Project Report No. RD-2006-3293/ LK0991 New Environmentally

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

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

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

3.6B Materials and Methods 73 3.6C Results 75 3.6D Conclusions 78 References 80

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. Further research to identify genetic and biochemical similarities in the ion channel targets of insects and molluscs would enable an informed and targeted approach to be followed for the development of molluscicidal fusion proteins.

The insecticidal fusion protein that exhibited the greatest detrimental effects on slugs was selected for production scale-up and efficacy testing via incorporation into baits by the industrial partners. The transfer of production methods from laboratory to pilot production scale was more problematic than anticipated and the production of large quantities of product was not achievable within the time constraints of the project. Initially it was believed that improvements in expression levels could be obtained using basic clone selection techniques and through the optimisation of fermentation conditions. Subsequently we demonstrated that significant increases in production levels were achievable but this required the use of more sophisticated molecular techniques to enable the incorporation of multiple copies of fusion protein cassettes into the yeast genome. Despite this, pilot scale fermentation (200 litre) by the industrial partners produced in excess of 3 grams of fusion protein for subsequent incorporation into slug baits. The biological activity of the product was verified in glass-house trials by Isagro Ricerca prior to supply to De Sangosse. Initially De Sangosse tested fusion protein produced by the academic partners in ‘forced oral route’ assays where the product was incorporated into an alginate gel. In the first assay a significant effect on mortality was observed. An effect on survival was not observed in the second assay and this may have been due to reduced activity following storage of the fusion protein containing gel. However, in both cases it was observed that the slugs did not feed following ingestion of the fusion protein containing gel. These results were comparable to the results obtained in laboratory scale assays carried out by the academic partners. However, a forced oral route carried out by De Sangosse using fusion protein produced by Isagro Ricerca did not show any significant effects on mortality and the slugs were seen to feed following ingestion of the gel. The reason for the discrepancy between results obtained with protein produced by academic and industrial partners are not clear. Further bait efficacy tests carried out using product supplied by Isagro Ricerca showed that the slugs consumed fusion protein containing pellets although in depth analysis of growth and consumption was not conducted. It is recognised that the fusion protein selected for scale-up and efficacy testing represents ‘proof of concept’ rather than the provision of a prototype for further development by industry. Further research is required to identify and develop fusion proteins that have greater efficacy against molluscan crop pests. Nevertheless it is concluded that potential for application of a biotechnological approach for the development of new molluscicidal products has been demonstrated.

Section 2: SUMMARY 2.1 Introduction/Background and Aims Molluscs, and particularly slugs, are a major problem for agriculture and horticulture in the UK. Reduced crop yields and the cost of applying treatments both have a significant economic impact upon industry. The destruction of seedling embryos and young plants by grey and black field slugs (Deroceras reticulatum and Arion hortensis, respectively) can result in considerable reductions in overall yield, both directly due to feeding, and also via the generation of gaps in growing crops allowing colonisation by invasive weed species. Indirect losses due to slug activity can also occur via the vectoring of parasites and plant diseases. The use of molluscicides is widespread, and has increased dramatically over the past 30 years (Brooks & Crook, 2006). Currently only six molluscicides (metaldehyde, methiocarb, thiodicarb, aluminium sulphate, copper silicate, ferric phosphate) are approved for the control of snails and slugs in the UK. The grey field slug, Deroceras reticulatum, is a major crop pest in the United Kingdom, with control by molluscicides requiring widespread treatment. The Pesticide Usage Survey conducted on UK arable crops by FERA, 2008, showed, for wheat alone, 882,555 out of a total of 2,068,104 hectares (43%) were treated with molluscicides. The two main agents, metaldehyde and methiocarb (representing 98% of molluscicides used), are toxic to non-target organisms and frequent contaminants of ground water. As a result, there is increasing interest in developing more environmentally-friendly alternatives. However, alternatives such as iron phosphate show lower efficacy and higher cost than metaldehyde-based crop protection methods (Spieser & Kistler 2002). Inclusion of chelating agents such as EDTA in iron phosphate baits affects earthworm viability (Edwards et al., 2009), and has been reported to cause toxic effects on mammals (Haldane & Davis 2009). "Natural" molluscicides have also been described, such as garlic (allicin) and copper plate but their effects are limited to temporary repellence (Schoeder et al., 2003). Caffeine has been shown to cause mortality but can also damage the leaves of crops such as lettuce (Hollinsgworth et al., 2002). The nematode species Phasmarhabditis hermaphrodita, a known parasite of D. reticulatum, has been developed as biological control agent with a commercial product now available (Nemaslug®). Field trials with biological control have been conducted on asparagus but the method has shown "poor results" when compared with chemical molluscicides for mainstream crop protection (Rae et al., 2009).

One of the main problems associated with the widespread use of molluscicides is their toxicity towards non-target organisms. The poisoning of wild mammals, domestic animals, agricultural livestock, and birds, has been attributed to metaldehyde and methiocarb slug pellets (Giles et al., 1984, Homeida & Cooke, 1982; Richardson et al., 2003; Greig-Smith et al., 1990; Fletcher et al., 1991: Fletcher et al., 1994). Toxic effects in non-target species can occur both indirectly as a result

7 of feeding upon slugs that have consumed molluscidal pellets, and directly as a result of feeding on the pellets themselves.

Public concerns about negative environmental impacts resulting from conventional molluscicide usage are reflected in current Government policy objectives. Directives aim to minimise the use of plant protection agents to provide a more environmentally sensitive approach to pest control, whilst retaining the sustainable modernisation of UK agricultural practices. As such, industry has been under increasing pressure to develop alternative approaches for the control of slugs, and during the past twenty years non-chemical control methods have been tested in both agricultural and horticultural sectors (i.e. biological and varietal control methods). However, to date, the uptake of such methods has been hindered by variability in efficacy and high costs, as compared to the use of chemical pesticides (Glen and Moens, 2002). For example, an environmentally benign alternative, biological control of slugs using parasitic nematodes (Phasmarhabditis hermaphrodita) has proved expensive and impractical on a large scale. Thus, there is a clear and immediate need for the development of novel, effective and environmentally safe molluscicides as alternatives to conventional chemical control methods for use in both agricultural and horticultural sectors.

This project was based on the exploitation of a novel approach that allows biologically active proteins, which have low, or no, toxicity when delivered orally to insects to be converted into effective and orally active insecticides. This patented technology has been developed through joint research carried out at the Food and Environment Research Agency (Fera) and Durham University. Recombinant techniques are used to link a gene encoding an insect-specific toxic peptide or protein to a gene encoding a carrier plant protein which is able to cross the gut epithelium and pass into the haemolymph. The recombinant gene encodes a fusion protein containing both components fused into a single polypeptide. The fusion protein is produced using a recombinant expression system, purified, and subsequently ingested by insects after spraying on plant material or as a component of diet. 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 haemolymph, from where it can access its target site. A schematic to illustrate the fusion protein concept is presented in Figure 2.1. Preliminary work suggested that fusion protein technology could also be utilised for the development of molluscicidal proteins. Feeding studies have demonstrated that an insecticidal fusion protein could be detected in the blood of slugs after oral ingestion.

The aim of this LINK project is to investigate the potential for the extension of this technology for the control of molluscs through the production of novel molluscicidal fusion proteins for the benefit of UK and European agriculture. Our aim in this project was to investigate the potential for the exploitation of naturally occurring molluscicidal proteins or peptides, using methodology based on proven patented fusion protein technology. To this end, a consortium consisting of government and 8 university research laboratories, representing the co-inventors of the fusion protein technology (Fera and Durham University), and industrial partners with experience in protein production (Isagro Ricerca) and in the production and testing of molluscicides (De Sangosse) was assembled. The central goal of the proposed project was the discovery and development of new target specific products to be formulated as components of baits for the control of slugs. The use of naturally derived proteins with specific molluscicidal activity offered the potential for the development of novel efficacious products with vastly improved environmental profiles. In collaboration with industry our aim was to investigate large-scale production technologies and to evaluate the potential of fusion proteins as the active component of slug baits. This project provided an opportunity to develop a novel technology that may lead to an improvement in crop yields for farmers and a reduction in the negative environmental impact of currently approved molluscicides.

2.2 Materials and Methods A wide range of materials and methods were used during this project. These included molecular (DNA cloning and sequencing); biochemical (SDS-PAGE; western blotting; protein purification; high liquid chromatography [HPLC]; fast liquid chromatography [FPLC]; mass spectrometry MALDI-TOF analysis); fermentation methodology (lab-scale and pilot scale [200 litre]) and physiological studies (laboratory scale bioassays; dissection and extraction).

Figure 2.1 Diagrammatic representation to illustrate the proposed use of fusion protein technology for the development of novel molluscicidal products.

2.3 Results Objective I: Production and testing of selected conotoxins for use in fusion proteins (Fera/DU). Two conotoxins were selected from the literature based on their reported molluscicidal activity. Initial attempts to express functional peptides using bacteria as an expression host were unsuccessful. Analysis of the purified proteins demonstrated that this was attributable to in-correct folding, known to be critical for biological activity. One of the selected conotoxins was successfully produced using the yeast Pichia pastoris as an expression system. This work has been published (Bruce et al., 2011) and represents the first report of the successful production of a recombinant conotoxin, using yeast as an expression host. Surprisingly the purified conotoxin did not show biological activity against D. reticulatum but did show activity against two insect species (lepidopteran larvae and dipteran adults). For full details please see technical sections 3.1A and B.

Objective II: Identification and testing of other mollusc-specific toxins (Fera/DU). Three peptides were selected from published literature based on reported specificity of activity towards molluscs. These peptides were reported to disrupt muscle contractions or disrupt the central nervous system of mollusc species. The three peptides were synthesised and tested by injection for activity towards D. reticulatum. Disappointingly none of the peptides exhibited any significant effects upon slugs even when injected at high doses. Unfortunately it was not possible to determine if the synthesised peptides contained correctly bridged di-sulphide bonds and thus the lack of activity observed in injection bioassays may have been attributable to incorrect folding of the peptides. For full details please see technical section 3.2.

Objective III: Design and production of fusion proteins containing plant protein carriers and molluscicidal toxins (DU/Fera). Two additional carrier proteins, one derived from garlic bulbs and a protein derived from chicken egg white were identified as potential candidates for incorporation into fusion proteins. These proteins were produced in yeast by bench-top fermentation and the purified products were subsequently detected in the circulatory system of slugs in following ingestion. This provided evidence that both proteins were transported across the gut epithelium of slugs demonstrating their potential use as alternative carrier proteins to the snowdrop lectin, GNA. For full details please see technical section 3.3.

The surprising results obtained for expressed conotoxins (objective 1 above) did not allow for the design and production of mollusc specific fusion protein(s). However, biological activity towards slugs was observed with two insecticidal fusion proteins (FP4 and FP5). FP4 and FP5 were produced in yeast by bench-top fermentation and more than 500 mg of protein was purified and 11 supplied for laboratory based bioassays. Injections of both fusion proteins caused slug mortality, which was significant for FP5. These results demonstrated that the neurotoxin components of FP4 and FP5 were able to disrupt the central nervous system of slugs. Significant reductions in growth of, and consumption by, slugs were observed in laboratory scale bioassays. These results were observed consistently in bioassays where the fusion proteins were coated onto leaf discs (1 mg/disc) or incorporated into wheat pellets (at a level of 1.3 %w/w). As for injection bioassays FP5 was had greater effects on slugs that FP4. Thus FP5 was selected as a candidate for production scale up and efficacy testing by the industrial partners. For full details please see technical section 3.4.

Objective IV: Large-scale production of selected molluscicidal fusion proteins (IR/DS/Fera/DU) FP5, a fusion protein that exhibits both insecticidal and molluscicidal activity was selected for production scale-up and was carried out by Actygea, as contracted by Isagro Ricerca. Actygea carried out fermentation of FP5 at a 200 litre pilot scale. Fermentation was successful, achieving production levels (40-50 mg/litre culture) that were comparable to laboratory scale fermentation that had been conducted by the academic partners. The FP5 product was purified from culture broths using methods that were based on protocols developed by the academic partners. In excess of 3 grams of purified product was purified and verified for biological activity in glass-house trials that demonstrated efficacy of the fusion protein towards Colorado Potato beetle. Actygea also developed a method to allow quantification of product in culture broths. The FP5 product was supplied to De Sangosse for efficacy testing. For full details please see technical section 3.5.

Objective V: Development of optimal bait formulations (DS/IR/DU/Fera) Due to the difficulties encountered in the production of large quantities of FP5 product De Sangosse carried out only a limited number of trials. In addition to the protein produced by Actygea, the academic partners produced in excess of 500 mg of purified FP5 for efficacy tests. In a preliminary ‘forced oral route’ oral assay where slugs were force fed an alginate based material containing FP5 (4.45 % w/w) significant mortality of slugs was recorded. However this result was not reproducible. A second trial did not result in slug mortality although the FP5 treatment did cause a cessation of feeding by the treated slugs indicating that at least some FP5 was present in active form in the gel. It is thought that storage of the FP5 containing alginate at low temperature for 3 weeks may have reduced the levels of biologically active product present in the alginate. No molluscicidal activity was observed in subsequent ‘forced oral route’ and bait trials that incorporated FP5 produced by Actygea into slug baits. Unfortunately the amount of product available for testing was limited and the bait trials conducted by De Sangosse were not designed to evaluate effects of the fusion protein containing baits on slug growth or consumption of diet. Thus a comparison with results obtained in laboratory scale assays carried out by the academic 12 partners was not possible. Further work is required to establish if the lack of activity observed in trials using FP5 containing baits was due to inactivation of the biological activity of FP5 by the pellet formulation process.

2.4 Discussion/Conclusions and Implications

The fusion protein approach was originally conceived and developed for the production of novel biopesticidal products for use against insect crop pests. This approach has shown great promise and continues to undergo development through a Technology Strategy Board (TSB) project that aims to develop a pre-registration package for a prototype insecticidal fusion protein. This work represents a truly novel biotechnological approach for the development of biopesticides and the potential for the development of a range of fusion protein products incorporating different toxin and carrier components has been identified. The current project aimed at extending the application of this platform technology for the control of molluscan crop pests.

One major factor limiting the exploitation of this technology for the control of mollusc pests is the limited information published in the field of molluscan biology. This significantly reduced the number of candidates available for testing as the toxin components of fusion proteins. By contrast, a large pool of toxins identified from arthropod venoms have been isolated, identified and their corresponding targets and biological activity towards a range of insect species from different orders have been characterised. What was surprising in this project was the finding that a peptide originally claimed to be mollusc specific was shown to be insecticidal. Furthermore we have shown that two fusion proteins with demonstrated insecticidal action exhibited toxicity by injection towards the grey field slug, Deroceras reticulatum. This suggests that toxins able to disrupt the insect central nervous system are also able to disrupt the integrity of the nervous system of distantly related mollusc species. It is important to note that the toxin component of both of the fusion proteins that were tested have been shown to have no activity against mammalian ion channels. From this we can conclude that with further research into the molecular and biochemical similarities between insect and mollusc ion channels a more targeted approach could be adopted to enable toxins with high levels of activity against slugs to be identified. It is anticipated that toxins showing activity against the grey field slug are also likely to disrupt ion channel function in other molluscs, such as garden slug Arion hortensis and snail Helix aspersa. The current project also successfully identified two additional proteins that could be used as carrier domains in molluscicidal fusion proteins. The possibility to improve take-up of attached toxins into the slug circulatory system following ingestion warrants further investigation.

Due to the finding that an expressed conotoxin was not in fact mollusc specific in its activity but was also toxic to insects and that insecticidal fusion proteins had molluscicidal activity the 13 possibility of producing a mollusc specific fusion protein was not realised. In fact it is believed that an extensive screening programme covering a wide range of purified toxins would be required to enable the identification of a mollusc specific molecule. Current commercially available molluscicides are not specific in their action and there are concerns over toxic effects of products not just upon insects but also upon bird and mammals. Candidate toxin and carrier components of fusion proteins are selected based on non-toxicity towards mammals, and as such the development of a fusion protein based product would offer distinct benefits over currently available molluscicides. It is noteworthy that neither of the two prototype insecticidal fusion proteins that have been investigated to date exhibit toxicity towards honeybees and no evidence for detrimental effects upon the second trophic level have been found (Wakefield et al., 2010).

An insecticidal fusion protein that was found to cause significant reductions in slug growth and concomitant reductions in feeding was selected for scale-up production and efficacy testing by the industrial partners. The ability to produce a large quantity of the selected fusion protein was limited largely by the expression level of the clones available at the time of pilot scale fermentation. Attempts to improve expression levels through clone selection on high antibiotic containing media and optimisation of fermentation parameters were largely unsuccessful. Research conducted in a complementary project demonstrated that expression levels could be significantly enhanced (> 10 fold) using molecular techniques to introduce multiple copies of the expression cassette into the yeast genome. High levels of expression and a simple downstream purification process are both necessary pre-requisites for the production of economically viable biopesticides. A current TSB project is focussed on achieving high levels of fusion protein production and a simple downstream process to enable evaluation of the cost of production of fusion protein products. Nevertheless, the industrial partners, Isagro Ricerca were able to produce in excess of 3 grams of fusion protein and validated the biological activity of the product in green house trials. Subsequently De Sangosse carried out trials against D. reticulatum using protein produced both by the academic and industrial partners. The academic partners were able to demonstrate that fusion protein was still biologically active after incorporation into baits, although determination of the concentration of active protein in the baits was not possible. Results obtained from preliminary ‘forced oral route’ trials using product supplied by the academic partners were promising and provided evidence for mortality (trial 1) and reduced feeding (trial 1 and 2) following the ingestion of alginate containing fusion protein. Unfortunately trials with product supplied by Isagro Ricerca did not corroborate the initial findings. It remains unclear as to why the ‘forced oral route’ assay using Isagro Ricerca produced fusion protein did not result in a cessation of feeding by the treated slugs. More in depth analysis of slug feeding and growth from the bait trials would have provided valuable data to enable comparisons with data obtained in the laboratory to be made. This was an extremely challenging component of the project. Despite this the industrial partner was able to successfully produce and purify active fusion protein by fermentation at a pilot production scale. Unfortunately the subsequent analysis of 14 molluscicidal activity in bait trials was hindered by the methods employed and the low efficacy of what was recognised as a ‘proof of concept’, rather than prototype product. It is concluded that a more effective prototype candidate must be developed in order to enable efficacy in slug bait trials to be evaluated.

