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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code PS2103

2. Project title

Manipulation of insect immune defences to optimize biological control

3. Contractororganisation(s)

Central Science LaboratorySand HuttonYorkYO41 1LZ          

54. Total Defra project costs £ 450,695.40

5. Project: start date................ 01 April 2003

end date................. 31 March 2006

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

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Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.The overall objective of the work is to devise improved and/or new crop protection strategies as effective alternatives to the use of broad-spectrum, neurotoxic pesticides. We aim to do this by selectively suppressing the key immune responses of target pest insects that ordinarily protect them from potential pathogens in their environment, and those used in bio-control strategies.

Briefly, the main objectives of the current phase of the project (as set out in the CSG7) were to continue our characterization of immunosuppressive factors present in venom from the endoparasitoid wasp, Pimpla hypochondriaca, particularly with regard to the presence and activity of proteases and/or peptidases, and to determine the range of activity of anti-haemocyte factors. We also sought to isolate a single immunosuppressive factor using biochemical techniques, and to confirm activity of the factor using in vitro and in vivo (concept-proving) assays. We then aimed to determine some N-terminal sequence for the protein, to perform a data base homology search to look for identity to previously published proteins (in order to get some idea of possible function), and finally to use this information to clone the gene for the factor from a venom gland cDNA library.

P. hypochondriaca venom has potent anti-haemocyte activity against haemocytes from the tomato moth, Lacanobia oleracea. We have also determined that it damages haemocytes from a number of other insects, including Spodoptera littoralis, S. exigua, Mamestra brassicae, etc, and against a human testis and bladder cancer cell line. These results (and others) raised the possibility that the anti-haemocyte activity may be due to a fairly non-specific molecule, such as a protease. Thus, the protease content of the venom was investigated. Using a colourimetric API ZYM kit and other assays, we determined that P. hypochondriaca venom contains 19 hydrolases. Since the greatest activity was determined to be due to acid phosphatase, we used specific inhibitors of this enzyme to investigate if it contributes to the anti-haemocyte properties of the venom. The results of a large number of experiments indicated that it did not. Furthermore, inhibition of venom protease activity using a protease cocktail inhibitor also did not reduce the As a first step in generating the ‘tools’ we need for practical application of our work, we sought to isolate and characterize at least one venom anti-haemocyte / immunosuppressive factor. This was done using a variety of biochemical techniques, including gel filtration, ion exchange, SDS polyacrylamide gel electrophoresis, etc. The isolated protein was then blotted over to nitrocellulose, and its N-terminal sequence determined. Seven N-terminal amino acids were identified, and these were submitted to a number of protein sequence sites in order to make a data base (BLAST) homology search. The results of this indicated that our sequence occurs in a wide variety of proteins, including one protein present in P. hypochondriaca venom. Using a PCR approach, we then cloned the gene for the anti-haemocyte factor, and placed it into vectors suitable for DNA sequencing (to confirm integrity of the gene) and for subsequent protein expression in bacteria (i.e. to manufacture recombinant protein). Using concept-proving assays, we

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then determined that whole venom, and the isolated anti-haemocyte factor, are both capable of increasing the bio-pesticidal properties the fungus, Beauvaria bassiana, a commercially available bio-control agent. This work represents the first time that a gene for a protein with proven anti-haemocyte and immunosuppressive properties (and potential to be used in bio-control strategies) has been cloned from the venom gland cDNA library of an endoparasitic wasp. In the next phase of the work, we plan to identify and clone the genes for more anti-haemocyte/immunosuppressive venom factors, and to use these to produce relatively large amounts of recombinant protein. These ‘tools’ will enable us to drive the work towards practical application, and to help us forge links with commercial companies and organisations.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Scientific Objectives.The scientific objectives as set out in the contract are as follows:

1) To extend the characterization of factors present in P. hypochondriaca venom which manipulate (suppress) immune defences in our model pest insect, L. oleracea. In particular, determine if proteases and/or peptidases are present in venom and if they have any effect on the potency of its anti-haemocyte/immunosuppressive activity. In addition, determine the ‘range’ of the venom anti-haemocyte activity by testing against haemocytes from other insect species, and mammalian cells.

2) To isolate a factor or factors with proven anti-haemocyte activity from the venom and confirm purity using selected biochemical techniques (this involves dissecting the venom glands from as many adult female P. hypochondriaca as possible in order to build up a pool of venom for use in subsequent isolation procedures). In addition, make a preliminary physicochemical characterization of the molecule(s) (particularly with regard to stability), determine N-terminal sequence, and perform a data base homology search. Information gained from this work will help in the design of experiments aimed at elucidating the mechanism of action of the active factor(s) as potential bio-control agents.

3) Using an isolated anti-haemocyte factor with proven immunosuppressive properties, to perform bio-assays to test the concept that targeted suppression of key immune responses can make an insect more susceptible to a suitable bio-control agent (for example, the fungus Beauveria bassiana).

4) To clone the gene(s) of isolated anti-haemocyte factor(s) and determine their DNA sequence(s). This data, and that obtained on the isolated protein(s), are necessary precursors to establishing LINK projects with suitable companies so that the research progresses towards practical uptake.

Note. All of the scientific objectives have been completed in full.

Scientific Objective 1. To extend the characterization of factors present in P. hypochondriaca venom which manipulate (suppress) immune defences in our model pest insect, L. oleracea. In particular, determine if proteases and/or peptidases are present in venom and if they have any effect on the potency of its anti-haemocyte/immunosuppressive activity. In

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addition, determine the ‘range’ of the venom anti-haemocyte activity by testing against haemocytes from other insect species, and mammalian cells.

Introduction.Our work on parasitoid-mediated suppression of insect immunity has demonstrated that venom from adult female Pimpla hypochondriaca (a solitary, pupal endoparasitoid which parasitizes many different species of insect) represents a rich source of immunosuppressive factors. For instance, P. hypochondriaca venom exerts potent, dose-dependent, adverse effects on haemocytes from pupae and larvae of the host species, L. oleracea (our model pest insect) (Richards and Parkinson, 2000; Parkinson et al., 2002a). At relatively low venom doses (approximately 1.6 ng/l), the formation of pseudopods by certain L. oleracea haemocytes (mainly plasmatocytes) is inhibited. As a result, haemocyte spreading and movement are reduced with the result that fewer haemocyte aggregates are formed in vitro (Richards and Parkinson, 2000). At higher doses (approximately 500 ng/l), the plasma membrane of some haemocytes disintegrates 32. A direct consequence of these venom-mediated effects on such immunocompetent haemocytes is that they are rendered less able, or totally unable, to execute certain key immune responses. For example, their ability to phagocytose bacteria in vitro and to encapsulate Sephadex beads in vivo is significantly suppressed Richards and Parkinson, 2000; Parkinson et al., 2002a).

