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aminopeptidase lepidoptera

Anne Bretschneider, David G Heckel, Yannick Pauchet
Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN)...
September 2016: Insect Biochemistry and Molecular Biology
Mingxing Feng, Zhenyu He, Yuanyuan Wang, Xiufang Yan, Jiwen Zhang, Zhaonong Hu, Wenjun Wu
Periplocosides, which are insecticidal compounds isolated from the root bark of Periploca sepium Bunge, can affect the digestive system of insects. However, the mechanism though which periplocosides induces a series of symptoms remains unknown. In this study, affinity chromatography was conducted by coupling periplocoside E-semi-succinic acid ester with epoxy amino hexyl (EAH) sepharose 4B. Sodium dodecyl sulfonate-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed to analyze the fraction eluted by periplocoside E...
2016: Toxins
Umut Toprak, Martin Erlandson, Doug Baldwin, Steve Karcz, Lianglu Wan, Cathy Coutu, Cedric Gillott, Dwayne D Hegedus
The peritrophic matrix (PM) is essential for insect digestive system physiology as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata (Lepidoptera: Noctuidae), a major pest of cruciferous oilseed crops in North America...
October 2016: Insect Science
Austin L Hughes
Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR...
July 2014: Molecular Phylogenetics and Evolution
Jorge W Arboleda Valencia, Maria Fátima Grossi de Sá, Arnubio Valencia Jiménez
The enzymatic activity of leucine aminopeptidase (EC from the intestinal tract of sugarcane giant borer (Telchin licus licus) was assayed by using a simple and sensitive spectrophotometric assay that uses L-leucyl-2- naphthylamide as substrate. In this assay, L-leucyl-2-naphthylamide is hydrolyzed to produce 2-naphthylamine and Lleucine. The product 2-naphthylamine reacts with Fast Black K and can be monitored using a continuous spectrophotometric measurement at 590 nm. The data on the kinetic parameters indicates that the Km for the L-leucyl-2- naphthylamide at pH 7...
June 2014: Protein and Peptide Letters
Ping Lin, Tingcai Cheng, Shengkai Jin, Liang Jiang, Chen Wang, Qingyou Xia
Aminopeptidases N (APNs), the receptors of Bacillus thuringiensis (Bt) toxin in the lepidopteran midgut, are involved in the Bt pathogen infection mechanism. In the present work, we screened 102 APNs from SilkDB, ButterflyBase and MonarchBase; 16 APNs were identified from the silkworm (Bombyx mori) and 24 from the monarch butterfly (Danaus plexippus). Syntenic and phylogenetic tree analysis showed that APN genes have developed multi-family genes before evolutionary divergence of the Lepidoptera. The tissue-expression pattern shows some BmAPNs are specifically or highly expressed in the midgut...
February 10, 2014: Gene
Samar Ramzi, Ahad Sahragard, Jalal J Sendi, Ali Aalami
Lectins are the heterogeneous proteins in plants that serve as storage proteins via defensive mechanisms against herbivores. In the current study, a lectin was extracted and purified from seeds of Citrullus colocynthis by Sepharose 4B-Galactose and DEAE-cellulose fast flow chromatographies. Different concentrations of the lectin were added to artificial diet of Ectomyelois ceratoniae larvae finding out its effect on some biological parameters, digestive physiology and amount of storage macromolecules. It was found that CCA (C...
2013: Frontiers in Physiology
Lina Xu, Zhenying Wang, Jie Zhang, Natalie Ferry, Martin G Edwards, Angharad M R Gatehouse, Kanglai He
The full-length cDNA of four Ofapn aminopeptidases were cloned and sequenced from susceptible and resistant Ostrinia furnacalis strains. Four sequences were identified as APN because they shared the common structural features with APN from Lepidoptera, including the signal peptide, GPI anchor signal, the zinc binding/gluzincin motif HEX2HX18E and the gluzincin aminopeptidase motif GAMEN. Compared with APN sequences from the susceptible strain, there were 9, 5, 10 and 12 amino acid variations in the deduced protein sequences from the resistant strain...
January 2014: Journal of Invertebrate Pathology
Brad S Coates, Douglas V Sumerford, Blair D Siegfried, Richard L Hellmich, Craig A Abel
Transgenic expression of Bacillus thuringiensis (Bt) crystalline (Cry) toxins by crop plants result in reduced insect feeding damage, but sustainability is threatened by the development of resistance traits in target insect populations. We investigated Bt toxin resistance trait in a laboratory colony of the European corn borer, Ostrinia nubilalis, selected for increased survival when exposed to Cry1Ab and correlated survival on Cry1Ab toxin with a constitutive ∼146.2 ± 17.3-fold reduction in midgut aminopeptidase N1 (apn1) transcript levels...
December 2013: Insect Biochemistry and Molecular Biology
Yanyuan Lei, Xun Zhu, Wen Xie, Qingjun Wu, Shaoli Wang, Zhaojiang Guo, Baoyun Xu, Xianchun Li, Xuguo Zhou, Youjun Zhang
To investigate the response of Plutella xylostella transcriptome in defending against a Bt toxin, high-throughput RNA-sequencing was carried out to examine Cry1Ac-susceptible and -resistant strains. The comparative analysis indentified over 2900 differentially expressed unigenes (DEUs) between these two strains. Gene Ontology analysis placed these unigenes primarily into cell, cell part, organelle, binding, catalytic, cellular process, metabolic process, and response to stimulus categories. Based on pathway analyses, DEUs were enriched in oxidoreductase activity and membrane lipid metabolic processes, and they were also significantly enriched in pathways related to the metabolic and biosynthesis of secondary metabolites...
