Abstract
BackgroundThe spider-venom peptide ω-hexatoxin-Hv1a (Hv1a) targets insect voltage-gated calcium channels, acting directly at sites within the central nervous system. It is potently insecticidal when injected into a wide variety of insect pests, but it has limited oral toxicity. We examined the ability of snowdrop lectin (GNA), which is capable of traversing the insect gut epithelium, to act as a “carrier” in order to enhance the oral activity of Hv1a.Methodology/Principal FindingsA synthetic Hv1a/GNA fusion protein was produced by recombinant expression in the yeast Pichia pastoris. When injected into Mamestra brassicae larvae, the insecticidal activity of the Hv1a/GNA fusion protein was similar to that of recombinant Hv1a. However, when proteins were delivered orally via droplet feeding assays, Hv1a/GNA, but not Hv1a alone, caused a significant reduction in growth and survival of fifth stadium Mamestra brassicae (cabbage moth) larvae. Feeding second stadium larvae on leaf discs coated with Hv1a/GNA (0.1–0.2% w/v) caused ≥80% larval mortality within 10 days, whereas leaf discs coated with GNA (0.2% w/v) showed no acute effects. Intact Hv1a/GNA fusion protein was delivered to insect haemolymph following ingestion, as shown by Western blotting. Immunoblotting of nerve chords dissected from larvae following injection of GNA or Hv1a/GNA showed high levels of bound proteins. When insects were injected with, or fed on, fluorescently labelled GNA or HV1a/GNA, fluorescence was detected specifically associated with the central nerve chord.Conclusions/SignificanceIn addition to mediating transport of Hv1a across the gut epithelium in lepidopteran larvae, GNA is also capable of delivering Hv1a to sites of action within the insect central nervous system. We propose that fusion to GNA provides a general mechanism for dramatically enhancing the oral activity of insecticidal peptides and proteins.
Highlights
Arthropod venoms contain a rich diversity of compounds including a significant number of neurotoxic disulphide-rich peptides
One such example is v-hexatoxin-Hv1a, the best-studied member of a family of 36–37 residue insecticidal neurotoxins isolated from the venom of the Australian funnel web spider Hadronyche versuta
Hva1 Retains Insecticidal Activity when Fused to GNA Hv1a is the most studied member of the v-HXTX-1 family of insecticidal toxins isolated from the venom of the Australian funnel web spider Hadronyche versuta [9]
Summary
Arthropod venoms contain a rich diversity of compounds including a significant number of neurotoxic disulphide-rich peptides. Most spiders prey exclusively upon insects and other arthropods and it is not surprising that many spider-venom peptides have been shown to modulate the activity of arthropod ion channels One such example is v-hexatoxin-Hv1a (formerly vatracotoxin-Hv1a; hereafter referred to as Hv1a), the best-studied member of a family of 36–37 residue insecticidal neurotoxins isolated from the venom of the Australian funnel web spider Hadronyche versuta. The spider-venom peptide v-hexatoxin-Hv1a (Hv1a) targets insect voltage-gated calcium channels, acting directly at sites within the central nervous system. It is potently insecticidal when injected into a wide variety of insect pests, but it has limited oral toxicity. We examined the ability of snowdrop lectin (GNA), which is capable of traversing the insect gut epithelium, to act as a ‘‘carrier’’ in order to enhance the oral activity of Hv1a
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