In the literature, studies with insect haemocytes and plasma indicate that proteinases and proteinase inhibitors may influence haemocyte behaviour, and cellular and humoral immune responses (Kanost, 1999). Furthermore, results obtained from random sequencing of a cDNA library made from P. hypochondriaca venom gland tissue indicated the presence of cDNAs encoding an arthropod-specific phenoloxidase, a serine protease, and a reprolysin-type protease (Parkinson et al., 2001; Parkinson et al., 2002b). Furthermore, a form of insect ACE has also been detected in the venom (Dani et al., 2003). Thus, in accordance with our desire to isolate specific venom immunosuppressive factors, such proteins represent prime candidate molecules. In view of this, one of the main aims of objective 1 was to determine the range of proteases and/or peptidases present in P. hypochondriaca venom, and then determine if they have any effect on the potency of the venom’s anti-haemocyte and immunosuppressive properties.

Methods.Venom toxicity assaysHaemocytes were prepared from a number of different insects and the anti-haemocyte activity of venom assessed as described previously (Richards and Parkinson, 2000). Similar assays were used to test for activity against a human testis and bladder cell line.

Enzyme Assays.Hydrolase activity in P. hypochondriaca venom was investigated using the colourimetric API ZYM kit (Bio-Mérieux UK limited, Basingstoke, Hampshire). The kit was used according to the manufacturer’s instructions. In brief, venom diluted in sterile distilled water (SDW) was added to each well of the test strip (40 to 62 g venom protein/well, or 0.22 to 0.34 venom sac equivalent (vse) per well). The test strip was then placed in a humid chamber and incubated at 37 oC for 4 h. The controls consisted of heat-treated venom (70 oC for 30 min, followed by centrifugation at 10 000 g for 30 min), and SDW. Following incubation, two kit reagents were added to each well and the colour reaction was monitored. Each well was scored from 0 to 5; 0 representing no colour change and 5 representing a strong colour reaction. Venom that had been pre-incubated for 20 min with a 1 in 75 dilution of cocktail inhibitor 2 (CI 2; a phosphatase inhibitor), was also applied to a test strip. The term “vse” is used to define the mean protein content from one venom sac (180 g protein/sac).

Acid phosphatase assayThe acid phosphatase assay utilised was based on the method of Andersch and Szcypinski (1947), modified by Xia et al. (2000), and presently adjusted for a 96-well microtitre plate format. For the standard assay, the following were added to each well; 10 L of venom diluted in SDW (0.55 g of venom protein which is equivalent to 0.0031 vse), 40 L of 0.1 M sodium acetate/acetic acid buffer pH 4.8 and 25 L of the synthetic substrate, 10 mM p-nitrophenyl phosphate (p-NPP) prepared in SDW. The effect of pH on venom acid phosphatase activity was determined using the following buffers: 0.1 M sodium acetate/acetic acid buffer (pH 3.8 to 5.6); 0.2 M Tris-maleate/NaOH buffer (pH 5.3 to 8.7); 0.1 M glycine-NaCl /NaOH buffer (pH 8.7 to 11.5). For inhibition studies the following were added to each well; 10 L of venom diluted in SDW and 10 L of phosphatase inhibitor CI 2, or 10 L of the acid phosphatase inhibitor NaF (Kneipp et al., 2003). The positive controls consisted of 10 L of diluted venom and 10 L of SDW. Forty L of 0.1 M sodium acetate/acetic acid buffer pH 4.8 was then added to each well. Venom and inhibitor were pre-incubated for 15 min in buffer before the addition of 25 L of 10 mM p-NPP. The reactions were performed in duplicate and incubated at 37 oC for 1 h with gentle shaking. The reaction was terminated by the addition of 150 L of 0.05 M NaOH. Blanks were also performed in duplicate and prepared concurrently. The blanks were prepared by adding 150 L of 0.05 M NaOH to diluted venom and buffer in the presence or absence of inhibitor, before the addition of substrate. The amount of p-nitrophenol released was measured using a microplate reader (Bio-Tek Instruments) at 405 nm. The absorbance reading of the blanks was used to determine the net absorbance, and the total and specific activities were calculated using a nitrophenol calibration curve produced using a standard solution of p-nitrophenol (Sigma-Aldrich). Under the above assay

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conditions, the reaction was linear with time. The inhibition of acid phosphatase activity was calculated using the formula (A-B)/A x 100 (where A was the absorbance in the absence of inhibitor, and B was the absorbance in the presence of inhibitor). The IC50 value for NaF was determined using the program FigP (Biosoft, Cambridge)

Haemocyte aggregation assayHaemocyte monolayers were prepared from sixth stadium L. oleracea larvae as described by Richards and Parkinson (2000), with the following modifications; 100 L of diluted venom or the phosphatase inhibitors NaF or CI 2, were added to the wells in the first column of a microtitre plate. Samples were then serially diluted 2-fold across the plate in 50 L of the insect tissue culture medium (TC-100 pH 6.6, with osmolarity adjusted to 307 nmols/kg and containing 100 g/mL ampicillin, 25 M PTC, and 0.005 % (w/v) methyl paraben, an antifungal agent). Then, 50 L of haemocyte suspension containing 3 x 106 cells/mL was added to each well. The final assay volume was 100 L/well. Control wells contained 50 L of the medium and 50 L of the haemocyte suspension. The plate was incubated overnight in a humid chamber at 20 oC and 65 % relative humidity. The medium was then carefully removed from each well and the attached haemocytes were stained with 0.125 % (w/v) Coomassie blue (50 L/well) for 45 min, and destained (40 % (v/v) methanol, 10 % (v/v) acetic acid) twice over a 1 to 2 h period. Finally, the monolayers were overlaid with sterile PBS. Under the above incubation conditions, for the cell controls, the haemocytes spread and aggregate into well defined clumps. The effect of serially titrated venom or inhibitors on haemocyte aggregation was observed and compared with the cell control using a Leitz inverted microscope at 40, 200 and 400 x magnification. In addition, the affect of CI 2 on the ability of venom to inhibit aggregation was investigated by serially titrating venom in a known dilution of CI 2 (a dilution determined not to affect the formation of haemocyte clumps in vitro, see above). Assays were also performed using the insect culture medium with the pH adjusted to 5.8 and 4.8 (the pH of TC-100 was adjusted using 0.5 M HCl).