Pressure upon the agrochemical industry to produce environmentally compatible crop protection products combined with the withdrawal of actives available for use by the farmer have fuelled research into the development of new approaches to pest control. This project has demonstrated ‘proof of concept’ for a biotechnological approach suitable for adoption for the development of novel biopesticides. The results obtained provide evidence to suggest that arthropod derived venom peptides represent a large hitherto untapped group of molecules that warrant further investigation for the development of molluscicidal fusion proteins. Indeed potential exists for the development of not just one, but a number of different products, incorporating different toxins and/or carrier proteins. Further investment however is required to develop a truly efficacious product and to enable production at an economically viable cost.

TECHNICAL DETAIL Section 3.1: Production of recombinant molluscicidal peptides 3.1A. Introduction Approximately 500 species of cone snails are known, which together produce venoms containing more than 50,000 unique peptides (Olivera, 1997). These peptides, containing 10-40 amino acid residues, are referred to as conotoxins or conopeptides, and are divided into two main groups, disulphide-rich, and the less abundant non-disulphide rich peptides (Terlau and Olivera, 2004). Some conopeptides have important pharmaceutical properties as a result of interactions with “targets” in humans and other mammals, and have been exploited for clinical applications in pain relief relief; the conotoxin Prialt (ziconotide) is an approved drug for severe chronic pain. They have further potential use in the treatment of human neurological diseases. In addition, their specificity in targeting ion channels, cell surface receptors, and neurotransmitter transporters makes them valuable tools in neuroscience and pharmacological research (reviewed by Wang and Chi, 2004: Terlau and Olivera, 2004). In contrast, the potential use of these proteins as pesticides has received comparatively little attention, although their high level of specificity and toxicity towards target species make them suitable candidates for exploitation. Conotoxins could be particularly useful in applications such as mollusc control, where alternatives to environmentally damaging chemical treatments are not available.

Although the subject of much scientific interest, conotoxin research and development has been hindered by difficulties in obtaining active peptides. Peptides from the natural sources are difficult to obtain and only small quantities can be collected. Alternative methods of obtaining conotoxins are all associated with certain drawbacks (reviewed by Becker and Terlau, 2008). Disulphide rich peptides require accurate bridge assembly that cannot be easily achieved in current systems. Chemical synthesis requires subsequent folding steps involving oxido-shuffling reagents (Rudolph and Lilie 1996) and recombinant expression in E. coli, though successful for a number of conopeptides, involves fusion with a soluble carrier protein or denaturation of insoluble inclusion bodies followed by refolding. This has been successfully achieved through fusion of glutathione-S- transferase (GST) to the conotoxin MVIIA (Xia et al., 2006) thioredoxin fusions to It7a and It6c (Pi et al., 2007; Wang et al., 2008) and by denaturing inclusion bodies and refolding in the case of Conk-S1 (Bayruber et al., 2006). Although not yet reported for a conotoxin, small disulphide rich peptides have also been produced in E. coli Origami strains, which have a more oxidizing cytoplasmic environment which aids correct folding of disulphide bridges, for example the peanut allergen Ara h2 (Lehman et al., 2003). An alternative biosynthetic option is expression in the yeast Pichia pastoris, which is well established for recombinant proteins and more suited to folding of disulphide-rich peptides (White et al., 1994; Cregg et al., 2000, Daly and Hearn, 2005). As a eukaryotic system, the cellular environment is able to process and fold disulphide-rich peptides

16 more easily than the prokaryotic E. coli. To date however, there are no reports of successful expression of a conotoxin in yeast. Additional alternatives using mammalian and insect cell lines have been used for biosynthesis of TxVI in COS7 cells (Conticello et al., 2003) and psalmotoxin 1 in Drosophila melanogaster S2 cells (Escoubas et al., 2003) but are less favoured options for large-scale production due to the high handling and maintenance costs.

Conus textile f. neovicarius, the cloth of gold cone shell, is a molluscivore and the venom would be expected to contain toxins active against molluscs. The conotoxin TxVIA (also known as -TxIA and KingKong peptide) is a major component in the venom. TxVIA is a 27 amino acid, disulphide- rich delta conotoxin of the O1 superfamily, containing six cysteine residues, which form disulphide bridges according to the typical O1 superfamily cysteine framework, I-IV, II-V, III-VI. Delta- conotoxins function in the initial “stun” phase of immobilization of prey, which is followed by a second phase involving neuromuscular block and complete paralysis (Terlau et al., 1996). TxVIA has been reported to have specific molluscicidal activity. Most notably, TxVIA causes convulsions in the garden snail, Helix aspersa, and paralysis in limpets, Patella caerula (Hillyard et al., 1989, Fainzibler et al., 1991). Altered behaviour was also noted upon injection of TxVIA into lobster (Hillyard et al., 1989). No toxic effects were observed in mammals (mouse and rat), fish (Cambusia), woodlice (Porcellio) or fleshfly (Sarcophaga) (Hillyard et al., 1989, Fainzibler et al., 1991, Fainzibler et al., 1994). TxVIA binds to neurons of the sea slug Aplysia inducing membrane depolarization, spontaneous repetitive firing and a sodium dependent prolonged action potential, which recovers within 30 minutes (Fainzibler et al., 1991, Hasson et al., 1993). Although non-toxic to vertebrates, TxVIA is known to bind to Na channels in the rat central nervous system (CNS) and acts as an antagonist against the conotoxins CsTx and NgVIA (Fainzibler et al., 1994, Fainzibler et al., 1995). In contrast to other Na channel neurotoxins, TxVIA binds to a novel site on voltage gated Na channels through a non-voltage dependant mechanism (Fainzibler et al., 1994).

A second conotoxin -GmVIA, that shares a similar cysteine framework to TxVIA, was previously isolated from the mollusc-hunting snail Conus gloriamaris, and shown by Hasson et al., 1995 to induce convulsive-like contractions when injected into the garden snail Helix aspersa, and to target sodium channels in the sea slug Aplysia, but no effects on mammals (mice) were observed.

TxVIA and -GmVIA were selected for expression as recombinant peptides and subsequent evaluation of activity against the terrestrial molluscan crop pest, Deroceras reticulatum. Initially these peptides were expressed using E. coli as an expression host (section 1A). Subsequently, due to problems encountered with the expression of correctly folded peptides using E.coli, yeast was used to express functional TxVIA (section 1B).

3.2A. Materials & Methods

3.2A.1 Assembly of synthetic genes encoding TxIA and -GmVIA

The TxIA amino acid sequence (Genbank accession no. P18511) was used as the basis for the assembly of a synthetic TxIA gene. Each strand (coding and complementary strands) of the sequence encoding the mature TxIA peptide (figure 3.1) was subdivided into 3 fragments, such that each fragment overlapped neighbouring fragments on the complementary strand by 12 bases. Six oligonucleotide primers were synthesised and used in an assembly reaction of the full mature TxIA coding sequence. All primers were individually phosphorylated (by incubating nucleotide with ATP and T4 DNA polynucleotide kinase for 30 min at 37 °C). An equimolar solution of phosphorylated nucleotides was prepared in T4 DNA ligase buffer containing no ATP or DTT, boiled for 10 min (to denature secondary structures) and cooled slowly at room temperature to allow nucleotides to anneal. After addition of ATP, DTT and T4 DNA ligase, a ligation reaction was carried out at 16 °C for 24 h. A PCR reaction was performed (using sense [C1] and antisense [NC1] primers at 5’ and 3’ ends of the gene) to obtain sufficient DNA for sub-cloning into the intermediate vector PCR2.1. The resulting clones were verified by sequencing.

The -GmVIA amino acid sequence (NCBI; accession AAB32042.1’ Figure 1) was used as the basis for the assembly of a synthetic gene that was assembled and verified essentially as described for TxIA.

3.2A.2. Assembly of thioredoxin-TxIA and thioredoxin- -GmVIA expression constructs and E. coll transformation Constructs encoding for TxIA or -GmVIA fused to thioredoxin were prepared for the expression of in E.coli using the bacterial expression vector pET32a. A schematic presentation of the constructs is shown in figure 3.1. TxIA or -GmVIA were amplified by PCR using primers to incorporate NcoI and XhoI restriction sites at 5’ and 3’ ends, respectively. A stop codon, preceeding the 3’ XhoI site was also introduced by PCR. After amplification the PCR products were digested with NcoI and XhoI, and ligated into pET32a (similarly digested). A control empty vector digestion reaction was also performed. Ligated pET32a-TXIA and pET32a- -GmVIA DNA was transformed into Top10 cells and a positive transformants were verified by sequencing. Sequenced clones were transformed into chemically competent Origami Bl21 DE3 expression host cells. Positive transformants, selected on ampicillin containing media (100 g/ml) were confirmed by colony PCR, and positive transformants were used for induction experiments.

Empty vector (pET32a) was also transformed into Origami BL21 DE3 to enable expression and purification of thioredoxin (TRX) for use as a control treatment in downstream injection bioassays. 18

Figure 3.1. Schematic presentation of thioredoxin(TRX)-TxIA and TRX- -GmVIA encoding construct in bacterial expression vector pET32a. Conotoxins were cloned just downstream of an enterokinase cleavage site and a stop codon was introduced before Xho I site to obtain native ended protein. A histidine tag is present in between thioredoxin and TxIA, which can be utilized for affinity purification. The predicted amino acid sequences of the conotoxins are given.

3.2A.3. Expression and purification of thioredoxin (TRX)-TxIA, TRX- -GmVIA and TRX recombinant peptides Overnight cultures of TRX-TxIA, TRX- -GmVIA or TRX were grown in 5 ml LB-ampicillin (100 g/ml medium) and at 37 °C. These cultures were diluted (1:50) into fresh LB medium and incubated at 37 °C until the OD at 600 nm was 0.8. Recombinant expression of fusion proteins and thioredoxin was then induced by the addition of IPTG (isopropyl -D-thiogalactoside) to a final conc. of 0.1 mM and cultures were subsequently grown at 210C for a further 6 hr. Cells were harvested by centrifugation (8000 rpm for 10 min), re-suspended in extraction buffer (10 ml of 50 mM Tris-Cl pH 8.0 containing 500 mM NaCl), sonicated and spun (12000 rpm for 10 min at 4°C). Collected supernatants were then applied to nickel-affinity (Ni-NTA) columns (1 ml, pre- equilibrated with extraction buffer) at 1 ml/min. After washing with 10 column volumes of extraction buffer, bound protein was eluted with extraction buffer containing 50 mM and 300 mM imidazole. Eluted fractions were run on SDS-PAGE gels to identify the presence and purity of recombinant proteins. Fractions containing purified proteins were pooled, diluted two times with 50 mM Tris-Cl pH 8.0, 500 mM NaCl and dialysed against 1X Phosphate buffer saline (PBS) overnight at 4°C. Dialysed protein samples were spun at 12 000 rpm for 10 min at 4°C and concentrated at least 10 fold using a 10 kDa MWCO centrifugal concentrator at 4°C. Concentrated protein samples were quantified by BCA protein assay using bovine serum albumin as a standard and stored at -20°C.

3.2.A.4 Mollusc and Insect Cultures Deroceras reticulatum cultures were maintained at the Food and Environmental Research Agency (Fera), York, UK. Mamestra brassicae (cabbage moth), originally obtained from Fera, was maintained at Biological and Biomedical Sciences Dept., Durham University, UK. All cultures were subject to a 16 h light, 8 h dark cycle. M. brassicae were maintained at 25 C, 40% relative humidity. Insects were maintained on a standard diet as described by Bown et al. (1997). D. reticulatum were derived from a laboratory culture, supplemented with field collected slugs, that was maintained at 10 C, 75% relative humidity on a diet of organic lettuce, carrot and wheat seeds.

3.2A.5. Biological activity: Deroceras reticulatum and Mamestra brassicae injection bioassays of thioredoxin (TRX)-TxIA, TRX- -GmVIA and TRX recombinant peptides Injection bioassays were carried out using newly eclosed fifth instar larvae of M. brassicae, and 12 week old D. reticulatum. In all cases, injections were carried out with varying doses of recombinant TxVIA or TRX- -GmVIA in PBS. Purified recombinant TRX-TxVIA and TRX- -GmVIA (50-100 g) re-suspended in PBS were injected into mature (0.5-1.0g) slugs (D. reticulatum) and survival monitored daily for 7 days. TRX was injected as a control treatment.

The M. brassicae larvae were monitored through development to pupation. Paralysis in D. reticulatum was determined by slug inversion assay and scored according to the slug’s ability to correct itself. Paralysis and mortality were scored at 10-30 mins, 24 and 48 h post injection.

3.2A.6. Cleavage of thioredoxin from TRX-TxVIA and MALDI-TOF analysis of TxVIA peptide In order to obtain native ended TxIA, enterokinase cleavage of the recombinant conotoxin from thioredoxin was conducted. Enzymatic cleavage of S-tag, His-tag and TRX was carried out with 80 g enterokinase per mg purified TxVIA-TRX fusion protein at 37 C for 3 hours. TxVIA was purified from cleaved TRX by size exclusion chromatography on a G50-F Sephadex column in PBS. Mass spectra were acquired using a Voyager DE STR MALDI-TOF mass spectrometer (Applied Biosystems, Warrington, UK) in positive ion reflector mode, using a matrix of ferulic acid (trans-4- hydroxy-3-methoxycinnamic acid, 99%; Sigma-Aldrich, U.K.) prepared at a stock concentration of 15 mg/ml in 50% acetonitrile/0.3% (v/v) aqueous TFA. A working dilution at 1:5 (v/v) was made by adding 0.1% TFA to the stock solution. Aqueous samples (approx. 1 mg ml-1) were mixed 1:3 to 1:5 with diluted matrix. Sample application was based on the method of Onnefjord et al.(1999). Seed layers were formed on the stainless steel target plate by adding 0.7µl stock solution diluted 1:15 in acetonitrile to each spot and allowing to air dry. Aliquots of the samples (0.5 – 0.7 µl) mixed with the 1:5 diluted matrix were added next, and allowed to air dry. Standards (bovine 20 insulin, thioredoxin from E. coli, horse apomyoglobin; Sequazyme peptide mass standards kit; Applied Biosystem U.K.) were added adjacent to samples. Spectra represent the resolved average [M+H]+ masses in the mass range m/z 2000–1500. Spectra are the accumulation of 5 x 20 laser shots. Theoretical masses were calculated using Protein Prospector software (University of California, San Francisco).

3.3A Results 3.3A.1. Synthetic gene assembly, expression and purification of TRX-TXIA, TRX- - GmVIA fusion proteins and TRX in E.coli Synthetic genes encoding a mature 27 amino acid TxVIA and a mature 29 amino acid -GmVIA conotoxin peptide were generated using a series of overlapping, complementary oligonucleotides. To aid the formation of di-sulphide bonds and expression of the peptides in soluble form the coding sequences were cloned into the pET32 expression vector to allow expression of the proteins as a fusion with thioredoxin. The predicted protein products also contained an enterokinase site to allow cleavage of TxVIA or -GmVIA from thioredoxin, and an N-terminal histidine tag to facilitate purification and detection by western analysis. On expression in E. coli BL21(DE3) cells, optimal production of soluble protein (induced by IPTG) was found to occur at 21 C, with yields of approx. 10 mg/l culture. Recombinant fusion proteins were purified by nickel-affinity chromatography under native conditions. Purified recombinant TRX-TxVIA and TRX- -GmVIA stained as single polypeptides of approx. 21 kDa on SDS-PAGE gels (figure 3.2), close to the respective predicted molecular masses of 20.3 and 20.6 kDa for the fusion proteins. When injected, purified TRX- TxVIA was not found to have any activity against terrestrial molluscs (mature D. reticulatum) or larvae of the lepidopteran M. brassicae (data not shown).

Thioredoxin was subsequently enzymatically cleaved from TxVIA by enterokinase cleavage and the mature TxVIA peptide (containing an additional N-terminal 3 amino acids, AMA) was separated by size exclusion on Sephadex columns. MALDI-TOF analysis of mature TxVIA revealed a single mass ion of 3316.2 (figure 3.3). This mass is in accordance with the predicted mass of 3315.91 for the reduced form of the peptide, suggesting that none of the 3 disulphide bonds present in active TxVIA had formed.

