The mechanisms of action of insecticidal lectins from snowdrop (GNA) and jackbean (concanavalin a) ontomato moth larvae

Abstract

Artificial diet bioassays were carried out to investigate the impact of GNA and Con A upon the development of L. oleracea larvae. GNA, at 2 % of total dietary protein, exerted a significantly detrimental effect upon larval development, growth and consumption, with little effect upon survival. Con A was shown to be the more toxic of the 2 lectins. When tested at concentrations of 2.0 %, 0.2 % and 0.02 %, Con A caused a significant decrease in survival and larval development, and caused greater reductions in larval growth and consumption compared to GNA. The potential for GNA and Con A to exert insecticidal effects via binding to the brush border membrane (BBM) and peritrophic membrane (PM) of L. oleracea larvae was investigated. Con A, which specifically binds a-D-mannopyranoside and a-D- glucopyranoside residues, was shown to bind in vitro to the majority of BBM and PM proteins. In contrast GNA, which exhibits strict specificity for a(l,3) and a(l,6)-linked D-mannose residues, bound to only 5 BBMV and 2 PM proteins. In agreement, higher levels of Con A, compared to GNA, were shown to accumulate in larval gut tissue after feeding the proteins in vivo. Despite this both lectins were shown to have a similar ability to disrupt the digestive capacity of the larval midgut. GNA and Con A stimulated similar short term elevations in BBM enzyme and soluble trypsin activities and a long-term reduction in a-glucosidase activity. Increases in levels of trypsin activity in faecal material collected from lectin-fed larvae suggested that the proteins may act by disrupting mechanisms of enzyme recycling. Aminopeptidase, an abundant and avidly binding BBM protein (120 kDa), was identified as a major Con A binding species in L. oleracea. A 98 kDa GNA-binding BBM protein was purified and amino acid sequence data was obtained from digest polypeptides allowing oligonucleotide primers to be designed. Subsequent attempts to amplify (by PCR and RT-PCR) fragments containing coding sequence corresponding to the 98 kDa protein were unsuccessful. This was attributed to oligonucleotide degeneracy together with the low abundance and relatively large size of the protein. The potential for GNA and Con A to exert systemic effects upon I. oleracea was demonstrated by the detection of both lectins in the haemolymph of larvae exposed to experimental diets. GNA was detected in haemolymph of larvae exposed to experimental diet for just 2 hours. In contrast, no Con A was detectable in haemolymph extracted from larvae fed for 24 hours, although it was present in the haemolymph after 5 days of exposure to the diet. That GNA and Con A may act directly upon organs other than the insect gut was indicated by the detection of lectins in vivo in malpighian tubule and fat body tissue extracts. A significant reduction in haemocyte abundance in haemolymph samples extracted from lectin-fed larvae suggested that both GNA and Con A may also act by disrupting the immune system of L. oleracea.