Measurement of the pH of pupal plasmaOne to three day old L. oleracea pupae were pierced with a sterile 21G 1 ½ inch needle through the cuticle in the abdominal region of the pupa. The needle was introduced horizontally to the pupa, and when the needle was withdrawn the fluid that was released (haemolymph) was collected using a capillary tube. The haemolymph was then placed into an ice-chilled eppendorf tube and centrifuged at 10 000 g for 20 min at 4 oC. The supernatant (plasma) was maintained on ice. The pH of pupal plasma at room temperature was measured using a Sentron pH meter.

Protein concentrationThe protein concentration of samples was estimated using the BioRad DC Protein Assay based on the method of Lowry et al. (1951), and using bovine serum albumin as the standard.

Results.Range of venom toxicity.P. hypochondriaca venom has potent anti-haemocyte activity against haemocytes from the tomato moth, Lacanobia oleracea. We have determined that it also damages haemocytes (although to a lesser degree) from a number of other insects, including Spodoptera littoralis, S. exigua, Mamestra brassicae, etc, and a human testis and bladder cancer cell line. These results (and others) raised the possibility that the anti-haemocyte activity may be due to a fairly non-specific molecule, such as a protease. Thus, the protease content of the venom was investigated.

Detection of hydrolase activity in venomAs shown in Table 1, the API ZYM kit detects the presence of 19 hydrolases.

Table 1. Enzymatic activity detecteda for three batches of P. hypochondriaca venom (A, B and C) using the colourimetric API ZYM kit. Enzymes are listed in order of relative activity.

Venom batch and the amount of venom/ well Controls

Enzyme A

40 μg

B

62.3 μg

C

40 μg

heated C C 40 μg + CI

2 (1/75)

SDWb CI 2c

(1/75)

acid phosphatase 5d 5 5 0 0 0 0

-glucosidase 5 5 5 0 5 0 0

esterase 4 4 5 0 4 0 0

-galactosidase 4 4 3 0 3 0 0

esterase lipase 3 3 4 0 3 0 0

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lipase 2 2 3 0 2 0 0

1 naphthol-AS-B1-

phosphohydrolase 2 2 2 2 2 1 0

alkaline phosphatase 1 1 0 0 0 0 0

leucine arylamidase 1 1 1 0 1 0 0

cystine arylamidase 1 1 1 0 1 0 0

-chymotrypsin 1 0 0 0 0 0 0

controle 0 0 0 0 0 0 0

a: no enzyme activity was detected for; valine arylamidase, trypsin, -galactosidase, -glucuronidase, -glucosidase, -mannosidase, -fucosidase, N-acetyl- glucosaminidase.b: sterile distilled waterc: cocktail inhibitor 2d: The numbers represent the intensity of colour detected in each well. 0 = no colour change and 5 represents the strongest colour changee: Test strip control

For all samples tested, no colour change was visible for the test strip control. For the experimental control samples (SDW and heat-treated venom), a pale blue colour was observed for naphthol-AS-B1-phosphohydrolase only. A weak colour reaction was observed for alkaline phosphatase, leucine arylamidase and cystine arylamidase activity, indicating that these hydrolases might be present in venom. The following enzyme activities were consistently detected for all batches of venom tested: acid phosphatase, -glucosidase, esterase, -galactosidase, esterase lipase and lipase. The most rapid and the most intense colour reaction was observed with acid phosphatase. Only a slight variation in enzyme activity was observed between the different batches of venom tested. In the presence of a phosphatase inhibitor, CI 2, acid phosphatase activity in venom was not detected.

pH dependence of venom acid phosphatase activityThe optimum pH and the pH dependence of the P. hypochondriaca venom acid phosphatase activity were determined by performing the assay with a variety of buffers with different and overlapping pH ranges. Thus, we investigated the effects of both pH and buffer composition on the activity of the acid phosphatase. The composition of the buffers did not appear to affect acid phosphatase activity. Acid phosphatase activity in P. hypochondriaca venom was highest at pH 4.8 with only a small decrease in activity occurring at pH 4.1 (reduced to 92 %). However, under more acidic conditions (pH 3.8), the acid phosphatase activity was reduced to 49 %. Relatively high levels of activity were measured at pH 5.2 (76 %), with activity decreasing to 20 % at pH 6.1. Under alkaline conditions i.e. pH 7.6 to pH 11.5, for both buffers utilized (Tris-maleate and glycine), very little to no activity was measured at the end of the incubation period. The specific activity of acid phosphatase for a single batch of P. hypochondriaca venom was 0.467 ± 0.039 (SEM) nmol p-nitrophenol/min/g of venom protein.

Inhibition of venom acid phosphatase activityThe acid phosphatase activity present in P. hypochondriaca venom was inhibited by CI 2 and by NaF. The acid phosphatase activity in 0.55 g of venom protein (0.0031 vse), was inhibited 63 % by a 1/10 000 dilution of CI 2, and 60 % by 0.476 mM NaF which had an IC50 value of 4.2 x 10-4 M..

Presence of acid phosphatase activity in venom fractionsPreviously, P. hypochondriaca venom was fractionated by fast pressure liquid chromatography gel filtration using a Sephacryl S-100 column (Dani et al., 2003). Fractions 1 to 20 were presently tested for acid phosphatase activity. Fractions 5, 7, 8, 9 and 10 possessed weak acid phosphatase activity following a 210 min incubation period with substrate. Interestingly, fractions 7 to 12 had previously been reported to possess antihaemocytic activity (Dani et al., 2003).

The effect of acid phosphatase activity in venom on monolayers of L. oleracea haemocytesAt certain concentrations, P. hypochondriaca venom reduces the spreading behaviour of L. oleracea haemocytes and abolishes the ability of these cells to migrate and form aggregates in vitro (Parkinson et al., 2002a). Therefore, as relatively high levels of acid phosphatase activity were presently detected in P. hypochondriaca venom, monolayers of L. oleracea haemocytes were used to investigate the effect of venom on these cells in the presence of an acid phosphatase inhibitor. Since the optimum pH of the venom acid phosphatase activity was found to be 4.8, and the pH of the medium (TC-100) used to maintain haemocytes in vitro is 6.6, the effect of the medium on venom acid phosphatase activity was determined using the acid phosphatase assay. In addition, the level of activity in medium adjusted to pH 5.8 and 4.8 was also determined. The acid phosphatase activity

