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EVID4 Evidence Project Final Report (Rev. 06/11) of 27

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Evidence Project Final Report

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Project identification

1. Defra Project code PS2134

2. Project title

Use of refuge traps to disseminate entomopathogenic fungi for the control of adult vine weevil

3. Contractor organisation(s)

ADAS UK Ltd ADAS Boxworth, Battlegate Road, Boxworth, Cambridge, CB23 4NN. University of Warwick School of Life Sciences, Gibbet Hill Road, Coventry CV4 8UW UK

54. Total Defra project costs £ 45,441

(agreed fixed price)

5. Project: start date ................ 27/10/2010

end date ................. 30/11/2011

mailto:[email protected]


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Please confirm your agreement to do so. ................................................................................... YES NO

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Executive Summary

7. 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.

Vine weevil (Otiorhynchus sulcatus) remains one of the most serious problems in both soft fruit and nursery stock industries. Despite non-chemical options for the control of vine weevil larvae, including use of entomopathogenic nematodes and the entomopathogenic fungus (EPF) Metarhizium anisopliae (Met52) control of adult weevils is currently reliant on insecticide applications. These applications are difficult, as they need to be applied at dusk, and are often incompatible with integrated pest management programmes. This project was completed as a proof of principle to test a novel control strategy targeted at adult vine weevils. The strategy is based on the fact that adult weevils are nocturnal and hide within suitable refuges during the day. In addition, adult weevils exhibit aggregation behaviour, thought to be in response to chemicals in the weevil droppings (frass). This means that weevils are often found in groups within refuges. These two aspects of adult vine weevil behaviour together with the fact that like the larval stages, adult vine weevils are also subject to attack by EPF means that refuges containing spores of a suitable EPF may provide effective control of this pest. However, to be effective this approach would require a simple robust artificial refuge trap into which the spores of the EPF can be held and for the EPF itself to effectively kill adult weevils and be disseminated throughout the weevil population.

Objective 1: Confirm that refugia have the potential to spread an entomopathogen throughout adult vine weevil populations through use of fluorescent powders Pilot experiments to develop a suitable simple trap design

Pilot experiments tested two simple plastic crawling insect/cockroach traps and a novel design made from yew wood. The novel yew wood trap was included as previous work has indicated that adult vine weevils respond positively to the odour of this plant. In a single trap (no choice) experiment each trap design was placed separately into a gauze cage (50 x 50 x 50 cm), which in turn was placed into a glasshouse compartment. Twenty weevils were then released into the gauze cage. The proportion of weevils found within a trap 24 and 48 hours after the weevils had been released was recorded. Similarly, a two-trap (choice) experiment was completed by placing two traps (one each of two different designs) into a cage. In both the single trap and two trap experiments the best performing design was the Roguard trap, a simple plastic crawling insect/cockroach trap. This design met the requirements of being relatively cheap, robust, waterproof and with a central dish is well suited to containing a spore formulation of an EPF.


Additional pilot experiments were completed using the Roguard trap design to test whether adding an odour to the trap increased its efficacy. Traps were coated with a paste of yew leaves, weevil frass or a combination of both yew paste and weevil frass. The yew and weevil frass were selected based on previous research with adult vine weevils, which has shown that vine weevil adults respond positively to these odours. Experiments were completed as described for the two-trap experiments. The addition of yew paste or weevil frass significantly increased the proportion of weevils found within Roguard traps compared to clean traps 48 hours after the weevils were released into cages. However, the addition of yew paste also significantly reduced the proportion of weevils found within Roguard traps compared to clean traps 24 hours after the weevils were released into cages. This suggests that the composition or concentration of the volatiles produced is important in determining behavioural responses to refuge traps. A combination of yew paste and weevil frass significantly increased the proportion of weevils found within Roguard traps 24 hours after the weevils were released into cages. In a final pilot experiment the effect of increasing the density of Roguard traps on the proportion of weevils found within all traps 24 hours later was investigated. Using the same experimental set-up a simple relationship was found where the proportion of weevils found within all traps placed into a cage increased with increasing number of traps (between one and four). Determine potential efficacy of refuge traps in spreading an EPF

Under semi-field conditions within large ‘tent’ cages (145 x 145 x 152 cm) in a ventilated polytunnel the potential efficacy of Roguard traps to disseminate an EPF was investigated. These experiments were completed using fluorescent powder placed into the traps in order to identify weevils that had either entered a trap or come into contact with weevils that had. Cages were prepared either to simulate a hardy nursery stock crop (potted Euonymus fortunei) or a soft fruit crop (strawberry plants in grow-bags). Twelve Roguard traps were placed into each cage (six on the floor and six on the plant pot or grow-bag. Forty weevils were released into each cage and the number of weevils coming into contact with the fluorescent powder was recorded after seven days. Results from these two experiments indicated that after seven days approximately 90% of weevils had either entered a trap or come into contact with a weevil that had. In an additional experiment using the same experimental design, 35 weevils were released into each cage. After 24 hours five weevils coated in fluorescent powder were also released into each cage. When all of the weevils were collected after seven days 75% of weevils not originally coated in powder were found to have come into contact with at least one of the five powder-coated weevils.

Objective 2: Assess a range of commercial and non-commercial EPF for efficacy, speed of kill and sub-lethal effects against adult vine weevil Initial screen The susceptibility of adult weevils to eight isolates of entomopathogenic fungi (EPF) were investigated under laboratory conditions in a replicated bioassay. Adult weevils were treated directly with a high dose (10

8 conidia ml

-1) and maintained within bioassay chambers at 20°C, 16:8 L:D for 28 days. Mortality was

assessed daily and egg production counted weekly. Adult weevils were susceptible to all of the isolates examined and six of the isolates killed 100% of the weevils treated within 24 days. Two isolates of Beauveria bassiana killed 90% of the adult weevils within 10 days of treatment. All of the isolates examined produced conidia on the adult cadaver. Trap experiment The effect on adult weevils of conidia of EPF dispensed within Rogard traps was investigated in a laboratory bioassay. Three isolates of fungi were evaluated in an experiment done over 28 days at 20°C, 16:8 L:D. Weevil mortality was assessed daily and egg production counted weekly. Fungal conidia were observed on the weevils within 4 hours. One isolate of Metarhizium anisopliae killed 50% of the weevil population within 14 days. There was evidence of avoidance behaviour by adult weevils with one isolate of Beauveria bassiana.

Objective 3: Confirm if sporulating EPF infecting adult vine weevils can infect healthy weevils within a refuge The ability of fungal infection to be acquired by healthy adult weevils from dead, sporulating weevils was investigated under laboratory conditions. One sporulating cadaver was placed within a bioassay chamber containing groups of healthy adult weevils. The chambers were maintained at 20°C, 16:8 L:D for 28 days. Weevil mortality was assessed daily and egg production counted weekly. Adult weevils acquired conidia from the cadavers in all of the treatments used in the experiment. The amount of sporulation on the cadavers was an important factor in determining the transmission of the fungus.


Options for new work The project has demonstrated the principle of using artificial refuge traps to disseminate an EPF for control of adult vine weevil. Further work is required to test the efficacy of identified EPF isolates under more realistic environmental conditions. Improved understanding of adult vine weevil behaviour in relation to refuges under different cropping environments is also required.

Project Report to Defra

8. 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 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 Exchange).


Background

Vine weevil (Otiorhynchus sulcatus) remains one of the most serious pest problems in both soft fruit and nursery stock industries. In strawberry crops approximately 8% of all insecticide use in the crop is targeted against the adult stage of this pest, while damage to raspberry and blackberry crops has increased considerably over recent years. Despite current controls against both adult and larval stages of this pest, losses are estimated to be in excess of £10 million/year for strawberry and as a result of pesticide regulation changes are estimated to rise to over £10 million/year for raspberry (Wynn, 2010). For hardy nursery stock more than 50% of the insecticide-treated area is for the control of vine weevil. Damage is caused both by the adults, resulting in characteristic leaf notching, and larvae, which feed on plant roots, corms and tubers. As the larvae are root pests and the adult weevils are nocturnal an infestation may pass unnoticed for some time until leaf notching is noticed or plant growth begins to flag or plants show signs of wilting by which time they will have been damaged beyond recovery. Vine weevils attacking nursery stock have for a number of years been controlled through the use of controlled-release applications of chlorpyrifos, thiacloprid or imidicloprid. However, these soil-applied insecticides are not approved for use on fruit crops. Autumn-applied drenches of chlorpyrifos may be applied to strawberry crops though for control of vine weevil larvae. Insecticide use against vine weevil in hardy nursery stock is extensive, accounting for more than 50% of the insecticide treated area (Pesticide Usage Survey Report 211). In addition to these chemical controls for vine weevil larvae, growers may also use biological control products based on the insect parasitic nematodes Heterorhabditis bacteriophora, H. megidis or Steinernema kraussei. The nematode products are used as a drench applied to vine weevil-infested soil and can be used in both soft fruit and nursery stock production. More recently the entomopathogenic fungus (EPF) Metarhizium anisopliae has been developed as a soil incorporated biological control against vine weevil larvae (Horticulture Link HL017; Shah et al, 2007). The control of vine weevil adults is currently limited to insecticide applications. In strawberry crops the most widely used insecticide has been the pyrethroid, bifenthrin, however, this active ingredient is not being supported for Annex 1 listing under 91/414/EEC and so will be unavailable to growers after 2011. In an attempt to address this problem, HDC are currently supporting ADAS evaluation of insecticides and insecticide mixtures for the control of adult vine weevil (SF HNS 112). However, insecticide application against adult vine weevil is made difficult by the nocturnal behaviour of this pest, which necessitates evening applications when the weevils become active. In addition, insecticide applications themselves may have a negative impact on biocontrol agents used against other pests and naturally occurring beneficials such as ground beetles that predate on vine weevil adults (Cross et al., 2001). Adult vine weevil behaviour may present opportunities with which to develop novel control strategies. In particular adult weevils are nocturnal and hide within suitable refuges during the day. Suitable refuges include the underside of plant pots, under the rims of pots, within leaf litter, under plastic mulches etc. In addition, adult weevils exhibit aggregation behaviour, thought to be in response to chemicals in the weevil frass (van Tol et al., 2004), and as such are often found in groups within a suitable refuge. Ongoing HDC-funded research is seeking to characterise this aggregation pheromone (project SF HNS 127). Adult vine weevils are also susceptible to infection by entomopathogenic fungi,(Moorhouse, 1990). This fact together with aggregation behaviour within refuges presents an opportunity to target adult vine weevils within artificial refuges containing an EPF. To be effective this approach would require development of an inexpensive refuge trap that is readily used by adult weevils, that is also robust and waterproof and able to hold a formulation of a suitable EPF. The approach would then work directly with weevils entering an artificial refuge where they would pick up spores of the EPF and subsequently die. However, the approach would also work indirectly as weevils coming into contact with fungal spores would not die immediately and so would have the potential to pass on spores to other weevils within other natural refuges. In addition, once dead the EPF infecting a weevil would begin to sporulate, further spreading the EPF within the weevil population. Direct and indirect dissemination of the pathogen through the use of refuge traps could then initiate an epizootic within the adult weevil population. This approach to controlling adult vine weevil would provide growers of both soft fruit and hardy nursery stock with a cost effective means of controlling this pest that is


both sustainable and compatible with integrated pest management programmes. To date the use of an EPF has not been investigated in field scale experiments for the control of vine weevil adults. However, work to control the German cockroach (Blatella germanica), another gregarious insect, has indicated that M. anisopliae can be rapidly spread through a population through horizontal transmission (Quesada-Moraga et al., 2004). The following study was, therefore, completed as a proof of principle investigation to test this potential means of controlling vine weevil adults through the use of artificial refuge traps containing a suitable EPF.

Aims & Objectives

1. Confirm that refuges have the potential to spread an entomopathogen within populations of adult vine weevils .

1.1. Pilot experiments to develop a suitable simple trap design

1.2. Determine potential efficacy of refuge traps in spreading an EPF

2. Assess a range of EPF (including isolates used in commercial products) for efficacy, speed of kill and sub-lethal effects against adult vine weevil.

3. Confirm if sporulating EPF infecting adult vine weevils can infect healthy weevils within a refuge.

Objective 1: Confirm that refuges have the potential to spread an entomopathogen throughout adult vine weevil populations through use of fluorescent powders

Materials & methods

Adult vine weevil culture

Field-collected adult vine weevils were initially supplied by S. J. Cockbill Vine Weevils (Herefordshire, UK) in January 2011. Additional field-collected adult vine weevils were collected from commercial strawberry and raspberry crops in May and June 2011. Adult vine weevils were kept in 1.5 l plastic pots. The lids of these pots were perforated in order to provide ventilation. The base of each pot was lined with tissue paper, an additional ball of damp tissue paper provided a source of moisture, a piece of corrugated cardboard provided a refuge and fresh yew leaves (Taxus baccata) provided a food source. Tweny-five to 30 weevils were placed into each pot, which in turn were placed in a controlled temperature laboratory at 21°C. Pots were cleaned once a week taking care to remove any dead or dying weevils. If a weevil was found to have been killed by an EPF the infected weevil was kept in order to identify the pathogen while the remaining weevils within the pot were discarded in order to maintain the health of the culture.

Pilot experiments to develop a suitable simple trap design

A series of experiments were completed in a research glasshouse compartment between January and April 2011 at ADAS Boxworth. The temperature within the glasshouse compartment was maintained at approximately 20°C by providing additional heat and shading/ventilation when required. Each experiment was completed using gauze cages (50 x 50 x 50 cm). Into these cages were placed one or more experimental traps, a damp cotton wool pad as a source of moisture, yew leaves as a food source and 20 vine weevil adults. Three simple refuge trap designs were tested in these experiments; two commercial crawling insect/cockroach traps (Roguard and Roachmaster) and a novel design made from yew wood (‘WeeVille’) (Figure 1). The three refuge trap designs were selected to be robust, waterproof, suitable for the addition of spores of EPF and relatively cheap. The novel design made from yew wood was included as adult vine weevils have been shown to respond positively to the odour of this plant (van Tol et al., 2002).


Figure 1. Simple vine weevil traps tested; a) Roguard trap (BASF), b) Roachmaster traps (Russell IPM), c) WeeVille trap

Single trap (no choice) experiment

Six gauze cages were prepared as previously described and placed into the glasshouse compartment. A single vine weevil trap was placed into each cage so that the arrangement within a cage was as illustrated in Figure 2. Twenty adult vine weevils collected from the culture were released from the centre of each cage during daylight hours. After 24 hours the number of weevils within each trap was counted. The trap was replaced for a further 24 hours when the number of weevils within each trap was again counted. The experiment was completed using a randomised block design testing the three traps; Roguard, Roachmaster and WeeVille. The six gauze cages were arranged in two blocks of three and the experiment was repeated on three dates.

50 cm

Refuge trap

Weevil release point &

damp cotton wool pad

Yew leaves

Gauze cage

Figure 2. Arrangement within each gauze cage for single trap experiment.

Two trap (choice) experiment

This experiment again used the gauze cages placed into the glasshouse compartment. Two different vine weevil traps were placed into each cage so that the arrangement within a cage was as illustrated in Figure 3. Twenty adult vine weevils collected from the culture were released from the centre of each cage during daylight hours. Numbers of weevils within each trap was again recorded 24 and 48 hours after the weevils were released.


50 cm

Refuge trap b



Yew leaves

Refuge trap a

Gauze cage

Figure 3. Arrangement within each gauze cage for two trap experiment.

Two experiments were completed using the Roguard trap as the best performing design from the single trap experiment; Roguard + Roachmaster and Roguard + WeeVille. Each experiment was replicated six times on two dates, 12 replicates in total. For each experiment the position of each trap design was alternated to avoid any directional bias.

Olfactory experiments

Roguard traps (the best performing trap design from the single and two-trap experiments) were used in these three experiments, which were completed using the previously described gauze cages placed in the glasshouse compartment. Roguard traps were used clean, coated in a yew paste, coated in weevil frass or coated in a combination of yew paste and weevil frass. To coat the traps in a yew paste, one gram of fresh yew leaves was first ground using a pestle and mortar. The yew paste was then used to coat the inside of the previously clean Roguard trap. To coat the traps in weevil frass, 10 adult vine weevils were collected from the culture and placed into a small ventilated plastic pot lined with filter paper. Fresh yew leaves were provided as a food source. The prepared pot was placed in a controlled temperature laboratory at 21°C for 48 hours. The frass produced by the weevil during this period (approx. 0.4 g) was used to coat the inside of the previously clean Roguard trap. Coating the traps with both the yew paste and weevil frass used the methods described but based on initial results the traps were first coated with the yew paste then after 24 hours the frass was added. For each experiment the arrangement within the cage was as illustrated in Figure 4. Again, 20 adult vine weevils collected from the culture were released from the centre of each cage during daylight hours. The number of weevils within each trap was recorded 24 and 48 hours after the weevils were released.

50 cm

Roguard trap coated with

yew paste, weevil frass or

yew paste + weevil frass



Yew leaves

Clean Roguard

trap

Gauze cage

Figure 4. Arrangement within each gauze cage for olfactory experiments.

Three olfactory experiments were completed using Roguard traps. In each experiment two Roguard traps were used (as shown in Figure 4) with one clean trap and one trap coated with yew paste, weevil


frass or both yew paste and weevil frass. The three experiments were; clean trap + yew paste coated trap, clean trap + weevil frass coated trap and clean trap + yew paste and weevil frass coated trap. Each experiment was replicated six times on two dates, 12 replicates in total. For each experiment the position of the clean and coated trap was alternated to avoid any directional bias.

Trap number experiment

This experiment was completed using Roguard traps placed into the gauze cages within the glasshouse compartment. Based on the number of traps (one, two, three or four) placed into each cage the arrangement within the cage was adapted in order to evenly space the traps and sources of food (Figure 5). Again, twenty adult vine weevils collected from the culture were released from the centre of each cage during daylight hours. The total number of weevils within the trap(s) was recorded 24 hours after the weevils were released. The experiment was completed using a randomised block design testing four treatments; one, two, three and four traps. The treatments were replicated on nine dates.

Approx. 50 cm

Roguard trap



Yew leaves

1 Trap

3 Trap 4 Trap

2 Trap

Figure 5. Arrangement within each gauze cage for the trap number experiment.

Determining potential efficacy of refuge traps in spreading an EPF

A series of experiments were completed in a ventilated polytunnel at ADAS Boxworth. Each experiment was completed using large gauze ‘tent’ cages (145 x 145 x 152 cm) see Appendix 1. Cages were prepared by first placing Euonymus fortunei (cv. Emerald Gaiety) plants in 1.5 l pots or strawberry (cv. Elsanta) plants in one meter grow-bags into the cages. Next adult vine weevils collected from the culture were released into the cage and allowed to acclimatise for 24 hours. Roguard traps were used in these experiments as the best performing design from the pilot experiments. Temperatures within one of the tent cages were recorded during each experiment using a TinyTag data logger.

The aim of these experiments was to identify whether each weevil released into the cage had either entered a trap during the day or night or come into contact with a weevil that had. To do this hydrophobic fluorescent powders (Swada, Stalybridge, UK) were used. The fluorescent powders were either added to Roguard traps (approx. 0.2 g to each trap) or used to coat weevils which were then released into the cage (see Appendix 2). Fluorescent powders were selected for use in these experiments in order to visualise potential spread of spores from an EPF. Any contact a weevil had with the fluorescent powder was usually clearly visible. However, by placing each weevil under a UV light even very small amounts of fluorescent powder on the insect’s body were clearly visible.


Euonymus fortunei experiment

This experiment was completed using 16 potted Euonymus fortunei plants arranged into four blocks of three plants and a central block of four plants within each cage (Figure 6). Forty adult vine weevils were released into the cage and allowed to acclimatise for 24 hours. Next 12 Roguard traps were placed within the cage. Six of the traps had been filled with a yellow powder and these were arranged on the floor of the cage. The other six traps were filled with a pink powder and these were arranged on the surface of the soil in six pots (Figure 6). Seven days after introducing the Roguard traps the weevils were carefully collected into separate tubes taking care not to contaminate them with fluorescent powder. Each weevil was then carefully inspected under a UV light for fluorescent powder. The experiment was replicated four times on two dates, eight replicates in total.

Trap on floor of

cage

Trap on plant pot

Euonymus plant

Approx. 145 cm

Figure 6. Arrangement of plant pots and Roguard traps filled with either yellow or pink fluorescent powder within each cage containing Euonymus plants.

Strawberry experiment

This experiment was completed using one meter grow-bags, each planted with five strawberry plants. Two grow-bags were placed within each cage (Figure 7). Forty adult vine weevils were released into the cage and allowed to acclimatise for 24 hours. Again 12 Roguard traps were placed into the cage with six traps filled with yellow powder placed on the floor and six traps filled with pink powder placed close to plants on the top of the grow-bags (Figure 7). Each weevil was inspected for fluorescent powder seven days after the traps were introduced. The experiment was replicated four times on one date and three times on a second date, seven replicates in total.

Strawberry grow-

bag

Approx. 145 cm

Trap on floor of

cage

Trap on grow-bag

Figure 7. Arrangement of plant pots and Roguard traps filled with either yellow or pink fluorescent powder within each cage containing strawberry grow-bags.

Weevil contacts experiment

This experiment was completed using 16 potted Euonymus fortunei plants as previously described (see


Figure 6). Twelve Roguard traps were arranged again as previously described but were not filled with fluorescent powder. Thirty-five adult vine weevils were released into the cage and allowed to acclimatise for 24 hours. Next, five adult vine weevils were coated with fluorescent powder and then released into the cage. Seven days after releasing the powder coated vine weevils all of the weevils within the cage were collected and inspected for any fluorescent powder as previously described. The experiment was replicated four times on two dates, eight replicates in total.

Analysis

The data were analysed using a generalized linear model, which calculated the proportion of weevils within traps or covered with fluorescent powder. The data were logit transformed before the analysis of variance was completed. This method corrected for any non-normality in the data.

Results & discussion

Pilot experiments to develop a suitable simple trap design

Single trap (no choice) experiment

Results are summarised in Figure 8. Trap design significantly affected the proportion of adult vine weevils within a trap during daylight hours both 24 (χ2 = 17.80, P < 0.001) and 48 hours (χ2 = 21.06, P<0.001) after the weevils were introduced. Individual comparisons between the treatments shows that a significantly higher proportion of weevils were found within the Roguard or Roachmaster traps compared to the WeeVille traps after 24 and 48 hours. There was no significant difference in the proportion of weevils found within the Roguard or Roachmaster traps after 24 hours. However, after 48 hours the proportion of weevils within Roguard traps was significantly higher than in Roachmaster traps.

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a

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a

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a

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Figure 8. Mean percent adult vine weevils within each trap design presented in a no-choice environment 24 and 48 hours after weevils were released into cages. Different letters indicate a significant difference (P < 0.05).

Two trap (choice) experiment

Results are summarised in Figure 9. A significantly higher proportion of weevils were found within Roguard traps than in Roachmaster traps when placed together within a cage. This difference was seen both after 24 (χ2 = 7.48, P = 0.006) and 48 hours (χ2 = 15.38, P < 0.001). Similarly, significantly more weevils were found within Roguard traps when placed together with WeeVille traps within a cage after 24 (χ2 = 55.02, P < 0.001) and 48 hours (χ2 = 58.01, P < 0.001).


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Figure 9. Mean percent adult vine weevils within; a) Roguard + Roachmaster traps, and b) Roguard + WeeVille traps presented in a choice environment 24 and 48 hours after weevils were released into cages. Different letters indicate a significant difference (P < 0.05).

It is not clear why adult vine weevils should show a clear preference for Roguard traps in both the single trap and two-trap experiments. However, the design of the Roguard trap does mean that the four entrance holes in this trap are much darker than in either the Roachmaster or WeeVille traps, which had a more open design. It is also the case that the plastic Roguard trap is robust, waterproof and is relatively inexpensive (<£0.50/trap). An additional advantage of the Roguard trap design is the central area surrounded by a raised plastic ridge providing a convenient container in which to place a EPF spore formulation. It was also the case that adult weevils entering a Roguard trap favoured the centre of the trap (see Appendix 3).

Olfactory experiments

Results are summarised in Figure 10. A significantly higher (χ2 = 26.62, P < 0.001) proportion of weevils was found within clean Roguard traps than in Roguard traps coated with yew paste 24 hours after the weevils were released into cages containing both trap types. However, 48 hours after releasing the weevils into the cage the opposite result was found with a significantly higher (χ2 = 6.68, P = 0.01) proportion of weevils within the traps coated with yew paste compared to clean traps. A higher proportion of weevils was found within frass coated Roguard traps than in clean Roguard traps 24 and


48 hours after the weevils were released into the cage. While there was no significant difference in the proportion of weevils within these traps after 24 hours there was after 48 hours (χ2 = 5.56, P = 0.018). Finally, a significantly higher (χ2 = 9.58, P = 0.002) proportion of weevils was found within yew and frass coated Roguard traps compared to clean traps 24 hours after the weevils were released into the cage. No significant difference was found after 48 hours.

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Figure 10. Mean percent adult vine weevils within; a) clean Roguard + yew coated Roguard traps, b) clean Roguard + frass coated Roguard traps, and c) clean Roguard + yew and frass coated Roguard traps presented in a choice environment 24 and 48 hours after weevils were released into cages. Different letters indicate a significant difference (P < 0.05).


The positive response to traps coated in weevil frass supports the work of van Tol et al. (2004) who suggested that the aggregation behaviour of adult weevils is connected to components in the weevil frass. Indeed, ongoing HDC-funded research is seeking to characterise the vine weevil aggregation pheromone (project SF HNS 127). The results for the yew paste coated traps were more variable than for the frass coated traps, with a negative effect seen after 24 hours and a positive effect after 48 hours. Without more detailed analysis of the chemicals produced by the yew paste or weevil responses to these chemicals it is only possible to speculate as to why results after 24 and 48 hours differed so markedly. However, it seems likely that the concentration and composition of volatile chemicals would be important in determining weevil behavioural responses. The fact that the yew paste increased the proportion of weevils found within traps after 48 hours does support the work of van Tol et al. (2002) who found that adult vine weevils respond positively to odours from this plant. Work is also underway to exploit behavioural responses of adult vine weevils to host-plants in a lure, which may be used in a weevil trap (van Tol pers. comm.).