3.3A.2. Biological activity: D. reticulatum and M. brassicae injection bioassays of thioredoxin (TRX)-TxIA, TRX- -GmVIA and TRX recombinant peptides

No obvious signs of paralysis or mortality were observed following the injection of mature slugs or fifth stadium M. brassicae larvae with purified recombinant TRX-TxIA (50-100 g) or TRX- - GmVIA. These results were consistent with MALDI-TOF analysis of TxVIA that suggested that the

21 correct di-sulphide bridge formation had not occurred when these peptides were expressed in E. coli. Correct di-sulphide bridge formation is known to be essential for biological activity of conotoxin

Figure 3.2: (i) Representative absorbance profile for purification of TRX and TRX-TXIA by nickel affinity (Ni-NTA) chromatography. A: in the figure indicates flow through, B: 20 mM imidazole wash, C: 100 mM imidazole wash, and D: 300 mM imidazole elution. Coomassie stained SDS- PAGE gels of (ii) TRX-TxIA and (iii) TRX purification fractions. Loading is as follows: M denotes molecular weight marker mix as annotated in both gels, (ii) Lane 1: load fraction, Lane 2: flow through (non-bound proteins), Lane 3 wash fraction, Lane 4: 20 mM imidazole wash, Lane 5: 100 mM imidazole wash, and Lane 6 300 mM imidazole elution; (iii) Lane 1: flow through, Lane 2: buffer wash, Lane 3: 20 mM imidazole wash, Lane 4: 100 mm imidazole wash, Lane 5: 300 mM imidazole elution. 10 l of 5 ml fractions were loaded in each lane.

Figure 3.3. Recombinant Expression of TxVIA in E. coli. Panel A, SDS-PAGE (15 % acrylamide gel) of thioredoxin-TxVIA purified by nickel-affinity chromatography from E. coli biofermentation. Molecular weight scale is derived from a marker protein mixture (SDS-7, Sigma) run on the same gel. Panel B, MALDI-TOF MS of TxVIA following enterokinase cleavage of thioredoxin. The predicted mass of the recombinant peptide when di-suplhide bonds are bridged is 3311.9, when bridges are reduced the predicted mass is 3315.9.

Section 3.1B: Expression of recombinant TxVIA using yeast as an expression host.

3.1B. Materials and Methods 3.1B.1 Yeast Expression constructs A construct encoding for the mature TxVIA coding sequence was initially created for expression in P. pastoris by PCR amplification of the sequence generated in pCR2.1 as described in section 1A. TxVIA was amplified with primers incorporating PstI and XbaI sites to allow insertion of the sequence into the yeast expression vector pGAPZ B in frame with the the N-terminal alpha factor secretion sequence and C-terminal myc epitope and 6 residue histidine tag. Following verification by sequencing, plasmid DNA (TxVIA-pGAPZ B) was amplified in TOP10 E. coli, purified and linearized with BlnI for integration into P. pastoris parental strain SMD 1168H. pGAPZ B was transformed into P. pastoris using an EasyComp Transformation Kit and selected on YPG agar plates containing zeocin (1% yeast extract (w/v), 2% peptone (w/v), 4% glycerol (v/v), 1.5% agar (w/v), 100 g/ml zeocin). Selected clones were checked for integration into the yeast genome by colony PCR using gene specific primers and vector primers against the flanking regions of the inserted TxVIA sequence. Positive clones were then analysed for the expression of TxVIA by western blotting of culture supernatant from three-day-old YPG-zeocin liquid cultures.

A second construct incorporating the PrePro region of the TxVIA peptide was subsequently generated for expression in P. pastoris. The entire PreProTxVIA gene sequence was synthesized with codon optimization for P. pastoris (MWG Eurofin) and supplied in pCR2.0 vector (ATG AAG TTG ACT TGC ATG ATG ATT GTT GCC GTT TTG TTC CTT ACA GCA TGG ACG TTT GCT ACT GCT GAT GAC CCA AGA AAT GGA CTA GGT AAC CTG TTT TCC AAT GCT CAT CAC GAG ATG AAG AAT CCT GAA GCC TCA AAG CTC AAC AAA CGT TGG TGT AAA CAG TCT GGT GAA ATG TGC AAC TTA TTG GAC CAA AAC TGC TGT GAT GGC TAC TGT ATC GTG CTT GTC TGT ACC TAA). ProTxVIA was amplified using the sense primer 5’- GCctgcagCCGATGACCCAAGAAATGGAC and anti-sense primer 5’- GGtctagaGCGGTACAGACAAGCACGATA with PstI and XbaI sites shown indicated by lower case. The ProTxVIA amplicon was digested and ligated into PstI and XbaI digested vector pGAPZ B in frame with the N-terminal alpha factor secretion sequence and C-terminal myc epitope and 6 residue histidine tag. Following sequence analysis, plasmid DNA was prepared and transformed into SMD1168H cells as described for TxVIA-pGAPZ B. P. pastoris lines expressing TxVIA were selected by western blotting supernatant from three day old YPG-zeocin liquid cultures.

3.1.B.2 Recombinant TxVIA expression and purification from yeast P. pastoris containing the integrated Pre-proTxVIA construct was grown in a 7.5 l BioFlo 110 bench-top fermenter (New Brunswick Scientific) as previously described (Fitches et al., 2004). Culture supernatant was adjusted to 20 mM sodium phosphate and 0.4 M sodium chloride pH 7.4. Recombinant TxVIA was purified by nickel affinity chromatography on 5 ml HisTrap crude nickel columns with a flow rate of 2 ml / min. Bound protein was eluted with 0.3 M imidazole, 20 mM sodium phosphate pH 7.4. Purified protein was concentrated and buffer exchanged to PBS pH 7.4 in Vivaspin 15R, 2000 Da MWCO columns followed by further purification through a G50-F Sephadex column with a flow rate of 0.5 ml / minute. TxVIA fractions in PBS were concentrated in Vivaspin 15R, 2000 Da MWCO columns then Vivaspin 500, 3000 Da MWCO columns. The concentration of TxVIA was estimated by comparison to known amounts of protein standards run on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels.

3.1.B.3 Insect and Mollusc Cultures Deroceras reticulatum and Mamestra brassicae cultures maintained as described in section 3.2A. Musca domestica cultures were maintained at the Food and Environmental Research Agency (Fera), York, UK, and were subject to a 16 h light, 8 h dark cycle. Musca domestica (house fly) adults were cultured at 22 C, 65% relative humidity with a diet of bran, grass meal, yeast, malt and milk powder as described by Barson et al. (1994).

3.1B.4 Injection bioassays Injection bioassays were carried out using newly eclosed fifth instar larvae of M. brassicae, 24 - 48 h old M. domestica adult flies, and 12 week old D. reticulatum. In all cases, injections were carried out with varying doses of recombinant TxVIA in PBS or C. textile total soluble proteins from crude venom in PBS. Controls were injected with PBS. M. brassicae larvae were monitored through development to pupation. Paralysis in D. reticulatum was determined by slug inversion assay and scored according to the slug’s ability to correct itself. Paralysis and mortality were scored at 10-30 mins, 24 and 48 h post injection. Data from duplicate or triplicate experiments are shown. Data were analysed using Prism (GraphPad, USA) Kaplan-Meier survival analysis. Lyophilised crude venom from Conus textile was supplied by Professor M. Fainzibler, Department of Biological Chemistry, Weismann Institute of Science, Israel and re-suspended in PBS at 10 mg / ml.

3.1B.5 Electrophoresis and Western blotting Proteins were routinely separated and analysed by SDS-polyacrylamide gel electrophoresis (SDS- PAGE). Samples were prepared by adding 5x SDS sample buffer (containing 10% 2- mercaptoethanol) and boiled for 10 mins prior to loading. Gels were either stained with Coomassie

Blue or transferred to nitrocellulose membrane using an ATTO HorizBlot semi dry electroblotting system according to manufacturer’s instructions. Western blotting was carried out as described previously (Fitches and Gatehouse, 1998), except for chemiluminescent reagents. Enhanced Chemiluminescence detection was carried out using coumaric acid (0.2 mM) and luminol (1.25 mM) in 1M Tris (pH 8.5) with the addition of 0.009% (v/v) hydrogen peroxide.

3.2B Results

3.2B.1 Production of recombinant TxVIA in yeast

Initially a construct containing the mature TxVIA peptide between an N-terminal secretion signal ( -mating factor prepro-peptide) and a C-terminal myc epitope and histidine tag was created in pGAPZ B for expression in yeast. Following transformation, integration of the plasmid into the SMD1168H genome was verified by colony PCR of antibiotic-selected clones. However, western analysis (using anti-His antibodies) of supernatants derived from small-scale cultures did not provide any evidence for the presence of recombinant TxVIA (results not shown). Subsequently, a second construct incorporating the 29 amino acid residue propeptide sequence of TxVIA was generated for expression in yeast (fig. 3.4). The predicted protein products also contain a C- terminal extension including a myc epitope and histidine tag, arranged in frame with an N-terminal alpha factor secretory signal. Following verification by sequencing the construct was transformed into a protease deficient strain of P. pastoris. Western analysis (using anti-his antibodies) of supernatant derived from small-scale cultures showed positivie immunoreactivity of two proteins corresponding in size to ProTxVIA (including the pro region; approx. 10 kDa) and mature TxVIA from which the pro-region had been cleaved (approx. 5 kDa). Following production by bench-top fermentation and purification by nickel affinity chromatography these two proteins were visible on SDS-PAGE gels and western blots (Figure 4 A and B). The ratio of the ProTxVIA to TxVIA bands varied between different preparations, but were typically approx. 2:1. Protein yield (ProTxVIA + TxVIA) after fermentation was estimated to be approx. 10 mg / l of culture supernatant.

MALDI-TOF analysis of recombinant TxVIA from yeast showed two heterogenous peaks centered around 5730 and 9000, in reasonable agreement with the predicted molecular weights for the recombinant ProTxVIA and TxVIA proteins of 9536.7 and 5721.4 (data not presented).

Figure 3.4. Recombinant expression of TxVIA in Pichia pastoris. Panel A, SDS-PAGE (17.5 % acrylamide gel) analysis of recombinant proteins purified by nickel affinity and size-exclusion chromatography. Molecular weight scale is derived from a marker protein mixture (Ultra-Low Range, Sigma) run on the same gel. Panel B, anti-HIS immunoblot. P = propeptide, M = mature toxin. Below, ProTxVIA amino acid sequence. Boxed = pro region and cysteine bridges are indicated.

3.3B.2 Injection Bioassays

3.3B.2.1 Effect of crude Conus textile venom on slugs and cabbage moth larvae

Mature grey field slugs (D. reticulatum) were injected with soluble proteins from C. textile crude venom (Figure 3.5, Table 3.1). Slugs injected with crude venom showed paralysis within 5 minutes at all doses tested. At the highest dose of 0.83 g/ mg biomass, persistent paralysis 24 hours post injection (hpi) was observed. In all cases paralysis was reversible and no acute effect on survival was found. Cabbage moth (M. brassicae) larvae injected with up to 13-fold higher dose showed no response to C. textile crude venom protein (Table 2).

Figure 3.5. SDS-PAGE (17.5% acrylamide gel) of total soluble protein from crude venom of C. textile. Lane 1, 150 μg and lane 2, 250 μg. Molecular weight scale is derived from a marker protein mixture (SDS-7, Sigma) run on the same gel.

Table 3.1 Paralytic activity of crude venom in D. reticulatum (dose per mg biomass).

C. textile crude venom dose Percentage paralysed (paralysed / total)

Per slug Per mg biomass 5 minutes 24 hours

0 0 0 (0/6) 0 (0/6)

62.5 g 0.21 g 100 (6/6) 0 (0/6)

125 g 0.42 g 100 (6/6) 0 (0/6)

250 g 0.83 g 100 (6/6) 100 (6/6)

3.2B2.2. Effect of recombinant TxVIA on cabbage moth, house fly and slug

The recombinant TxVIA/ProTxVIA mixture produced in yeast was injected into 5th instar larvae of cabbage moth (M. brassicae) and adult houseflies (M. domestica) at doses ranging from 0.6 - 3 g/mg body weight. Percent survival was measured at 24 h (Figure 3.6A and B). Injection with TxVIA had a significant, dose-dependent effect on survival rate in both insect groups tested (M. brassicae and M. domestica) as determined by chi-squared T-test (p < 0.05). Survival at 48 h was similar to that at 24 h (data not shown). At the highest dose tested in M. brassicae larvae (3 g/mg body weight), immediate paralysis was observed during injection. Mortality of approx. 20% was observed in M. brassicae larvae at doses as low as 0.75 g/mg body weight; the lowest dose tested with M. domestica adults (0.86 g/mg body weight) resulted in over 50% mortality. Results were consistent over different batches of recombinant TxVIA containing differing ratios of ProTxVIA and mature TxVIA. In contrast, injections of 12 week-old slugs (D. reticulatum) with

28 recombinant TxVIA at doses up to 1.7 g/mg body weight did not cause paralysis or affect survival (Table 3.2).

Figure 3.6. Survival of (A) Mamestra brassiace (cabbage moth) larvae and (B) adult Musca domestica (house fly) 24 h after injection of various doses of recombinant TxVIA. or PBS control (n=10 per treatment).

Table 3.2 Comparison of C. textile venom proteins and TxVIA activity across species.

Protein injected Biological activity in

M. brassicae M. domestica D. reticulatum (grey field slug) (cabbage moth) (housefly) adults larvae

C. textile crude No effect at 2.86 g / Not tested Paralysis within 5 min at 0.21 g venom mg biomass / mg biomass. No acute mortality Recombinant Immediate paralysis 80% mortality at No effect at 1.7 g / mg biomass TxVIA and 24 hpi with 1.7 g 44% mortality at 24 / mg biomass hpi with 1.5 g / mg biomass

3.3B Discussion (section 3.1A and 3.1B) TxVIA, one of three conotoxins originally isolated from the venom of the molluscivorous snail C. textile (Hillyard et al., 1989) and shown to have paralytic activity towards limpets (Patella caerula) (Fainzibler et al., 1991), has been investigated as a potential molluscicide towards the grey field slug (D. reticulatum), a serious pest of crops in Northern Europe. Similarly a second conotoxin, - GmVIA isolated from the mollusc-hunting snail Conus gloriamaris, previously shown by Hasson et al., 1995 to induce convulsive-like contractions when injected into the garden snail Helix aspersa, and to target sodium channels in the sea slug Aplysia, was investigated as a potential molluscicidal peptide towards D. reticulatum. The aim of this work was to identify a suitable candidate molluscicidal toxin for incorporation into a fusion protein.

Fainzibler et al., (1991) estimated that TxVIA (previously TxIA), TxVIB (previously TxIB) and TxVIIA (previously TxIIA) combined, represent around 1.2% of venom duct protein yet are responsible for 53% of the paralytic activity observed against limpets (approx. 17% activity is due to TxVIA alone). Approximately 0.5% of total protein from C. textile venom is TxVIA. This low abundance and poor recovery rates from the natural source requires alternative methods for generating larger quantities of the conotoxin.

Initial attempts to produce functional recombinant TxVIA and -GmVIA using E. coli as an expression host were unsuccessful. Despite the use of thioredoxin as a fusion partner and Origami cells, and the expression of both proteins in soluble form, MALDI-TOF analysis indicated that none of the three di-sulphide bridges present in the naturally occurring mature TxVIA peptide were present in recombinant peptide purified from E. coli. Furthermore neither purified recombinant TxVIA or -GmVIA exhibited any biological activity when injected into invertebrates. As yeast contains the cellular machinery necessary to correctly fold eukaryotic disulphide-rich peptides, we subsequently focused on the use of P. pastoris as an expression host for the production of recombinant TxVIA. A number of disulphide-rich peptides have been successfully produced in P. pastoris, including Pla l 1, the major allergen from Plantago lanceolata (Calabozo et al., 2003), and defensins Psd1 and AX2 from Pisum sativum and Beta vulgaris, respectively (Cabral et al., 2003, Kristensen et al., 1999), as well as insecticidal toxins from arthropods (Fitches et al., 2004; Trung et al., 2006).

A construct containing only the mature TxVIA sequence was introduced into yeast, but no expression of recombinant protein was detectable. This may be attributable to C-terminal cleavage of the myc and histidine tags during expression, despite use of a protease-deficient P. pastoris strain. Another possibility is that the recombinant peptide may have been targeted for degradation during passage through the yeast translationary pathway. As part of the regulatory machinery of

30 the endoplasmic reticulum (ER), misfolded proteins are recognized by their exposed hydrophobic residues, retained in the ER and targeted for degradation (Ellgaard et al., 1999). The three- dimensional solution structure of TxVIA reveals a high level of hydrophobicity in the mature folded peptide (Kohno et al., 2002), making the mature TxVIA toxin potentially sensitive to ER retention and degradation.

Transformation of a second construct incorporating the Pro- region of the precursor for TxVIA and a C-terminal histidine tag into P. pastoris resulted in the expression of two immunoreactive recombinant proteins. Conticello et al. (2003) demonstrated the importance of the pro- region in facilitating efficient secretion of a hydrophobic conotoxin, TxVI, from the ER in a COS7 mammalian cell line. Enhanced secretion of TxVI is mediated by the pro- region via interaction with sortilin protein family sorting receptors allowing the TxVI-sortilin complex to be trafficked out of the ER and into the golgi aparatus. It is possible that the pro region of TxVIA, differing at only four of 29 amino acid residues compared to TxVI, may also facilitate enhanced secretion from the ER in P. pastoris.

