CSG 15 Research and Development Final Project Report (Not to Be Used for LINK Projects)

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MINISTRY OF AGRICULTURE, FISHERIES AND FOOD CSG 15 Research and Development Final Project Report (Not to be used for LINK projects)

Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit MAFF, Area 6/01 1A Page Street, London SW1P 4PQ An electronic version should be e-mailed to [email protected]

Project title Integrated management of pest and beneficial insects on oilseeds (prev. AR0105)

MAFF project code AR0203

Contractor organisation IACR-Rothamsted, and location Harpenden, Herts AL5 2JG

Total MAFF project costs £ 1142350

Project start date 01/04/94 Project end date 31/03/00

Executive summary (maximum 2 sides A4)

The overall objective of Project AR0203 was to develop Integrated Pest Management (IPM) strategies for oilseed rape based on a sound knowledge of the ecology and behaviour of pest and beneficial insects and their interactions with the crop.

Oilseed rape (Brassica napus L.) is the most important non-cereal crop in the UK. In 1997, 445,000 hectares were grown (7% of total arable land), producing 1.51 million tonnes of seed with a value of £408 million. Insecticide is widely used on the crop to kill insect pests, of which the cabbage stem flea beetle, the pollen beetle, the cabbage seed weevil, the cabbage aphid and the brassica pod midge cause most concern to growers. In 1996, crops received on average 1.5 insecticide sprays (Pesticide Usage Survey Report); the area treated has increased by 49% since 1994 and by 487% since 1982. The project focused on those pests, named above, that cause reduction in yield and quality of rape, and against which insecticides are targeted. It addressed MAFF policy ‘that pesticide use should be limited to the minimum necessary for the effective control of pests compatible with the protection of human health and the environment (1990, White Paper, The Common Inheritance). It sought ways to reduce overall use of pesticide and effects on non-target species, while maintaining production and effective control of pests.

The project involved strategic research on:

1. Insect-insect interactions, particularly semiochemical-mediated behaviour associated with pest reproductive biology with potential for manipulation in pest management strategies.

CSG 15 (Rev. 12/99) 1 Project Integrated management of pest and beneficial insects on MAFF AR0203 title oilseeds (prev. AR0105) project code

The pod-marking behaviour of the cabbage seed weevil following egg laying was described. The glandular source of the oviposition-deterring pheromone of the weevil was located and its mode of perception by contact chemoreceptors determined. Chemical characterisation of the pheromone is progressing.

Electron microscopy of the abdomen of the cabbage stem flea beetle has located an abdominal gland present in females by not in males; its role in their courtship behaviour is under investigation.

2. Insect-plant interactions, particularly pest responses to plant genotypes with potential to provide resistance to insect attack.

The glucosinolates (GS) and their metabolites in oilseed rape are important mediators of insect-plant interactions. No relationship was found however, between cabbage stem flea beetle feeding and GS concentration in tests using breeders’ lines of rape with similar GS profiles. Tests with lines with similar GS concentration but with differing GS profile were discontinued as plants did not express as expected.

Cabbage stem flea beetles neither fed nor developed differently on control and transformed plants expressing protease inhibitors designed to confer resistance to insect herbivores.

3. Natural enemies of pests, particularly their identity, status, potential for bio-control, ways of enhancing their effectiveness and of managing their populations.

Three parasitoids were found to attack the cabbage seed weevil in the UK, of which T. perfectus was the most widespread and abundant. Four parasitoids were reared from the brassica pod midge, one a new species record for the UK. Species of endoparasitoid were reared from cabbage seed weevil and cabbage stem flea beetle adults, one is a new record for the UK and the other a previously undescribed species. Studies into the biology of the key species have been initiated. New methods for selectively sampling and monitoring parasitoids were devised.

A novel method, using honey bees to disseminate the entomopathogenic fungus, Metarhizium anisopliae, to the flowers of oilseed rape to kill pollen beetles, has been devised and tested in field cages with encouraging results; mortality of beetles was high.

4. Population ecology and dynamics of pest and beneficial insects, particularly their spatial and temporal distributions on the crop to enable more effective targeting of pests by insecticides.

The cabbage seed weevil and, simultaneously its parasitoid T. perfectus have been sequentially sampled in two rape crops using traps in two-dimensional arrays of spatially-referenced sampling points. Parasitoid adults immigrated later than weevil adults and did not aggregate at the edge of the crop except briefly during the early phase of immigration. Seed weevil larvae and parasitoid larvae were both aggregated near crop edges and were associated with the spatial distribution of adult female weevils.

Work has been initiated into the feasibility of using microsatellite primers to separate populations of parasitoids of rape pests for studies into the movement patterns of individuals and populations.

The implications of the research results for IPM strategies are discussed and aspects that require further research are indicated. A full publication list is included.

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Scientific report (maximum 20 sides A4)

Table of contents

1.0 Overall scientific objective

2.0 Introduction

3.0 Insect-insect interactions 3.1 The oviposition-deterring pheromone of the cabbage seed weevil 3.1.1 Pod-marking behaviour 3.1.2 Glandular source 3.1.3 Perception 3.1.4 Effect on males 3.1.5 Chemical characterisation 3.1.6 Pheromone supply 3.2 Courtship behaviour in the cabbage stem flea beetle 3.3 Weevil-midge interactions 3.4 Midge-parasitoid interactions 3.5 Future work

4.0 Insect-plant interactions 4.1 Glucosinolates 4.2 Jasmonic acid 4.3 Isothiocyanates 4.4 Hybrid cultivars 4.5 Resistant cultivars 4.6 Future work

5.0 Natural enemies 5.1 Parasitoids 5.1.1 Parasitoids of the cabbage seed weevil 5.1.1.1 Trichomalus perfectus 5.1.1.2 Microctonus sp. 5.1.2. Parasitoids of the brassica pod midge 5.1.2.1 Platygaster subuliformis 5.1.2.2 Omphale clypealis 5.1.3. Parasitoids of the cabbage stem flea beetle 5.1.4 Sampling methods for parasitoids 5.1.4.1 Omni-directional flight trap 5.1.4.2 Malaise trap with time-sorter 5.2 Pathogens 5.2.1 Metarhizium anisopliae 5.3 Future work

6.0 Population ecology and dynamics 6.1 Spatial and temporal distributions 6.2 Population genetics 6.3 Future work

7.0 Acknowledgements

8.0 Technology transfer

9.0 Publications

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1.0 Overall scientific objective To develop an integrated pest management (IPM) strategy for oilseed rape based on a sound knowledge of the ecology and behaviour of pest and beneficial insects and their interactions with the crop.

2.0 Introduction Oilseed rape (Brassica napus L.) is the most important non-cereal crop in the UK. In 1997, 445,000 hectares were grown (7% of total arable land), producing 1.51 million tonnes of seed with a value of £408 million. Insecticide is widely used on the crop to kill insect pests, of which the cabbage stem flea beetle (Psylliodes chrysocephala), the pollen beetle (Meligethes aeneus), the cabbage aphid (Brevicoryne brassicae), the brassica pod midge (Dasineura brassicae) and the cabbage seed weevil (Ceutorhynchus assimilis) cause most concern to growers. In 1996, crops received on average 1.5 insecticide sprays (Pesticide Usage Survey Report); the area treated has increased by 49% since 1994 and by 487% since 1982.

