Research article

Bacillus thuringiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of four Aminopeptidase N genes

Salvador Herrero^{* 1,2} , Tsanko Gechev^{* 1} , Petra L Bakker¹ , William J Moar³ and Ruud A de Maagd¹

¹Business Unit Bioscience, Plant Research International B.V., Wageningen University and Research Center, P.O. Box 16, 6700 AA Wageningen, The Netherlands

²Laboratory of Virology, Department of Plant Sciences, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands

³Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama 36849, USA

author email corresponding author email* Contributed equally

BMC Genomics 2005, 6:96doi:10.1186/1471-2164-6-96

Published:	24 June 2005

Abstract

Background

Insecticidal toxins from Bacillus thuringiensis bind to receptors on midgut epithelial cells of susceptible insect larvae. Aminopeptidases N (APNs) from several insect species have been shown to be putative receptors for these toxins. Here we report the cloning and expression analysis of four APN cDNAs from Spodoptera exigua.

Results

Suppression Subtractive Hybridization (SSH) was used to construct cDNA libraries of genes that are up-and down-regulated in the midgut of last instar larvae of beet armyworm, S. exigua exposed to B. thuringiensis Cry1Ca toxin. Among the clones from the SSH libraries, cDNA fragments coding for two different APNs were obtained (APN2 and APN4). A similar procedure was employed to compare mRNA differences between susceptible and Cry1Ca resistant S. exigua. Among the clones from this last comparison, cDNA fragments belonging to a third APN (APN1) were detected. Using sequences obtained from the three APN cDNA fragments and degenerate primers for a fourth APN (APN3), the full length sequences of four S. exigua APN cDNAs were obtained. Northern blot analysis of expression of the four APNs showed complete absence of APN1 expression in the resistant insects, while the other three APNs showed similar expression levels in the resistant and susceptible insects.

Conclusion

We have cloned and characterized four different midgut APN cDNAs from S. exigua. Expression analysis revealed the lack of expression of one of these APNs in the larvae of a Cry1Ca-resistant colony. Combined with previous evidence that shows the importance of APN in the mode of action of B. thuringiensis toxins, these results suggest that the lack of APN1 expression plays a role in the resistance to Cry1Ca in this S. exigua colony.