Trap number experiment

Results are summarised in Figure 11. The proportion of weevils found within traps placed within each cage was significantly affected (χ2 = 21.07, P < 0.001) by the number of traps. The trend was for a higher proportion of weevils to be found within traps when the number of traps was also increased. Indeed, individual comparisons between treatments supports this with significantly more weevils caught by two traps compared to one trap and significantly more weevils caught by four traps than two.

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Figure 11. Mean numbers of adult vine weevils caught when different numbers of Roguard traps were placed into cages. Different letters indicate a significant difference (P < 0.05).

Determining potential efficacy of refuge traps in spreading an EPF

Euonymus fortunei experiment

Seven days after introducing the Roguard traps containing the fluorescent powder 94% of adult vine weevils were recovered from the cages. Of those recovered 88% had come into contact with fluorescent powder. Of those weevils coming into contact with fluorescent powder, 17% had only yellow powder, 49% had only pink powder and 34% had both yellow and pink powder. The number of weevils with pink fluorescent powder, either on its own or together with yellow powder was significantly higher (χ2 = 47.37, P < 0.001) than the number of weevils with yellow powder, again on its own or together with pink powder. Mean temperatures during block one of this experiment were 23.5°C (daytime) and 11.5°C (night time) and in block two 26.3°C (daytime) and 13.0°C (night time).

Strawberry experiment

Seven days after introducing the Roguard traps containing the fluorescent powder 92% of adult vine weevils were recovered from the cages. Of those recovered 94% had come into contact with fluorescent powder. Of those weevils coming into contact with fluorescent powder 36% had only yellow


powder, 13% had only pink powder and 51% had both yellow and pink powder. The number of weevils with yellow fluorescent powder, either on its own or together with pink powder was significantly higher (χ2 = 23.23, P < 0.001) than the number of weevils with pink powder, again on its own or together with yellow powder. Mean temperatures during block one of this experiment were 22.8°C (daytime) and 11.0°C (night time) and in block two 24.1°C (daytime) and 13.3°C (night time). It is interesting to note that in the Euonymus experiment the traps placed close to the plants appear to have been more heavily used than those placed on the floor while in the strawberry experiment the reverse appears to be true. This suggests crop specific weevil behaviour in terms of movement between the plant and suitable refuges. Understanding these differences would be important in order to use optimal placement of artificial refuges within different cropping environments.

Weevil contacts experiment

Seven days after introducing the adult vine weevils coated in fluorescent powder 75% of the adult vine weevils recovered (excluding those originally coated in powder) had come into contact with fluorescent powder. Mean temperatures during block one of this experiment were 26.7°C (daytime) and 14.9°C (night time) and in block two 28.0°C (daytime) and 13.6°C (night time). Results from this experiment suggest that once a weevil has entered a refuge trap containing spores of the EPF it has the potential to pass those spores on to other weevils. It is likely, although not confirmed in this experiment, that this transmission occurs in shared use of other refuges. The extent to which spores are passed from weevil to weevil would depend on the speed of kill of the fungus as well as any sublethal effects that fungal infection may have on weevil behaviour. Previous studies with different species of insect have for example shown that following infection with an EPF feeding and oviposition may be affected (Noma & Strickler, 2000; Quesada-Moraga et al., 2004; Torrado-León et al., 2006). Work with another gregarious species of insect, the German cockroach, has indicated that individuals infected and killed by an EPF may also effectively spread the pathogen to previously healthy individuals (Quesada-Moraga et al., 2004).

Conclusions

The Roguard trap was the most effective design tested for use as an artificial adult vine weevil refuge. This design is robust, waterproof, inexpensive and capable of carrying spores of a suitable EPF.

Olfactory cues, either weevil frass or host plant volatiles, have potential to improve the efficacy of Roguard traps.

Under semi-field conditions Roguard refuge traps disseminated a fluorescent powder to approximately 90% of a weevil population within seven days.

Weevil behaviour in relation to artificial refuge traps appears to differ depending on the cropping environment.

Objective 2: Assess a range of EPF (including isolates used in commercial products) for efficacy, speed of kill and sub-lethal effects against adult vine weevil.

Materials & methods

Adult vine weevil culture

Adult weevils were obtained from the ADAS culture as described in Objective 1. Adult vine weevils were kept in 1.5 l plastic pots and maintained as described previously.


Fungal culture – storage and production

Stock cultures of the fungal isolates were stored on porous plastic beads in liquid nitrogen vapour (Chandler, 1994). Laboratory cultures were grown on Sabouraud dextrose agar (SDA) slopes and maintained in a refrigerator at 4°C for up to six months. Subcultures for laboratory experiments were

prepared on SDA from the slope cultures and incubated at 23 1°C for 10-12 d in the dark. For the initial screen conidia were harvested in sterile 0.05% Triton X-100 and filtered through sintered glass thimbles (40 - 100 μm pore). Conidia were then enumerated using an improved Neubauer haemacytometer and aliquots (10 ml) were prepared at a concentration of 108 conidia.ml-1. For the trap experiment (2.2, below) conidia were grown on SDA plates as described previously and conidia were scraped from the plates as a powder. The number of conidia per dry weight of powder was calculated by adding 0.1g of conidia powder to 10ml of 0.05% Triton X-100 and the suspensions enumerated using an improved Neubauer haemacytometer.

2.1: Initial screen

The susceptibility of adult vine weevils to eight isolates of entomoptahogenic fungi was measured in a single dose laboratory bioassay. The isolates (Table 1) were selected from the WHRI culture collection based on their availability as commercial biopesticides or virulence predictions in bioassays against larval O. sulcatus (Moorhouse,1990).

Table 1. Fungal isolates used in the initial screen.

Isolate† Species Host/Substrate Collection site

432.99a Beauveria bassiana Anthonomus grandis USA 433.99b Beauveria bassiana - - 342.92 Beauveria bassiana Otiorhynchus sulcatus UK 1749.11 275.86c

Beauveria bassiana Metarhizium anisopliae

Otiorhynchus sulcatus Cydia pomonella

UK Germany

535.02 Metarhizium anisopliae Otiorhynchus sulcatus Germany 534.02 Metarhizium anisopliae Otiorhynchus sulcatus UK 528.02 Metarhizium anisopliae Otiorhynchus sulcatus UK

†Isolate number in the WHRI culture collection

(a) Isolate forms the active ingredient in the proprietary mycopesticide ‘Naturalis’ (Troy Biosciences Inc., 113 South 47th Ave., Phoenix, AZ

850433, USA). (b) Isolate forms the active ingredient in the proprietary mycopesticide ‘BotaniGard’ (Mycotech Corporation, PO Box 4109, Butte, MT 59702, USA). (c) Isolate forms the active ingredient in the proprietary mycopesticide ‘Met52’ (Novozymes, Hallas Allé 4400 Kalundborg. Denmark).

