Project Report No. RD-2006-3293/ LK0991 New environmentally-friendly technologies for slug control based on orally-delivered fusion proteins containing specific molluscicidal toxins. by E. Fitches1, F. Beltrametti2 and H. Caruel3, J.A. Gatehouse4 1Fera, Sand Hutton, York, YO41 1LZ 2Isagro Ricerca S.r.l.,Via Fauser, 4, 28100 Novara (NO), Italy 3 De Sangosse, Centre Recherche et Developpement De Sangosse, ZA Malère-Av. Jean Serres, 47 480 Pont du Casse, France. 4 School of Biological and Biomedical Science, South Road, Durham, DH1 3LE May 2012; Cost £600 000 This is the final report of a 54 month project (RD-2006-3293) which started in September 2007. The work was funded by LINK and a contract for £150, 000 from HGCA. While the Agriculture and Horticulture Development Board, operating through its HGCA division, seeks to ensure that the information contained within this document is accurate at the time of printing no warranty is given in respect thereof and, to the maximum extent permitted by law, the Agriculture and Horticulture Development Board accepts no liability for loss, damage or injury howsoever caused (including that caused by negligence) or suffered directly or indirectly in relation to information and opinions contained in or omitted from this document. Reference herein to trade names and proprietary products without stating that they are protected does not imply that they may be regarded as unprotected and thus free for general use. No endorsement of named products is intended, nor is any criticism implied of other alternative, but unnamed, products. HGCA is the cereals and oilseeds division of the Agriculture and Horticulture Development Board. Table of Contents Page No. Section 1: Abstract 5 Section 2: Summary 7 2.1 Introduction/Background and Aims 7 2.2 Materials and Methods 9 2.3 Results 11 2.4 Discussion/Conclusions & Implications 13 Section 3: Technical Report 16 3.1: Production of recombinant molluscicidal peptides 16 3.1A. Introduction 16 3.2A. Materials & Methods 18 3.2A.1 Assembly of synthetic genes encoding TxIA and -GmVIA 18 3.2A.2. Assembly of thioredoxin-TxIA and thioredoxin- -GmVIA expression constructs and 18 E. coll transformation 3.2A.3. Expression and purification of thioredoxin (TRX)-TxIA, TRX- -GmVIA and TRX 19 recombinant peptides 3.2.A.4 Mollusc and Insect Cultures 20 3.2A.5. Biological activity: Deroceras reticulatum and Mamestra brassicae injection 20 bioassays of thioredoxin (TRX)-TxIA, TRX- -GmVIA and TRX recombinant peptides 3.2A.6. Cleavage of thioredoxin from TRX-TxVIA and MALDI-TOF analysis of TxVIA 20 peptide 3.3A Results 21 3.3A.1. Synthetic gene assembly, expression and purification of TRX-TXIA, TRX- -GmVIA 21 fusion proteins and TRX in E.coli 3.3A.2. Biological activity: D. reticulatum and M. brassicae injection bioassays of 21 thioredoxin (TRX)-TxIA, TRX- -GmVIA and TRX recombinant peptides 3.1B: Expression of recombinant TxVIA using yeast as an expression host. 24 3.1B. Materials and Methods 24 3.1B.1 Yeast Expression constructs 24 3.1.B.2 Recombinant TxVIA expression and purification from yeast 25 3.1.B.3 Insect and Mollusc Cultures 25 3.1B.4 Injection bioassays 25 3.1B.5 Electrophoresis and Western blotting 25 3.2B Results 26 1 3.2B.1 Production of recombinant TxVIA in yeast 26 3.2B.2 Injection Bioassays 27 3.2B2.2. Effect of recombinant TxVIA on cabbage moth, house fly and slug 28 3.3B Discussion (section 3.1A and 3.1B) 30 Section 3.2: Chemical synthesis and testing of mollusc-specific 33 peptides 3.2A Introduction 33 3.2B Materials & Methods 34 3.2.B.1 Selection and synthesis of candidate molluscicidal peptides 34 3.2B.2 Mollusc cultures 34 3.2.B.3 Biological activity: Deroceras reticulatum Injection bioassays 34 3.2C Results 34 3.2.C.1 Biological activity: Deroceras reticulatum Injection bioassays 34 3.2D Conclusions 35 Section 3.3: Evaluation of carrier proteins 36 3.3A Introduction 36 3.3B. Materials and Methods 36 3.3B.1 Production and purification of recombinant GNA 36 3.3B.2 Production and purification of recombinant ASA II 37 3.3B.3. Production and purification of recombinant avidin 37 3.3B.4. Antibodies to recombinant GNA, ASA II, and avidin 37 3.3B.5. Haemagglutination assays 38 3.3B.6 Recombinant avidin; In vitro activity 38 3.3B.7 Mollusc D. reticulatum Culture 38 3.3B.8. Feeding assays D. reticulatum and sample extraction 38 3.3B.9. Western analysis of D. reticulatum samples 39 3.3C. Results 39 3.3C.1. Production and purification of recombinant GNA, ASA II, and avidin 39 3.3C.2. Haemagglutination assays 40 3.3C.3. In vitro activity of recombinant avidin 41 3.3C.4. Detection of recombinant GNA, ASA II and avidin in slugs after feeding 41 3.3D Discussion 43 Section 3.4: Evaluation of insecticidal fusion protein(s) 44 3.4A Introduction 44 3.4B Materials