This project: · focuses on those pests, named above, that cause reduction in yield and quality of rape, and against which insecticides are targeted. · addresses MAFF Policy ‘that pesticide use should be limited to the minimum necessary for the effective control of pests compatible with the protection of human health and the environment (1990, White Paper, The Common Inheritance). · seeks to reduce overall use of pesticide and effects on non-target species, while maintaining production and effective control of pests.

This project involves strategic research on: · insect-insect interactions, particularly behaviour with potential for manipulation for control · insect-plant interactions, particularly pest responses to plant genotypes with potential to provide resistance to insect attack · natural enemies of pests, particularly their identity, status, potential for control, ways of enhancing their effectiveness and of managing their populations · population ecology and dynamics of pest and beneficial insects, particularly their spatial and temporal distributions on the crop to enable more effective targeting of pests by insecticides

The project forms a basis for a more integrated and sustainable management of oilseed rape pests substituting pesticide usage with effective control by natural enemies and more focussed control. This approach is expected to bring environmental benefits and help to prevent and delay the onset of pesticide resistance in pest populations. By reducing inputs and unnecessary costs to the farmer it will also improve competitiveness and contribute to wealth creation.

This project was started (as AR0105) on 1 April 1991 and finished on 31 March 2000. Research progress for 1991-1994 was reviewed on 21 December 1994 and the project recommissioned from April 1995 as project AR0203. Research progress for 1995-1999 was reviewed on 20 January 1999. This final report reports research progress for 1995-2000, indicating where this builds on and continues research initiated during 1991-1995.

3.0 Insect-insect interactions Both intra- and inter-specific interactions between insects on the oilseed rape crop are largely mediated by behaviour-modifying chemicals, particularly pheromones and kairomones. These have potential for use in IPM strategies for insect monitoring or for altering insect behaviour and/or location on the crop. Recent work on the pheromones of rape pests builds on earlier studies, which focussed on the sex pheromone of the brassica pod midge, the oviposition-deterring pheromone of the seed weevil and the antennal pheromone glands of the

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cabbage stem flea beetle.

3.1 The oviposition-deterring pheromone (ODP) of the cabbage seed weevil Oviposition-deterring pheromones in insects increase the fitness of immature individuals by reducing or eliminating conspecific competition for a restricted food resource. The female cabbage seed weevil lays her eggs singly into rape pods through punctures bored with her mouthparts and rostrum. The larvae feed on developing seeds, consuming 4-5 seeds before exiting the pods to pupate in the soil. Earlier work showed that the female prefers to lay in rape pods not recently used for egg-laying, that she regulates the distribution of her eggs by applying an ODP to the pod after egg-laying and that the deterrent effect lasts 1-2 h. The ODP of the weevil has potential for controlling egg-laying of this pest, and also that of the pod midge which uses weevil punctures for egg-laying. If the pheromone could be identified, synthesised and applied to crops, possibly using a slow release formulation, egg laying by the weevil and the midge may be deterred. Recent work has focussed on the pod-marking behaviour of the female weevil, on locating the glandular source of the pheromone, on determining its mode of perception and on its chemical characterisation.

3.1.1 Pod-marking behaviour Behavioural observations and analyses of video recordings showed that the mean time from the start of pod marking (application of the pheromone to the pod) to the departure of the female weevil from the pod was 74s and that pod brushing occupied 87% of this time (Ferguson et al., 1999a). The weevil usually made three traverses of the pod, spending the remainder of the time grooming or at rest. Pod brushing was discontinuous, consisting of a number of successive periods of movement along the pod during which the brush-like caudal setae (hairs) of the eighth urotergite were extended and were applied to the pod while the weevil moved her abdomen from side to side and progressed slowly forward. These periods were separated by shorter periods during which the brush was retracted into the abdomen and extended again while the weevil hesitated in her progress along the pod; a mean of 27 retractions/extensions were made per pod.

3.1.2 Glandular source Behavioural observations, anatomical, histological and ultrastructural investigations, have now demonstrated that the ODP of the weevil is produced by secretory epidermal cells opening onto a fold of the seventh urotergite of the abdomen in overwintered, sexually mature, gravid females (Ferguson et al., 1999a). Retraction of the eighth urotergite into the abdomen during pod marking behaviour, brings the caudal setae into contact with the opening of the glandular cells of the seventh urotergite and they acquire secretion from the cells. On extension of the eighth urotergite from the abdomen, the caudal setae contact the pod wall and the secretion is applied to the pod. Histological studies of excised glands, using transmission electron microscopy, revealed that the cells of the seventh urotergite gland are hypertrophic, showing intense secretory activity. Extracts from such glands elicited strong electrophysiological responses from antennal club gustatory sensilla and deterred oviposition. In pre-diapause, sexually immature females, the cells of the seventh urotergite gland were neither hypertrophic nor active and an extract of their urotergites elicited no significant electrophysiological or behavioural response.

3.1.3 Perception Egg laying by the female weevil is preceded by intense antennation of the pod. The ODP is sensed primarily by contact chemoreception by sensilla chaetica of the antennal club; females with their antennal clubs amputated showed no behavioural response to ODP (Ferguson et al., 1999b). Females approaching a freshly ODP-marked pod brought their antenna within 1 mm of the pod but usually did not antennate it before rejecting it for egg laying. Extracts of volatiles, air-entrained from egg-laying weevils, failed to inhibit egg laying and air passed over behaviourally active extract of ODP did not elicit a detectable electroantennogram response. By contrast, when presented as a gustatory stimulus to the sensilla chaetica of the antennal club, a behaviourally active extract of ODP from postdiapause, gravid females elicited a strong electrophysiological response; extract from prediapause, sexually immature females elicited neither behavioural nor electrophysiological contact responses.

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3.1.4 Effect on males Adult and larval weevils both feed on the seeds within rape pods. The ODP could be used as a cue to avoid direct intraspecific competition between adults and larvae for the same resource. Preliminary analyses of experiments indicates, that on ODP-marked pods, the frequency and the total duration of feeding by adult males was reduced. Males presented with ODP-marked pods were more active than those with unmarked pods, suggesting that ODP may also have a role as a sex pheromone.

3.1.5 Chemical characterisation Work on the chemical composition of ODP, isolated from glass tubes marked by the weevil and from extracts of its dissected seventh urotergite, and identified by gas chromatography-mass spectrometry, showed that it contained iso- and n- alkanes, dimethylalkanes, alkenes, fatty acids, 15-nonacosanone, 15-nonacosanol, and cholesterol. However, none of these organic components were behaviourally active (with MAFF Project P10308; Mudd et. al., 1997). ODP was shown to have low volatility. Activity was recovered by liquid chromatography; HPLC fractionation located activity at the solvent front indicating that ODP is probably polar and of high molecular weight. Further fractionation by HPLC located bioassay activity in the most polar of 30 fractions. Studies are in progress to optimise the chromatographic separation of the active constituent and to identify it by spectroscopic methods.

3.1.6 Pheromone supply The cabbage seed weevil has only one generation per year and overwinters in the soil for about seven months of the year. It is reproductively active for only about 6-8 weeks, limiting the time during which behavioural bioassays with field-collected sexually, mature gravid females can be conducted. Ability to continuously culture, overwinter artificially or terminate the diapause of weevils, would overcome the problem of seasonal supply. So far, however, overwintering in the laboratory or insectary under controlled environment or semi- natural conditions, has resulted in high mortality and abnormal mating; overall only 4% of males and 23% of females survived diapause. When field-collected males attempted to mate with overwintered virgin females they were usually rejected.

