Targeting of an expressed neurotoxin by its recombinant baculovirus.

AaIT, an insect-selective neurotoxic polypeptide derived from scorpion venom, has recently been used to engineer recombinant baculoviruses for insect pest control. Lepidopterous larvae infected with an AaIT-expressing baculovirus reveal symptoms of paralysis identical to those induced by injection of the native toxin. However, the paralyzed larvae treated by the recombinant virus possess an approximately 50-fold lower hemolymph toxin concentration than insects paralyzed by the native toxin. The mechanism of this potentiation effect was studied using immunocytochemistry, electrophysiology and toxicity assays. (i) Light microscopy, using peroxidase-conjugated antibodies, revealed the presence of toxin in virus-susceptible tissues, including tracheal epithelia located close to the central nervous system and beyond its lamellar enveloping sheath. (ii) High-resolution immunogold electron microscopical cytochemistry clearly revealed the presence of recombinant AaIT toxin inside the thoracic and abdominal ganglia on neuronal cell bodies and axonal membranes. (iii) Ventral nerve cords dissected from silkworm larvae infected with the recombinant baculovirus exhibited a high degree of excitability, expressed as enhanced frequency and bursting mode of their spontaneous activity, when compared to nerve cords infected with the wild-type virus. We conclude that the recombinant-virus-infected tracheal epithelia, outbranching in the body of an infected insect, (i) locally supply a continuous, freshly produced toxin to its neuronal receptors and (ii) introduce the expressed toxin to the insect central nervous system, thus providing it with critical target sites that are inaccessible to the native toxin.

AaIT: from neurotoxin to insecticide.

AaIT is a single chain neurotoxic polypeptide derived from the venom of the Buthid scorpion Androctonus australis Hector, composed of 70 amino acids cross-linked by four disulfide bridges. Its strict selectivity for insects has been documented by toxicity, electrophysiological and ligand receptor binding assays. These last have shown that various insect neuronal membranes possess a single class of non-interacting AaIT binding sites of high affinity (K(D) = 1-3(n)M) and low capacity (0.5-2.0 pmol/mg prot.). The fast excitatory paralysis induced by AaIT is a result of a presynaptic effect, namely the induction of a repetitive firing in the terminal branches of the insect's motor nerves resulting in a massive and uncoordinated stimulation of the respective skeletal muscles. The neuronal repetitive activity is attributed to an exclusive and specific perturbation of sodium conductance as a consequence of toxin binding to external loops of the insect voltage-dependent sodium channel and modification of its gating mechanism. From a strictly agrotechnical point of view AaIT involvement in plant protection has taken the following two complementary forms: firstly, as a factor for the genetic engineering of insect infective baculoviruses resulting in potent and selective bio-insecticides. The efficacy of the AaIT-expressing, recombinant baculovirus is attributed mainly to its ability to continuously provide and translocate the gene of the expressed toxin to the insect central nervous system; secondly, based on the pharmacological flexibility of the voltage-gated sodium channel, as a device for insecticide resistance management. Channel mutations conferring resistance to a given class of insecticidal agents (such as the KDR phenomenon) may greatly increase susceptibility to the AaIT expressing bioinsecticides. Thus the AaIT is a pharmacological tool for the study of insect neuronal excitability and chemical ecology and the development of new approaches to insect control.