Two proteins similar in molecular mass to ProTxVIA and mature TxVIA peptide were obtained following bench-top fermentation and purification by nickel affinity chromatography. Approximately one third of protein produced was successfully cleaved to mature TxVIA. Incomplete cleavage may reflect differences in protein sorting enzymes between cone snail and yeast and/or a lower abundance of protein disulphide isomerase (PDI) in P. pastoris as compared to C. textile, where PDI is the most abundant protein found in the venom duct (Bulaj et al., 2003). The heterogeneity of the recombinant protein produced in P. pastoris on MALDI-TOF analysis is typical of proteins produced in this host, and reflects incomplete processing of precursors, and proteolytic activity in the culture supernatant. It is not known whether the toxicity of the TxVIA preparation was solely due to the content of mature TxVIA, or whether pro-TxVIA is also toxic. It is also possible that further processing of pro-form to mature toxin occurs in vivo after injection.

The paralysis observed following the injection of recombinant TxVIA into invertebrates provided additional evidence to suggest that the presence of the pro- region was also critical for correct folding of the recombinant conotoxin. The conotoxin pro- region has previously been reported to be essential for the interaction of precursors with PDI (Buczec et al., 2004), which catalyzes rearrangement of conotoxin disulphide bonds and correct folding of the peptide. In C. textile the pro- region is enzymatically cleaved from the conotoxin precursor, resulting in mature toxin. Although the prodomain is not always necessary for correct folding, eg. -MVIIa (Price-Carter et al., 1996) in some cases it is crucial, eg. contoxin GI (Buczec et al., 2004). In our study the pro- region was found to be critical for both expression and correct folding of -TxVIA, since constructs lacking this region either produced no detectable recombinant protein (in yeast) or did not fold correctly to give biologically active product (in E. coli). 31

TxVIA was previously reported to be a mollusc-specific toxin due to its paralytic activity on the marine mollusc P. caerula and inactivity on crustacean, fish and insect species tested (Fainzibler et al., 1991). By contrast, we observed activity of recombinant TxVIA against the lepidopteran M. brassicae and the dipteran M. domestica. This discrepancy is probably due to the much higher doses of TxVIA injected in our study (approx. 40x) as compared to Fainzilber et al. 1991. No activity against M. brassicae was observed following injections of crude C. textile venom reflecting the low abundance of TxVIA in crude samples. Venom injections of approx. 3 g/mg would contain approx. 0.15 g of native TxVIA, much lower than the 0.75 g/mg of recombinant TxVIA shown to induce mortality in M. brassicae or M. domestica. On the contrary, injections of 0.2 – 0.8 g/mg of crude venom from C. textile into D. reticulatum induced reversible paralysis, whereas no effects were observed when slugs were injected with doses of up to 1.7 g/mg of recombinant TxVIA. Given that crude venom injections contained lower levels of TxVIA (approx. 0.01 - 0.04 g/mg) compared to injections of purified recombinant protein, it can be concluded that TxVIA is inactive against this species, and the response to crude venom injection observed in D. reticulatum must therefore result from other peptides in the Conus venom. TxVIA was inactive against slugs even at doses far exceeding that observed to paralyze the marine mollusc P. caerula (Fainzilber et al., 1991). This may reflect the evolutionary distance between marine and land molluscs although further studies of TxVIA toxicity would be necessary to establish more generally applicable predictions of specificity for this toxin.

Taken together, the insect and mollusc bioassay data presented here contradict the claim that TxVIA is a mollusc-specific peptide. Cone snail venoms have evolved to target ion channels in their three main prey groups; fish, worms and molluscs and conservation of ion channels has meant that in many cases Conus venom peptides are active in taxa other than their target prey, including mammals and insects. Examples of conotoxin activity against insects include the omega conotoxins GVIA from C. geographus, and MVIIC from C. magus, which have been shown to block calcium channels in the dorsal neurons of Periplaneta americana (cockroach) (Wicher and Penzlin 1998) and the kappa conotoxin -PVIIA from C. purpurascens which is known to inhibit the Drosophila potassium channel, Shaker (Shon et al., 1998). To date, whole organism bioassays investigating the activity of conotoxins towards different taxa are limited and further studies are required to elucidate the level of promiscuity of Conus venom peptides. The successful production of TxVIA as a recombinant peptide using the host P. pastoris reported in this study provides a starting point for the development of additional yeast-based expression of conotoxins. Unfortunately these results led to the conclusion that neither TxVIA or -GmVIA were suitable candidates for incorporation into molluscicidal fusion proteins and o further work was conducted with these peptides.

Section 3.2: Chemical synthesis and testing of mollusc-specific peptides 3.2A Introduction Many invertebrate-derived peptides (eg. conotoxins; neurohormones; myoregulatory peptides) have potential application as pesticides but in nature are post-translationally modified, a process that cannot yet be reproduced using heterologous expression systems. An alternative method for the creation of insecticidal fusion proteins via exploitation of the ability of avidin to bind biotin was previously demonstrated (LINK Project No 09110 “Improvements in the efficacy and production of insecticidal fusionproteins for environmentally benign pest control”). Avidin was previously identified as an alternative carrier protein for use in insecticidal fusion proteins where a synthesized biotinylated myoinhibitory peptide leucomyosuppressin (LMS) was linked to avidin and shown to have significant aphicidal activity as compared to the individual components (i.e. avidin or LMS alone; unpublished data). In this project avidin has been identified as an alternative carrier protein to GNA for use in molluscicidal fusion proteins (see section 3.3). Thus the aim of this work was to identify candidate molluscicidal peptides for incorporation into avidin based fusion proteins.

Three candidate peptides were selected from previously published literature as having potential for use as components of fusion proteins whereby the molluscicidal peptide could be linked ‘chemically’ to the newly identified carrier protein, avidin. The process of selecting suitable candidate peptides was greatly inhibited by the relative paucity of relevant published literature for molluscs as compared to arthropods, reflecting the lack of research that has been conducted in this area. Nevertheless three candidate peptides were identified. Firstly a myoinhibitory peptide GAPRVamide was selected based on previous data reporting significant suppression of oesophageal and body wall contractions of the great pond snail Lymnea stagnalis following injection of peptide purified from L. stagnalis tissue (Li et al., 1996). Two conotoxins containing post-translationally modified C-termini were also selected, namely PnIA and PnIVB. Originally purified from the molluscivorous snail Conus pennaceus, PnIA has previously been shown to cause paralysis of snails and to block acetylcholine receptors of cultured Apylsia neurons (Fainzilber et al., 1994). A PD50 of 14.6 pmol/100 mg body mass of Patella was reported whereas injection doses of more than 100x this dose produced no paralytic effects in insects (fly, Sarcophaga) or fish (Gambia). A second conotoxin purified from C. pennaceus, designated PnIVB was selected on the basis of paralytic activity towards Mytilus (ED50 21.9 pmol/mg body mass) and reported blockage of molluscan (Lymneae) sodium channels (Fainzilber et al., 1995). Like PnIA, PnIVB was shown to be inactive against insects and fish.

3.2B. Materials & Methods

3.2B.1 Selection and synthesis of candidate molluscicidal peptides

Three candidate peptides were synthesized by Cambridge Research Biochemicals (www.crbdiscovery.com). Approximately 10 mg of each HPLC purified (>95 %) peptide was supplied. All three peptides were C-terminally amidated and incorporated an N-terminal biotin to enable downstream linkage to avidin. The myoinhibitory peptide GAPRVamide (Biotin-Gly-Ala-Pro-

Arg-Phe-Val-NH2) was synthesised based on the sequence from the great pond snail Lymnea stagnalis characterised by Li et al., 1996. The conopeptide PnIA GCCSLPPCAANNPDYC-NH2

(Biotin-Gly-Cys-Cys-Ser-Leu-Pro-Pro-Cys-Ala-Ala-Asn-Asn-Pro-Asp-Tyr-Cys-NH2)was synthesised based on the sequence from molluscivorous snail Conus pennaceus characterised by Fainzilber et al.1994. A second conopeptide PnVIB CCKYGWTCWLGCSPCGC-NH2 (Biotin- Cys-Cys-Lys-Tyr-

Gly-Trp-Thr-Cys-Tyr-Leu-Gly-Cys-Ser-Pro-Cys-Gly-Cys- NH2) was synthesised based on the sequence from the cone snail C. pennaceus characterised by Fainzilber et al.1995.

3.2.B.2 Mollusc Cultures Deroceras reticulatum cultures maintained as described in section 2A.

3.2B.3. Biological activity: Deroceras reticulatum injection bioassays Injection bioassays were carried out using mature (0.5-1.0 g) slugs (D. reticulatum). In all cases, injections were carried out with three doses (10, 25 and 50 g) of each of synthesised GAPRVamide, PnIA, or PnVIB re-suspended in PBS. Ten slugs were injected per dose for each peptide. Injected slugs were analysed for evidence of paralysis for 24 hours post injection. Survival and feeding behaviour (consumption of lettuce discs) was monitored for 7 days post injection. PBS was injected as a control treatment.

3.2C Results

3.2C.1 Biological activity: Deroceras reticulatum injection bioassays

No effect on the survival of slugs was observed following the injection of GAPRVamide, PnIA, or PnVIB at doses of up to 50 g of synthesised peptides. Survival was comparable to control injected slugs and was more than 70% for all treatments. Of the three peptides injected only PnIA was found to result in a non-significant temporary inhibition of feeding (0-3 days post injection) by slugs.

3.2D Conclusions

Three peptides, a myoinhibitory peptide and two conopeptides were selected from the available published literature on the basis of reported activity towards mollusc species and non toxicity towards mammals, fish or insects. These peptides were synthesised and subsequently tested (by injection) for activity towards mature slugs (D. reticulatum). Unfortunately, when injected none of the tested peptides were found to have any effects upon the survival or feeding of D. reticulatum. It is noted for the two tested conotoxins ( PnIA and PnIVB) that following synthesis, the peptides were air oxidised, and thus correct di-sulfide bridge formation could not be guaranteed. Correct bridge formation (and thus the generation of correctly folded peptides) is critical for biological activity. However, to conduct bridge formation of synthesised peptides is an extremely costly process and beyond the available budget for this programme. Given the lack of evidence for activity against slugs no further work was carried out with these peptides. It is concluded that further fundamental research is required to enable the identification and characterisation of peptides with a suitable level of activity against D. reticulatum is required before progress with the development of chemically linked fusion proteins can be made.

Section 3.3: Evaluation of carrier proteins 3.3A Introduction The development of orally active insecticidal fusion proteins has been dependent on the availability of suitable "carrier" proteins". Snowdrop lectin (Galanthus nivalis agglutinin; GNA), the homodimeric mannose-binding lectin from garlic (Allium sativum agglutinin; ASA II), and avidin, a protein derived from chicken egg white, have all been shown to bind to the insect gut epithelium, and subsequently to be transported across the gut and into the systemic circulation, following ingestion. A similar strategy has been explored in molluscs; previous studies have shown that snowdrop lectin, following ingestion by D. reticulatum, is transported across the gut epithelium and can subsequently be detected in the circulatory system. The aim of this component of the project was to establish if garlic lectin (ASA II) and / or avidin were transported to the circulatory system of slugs following ingestion of these proteins, hereby identifying further potential carrier proteins for use in the development of novel molluscicidal fusion proteins. Recombinant ASA II and avidin were produced by expression in Pichia pastoris, and purified proteins were utilised in feeding studies. Slug gut and haemolymph samples were analysed by western blotting for the presence of ASA II and avidin following ingestion of purified proteins.

3.3B. Materials and Methods

3.3B.1 Production and purification of recombinant GNA

A GNA expression construct, generated as described previously (Raemakers et al., 1999) was used routinely for the production of recombinant GNA in the yeast P. pastoris. The sequence encoding GNA omitted the C-terminal extension removed from the protein in planta; this truncated form of GNA has been shown to be fully active as a lectin (Longstaff et al., 1998). For protein overproduction P. pastoris cells containing the GNA, were grown in benchtop fermenters as described in section 1B. Following the addition of salt to culture supernatant (2M NaCl) recombinant GNA was purified by hydrophobic interaction chromatography on phenyl-Sepharose columns. Folowing loading (1-3 ml/min) and washing with 2M NaCl recombinant GNA eluted at the end of the salt gradient in water and fractions were checked for purity by SDS-PAGE. A second clean-up gel filtration step was conducted to remove high molecular weight contaminating yeast proteins. Gel filtration fractions, again analysed for purity by SDS-PAGE, were pooled, dialysed against 5mM ammonium bicarbonate, and freeze-dried. Prior to all subsequent assays the concentration of recombinant proteins was estimated by comparison with known amounts of standard proteins and known amounts of commercial GNA (Vector Labs) by SDS-PAGE.

3.3B.2 Production and purification of recombinant ASA II

The cloning of a construct for the expression of ASAII in P. pastoris has been described previously (LINK 0948 final report). Following fermentation (carried out in benchtop fermenters, as previously described in section 1B) recombinant ASA II was purified by cation-exchange chromatography. Culture supernatant was diluted ten-fold (to decrease the salt concentration) and buffered with 50 mM sodium acetate (pH 4.0 with acetic acid). This was loaded onto S-sepharose columns (13 cm height; 2 cm diameter) at 2 ml/min, and a salt gradient (0 – 0.5M NaCl) was applied over a minimum of 100 ml. Following application of the salt gradient ASAII eluted from S-sepharose columns in the range 0.15 - 0.3M NaCl. As ASA II precipitated rapidly in buffers containing salt, eluted fractions were immediately dialysed into water and freeze-dried. Following freeze-drying, a second gel filtration purification step (as previously described in section 3B.1) was carried out, where necessary, to remove higher molecular weight contaminating yeast proteins. Prior to all subsequent assays, the concentration of recombinant ASAII was estimated by comparison with known amounts of standard proteins by SDS-PAGE.

3.3B.3. Production and purification of recombinant avidin

The cloning of a construct for the expression of avidin in P. pastoris has been described previously (LINK 0948 final report). As the avidin construct encodes for a C-terminal histidine tag, purification (following bench-top fermentation), is carried out by nickel-affinity (Ni-NTA) chromatography. Following centrifugation, the culture supernatant was brought to pH4.0 in 50 mM sodium acetate (with acetic acid) and applied to HisTrap columns (5 ml, Amersham Pharmacia) at 1-2 ml/min. Following washing (50 mM sodium acetate, pH 4.0, 0.3 M NaCl, containing 10 mM imidazole), avidin was eluted with 300 mM imidazole in 50 mM sodium acetate pH 7.4, 0.3 M NaCl. Elution volumes for nickel affinity chromatography are typically less than 10 ml, resulting in very high concentrations of protein in the elution solution that can precipitate upon dialysis. To overcome this problem eluted samples were diluted approx. 10-fold with distilled water prior to dialysis against 5 mM ammonium bicarbonate. Dialysed samples were freeze-dried. Prior to all subsequent assays the concentration of recombinant avidin was estimated by comparisons with known amounts of standard proteins and commercial avidin (Sigma) by SDS-PAGE.

3.3B.4. Antibodies to recombinant GNA, ASA II, and avidin

Polyclonal anti-GNA antibodies, raised in rabbits, were prepared by Genosys Biotechnologies, Cambridge, UK. Anti-avidin antibodies were purchased from Sigma. For ASA II, polyclonal antibodies were raised in rabbits via immunisation with SDS-PAGE gel slices containing purified ASA II. Anti-ASA II antibodies were produced by Eurogentec (www.eurogentech.com). Anti-sera

37 was evaluated for immunoreactivity by Western blotting SDS-PAGE separated samples of known amounts of recombinant ASA II.

3.3B.5. Haemagglutination assays

Haemagglutination of rabbit erythrocytes was used as a functional test for recombinant GNA and ASA II lectins. Microtitre-based assays (described by Raemakers et al., 1999) were conducted using washed rabbit erythrocytes incubated in PBS in U-shaped wells with a series of dilutions of each test protein. A total volume of 100 l was used in each well: 50 l aliquots of serial two-fold dilutions of standard GNA, recombinant GNA, or recombinant ASA II and 50 l of 2 % erythrocyte suspension in PBS were incubated for 2-3 h at room temperature. The lowest concentration required to completely agglutinate the red blood cells was determined visually.

3.3B.6 Recombinant avidin; In vitro activity The ability of recombinant avidin to bind biotin was assayed in vitro using biotin-agarose (Thermo Scientific). Matrix (1:1 suspension in 100µl PBS) was incubated with 5µg of recombinant avidin at room temperature for 30 mins. Following centrifugation (1 min at 2000 x g) the pellet was washed two times with PBS. Avidin bound to biotin was eluted from the matrix by boiling, and fractions were analysed by SDS-PAGE.

3.3B.7 Mollusc D. reticulatum Culture A Deroceras reticulatum culture maintained as described in section 3.2A.

3.3B.8. Feeding assays D. reticulatum and sample extraction Mature slugs (0.5-1.0 g) were fed on lettuce discs (diam. Ca. 20 mm) coated on one side with recombinant GNA, ASA II or avidin at a concentration of 1mg/disc. Six slugs were fed for each test protein with fresh discs provided as required. After 5 – 7 days of feeding gut (containing contents) and haemolymph samples were extracted from individual slugs. To this end slugs were chilled on ice prior to the extraction of haemolymph which was carried out using a Hamilton syringe. To prevent oxidation haemolymph samples were placed into eppendorfs containing phenylthiocarbamide. The digestive tract was subsequently dissected and individual gut samples homogenised in chilled PBS. The homogenate was then centrifuged (4 C at 12 000 rpm for 5 mins) and supernatants analysed for protein content by BCA assay using BSA as a standard protein. Control samples were prepared from slugs fed on lettuce discs without additional protein.

