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SID 5 Research Project Final Report

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

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Project identification

1. Defra Project code PS2715

2. Project title

Monitoring of insecticide resistance and viruses in Myzus persicae in the UK

3. Contractororganisation(s)

Rothamsted Research, Harpenden, Herts AL5 2JQ, UK

                         

54. Total Defra project costs £ 59,238(agreed fixed price)

5. Project: start date................ 01 July 2008

end date................. 31 March 2009

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

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 In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain.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

(b) If you have answered NO, please explain why the Final report should not be released into public domain

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

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.The peach-potato aphid, Myzus persicae, is a major pest of many arable and horticultural crops in the UK. It is a potent vector of plant viruses and therefore needs to be controlled effectively through a combination of tactics including application of insecticides. This species also exemplifies the problems that can arise through the development of resistance to insecticides by insect pests. It has repeatedly evolved resistance to compounds used in the past, highlighting the importance of ensuring that newer insecticides, such as the neonicotinoid class of chemicals, retain their effectiveness for the longest period possible.

This project aimed to support nationwide initiatives to combat risks of neonicotinoid resistance by a continuation of monitoring, in the laboratory, of the responses of aphid samples collected from different crops during 2008 and early 2009. The primary objective was to characterise field samples of M. persicae for their response to neonicotinoids (exemplified by imidacloprid), and to combine the new data with those collected previously to investigate any potential directional shifts in susceptibility. These samples were also tested, using DNA-based assays, for the presence of the MACE and kdr mechanisms, conferring resistance to pirimicarb and pyrethroids, respectively. Two secondary objectives were: (i) to develop and validate a new bioassay for measuring aphid responses to neonicotinoids delivered to plants through systemic uptake (as opposed to topical application directly to aphids); and (ii) a pilot study monitoring the presence of aphid-transmitted viruses in M. persicae on crop and non-crop plants during the period Autumn 2008 – Spring 2009.

20 samples of M. persicae were characterised for resistance to imidacloprid and for their MACE and kdr genotypes. Data for imidacloprid were added to a time series extending back to autumn 2004, and showed no directional trend towards an increased frequency of aphids carrying reduced sensitivity to imidacloprid (up to ~ 10 fold resistance) or any aphids with significant resistance that may compromise control by neonicotinoids. Individuals with the former phenotype continue to be detected but seem of little or no practical importance under prevailing practices of neonicotinoid use. The frequencies of aphids with MACE and kdr remained high during 2008 despite evidence for a gradual decline in the latter in recent years, probably mediated through reduced use of pyrethroids against M. persicae. Both the MACE and kdr genes were only present in heterozygous form, implying that fitness costs constrain the build-up and/or Two bioassays were evaluated for measuring responses to systemically-applied neonicotinoids. One (‘excised leaf bioassay’) tested the response of aphids feeding directly on whole excised leaves of Chinese cabbage with their petioles immersed in insecticide solution throughout the bioassay. The other (‘leaf-disc bioassay’) tested the response of offspring of aphids placed on leaf discs cut from excised leaves previously placed in insecticide solution. Both these approaches yielded good dose-response relationships, but the excised leaf bioassay is being adopted as the method of choice due to its greater versatility, allowing behavioural responses to be investigated as well as survival per se.

It was notable that both systemic bioassays gave resistance factors for standard clones to neonicotinoids that were markedly lower those obtained using topical application. Therefore, the method of treatment (topical versus systemic) appears to affect the strength of selection and ‘resistance risk’. If extrapolated to the field, this implies that the current (unresolved) mechanism(s) of resistance will be more manifest in

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response to neonicotinoids applied as foliar sprays rather than seed or soil treatments. The previously exclusive use of systemic applications of neonicotinoids in the UK may have been a potent factor precluding the selection of resistance. If true, this scenario is threatened by the ongoing proliferation of foliar neonicotinoid registrations in the UK (and ultimately abroad), as well as indirect changes such as recommendations to spray thiacloprid as an alternative to pyrethroids for pollen beetle control on oilseed rape. The number of aphids caught in water pans in oilseed rape fields in the autumn of 2008 was far lower than in the two previous years and was too low to draw conclusions about the incidence of overwintering aphids and their virus content. Aphids were collected on weed species at four of 10 sites surveyed in Norfolk, Suffolk and Bedfordshire, and five sites yielded weed plants infected with TuYV. The weeds most commonly containing virus were Plantago spp., Senecio vulgaris and Viola arvensis. At present, the role of TuYV in constraining rape production is controversial but there is growing concern that warmer conditions during autumn and winter will encourage greater build up and survival of aphids throughout this period and an increase in potential virus sources leading to further reliance on insecticides; this could enhance the insecticide resistance risks. Results were used to inform and strengthen recommendations for preventing resistance that are being co-ordinated by the Pesticide Safety Directorate in close consultation with the UK's Insecticide Resistance Action Committee (IRAG-UK). The research on this topic from 2009 onwards has been subsumed into a new Sustainable Arable LINK project with participation from a large consortium of agrochemical companies and crop commodity organisations.

