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.

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The Drosophila melanogaster (fruit fly) gene Diap1 encodes a protein referred to as DIAP1 (D rosophila Inhibitor of Apoptosis Protein 1) that acts to supress apoptosis in “normal” cells in the fly. In this study we investigate the use of RNA interference (RNAi) to control two dipteran pests, Musca domestica and Delia radicum, by disrupting the control of apoptosis. Larval injections of 125–500 ng of Diap1 dsRNA resulted in dose-dependent mortality which was shown to be attributable to down-regulation of target mRNA. Insects injected with Diap1 dsRNA have approx. 1.5-2-fold higher levels of caspase activity than controls 24 hours post injection, providing biochemical evidence that inhibition of apoptotic activity by the Diap1 gene product has been decreased. By contrast adults were insensitive to injected dsRNA. Oral delivery failed to induce RNAi effects and we suggest this is attributable to degradation of ingested dsRNA by intra and extracellular RNAses. Non-target effects were demonstrated via mortality and down-regulation of Diap1 mRNA levels in M. domestica larvae injected with D. radicum Diap1 dsRNA, despite the absence of 21 bp identical sequence regions in the dsRNA. Here we show that identical 15 bp regions in dsRNA are sufficient to trigger non-target RNAi effects.

Herein, we report the production of a recombinant Tepary bean lectin (rTBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. rTBL-1 was expressed in Pichia pastoris, yielding 316 mg per liter of culture, and was purified by nickel affinity chromatography. Characterization of the protein showed that rTBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca2+ and Mn2+) attached to each subunit, confirmed in its 3D structure solved by X-ray diffraction at 1.9 Å resolution. Monomers also presented a ~2.5 kDa N-linked glycan located on the opposite face of the binding pocket. It does not participate in carbohydrate recognition but contributes to the stabilization of the interfaces between protomers. Screening for potential rTBL-1 targets by glycan array identified 14 positive binders, all of which correspond to β1-6 branched N-glycans’ characteristics of cancer cells. The presence of α1-6 core fucose, also tumor-associated, improved carbohydrate recognition. rTBL-1 affinity for a broad spectrum of mono- and disaccharides was evaluated by isothermal titration calorimetry (ITC); however, no interaction was detected, corroborating that carbohydrate recognition is highly specific and requires larger ligands for binding. This would explain the differential recognition between healthy and cancer cells by Tepary bean lectins.

Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the “carrier” snowdrop lectin (GNA) to confer oral toxicity.The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th – 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide.