Digestive enzymes of vine weevil (Otiorhynchus sulcatus) as potential targets for insect control strategies.

Over the previous quarter century the vine weevil (Otiorhynchus sulcatus) has become a pest of horticultural and agricultural plants. The vine weevil is a polyphagous coleopteran insect and is able to attack over one hundred different plant species. Its spread has been limited by its lack of flight but modern world trade in live container grown plants has spread the insect to new habitats. Damage to plants caused by vine weevil is two fold, with the larvae destroying root balls while the adults attack the, leaves. The larval stage, in particular is difficult to treat with conventional insecticides unless environmentally undesirable soil treatments are used. The current lack of defence against the vine weevil has opened the door for methods of crop protection through the generation of genetically modified plants. The design of an efficient GM approach to control the vine weevil requires a sound knowledge of the insect’s digestive enzymes, which may be used as potential targets for insecticidal proteins. This approach was achieved for the vine weevil through analysis of active digestive proteases in the insects gut and the identification of suitable proteinase inhibitors which would reduce the overall level of protein hydrolysis. Using this method it was discovered that the vine weevil contained both serine and cysteine proteases in addition to a range of other digestive hydrolases. This biochemical data was supported by a molecular approach to isolate cDNA clones associated with the insect's digestive tract. Using a gut specific cDNA library clones encoding a cathepsin B protease, two trypsin proteases, a pectinesterase, a lipase and a cellulase were isolated and characterised. The cellulase isolated from vine weevil has been shown to originate from the insect genome as shown through Southern Blot analysis and sequencing across several intronic regions. Evidence presented herein shows that the vine weevil gut extract hydrolyses both cellulose and cellobiose. Similar results were observed with recombinant protein expressed in the eukaryotic yeast P.pastoris. Furthermore data presented here shows that the vine weevil has the full complement of enzymes needed for the complete digestion of crystalline cellulose, which was until recently believed to be the sole domain of several species of bacteria and yeast. In addition a cDNA clone encoded a vine weevil endogenous chitinase was isolated from the cDNA library. This chitinase cDNA and one encoding the proteinase inhibitor Oryzacystatin-I were used to generate transgenic tobacco plants which have been shown to express the transgene. These transgenic plants are the first step in developing a strategy for plant protection against vine weevil based on genetic modification