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

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

1. Background

Management policy for insecticide resistance needs strong scientific underpinning to inform regulatory processes and formulate risk mitigation tactics. Under EU Pesticide Directorate 91/414, resistance risk analyses have become an integral component of the approval process, providing greater scope for proactive measures to restrict the use of products in line with perceived risks of resistance developing. The development and implementation of monitoring programmes is an integral part of this process, alongside work to interpret any changes in the susceptibility of target pests and to evaluate management options.

Neonicotinoids (including imidacloprid, thiacloprid, thiamethoxam, clothianidin and acetamiprid) are the most important group of insecticides to be developed since the pyrethroids. The scale of their uptake has already led to damaging outbreaks of resistance in key pests such as the tobacco whitefly (Bemisia tabaci) and the rice brown planthopper (Nilaparvata lugens) (Nauen & Denholm, 2005). The major target pests in the UK are aphids including the highly adaptable peach-potato aphid, Myzus persicae. This attacks field and glasshouse crops including potatoes, sugar beet, brassicas, lettuce and ornamentals. Use of insecticides against M. persicae has selected for three resistance mechanisms: elevated carboxylesterase, modified acetylcholinesterase (MACE) and knock-down resistance (kdr and

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super-kdr) that jointly render carbamates, pyrethroids and organophosphates (OPs) ineffective (Anstead et al., 2008). Neonicotinoids, as well as pymetrozine and flonicamid, circumvent all three mechanisms. Given problems encountered in the past, urgent attention is being given to sustaining these compounds for the broad range of crops to which they are now applied.

A recently-completed SA-LINK project (LK 0953: Stewardship of neonicotinoid insecticides) provided an acclaimed example of proactive research to anticipate and combat risks of neonicotinoid resistance in M. persicae. Major outcomes of relevance to the current proposal were:

1) Confirmation of widespread, but consistent, variation in the sensitivity of M. persicae affecting all neonicotinoid molecules used in the UK. Despite increasing usage, there was no overall upward trend in the frequency of aphids showing reduced sensitivity between late 2004 and the end of 2007. There was therefore no economically-significant resistance to neonicotinoids present at that time in M. persicae in the UK. However, increased resistance to imidacloprid in M. persicae collected in northern Greece was a development with important implications given that MACE resistance to pirimicarb first developed in southern Europe and spread subsequently to the UK. Two Greek clones showed the strongest resistance known to date with one having a response factor of ~50-fold (relative to a susceptible standard, Nic-S, in a topical bioassay), which was significantly greater than the previously highest resistance factor of ~10-fold.

2) In parallel with increasing reliance of neonicotinoids, there have been important changes in the status of established mechanisms of resistance in M. persicae. Elevated carboxylesterase, MACE and kdr are still present, but their recent dynamics suggests opportunities for continuing to exploit older chemistry alongside new molecules in strategies for managing resistance.

On many crops, the main threat posed by M. persicae is the transmission of plant viruses. Currently, the control of aphid-transmitted viruses in arable crops is largely (cereals, potatoes) or wholly (oilseed rape, sugar beet) dependent on the use of insecticides, often applied prophylactically as neonicotinoid seed treatments. The impact of warmer autumn/winters will be for aphids to fly throughout the year, to continue to reproduce on appropriate crop plants and weed species and spread viruses such as the Turnip yellows virus (TuYV) formerly known as Beet western yellows virus (BWYV); a virus with a wide host range including brassica crops and lettuce. The increase of TuYV may be a major contributory factor in the variable yield performance of oilseed rape in recent years and its impact will be further exacerbated by climate change. Consequently, there will be more pressure for increased insecticide usage leading to the subsequent threat of (a) loss of efficacy of remaining classes of insecticides, (b) further insecticide resistance in aphid populations (either heightening existing resistance problems or leading to the development of new resistance mechanisms) and (c) the environmental implications of increased pesticide usage. An improved knowledge of the incidence of viruses on crop and non-crop plants is needed to anticipate the scale of this threat and to formulate counter-measures.

Pending the start of a new SA-LINK project in January 2009, the current research aimed to ensure continuity of data on resistance in M. persicae and accommodate a pilot study on the incidence of virus in over-wintering aphids. A final goal was to develop, for the first time, a reliable bioassay for exposing aphids and quantifying their resistance to systemic applications of neonicotinoids, enabling a direct comparison with resistance factors measured using topical application of insecticide.

2. Scientific objectives

Objective 1: Continued monitoring of the susceptibility of M. persicae to neonicotinoid insecticides through topical application bioassays of field and glasshouse samples with imidacloprid, and monitoring of established resistance mechanisms (MACE and knockdown resistance).

For this objective, ADAS were subcontracted by Rothamsted to provide aphid samples from a range of localities. Additional samples were provided by colleagues including crop consultants. Successful

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collections were reared for at least one generation to obtain sufficient numbers for bioassays with imidacloprid and DNA-based testing for other resistance mechanisms (MACE, kdr and super-kdr).

Objective 2: Development and validation of a bioassay for quantifying the response of aphids to systemic applications of neonicotinoids (and any other compounds exhibiting systemicity).

