Abstract Bacillus thuringiensis Cry proteins have been widely used over the past decades for many different insect pests, which are safe for users and the environment. The coleopteran-specific Cry3Aa toxin from B. thuringiensis exhibits toxicity to the larvae of Monochamus alternatus. Receptors play a key role in the mechanisms underlying the toxic action of Cry. However, the binding receptor for Cry3Aa has yet to be identified in the midgut of M. alternatus larvae. Therefore, the aim of this study was to identify the receptor for Cry3Aa toxin in the brush border membrane vesicles (BBMVs) of M. alternatus larvae. Our results indicate that the Cry3Aa toxin binds to the BBMVs (Kd = 247 nM) of M. alternatus via a 107 kDa aminopeptidase N (APN) (Kd = 57 nM). In silico analysis of the APN protein predicted that an 18 amino acid sequence in the N-terminal acted as a signal peptide, and that the Asn residue, located at position 918 in the C-terminus is an anchored site for glycosyl phosphatidyl inositol. Further analysis showed that M. alternatus APN exhibits 75% homology to the APN from Anoplophora glabripenis. Our work, therefore, confirmed that APN, which is localized in the BBMVs in the midgut of M. alternatus larvae, acts as a binding protein for Cry3Aa toxins.

Monochamus alternatus alternatus is the major vector of pinewood nematode, Bursaphelenchus xylophilus, in Asia. The length of the complete mitochondria genome of M. alternatus alternatus was 15,880 bp with 21% GC content, including 39.7% A, 12.3% C, 8.7% G and 39.3% T. There were 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and one AT-rich region. This study provides a useful genetic information for subsequent study of the differences between M. alternatus subspecies.

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The red palm weevil (RPW), Rhynchophorus ferrugineus (Oliver) is an important pest of palms that causes significant damage by boring into and feeding within palm stem tissues. Here, we studied the proteolytic process of Cry3Aa in the RPW to understand the mechanism of Cry toxicity. The bioassays showed that Cry3Aa toxin is weakly toxic to the RPW. Proteolytic activation assays indicated the Cry3Aa protein is digested into smaller fragments than the 55-kDa activated fragments under different conditions. In particular, at higher mass ratios of gut protease and Cry3Aa protein (5:1, 2:1, and 1:1, respectively), and at 36.9°C for 16 h in a solution of pH 8.6, the Cry3Aa protoxin is over-digested by the gut proteases of weevil larvae. Moreover, the zymogram analysis of the gut proteases revealed the RPW larvae harbors intestinal digestive enzymes mainly composed of serine proteases. This study describes the proteolytic activation process of Cry3Aa in the midgut of RPW larvae.

Trictenotoma davidi Deyrolle, 1875 is a beetle of the Trictenotomidae family. The length of the complete mitochondria genome of T. davidi was 15,910 bp with 24.1% GC content, including 39.9% A, 15.1% C, 9.0% G, and 36.0% T. The genome encoded 13 protein-coding genes, 22 tRNAs, and 2 rRNAs. Phylogenetic analysis showed that T. davidi was closely related to Vincenzellus ruficollis. This study provided useful genetic information for the evolution of T. davidi and Trictenotomidae insects.