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measured in TC-100 was greatly reduced compared with the activity measured with sodium acetate buffer, pH 4.8 (TC-100 at pH 4.8 reduced activity to 32.7 %, TC-100 at pH 5.8 reduced activity to 11.6 %, and for TC-100 at pH 6.6 activity was reduced to 3.3 %). However, as there was residual acid phosphatase activity in TC-100 at pH 6.6, the effect of venom on haemocytes in vitro, in the presence of an acid phosphatase inhibitor was investigated. The lowest dilution of inhibitor that had no apparent effect on either the morphology or ability of haemocytes to form aggregates, was found to be 0.186 mM NaF and a dilution range of 1/400-1/800 of CI 2 (data not shown). Since we found that CI 2 could be used at a lower dilution than NaF without any adverse effects on the haemocytes in culture, we decided to use this inhibitor in subsequent assays. Therefore, P. hypochondriaca venom was serially titrated against a 1/600 dilution of CI 2 before the addition of L. oleracea haemocytes. Following an overnight incubation, there was no apparent difference between monolayers incubated with venom in the presence or absence of CI 2. Thus, the inhibitor did not reduce or abolish the anti-haemocytic properties of P. hypochondriaca venom. This result suggests that under the present assay conditions, acid phosphatase does not play a role in inhibiting haemocyte aggregation.

The pH of the tissue culture medium was adjusted from 6.6 to 5.8 and 4.8 and the suitability of the pH-adjusted media for the maintenance of haemocytes in vitro was tested. After an overnight incubation, the monolayers were assessed and it was found that the decrease in pH had a detrimental effect on the haemocytes. At a pH of 5.8, fewer cells were present in the monolayer compared with the control, and haemocyte aggregates were slightly smaller and less tightly packed (data not shown). At a pH of 4.8, very few haemocytes were attached to the surface of the well, little contact existed between these cells (i.e., due to the lack of extended pseudopods) and generally haemocytes appeared in a rounded configuration. Therefore, the pH-adjusted media were unfavourable for the maintenance of haemocytes in vitro and could not be used to evaluate if acid phosphatase, under more acidic conditions, is involved in the anti-haemocytic activity of P. hypochondriaca venom previously reported (Richards and Parkinson 2000; Parkinson et al., 2002a).

Discussion.P. hypochondriaca venom is capable of damaging/killing haemocytes from a range of insets. It also has activity, but to a much lesser degree, against a human testis and bladder cancer cell line. These results raised the possibility that, a non-specific factor, such as a protease or peptidase may be involved. To further investigate this, the presence of such molecules in P. hypochondriaca venom was examined. In the venom of P. hypochondriaca, we found evidence for the presence of several hydrolases using the colourimeteric API ZYM detection kit. We found activity (albeit weak) for an alkaline phosphatase, leucine and cystine arylamidase . Previously we have detected weak aminopeptidase activity against Leu-AMC using an assay that is more sensitive than the API ZYM detection kit (Dani et al., 2003). The following six hydrolases were consistently detected: acid phosphatase, β-glucosidase, esterase, β-galactosidase, esterase lipase and lipase. The activity of the enzymes disappeared when the venom was heated to 70 oC for 30 mins, indicating they are proteinaceous. The quickest and most intense colour reaction was recorded for acid phosphatase. In the presence of CI 2, an acid and alkaline phosphatase inhibitor, acid phosphatase activity was abolished, whilst activity of the five other major hydrolases was unaffected. This result clearly indicates that the enzyme activities detected were not due to a single enzyme with broad substrate specificity. Since acid phosphatase activity was the highest detected, further analysis of this venom enzyme was made. The acid phosphatase substrate presently used (p-NPP) is hydrolysed by acid and alkaline phosphatase. However, the acid phosphatase activity in P. hypochondriaca venom had a pH optimum of 4.8 with slight activity detected under alkaline conditions. This result suggests that the activity measured in P. hypochondriaca venom is due to an acid dependent phosphatase. In addition, the acid phosphatase inhibitor NaF inhibited the acid phosphatase activity. The specific acid phosphatase activity in P. hypochondriaca venom is much higher than the activity of proteolytic enzymes previously recorded for this venom (Dani et al., 2003).

Utilizing larval L. oleracea haemocyte monolayers, P. hypochondriaca venom exhibited antihaemocytic activity (Richards and Parkinson, 2000; Parkinson et al., 2002a). Presently, weak acid phosphatase activity was detected for venom fractions that had previously been shown to possess antihaemocytic activity measured by inhibition of haemocyte aggregation in vitro (Dani et al., 2003). The haemolymph of insects infected by fungi contain fungal acid phosphatase (Xia et al., 2000, 2001). It has also been proposed that the fungal acid phosphatases in the haemolymph of the host insect might play a role in hampering the host insect immune response by dephosphorylating immune proteins (Xia et al., 2000). Thus, the ability of P. hypochondriaca venom to exert antihaemocytic activity and inhibit haemocyte aggregate formation in the presence of the phosphatse inhibitor, CI 2, was investigated. The results indicated no apparent difference between the behaviour of haemocyte monolayers in the presence of venom only, or venom and CI 2. Thus, the antihaemocytic activity of P. hypochondriaca venom was not abolished or even reduced by an inhibitor of acid phosphatase. The pH of haemolymph from L. oleracea larvae and the medium used for maintaining haemocytes in vitro is approximately 6.6. The pH curve in figure 2 shows that at a pH of 6.1 the acid phosphatase activity in venom is reduced by 80 % compared to the activity measured at pH 4.8. The fact that activity of the phosphatase is optimum at pH 4.8 suggests that this enzyme would not be very active in the haemolymph of larval L. oleracea nor under the conditions used to maintain haemocytes in vitro. It was not possible to determine the pH of venom due to the

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small quantities that can be harvested, and the viscous nature of the venom. However, it seems likely that the pH of the venom would be neutral or alkaline to prevent the acid phosphatase from having any deleterious effect during storage in the venom sac. Under natural conditions, an adult female P. hypochondriaca wasp injects venom into a suitable pupal host during oviposition. Therefore, to establish whether venom acid phosphatase would be active within the haemocoel of a natural host, the pH of haemolymph, collected from 1-4 day old L. oleracea pupae, was measured. Pupal haemolymph had a pH of 6.7 suggesting that the venom acid phosphatase would not be very active in this sub-optimal condition.

It is possible that P. hypochondriaca venom acid phosphatase is present in a secretory pathway in the venom gland, as reported for the snake venom from B. jararaca. Alternatively, acid phosphatase might be released from cells undergoing cell death. The in vivo substrate for venom acid phosphatase is not known and due to the acid dependence of this enzyme it is highly unlikely to be molecules within the host haemolymph. Elucidating the sites of synthesis and activity will provide vital clues towards identifying potential substrates and possible function(s) for this enzyme in parasitoid venom.