A previous preliminary study (done as part of SA-Link project LK 0953) suggested that resistance factors measured by topical application of imidacloprid are reduced when aphids are exposed to residues delivered to plants through systemic uptake. However, a method employed (with aphids placed directly onto leaf discs cut from excised Chinese cabbage leaves whose petioles had been immersed in dilutions of formulated imidacloprid for 24 h) was not ideal for this purpose. We therefore proposed to explore an improved systemic bioassay that could henceforth be used to characterise resistance to neonicotinoid insecticides, providing a ‘bridge’ between topical application (still the preferred method for routine monitoring) and whole plant experiments included in a forthcoming SA-LINK project.

Objective 3: Monitoring of M. persicae through autumn, winter and early spring (Sept 2008-March 2009) to determine the extent of virus threats within the UK

It was proposed to monitor aphids through the autumn, winter and early spring in weeds and crops such as autumn-sown oilseed rape crops to determine the extent of the main virus threats in the UK (concentrating on the indicator virus Turnip yellows virus (TuYV)), alongside establishing the insecticide resistance status of aphids (Objective 1). Samples were to be collected from oilseed rape crops in November 2008 and March 2009 where aphids were trapped in yellow water trays and also inspect leaves for the presence of M. persicae. In addition, weed samples were collected from 10 oilseed rape crops in March and were inspected for aphids and TuYV.

3. Materials and methods

3.1 Monitoring of insecticide resistance

3.1.1 Collection and rearing of M. persicae samples

Whenever possible, aphids were collected, along with their supporting leaves, from plants at scattered positions throughout a collection site. Samples were immediately transported by post or by hand to Rothamsted either in staple-sealed plastic bags or Petri dishes inside a robust box. Each sample was accompanied by a record of host plant, insecticide treatment history, and place and date of origin. Place of origin was subsequently converted into longitude and latitude coordinates using the Google Earth program.

On arrival at Rothamsted, M. persicae were sorted from each sample and placed as adults or nymphs onto excised Chinese cabbage leaves in small box cages to produce subsequent generations. This was done to avoid any possible effects of prior insecticide exposure in the field. Boxes were checked regularly for the presence of aphids that had succumbed to parasitoid or fungal attack and these individuals were removed. Normally, when aphids in the next generation had become young adults, apterous individuals were selected for the screening bioassays. However, depending on sample size and rearing success, some samples needed to be reared for a further generation. Rearing and bioassay was done at 21°C and under a 16/8 light/dark photoperiod. Despite aphid numbers being low in 2008, 20 field samples were successfully reared and screened for insecticide resistance. The initial samples ranged in size from one to over sixty aphids although the majority consisted of at least 20 individuals. 3.1.2 Testing for insecticide resistance

Bioassays with imidacloprid involved transferring young adult apterae to the abaxial surface of 4 cm-diameter leaf discs cut from Chinese cabbage (Brassica napus L var chinensis cv Tip-Top) that had been

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grown from seed in a glasshouse for about four weeks (10 aphids per leaf disc) (Foster et al., 2003, 2008). The discs were held on 2% agar in small plastic tubs (also 4 cm in diameter). Clean inverted tubs were placed onto each replicate, held in place by small pieces of Bluetak (Bostick, UK), to prevent any aphids from escaping and stop the leaf discs from drying out. Aphids were left for at least 2 h to settle and then dosed individually with a 0.25 μl droplet of acetone containing 2.5 ng of technical-grade imidacloprid (10 ppm concentration) using a micro-applicator (Burkard Manufacturing Ltd., UK). In each sample at least 27 aphids were tested. Aphid response was assessed after 72 h. Aphids that were dead or showed symptoms of irreversible poisoning (incapable of coordinated movement after being prodded gently with a fine paint brush) were classed together as ‘affected’. Those that were able to walk short distances were scored as being ‘mobile’. The very few aphids that fell into the mobile category and were also producing nymphs were transferred to excised leaves in a box cage, allowed to reproduce further, and their offspring re-tested to check that the result was not due to a mis-dosing, ie. the aphids had either not been dosed or had not received a complete droplet. Control bioassays, applying the screening dose and acetone alone to Nic-S (susceptible) and Nic-R (resistance factor of 10-fold) standard clones were also done on a regular basis throughout the course of sample screening. A subset of between 1 and 6 aphids from each (depending on initial sample size) was also tested for their MACE, kdr and super-kdr genotypes using DNA-based techniques (Anstead et al., 2008).

3.2 Development of a systemic bioassay for neonicotinoids

3.2.1 Clones of Myzus persicae

Three standard M. persicae clones were included in these experiments (Table 1). Nic-S was fully susceptible to insecticides. Nic-R and Nic-R+ showed progressively greater resistance to imidacloprid in a topical application bioassay (resistance factor of 10- and ~50-fold respectively). Each clone had been established originally from a single parthenogenetic female. All rearing and bioassay was done at 21°C and under a 16/8 light/dark photoperiod.