3.2 Courtship behaviour in the cabbage stem flea beetle The courtship behaviour of the cabbage stem flea beetle was investigated to determine whether the behaviour associated with male-female interactions has potential for manipulation for control. Scanning electron microscopy studies have revealed two types of gland ductule in the abdomen of both male and female beetles. Their distribution was mapped. Long ductules were widely distributed, but short ductules were generally less common and did not occur on the chitinous infolding of the terminal abdominal segment of the male. Transmission electron microscopy has confirmed that there is a gland associated with the terminal abdominal segment of the female that is absent in males. The role of these glands in the courtship or mating behaviour of the beetle needs to be investigated.

3.3 Weevil-midge interactions The ovipositor of the brassica pod midge is too weak to pierce the rape pod wall for egg-laying so the midge most frequently uses the punctures made for feeding and egg-laying by the weevil; larvae of the weevil and the midge are frequently found in the same rape pod and thus may compete for resources. Cage studies showed that the midge preferred weevil egg-laying punctures to weevil feeding punctures for its egg laying but there was no evidence that it used ODP as a cue to their location. Midge larvae hatch and develop more rapidly than do weevil larvae. Mortality of weevil eggs was 32% in plants caged with midges and 0% in plants without midges; this suggest that the midge larvae damage weevil eggs and may feed upon them. Because they preferred to lay through weevil egg-laying sites, we infer that feeding on weevil eggs represents predatory behaviour (Ferguson et al., 1995).

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3.4 Midge-parasitoid interactions Earlier studies have shown that virgin female brassica pod midge release a sex pheromone from their ovipositors that attracts conspecific males in the laboratory (in olfactometers) and in the field (in traps baited with live females or ovipositor extracts). Field experiments were conducted in 1997 to determine whether Platygaster subuliformis, an egg-larval parasitoid of the midge, is attracted to traps baited with either live virgin females or extracts of virgin female midge. Results were inconclusive as few parasitoids were caught. The numbers of male midge caught were also small; this was probably because the inclement weather during the experiments deterred midge and parasitoid flight, precluding effective testing of the pheromone.

3.4 Future work Behavioural and morphological aspects of the ODP have now been completed; some work on ethological studies of the influence of ODP on resource-partitioning by egg-laying females and on weevil-midge interactions remains to be published. Future emphasis will be on chemical characterisation under MAFF Project P10308 led by Prof. J.A. Pickett at IACR-Rothamsted. We will continue to supply biological material (glands) and to bioassay extracts and candidate compounds for activity, as required, to progress the chemical characterisation work. The collection of sufficient pheromone and the short time (often only 6 weeks per year) for which egg-laying female weevils are available for behavioural bioassays have been major constraints to progress with chemical analysis of the ODP secretion. Location of the source of the ODP now allows its extraction into solvents directly from seventh urotergites dissected from female weevils; since 1995, >30,000 weevils have been collected for this purpose. Determination of the mode of ODP perception should enable the screening of chemical fractions throughout the year and in smaller quantities by electrophysiological bioassay, rather than by more time-consuming behavioural bioassay. Bioassays to manipulate populations of weevils in semi-field experiments are envisaged to follow successful identification and synthesis of the ODP.

Further work on the courtship behaviour of the cabbage stem flea beetle will also transfer to MAFF P10308.

4.0 Insect-plant interactions Many insect-cruciferous plant interactions are mediated by plant chemistry. Different cruciferous plant species and cultivars of the same species differ in their relative attractiveness to insects, both pest and beneficial. Plant breeding and genetic engineering can be used to alter the semiochemical composition of the plant to make it less attractive and susceptible to insect pests and more attractive to beneficial insects. For example, there is potential to breed/engineer plants that emit lower levels of attractants for pests, making it more difficult for them to find their host plant. Such plants could perhaps be used as part of an integrated pest management system, such as the stimulo-deterrent diversionary strategy.

Plant breeders have also produced hybrid rape comprising male-fertile and male-sterile lines. The latter produce no pollen, an important food for some of the summer pests of oilseed rape; these lines may provide a poorer quality host plant for such pests.

Novel breeders’ lines have offered several opportunities for investigating insect-plant interactions with potential for manipulation for integrated pest management.

4.1 Glucosinolates The secondary metabolite chemistry of crucifers, such as oilseed rape, is characterised by the presence of glucosinolates. The glucosinolates (GS) and their metabolites, such as the isothiocyanates, are important mediators of insect-rape plant interactions, particularly host location and recognition (Bartlet, 1996; Bartlet et al., 1997). Cultivars with altered GS content have potential for reducing pest colonisation and damage (Bartlet, Williams & Pickett, 1999).

Previous field trials with commercial cultivars of single low (high seed GS) and of double low (low seed GS)

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rape showed no consistent differences in cabbage stem flea beetle damage to the cotyledons; differences in GS content of the leaves of the cultivars tested may have been insufficient to influence feeding. The effect on pest damage of varying GS concentration or GS profile (relative proportion of GS types) of oilseed rape was investigated. Field trials by collaborators at the John Innes Centre had previously found a positive correlation between cabbage stem flea beetle damage to the leaves and total leaf GS concentration, but these tests used lines which varied greatly in the proportion of isothiocyanate-releasing GS they contained. No relationship was found between beetle feeding and GS concentration in laboratory tests using breeder's lines of rape with similar GS profiles (Bartlet, Mithen & Clark, 1996).

Some GS types, such as the indolylGSs, do not metabolise to produce stable isothiocyanates and lines containing predominantly these GS types, may be less attractive to insect pests. To test this further, feeding tests were conducted to compare the relative attractiveness of breeders’ lines (of Chinese origin) with similar GS concentration but differing GS profile to cabbage stem flea beetle. However, these experiments were discontinued as the plants did not express the profiles predicted by the breeder when grown in our glasshouses.

Recent GS analysis of rape lines (in MAFF Project P10309) has revealed two that lack detectable levels of any isothiocyanate-releasing GSs and these will be tested for their attractiveness to rape pests.

4.2 Jasmonic acid Biochemical studies under MAFF Project P10309 have shown that the glucosinolate composition of the oilseed rape plant is altered when the plant is damaged, the indolylGS content increasing, usually resulting in a net increase in total glucosinolates (Bartlet et al., 1999). Jasmonic acid is a naturally-occurring plant compound involved in the damage response of rape. Preliminary laboratory experiments have now revealed that exogenous application of small (nmoles) amounts of jasmonic acid to the rape plant reduced damage from both slugs and cabbage stem flea beetles.

4.3 Isothiocyanates Host location in phytophagous insects is mediated by a combination of visual and olfactory cues and secondary plant chemicals provide orientation and feeding cues for specialized insect feeders, which are often specific to a restricted range of plants. Parasitoids also often use plant-derived chemicals to locate host habitats. On tissue damage, the glucosinolates are catabolised to isothiocyanates and other compounds. Previous studies have shown that isothiocyanates are key cues for host plant location by some rape pests, particularly the cabbage seed weevil and the pollen beetle (Bartlet et al., 1997). In collaborative studies with scientists in Poland, we have also now shown that traps baited with 2-phenylethyl isothiocyanate catch about three times as many of the stem weevils, Ceutorhynchus pallidactylus and Ceutorhynchus napi than traps without.

Some of the parasitoids attacking rape pests are also attracted by isothiocyanates. The responses of the brassica pod midge and its parasitoids P. subuliformis and O. clypealis to isothiocyanates, was investigated using the newly designed omni-directional flight trap, described below (Murchie, Smart & Williams, 1997).

Traps baited with allyl isothiocyanate caught more male and more female midge and more female O. clypealis (during one week only, not overall) than traps baited with 2-phenylethyl isothiocyanate or unbaited traps; traps baited with 2-phenylethyl isothiocyanate caught more male and more female P. subuliformis than traps baited with allyl isothiocyanate or unbaited traps (Table 1). Although not effective when presented in flight traps singly, allyl isothiocyanate synergised the response of P. subuliformis to 2-phenylethyl isothiocyanate. Catches of some pollen beetle and stem weevil parasitoids were also enhanced when traps are baited with isothiocyanates; flight trap catches of Phradis interstitialis and Phradis morionellus, parasitoids of pollen beetle larvae were enhanced by 2-phenylthethyl isothiocyanate, whereas Tersilochus obscurator, a parasitoid of the stem weevil, C. pallidactylus, was attracted to allyl isothiocyanate.

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Table 1. Mean numbers of brassica pod midge, Platygaster subuliformis and Omphale clypealis caught per trap

Allyl isothiocyanate 2-Phenylethyl isothiocyanate Unbaited Midge male 46.2 0.6 0.6 female 0.6 0.2 0 P. subuliformis male 8.4 341.2 5.6 female 0.8 43.0 0.2 O. clypealis male 0.4 0.2 0.2 female 3.2 3.8 0.8

The isothiocyanates therefore appear to have a role in host habitat location for the parasitoids as well as a role in host plant location for the pests. The parasitoid species responded differently to different isothiocyanates and synergy between isothiocyanates occurred. This response to isothiocyanates may lead to ways in which field parasitization rates could be enhanced by manipulation of the chemical environment of the crop.

4.4 Hybrid cultivars Synergy, a composite hybrid winter oilseed rape is a varietal association of 80% male sterile hybrid plants (cv. Falcon x Samouri) which produce no pollen and 20% male fertile pollinator plants (cv, Falcon). Adult and larval pollen beetles both feed on pollen from oilseed rape flowers and thus pose a particular threat to Synergy; growers are advised to control pollen beetle when there is an average of only one beetle per five plants at the green/yellow bud stage. The effects of pollen presence/absence on adult infestation, egg-laying and larval development were investigated (Cook, Murray & Williams, 1999). Adult infestation was greater on fertile plants than sterile ones. Although more sterile than fertile buds were devoid of eggs and larvae despite egg- laying damage, there was no difference in the mean number of eggs laid per bud, indicating that females do not adjust clutch size in response to host quality assessments. More second instar larvae were present on fertile than on sterile plants, indicating a greater rate of survival, although fitness (weight) of larvae found on each plant type did not differ. Since more adults and second instar larvae were found on fertile plants than sterile, the greater need for pollen beetle control to ensure adequate pollination of hybrid crops, such as Synergy, was confirmed.

4.5 Resistant cultivars Proteinase inhibitor gene sequences have been inserted into oilseed rape plants in an attempt to create transformed plants resistant to insect pests. Proteinase inhibitors bind specifically to target gut proteinases and thus inhibit the ability of the insect to digest proteins, which may result in reduced growth rate and death. Four families of insect proteinases are known: serine proteinases, cysteine proteinases, aspartyl proteinases and carboxypeptidases. Emphasis to date has been placed on the use of inhibitors of serine proteinases since this class constitutes the major proteolytic activity present in Lepidoptera. Coleoptera, on the other hand, predominantly use cysteine proteinases.

The resistance of a transgenic line expressing constitutively the cysteine proteinase inhibitor oryzacystatin I (OCI) was assessed against the cabbage stem flea beetle. The levels of OCI expression in the transformed line averaged 0.2% and 0.05% of total soluble protein in leaves and petioles, respectively. Adults fed identically on leaf discs from control and transformed plants and larvae showed greater weight gain when reared on transformed than control plants. In vitro analyses showed that the larvae use both cysteine and serine poteinases for protein digestion, and that all the cysteine proteolytic activity is OCI- sensitive. Larvae from transgenic plants exhibited increased cysteine and serine proteolytic activity in response to OCI. Rape co-expressing high

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levels of both cysteine and serine inhibitors may be more effective (Girard et. al. 1998).

4.6 Future work Our studies into the role of glucosinolates and their metabolites on rape pests suggest two strategies for improving the glucosinolate content of oilseed rape for pest resistance. The first involves rape lines with low constitutive but high induced glucosinolate levels. The second involves rape lines with a high proportion of glucosinolate types that do not catabolise to isothiocyanate, particularly the higher alkenyl isothiocyanates (Bartlet, Williams & Pickett, 1999). Opportunities to test plant material differing in their glucosinolate profiles will be sought. Recent GS analysis of rape lines (in P10309) has revealed two that lack detectable levels of any isothiocyanate-releasing GSs and these will be tested for their attractiveness to rape pests.

5.0 Natural enemies There are potential benefits to be gained from incorporating the conservation of natural enemies of pests into Integrated Pest Management Strategies and of finding ways of enhancing their activity. IPM requires that any insecticides used should exert maximal control over targeted pests but minimal effects on the environment and, in particular, on the natural enemies of the pests against which they are applied. The study of the natural enemies of the pests of oilseed rape in the UK is in its infancy; little is known about the status, biology or potential for control of the parasitoids, the predators or the pathogens that attack the pests.

5.1 Parasitoids Study of the parasitoids attacking the pod pests in UK rape crops, was initiated in 1991 through the LINK Programme (LK 0402 co-funded by MAFF and HGCA): Parasitoids of seed weevil and pod midge on winter oilseed rape, a collaborative project with Central Science Laboratory and Advisory and Development Agriculture Service (Alford et al., 1995) and subsequently continued in this MAFF Project. Studies have focussed on the identity, biology and phenology of the parasitoids of the cabbage seed weevil and the brassica pod midge.

5.1.1 Parasitoids of the cabbage seed weevil The LINK study found three parasitoid species that attacked the cabbage seed weevil in the UK, namely: Trichomalus perfectus and Mesopolobus morys (Hymenoptera: Pteromalidae), both ectoparasitoids of the larvae, and Microctonus sp. (Hymenoptera: Braconidae), an endoparasitoid of the adults.

5.1.1.1 Trichomalus perfectus The parasitic chalcid wasp, T. perfectus, was the most widespread, abundant and effective parasitoid attacking the weevil, killing up to 71% of its host in some winter rape crops (Murchie, 1996). The effects on T. perfectus of commercial applications of alphacypermethrin and of triazophos to control the weevil were investigated over two years. Triazophos had a detrimental effect on T. perfectus. It was applied at the time that parasitoids were searching for weevil larvae in which to lay their eggs, and it killed both adult T. perfectus and their host larvae. Alphacypermethrin was less harmful than triazophos, probably because it was applied before the main migration of T. perfectus into the crop. Thus temporal targeting of insecticide to avoid the peak flight period of the parasitoid enhances its potential for bio-control (Murchie, Williams & Alford, 1997).

5.2.1.1 Microctonus sp. A male endoparasitoid was reared from a field-collected cabbage seed weevil adult. The specimen was sent to the taxonomist Dr. M. Shaw, Royal Museum of Scotland for identification and description; it was identified as a species of Microctonus, tentatively either Microctonus melanopus or Microctonus aethiops, but no conclusive identification was possible without a female specimen. If the species reared is M. melanopus it is a new species record for the UK.

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5.1.2 Parasitoids of the brassica pod midge We have reared four parasitoids from the pod midge, belonging to the families Eulophidae (Omphale clypealis), Ceraphonidae (Aphanogmus and Ceraphron spp.) and Platygastridae (Platygaster subuliformis).

5.1.2.1 Platygaster subuliformis Platygaster subuliformis, the most abundant parasitoid of the brassica pod midge at Rothamsted is a new species record for the UK (Murchie, Polaszek & Williams, 1999). At Rothamsted, it parasitises up to 74% of overwintered midge cocoons (Murchie, 1996); its status elsewhere in the UK is not known. The high percentage parasitism suggests that this species has considerable impact on midge populations and thus potential for incorporation into IPM strategies for the crop.

Observations on the life-history of P. subuliformis have shown that it is an egg-larval parasitoid attacking midge eggs within the pods of oilseed rape. Each female parasitises several eggs inside the infested pod, laying a single egg in each and several females can parasitise the same egg cluster. The parasitoid is a koinobiont as its egg develops only after the host is nearly full-grown and at the prepupa/pupa stage within a cocoon in the soil. On maturity, the parasitoid pupates within the host’s larval skin. A part of the parasitoid population emerges the same season whereas the remainder enters diapause. Diapause is spent in the soil inside midge cocoons. Mating occurs soon after emergence and females migrate to rape crops (Murchie, Polaszek & Williams, 1999).

Platygaster subuliformis have been reared from parasitized midge larvae collected from winter rape at weekly intervals during June. At 180C, adult midge emerged ca. 14 days after their larvae burrowed into sand to pupate while P. subuliformis emerged after 50 days. The proportions of midge and parasitoid larvae, from the weekly samples, that entered diapause, increased with later collection of the midge larvae: for example, from one crop, midge larvae entering diapause increased from 5 to 11 to 16 to 46% and similarly P. subuliformis increased from 52 to 63 to 75 to 93%. The greater proportion of P. subuliformis entering diapause on any given date is probably the result of the longer development time for the parasitoid than for the host and explains why the ratio of P. subuliformis to midge emerging is greater post- than pre-diapause. As midge larval development in the pod is about 14 days, egg to adult development for the parasitoid is about 64 days. Emergence of P. subuliformis from spring rape, pre-diapause, took ca. 41 days. Post-diapause, the midge and female parasitoid emerged at the same time, 21 days after being transferred from overwintering at 50C to 180C, whereas male parasitoids emerged about 8 days earlier. Thus it would appear that the parasitoid is one generation out of synchrony with its host pre-diapause but regains synchrony post-diapause (Murchie, Polaszek & Williams, 1999).

Assessing the impact of P. subuliformis on midge populations is thus complicated; both host (and possibly the parasitoid) have up to three generations each year and, as the season progresses, an increasing proportion of host larvae and parasitoids enter diapause. The number of generations of P. subuliformis attacking the midge remains uncertain; rearing studies showed that the parasitoid takes more than three times as long as its host to emerge pre-diapause suggesting that it may have fewer generations per year than its host. However, in the field spring emergence of P. subuliformis was well synchronised with that of its host, both appearing in the first week of May; the parasitoid increased through May and early June, decreased during mid-June and increased again in late June and early July (Murchie, 1996; Murchie, Polaszek & Williams, 1999).

The increased ratio of parasitoids to midges, post-diapause, may limit midge population expansion early in the next season; this avoids the criticism often leveled at natural enemies, that their impact on pests comes too late to prevent damage to the crop.

5.1.2.2 Omphale clypealis Omphale clypealis is a larval endoparasitoid of the brassica pod midge but its biology is poorly known. The number of generations on rape crops in UK is uncertain but it is probably multivoltine like its host. It overwinters within midge larval cocoons in the soil and emerges in the spring. The females lay their eggs into

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mature midge larvae through the wall of the rape pod and the parasitoid larva feeds within the host during its larval and pupal stages. Rearing studies showed that, at 180C, some adults emerged 31 days after host larvae from spring rape had burrowed into the soil while others emerged post-diapause, emerging 42 days after their transfer from 50C for overwintering to 180C ; pre-diapause emergence from winter rape samples took 28 days, while post-diapause emergence took 37 days. Sex ratio was strongly female biased; 97% of parasitoids reared from spring rape and all those reared from winter rape were female (Murchie, 1996).

5.1.3 Parasitoids of cabbage stem flea beetle Information on the parasitoids that attack the cabbage stem flea beetle is particularly sparse. A species of Microctonus (both male and female specimens), has been reared from the adults, and specimens have been sent to taxonomists (Dr. E. Haeselbarth, Munich via Dr. Shaw, Royal Museum of Scotland) for identification and description; although superficially similar to Microctonus aethiops it appears to be a previously undescribed species.

5.1.4 Sampling methods for parasitoids

5.1.4.1 Omni-directional flight trap New methods for selectively sampling, monitoring and manipulating the parasitoids have been investigated. A new selective omni-directional flight trap was designed to catch small flying insects in a preservative to eliminate the time-consuming sorting and deterioration of small insects from water traps. The trap has three main parts: yellow PVC fins leading into a funnel unit capable of housing a volatile dispenser and surmounted by the collecting jar containing alcohol. The trap was used to investigate the responses of pests and parasitoids to host plant volatiles (section 4.3).

5.1.4.2 Malaise trap with time-sorter A novel mechanism that time-sorts insect catches into two-hourly samples has also been developed (Murchie et al., 2000). It uses a 24 h-rotation quartz clock motor powered by a single 1.5V battery to rotate a funnel directing positively phototactic insects from a malaise trap into consecutive sample tubes. The mechanism was used to derive the diel flight periodicity of brassica pod midge (Murchie et al., 2000). It also caught numerous parasitoid species; these samples still need to be processed.

5.2 Pathogens There is potential for the incorporation of pathogenic organisms into integrated pest management strategies for oilseed rape.

5.2.1 Metarhizium anisopliae The entomopathogenic fungus, Metarhizium anisopliae, has been shown in the laboratory and in semi-field studies at Rothamsted (MAFF Project P10309) to be pathogenic for cabbage stem flea beetle, cabbage seed weevil and pollen beetle; it is much less pathogenic to beneficial insects, such as the honeybee. Metarhizium anisopliae is therefore a potential control agent for these pests if an efficient means of dissemination of fungal spores to the target insects could be developed.