and Methods 44 2 3.4B.1. Production of RST/GNA (FP4) and omega/GNA (FP5) 44 3.4B.2. Mollusc culture 46 3.4B.3. Injection assays 46 3.4B.4 Feeding assays: Coated lettuce disc assays 46 3.4B.5. Fusion proteins: Stability to heat treatment 46 3.4B.6. Wheat pellet assays 46 3.4B.7. Stability of fusion proteins in feeding assays 47 3.4B.8. Oral delivery of omega/GNA to slug circulatory system 47 3.4C Results 47 3.4C.1. Production of RST/GNA (FP4) and omega/GNA (FP5 + and – His) 47 3.4C.2. Biological activity by injection: D. reticulatum 49 3.4C.3. Biological activity by ingestion: leaf disc assays D. reticulatum 50 3.4C.4. Stability of FP5 to heat treatment 52 3.4C.5. Biological activity by ingestion: wheat pellet assays D. reticulatum 52 3.4C.6. Stability of FP5 when coated onto discs or incorporated into pellets 54 3.4C.7. Delivery of FP5 to slug circulatory system following ingestion 56 3.4D Discussion and Conclusions 57 Section 3.5: Production of molluscicidal fusion proteins by 59 industry 3.5A Introduction 59 3.5B Materials and Methods 59 3.5B.1 Growth and fermentation in flask 60 3.5B.2 Fermentation at 200 liter scale 60 3.5B.3 Monitoring of the expression of the FP5 and of total secreted proteins 62 3.5B.4 Quantification of the insoluble and soluble FP5 complex fractions 62 3.5B.5 Development of an HPLC method for the quantification of the FP complex 62 3.5B.6 Purification of the FP5 complex with Ni conjugated resins 62 3.5C Results and Discussion 63 3.5C.1 Bioreactor fermentation 63 3.5C.2 Downstream processing 64 3.5C.3 Purification of the FP5 protein complex on Ni-conjugated resins. 64 3.5D Conclusion 69 3.5E Appendix 69 Section 3.6: Efficacy evaluation by industry 73 3.6A Introduction 73 3 3.6B Materials and Methods 73 3.6C Results 75 3.6D Conclusions 78 References 80 4 Section 1. ABSTRACT The aim of this project was to investigate the potential for a novel technology, originally conceived and developed for the control of insect pests, to be extended for the control of mollusc crop pests, with focus on the grey field slug Deroceras reticulatum. This technology allows naturally occurring proteins, which have low, or no, toxicity when delivered orally to be converted into effective and orally active pesticides. The approach, patented by the academic partners uses genes encoding fusion proteins that contain a plant derived protein carrier (Galanthus nivalis agglutinin; GNA) linked to an insect derived peptide (neurohormone, venom toxin, etc.) that must normally be delivered to the blood in order to be active (e.g. via a sting). Whereas neither component of the fusion is toxic when fed alone, or as a mixture, the fusion protein shows oral toxicity as a result of the carrier transporting the active peptide across the insect gut and delivering it to the blood, from where it can access the central nervous system. The proteins are produced in bacterial or yeast expression systems, and delivered orally as a component of diet. Our primary aim was to investigate if peptides derived from the venom of mollusc hunting Cone snails (Conus spp.), that had previously been reported to have mollusc specific activity, could be exploited for the generation of molluscicidal fusion proteins. Considerable difficulties were encountered with the expression of functional cone snail derived peptides. Nevertheless we reported the first successful production of a conopeptide using a yeast- based system (Bruce et al., 2011). Contrary to published claims this conopeptide was found to have insecticidal activity and exhibited no toxicity towards slugs. The limited research that has been conducted with molluscs significantly restricted the number of candidates available for exploitation using fusion protein technology. However, we have identified for the first time the potential for the use of arthropod derived peptides to target the central nervous system of slugs. Two insecticidal fusion proteins incorporating spider venom peptides known to target insect ion channels were found to cause mortality when injected into slugs. Furthermore “proof of concept” for the delivery system was provided by assays that demonstrated significant reductions in the growth of slugs fed on coated discs or fusion protein containing pellets. Reduced growth was shown to be attributable to significantly reduced consumption by the treated slugs. Thus, despite the evolutionary distance between arthropods and molluscs, our results suggest that sufficient similarities in the genetic make up of the central nervous systems exist to enable exploitation of arthropod derived toxins for the control of pestiferous molluscs. A vast range of arthropod derived venom peptides have been isolated to date and in many cases the insect ion channels that they target have been identified.