3.2.2 Excised leaf bioassay

Two new bioassay methods were assessed for measuring the response of M. persicae to systemic insecticide applications. The first tested the response of aphids feeding directly on whole excised leaves supported by their petioles immersed in insecticide solution, using imidacloprid (Confidor, 20% SL, Bayer CropScience) and clothianidin (Deter, 25% FS, Bayer CropScience) diluted in de-ionised water (with a neutral PH). Replicates were set up using primary leaves cut from Chinese cabbage plants that had been grown from seed in a glasshouse for about three weeks. Each leaf was trimmed slightly at the lower leaf edges using a razor blade and placed, abaxial side uppermost, into small box cages supported by wet sponges. Up to five young apterous adults (between 10 and 12 days old) from each M. persicae clone were transferred to the abaxial leaf surface using a fine paint brush. Cages and leaves were held upright in de-ionised water-filled trays for one day before the adults were removed leaving a synchronized cohort of up to 50 offspring primarily on the underside of each leaf. After a further three days each leaf plus nymphs (at a maximum of four days old) was removed from its box and inserted individually into a 7 ml glass vial (VWR, UK) full of insecticide solution and covered by Parafilm (Pechiney, USA). Each vial and its supported leaf plus aphids was placed upright into the centre of a circular plastic container (17 cm in diameter x 6 cm high), the lips of which were coated with insect-trapping adhesive (Oecotak) (Oecos, UK) to prevent any aphid escape. Containers were then held for 72 h. Each bioassay was evaluated by removing the leaves from the vials, using fine forceps, and counting, under a binocular microscope, the healthy live aphids (capable of walking at least short distances), those that were moribund (incapable of walking and therefore irreversibly poisoned) and dead individuals. Any aphids that had moved onto the vial or into the container were also counted and evaluated. The lips of the containers were checked for any aphids that had become trapped by the adhesive.

Four or five separate bioassays were done for each clone. In each bioassay up to three replicates per clone were tested at up to seven insecticide concentrations plus a control. Aphids that were dead or

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moribund were classed together as ‘affected’. LC50 values were calculated by probit analysis on pooled data using the POLO program (Leora Software, Petaluma, California). Less than 1% of aphids in each clone tested with imidacloprid or clothianidin were found to have become trapped in the adhesive on the lips of the plastic containers. Every one of these few trapped aphids were dead and included in the analysis in the ‘moribund plus dead’ category.

As the excised leaf experiments proceeded, it was realised that aphids could also be analysed for their behavioural response to insecticide in the form of their location at the end of the bioassays. Those that were either on the vial or in any part of the container at the end of each bioassay were classed as being ‘off the leaf’ (irrespective of their condition). MC50 values were calculated by probit analysis using the POLO program (Leora Software, Petaluma, California).

3.2.3 Leaf-disc bioassay

The second bioassay method tested the response of aphids placed on leaf discs that had been cut from excised leaves previously placed in insecticide solutions. These bioassays used imidacloprid (Confidor) diluted in de-ionised water (with a neutral PH) and, initially, a similar approach to the excised leaf bioassays. Chinese cabbage leaves were inserted individually into 7 ml glass vials (VWR, UK) containing insecticide solution and covered with Parafilm. However, these leaves did not support any aphids and were left to take up the solutions for 24 h. 3 cm diameter leaf discs were then cut from them and placed, abaxial side up, on a bed of 2% agar in small plastic tubs (4 cm in diameter). Four young apterous adults from each aphid clone were transferred to each disc using a fine paintbrush. Clean inverted tubs were placed onto each replicate tub to prevent aphid escape, and held in place with small pieces of Bluetak (Bostick, UK). The aphids were left for 24 h to produce offspring and then removed leaving a cohort of up to 40 first instar nymphs on each leaf disc. After a further 72 h each replicate was evaluated under a binocular microscope for the healthy live aphids (capable of walking at least short distances), those that were moribund (incapable of walking and therefore irreversibly poisoned) and dead individuals.

Three or four separate bioassays were done for each M. persicae clone. In each bioassay two replicates per clone were tested at up to eight insecticide concentrations plus a control. Aphids that were dead or moribund were classed together as ‘affected’. LC50 values were calculated by probit analysis on pooled data using the POLO program (Leora Software, Petaluma, California).

3.3 Monitoring of virus incidence

3.3.1 Aphid infectivity monitoring via water pans.

Three yellow water traps were set up 15 metres apart in two oilseed rape crops at Broom’s Barn and Ousden, Suffolk respectively. Insect catches were collected twice weekly from 10 September until the end of November 2008. All M. persicae and Macrosiphum euphorbiae (potato aphids) were then identified from the insect samples and tested for TuYV using ELISA. Fifty oilseed rape leaf samples were collected at the end of March from both sites, assessed for aphids and tested for the presence of TuYV by ELISA.

3.3.2 ELISA

Microtitre plates were coated with BMYV immunoglobulins (IgGs) diluted in carbonate buffer. Plates were incubated for 1 h at 37°C, washed with phosphate buffered saline + Tween (PBSt) and then blocked using PBS containing 0.1% non-fat dried milk (Marvel). Aphid extracts, produced by grinding individuals in PBSt + 0.1% milk using a hand-held insect homogeniser (Burkard Scientific, UK), were added to selected wells and plates then incubated overnight at 4°C. Similarly, all leaf samples were ground in the same buffer

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(1/10 w/v). Alkaline-phosphatase conjugated IgGs were diluted in PBSt + 0.1% milk, added to the wells and incubated for 2.5 h at 37°C. Either para-nitrophenyl phosphate (p-NPP) or the enzyme amplification system (AMPAK) were used as substrates for plant and aphids respectively; p-NPP was diluted in substrate buffer and used at a concentration of 0.5 mg/ml. Plates were incubated at room temperature, and absorbance values at 405 nm were recorded after 1 h and 18 h, using a colorimeter. The enzyme amplification system was used according to the manufacturer’s instructions, and after the addition of the stop solution, absorbance values at 490 nm were taken.

3.3.3 Assessment of weeds for aphids and virus.

Weeds were collected from 10 sites in East Anglia in March 2009 (seven in Suffolk, two in Norfolk and one in Bedfordshire). Up to 5 randomly selected weeds within a one metre square quadrat were taken at 15 locations around the field margins; each sampling point was at least 20 metres apart. All harvested plants were brought back to Broom’s Barn and examined for the presence of aphids and these plants were then tested for the presence of TuYV using the ELISA protocol.

4. Results

4.1 Resistance monitoring

Results for the 20 M. persicae field samples tested with imidacloprid in 2008 were added to the spatio-temporal database developed in the LK0953 project. Temporal changes in the proportion of mobile aphids were analysed using a Generalized Linear Model (GLM) with binomial error and logit link (Figure 1). This analysis takes into account the varying numbers of aphids tested in each sample (ranging from 5 – 93) and assumes the number of mobile aphids follows a binomial distribution. Cubic splines in day number were fitted with up to 8 df to investigate the underlying time trend in the data. This revealed no evidence of any increase in the frequency of aphids showing reduced sensitivity to imidacloprid (resistance factors up to 10-fold). Indeed, no such aphids were found in the latter part of 2008. Furthermore, aphids with resistance factors up to 50-fold, which have recently been shown to occur in southern Europe, have yet to be found in the UK.

Frequencies of field samples that contained aphids with the MACE and kdr mechanisms are shown in Figure 2, alongside comparative data collected since 1996. MACE frequency remained high in 2008. However, samples containing kdr have continued to fall since 2003. All of the MACE and kdr aphids tested were heterozygotes and none carried super-kdr. This is consistent with the findings in previous years.

4.2 Development of systemic bioassays

4.2.1 Excised leaf bioassay

Dose-response survival data for Nic-S, Nic-R and Nic-R+ clones tested with imidacloprid and clothianidin are shown in Figures 3-8, with probit statistics listed in Table 2. For imidacloprid, the Nic-R clone showed a much lower resistance factor (2.1) than recorded by topical application (ca. 10). The Nic-R+ clone was significantly less sensitive to imidacloprid than the Nic-S clone (resistance factor = 2.9), but the differential was again substantially lower than seen with topical application. Variation between LC50 values to clothianidin was greater than with imidacloprid, with the Nic-R and Nic-R+ clones both showing significantly higher LC50s than the susceptible clone (i.e. their 95% confidence limits did not overlap).

Data on the location of aphids at the end of a bioassay proved amenable to probit analysis, yielding MC50 values (related to movement rather than survival) and equivalent response ratios (Table 3). Behavioural response ratios were greater using the data on movement rather than survival, and response rations were again higher for clothianidin compared to imidacloprid.

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4.2.2 Leaf-disc bioassay

Pooled data for each clone showed a clear dose-response relationship for imidacloprid that could be calculated using the POLO program. The probit-transformed data for each clone are shown in Figures 9-11 and the resulting LC50 values are shown in Table 2. The pattern of response was statistically identical to the excised leaf bioassays although the response ratios were higher and the slopes were not as steep.

4.3 Virus incidence

4.3.1 Aphids in yellow water pans.

Only two individuals of M. persicae were caught in the yellow water pans at Broom’s Barn (one on the 29th September and a second on the 16th October 2008). No M. persicae were caught at the Ousden site and no M. euphorbiae were trapped at either site during the autumn period. One of the two M. persicae contained TuYV when tested by ELISA. None of the oilseed rape leaves collected from either site was infected with TuYV at the end of March 2009.

4.3.2 Assessment of weeds for aphids and virus.

Up to 54 weeds were sampled from each of the 10 locations in East Anglia (Table 4). On inspection, aphids were found on weeds at four of the 10 sites (e.g. 36 aphids were identified at Eye, Suffolk). However, further analysis showed that none of these aphids were M. persicae; the predominant species were Myzus ascolonicus and Aphis species. Infection of weeds with TuYV was found at five sites, (two of these sites also contained aphids). Up to 15% of weeds were found to be infected with TuYV per site and the most common weeds with TuYV were Plantago species (6/23), Senecio vulgaris (5/65) and Viola arvensis (2/6) (Table 5).

5. Discussion and implications

Results reported here extend and strengthen the findings of our previous SA-Link project (LK 0953). Reduced sensitivity to neonicotinoids, giving resistance to imidacloprid in topical bioassays of up to 10-fold, continues to be encountered in the UK but remains of little importance in practice. Furthermore, there has been no directional increase in the frequency of these aphids with time, or evidence of any greater resistance likely to result in control problems. Therefore, in the UK at least, there is no evidence for the evolution of resistance to neonicotinoids that would affect their field performance. This is supported by no confirmed reports of control failures attributable to resistance. However, our detection of the Nic-R+ phenotype in northern Greece has potentially important implications, bearing in mind that MACE resistance to pirimicarb developed in southern Europe and then spread quickly to the UK.

The frequency of samples containing MACE (conferring immunity to pirimicarb) remained high in 2008. This continues to have major practical implications, particularly for beet growers, because there are currently no viable control alternatives if neonicotinoid resistance should appear and spread. Interestingly, all aphids with MACE continue to be heterozygotes, suggesting that homozygotes suffer a fitness cost. Collaborative research with SCRI (Kasprowicz et al., 2008) has shown that in Scotland, MACE is now being found in clones with a new genetic background (O- and P-types) that contain neither kdr nor high carboxylesterase resistance. Thus, MACE aphids are no longer handicapped by an association with these other insecticide resistance mechanisms, and may be better-adapted to overwintering climates in the UK.

Aphids with kdr (to pyrethroids) remain relatively common although it appears that the frequency of samples containing aphids with this mechanism has fallen gradually since 2003 when over 80% of samples contained kdr aphids. This probably reflects reduced selection pressures by pyrethroids against this pest resulting from updated management guidelines aimed at M. persicae. No aphids with super-kdr

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were collected in 2008. As with MACE, all aphids with the kdr allele were heterozygotes, supporting the hypothesis of fitness costs conferred by kdr and super-kdr alleles in the homozygous form. The prevalence of kdr and MACE in M. persicae in the UK reinforces the importance of maintaining the effectiveness of neonicotinoids and other novel insecticides including pymetrozine and flonicamid for aphid control.

Given the preponderance of systemic applications of neonicotinoids in the field, two new systemic bioassay methods were evaluated for testing the response of insecticides present in plant tissues. Both involved aphids that had been produced by adult apterae directly onto the leaf surfaces, and produced clear dose-response relationships with slopes steeper than 1 when calculated using the POLO program. However, the ‘excised leaf’ method (as opposed to the leaf disc method) proved to be the most versatile as more individuals could be tested in each replicate (up to 50) and aphid behaviour, in the form of position on or off the leaf at the end of the bioassay, could also be measured. Furthermore, preliminary observations suggest that this method will also allow the measurement of aphid movement from the leaves in the first several hours of the bioassay and therefore a means of quantifying short term repellence by any systemic-acting insecticide, a criterion that is closely linked with virus transmission success.

The systemic bioassays (which included the newly acquired Nic-R+ clone), consistently yielded lower resistance factors than topical application. Therefore, the method of treatment (topical versus systemic) appears to affect the strength of selection and ‘resistance risk’. If extrapolated to the field, this implies that the current (unresolved) mechanism(s) of resistance will be more manifest in response to neonicotinoids applied as foliar sprays rather than seed or soil treatments. The previously exclusive use of systemic applications of neonicotinoids in the UK may have been a potent factor precluding the selection of resistance. If true, this scenario is threatened by the ongoing proliferation of foliar neonicotinoid registrations in the UK (and abroad), as well as indirect changes such as recommendations to spray thiacloprid as an alternative to pyrethroids for pollen beetle control on oilseed rape.

The cooler/wetter autumn and winter period in 2008 and early 2009 clearly influenced the abundance of aphids and their subsequent survival during this study. This contrasted with the previous two years, when aphids built up to much higher numbers and up to 72% of M. persicae contained TuYV. Industry surveys of oilseed rape crops in the springs of 2007 and 2008 showed that many crops had significant TuYV infection, and several fields, south of the Humber estuary, were 100% infected with this virus. In previous studies, losses of up to 26% have been recorded due to TuYV infection in UK oilseed rape, and insecticide treatments gave yield responses of up to 22% in 2008. However, the disease often only shows mild symptoms, usually in the following spring, of leaf reddening/purpling and such symptoms are often mistaken for stress/nutrient deficiencies. In mild autumns M. persicae will migrate into brassica crops and transmit and spread TuYV during the early stages of crop establishment. The occurrence of TuYV is a likely contributory factor in the poor performance of oilseed rape crops during the last decade, and is currently being investigated in an HGCA-funded project at Broom’s Barn..Currently, control of aphid-transmitted viruses in arable crops is largely (cereals, potatoes) or wholly (oilseed rape, sugar beet) dependent on the use of insecticides, often applied prophylactically as neonicotinoid seed treatments. The impact of warmer autumn/winters will be for aphids to continue to fly throughout the year, continue to reproduce on appropriate crop plants and weed species and spread viruses such as TuYV. In fact, the increase of TuYV may be a major contributory factor in the variable yield performance of oilseed rape in recent years and its impact will be further exacerbated by climate change. Consequently, there would be more pressure for increased insecticide usage and the subsequent threats of the loss of efficacy of remaining classes of insecticides, insecticide resistance in aphid populations (either heightening existing resistance problems or leading to the development of new problems) as well as the environmental implications of increased pesticide usage.