ReferencesAndersch, M.A., Szcypinski, A.J., 1947. Use of p-nitrophenolphosphate as the substrate in determination of serum acid phosphatase. Am. J Clin. Pathol. 66, 287-292.

Dani, M.P., Richards, E.H., Isaac, R.E., Edwards, J.P., 2003. Antibacterial and proteolytic activity in venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae). J. Insect Physiol. 49, 945-954.

Kanost MR 1999. Serine proteinase inhibitors in arthropod immunity. Developmental and Comparative Immunology. 23: 291-301.

Parkinson, N., Smith, I., Weaver, R., Edwards, J.P., 2001. A new form of arthropod phenoloxidase is abundant in venom of the parasitoid wasp Pimpla hypochondriaca. Insect Biochem. Mol. Biol. 31, 57-63.

Parkinson, N., Richards, E.H., Conyers, C., Smith, I., Edwards, J.P., 2002a. Analysis of venom constituents from the parasitoid wasp Pimpla hypochondriaca and cloning of a cDNA encoding a venom protein. Insect Biochem. Mol. Biol. 32, 729-735.

Parkinson, N., Conyers, C., Smith, I., 2002b. A venom protein from the endoparasitoid wasp Pimpla hypochondriaca is similar to snake venom reprolysin-type metalloproteases. J. Invertebr. Pathol. 79, 129-131.

Richards, E.H. Parkinson, N.M., 2000. Venom from the endoparasitic wasp Pimpla hypochondriaca adversely affects the morphology, viability and immune-function of haemocytes from larvae of the tomato moth, Lacanobia oleracea. J. Invertebr. Pathol. 76, 33-42.

Xia, Y., Dean, P., Judge, A-J., Gillespie, J.P., Clarkson, J.M., Charnley, A.K. 2000. Acid phosphatase in the haemolymph of the desert locust, Schistocera gregaria, infected with the entomopathogenic fungus Metarhizium anisopliae. J. Insect Physiol. 46, 1249-1257.

Scientific Objective 2.To isolate a factor or factors with proven anti-haemocyte activity from the venom and confirm purity using selected biochemical techniques (this involves dissecting the venom glands from as many adult female P. hypochondriaca as possible in order to build up a pool of venom for use in subsequent isolation procedures). In addition, make a preliminary physicochemical characterization of the molecule(s) (particularly with regard to stability), determine N-terminal sequence, and perform a data base homology search. Information gained from this work will help in the design of experiments aimed at elucidating the mechanism of action of the active factor(s) as potential bio-control agents.

Introduction.(NB For references, see list above).Our previous work has clearly demonstrated that P. hypochondriaca venom contains a number of anti-haemocyte and immunosuppressive factors (Richards and Parkinson, 2000; Parkinson et al., 2002a and b). In order to drive the work forward, towards practical uptake, it is essential that at least some of these factors are isolated and characterized, both functionally and bio-chemically, in detail.

Methods.Final Isolation Procedure.P. hypochondriaca venom is extremely complex and is composed of a large number of proteins and polypeptides, with molecular weight estimates ranging from less than 10 kDa to over 200 kDa. Thus, in order to isolate a single

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immunosuppressive factor, a number of different protein separation techniques were tested. Ultimately, a multi-step procedure was optimized, that included gel filtration, anion exchange and cation exchange chromatography.

Determination of N-terminal sequence.In order to obtain some N-terminal sequence for the isolated immunosuppressive factor, the protein was run on a 12 % SDS polyacrylamide gel, and then blotted onto a nitrocellulose membrane as described by Dani et al., (2003). After staining the membrane in sulphorhodamine B, the protein was cut out and sent off for N-terminal sequencing, using Edman degradation (protein sequencing facility, Leeds University).

Results and Discussion.Biochemical isolation of an immunosuppressive venom protein.Following gel filtration, anion exchange and then cation exchange, a single peak containing protein with anti-haemocyte activity was obtained (Figure 1).

When the protein in this peak (P) was processed on a 12 % SDS polyacrylamide gel under reduced conditions, two bands were visualized following silver staining of the gel (Figure 2). 20

mAU

Figure 1 Figure 2

These 2 bands could not be separated further, suggesting that they either represent the same protein occurring in different isoforms (for example, with slightly different molecular weights due to the presence of different groups, such as carbohydrate), or they represent 2 different proteins. To help determine which, both bands were blotted onto nitrocellulose, cut out and then sent off for N-terminal sequencing. The results of this indicated that both bands have the same N-terminal sequence. Thus, we have successfully isolated an immunosuppressive protein, and that this exists as 2 isoforms. Data Base Homology Searches.The seven N-terminal amino acids sequenced for each protein band were submitted to a number of protein sequence sites in order to make a data base (BLAST) homology search. The results of this indicated that our sequence occurs in a wide variety of proteins, including one protein present in P. hypochondriaca venom (the amino acid sequence of this protein was derived from the DNA sequence of the gene coding for the protein). Note that this gene had been cloned as part of a random DNA sequencing exercise, and, until this recent work, no function had been attributed to the protein produced from this gene.

Scientific Objective 3.Using an isolated anti-haemocyte factor with proven immunosuppressive properties, to perform bio-assays to test the concept that targeted suppression of key immune responses can make an insect more susceptible to a suitable bio-control agent (for example, the fungus Beauveria bassiana).

Introduction.Our work with P. hypochondriaca venom clearly indicates that it is a rich source of potent anti-haemocyte/immunosuppressive factors. As such, it presents us with the opportunity to not only further our understanding of insect immune systems and host-parasitoid interactions, but also to utilize the factors present in bio-control strategies based on suppression of insect immune responses. As a first step in determining if anti-haemocyte factor(s) in P. hypochodriaca venom can make an insect more susceptible to a pathogen, we performed bio-assays using L. oleracea as our model pest insect and a variety of pathogens, including bacteria (E. coli and B. cereus), and a fungus (Beauveria bassiana) (Dani et al., 2004). Basically, these assays involved exposing larvae to known doses of venom, isolated immunosuppressive factor, or buffer (as a control), and the potential pathogen (usually LD50) or buffer (as a control). The larvae were then monitored on a daily basis until they died or pupated, with a number of criteria being recorded, including larval weight, production of frass, mobility, cuticle colour, and body ‘posture’ (whether turgid or flaccid).