3.3B.9. Western analysis of D. reticulatum samples Gut and haemolymph samples extracted from slugs fed on coated lettuce discs were analysed for the presence of recombinant proteins by western blotting as desribed in section 3.2B.5.

3.3C. Results

3.3C.1. Production and purification of recombinant GNA, ASA II, and avidin

Recombinant proteins were produced by expression in P. pastoris, using a benchtop fermentation system. Recombinant GNA was purified by hydrophobic interaction chromatography on phenyl- Sepharose, followed by gel filtration to remove high molecular weight contaminating yeast proteins. Recombinant ASA II was purified by cation-exchange chromatography using S-Sepharose at pH 4.0; where necessary, as described for GNA, a further clean-up gel-purification step was carried out. Recombinant avidin was purified by nickel-affinity chromatography. Figure 3.7 shows SDS- PAGE and Western analysis of purified recombinant GNA (figure 3.7 (i)), ASAII (figure 3.7 (ii)), and avidin (figure 3.7 (iii)). In all cases proteins were purified to more than 90% homogeneity. Recombinant GNA was immunoreactive with anti-GNA antibodies and stained as a band of approx. 14 kDa, which is larger than the predicted molecular mass of approx. 12.1 kDa. Recombinant ASAII was immunoreactive to polyclonal antibodies raised against the recombinant lectin but did not react with anti-myc or anti-His antibodies indicating cleavage of the C-terminal tags during expression (results not shown). ASAII stains as a band of approx.17 kDa on SDS- PAGE gels, which is in agreement with the predicted molecular mass of 17 kDa for recombinant ASAII containing the myc and his tags. However, cleavage of the C-terminal tags (as indicated by immunoanalysis) suggests that, as is the case for GNA, ASAII runs as a larger protein than predicted on SDS-PAGE gels. Recombinant avidin was immunoreactive with polyclonal antibodies raised against native avidin, and stains as a band of approx. 20 kDa on SDS-PAGE gels, again larger than the predicted mass of 15.4 kDa. Typical yields of GNA, ASAII, and avidin, from benchtop fermentation were estimated to be 100-150 mg/l; 10-25 mg/l; and 50-60 mg/ l culture supernatant, respectively. In all cases purified lyophilised proteins were analysed for purity and quantity by SDS-PAGE using standard or native proteins for quantification. Lyophilised samples typically contained 20-60% dry weight of recombinant protein, the remaining dry weight being accounted for by yeast carbohydrate. Samples of GNA, ASAII, and avidin were produced for feeding bioassays against slugs to assess uptake of protein into the circulatory system following ingestion.

Figure 3.7: SDS-PAGE (a) and Western analysis (b) of recombinant (i) GNA, (ii) ASAII, and (iii) avidin purified from yeast culture supernatant after fermentation. M denotes standard protein mix (SDS-7) as denoted. (i) Lanes 1 & 2 are loadings of 2.5 and 5 g GNA, respectively and lanes 3 & 4 show 25 and 50 ng GNA, respectively. (ii) Lanes 1&2 are loadings of 5 and 10 g ASAII, respectively and lanes 3 & 4 show 25 and 50 ng ASAII, respectively. (iii) Lanes 1 – 5 show successive loading of 0.5, 1.0, 2.5, 5.0, & 10 g of recombinant avidin and lanes 6,7,& 8 are 1.0, 2.5, & 5 g of commercial (Sigma) avidin. Lanes 1,2, & 3 in (iii)(b) are negative control, 10 & 20 ng of recombinant avidin.

3.3C.2. Haemagglutination assays

The end-point of agglutination (lowest concentration required for agglutination) was found to be approx 10 g/ml for both purified recombinant GNA and commercially available GNA purified from snowdrop bulbs. Recombinant ASAII was able to agglutinate fresh rabbit erythrocytes down to a dilution of approx. 0.5 mg/ml (results not shown).

3.3C.3. In vitro activity of recombinant avidin

The ability of recombinant avidin to bind to biotin was confirmed in vitro by incubation with a molar excess of immobilised biotin. SDS-PAGE analysis showed the presence of avidin in biotin-bound fractions and absence of protein in wash fractions, suggesting that 100% of the purified protein was able to bind to biotin, and that the recombinant protein is fully biologically active (results not shown).

3.3C.4. Detection of recombinant GNA, ASA II and avidin in slugs after feeding

To determine whether either ASA II or avidin were transported across the gut epithelium and into the circulatory system of slugs after feeding, haemolymph samples were extracted from slugs fed on recombinant proteins. In addition, to examine the stability of ASA II and avidin to digestive proteolysis in the digestive tract, gut samples (containing gut contents) were also dissected and extracted. Protein extracts were analysed for the presence of intact recombinant proteins by western blotting. As a positive control, GNA was also fed to slugs and samples analysed as for ASA II and avidin. Control samples were prepared from slugs fed on lettuce discs without additional protein. Figure 3.8 shows representative western analysis of haemolymph and gut tissues extracted from D. reticulatum fed (A) GNA, (B) ASA II, and (C) avidin. As previously shown, a band immunoreactive with anti-GNA antibodies corresponding in molecular weight of GNA standards is detected in gut and haemolymph samples of GNA fed slugs. No cross reactivity of the GNA antibodies is evident in samples extracted from control (no additional protein) fed slugs. This is indicative of stability to gut proteolysis and transport of intact GNA across the slug gut and into the circulatory system. Similarly the analysis of slug samples extracted from ASA II fed slugs also shows the presence of proteins immunoreactive with anti-ASA II antibodies in both gut and haemolymph samples. This suggests that, like GNA, ASA II is also transported across the gut and into the circulatory system of slugs after feeding. However the two immunoreactive bands in both gut and haemolymph samples are of a lower molecular weight as compared to the ASA II standard suggesting that ASA II is not as stable as GNA and is prone to proteolytic cleavage during digestion. Western analysis of samples from slugs fed on avidin also shows positive immunoreactivity of proteins with anti-avidin antibodies. As observed for ASA II, some cleavage of the recombinant protein is indicated by the presence of two immunoreactive bands that are of a lower molecular weight than the avidin standard. Nevertheless, intact avidin is present in both gut and haemolymph samples extracted from avidin fed slugs.

Figure 3.8: Western analysis of slug gut and haemolymph samples extracted from (A) GNA, (B) ASA II, and (C) avidin fed slugs using (A) anti-GNA, (B) anti-ASA II, and (C) anti-avidin antibodies. C denotes control samples extracted from slugs fed on control (no added protein) diet in all cases. St. denotes standards of 10 and 25 ng of recombinant GNA and ASA II, and 10 & 20 ng of recombinant avidin. 1 and 2 denotes samples extracted from individual slugs. Ten l of haemolymph was loaded in (A) and (B), and two loadings of 5 and 10 l for each haemolymph sample in (C). Approx. 40 g total gut proteins were loaded in all cases. The location of standard protein mix (SDS-7) run on the same gel is denoted.

3.3D Discussion The aim of this component of the project was to identify alternative candidate carrier proteins to the mannose binding snowdrop lectin, GNA. To this end two proteins, garlic lectin (ASA II) and avidin, both of which have been shown, following ingestion, to bind to the gut and be transported to the circulatory system of insects (lepidopteran and dipteran larvae, unpublished results) were selected for evaluation. Like GNA, ASA II is a mannose-binding lectin and is therefore likely to bind to the epithelium of slug digestive tract through interaction with glycosylated polypeptides associated with cell membranes, containing mannose residues. Avidin, a protein found in the egg white of birds, reptiles and amphibians, is thought to function as an antibacterial, host-defence protein, by virtue of its ability to bind biotin, an essential vitamin for most organisms. When fed to insects it is thought that avidin is able to bind to biotinyated polypeptides present in the gut epithelium, and a similar mode of action may occur in the slug digestive tract.

Recombinant GNA, ASA II and avidin were successfully produced by bench-top fermentation and purified from culture supernatants by liquid chromatography. In excess of 40 mg of purified proteins were produced for feeding trials. In vitro analysis demonstrated biological activity, either by the ability to agglutinate rabbit red blood cells (GNA and ASA II), or the ability to bind biotin (avidin). When coated onto lettuce discs and fed to slugs ASA II and avidin were both detectable by western analysis in gut and haemolymph samples. This suggests that like GNA, both ASA II and avidin are transported across the gut and into the circulatory system of slugs following ingestion. As such, both proteins are considered to be potential candidates for use as the carrier component of molluscicidal fusion proteins. However, it is noted that ASA II and, to a lesser degree avidin, appeared to be less stable than GNA, as both were prone to a degree of proteolysis in the slug digestive tract. By contrast, GNA is highly stable to breakdown in the slug digestive tract and remains fully intact following transport into the circulatory system. Nevertheless, a proportion of ingested ASA II and avidin remained intact in the gut and following transport to the circulatory system providing clear evidence for the potential use of these proteins as alternative candidate carrier proteins to GNA.

Section 3.4: Evaluation of insecticidal fusion protein(s) 3.4A Introduction The aim of this component of the programme was to investigate if an insecticidal fusion protein previously developed by Fera/Durham University had any molluscicidal activity when tested against D. reticulatum. The fusion protein RST/GNA or FP4 is composed of a venom protein derived from the scorpion Buthus tamulus, linked to the snowdrop lectin carrier GNA and has been shown to be insecticidal towards a range of pests from different insect orders (Trung et al., 2006; Fitches et al., 2010). The RST peptide is known to disrupt ion channel function and belongs to a group of venom peptides thought to affect insect voltage-dependent potassium channels and conductance calcium-activated potassium channels (Carbone et al., 1982; Miller et al., 1985). The potential for disruption of molluscan ion channel function by RST has not been previously reported. To this end RST/GNA was produced and assayed for activity against D. reticulatum by injection and feeding assays.

As the results obtained from injection and feeding assays with RST/GNA provided evidence for biological activity against slugs, a second insecticidal fusion protein (omega/GNA or FP5) comprised of a peptide derived from the funnel web spider Hadronyche versuta -hexatoxin-Hv1a (Hv1a) linked to GNA was produced and tested for activity against D. reticulatum. The Hv1a peptide is known to target insect voltage-gated calcium channels, acting directly at sites within the central nervous system (Fletcher et al., 1997; Tedford et al., 2004; Chong et al., 2007). As for RST the potential for disruption of molluscan ion channel function by Hv1a has not been previously reported.

3.4B Materials and Methods 3.4B.1. Production of RST/GNA (FP4) and omega/GNA (FP5)

The generation of a construct encoding a synthetic RST peptide derived from the scorpion Buthus tamulus fused to the snowdrop lectin, GNA has previously been reported (Trung et al., 2006). Similarly the generation of a construct encoding the Hv1a toxin linked to GNA has previously been described (Fitches et al. in submission). In addition to the omega/GNA fusion protein an additional construct encoding for FP5 with an additional C-terminal histidine tag was generated using standard restriction and ligation techniques. A diagrammatic representation of the FP5 constructs used to produce protein for slug bioassays is presented in figure 3.10. Both fusion proteins were produced by bench-top fermentation carried out as previously described (Fitches et al., 2004), using clones expressing either FP4 or FP5. For proteins containing histidine tags (FP4 and FP5+His) purification from culture supernatants was carried out by nickel affinity chromatography. To this end supernatants were diluted 1 in 4 with 4x Binding buffer (BB; 0.08M sodium phosphate; 44

1.6M NaCl) and loaded onto nickel affinity columns columns (5 ml HisTrapFF columns). Typically 2 x5 ml columns were linked and loaded at 3-4 ml/min for 3-5 hours with cycling. After loading the columns were washed with 1xBB (0.02M sodium phosphate; 0.4M NaCl, pH7.4) and protein was eluted from the columns with BB containing 0.2M imidazole. For FP5 (no histidine tag) purification from culture supernatants was carried out by hydrophobic interaction chromatography followed by a second gel filtration purification. To this end NaCl was added to supernatants to a final concentration of 2 M and loaded on phenyl-Sepharose (Amersham Pharmacia Biotech) columns (1 cm dia., 25 ml), run at 2 ml/min. After loading, the phenyl-Sepharose column was washed with 2 M NaCl and a linear salt gradient (2 M–0 M NaCl) applied over 60 min. Recombinant FP5 eluted at ~1 M NaCl. Fractions containing purified proteins (analysed by SDS-PAGE) were then pooled, dialysed against distilled water and lyophilised. Lyophilised fusion protein was then subjected to gel filtration on Sephacryl S-200 columns (1.6 cm diameter, 90 cm length, flow rate 0.3 ml/min) to remove high molecular weight yeast proteins as described previously (Trung et al. 2006).

Peak fractions were analysed for the presence and purity of FP4 or FP5 (+ and – his tag) by SDS- PAGE (17.5% acrylamide gels) and then diluted (50:50 with dist. water) and dialysed against distilled water. Dialysed protein was then lyophilised and stored at 4 C. Prior to use in injection or feeding assays the quantity and purity of recombinant proteins (FP4 and FP5) was estimated by comparison with known amounts of standard GNA on SDS-PAGE gels.

Figure 3.10. Diagrammatic representation of constructs created for the expression of the omega/GNA (FP5) fusion protein. The position of the N-terminal -factor signal sequence and presence of C-terminal tags are shown. Predicted molecular weight of the expressed fusion protein is depicted. 45

3.4B.2. Mollusc culture Deroceras reticulatum were maintained as described in section 3.2A.

3.4B.3. Injection assays Injection bioassays were carried out using 12 week old D. reticulatum. In all cases, injections were carried out with varying doses of recombinant FP4 or FP5. Purified recombinant FP4 (100 g) and FP5 (50 and 100 g) re-suspended in PBS were injected into mature (0.5-1.0g) slugs (D. reticulatum) and survival monitored daily for 7 days. PBS was injected as a negative control treatment.

3.4B.4 Feeding assays: Coated lettuce disc assays To evaluate FP4 or FP5 for oral activity towards D. reticulatum, immature slugs (10-40 mg) were fed on lettuce discs (ca. 20 mm) coated with purified recombinant proteins. Proteins were re- suspended in PBS/ 0.01 % Tween and the uppermost side of each disc was coated with 1mg of protein. Slugs were maintained for 14 days in petri dishes (5 slugs per dish) lined with damp tissue to maintain moisture at 10 C, 75 % relative humidity (RH). Fresh discs were replaced as required. Survival was monitored every 1-2 days and weights were recorded at day 0, day 7, and day 14. The consumption of leaf material in assays with FP5 coated discs was analysed by scanning leaf material (reference control discs not exposed to slugs and discs from FP5 and control treatments) removed from the petri dishes and area of leaf consumed measured using Image J software.

3.4B.5. Fusion proteins: Stability to heat treatment Prior to the preparation of wheat pellets incorporating fusion proteins, the stability of FP5 to heat treatment was investigated. Samples of lyophilised FP5 or FP5 re-suspended in water were subjected to heat treatment (50 C for 5 h). FP5 is known to cause significant mortality of lepidopteran (Mamestra brassicae) larvae and thus an injection bioassay was used to assess if FP5 was stable to heat treatment. Two doses of FP5 (20 and 40 g; before and after heat treatment) were injected into newly eclosed fifth stadium larvae (10 per treatment) and survival recorded daily for 3 days post injection.

3.4B.6. Wheat pellet assays Wheat pellets were prepared as follows: Flour was heat-treated (overnight at 80° C) prior to mixing with freeze dried fusion protein. Distilled water was added (0.5ml water /gram dry mix) and the mix was then placed into eppendorf lids and oven dried at 50°C for three hours. Resulting pellets (pellet wt. approx 80 mg) were then stored at room temperature until use. FP5 was incorporated at a single dose of 1.3% w/w. Two cohorts of slugs (10-20 mg and 20-40 mg) were starved 24 hours

46 before exposure to wheat pellets. Five slugs per replicate (maintained in petri dishes lined with moist paper towel) were treated and survival monitored daily. Growth was recorded at day 0, and 7 and 14 days after exposure to the treatments. Metaldehyde treatments comprised of (ASDA generic pellets containing 3 % metaldehyde or De Sangosse pellets containing 5 % metaldehyde). Fresh pellets were provided as required or, if not consumed every 2-3 days to prevent contamination.

3.4B.7. Stability of fusion proteins in feeding assays Lettuce discs were coated with 1mg/disc of FP5 and incubated in petri dishes (10 C, 75% RH) either alone, or in the presence of slugs. Samples of leaf were taken at time 0, day 1, 3 and day 5. Leaf samples of known weight were subsequently homogenised in PBS and comparable aliquots run on SDS-PAGE gels were analysed for the presence of intact and cleaved FP5. Similarly wheat pellets containing FP5 were incubated in the presence and absence of slugs and samples (taken at time 0, day 1, 3, 5, and 7) were re-suspended in PBS buffer and analysed for the presence of intact and cleaved FP5 by western blotting using anti-GNA antibodies.

3.4B.8. Oral delivery of omega/GNA to slug circulatory system Haemolymph and gut tissue samples were extracted from slugs fed on FP5 coated lettuce discs and FP5 containing wheat pellets and analysed for the presence of FP5 by western blotting as described previously in section 3.3C.4.