6. Opportunities for future work

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Our finding of no directional change in susceptibility of M. persicae to neonicotinoids means that this class of insecticides can continue to play a key role for controlling this pest species. However, there is no room for complacency since two resistance mechanisms to other compounds (MACE and kdr) remain prevalent and widely distributed in the UK population and our bioassays suggest that foliar applications may favour the evolution of significant neonicotinoid resistance in aphids similar to that already seen in several other insect pests. The over-riding question is whether the existing mechanism in M. persicae conferring reduced sensitivity to neonicotinoids (exemplified by Nic-R and Nic-R+ clones), whatever its nature, can be progressively enhanced, or whether a different mechanism is needed to compromise efficacy under field conditions. Continued effort is therefore required to monitor for further upward shifts in response in this species, both in the UK and abroad, and tailor management recommendations accordingly at an early enough stage to counter any spread of potential resistance. This effort will continue through a new SA-Link project (LK 09114), commencing in January 2009, which will include full characterisation of the response of the Nic-R+ phenotype to seed and foliar applications using field simulators, and use of the systemic excised leaf bioassay to assess the likely practical importance of shifts in response detected throughout routine use of the topical bioassay application method.

Monitoring aphids and their infectivity through the autumn, winter and early spring provides information that can be used to predict the likely risks of virus infection as well as aid in the development of strategies to combat the threat of virus infection in the future. For example, the deployment of broad-spectrum, durable and highly-expressed resistance to plant viruses in a wide range of arable crops offers considerable potential for reductions in pesticide use, with its associated economic and environmental benefits, and for more sustainable cropping systems.

7. Actions

As with LK0953, results of this project have been communicated rapidly to relevant stake-holders including the agrochemical industry, growers and advisors, and the UK Chemical Regulation Directorate. The primary forum for this has been twice-yearly meetings of the UK Insecticide Resistance Action Group, which reviews new data and their implications and revises guidelines accordingly. Members of the project team have continued to maintain close contacts with individual organisations including presentations on resistance monitoring and management in M. persicae.

8. References

Anstead JA, Williamson MS & Denholm I (2008) New methods for the detection of insecticide-resistant Myzus persicae in the UK suction trap network. Agricultural and Food Entomology 10, 291-295.

Foster SP, Denholm I. & Thompson R (2003) Variation in response to neonicotinoid insecticides in peach-potato aphids, Myzus persicae (Hemiptera: Aphididae). Pest Management Science, 59, 166-173.

Foster SP, Cox D, Oliphant L, Mitchinson S & Denholm I (2008) Correlated responses to neonicotinoid insecticides in the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae). Pest Management Science 64, 1111-1114.

Kasprowicz L, Malloch G, Foster S, Pickup J, Zhan J & Fenton B (2008) Clonal turnover of MACE-carrying peach-potato aphids (Myzus persicae (Sulzer), Homoptera: Aphididae) colonizing Scotland. Bulletin Entomological Research 98, 115-124.

Nauen R & Denholm I (2005) Resistance of insect pests to neonicotinoid insecticides: current status and future prospects. Archives of Insect Biochemistry and Physiology 58, 200-215.

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9. Tables and figures

Table 1. Neonicotinoid status and origins of standard Myzus persicae clones. ___________________________________________________________________

Clone Resistance factor Country of Year Imidacloprid clothianidin origin collected ___________________________________________________________________

Nic-S 1 1 Scotland 2000Nic-R 11 100 Greece 1990Nic-R+ 50 not tested Greece 2007 ___________________________________________________________________

Table 2. LC50 responses of standard M. persicae clones in systemic bioassays applying imidacloprid (leaf disc and excised leaf bioassays) and clothianidin (excised leaf bioassays)._____________________________________________________________________

Clone Na LC50

b 95% CLc Slope Resistance factord

_____________________________________________________________________Imidacloprid

Leaf discsNic-S 218 0.019 0.011-0.038a 1.5 1Nic-R 587 0.067 0.038-0.106ab 1.4 3.5Nic-R+ 467 0.117 0.049-0.728b 1.3 6.2

Intact excised leavesNic-S 1159 0.106 0.071-0.155a 1.8 1Nic-R 738 0.217 0.153-0.313ab 1.9 2.1Nic-R+ 1305 0.304 0.215-0.428b 1.8 2.9

ClothianidinIntact excised leaves

Nic-S 467 0.092 0.050-0.154a 2.1 1Nic-R 600 0.391 0.283-0.549b 1.9 4.3Nic-R+ 540 0.695 0.493-1.061b 2.3 7.6_____________________________________________________________________a Total number of aphids tested (including controls).

b Concentration resulting in 50% aphids dead or irreversibly poisoned.c Confidence limits at 95%; values followed by the same letter do not differ significantly within each study (i.e. they overlap).d Ratio of clone LC50/LC50 for Nic-S.