In field-cage experiments in winter- and spring-sown crops of oilseed rape, we have now shown that honeybees will effectively disseminate M. anisopliae spores to the flowers of oilseed rape to infect pollen beetles feeding from the flowers (Butt et al.,1998). The honeybees foraged on flowering oilseed rape from hives fitted with inoculum dispensers containing dry conidia (from isolate V245, isolated from Finnish soil) mixed with Biobeads. A greater mortality of pollen beetles occurred in treated plots than in control plots (Table 2). The mortality (61% on winter rape, 100% on spring rape) was greatest during peak flowering, when the feeding activity of both bees and beetles from the flowers was maximal, providing optimal conditions for inoculum dissemination and infection. Conidial sporulation occurred on a significant proportion of the dead pollen

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beetles. There was no evidence of any adverse effect on the honeybee colonies.

Table 2. Mortality (%) of pollen beetles on spring oilseed rape when exposed to honey bees with and without the entomogenous fungus M. anisopliae

Sample date Beetles only Beetles + Bees Beetles + Bees + Fungus 24.6.97 23 23 99 1.7.97 10 8 69 8.7.97 8 3 27 15.7.97 7 8 60

5.3 Future work Relatively little is known about the role of natural enemies in controlling the pests of oilseed rape. Research to date indicates that some pests can have a high incidence of parasitism suggesting potential for integrating natural control into IPM strategies for the crop. Future research will continue to identify the key natural enemies, primarily hymenopterous parasitoids and predatory carabids, for each of the five target pests of oilseed rape in the UK. It will seek to improve knowledge of their biology, ecology and phenology, assess their potential for control, improve knowledge of their population genetics and dynamics at various spatial scales and develop ways of conserving their populations and enhancing their effectiveness. Like their hosts, parasitoids migrate into the rape crop from their overwintering sites each spring but parasitoid phenology on the crop may differ from that of the host. Appropriate temporal targeting of insecticides can conserve parasitoids. The phenology of the flight periods of all key parasitoid species in relation to crop growth stage and weather conditions is needed to help define spray windows for insecticide applications timed to minimise mortality of key parasitoids. For IPM, particularly for manipulation of parasitoid behaviour to enhance efficacy, the effect of chemical cues on habitat and host location by parasitoids needs further investigation.

6.0 Population ecology and dynamics The pests of oilseed rape and their parasitoids migrate into the crop from their overwintering sites each spring or autumn but there is little information about either on the landscape scale. Although there have been publications in recent decades on the spatio-temporal distributions of the pests of oilseed rape, in most studies, the spatial information collected was limited, statistical analysis was minimal and parasitoids were not included. The distances over which they disperse to or from their overwintering sites is not known; neither is the effect of landscape structure on parasitization rates.

6.1 Spatial and temporal distributions Targeting insecticide treatments to crop area, as well as in time, offers potential for parasitoid protection. In the UK, most crops of winter rape are treated with insecticide to kill the cabbage seed weevil, either prophylactically or when threshold numbers are reached, and most treatments are applied over the entire area of the crop. Our previous studies of the distribution of the weevil on winter rape, by sampling along line transects, showed that, during immigration to the crop in April and May, the weevil infests crop edges more than crop centres and that the proportion at the crop edge gradually diminishes as adults spread further into the crop during June and July. It has been suggested that their numbers could be effectively reduced by applications of insecticide to crop borders only. We have shown that the most important parasitoid to attack the weevil in the UK, T. perfectus, migrates to winter rape during May, 2-4 weeks later than its host, but its spatial distribution in the crop, relative to that of its host, was unknown. Further, previous spatial information collected on the pests was limited and statistical analysis was minimal. More detailed information on the spatio-temporal distributions of pest and parasitoid were needed.

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The cabbage seed weevil and, simultaneously its parasitoid T. perfectus have now been sequentially sampled in two winter oilseed rape crops, using traps in two-dimensional arrays of spatially-referenced sampling points.

In the winter rape crop (Murchie, Williams & Perry, 1999), the distributions of pest and parasitoid within the crop were investigated between May and July using yellow water-traps arranged on a grid system. A randomisation test, which compared the observed arrangement of trap counts with permuted arrangements, was used to quantify the observed tendency of the weevil to congregate at the edges of the crop as it migrated into the crop; this tendency declined during the season. The weevil was distributed at the edge of the crop only during the early phase of its immigration in May. Parasitoid adults immigrated later than weevil adults and did not aggregate at the edge of the crop except briefly during the early phase of immigration. No association between distributions of pest adults and parasitoid adults was found. The distributions suggest that application of insecticide to the crop borders alone, early during flowering, could be an effective way of spatially targeting the densest part of the weevil population on the crop.

In the second winter rape crop, the methodology of SADIE (Spatial Analysis by Distance IndicEs), is being used to characterise and compare spatial patterns of pest and parasitoid distributions across time, between species, between sexes and between adults and larvae. Spatio-temporal patterns of seed weevil distribution appear to be complex. Preliminary analyses suggest that weevil larvae and parasitoid larvae are both aggregated near field edges and are associated with the spatial distribution of adult female weevils. Relationships between the spatial distributions of pests and their associations with plant growth and yield have also been determined and await analysis.

6.2 Population genetics The understanding of spatio-temporal patterns of pest and parasitoid distributions within the agricultural environment is limited by the difficulty of tracking the movement of individuals or populations, which underlie these patterns. Genetic techniques and primers, developed for the aphid parasitoid Diaeretiella rapae, under MAFF Project CE 0405, may have potential for the study of the genetic variability within species of parasitoid of some rape pests. An investigation was therefore initiated into the feasibility of using these markers to define local populations of parasitoids and to study their movements within the landscape. Microsatellite primers are being tested to establish whether these loci are variable and have potential to separate populations; to date four primer pairs have been tested with promising results. Further work will test ten specimens each of each parasitoid (P. subuliformis, O. clypealis, P. morionellus, P. interstitialis, Tersilochus heterocerus) for polymorphism of these microsatellites.

6.3 Future work Further information on the spatio-temporal distributions and movements of all key parasitoids in relation to those of their hosts, both within-field and on a landscape scale, would help improve our understanding of parasitoid ecology and lead to ways of enhancing their effectiveness. There is no information about the population dynamics of key parasitoids on a landscape scale, for example, about the distance over which they disperse to or from their overwintering sites. Landscape structure has been shown to affect parasitisation rates and proximity of field margin strips and fallow to increase parasitisation of pollen beetle. There appears to be potential for on-farm management of parastoid populations but more information on their population dynamics is first needed.