3.4C Results 3.4C.1. Production of RST/GNA (FP4) and omega/GNA (FP5 + and – His)

A previously prepared construct for the expression of a fusion protein (FP4 or RST/GNA) comprised of a scorpion venom peptide (RST), fused to the N-terminus of snowdrop lectin was used for protein production in bench-top fermenters. Recombinant RST/GNA was purified by nickel affinity chromatography. Following purification, dialysis and freeze-drying typical yields of RST/GNA from benchtop fermentation were estimated to be 25-50 mg/l culture supernatant. The resulting lyophilisate typically contained 20 - 60% dry weight recombinant fusion protein, the remaining dry weight being accounted for by yeast carbohydrate with minimal contamination (< 10%) by high molecular weight yeast proteins. Figure 3.11 shows representative SDS-PAGE analysis of purified RST/GNA. The recombinant fusion protein stains as two major bands on SDS- PAGE gels; the largest at approx. 18.5 kDa is close to the predicted molecular weight of 19.4 kDa and a smaller band of approx. 17 kDa. Both bands were immunoreactive with anti-GNA antibodies but only the larger band reacted with anti-his antibodies. The indicated size and immunoreactivity of these polypeptides suggests that the larger is full-length RST/GNA with an intact C-terminal

47 extension and the smaller is RST/GNA from which the C-terminal his tag has been removed. In excess of 100 mg purified RST/GNA was produced for bioassays against D. reticulatum.

Two versions of second insecticidal fusion protein (FP5 or omega/GNA) with and without a C- terminal histidine tag were used for production in bench-top fermenters. FP5 that did not contain a C-terminal tag was purified by hydrophobic interaction chromatography followed by a second gel- filtration clean-up step. FP5 containing a histidine tag was purified by nickel affinity chromatography. As for FP4, following purification, dialysis and freeze-drying typical yields of FP5 (+ and – His) from bench-top fermentation were estimated to be 50-100 mg/l culture supernatant, respectively. The resulting lyophilisate typically contained 10 - 50% dry weight recombinant fusion protein, the remaining dry weight being accounted for by yeast carbohydrate with minimal contamination (< 10%) by high molecular weight yeast proteins. Figure 3.12 shows representative SDS-PAGE analysis of purified FP5. Both versions of fusion protein (FP5 + and – His) show a degree of proteolytic cleavage with two major bands present in purified fractions. The larger molecular weight bands (approx 20 kDa) are close to the predicted sizes for both omega/GNA variants (15.93 & 16.77 kDa for minus and plus His tag, respectively) and these bands correspond to intact fusion protein as confirmed by western analysis (results not shown). The lower molecular weight protein corresponds to GNA from which the omega peptide has been cleaved as demonstrated by positive immunoreactivity of this protein with GNA, but not with anti-omega antibodies (results not shown). The ratio of intact to cleaved protein for omega/GNA and omega/GNA/His protein is approx. 1:2. In excess of 300 mg purified FP5 (+ and- His) was produced for bioassays against D. reticulatum.

Figure 3.10: SDS-PAGE analysis (17.5% acrylamide gel) of recombinant RST/GNA (FP4) purified by nickel affinity from yeast culture supernatant. M shows standard protein mix (SDS 7) as denoted. Lanes 1-4 and 5-8 are successive loadings of 2.5, 5.0, 10.0, & 20 g of two different FP4 samples, and lanes 9,10 & 11, are recombinant GNA standards of 0.5, 1.0, & 5.0 g, respectively.

Figure 3.11. SDS-PAGE (17.5% acrylamide gel) analysis of omega/GNA variants after purification and lyophilisation. Loading as follows 1 denotes omega/GNA/His and 2 omega/GNA. Loading for each lane is given as total dry weight ( g) of lyophilised sample and loading of GNA standards is given in g recombinant protein.

3.4C.2. Biological activity by injection: D. reticulatum Slugs injected with 100 g of FP4 (equivalent to approx 50 g of toxin/g slug) showed a 20% reduction in survival as compared to control PBS injected treatment (n=18 per treatment). As observed in table 4 a dose dependent decrease in survival was observed in slugs injected with purified FP5, such that mortality of slugs injected with doses of toxin equivalent to 50 g/g and 25 g/g slug was 70 % and 23 % respectively. Results obtained following the injection of lepidopteran larvae (Mamestra brassicae) are included to enable a comparison of biological activity of the omega toxin towards molluscs and insects to be made. It is worth noting that slugs excessive sliming of slugs immediately post injection may enable the slugs to expel toxin from their system although this has not been verified experimentally.

Table 4 Mortality of slugs (D. reticulatum) and lepidopteran (Mamestra brassica) larvae recorded following the injection of different doses of FP5.

Injections of slugs (D. reticulatum) Treatment Dose FP5 Dose ( g/slug) Mortality Sample Dose ug ( g/slug) (omega (%) No. omega/g equivalents) control - - 3 18 - Omega/GNA 100 25 70 27 50 Omega/GNA 50 12.5 23 18 25 Based on mean slug weight at injection of 0.5 g

Injections of lepidopteran larvae (M. brassicae) Treatment Dose FP5 Dose Mortality Sample Dose ug (%) No. omega/g ( g/insect) ( g/insect) (omega insect equivalents) control - - 0 20 - Omega/GNA 20 5 90 20 100 Omega/GNA 10 2.5 45 20 50 Omega/GNA 5 1.25 0 20 25 Based on mean larval weight at injection of 50 mg

3.4C.3. Biological activity by ingestion: leaf disc assays D. reticulatum Survival and growth recorded for juvenile slugs fed on lettuce discs coated with purified RST/GNA (FP4; 1mg/disc) is presented in Figure 3.13. Growth and consumption of juvenile slugs fed on lettuce discs coated with purified omega/GNA (FP5; 1mg/disc) is presented in Figure 3.14. Feeding on either RST/GNA or omega/GNA was found to have a small but non-significant reduction in survival (ca. 10-20 % reduced as compared to control fed slugs). However, both fusion proteins were found to significantly reduce slug growth. The mean weight of slugs fed on RST/GNA was 20% lower as compared to controls on day 14 of the assay. By comparison, a greater reduction in growth was observed for omega/GNA fed slugs by day 7 of the assay where the mean weight treated slugs was 33% lower than that recorded for the control group. Analysis of leaf consumption in assays with omega/GNA confirmed that reduced growth was attributable to a significant reduction in consumption, such that cumulatively the treated slugs consumed approx. 50% of the leaf area that was consumed by the control group.

Figure 3.13. (A) Survival and (B) Growth of juvenile D. reticulatum fed on lettuce discs coated with RST/GNA (FP4). * denotes a marginally significant difference (t-test; P=0.06) between FP4 and control (no added protein) treatments. N= 30 per treatment

(A) Growth (B) Consumption

Figure 3.14. (A) Growth of juvenile D. reticulatum fed on lettuce discs coated with omega/GNA (FP5) N= 30 per treatment, (t-tests P<0.001 day 7; P=0.066 day 14). (B) Cumulative consumption recorded for slugs in (A) * denotes significant differences between omega/GNA and control (no added protein) treatments (t-test P<0.001), N= 6 per treatment. Bars are standard errors of mean.

3.4C.4. Stability of FP5 to heat treatment Prior to the development of a wheat pellet assay the stability of omega/GNA to heat treatment was evaluated. Samples of heat-treated (50 C for 5 h) omega/GNA were compared to non heat- treated samples for biological activity by injection into lepidopteran (M. brassicae) larvae. As shown in Figure 3.15 the survival of larvae injected with equivalent doses of heat treated or non heat was similarly reduced as compared to controls, such that 25% larvae survived following the injection of non heat treated samples as compared to 20% survival recorded for larvae injected with heat treated omega/GNA. These results confirm that the omega toxin component of the fusion protein is stable to heat treatment.

Figure 3.15. Survival of M. brassicae larvae recorded 72 hours following injection of two equivalent doses of non heat-treated (NHT) and heat-treated (HT; 50 C 5 h) samples of omega/GNA (n = 10 per treatment). Survival was significantly reduced by both treatments as compared to the control group Kaplan-Meier (P<0.05) but not significantly different between heat-treated and non-heat treated samples.

3.4C.5. Biological activity by ingestion: wheat pellet assays D. reticulatum Several bioassays were conducted to evaluate the effects of omega/GNA upon juvenile slugs following incorporation of the fusion protein into wheat pellets ( 1.3 % w/w). Representative data is presented herein. Negative control treatments in these bioassays included wheat pellets containing no added protein and “normal” diet (lettuce and chinese cabbage leaf and oat flakes). Positive control treatments were commercial metaldehyde pellets and a no diet treatment. Figure 3.16 shows survival and mean weights of two cohorts of slugs (10-20 mg and 20-40 mg recorded at day 0) exposed to control and omega/GNA treatments. A comparison of the results obtained for control no added protein wheat pellet with “normal” mixed diet shows improved survival and growth of the wheat pellet treatment indicating suitability of the wheat pellets for use in slug bioassays. The results obtained for slugs exposed to commercial metaldehyde-containing pellets were variable. As 52 shown in Figure 3.16, a significant decrease in survival was observed for the smaller cohort of slugs (10-20 mg) fed for 7 days on commercial pellets but, surprisingly, no reduction in survival was observed for the 20-40 mg cohort. Whilst omega/GNA did not affect survival, a significant reduction in mean weight, as compared to the control treatments (wheat pellet and mixed diet), was observed for both cohorts after 7 days. Figure 3.17 shows mean weight data obtained when the two cohorts of slugs were exposed for a further 7 days to omega/GNA wheat pellets, control pellets or no diet. After 14 days the slugs were then placed on a normal mixed diet to enable recovery from the omega/GNA and no diet treatments to be evaluated. The reduced growth of slugs fed on omega-GNA pellets is maintained over the 14 day assay period with mean weights comparable to the no diet treatment. This provides further evidence for reduced feeding by slugs exposed to omega/GNA containing pellets. An increase in mean weight is observed for the omega/GNA and no diet groups following transfer of the slugs after 14 days to a mixed diet. This is indicative of a return to feeding, although mean weights for omega/GNA and starved treatments after 7 days on “normal” mixed diet remain lower than the group fed for 14 days on control wheat pellets.

(A) Survival (B) Growth

Figure 3.16. (A) Survival and (B) mean weight of juvenile D. reticulatum recorded 7 days after exposure to wheat pellets containing omega/GNA (1.3% w/w); control wheat pellets (no added protein); “normal” mixed diet; no diet; and commercial metaldehyde pellets (3-5% metaldehyde). Two cohorts of slugs (10- 20 mg) and 30-40 mg) were assayed.

(A) 10-20 mg (B) 20-40 mg

Figure 3.17. Mean weight of juvenile D. reticulatum (A) 10 -20 mg (N=15 per treatment) and (B) 20- 40mg (N=10 per treatment) recorded at day 0, day 7, day 14 and day 21. Treatments were control (no added protein) wheat pellets, omega/GNA containing pellets and no diet ‘starved’ for 14 days followed by 7 days of feeding on ‘normal’ mixed diet.

3.4C.6. Stability of FP5 when coated onto discs or incorporated into pellets SDS-PAGE analysis was used to verify the persistence of omega/GNA in feeding assays where the protein was coated onto the surface of leaf discs. It was anticipated that the concentration of omega/GNA may be reduced with time due to the travel of slugs across the coated discs. To this end leaf samples that had or had not been exposed to slugs were taken and equivalent samples analysed by SDS-PAGE for the concentration of omega/GNA. As shown in Figure 3.18 the presence of omega/GNA is lower in samples taken after 3 days of exposure to slugs, whereas the presence of fusion protein is maintained in samples not exposed to slugs. This is indicative of the loss of protein from coated leaf discs.

The stability of omega/GNA following incorporation into wheat pellets and exposure to slugs was also analysed. In this case proteins were extracted from wheat pellets and equivalent amounts analysed by western blotting using anti-GNA antibodies. As shown in Figure 3.19 the concentration of omega/GNA remains constant with time and in the presence or absence of feeding slugs. This suggests that the developed wheat pellet assay system is the preferred option for testing the effects of fusion proteins against D. reticulatum.

Figure 3.18. SDS-PAGE (17.5% acrylamide gel) analysis of omega/GNA coated leaf disc samples taken at different time points without exposure to slugs (-S) and (+S) following exposure to slugs. Equivalent loadings of samples taken at different time points (D= day) were loaded. Loading of omega/GNA standards (St.) is given in g purified recombinant protein.

Figure 3.19. Western blot analysis (anti-GNA antibodies) of wheat pellets showing the presence and persistence of omega/GNA with time and in the presence and absence of slugs. Image on the left depicts slugs feeding on wheat pellets. D denotes time of exposure in days. Stds. and Std. denotes standards of purified omega/GNA (50 and 75 ng). In all pellet samples loading was equivalent to 70 ng omega/GNA, based on the initial concentration of fusion protein incorporated into the pellets.

3.4C.7. Delivery of FP5 to slug circulatory system following ingestion The presence of omega/GNA in slug gut and haemolymph samples following ingestion of either coated leaf discs coated fusion protein containing wheat pellets was assessed by immunoblot analysis, using anti-GNA antibodies. Figure 3.20 shows representative analysis of samples extracted from slugs fed on omega/GNA containing pellets. The fusion protein is clearly present in gut samples taken after 1 and 4 days of exposure to omega/GNA containing pellets, with GNA immunoreactive bands corresponding to the bands in the omega/GNA standard. A degree of proteolysis is present in gut tissues and evident as immunoreactive bands of a smaller molecular weight as compared to intact omega/GNA. Similarly GNA immunoreactive bands are present in haemolymph samples taken from fusion protein fed slugs providing evidence for the delivery of omega/GNA to the circulatory system following ingestion. As for gut samples the presence of a number of bands that reactive with anti-GNA antibodies is indicative of a degree of proteolytic cleavage that may occur either before or after transport of the protein across the gut and into the circulatory system. Similar results were obtained for slugs fed on coated leaf discs or omega/GNA containing wheat pellets.

Figure 3.20. Western blot analysis (anti-GNA antibodies) of slug gut and haemolymph samples taken from control-fed (C= no added protein) or omega/GNA containing wheat pellets. For gut samples a total of 50 g total protein (as estimated by BSA assay) was loaded. The volume of haemolymph samples is depicted in the figure. D denotes time of exposure in days. St denotes 50 ng purified omega/GNA standard.

3.4D Discussion and conclusions Two insecticidal fusion proteins have been produced and purified proteins evaluated for activity towards the grey field slug Deroceras reticulatum. The fusion protein RST/GNA, contains a peptide (red scorpion toxin; RST) that is derived from the scorpion Buthus tamulus, linked to the snowdrop lectin carrier GNA and has previously been shown to be insecticidal towards a range of pests from different insect orders (refs). The omega/GNA fusion protein contains a peptide derived from the funnel web spider Hadronyche versuta -hexatoxin-Hv1a (Hv1a) linked to the carrier GNA and has also been shown to be insecticidal to a range of insect pest species. Neither of these fusion proteins has previously been tested for activity towards mollusc species.

Both RST/GNA and omega/GNA, when injected, were found to cause mortality of mature slugs with omega/GNA found to be the more molluscicidal of the two fusion proteins. This is broadly in agreement with previous results obtained in insects, although variability in the susceptibility of different insect species to the tewo different fusion proteins has been observed (unpublished results). In fact, in the case of omega/GNA, the levels of mortality were similar for slugs and insects when expressed as dose of toxin (omega peptide) per gram of slug or insect. The omega peptide is known to target insect voltage-gated calcium channels, acting directly at sites within the central nervous system and the results presented here suggest that the omega peptide is also able to disrupt ion channel function within the nervous tissue of the mollusc D. reticulatum. This is the first evidence for biological activity of venom peptides known to target insect ion channels with molluscan ion channels suggesting that such so called ‘insect-specific’ neurotoxins have potential use for the development of strategies to control of molluscan pest species. Further evidence is required to verify the indirect evidence obtained from injection assays described herein. Unfortunately the shortage of molecular information available for molluscs species generally, and in particular for the crop pest D. reticulatum, currently prevents clear identification of potential ion channel targets in molluscs.

Both fusion proteins were also found to have a degree of oral molluscicidal activity. Whilst neither of the fusion proteins caused significant reductions in survival at the doses tested (1mg/disc in leaf assays; 1.3 % w/w in wheat pellet assays) both were consistently effective in causing significant reductions in growth as compared to control treatments. That reduced growth was attributable to reduced consumption was demonstrated directly through the analysis of consumption in leaf assays where total cumulative consumption was shown to be significantly lower for fusion protein fed slugs as compared to controls. As observed in injection assays omega/GNA was shown to be the more effective of the two fusion proteins, causing greater reductions in slug growth and consumption as compared to RST/GNA. A wheat pellet assay has been developed that enables fusion protein treatments to be compared more directly with the efficacy of commercial 57 metaldehyde containing pellets. Prior to oral pellet tests the omega/GNA fusion protein was evaluated for stability to heat treatment and no detrimental effects on biological activity of the peptide toxin were observed. This suggests that recombinant fusion proteins have potential to be incorporated into commercial pellets as exposure to heat occurs during the production process. Further analysis also demonstrated that fusion proteins remained stable following incorporation into wheat pellets, whereas reductions in the levels of fusion protein were observed in assays where slugs were exposed to leaves coated with protein and this was thought to be due to the movement of slugs across the leaf material during periods of feeding. Surprisingly, the effects of feeding commercial metaldehyde pellets (generic and De Sangosse pellets) to D. reticulatum were highly variable. In some cases no mortality was observed following exposure for up to 14 days, whereas in other cases mortality was in excess of 50%. These results are, in part, thought to be attributable to differences in the levels of metaldehyde present in the pellets. However, the data obtained also demonstrates the difficulty in assessing molluscicidal activity of any new active ingredient. In contrast to most insect species, slugs are able to survive long periods without feeding and therefore exposure to toxins such as metaldehyde can be avoided.