Methods.Chemicals

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P V

Chemicals were purchased from Sigma Aldrich (Poole, Dorset, U.K.) unless stated otherwise.

InsectsLacanobia oleracea were reared according to the method of Corbitt et al. (1996) on an artificial diet (Bioserv, New Jersey), at 20 oC, 65 % relative humidity and under a 16-h light, 8-h dark photoperiod. Larvae on day 3 of the fifth stadium (weighing between 141 and 240 mg) were used for all the experiments.

Preparation of the fungus, Beauveria bassianaFreeze-dried B. bassiana conidia, isolate IMI 173201 (batch date 14/3/89), were obtained from CABI Bioscience, Egham, Surrey, UK.

A known weight of freeze-dried conidia was resuspended in 0.05 % Tween 80 (prepared from a 0.5 % sterile stock solution) by vortexing the suspension for 1 m followed by sonication for 30 s. Since the fungal conidia are hydrophobic, the presence of Tween 80 aids the suspension of the freeze-dried conidia. This procedure was repeated 3 times and the suspension then passed through a 30 G ½’’ needle. If the conidia have not gone into suspension, the process is repeated. The total conidial count is determined using a counting chamber. The conidial concentration is adjusted to the appropriate concentration for the experiment using the figure obtained for the total count. A viable count is also determined by plating out 10-fold serial dilutions of the prepared suspension onto potato dextrose agar (PDA) plates. After a 96 h incubation in the lab, the number of colony forming units is counted, and subsequently compared with the initial total count on which the “dipping dose” is based. The counts are performed prior to the addition of Codacide (see below) since the oil interferes with the count.

Preparation of Humid chambersHigh humidity (> 90 %), is necessary for conidia to germinate and penetrate the larval cuticle. Humid chambers were prepared using deli pots (Autobar, Hemel Hemstead, UK) containing a single disc of 3 MM Whatman paper soaked in sterile water and placed into the lid of the pot. The humid chambers are prepared 24-48h prior to dipping the larvae (see below) so that the humidity can build up inside them. Whilst the larvae are in such chambers, they are supplied with a small block of diet but no tissue paper.

Topical application of the fungus. To topically apply the fungus, larvae were completely immersed (dipped) for 10 s in 1 ml of 0.05 % Tween 80, 1:100 Codacide (a commercial carrier oil, containing 50 ml/l of emulsifier in rapeseed oil), or 1 ml of known concentration of Beauveria bassiana conidia resuspended in 0.05 % Tween 80, 1:100 Codacide. The larvae were then placed individually in humid chambers (deli pots supplied by Autobar, Hemel Hemstead, UK) for 20-24 h (in order for the fungus to work its way into the insect haemocoel). At day two post-dipping (third day of the fifth stadium), larvae were weighed in the morning and then, in the afternoon, injected (see below) with sterile DPBS isolated protein. Following injection, larvae were returned to their humid chambers with fresh food. All larvae were then assessed and weighed daily for up to 15 days post dipping.

Injection procedureLarvae were anesthetized by submerging them in water for 5 min, then blotted dry with tissue paper. L. oleracea larvae were injected, through the base of a proleg using a nanolitre injector (World Precision Instruments, Inc. Hertfordshire, UK), or a Hamilton syringe. In early experiments, some larvae were anesthetized as described above, and then stabbed with a sterile needle (sham injection), whilst others were only submerged in water. Since neither the anesthetizing procedure nor the sham injection resulted in any substantial larval mortality, these particular controls were omitted in subsequent experiments. Following injection, larvae were housed individually in plastic containers containing tissue paper and artificial diet, and cultured under the conditions described above.

Assessment of fungal mortality.Larvae, which died from treatment with B. bassiana, were rigid and, in the majority of cases, their cuticle exhibited a red pigmentation. Following surface sterilisation of the cuticle of these cadavers and incubation in a humid environment, fungal growth was observed on their external surface confirming the presence of a viable fungus.

StatisticsThe data from the mortality studies have been analysed with a Cox proportional hazards model, using the survival package in R. Test statistics and p-values are taken from the sequential analysis of deviance table of the Cox proportional hazards model, analysing the factorial treatment structure and adjusting for replicates. Mortality estimates with standard errors are also derived from that model. Statistical significance of the larval weights was calculated using analysis of variance followed by Tukey paired comparisons.

Results.P. hypochondrica venom (V) increases the susceptibility of L. oleracea larvae to both bacteria (B. cereus; B) (Figure 1), and fungus (B. bassiana; F) (Figure 2). The effect of the injection of venom (F1,204 =6.40, p< 0.011) or

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B. cereus (F1,275 = 9.24, p=0.002) on mortality of L. oleracea larvae was significant. The non-significant effect of their interaction (F1,274 = 0.19, p>0.05) indicated that the effect on mortality of the injection of venom and then B. cereus was additive. The effect of venom (F1,204 = 16.10, p<0.001) or the effect of B. bassiana (F1,204 = 97.06, p< 0.001) on the mortality of L. oleracea larvae was significant. Their interaction was non-significant (F1,203 = 2.98, p>0.05), indicating that the effect on mortality was additive when larvae received an injection of both venom and B. bassiana.

Figure 1 Figure 2

X: V/B, ■:C/B, ▲: V/C, ●: C/C. X: V/F, ■: C/F, ▲: V/C, ●: C/C

Summary Of Results Obtained With Isolated Immunosuppressive Protein.

Experiment 1.Specific Conditions.0.0087g of B. bassiana freeze dried conidia, isolate IMI 386367, resuspended in 8 mls 0.05 % Tween 80.

Conidial concentration for the expt: 7 x 107 conidia/ml -dilution based on total count. Viable count = 5.13 x 107 CFU/ml (73.3%)Insects: newly moulted fifth instar larvae weighing between 61-100 mg

Isolated ProteinPrepared as described above in objective 2. Basically, flow through from a cation exchange column were concentrated and then ran through an anion exchange column. The most potent fractions containing the isolated protein were pooled, then concentrated by spinning through a sterile filter with a molecular weight cut off of 10 kDa (Amicon). Treatment groups: No treatment (13 larvae); dipped in 0.05% Tween 80, 1:100 Codacide/injection of 5µl DPBS (12 larvae); dipped in 0.05% Tween 80, 1:100 Codacide/injected with 5 µl fraction 32 (11 larvae); dipped in fungus/injected with 5µl DPBS (13 larvae); dipped in fungus/injected with 5µl fraction 32 (12 larvae).

