Identification of two Bacillus thuringiensis Cry3Aa toxin-binding aminopeptidase N from Rhynchophorus ferrugineus (Coleoptera: Curculionidae).

Rhynchophorus ferrugineus is a quarantine pest that mainly damages plants in tropical regions, which are essential economic resources. Cry3Aa has been used to control coleopteran pests and is known to be toxic to R. ferrugineus. The binding of the Cry toxin to specific receptors on the target insect plays a crucial role in the toxicological mechanism of Cry toxins. However, in the case of R. ferrugineus, the nature and identity of the receptor proteins involved remain unknown. In the present study, pull-down assays and mass spectrometry were used to identify two proteins of aminopeptidase N proteins (RfAPN2a and RfAPN2b) in the larval midguts of R. ferrugineus. Cry3Aa was able to bind to RfAPN2a (Kd = 108.5 nM) and RfAPN2b (Kd = 68.2 nM), as well as midgut brush border membrane vesicles (Kd = 482.5 nM). In silico analysis of both RfAPN proteins included the signal peptide and anchored sites for glycosyl phosphatidyl inositol. In addition, RfAPN2a and RfAPN2b were expressed in the human embryonic kidney 293T cell line, and cytotoxicity assays showed that the transgenic cells were not susceptible to activated Cry3Aa. Our results show that RfAPN2a and RfAPN2b are Cry3Aa-binding proteins involved in the Cry3Aa toxicity of R. ferrugineus. This study deepens our understanding of the action mechanism of Cry3Aa in R. ferrugineus larvae.

Proteolytic Activation of Bacillus thuringiensis Cry3Aa Toxin in the Red Palm Weevil (Coleoptera: Curculionidae).

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.

Characterization of Bacterial Communities Associated with Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) and its Host Phoenix sylvestris.

This study measured the changes of microorganisms in the midgut and habitat niche of Rhynchophorus ferrugineus Olivier, an invasive quarantine pest, by Illumina sequencing. The bacterial diversity in the R. ferrugineus larvae midgut and their habitat niche was compared to the uninfected P. sylvestris. The Proteobacteria and Firmicutes occupied a dominant position in the R. ferrugineus midgut and infected P. sylvestris, while in the uninfected P. sylvestris the predominant bacterial phylum was the Cyanobacteria. Enterobacter, Dysgonomonas, and Entomoplasma were the dominant bacterial genera in R. ferrugineus midgut and also within the infected trees and uninfected trees with low relative abundance. These bacteria could be exploited as the biopesticide vector to control R. ferrugineus population. Besides, Sphingobacterium, Shinella, and Rhodobacter genera had the same distribution pattern in the infected and uninfected P. sylvestris, and these bacteria were not found in the midgut of R. ferrugineus. Interestingly, Paludibacter and Parabacteroides were only distributed in the wood fiber of the infected P. sylvestris, which could be used as potential microbial markers to detect if the palm plants are damaged by the R. ferrugineus. The results of this study will be beneficial to the development of control strategies for R. ferrugineus.

The complete mitochondrial genome of Monochamus alternatus alternatus (Coleoptera: Cerambycidae).

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.

[Monthly changes in caloric values of five shrubby Palmae species leaves].

Studies on the monthly changes in the ash contents and caloric values of 5 shrubby Palmae species (Chamaerops humilis, Rhapis gracilis, Sabal minor, Chamaedorea cataractarum, Chamaedorea brachypoda) leaves showed that annual average ash content of the five shrubby species was 4.87% +/- 1.37% for Chamaerops humilis, 8.33% +/- 0.89% for Rhapis gracilis, 7.85% +/- 2.64% for Sabal minor, 9.20% +/- 1.35% for Chamaedorea cataractarum, and 12.42% +/- 1.78% for Chamaedorea brachypoda. The monthly changes of gross caloric value were different for Chamaerops humilis, Rhapis gracilis and Sabal minor, but similar to each other for Chamaedorea cataractarum and Chamaedorea brachypoda, and the annual average gross caloric value of the five shrubby species was 20.50 +/- 0.32 kJ x g(-1) for Chamaerops humilis, 20.04 +/- 0.50 kJ x g(-1) for Rhapis gracilis, 20.21 +/- 0.68 kJ x g(-1) for Sabal minor, 20.52 +/- 0.48 kJ x g(-1) for Chamaedorea cataractarum and 18.90 +/- 0.47 kJ x g(-1) for Chamaedorea brachypoda. The gross caloric values were correlated remarkably with ash contents for Chamaedorea cataractarum and Sabal minor (P < 0.05), but there was no significant correlation between gross caloric values and ash contents for other three species (P > 0.05). Rhapis gracilis and Sabal minor, and Chamaedorea cataractarum and Chamaedorea brachypoda had similar monthly changes in ash free caloric values, respectively. The average ash free caloric value of the five shrubby species was 21.55 +/- 0.53 kJ x g(-1) for Chamaerops humilis, 21.87 +/- 0.46 kJ x g(-1) for Rhapis gracilis, 21.84 +/- 0.53 kJ x g(-1) for Sabal minor, 22.60 +/- 0.81 kJ x g(-1) for Chamaedorea cataractarum, and 21.59 +/- 0.63 kJ x g(-1) for Chamaedorea brachypoda. Chamaedorea cataractarum had a higher ash free caloric value than other four species (P < 0.05), and the ash free caloric values of Chamaerops humilis, Rhapis gracilis, Sabal minor and Chamaedorea brachypoda were similar, the differences being not significant by t test (P > 0.05).