1. Introduction
Biopesticides offer an eco-friendly alternative to chemical
pesticides, which are dangerous to humans, beneficial insects
and the environment, and present the problem of resistance
developed by crop pests 1,2. Spider venom derived neurotoxins
have been proven to be toxic towards various species of insects,
including pest species from the orders lepidoptera, coleoptera,
diptera, orthoptera and dictyoptera3. However, whilst injected
toxins are effective, oral absorbance is reduced4. Fusion-protein
technology can offer a solution5, since the production of a spider
toxin joined to a “carrier” protein can enhance absorbance in the
insect guts, passage to the hemolymph and reaching of the
central nervous system (CNS), where the venom can deliver its
effects (Fig.1).
Aims
The aim of this project is to produce and test oral activity of the
following fusion protein:
Toxin: ω-hexatoxin-Hv1a (hereafter named Hv1a) from the
Australian funnel-web spider Hadronyche versuta.
Carrier protein: carrier protein A* (hereafter named A), which has
previously proved to have insecticidal properties6 and to be
transported into the insects hemolymph7.
* The name of the protein cannot be disclosed due to intellectual property
restrictions.
Methods
The fusion protein Hv1a/A was produced using recombinant
technologies (Fig. 2) and expressed in the yeast Pichia pastoris.
Protein production was carried out using a BioFlo 110 laboratory
fermentor unit. Purification was carried out by his-tag Ni2+ affinity
chromatography, and the protein was de-salted via dialysis and
lyophilised by freeze-drying.
Hv1a was tested in lepidopteran larvae (Mamestra brassicae) by
droplet feeding assays over 24 and 48 hrs. The presence of the
protein in the blood and guts was detected via Western blotting
analysis.
Fig. 1. The path followed by the fusion protein Hv1a/A once administered
to the insects.
Work in progress
• Testing the efficacy of Hv1a/A through injections in M. Brassicae
and control of mortality in a 7-days period.
• Testing transport and toxicity of other fusion proteins obtained by
combining Hv1a with alternative carrier proteins.
Acknowledgments
Thanks to the Radhika V. Sreedhar Scholarship Fund for providing
financial support.
References
1. Raymond-Delpech, V., Matsuda, K., Sattelle, B.M., Rauh, J.J. and Sattelle, D.B. 2005. Ion channels: molecular targets of
neuroactive insecticides. Invertebr. Neurosci., 5: 119–33.
2. Beard, J. 2006. DDT and human health. Sci. Total Environ., 355: 78-89.
3. Fitches, E.C., Pyati, P., King, G.F. and Gatehouse, G.A. 2012. Fusion to snowdrop lectin magnifies the oral activity of
insecticidal v-Hexatoxin-Hv1a peptide by enabling its delivery to the central nervous system. PLoS ONE, 7(6): e39389.
4. Fitches, E.C., Bell, H.A., Powell, M.E., Back, E. Sargiotti, C. Weavera, R.J. and Gatehouseb, J.A. 2009. Insecticidal
activity of scorpion toxin (ButaIT) and snowdrop lectin (GNA) containing fusion proteins towards pest species of
different orders. Pest Manag. Sci., 66: 74-83.
5. Beckage, N.E. and Gelman, D.B. 2004. Wasp parasitoid distruption of host development: Implications for new
biologically based strategies for insect control. Annu. Rev. Entomol., 49: 299–330.
6. Christeller, J.T., Laing, W.A., Shaw, B.D. and Burgess, E.P.J. 1990. Characterisation and partial purification of the
digestive proteases of the black field cricket, Teleogryllus commodus (Walker): elastase is a major component. Insect
Biochem., 20: 157-164.
7. Keenan, T., Fitches, E., Powell, M. and Pyati, P. 2013. The use of recombinant fusion protein technology in the
development of novel Bioinsecticides. Fera Student Conference 2013.
Hv1a Protein A
Fig. 2. Recombinant technology used to obtain Hv1a/A.
Hv1a GNA
pGAPZαB
Pro Protein A
pGAPZαB
Digestion with PstI and NotI Ligation with T4
Hv1a Protein A
pGAPZαB
The results demonstrate the successful transport of Hv1a into the
insects’ guts and blood 24 hrs after oral delivery. At 48 hrs the
protein concentration in the blood is reduced.
Results and discussion
Figure 3. Western blot of M. brassicae larvae homogenised guts (left) and
blood (right) 24 and 48 hrs after oral delivery of Hv1a/A. Anti-protein A
antibodies were used (1:5000).
Hadronyche versuta
Novel biopesticides: can fusion protein technology
enhance oral absorption of spider venom toxins?
Giovanna Pesante1, Elaine Fitches1, Michelle Powell1, Hannah Bradish1, Prashant Pyati2
1The Food and Environment Research Agency, Sand Hutton, York, YO41 1LZ, UK
2School of Biological and Biomedical, Sciences, University of Durham, Durham, United Kingdom
PstI
NotI
PstI PstINotI NotI
Mw
kDa
30
25
20
15
Negative
control
24hrs R1
10 40
24hrs R2
10 40
48hrs R1
10 40
48hrs R2
10 40c