The effect of various treatments on the mortality of L. oleracea larvae. Larvae were dipped for 10s in 1 ml of 0.05% Tween 80, 1:100 Codacide or in 1 ml of 7 x 107 B. bassiana conidia (IMI 386367) resuspended in 0.05% Tween 80, 1:100 Codacide. Larvae were maintained individually in a humid chamber overnight with food. Then larvae were transferred to a fresh deli pot containing tissue paper and diet. Two days post dipping larvae were injected with 5 µl of DPBS or 5 µl protein concentrated about 1:8.

Days post

dipping

NT(n =13)

0.05% Tw80, 1:100 Codacide/

DPBS(n = 12)

0.05% Tw80, 1:100

Codacide/P(n = 11)

Fungus in diluent/DPBS

(n = 13)

Fungus in diluent/P(n = 12)

3 0 0 0 0 06 0 0 9* 23 16.77 0 0 27.3 23 258 0 0 36.4 23 50

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9 0 0 54.5 30.8 58.310 0 0 72.7 30.8 7513 0 0 81.8 38.5 83.314 0 0 81.8 46.2 83.315 0 0 81.8 46.2 83.3

* Percentage mortalityNo mortality was recorded for no treatment and diluent dipped/diluent injected treatment groups. At each time point, from day 7 post-dipping, a lower level of mortality was recorded for fungus dipped/diluent injected than diluent dipped/fraction injected or fungus dipped/ fraction injected groups.

This result indicates that at high concentrations the isolated anti-haemocyte factor exhibits some toxicity towards L. oleracea larvae, and that this masks its immunosuppressive effects. Nevertheless, it raises the possibility that relatively high concentrations of the protein may be used on its own to kill pest insects.

The effect of various treatments on the weight gain of L. oleracea larvae. Larvae were treated as described above for effects on mortality.

Larvae belonging to the no treatment group (NT), diluent dipped/diluent injected (C/C), and fungus dipped/diluent injected (C/F) groups, showed very similar trends for average larval weight gain. From day 3 little increase in average weight gain was recorded for the two treatment groups that also had the highest levels of mortality recorded i.e. diluent dipped and protein injected (C/P) and fungus dipped and protein injected (F/P).

Experiment 2.Specific Conditions.0.0066g of B. bassiana freeze dried conidia, isolate IMI 386367, resuspended in 5 mls 0.05 % Tween 80.Viable count = 1.3 x 108 CFU/ml

Insects: newly moulted fifth instar larvae weighing between 40-80 mg

The effect of various treatments on the mortality of L. oleracea larvae. Larvae were dipped in fungus as described above. Two days post dipping larvae were injected with 3 µl of 1:2 buffer (C, control) or 3 µl 1:2 isolated protein (P)

Days post

dipping

NTn=13

0.05% Tw80, 1:100

Codacide / C(n = 8)

0.05% Tw80, 1:100 Codacide /

P(n = 8)

F / C(n = 6)

F / P (n = 7)

3 0 0 0 0 06 0 11.1% 0 33.3% 07 0 11.1% 0 33.3% 14.3%8 0 11.1% 12.5% 33.3% 28.6%9 0 11.1% 12.5 33.3% 42.9%

10 0 11.1% 25% 33.3% 42.9%13 0 22.2% 25% 50% 57%14 0 22.2% 25% 50% 71.4%15 0 22.2% 37.5% 66.6% 71.4%

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All the larvae survived in the no treatment control (NT). The highest level of mortality occurred for the fungus and protein (F/C) treatment group. However, a relatively high level of mortality also occurred for the fungus only group (F/C) indicating that fewer fungi may need to be used in future experiments.

The effect of various treatments on the weight gain of L. oleracea larvae. Larvae were dipped for 10s in 1 ml of 0.05% Tween 80 and 1:100 Codacide (C, control) or in 1 ml of 7 x 10 7

conidia resuspended in 0.05% Tween 80 and 1:100 Codacide (F, fungus).Two days post dipping larvae were injected with 3ìl 1:2 diluent (C, control) or with 3ìl 1:2 Venom protein (VP).

The trend in larval weight gain was similar to that for larval mortality (see above). The least gain in larval weight (indicating the least amount of food eaten) occurred for larvae treated with fungus dipped and then isolated immunosuppressive protein (F/P).

Discussion.

During the course of work performed to address objective 3, our culture of P. hypochondriaca underwent a severe decline in numbers, to the point where the whole culture was nearly permanently lost. A detailed report was written, and submitted to Defra towards the end of 2004. As explained in this report, the reduction in the number of wasps greatly reduced the amount of venom available, and thus the amount of isolated anti-haemocyte factor which could be produced. This had the greatest negative impact on work objective 3, where relatively large amounts of the protein were required for bio-assay work. Despite this, our work with whole venom (Dani et al., 2004) and that with the isolated anti-haemocyte factor (summarized above), clearly indicates that these ‘agents’ are both capable of increasing the biopesticidal properties of a potential pathogen, such as the commercially available fungus Beauveria bassiana.

The work performed for objective 3 has also made it clear that large amounts of isolated immunosuppressive protein are required to perform bio-assays and that obtaining them from P. hypochondriaca venom via conventional biochemical techniques (as described in objective 2), is relatively inefficient. Thus, for future work, we will concentrate on identifying and cloning the genes for the immunosuppressive factors we need, and then use these to ‘manufacture’ recombinant protein, which will subsequently be used in bio-assays.

Scientific Objective 4.

To clone the gene(s) of isolated anti-haemocyte factor(s) and determine their DNA sequence(s). This data, and that obtained on the isolated protein(s), are necessary precursors to establishing LINK projects with suitable companies so that the research progresses towards practical uptake.

Introduction.As indicated in the summary for objective 3, relatively large amounts of (immunosuppressive) protein are required for the bio-assay work. Since production via conventional biochemistry techniques is inefficient, and requires large amounts of venom, we will produce the protein required using a molecular approach. As a first step in this procedure, we need to clone the gene for the immunosuppressive factor we have identified using a biochemical approach, and then insert it into a suitable vector(s) for DNA sequencing and, subsequently, protein expression in bacteria, insect cells and/or yeast. The production of such ‘tools’ will subsequently allow us to forge links with bio-tech companies which may be interested in sponsoring the work towards practical uptake.

Methods.

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PCR Reaction For Cloning Genes.Forward and reverse primers were prepared to match the cDNA sequence of the gene for the isolated protein. These were then used to clone the gene for the protein out of the P. hypochondriaca venom gland library using a PCR approach.