January 1, 2014: Gene
Purushottam R Lomate, Bhakti R Jadhav, Ashok P Giri, Vandana K Hivrale
Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP...
2013: PloS One
Sek Yee Tan, Bonifacio F Cayabyab, Edwin P Alcantara, Fangneng Huang, Kanglai He, Kenneth W Nickerson, Blair D Siegfried
The European (Ostrinia nubilalis Hübner) and Asian corn borers (Ostrinia furnacalis Guenée) are closely related and display similar sensitivity to Cry1 toxins. In this study, we compared the binding patterns of Cry1Ab and Cry1F toxins between both Ostrinia spp., as well as the expression of putative cadherin- and aminopeptidase-N (APN)-like protein receptors. Additionally, cDNA sequences of these putative toxin receptors from both Ostrinia species were compared. Ligand blots for both species indicated a similar binding pattern for Cry1Ab with the strongest immunoreactive band at 260 kDa in both species...
November 2013: Journal of Invertebrate Pathology
Cristina M Crava, Yolanda Bel, Agata K Jakubowska, Juan Ferré, Baltasar Escriche
Aminopeptidase N (APN) isoforms from Lepidoptera are known for their involvement in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis. These enzymes belong to a protein family with at least eight different members that are expressed simultaneously in the midgut of lepidopteran larvae. Here, we focus on the characterization of the APNs from Ostrinia nubilalis (OnAPNs) to identify potential Cry receptors. We expressed OnAPNs in insect cells using a baculovirus system and analyzed their enzymatic activity by probing substrate specificity and inhibitor susceptibility...
October 2013: Insect Biochemistry and Molecular Biology
Estefanía Contreras, Michael Schoppmeier, M Dolores Real, Carolina Rausell
Understanding how Bacillus thuringiensis (Bt) toxins interact with proteins in the midgut of susceptible coleopteran insects is crucial to fully explain the molecular bases of Bt specificity and insecticidal action. In this work, aminopeptidase N (TcAPN-I), E-cadherin (TcCad1), and sodium solute symporter (TcSSS) have been identified by ligand blot as putative Cry3Ba toxin-binding proteins in Tribolium castaneum (Tc) larvae. RNA interference knockdown of TcCad1 or TcSSS proteins resulted in decreased susceptibility to Cry3Ba toxin, demonstrating the Cry toxin receptor functionality for these proteins...
June 21, 2013: Journal of Biological Chemistry
Guillaume Tetreau, Krishnareddy Bayyareddy, Christopher M Jones, Renaud Stalinski, Muhammad A Riaz, Margot Paris, Jean-Philippe David, Michael J Adang, Laurence Després
BACKGROUND: Bacillus thuringiensis var. israelensis (Bti) is a natural larval mosquito pathogen producing pore-forming toxins targeting the midgut of Diptera larvae. It is used worldwide for mosquito control. Resistance mechanisms of an Aedes aegypti laboratory strain selected for 30 generations with field-collected leaf litter containing Bti toxins were investigated in larval midguts at two levels: 1. gene transcription using DNA microarray and RT-qPCR and 2. differential expression of brush border membrane proteins using DIGE (Differential In Gel Electrophoresis)...
2012: BMC Genomics
Silvia Caccia, William J Moar, Jayadevi Chandrashekhar, Cris Oppert, Konasale J Anilkumar, Juan Luis Jurat-Fuentes, Juan Ferré
Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible...
August 2012: Applied and Environmental Microbiology
Weihua Ma, Zan Zhang, Chuanhua Peng, Xiaoping Wang, Fei Li, Yongjun Lin
The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt) toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp...
2012: PloS One
Xin Zhang, Kasorn Tiewsiri, Wendy Kain, Lihua Huang, Ping Wang
Alteration of binding sites for Bacillus thuringiensis (Bt) toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1) gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2) gene...
2012: PloS One
Santosh Kumar Upadhyay, Krishnappa Chandrashekar
Feeding of Helicoverpa armigera larvae on semi-synthetic diet containing Soybean trypsin inhibitor (STI) resulted in disappearance of STI sensitive protease in salivary and midgut protease extract. This might be due to in situ inhibition by dietary STI. STI was largely degraded within 1 h of incubation with total salivary protease (1:1). Degradation was relatively low in midgut proteases. STI interacting proteins were isolated from saliva and midgut extracts of larvae fed on STI supplemented diet using affinity column...
March 2012: Protein Journal
Brad S Coates, Douglas V Sumerford, Miriam D Lopez, Haichuan Wang, Lisa M Fraser, Jeremy A Kroemer, Terrence Spencer, Kyung S Kim, Craig A Abel, Richard L Hellmich, Blair D Siegfried
The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is an introduced crop pest in North America that causes major damage to corn and reduces yield of food, feed, and biofuel materials. The Cry1F toxin from Bacillus thuringiensis (Bt) expressed in transgenic hybrid corn is highly toxic to O. nubilalis larvae and effective in minimizing feeding damage. A laboratory colony of O. nubilalis was selected for high levels of Cry1F resistance (>12,000-fold compared to susceptible larvae) and is capable of survival on transgenic hybrid corn...
August 2011: Genetica
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