Biological activity and binding site characteristics of the PA1b Entomotoxin on insects from different orders

Abstract

The aim of this work was to investigate both the biological activity of an entomotoxin, the pea albumin 1b (PA1b), and the presence or absence of its binding site within an array of insect species. The data obtained showed that insect sensitivity was not related to its taxonomic position. Moreover, PA1b was not toxic to several tested microorganisms. However, the binding site was found to be conserved among very different insects, displaying similar thermodynamic constants regardless of the in vivo species sensitivity. The binding site alone was, therefore, not sufficient for toxicity. One exception was the pea weevil, Bruchus pisorum, which was the only tested species without any detectable binding activity. These findings indicate that the binding site probably has an important endogenous function in insects and that adaptation to pea seeds resulted in the elimination of the toxin binding activity in two independent insect lineages. Other mechanisms are likely to interact with the toxin effects, although they are still largely unknown, but there is no evidence of any specific degradation of PA1b in the midgut of insects insensitive to the toxin, such as Drosophila melanogaster or Mamestra brassicae.

Figure 1.

Effects of PA1b on the lady beetle H. axyridis. Tests were done with artificial diet supplemented with the toxin (250 µg/g), and the development of the insects was monitored daily. First bar, control insects; second bar, tested insects. P, Pupae; pP, Pre-pupae; L₄,4^th instar larvae; L₃, 3^rd instar larvae.

Figure 2.

Effects of PA1b on three aphid species. Tests were carried out with artificial diet supplemented with the toxin at increasing concentrations, and mortality of the insects was monitored daily (relative larval survival is used in the graph). Circle : A. pisum ; Square : M. persicae; Triangle : A. gossypii.

Figure 3.

Effects of A. gossypii protein extracts on PA1b. PA1b was incubated with 200 µg of A. gossypii proteins in a solution buffered from 3 to 9. Degradation of PA1b was monitored after incubation by reverse-phase HPLC. Control assays were done using heat-denaturated insect protein.