A large number of pre-tests were performed in order to optimise the PCR procedure. Ultimately, all PCR reactions were performed using a proof-reading Taq polymerase in a 20μl reaction volume. Basically, the reaction mix was composed of 1 x Phusion HF buffer (gives 1.5 mM MgCl2 in final reaction volume), 200 mM each dNTP 10 pmol of forward and reverse primers, 1 ml of cDNA (or diluted cDNA), 0.002U Phusion High Fidelity Polymerase. Final cycling conditions were as follows:

1. Temp 98 oC 2 mins2. Temp 98 oC 10 sec3. Temp 60 oC 30secs 4. Temp 72 oC 30secs go back to step 2 for 29 cycles5. Temp 72 oC 5 mins

Step 1 is the initial denaturation step, step 2 denaturation step for the cycle, step 3 is the annealing step, step 4 is the extension and step 5 is the final extension step.VP3 primer mature forward = 28 bases, Tm = 65.1oC, reverse primer = 24 bases, Tm = 55.9 oC.

The cloned genes were finally inserted into a vector, ready for DNA sequencing. In total, 14 bacterial colonies were picked, then screened again by PCR to confirm the presence of an insert of correct size. Three clones were finally sent for sequencing.

Data Base Homology Searches.DNA homology searches were performed by entering the DNA sequence of the cloned gene (or part of it) into a number of DNA databanks, and performing homology searches according to the specific instructions given by each site.

Results.In total, 3 clones containing genes for the isolated protein were produced from the PCR work. The DNA sequences of all three clones were determined in both directions. Two of the clones possessed DNA sequences which contained errors i.e. sequences which differed to the published sequence. The DNA sequence for the third clone corresponded exactly to the published sequence. This clone was selected for all future work.

Discussion.The ultimate aim of this work is to optimise bio-control strategies for insect pests through manipulation (suppression) of their immune responses. In order to achieve this, we need to have certain ‘tools’ at our disposal. This includes proteins or polypeptides with proven anti-haemocyte/immunosuppressive properties, and the genes for these factors. Successful completion of objective 4, represents a crucial first step along this path.

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References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

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Richards, E.H., Manderyck, B., Edwards, J.P. (2005). Partial amino acid sequence and physiological effects of a 27 kDa parasitism-specific protein present in the plasma of parasitized Lacanobia olercea(Noctuidae).

Dani, M. P., Edwards, J. P., Richards, E. H. (2005). Hydrolase activity in the venom of the pupal endoparasitic wasp, Pimpla hypochondriaca. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology 141, 373-381.

Dean, P., Potter, U., Richards, E. H., Edwards, J. P., Charnley, A. K., Reynolds, S. E., (2004). Hyperphagocytic haemocytes in Manduca sexta. Journal of Insect Physiology 50, 1027-1036.

Parkinson, N. M., Conyers, C., Keen, J., MacNicoll, A., Smith, I., Audsley, N., Weaver, R. (2004). Towards a comprehensive view of the primary structure of venom proteins from the parasitoid wasp Pimpla hypochondriaca. Insect Biochemistry and Molecular Biology 34, 565-571.

Dani, M. P., Richards, E. H., Edwards, J. P. (2004). Venom from the pupal endoparasitoid, Pimpla hypochondriaca, increases the susceptibility of larval Lacanobia oleracea to the entomopathogens Bacillus cereus and Beauveria bassiana. Journal of Invertebrate Pathology 86, 19-25.

Dean, P., Richards, E. H., Edwards, J. P., Reynolds, S. E., Charnley, K. (2004). Microbial infection causes the appearance of hemocytes with extreme spreading ability in monolayers of the tobacco hornworm Manduca sexta. Developmental and Comparative Immunology 28, 689-700.

Dani, M. P., Richards, E. H., Isaac, R. E., Edwards, J. P. (2003). Antibacterial and proteolytic activity in venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera : Ichneumonidae). Journal of Insect Physiology 49, 945-954.

Parkinson, N. M., Conyers, C. M., Keen, J. N., MacNicoll, A. D., Smith, I., Weaver, R. J. (2003). cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis. Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 134, 513-520.

Parkinson, N., Smith, I., Audsley, N., Edwards, J. P. (2002). Purification of pimplin, a paralytic heterodimeric polypeptide from venom of the parasitoid wasp Pimpla hypochondriaca, and cloning of the cDNA encoding one of the subunits. Insect Biochemistry and Molecular Biology 32, 1769-1773.

Richards, E. H., Edwards, J. P. (2002). Larvae of the ectoparasitic wasp, Eulophus pennicornis, release factors which adversely affect haemocytes of their host, Lacanobia oleracea. Journal of Insect Physiology 48, 845-855.

Dean, P., Gadsden, J. C., Richards, E. H., Edwards, J. P., Charnley, A. K., Reynolds, S. E. (2002). Modulation by eicosanoid biosynthesis inhibitors of immune responses by the insect Manduca sexta to the pathogenic fungus Metarhizium anisopliae. Journal of Invertebrate Pathology 79, 93-101.

Parkinson, N., Conyers, C., Smith, I. (2002). A venom protein from the endoparasitoid wasp Pimpla hypochondriaca is similar to snake venom reprolysin-type metalloproteases. Journal of Invertebrate Pathology 79, 129-131.

Parkinson, N., Richards, E. H., Conyers, C., Smith, I., Edwards, J. P. (2002). Analysis of venom constituents from the parasitoid wasp Pimpla hypochondriaca and cloning of a cDNA encoding a venom protein. Insect Biochemistry and Molecular Biology 32, 729-735.

Richards, E. H., Edwards, J. P. (2002). Parasitism of Lacanobia oleracea (Lepidoptera) by the Ectoparasitic wasp, Eulophus pennicornis, disrupts the cytoskeleton of host haemocytes and suppresses encapsulation in vivo. Archives of Insect Biochemistry and Physiology 49, 108-124.

Richards, E. H., Edwards, J. P. (2001). Proteins synthesized and secreted by larvae of the ectoparasitic wasp, Eulophus pennicornis. Archives of Insect Biochemistry and Physiology 46, 140-151.

Parkinson, N., Smith, I., Weaver, R., Edwards, J. P., (2001). A new form of arthropod phenoloxidase is abundant in venom of the parasitoid wasp Pimpla hypochondriaca. Insect Biochemistry and Molecular Biology 31, 57-63.

Richards, E.H., Edwards, J.P. (2000). Parasitism of Lacanobia oleracea (Lepidoptera) by the ectoparasitoid, Eulophus pennicornis, is associated with a reduction in host haemolymph phenoloxidase activity. Comparative Biochemistry and Physiology B, 127, 289-298.

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