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7.0 Acknowledgements

This project has: 1.benefitted scientifically from collaboration with the following: · University of Perugia (insect morphologists: N. Isidoro, R. Romani, M. Solinas, P. Scubla) funded by the Ministry for University and Scientific Technological Research (MURST), Italy; a Bilateral Project between Consiglio Nazionaly delle Recherche, Italy, and The British Council (Scienze Agrarie 06), the European Union (ERBAGROTIND Contract no. AIR-CT94-8752); Royal Society; International Scientific Interchange Scheme · Wroclaw Agricultural University (entomologists: A. Dudzic, M. Kelm, Z. Klukowski, B. Walczak) funded by Wroclaw University Special Grant, Rothamsted International Fellowship) · INRA, Laboratoire de Biologie Cellulaire, INRA de Versailles (molecular biologists: M. Bonado-Bottino, C. Girard, L. Jouanin, B. Zaccomer) and Laboratoire de Neurobiologie Comparee des Invertebres, INRA de Bures sur Yvette (entomologists: M. Le Metayer, M. Pham-Delegue) funded by the Fondation d'Entreprise Limagrain, France. · Max-Planck Institut fuhr Verhalten Physiologie, Seeweisen (J. Ziesmann) EU Fellowship (AIR CT94-8752) · CABI Bioscience (insect taxonomist: A. Polaszek) • John Innes Centre (molecular biologist: R. Mithen)

2. been underpinned by MAFF (PSD) projects : · P10308 Semiochemical-based control of pests and diseases of oilseed rape (pheromone chemists: M. Blight, A. Mudd, J.A. Pickett, L. Smart, L. Wadhams, C. Woodcock & insect pathologists: T. Butt, L. Ibrahim) · P10309 Regulation of secondary metabolism in oilseed rape for semiochemical-based pest and disease control (biochemists: Kiddle, R. Wallsgrove)

3. been complemented by MAFF (Arable Crops) projects : · P10307 Insect behaviour control using semiochemicals · LK 0402 MAFF-HGCA The status and potential of parasitoids of seed weevil and pod midge on winter oilseed rape ADAS (D. Alford, B. Emmett, Green, Raw) and CSL (Walters) · CE 0405 Study of the movement and gene flow between local populations of aphids and hymenopterous parasitoids using molecular markers (allozymes and DNA) (insect molecular biologists: C. Brookes, H. Loxdale)

4. been supported by basic science from BBSRC-funded programmes at IACR-Rothamsted on biomanagement, insect behaviour and biomathematics (N. Carreck, S. Clark, M. Kenward, P. Lane, M. Martin, M. Mugglestone, J. Perry)

8.0 Technology transfer

Information from this project has also been disseminated via: 1. The Home-Grown Cereals Authority The work on natural enemies of pests in UK rape, initiated through the LINK Programme (LK 0402) co-funded by MAFF and HGCA: Parasitoids of seed weevil and pod midge on winter oilseed rape, and continued in this project, showed that T. perfectus is an effective bio-control agent against cabbage seed weevil larvae and that temporal targeting of insecticide to avoid the peak flight period of the parasitoid would enhance their potential for control (Murchie, Williams & Alford, 1997). As a result of this work, we proposed a commercially viable low-cost strategy for the management of seed weevil populations on winter rape, that minimises insecticide use and avoids post-flowering insecticide treatment (Alford et al. 1996). This strategy and information from this Project promoting conservation of parasitoids was publicised in the HGCA magazine for growers ‘Crops: Theme Review Pests’ April 1995 and is now incorporated in the HGCA Guidelines to Oilseed Rape Growers,

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published in 1999.

2. Decision Support System for Oilseed Rape Pests developed by CSL (MAFF Project AR0201) Data from AR0203 and LK 0402 have been incorporated into the Decision Support System for Oilseed Rape Pests (DORIS) developed by CSL (MAFF Project AR 0201) which advocates pesticide use compatible with minimum impact on the parasitoids of the weevil. DORIS contains encyclopaedic information on the biology, ecology, pesticides and suitable control tactics for each pest, partly derived from AR0203.

3. EU Concerted Action (1997-2000) ‘Biocontrol of Oilseed Rape Insect Pests’ (BORIS) This sought to minimise pesticide use and environmental impact by the development and promotion of biocontrol strategies involving parasitoids, predators and pathogens. It provided an excellent forum for the exchange of information between participants from the main rape-growing countries in Europe. It held winter workshops and summer meetings on technological exchange, disseminated information widely through its Web page and Newsletters and produced a database on biocontrol agents in rape accessible via the Internet (http://www.entom.slu.se/res/boris.html). The project leader of AR0203 collated the information on parasitoids Europe-wide for this database; this includes information from AR0203.

4. IOBC/wprs Working Group on Integrated Control in Oilseed Crops This holds workshops every two years, which provide a forum for information exchange. The Project Leader is convenor of the entomologists in this group.

5. IOBC/wprs Working Group on Breeding for Resistance to Insects and Mites This holds workshops every two years, which provide a forum for information exchange. Elspeth Bartlet from Rothamsted is a member of this group.

6. Presentations The list of Proceedings papers indicates national and international meetings where oral and poster papers were presented. Presentations were also made regularly to the media/farming press, visiting scientists, agronomists, agricultural consultants, farmers at open days and field visits at Rothamsted, The Royal Agricultural Show, Stoneleigh and elsewhere.

9.0 Publications

Refereed research & review papers

Bartlet, E. (1996) Chemical cues to host-plant selection by insect pests of oilseed rape. Agricultural Zoology Reviews 7, 89-116.

Bartlet, E., Blight, M.M., Lane, P. & Williams, I.H. (1997) The responses of the cabbage seed weevil Ceutorhynchus assimilis to volatile compounds from oilseed rape in a linear track olfactometer. Entomologia experimentalis et applicata 85, 257-262.

Bartlet, E., Mithen, R. & Clark, S.J. (1996) Feeding of the cabbage stem flea beetle, Psylliodes chrysocephela on high and low glucosinolate cultivars of oilseed rape. Entomologia experimentalis et applicata 80, 87-89.

Bartlet, E., Kiddle, G., Martin, A., Williams, I.H. & Wallsgrove, R. (1999) Wound-induced increases in the indolyl glucosinolate content of oilseed rape and their effect on subsequent herbivory. Entomologia experimentalis et applicata 91, 163-7.

Butt, T.M., Carreck, N.L., Ibrahim, L. & Williams, I.H. (1998) Honeybee mediated infection of pollen beetle

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(Meligethes spp.) by the insect-pathogenic fungus, Metarhizium anisopliae. Biocontrol Science and Technology 8, 533-538.

Ferguson, A.W., Solinas, M., Ziesmann, J., Isidoro, N., Williams, I.H. , Scubla, P., Mudd, A., Clark, S.J. & Wadhams, L.J. (1999a) Identification of the gland secreting oviposition-deterring pheromone in the cabbage seed weevil, Ceutorhynchus assimilis, and the mechanism of pheromone deposition. Journal of Insect Physiology 45,687-699.

Ferguson, A.W., Ziesmann, J., Blight, M.M., Williams, I.H., Wadhams, L.J., Clark, S. J., Woodcock, C.M. & Mudd, A. (1999b) Perception of oviposition-deterring pheromone by the cabbage seed weevil (Ceutorhynchus assimilis). Journal of Chemical Ecology 25(7), 1655-1670.

Girard, C., Le Metayer, M., Zaccomer, B., Bartlet, E., Williams, I., Bonade-Bottino, M., Pham-Delegue, M. & Jouanin, L. (1998) Growth stimulation of beetle larvae reared on a transgenic oilseed rape expressing a cysteine proteinase inhibitor. Journal of Insect Physiology 44, 263-270.

Mudd, A. Ferguson, A.W., Blight, M.M., Williams, I.H., Scubla, P., Solinas, M. & Clark, S.J. (1997) Extraction, isolation and composition of the oviposition-deterring secretion of cabbage seed weevil Ceutorhynchus assimilis. Journal of Chemical Ecology 23, 2227-2240.

Murchie, A. K., Smart, L.E. & Williams, I.H. (1997) Responses of Dasineura brassicae Winn. (Diptera: Cecidomyiidae) and its parasitoids Platygaster subuliformis Kieffer (Hymenoptera: Platygastridae) and Omphale clypealis Thomson (Hymenoptera: Eulophidae) to traps, baited with organic isothiocyanates, in the field. Journal of Chemical Ecology 23, 917-926.

