (Walker) (Tortricidae) (Simpson et al., 2007), and B. mori (Kajiwara et al., 2005). This homology along with our finding that transcript abundance of similar MG genes also increased in European corn borer fed on Mp708 suggests that transcriptional responses of fall armyworm to the IIM-degrading Mir1-CP toxin are common across lepidopteran pests in general. "
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    ABSTRACT: Understanding the molecular mechanisms underlying insect compensatory responses to plant defenses could lead to improved plant resistance to herbivores. The Mp708 inbred line of maize produces the maize insect resistant 1-cysteine protease (Mir1-CP) toxin. Reduced feeding and growth of fall armyworm larvae fed on Mp708 was previously linked to impairment of nutrient utilization and degradation of the midgut (MG) peritrophic matrix (PM) by Mir1-CP. Here we examine the biochemical and transcriptional responses of fall armyworm larvae to Mir1-CP. Insect Intestinal Mucin (IIM) was severely depleted from pure PMs treated in vitro with recombinant Mir1-CP. Larvae fed on Mp708 midwhorls excrete frass largely depleted of IIM. Cracks, fissures and increased porosity previously observed in the PM of larvae fed on Mp708 midwhorls could ensue when Mir1-CP degrades the IIM that cross-links chitin fibrils in the PM. Both targeted and global transcriptome analyses were performed to determine how complete dissolution of the structure and function of the PM is prevented, enabling larvae to continue growing in the presence of Mir1-CP. The MGs from fall armyworm fed on Mp708 upregulate expression of genes encoding proteins involved in PM production as an apparent compensation to replace the disrupted PM structure and restore appropriate counter-current MG gradients. Also, several families of digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on diets lacking Mir1-CP (artificial diet, midwhorls from Tx601 or B73 maize). Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function.
    Insect biochemistry and molecular biology 01/2013; 43(3). DOI:10.1016/j.ibmb.2012.12.008 · 3.45 Impact Factor
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    • "The target site for Bt toxins is believed to be the midgut, a dynamic tissue which plays a vital role in metabolism, digestion, and detoxification. In Lepidoptera, previous studies have focused on the role of proteases, lipases and carbohydrases in digestion, and carboxylesterases, glutathione-s-transferases and cytochrome P450s in xenobiotic metabolism in the midgut 13, 14, 15. With the advent of genomics and its “omics” tools, current research looked more closely at the physiological and toxicological changes at a global level instead of focusing on individual genes in the midgut. "
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    ABSTRACT: The larval midgut of diamondback moth, Plutella xylostella, is a dynamic tissue that interfaces with a diverse array of physiological and toxicological processes, including nutrient digestion and allocation, xenobiotic detoxification, innate and adaptive immune response, and pathogen defense. Despite its enormous agricultural importance, the genomic resources for P. xylostella are surprisingly scarce. In this study, a Bt resistant P. xylostella strain was subjected to the in-depth transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes in the P. xylostella larval midgut. Using Illumina deep sequencing, we obtained roughly 40 million reads containing approximately 3.6 gigabases of sequence data. De novo assembly generated 63,312 ESTs with an average read length of 416bp, and approximately half of the P. xylostella sequences (45.4%, 28,768) showed similarity to the non-redundant database in GenBank with a cut-off E-value below 10-5. Among them, 11,092 unigenes were assigned to one or multiple GO terms and 16,732 unigenes were assigned to 226 specific pathways. In-depth analysis indentified genes putatively involved in insecticide resistance, nutrient digestion, and innate immune defense. Besides conventional detoxification enzymes and insecticide targets, novel genes, including 28 chymotrypsins and 53 ABC transporters, have been uncovered in the P. xylostella larval midgut transcriptome; which are potentially linked to the Bt toxicity and resistance. Furthermore, an unexpectedly high number of ESTs, including 46 serpins and 7 lysozymes, were predicted to be involved in the immune defense. As the first tissue-specific transcriptome analysis of P. xylostella, this study sheds light on the molecular understanding of insecticide resistance, especially Bt resistance in an agriculturally important insect pest, and lays the foundation for future functional genomics research. In addition, current sequencing effort greatly enriched the existing P. xylostella EST database, and makes RNAseq a viable option in the future genomic analysis.
    International journal of biological sciences 09/2012; 8(8):1142-55. DOI:10.7150/ijbs.4588 · 4.51 Impact Factor
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