Direct evidence for the delivery of fusion proteins to the circulatory system of slugs following ingestion was obtained by western analysis of samples extracted from omega/GNA fed slugs. Taken together with the bioassay data, demonstrating oral molluscicidal activity towards D. reticulatum, it is concluded that this technology offers potential for the generation of new molluscicidal products. Whilst a prototype fusion protein has not been produced the results obtained provide ‘proof of concept’ for this technology. Further molecular work to enable identification of ion channel targets in mollusc pests is required to facilitate the selection of suitable neurotoxins for incorporation into molluscicidal fusion proteins.

Section 3.5: Production of molluscicidal fusion proteins by industry 3.5A Introduction Research conducted as described previously in sections 1-4, provided the basis for the selection of the omega/GNA fusion protein for production scale-up. Laboratory scale studies (Fera/University of Durham) had demonstrated that omega/GNA when incorporated into wheat pellets had significantly detrimental effects on the growth of D. reticulatum. The issues to be encountered with the large-scale production of fusion proteins are likely to be similar for different fusion proteins and therefore this work was considered as a feasibility study to elucidate if fusion proteins could be produced on an industrial scale. This work was carried out by Actygea (Microbial Fermentation Department of Actygea, Gerenzano, Italy), as contracted by Isagro Ricerca. The following is a report describing work to produce gram quantities of the omega/GNA fusion protein (FP5) for tests to be carried out by the industrial partner De Sangosse. Prior to provision of material to De Sangosse glasshouse assays against Colorado Potato Beetle (Leptinotarsa decemlineata) were conducted to verify biological activity of the fusion protein.

The present report (provided by Fabrizio Beltrametti, Actygea) describes the production at 200 liter scale of the FP5 complex by use of the recombinant Pichia pastoris strain omega/GNAHIS #3. After fermentation, the supernatant was separated from the insoluble fraction (which contained a small amount of insoluble -and inactive- protein) and the pH was corrected to 8.5 with the addition of Tris-HCl buffer 100 mM and NaOH. The soluble protein was purified with Ni-conjugated resins and was active in bioassays. Soluble protein recovery with this method was ca 12.5 mg/L (referred to the volume at harvest), which was in line with previous observations. Additionally, an HPLC method was developed which allowed the identification and quantification of the GNA component of the FP5 complex, along all the fermentation/purification process.

3.5B Materials and Methods Information on fermentation and on protein FP5 is detailed in Tables below. Table 5.1. Information concerning the P. pastoris omega/GNA/HIS #3 strain and expression system

Notes Genus Pichia Species Pastoris Strain SMD1168 Expression system pGAPZ A Strain was routinely maintained without Selectable marker Zeocin selection with Zeocin System of transformation Invitrogen 59

Table 5.2. Information concerning the protein

Notes Protein name FP5 Spider Toxin conjugated Protein type with Gna, His-Tag Protein localization secreted Protein appears in SDS as two bands 17016 Da (FP5) attributed to FP5 and Gna. The two Protein dimension 12908 Da (Gna) proteins are collectively indicated as FP5 complex Analytical system HPLC Quantitative for Gna Other analytical system SDS-PAGE Qualitative Analytical standard FP5 complex Amount expressed ca 50 mg/L Reported by Elaine Fitches Percentage among unknown extracellular proteins Purification suggested Ni-conjugated resins

Table 5.3. Information concerning fermentation.

Notes Medium Available Fermentation process Batch/fed-batch Invitrogen protocol Induction of expression Glycerol (constitutive)

3.5B.1 Growth and fermentation in flask The strain was cultivated in 500 ml flasks containing 100 ml of the vegetative medium YPG (in grams per liter: yeast extract, 10; Bacto-Peptone, 20; glycerol, 40). Inoculum was performed by suspending a loop of cells collected from a slant or plate, into 10 ml of physiologic saline (0.8 g of NaCl in 1 liter deionized water). 5 ml of the above suspension were inoculated into 500 ml flasks containing 100 ml vegetative medium and the culture was incubated at 30°C with shaking. Growth was monitored as increase in OD600 for up to 96 hours (not shown). Alternatively, growth was in fermentation basal salt medium added with PTM1 trace salts and brought to pH 5.6 with NH4OH (Table 4).

3.5B.2 Fermentation at 200 litre scale The 200 liter fermentor was prepared and run according to the protocol received from Elaine Fitches, and verified at the 30 liter scale as described in report # 01/2011.

Table 5.4. Media and fermentation parameters for 30 liter fermenter.

Fermentation Basal Salts Notes Medium Phosphoric acid, 85% 26.7 ml/L Calcium sulfate 0.93 g/L Potassium sulfate 18.2 g/L

Magnesium sulfate-7H2O 14.9 g/L Potassium hydroxide 4.13 g/L Glycerol 40.0 g/L Water to 1 liter Antifoam Mix 0.0 ml Added on demand PTM1 Trace Salts 4.35 ml/L Added after sterilization PTM1 Trace Salts Notes Cupric sulfate-5H2O 6.0 g/L Sodium iodide 0.08 g/L Manganese sulfate-H O 3.0 g/L 2 Sodium molybdate-2H2O 0.2 g/L Boric Acid 0.02 g/L Cobalt chloride 0.5 g/L Zinc chloride 20.0 g/L Ferrous sulfate-7H2O 65.0 g/L Biotin 0.2 g/L Sulfuric Acid 5.0 ml/L Water To 1 liter Solutions were filter sterilized Feeding solution Notes Glycerol 500.0 g/L

PTM1 Trace Salts 4.35 ml/L Added after sterilization Water To 1 liter 0.2 liters/liter of fermentation Feed rate Basal Salts Medium (indicative Start when oxygen spikes 0.2 litres/litres media

The vegetative inoculum was prepared as follows. Ten 1000 ml baffled flasks, containing 0.3 liters of YPG each (total 3 liters) (no antibiotic selection was used) were inoculated from fresh P. pastoris slants and growth was allowed at 30 °C with shaking (220 rpm) for 48 hours. 27 liters of YPG, were sterilized in a 30 liter stirred fermentor, and the fermentor was inoculated with 3 liters of the YPG grown culture. The culture was grown as above for 24 hours. 20 liters of the above culture were then used to inoculate 180 liters of fermentation basal salt medium added with PTM1 Trace Salts. Initial glycerol exhaustion was monitored through the oxygen level. Oxygen decreased for 10 to 16 hours and then started to increase. When dissolved oxygen started to grow, the feeding was started. Feeding was calibrated so that an oxygen concentration of 30% was maintained during the whole fermentation. After 72 hours feeding was stopped.

3.5B.3 Monitoring of the expression of the FP5 and of total secreted proteins Total secreted proteins, and proteins from semi-purified and purified samples, were monitored according to standard methods (Bradford). FP5 production was monitored by qualitative SDS- 61

PAGE analysis performed according to standard methods. An HPLC method was also used for quantitative FP5 complex estimation (see below, Appendix 1). The FP5 and GNA proteins appear as two distinct bands in SDS-PAGE or two peaks in HPLC, and are herein collectively referred as FP5 complex.

3.5B.4 Quantification of the insoluble and soluble FP5 complex fractions

Our previous studies (report #01-2011) demonstrated that insoluble FP5 complex is not active in in-vivo studies. In order to optimize the preparation of the FP5 active complex, quantification of the insoluble protein was performed. The FP5 complex was either solubilized (when insoluble) or maintained into solution with 100 mM Tris-HCl buffer at pH 8.5. Quantification of the insoluble protein (inactive) was performed prior to the addition of the buffer as follows. Samples from the fermentation were collected and centrifuged at 6000 x g. The supernatant was recovered and further centrifuged at 16200 x g. The pellet, containing the insoluble FP5 was suspended in 100 mM Tris-HCl buffer pH 8.5 and analyzed as detailed below.

3.5B.5 Development of an HPLC method for the quantification of the FP5 complex

The HPLC method for the identification and quantification of the GNA component of the FP5 complex was developed, and is described in detail in Appendix 1.

3.5B.6 Purification of the FP5 complex with Ni conjugated resins Purification of the FP5 complex on Ni-conjugated resins was performed, as described before (report #01-2011), on the supernatant of the fermentation. The supernatant was kept at 4 °C for up to 48 hours, or stored at –80°C for longer. Purification was carried out at room temperature. Samples were prepared, before loading onto column, with the addition of Tris-HCl pH 8.5 100 mM and the correction of pH to 8.5 with NaOH. Tris-HCl pH 8.5 was used as the binding, washing and elution buffer, at the final concentration of 100 mM. 250 ml of resins were loaded at a flow rate of 0.2 bed volumes per minute. After loading, the column was washed with 2-3 bed volumes of Tris- HCl pH 8.5 100 mM and the protein was eluted from the column with 1 bed volume of Tris-HCl pH 8.5 100 mM, added with 0.3 M imidazole. The eluted samples were immediately diluted 1:1 with water (MilliQ quality), and dialysed against Tris-HCl pH 8.5 100 mM. Dialysed proteins were freeze-dried and stored at 4°C until use for in-vivo studies.

3.5C Results and Discussion

3.5C.1 Bioreactor fermentation

Production was scaled to the 200 liter bioreactors according to the conditions verified at the 30 liter scale and described in the section Materials and Methods and in report # 01-2011. Growth and fermentation details are reported in Figure 5.3.1.

Analysis of FP5 complex expression and estimation of total secreted proteins was determined as described in the section Materials and Methods.

Table 5.5. Initial fermentation parameters

Fermentation parameters Notes

Controlled at 30% saturation Dissolved oxygen 4.6 control with NH4OH 28% pH Agitation 200 rpm Variable with oxygen concentration Aeration 1.0 vvm Variable Antifoam the minimum needed to eliminate foam Carbon source (feeding) 0 Detailed in Table 5.4

Figure 5.1. Fermentation of P. pastoris GnaHIS #3 in 200 liter tank.

3.5C.2 Downstream processing Separation of the supernatant from cells and recovery of the precipitated protein complex by centrifugation. Cells were separated from the supernatant through centrifugation with a Sorvall RC12BP centrifuge operated at 3220 x g for 20 minutes. At that speed, the supernatant appeared cloudy (as already reported in report #01-2011), and was further centrifuged in a Beckman J2-21 centrifuge equipped with a JA10 rotor, operated at 14300 x g, for 60 minutes. The supernatant resulted then clarified. For the analysis of the precipitated FP5 protein, the pellet from the last centrifugation was suspended in Tris-HCl 100 mM pH 8.5 and analyzed. The precipitated protein was below 10 mg/L of the original fermentation broth (not shown). Quantitative data concerning centrifugation are reported in Table 6. After centrifugation, the supernatant was added with Tris-HCl pH8.5 at the final concentration of 100 mM. The pH was further corrected at 8.5 with NaOH. The resulting solution was directly loaded onto Ni-conjugated resins.

Table 5.6. Downstream data

Weight (Kg) Notes Weight at inoculum (Kg) 180 Feeding (Kg) 54 In ca 70 hours Weight at harvest (Kg) 230 Centrifugation (Sorvall RC12BP) Supernatant (kg) 150 3220 x g, 20 minutes Centrifugation (Beckman J2-21, JA10 rotor, Supernatant (kg) 148 14300 x g, 9000 rpm) 14300 x g, 60 minutes Supernatant (kg) 148 Loading on 250 ml of Ni-conjugated resins

3.5C.3 Purification of the FP5 protein complex on Ni-conjugated resins. 148 liters of the supernatant obtained as described above, were applied to 250 ml of Ni- conjugated resins (Merck), as described in the section Materials and Methods. The resin was then washed with 3 volumes of 100 mM Tris-HCl buffer at pH 8.5 and eluted with 1 volume of imidazole 300 mM in 100 mM Tris-HCl buffer at pH 8.5. Finally, the fractions containing the FP5 were dialyzed against 100 mM Tris-HCl buffer at pH 8.5 for 24-36 hours and lyophylized. Approximately 5.4 grams of material (Sample B) were produced after purification. 10 mg of material, were dissolved in water (MilliQ quality) and analyzed by SDS-PAGE and HPLC (Figures 5.2 and 5.3). The protein content of the purified sample was estimated with standard methods and is reported in Figure 5.2 (Sample B).

In order to investigate if the degree of aggregation had any influence on the in-vivo activity, the small amount of FP5 which permeated through a 0.1 mm membrane was purified as described above. A sample of ca. 300 mg of crude was produced with a content of FP5 complex of 21% of total protein (Figure 2, Sample A). Total proteins were, in both cases, mainly attributed to the FP5 complex.

Samples A and B were delivered to Isagro Ricerca for in-vivo testing. Results of activity against L. decemlineata are reported in Tables 5.7 and 5.8.

Sample Sample Protein content Microliters loaded Micrograms of concentration (%) on gel total protein (mg/ml) loaded on gel 1 10 21.0 20 42 2 10 21.0 15 31.5 Sample 3 10 21.0 10 21 A 4 10 21.0 5 10.5 5 10 21.0 2 4.2

6 10 49.5 20 99

7 10 49.5 15 74.3 Sample 8 10 49.5 10 49.5 B 9 10 49.5 5 24.8

10 10 49.5 2 9.9

Figure 5.2. SDS-PAGE (18% acrylamide gel) of Ni-purified FP5complex. Protein concentrations are indicated in table. FP5 and GNA bands are indicated by arrows. See text for further details.

Figure 5.3. HPLC analysis of the fermentation supernatant at harvest (A) and of the purified FP5 complex (B). Sample B of Figure 5.2 is reported as an example of the purified samples. Putative FP5 and GNA peaks are indicated by arrows. See text for further details.

I 14_11Actygea FP5Novodor- sp. Leptinotarsa decemlineata DIRECT ACTION (Plant) 3 days old larvae Le treatment 29/06/11

PROD. APPL. RATE ppm LARVAE 5 DAT averag % LARVAE 7 DAT averag % e eaten e eaten leaf. leaf. TOT ALIVE DEAD % MORT ALIVE DEAD % MORT.

UNTREATED 1a 10 10 0 0 0 100

1b 10 10 0 0 100

NOVODOR 2 l/ha 2a 10 3 7 70 75 12 1 9 90 95 15

2b 10 2 8 80 10 0 10 100 10

FP5 sample A 350 3a 10 10 0 0 0 85 9 1 10 5 100

3b 10 10 0 0 80 10 0 0 100

FP5 sample B 350 4a 10 10 0 0 0 65 10 0 0 0 100

4b 10 10 0 0 75 10 0 0 100

Plants were sprayed with 10 ml of FP - Novodor water solution. Sample A (microfiltered), product at 20%: 17,5 mg in 10 ml of distilled water; Sample B, product at 49% (sample for DeSangosse): 7,2 mg in 10 ml. Novodor: 1 ml in 500 ml of distilled water. The plants present corky leaves. FP5 sample A: microfiltered product, 21% of protein inside; FP5 sample B: product for DeSangosse, 49% of protein inside.

Table 5.7. In-vivo activity of the FP5 complex produced through 200 liter fermentation. No mortality was observed taking as reference the protein content of the FP5 complex samples. Samples A and B are described in Figure 5.2.

Table 5.8. In-vivo activity of the FP5 complex produced through 200 liter fermentation. Positive results were obtained by increasing the amount of FP5 complex ca 2.5 fold in respect to the amount applied as in Table 5.7. Sample B is described in Figure 5.2.

I 15_11 ActygeaFP5Novodor-Le sp. Leptinotarsa DIRECT ACTION (Plant) 3 days old larvae treatment 29/06/11 decemlineata

PROD. APPL. RATE LARVAE 5 DAT avera % LARVAE 6 DAT averag % LARVAE 7 DAT avera % ppm ge eaten e eaten ge eaten leaf. leaf. leaf. TOT ALIVE DEAD % MORT ALIVE DEAD % MORT ALIVE DEAD % MORT.

UNTREATED 1a 10 10 0 0 0 100

1b 10 10 0 0 100

NOVODOR 2 l/ha 2a 10 3 7 70 70 10 0 10 100 85 10 95

2b 10 3 7 70 10 3 7 70 12 1 9 90 12

FP5 sample B 350 3a 10 6 4 40 45 20 2 8 80 80 22 1 9 90 90 22

3b 10 5 5 50 35 2 8 80 37 1 9 90 38

Plants were sprayed with 10 ml of FP - Novodor water solution. Sample B, product at 20%: 17,5 mg in 10 ml. Novodor: 1 ml in 500 ml of distilled water.