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Table 3. Analysis of movement behaviour of standard M. persicae clones in systemic bioassays applying imidacloprid and clothianidin (using excised leaves).______________________________________________________________________

Clone Na MC50

b 95% CLc Slope Resistance factord

______________________________________________________________________Imidacloprid

Nic-S 1046 0.072 0.045-0.114a 1.3 1Nic-R 738 0.125 0.087-0.183a 1.7 1.7Nic-R+ 1132 0.287 0.202-0.408b 1.4 4.0

ClothianidinNic-S 467 0.099 0.007-0.342a 1.0 1Nic-R 600 0.572 0.312-1.085ab 1.5 5.8Nic-R+ 570 1.145 0.607-2.408b 1.5 11.6______________________________________________________________________a Total number of aphids tested (including controls).

b Concentration resulting in 50% aphids found off the leaf at 72 h endpoint.c Confidence limits at 95%; values followed by the same letter do not differ significantly (i.e. they overlap).d Ratio of clone MC50/MC5 0 for Nic-S.

Table 4. Details of sample sites and the number of weed plants with aphids and/or TuYV

NAME OF SITE GRID REF NO. OF PLANTS

NO. OF APHIDS

NO. OF PLANTS TuYV+

BARHAM, SUFFOLK TM 142 511 14 2 0BROOM'S BARN, HIGHAM, SUFFOLK: BEET POLYTUNNEL TL 752 656 54 0 3BROOM'S BARN, HIGHAM, SUFFOLK: WOSR FIELD MARGINS TL 752 656 54 0 8CASTON, NORFOLK TL 951 978 20 0 3EYE, SUFFOLK TM 135 730 13 36 0HIGHAM, BURY ST EDMUNDS SUFFOLK TL 751 668 54 3 4NORTH BURLINGHAM, NORWICH NORFOLK TG 377 095 17 0 0OUSDEN,SUFFOLK WOSR FIELD TL 745 588 54 0 0SUTTON, SANDY, BEDFORDSHIRE TL 209 485 19 0 0 WAXHAM,SEA PALLING, NORFOLK TG 439 263 21 0 0 THORNEY GREEN,STOWUPLAND, SUFFOLK TM 060 603 12 21 1TOTALS 332 62 19

Table 5. Weed species sampled that were confirmed to contain TuYV

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WEED SPECIESCOMMON NAME

NO. OF PLANTS TESTED

NO. OF PLANTS TuYV+

Capsella bursa-pastoris Shepherds purse 22 1Galium aparine Cleavers 30 1Plantago sp Plantain 23 6Rubus fruiticosus Bramble 1 1Senecio vulgaris Groundsel 65 5Sinapsis arvensis Charlock 13 2Urtica urens Small nettle 10 1Viola arvensis Field pansy 6 2

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Figure 1. Proportion of mobile aphids in each field sample screened with imidacloprid during LK0953 and the current project.

Figure 2. Frequency of M. persicae field samples containing individuals with the MACE and kdr mechanisms.

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Figure 3. Dose response of Nic-S M. persicae clone in an excised leaf bioassay with imidacloprid.

Figure 4. Dose response of Nic-R M. persicae clone in an excised leaf bioassay with imidacloprid.

Figure 5. Dose response of Nic-R+ M. persicae clone in an excised leaf bioassay with imidacloprid.

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Figure 6. Dose response of Nic-S M. persicae clone in an excised leaf bioassay with clothianidin.

Figure 7. Dose response of Nic-R M. persicae clone in an excised leaf bioassay with clothianidin.

Figure 8. Dose response of Nic-R+ M. persicae clone in an excised leaf bioassay with clothianidin.

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Figure 9. Dose response of Nic-S M. persicae clone in a leaf disc bioassays with imidacloprid.

Figure 10. Dose response of Nic-R M. persicae clone in a leaf disc bioassay with imidacloprid.

Figure 11. Dose response of Nic-R+ M. persicae clone in a leaf disc bioassay with imidacloprid.

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

published material generated by, or relating to this project.

Resistance AlertsIRAG-UK Alert: MACE resistance in peach-potato aphid (November 2008).

Management GuidelinesGuidelines for controlling aphids in brassica crops and managing insecticide resistance in the peach-

potato aphid, Myzus persicae (2008).

Guidelines for preventing and managing insecticide resistance in aphids on potatoes (2008).Both down-loadable from the website: http://www.pesticides.gov.uk/committees/resistance/index.htm.

Farming PressNo room for complacency (Potato Review, May/June 2009).

Knowledge key to controlling potato viruses (Potatosafe News, March 2009).

Stewardship guidelines protect aphid control (Potato Council Grower Gateway, October 2008).

Summer pest control strategies for brassicas (Syngenta Specialist Crops Technical Update, July 2008).

PresentationsThe current and potential future situation regarding insecticide resistance in aphids. Belchim Potato

Seminar. Wyboston, March 2009.

Resistance to neonicotinoids in peach-potato aphids in the UK: good news, bad news and ugly challenges ahead. Plant and Invertebrate Ecology Research Day. Rothamsted Research, Harpenden. November 2008.

Nicotinic acetylcholine receptors and resistance to neonicotinoids. Agrochemicals Award Symposium for David Soderlund, ACS National Meeting, Philadelphia, USA, August 2008.

Are neonicotinoid insecticides resistant to the evolution of resistance in aphids? International Congress of Entomology. Durban, South Africa, July 2008.

Aphid control with neonicotinoids: a sustained success story (but for how much longer?). Stewardship of neonicotinoid insecticides, Honolulu, Hawaii, June 2008.

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