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Durham University

Department of Biosciences

Academic Staff

Publication details for Professor John A. Gatehouse

Fitches, E.C., Pyati, P., King, G.F. & Gatehouse, J.A. (2012). Fusion to snowdrop lectin magnifies the oral activity of insecticidal omega-Hexatoxin-Hv1a peptide by enabling its delivery to the central nervous system. PLoS ONE 7(6): e39389.

Author(s) from Durham

Abstract

Background: 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.
Methodology/Principal Findings: A 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/Significance: In 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.