3.5D Conclusion

The FP5 complex was successfully produced in the 200 liter industrial pilot fermentation at 50 to 70 mg/L of fermentation broth. In excees of 3 grams of fusion protein were supplied to De Sangosse. Precipitated proteins represented below 10 mg/L of the total, and soluble protein (which was in the range 50-60 mg/L). The soluble fraction of the FP5 complex was maintained in solution during the whole purification process with Tris-HCl 100 mM and a pH of 8.5. The final recovery of soluble protein was ca 12.5 mg/L of fermentation broth. The purified protein was used in in-vivo assays and was active against L. decemlineata. Although the whole process was easily scaled to 200 liters volume, the resulting protein complex was not as effective as expected in in-vivo assays. Further work is required to determine if the activity of the protein could be increased and better reproduced.

An HPLC method was developed and was used during the whole production process to monitor protein presence and levels. Although, the HPLC method quantitatively only monitors the Gna component of the FP5 complex, it resulted useful since the two proteins show a fairly constant ratio during fermentation and purification (as evidenced before in SDS-PAGE experiments). The purification of the different components of the FP5 complex by preparative HPLC are in progress.

Additional comment (Fera/Durham University) Studies carried out during a complementary LINK programme to develop insecticidal rather than molluscicidal fusion proteins demonstrated that expression levels of fusion proteins can be improved (>10 fold) through the insertion of multiple copies of fusion protein cassettes into the yeast genome.

5.3E Appendix

5.3E.1 FP5 HPLC Method – reverse phase column (C4) SUMMARY The initial method for FP5 analysis was based on reversed phase Vydac C4 column, eluted with a TFA/acetonitrile gradient. During purification of the FP5 protein, data showed that the protein was better dissolved in slightly alkaline solutions. Among alkaline solutions, the best was Tris-HCl 100 mM at pH 8.5. Therefore, we developed an HPLC method using as buffer for the mobile phase Tris-HCl. The method developed was suitable for the identification and the quantification of the Gna component of the FP5. FP5 itself gave a shoulder in HPLC rendering quantification impossible (as already observed for the FP4 protein). Since the ratio between Gna and FP5 itself appears constant in different protein preparations (as observed in SDS-PAGE), we consider the following method good for the in-process quantification of the FP5 complex.

EQUIPMENT High performance liquid chromatograph (HPLC) capable of performing binary gradient elution, equipped with a sample injector, a variable wavelength UV detector and a data processing system with integration capabilities. Volumetric flasks. Analytical balance, accurate to +/- 0.00001 g. Analytical column Vydac Protein C4, 10 µm, 4.6 x 250 mm, or equivalent.

REAGENTS Acetonitrile, HPLC grade. Water, HPLC grade. Tris (AR) grade. HCl 37% (AR) grade. FP5 standard protein

SAFETY PRECAUTIONS Acetonitrile is flammable and toxic. Pour in fume hood. HCl is corrosive. Wear gloves when pouring.

STANDARD SOLUTION PREPARATION Prepare standard solution as follows: a) Accurately weigh about 50 mg of crude FP5 powder into a 5 mL volumetric flask b) Dissolve and dilute to volume with mobile phase A (see Paragraph 7) and mix well to obtain the standard solution (about 2.2 mg/mL as powder concentration).

SAMPLE PREPARATION Prepare sample as follows: a) Centrifuge 2 mL of fermentation broths at 6000 x g for 10 minutes b) pour supernatant into a clean 2 ml Eppendorf tube, and centrifuge at 16200 x g for 10 minutes. c) Recover supernatant and proceed to HPLC analysis (soluble FP5 complex) d) dissolve the pellet of step b) in 0.2 mL of Tris-HCl 100 mM pH8.5, filter clear supernatant through 0.22 µm filter, and proceed to HPLC analysis (insoluble FP5 complex)

CHROMATOGRAPHY Mobile Phase A: 50 mM Tris-HCl pH 8.5 Mobile Phase B: 50 mM Tris-HCl pH 8.5 in 80 % acetonitrile Gradient Elution Program (FP4TRS13):

Time % mobile phase (min) A B 0 88 12

15 88 12 25 30 70 50 0 100 60 0 100

61 100 0

Flow Rate: 0.5 mL/min Injection Volume: 20 mL

Column Temperature: 20°C Detection: UV detection, 280 nm Run Time: 60 min

Sample chromatogram of a FP5 standard sample is reported in Figure A.1. A response plot of the FP5 protein standard is reported in Figure A2. A typical chromatogram of the fermentation broth processed as described in paragraph 6 is reported in Figure A.3.

Based on previous observations on the FP4 protein (not reported), we tentatively assigned to the peaks the names GNA and FP5. The assignment has to be verified through purification of the peaks evidenced in the chromatogram (by preparative HPLC), and subsequently by analysing through SDS-PAGE.

Figure A.1. HPLC chromatogram of a Ni-purified FP5 sample (Sample B of figure 2 of this report). The purified FP5 complex (ca 10 mg total protein content) was dissolved in 1 mL of Tris-HCl 50 mM at pH 8.5. GNA and FP5 components of the complex are putatively indicated

Figure A2. Response plot of the GNA component of the FP5 complex. The absorbance of the protein in HPLC analysis (mAU, Peak area) is plotted against the protein concentration in mg/mL. The response is linear up to ca 5 mg/mL concentration. The limit of detection was approximately 0.1 mg/mL. Increased sensitivity could be achieved by use 220

7000 6000

5000 y = 559.28x - 145.01 4000

3000

2000

area) Peak (Gna mAU 1000

-1000 0 1 2 3 4 5 6 7 8 9 FP5 complex (mg/ml)

Figure A3. Typical HPLC chromatogram of the P. pastoris fermentation supernatant (concentrated 16.2 fold by ultrafiltration) containing the FP5 complex. The Gna component of the complex is putatively indicated.

Section 3.6: Efficacy evaluation by industry Objective V: Development of optimal bait formulations

Confidential report: prepared by H. Caruel (De Sangosse) 3.6A INTRODUCTION

DE SANGOSSE is involved in this project at the final stage: Objective V, work package 8. The aim of this WP8 was to evaluate the molluscicide potential of pellets formulated with fusion protein against grey slugs (Deroceras reticulatum), to develop an optimised bait formulation assessing palatability, efficacy and persistence of action. These actions were planned to start at the delivery of fusion protein in quantity and quality allowing the formulation development.

To manage these actions DE SANGOSSE requested fusion protein with a high level of purity, to be able to validate the molluscicide action of the active and a sample close to 50g to be able to produce baits pellets.

Comment by Fera/Durham University Expression levels of the omega/GNA fusion protein at the time of clone transfer to Isagro Ricerca were approx. 50 mg/litre of culture supernatant. Whilst the consortium recognised that large (gram) quantities of fusion protein would enable extensive trials against D. reticulatum to be carried out this was not possible during the term of the project. Work to develop an optimal production and downstream process for omega/GNA was conducted by Isagro Ricerca (see section 5) and in excess of 3 grams of fusion protein were supplied to De Sangosse for efficacy trials. In fact Isagro committed considerable extra work and incurred extra “in kind’ costs during this project but further development is necessary before the production of 50 gram quantities (50g) can be achieved. De Sangosse were advised throughout the project that 50 g was not an achievable target.

The Fusion proteins (FP) were tested à 3 different levels: - Forced oral route, - Bait toxicity in lab, - Bait toxicity in external conditions.

3.6B Materials and Methods

DE SANGOSSE received the following samples of FP:

Purity Production Reception date Samples Quantity (estimated)% scale FP5+HIS ABC – 06/10 545+465+320=1330mg 10 Lab 06 Oct. 2010 FP5+HIS ABCD – 08/10 296+95+347+335=1073mg 20 Lab 06 Oct. 2010 FP5 ISAGRO sample B 2.6g 49 Pilot 08 Aug 2011 FP5 ISAGRO sample D 3.4g 21 Pilot 08 Aug 2011

These samples were delivered in very few quantities according the expected ones and their purity levels were also very low. Resulting to these observations, it was difficult for DE SANGOSSE to evaluate the mollusicicide effect of fusion protein alone. The preparations were produced with the received fusion proteins according to the estimated purity and so the molluscicide effect was evaluated with the impurities.

Comment from Fera/University of Durham Samples supplied by Isagro were >95% pure with respect to the presence of any contaminating proteins (see section 5, Figure 5.2), the remaining impurities are carbohydrates derived from yeast fermentation and have been shown to have no toxicity to invertebrates.

The different types of tests were performed in the following ways:

- Forced oral route: A gel containing fusion protein and a placebo gel without fusion protein are prepared.

For each test, 40 slugs (BW ~800mg) were anaesthetized with CO2 and then, 10µL of gel was injected in their mouth. 10 slugs received gel with fusion protein, 10 received placebo gel (blank), 10 received a reference toxic gel and 10 were only anaesthetized. They were put in humid boxes (one box per modality) with wheat flour and observed for 4 days in a climate chamber: - the dead ones counted, - Do they start feeding again?

The test is validated if the dead slugs do not exceed 2/10 in the blank and in the only anaesthetized group and if the dead slugs in the reference group are at least of 8/10.

- Bait toxicity in lab: Pellets containing fusion protein and placebo pellets without fusion protein have to be prepared by mixing wheat flour, fusion protein (or not) and water. The mix is then extruded in the way to obtain wet pellets which are dried at RT.

For each test, 75 slugs (300 mg ≤ BW ≤ 600 mg) were put in individual humid box with one pellet. 25 slugs with placebo pellet, 25 slugs with pellets containing fusion protein and 25 slugs with reference pellets. The boxes were kept in a climate chamber for a week. The slugs were observed during 8 days: - the dead ones counted, - the pellets uptake evaluated.

The test is validated if the dead slugs with placebo pellets do not exceed 20% and if the dead slugs of the group with reference are at least 70%.

- Bait toxicity in external conditions: The trial is performed using 12 cages of 1 m² each set externally on the grass around the DE SANGOSSE laboratory. A layer of soil is set at the bottom of each cage and a tile, used as shelter, is installed on the ground of each cage. Seedling lettuces are set in the cages and pellets are spread manually. 25 slugs were set per cage (25slugs / m²) Each modality is four times repeated: - Modality 1: without treatment (to validate the slugs’ activity) - Modality 2: 35 pellets/m². - Modality 3: 35 pellets of reference/m². The dead slugs are counted at 3 and 7 DAT. The test is validated if the activity of the slugs is enough during the test (consumption of at least the half of the seedling lettuces) and if the dead slugs do not exceed 20% in modality 1 and if the percentage of dead slugs of modality 3 is at least of 60%.

3.6C Results - Forced oral route tests 3 tests were performed. For the 2 first ones, the FP5+HIS ABC 06/10 and 08/10 were mixed to obtain a mix of FP5+HIS powder at 13.9% of purity in FP5+HIS. A gel was prepared at 4.45% of FP5+HIS. It was injected in the mouth of 10 slugs. The ingested quantities were of 0.5 mg of FP5+HIS/ g BW for the first test and of 0.6. mg of FP5+HIS/ g BW (according to the mean weigh of the used slugs) for the second test.

Test 1 Eat again Eat again Test Gels Death 1DAT Death 4 DAT 1 DAT 4 DAT validation Placebo 0 No 1 Yes Yes FP5+HIS 0 No 7 No / Reference 2 No 8 No Yes

Anaesthesia 0 No 2 Yes Yes

The gel used in test 1 and in test 2 was the same preparation. It was used for test 1 and kept in the fridge for two weeks before being used in test 2

Test 2 Gels Death 1DAT Eat again 1 Death 4 DAT Eat again 4 Test DAT DAT validation Placebo 0 No 2 Yes Yes FP5+HIS 0 No 2 No / Reference 2 No 8 No Yes Anaesthesia 0 No 1 Yes Yes

The results on test 1 were very encouraging. The test was validated and we observed a good level of efficacy with a cumulative of 7 dead slugs 4 DAT. Unfortunately, it was not repeatable. In the second test we observed only 2 cumulative dead slugs. This was the same level of mortality as in the placebo group. The only thing we can say is that the slugs didn’t eat again after gel feeding even if they had access to flour.

At that time, it was supposed that the FP5+HIS could be instable and it degraded in the gel during storage for 2 weeks in the fridge. A part of the remaining sample was sent to the FERA team to be analysed in the way to define if it was or not degraded. The analysis result shown the presence of FP5+HIS in the gel but it was not possible to define the FP5+HIS content. So, we do not know if the active degrade itself during storage and why the test results were not repeatable.

A third test was performed with a new gel prepared with a new FP5 sample supplied by ISAGRO. This sample was produced with the pilot scale plan. A new gel was prepared at 20% of FP5 ISAGRO sample B (49% of purity) so at 10% of FP5. For this test, the ingested quantities (mg FP5/ g BW) by the slugs were two times the ones ingested in the tests 1 and 2.

Test 3 (performed according to the reception date of samples). Gels Death 1DAT Eat again 1 Death 4 DAT Eat again 4 Test DAT DAT validation Placebo 0 No 0 Yes Yes FP5 ISAGRO 0 No 0 Yes / Reference 2 No 2+5 No Yes Anaesthesia 0 No 1 Yes Yes

The test was also validated, even if the reference shown only 2 dead slugs 4 DAT because 5 others were moribund No dead slugs were observed with the FP5 ISAGRO, even if the dose was 2 times (1.25 mg of FP5 ISAGRO sample D/g BW) than in the first tests (0.6 mg FP+HIS/g BW). We also observed that the slugs eat the flour again 4 DAT.

Bait toxicity in lab: A very few quantity of pellets was produced at the lab scale with the FP5+HIS sample D 08/10 in order to see if the active should support the manufacturing process. We also wanted to know if the slugs will eat or not a bait with FP5+HIS (even if the content was very low). According to the FP5+HIS availability at that time, the pellets were produced with 0.01% of FP5+HIS. The flour, the FP5+HIS D 08/10 and water were mixed and the extruded and dried at RT. One test was performed with these pellets. The results were as follows:

Pellets Death 8 DAT Test validation Placebo 5/25 Yes FP5+HIS D 08/10 (0.01% w/w) 4/25 / Reference 21/25 Yes

The test was validated. We observed that all the pellets were consumed and so, at 0.01% the FP+HIS is not a problem for the palatability of pellets. 4 dead slugs were observed 8 DAT. This was less than in blank reference, so it is not possible to say that there is some efficacy.

Some pellets were sent to the DEFRA team for analyse. There again, it was possible to identify the presence of the active ingredient, but not to define the quantity in the pellets. The assay of FP5 was made difficult due to presence of the wheat proteins. So we can say that it stays some FP5 after the process, but we do not know if there is any degradation during the production.

Another test was performed with pellets obtained with the FP5 ISAGRO sample B. Due to the low quantity of active ingredient; it was not possible to extrude the dough. It was hand flattened, cut and dried at RT. The pellets were prepared with 5% (w/w) of FP5 ISAGRO sample B. The test results were as follow:

Pellets Death 8 DAT Test validation Placebo 0/25 Yes FP5 ISAGRO B (5% w/w) 0/25 / Reference 16/25 Yes

The test was validated. All the slugs consumed the bait. The mean consumption was of 2 mg of FP ISAGRO sample B /g BW. This was more than with the forced oral route, but no mortality was observed.

As we didn’t observe any efficacy with the fusion protein we received with the forced oral route tests and with bait toxicity in lab tests, we decided to perform one test on bait toxicity in external conditions in order to validate the lack of efficacy.

Bait toxicity in external conditions: The 5% FP5 ISAGRO B pellets were used for this test at the rate of 35 pellets/m². The results are as follow:

Pellets Death 8 DAT Test validation Placebo 4/25 Yes FP5 ISAGRO B (5% w/w) 2/25 / Reference 22/25 Yes

The test was validated. No different mortality was observed comparing to placebo. So once again, it was not possible to observe any efficacy of the baits produced with the fusion protein.

3.6D Conclusions

3 different level of test were performed by DE SANGOSSE in the way observe the efficacy of the new environmentally-friendly specific molluscicidal toxins formulated in bait pellet to fight against slugs by ingestion. 6 tests in controlled conditions were performed: 3 forced oral route tests, 3 tests on bait toxicity in lab.

1 test in semi controlled conditions (bait toxicity in external conditions).

In any case we observed mortality different than the blank, except for the first test on forced oral route, but it was not repeatable.

In conclusion, the new active ingredient we received, when formulated in a gel or in pellets, is not active against slugs in lab or with external conditions.

To be able to evaluate a new active ingredient, we should use a product with a high level of purity. Today we do not know exactly what was evaluated. In the best case the purity was close to 50%. What are the other 50%? Is it possible to improve this purity on the pilot scale production?

Comment from Fera/University of Durham Please see previous comments; the level of purity with respect to protein was >95% and the remaining contaminating material is non-toxic carbohydrate. It was encouraging to see mortality in the first alginate test. The results from the second oral test were disappointing but it may be that the fusion protein had precipitated out of the gel during 3 week storage at 4 C. We have observed precipitation of recombinant proteins when stored in water at 4 C for more than 7 days. Nevertheless the cessation of feeding following ingestion of the gel in the second assay suggest that at least a proportion of the fusion protein had remained in soluble, active form.

Results from assays using protein supplied by Isagro Ricerca were disappointing. The activity of the supplied protein was verified by Isagro (see section 5). Fera/Durham were able to re-extract protein from the pellets and show functionality by injection assay. However, it was impossible to determine the level of active protein in the pellets due to high losses of protein during the re- extraction process. The results obtained by De Sangosse suggest that the levels of biologically active protein in the pellets were insufficient to exert any activity against slugs. Further work is required to evaluate if the fusion proteins retain full biological activity following incorporation into baits using a commercial process.

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