Groups of five adult weevils were inoculated by immersion in suspensions of conidia (108 ml-1, prepared as described previously) for 10 s. Excess suspension was removed by filtration through filter paper under vacuum. The weevils were left to air dry on the filter paper for 1 h and then transferred to a bioassay chamber (see Appendix 4) consisting of a ventilated plastic box (180mm long x 140mm high x 120 mm deep) lined with tissue paper and containing a Roguard cockroach trap, and damp tissue paper and Taxus baccata (yew) leaves. The chambers were maintained at 20°C, 16:8 light:dark and high (> 90%) relative humidity and the yew leaves and damp tissue replaced ad libitum. Numbers of living and dead weevils were counted daily for a total of 28 days. Dead weevils were removed and incubated on damp filter paper within Petri dishes at 23°C, and the production of fungal conidia on these cadavers was recorded. The numbers of eggs laid were counted weekly for the duration of the experiment. The viabilities of conidia of the fungal isolates were measured in germination tests on nutrient agar prior to bioassays. The experiment was done according to a randomised block design. Each block comprised eight fungal isolates, with three blocks in total. Each block contained two control chambers (treated with 0.05% Triton X-100). 2.2:Trap Experiment

The susceptibility of adult weevils to EPF when applied as a conidia powder within the traps selected in Objective 1 was measured in a replicated laboratory bioassay. The isolates (Table 2) were selected on


their virulence in the initial screen and their availability as commercial products.

Table 2. Fungal isolates used in the trap experiment.

Isolate† Species Host/Substrate Collection site

433.99a Beauveria bassiana - - 1749.11 275.86b

Beauveria bassiana Metarhizium anisopliae

Otiorhynchus sulcatus Cydia pomonella

UK Germany

†Isolate number in the WHRI culture collection

(a) Isolate forms the active ingredient in the proprietary mycopesticide ‘BotaniGard’ (Mycotech Corporation, PO Box 4109, Butte, MT 59702, USA). (b) Isolate forms the active ingredient in the proprietary mycopesticide ‘Met52’ (Novozymes, Hallas Allé 4400 Kalundborg. Denmark).

The conidia powders were added to Roguard traps (0.4 g to each trap). Groups of five adult weevils were removed from the culture and then transferred to a bioassay chamber (Figure 15) consisting of a ventilated plastic box (180 mm long x 140 mm high x 120 mm deep) lined with tissue paper and containing a treated Roguard trap, and damp tissue paper and yew leaves. The chambers were maintained at 20°C, 16:8 light:dark and high (> 90%) relative humidity and the yew leaves and damp tissue replaced ad libitum. Numbers of living and dead weevils were counted daily for a total of 28 days. Dead weevils were removed and incubated on damp filter paper within Petri dishes at 23°C, and the production of fungal conidia on these cadavers was recorded. The numbers of eggs laid were counted weekly for the duration of the experiment. The experiment was done according to a randomised block design. Each block comprised three fungal isolates, with three blocks in total. Each block contained two control chambers (traps treated with talc).

Analysis

The analyses of variance/deviance were carried out separately for each day due to the fact that data between days may be correlated. The percentage mortality data was angular transformed to stabilize the variation prior to analysis of variance. Regression analyses were also carried out where appropriate. Data were fitted to percentage mortality at each time point for each treatment using a Gompertz curve. A LT50 and a LT90 value along with a lag time were calculated using the parameters from each fitted equation. An ANOVA was carried out on the LT50s, LT90s and Lag times and LSDs used to compare means. The rate of egg production was estimated by dividing the total number of eggs laid by the total number of weevil days. Weevil days were defined as the total number of days on which individual weevils were alive. The total weevil days are therefore the sum of the weevil days from each individual for each treatment over the whole time course of the experiment. An ANOVA was carried out on the rate of egg production and LSDs used to compare the means.


2.1: Initial Screen

All of the isolates caused greater mortality of adult weevils than controls (Figure 12, Table 3). After 28 days only two (Beauveria bassiana 342.92 and 432.99) of the eight isolates tested did not result in 100% mortality of adult weevils. There was no significant difference (P<0.05) in the lag time between the isolates but two of the isolates (B. bassiana 433.99 and 1749.11) killed 50% and 90% of the population significantly (P<0.05) faster than the other isolates in 6.82, 7.52 and 9.20, 9.55 days respectively (Table 4). All of the isolates tested produced conidia on adult cadavers (see Appendix 5). The majority of sporulation occurred between the body segments and leg joints. Egg laying in control chambers was consistent throughout the experiment with 8-10 eggs per weevil being laid weekly (Figure 13A). The total number of eggs counted per bioassay chamber was significantly (P<0.05) reduced when weevils were treated with three of the isolates (528.02, 535.02 and 275.86) at day 7. Mean rate of egg laying increased with the two most virulent isolates (Figure 13B),


which suggests that weevils continue egg laying until death occurs. These results are difficult to interpret as it could be taken as evidence that fungal infection increases egg production per weevil, however, it is difficult to get conclusive evidence of the effect of fungal infection on egg production where weevils are maintained within a group. The only way to do this would be to house weevils individually and count egg production daily.

Figure 12. The cumulative daily mean % mortality of O. sulcatus treated with 8 isolates of entomopathogenic fungi. Table 3. Pathogenicity of 8 isolates of entomopathogenic fungi applied to O. sulcatus at 1 x 108 ml-1, 7, 14, 21 and 28 days post inoculation (dpi). Numbers in parenthesis represent the transformed data.

Treatment Estimated % mortality

Isolate 7 dpi 14 dpi 21 dpi 28 dpi

528.02 19.4 (26.15) 97.6 (81.14) 100 (90) 100 (90)

534.02 9.2 (17.71) 73.8 (59.21) 97.6 (81.14) 100 (90)

535.02 5.1 (13.08) 60.6 (51.14) 100 (90) 100 (90)

275.86 9.2 (17.71) 90.7 (72.29) 100 (90) 100 (90)

1749.11 40.0 (39.23) 100 (90) 100 (90) 100 (90)

433.99 60.0 (50.77) 100 (90) 100 (90) 100 (90)

342.92 9.2 (17.71) 54.0 (47.3) 60.6 (51.14) 67.7 (55.37)

432.99 9.2 (17.71) 46.6 (43.08) 46.6 (43.08) 67.7 (55.37)

Control 0.6 (4.43) 0.6 (4.43) 2.4 (8.86) 7.0 (15.39)

LSD (p < 0.05; df=19)

(between isolates) (between control)

24.66 21.36

21.40 18.53

16.89 14.62

18.39 15.93


Table 4. Lag, median and 90% lethal time of 8 isolates of entomopathogenic fungi applied to O. sulcatus at 1 x 108 ml-1.