Murchie, A.K. Williams, I.H. & Alford, D.V. (1997) Effects of commercial insecticide treatments to winter oilseed rape on parasitism of Ceutorhynchus assimilis Paykull (Coleoptera: Curculionidae) by Trichomalus perfectus (Walker) (Hymenoptera: Pteromalidae). Crop Protection 16, 199-202.

Murchie, A.K., Polaszek, A. & Williams, I.H. (1999) Platygaster subuliformis (Kieffer) (Hymenoptera: Platygastridae) new to Britain, an egg-larval parasitoid of the brassica pod midge Dasineura brassicae (Diptera: Cecidomyiidae). Entomologists' Monthly Magazine 135, 217-222.

Murchie, A.K., Williams, I.H. & Perry, J. (1999) Edge distributions of Ceutorhynchus assimilis Paykull (Coleoptera:Curculionidae) and its parasitoid Trichomalus perfectus (Walker) (Hymenoptera: Pteromalidae) in a crop of winter oilseed rape (Brassica napus L.). Biocontrol, 44(4), 379-390.

Murchie, A.K., Burn, D.J., Kirk, W.D.J. & Williams, I.H. (2000) A novel mechanism for time-sorting insect catches, and its use to derive the diel flight periodicity of brassica pod midge Dasineura brassicae (Diptera: Cecidomyiidae) (submitted for publication).

Proceedings papers Alford, D.V., Emmett, B.J., Green, M.R., Murchie, A.K., Williams, I.H., Raw, K.A. & Walters, K.F.A. (1995) Field experiments to assess the status and importance of Trichomalus perfectus, a parasitoid of seed weevil on winter oilseed rape in the UK. Proceedings of the 9th International Rapeseed Congress, Cambridge, UK, 4-7 July1995, pp.1301-1303.

Alford, D.V., Murchie, A.K. & Williams, I.H. (1995) Observations on the impact of standard insecticide treatments on Trichomalus perfectus, a parasitoid of seed weevil in winter oilseed rape in the UK. OILB/wprs Bulletin 18(4),122-126.

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Alford, D.V., Walters, K.F.A. , Williams, I.H. & Murchie, A.K. (1996) A commercially viable low-cost strategy for the management of seed weevil populations on winter oilseed rape in the UK. Proceedings of the British Crop Protection Conference, Pests & Diseases, Brighton of the, pp. 609-614.

Bartlet, E., Williams, I.H. & Pickett, J. A. (1999) The ideal glucosinolate profile for pest resistance in oilseed rape. OILB/wprs Bulletin 22(10), 13-17.

Butt, T.M., Carreck, N.L. Ibrahim, L. & Williams, I.H. (1999) Honey bee mediated infectin of pollen beetle (Meligethes aeneus Fab.) by the insect-pathogenic fungus, Metarhizium anisopliae. International Symposium on Biological Control Agents in Crop and Animal Protection, 24-8 August 1999. The University of Wales, Swansea. (Abstract).

Carreck, N.L., Butt, T.M., Ibrahim, L., Pell, J.K. & Williams, I.H. (1999) The use of honey bees to disseminate an insect pathogenic fungus for control of insect pests on oilseed rape. Proceedings of the XXVI International Congress of Apimondia, Vancouver, Canada 12-17 September 1999, 236-237.

Cook, S., Murray, D. A. & Williams, I.H. (1999) Pollen beetle, Meligethes aeneus Fabricius, incidence in the composite hybrid winter oilseed rape, Synergy. 10th International Rapeseed Congress 26-29 September, Canberra, Australia.

Ferguson, A.W., Kenward, M., Williams, I.H., Clark, S.J., Kelm, M. & Dudzic, A. (1995) Interactions between the cabbage seed weevil (Ceutorhynchus assimilis Payk.) and the brassica pod midge (Dasineura brassicae Winn.) infesting oilseed rape pods. Proceedings of the 9th International Rapeseed Congress, Cambridge, UK, 4-7 July 1995, pp. 679-681.

Inyang, E.N., Butt, T.M., Bartlet, E., Ibrahim, L. & Archer, S. (1998) Factors affecting the susceptibility of Phaedon cochleariae to the entomogenous fungus Metarhizium anisopliae. Proceedings of the International Symposium of the British Mycological Society, 5-9 april 1998, Southampton, p.62.

Kelm, M., Walczak, M. & Williams, I.H. (1995) Changes in growth and yield parameters of winter rape as a result of injury by the stem weevil (Ceutorhynchus quadridens). Proceedings of the 9th International Rapeseed Congress, Cambridge, 4-7 July 1995, pp. 673-675.

Murchie, A.K., Williams, I.H. & Smart, L.E. (1995) Responses of brassica pod midge (Dasineura brassicae) and its parasitoid (Platygaster sp.) to isothiocyanates. Proceedings of the 9th International Rapeseed Congress, Cambridge, UK, 4-7 July 1995, pp. 1028-1030.

Murchie, A.K. & Williams, I.H. (1998) A bibliography of the parasitoids of the cabbage seed weevil (Ceutorhynchus assimilis Paykull) IOBC/wprs Bulletin, Integrated control in Oilseed Crops, 21(5), 163-9.

Murchie, A.K. & Williams, I.H. (1998) Effect of host size on the sex of the parasitoid Trichomalus perfectus (Walker) (Hymenoptera: Pteromalidae). IOBC/wprs Bulletin, Integrated control in Oilseed Crops, 21(5), 177- 181.

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Pickett, J.A., Butt, T.M., Doughty, K.J., Wallsgrove, R.M. & Williams, I.H. (1995) Minimising pesticide input in oilseed rape by exploiting natural regulatory processes. Proceedings of the 9th International Rapeseed Congress, Cambridge, 4-7 July 1995, pp. 565-571.

Walczak. B., Kelm, M., Klukowski, Z., Smart, L.E., Ferguson, A. W. & Williams, I.H. (1998) The effect of trap design and 2-phenylethyl isothiocyanate on catches of stem weevils (Ceuthorynchus pallidactylus Marsh. and C. napi Gyll.) in winter oilseed rape. IOBC/wprs Bulletin, Integrated control in Oilseed Crops, 21(5), 141- 146.

Williams, I.H. (1998) Insect-plant interactions: pollinators and pests. In Interactions insectes-plantes. Actes des 6e journées du groups de travail relations insectes-plantes. ed. Bournville, R., 4-5 June 1997, Lusignan, France, pp. 9-12.

Technical Reports Alford, D.V., Williams, I.H., Murchie, A.K. & Walters, K.F.A. (1995) The status and potential of the parasitoids of the seed weevil and pod midge on winter oilseed rape. HGCA Oilseeds Research Project no. OS 14 (Project no. OS15/1/91), August 1995, 89pp.

Popular articles Williams, I.H.& Murchie, A.K. (1995) The role of parasitoids. Agronomist, Spring 1995, 13-15.

Theses Bartlet, E. (1995) Semiochemicals and behaviour of the oilseed rape pests Ceutorhynchus assimilis and Psylliodes chrysocephela. University of London.

Murchie, A.K. (1996) Parasitoids of cabbage seed weevil and brassica pod midge in oilseed rape. University of Keele.

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