Isolate Lag time LT50 LT90

528.02 6.13 10.29 15.38

534.02 5.96 10.30 15.55

535.02 7.64 12.28 17.91

275.86 6.99 10.25 15.16

1749.11 6.03 7.52 9.55

433.99 5.07 6.82 9.20

342.92 5.37 12.44 > 28

432.99 5.04 11.28 > 28

Control 5.11 >28 > 28

LSD 3.725

(16 d.f., p =0.05)

4.436

(13 d.f., p=0.05)

4.330

(9 d.f., p=0.05)

A

B

Figure 13. Egg production per bioassay chamber over 28 days (A) and the mean rate of egg production. (B) by weevils treated with 8 isolates of entomopathogenic fungi applied at 1 x 108 ml-1.


(LSD= 1.248 (between treatments); LSD= 1.080 (between controls); P<0.05; df=19).

Trap experiment

All of the isolates examined caused significantly (P<0.05) greater mortality of adult weevils than controls after 28 days post infection (dpi). (Figure 14, Table 5). However only two of the isolates (M. anisopliae 275.86 and B. bassiana 433.99) resulted in significantly greater mortality at 14 dpi and only one isolate (M. anisopliae 275.86) at 7 dpi. Weevils were seen to be covered in fungal spores within four hours of setting up the experiment and fungal spores were seen on the floor of the bioassay chamber, indicating that they had been carried out of the trap by weevils. Isolate 1749.11 was not as effective in the traps as we had been expecting, based on the data from the initial screen. There was little evidence that weevils had visited the traps containing spores of isolate 1749.11 to the same extent as traps containing spores of the other fungal isolates, as indicated by the amount of weevil frass in the traps and the amount of conidial powder on the floor of the bioassay chamber outside the trap (see Appendix 6). Insect avoidance of pathogenic fungi has been described in other systems. Meyling & Pell (2006) demonstrated that the anthocorid bug, Anthocoris nemorum L. avoided leaf surfaces inoculated with the fungus B. bassiana. Coccinella septempunctata adults also exhibited avoidance behaviours in response to the fungus B. bassiana on leaf surfaces, in soil and in mycosed C. septempunctata (Ormond et al, 2011). Termites can detect the fungus M. anisopliae in soil and in infected conspecifics and it has been demonstrated that the detection and avoidance of B. bassiana and M. anisopliae by termites (Macrotermes michaelseni) depends on the virulence of the isolate; infective isolates are recognized and avoided (Mburu et al. 2009). It is likely that chemical cues are important in the detection of B. bassiana, which suggests the possibility of developing a fungus-based chemical repellent. Egg laying in control chambers decreased throughout the duration of the experiment from 18-20 eggs per chamber per week to six eggs per week by day 28 and so this may have masked any effect of treatment on egg laying (Figure 15A). No significant reduction (P<0.05) on weekly egg laying was observed until day 21 and 28 compared to the controls with all of the treatments with the exception of the low dose treatment of isolate 1749.11. No effect was observed on mean rate of egg production (Figure 15B).

Figure 14. The cumulative daily mean % mortality of O. sulcatus in the presence of entomopathogenic fungal baited Roguard traps.


Table 5. Pathogenicity of 4 isolates of entomopathogenic fungi applied as conidial powders within Roguard traps to O. sulcatus, 7, 14, 21 and 28 days post inoculation. Numbers in parenthesis represent the transformed data.



275.86 18.9 (25.78) 46.65 (43.08) 91.53 (73.08) 97.63 (81.14)

1749.11 (0.1) 0 (0) 9.25 (17.71) 26.20 (30.79) 26.2 (30.79)

1749.11 (pure) 0 (0) 2.37 (8.86) 5.12 (13.08) 18.91 (25.78)

433.99 0 (0) 26.20 (30.79) 46.65 (43.08) 75 (60)

Control 0 (0) 0 (0) 0 (0) 0.60 (4.43)

LSD (p < 0.05; df=11)

(between isolates) (between control)

17.79 15.38

32.70 28.32

28.61 24.78

31.92 27.65

A

B

Figure 15. Egg production per bioassay chamber over 28 days (A) and the mean rate of egg production per weevil with 3 isolates of entomopathogenic fungi applied as conidial powders within Rogard traps. (LSD= 0.386 (between treatments); LSD= 0.335 (between controls); P<0.05; df=11).


Conclusions

Adult vine weevils were susceptible to a range of entomopathogenic fungal isolates.

Six of the eight isolates tested killed 100% of the adult weevils treated within 28 days.

Two isolates of Beauveria bassiana were able to kill 90% of the weevils treated within 9.2 and 9.55 days respectively.

Results suggest that weevils continue egg laying until the point of death.

Entomopathogenic fungal conidia were easily applied to Roguard traps.

Under laboratory conditions entomopathogenic fungal conidia applied to Roguard traps gave 26% -97% control.

Weevil behaviour in relation to entomopathogenic fungal baited refuge traps appears to differ depending on the entomopathogenic fungi used.

Objective 3: Confirm if sporulating EPF infecting adult vine weevils can infect healthy weevils within a refuge

The aim of this experiment was to measure the ability of EPF infected weevil cadavers to spread infection to uninfected weevils. This is referred to as the “epizootic potential” of the fungus. The ability to spread infection from dead weevils could improve the efficacy and the persistence of a fungal pathogen and so would be a valuable attribute for a biocontrol fungus. The epizootic potential of EPF depends on their infectivity together with the amount of sporulation on the host cadaver, and on the subsequent ability of the fungus to spread to healthy insects (Carruthers & Soper, 1987). Fungi that cause natural epizootics rely on this ability to spread and persist in the host population, and it can also be important to microbial control with fungal pathogens (Hall & Papierok, 1982).

Materials & methods

Using the method described previously (2.1) weevils were infected with isolates of Beauveria bassiana (433.99 or 1749.11) or Metarhizium anisopliae (275.86). One sporulating cadaver per isolate was placed individually into a bioassay chamber (see Appendix 7) consisting of a ventilated plastic box (150 mm long x 125 mm high x 80 mm deep) lined with tissue paper and damp tissue paper and yew leaves as a water and food source and containing five healthy weevils. The chambers were maintained at 20°C, 16:8 light:dark and high (> 90%) relative humidity and the yew leaves and damp tissue replaced ad libitum. Numbers of living and dead weevils were counted daily for a total of 28 days. Dead weevils were removed and incubated on damp filter paper within Petri dishes at 23OC, and the production of fungal conidia on these cadavers was recorded. The number of spores produced on a cadaver were counted by placing sporulating individuals into aliquots (1 ml) of 0.05% Triton X-100 in 1 ml microcentrifuge tubes. Tubes were vortex-mixed for approximately 20 seconds to dislodge conidia from the integument. Conidia in the suspension were enumerated using an improved Neubauer haemacytometer. The numbers of eggs laid were counted weekly for the duration of the experiment. Controls contained only healthy weevils. The treatments were assessed in a randomised block design, with three blocks. Each block consisted of all four treatments.

Analysis

The analyses of variance/deviance were carried out separately for each day due to the fact that data between days may be correlated. The percentage mortality data was angular transformed to stabilize the variation prior to analysis of variance (ANOVA). A generalised linear model with a Poisson distribution was used to analyse the egg counts. The rate of egg production was estimated by dividing the total number of eggs laid per chamber by the total number of weevil days. Weevil days were defined as the total number of days on which individual weevils were alive. The total weevil days are therefore the sum of the weevil days from each individual


for each treatment over the whole time course of the experiment. An ANOVA was carried out on the rate of egg production and LSDs used to compare the means.


Only one of the isolates examined (Beauveria bassiana 1749.11) caused significantly (P<0.05) greater mortality than the control after 28 days (Figure 16, Table 6). There was evidence that the other isolates were able to kill healthy weevils in this way, however the considerable variation within the experiment masked this effect. This was probably a result of the reliance of healthy weevils having to come into contact with the infected cadaver before they can become infected and the fact that isolate 1749.11 often produced a greater number of spores on infected cadavers than the other isolates examined.

Egg laying in control chambers was initially low in this experiment (Figure 17A). All of the treatments had significantly (P<0.05) increased the number of eggs per chamber at day 7 compared with the control but there was no subsequent difference observed throughout the experiment. Weevils in the presence of a sporulating cadaver of isolate 433.99 had a significantly (P<0.05) higher rate of egg production than the control (Figure 17B) and may be indicative of the viability of the weevils in these chambers as higher control mortality was also observed.

Figure 16. The cumulative daily mean % mortality of O. sulcatus in the presence of one entomopathogenic fungal infected cadaver.

Table 6. Estimated % mortality of healthy weevils in the presence of one sporulating cadaver 7, 14, 21 and 28 days post inoculation. Numbers in parenthesis represent the transformed data.



275.86 0 9.25 (17.71) 61.98 (51.93) 80.57 (63.85)

1749.11 0 26.20 (30.79) 75 (60) 94.88 (76.92)

433.99 0 13.95 (21.93) 67.09 (54.99) 86.05 (68.07)

Control 0 9.25 (17.71) 32.91 (35.01) 32.91(35.01)

LSD

(p < 0.05; df=6)

* 33.01 42.51 39.55


Table 7. Lag and median lethal time of healthy weevils in the presence of one sporulating cadaver.

Isolate Lag time LT50

1749.11 12.32 17.8

433.99 11.73 17.4

275.86 9.49 18

Control 11.22 >28

LSD 6.557

(2 d.f., p =0.05)

13.520

(2 d.f., p=0.05)

Figure 17. Egg production per bioassay chamber over 28 days (A). The mean rate of egg production (B) by weevils in the presence of one sporulating cadaver. (LSD= 0.0.1029; P<0.05; df=6).


Conclusions

Sporulating cadavers produced 107 – 108 conidia per weevil.

Infected cadavers were able to transfer infection to healthy weevils and death and subsequent infection was observed within 10-14 days.

Results suggest that weevils continue egg laying until the point of death.

Presentation of results to scientific community

Tom Pope gave a presentation on the project results at the AAB conference ‘Advances in biological control’, Old Barn Hotel, Lincolnshire, 30 November 2011.

Possible future work

The project has demonstrated the principle of using artificial refuge traps to disseminate an EPF for control of adult vine weevil. Further work is required to test the efficacy of identified EPF isolates under more realistic environmental conditions as well as determining the number for traps per unit area required. In addition, the relative importance of the direct efficacy of refuge traps in disseminating an EPF as well as indirect effects such as weevil to weevil contacting passing on spores and sporulation from dead weevils needs to be understood. This information will provide information on the required density of refuge traps that may be required and the ability of this approach to initiate epizootics within vine weevil populations. More investigation is required to identify any sub-lethal effects of EPF as the results were not conclusive or not investigated in our initial experiments. In particular, more work is required to record the effect of EPF on oviposition rates per weevil. There are additional questions relating to adult vine weevil behaviour in relation to refuges under different cropping environments and response to different EPF isolates, such as potential repellent effects, is also required. Improved understanding of weevil behaviour within different crops is required if refuge traps are to be placed where they will be most effective.

Acknowledgements

ADAS fruit consultants for help with collecting adult vine weevils from commercial soft fruit farms.


References to published material

9. 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.

Carruthers, R. R. & Soper, R. S. (1987) Fungal diseases. In Epizootiology of insect diseases (Fuxa, J. R. & Tanada, Y. Eds.) New York, USA: John Wiley & Sons, Inc. pp. 357 –416

Chandler, D. (1994) Cryopreservation of fungal spores using porous beads. Mycological Research. 98: 525-526.

Cross, J. V., Easterbrook, M. A., Crook, A. M., Fitzgerald, J. D., Innocenzi, P. J., Jay, C. N. & Solomon, M. G. (2001) Review: natural enemies and biocontrol of pests of strawberry in northern and central Europe. Biocontrol Science and Technology. 22: 165-216.

Hall, R. A. & Papierok, B. (1982) Fungi as biological-control agents of arthropods of agricultural and medical importance. Parasitology. 84: 205-240.

Meyling, N. & Pell, J.K. (2006) Detection and avoidance of an entomopathogenic fungus by a generalist insect predator. Ecological Entomology 31: 162–171.

Moorhouse, E.R. (1990) The potential of the entomopathogenic fungus Metarhizium anisopliae as a microbial control agent of the black vine weevil Otiorhynchus sulcatus. Phd Thesis, University of Bath.

Mburu, D.M., Ochola, L., Maniania, N.K,, Njagi, P.G.N., Gitonga, L.M., Ndung’u, M.W.,,Wanjoya, A.K. & Hassanali, A. (2009) Relationship between virulence and repellency of entomopathogenic isolates of Metarhizium anisopliae and Beauveria bassiana to the termite Macrotermes michaelseni. Journal of Insect Physiology 55: 774–780.

Noma, T. & Strickler, K. (2000) Effects of Beauveria bassiana on Lygus hesperus (Hemiptera: Miridae) feeding and oviposition. Environmental Entomology. 29: 394-402.

Ormond, E.L., Thomas, A.P.M, Pell, J.K., Freeman, S.N. & Roy, H.E. (2011). Avoidance of a generalist entomopathogenic fungus by the ladybird, Coccinella septempunctata. FEMS Microbiology Ecology 77: 229–237

Quesada-Moraga, E., Santos-Quirós, R., Valverde-Garcia, P. & Santiago-Alvarez, C. (2004) Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (Anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). Journal of Invertebrate Pathology. 87: 51-58.

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