RESISTANCE IN APHIDS Dr Grant A. Herron NSW DPI, Elizabeth Macarthur Agricultural Institute (EMAI), PMB 4008 Narellan 2567, Australia grant.herron@dpi.nsw.gov.au SUMMARY  The frequency of neonicotinoid resistant Aphis gossypii Glover populations has markedy decreased from a high of 94% to a moderate 22% in a single season.  The decrease in the frequency of neonicotinoid aphids likely relates to a subtle change in the way foliar neonicotinoids are being used against pests other than aphids and where aphids are accidently being selected by that use rather than a reduction in neonicotinoid seed dressing consumption or an increase in seed dressing efficacy (it was shown not to completely control resistant A. gossypii).  Pirimicarb resistant A. gossypii remain infrequent and the chemical continues to be a relaible IPM compatible method of aphid control.  A new quantitative real-time PCR (qPCR) method under development to estimate pirimicarb resistance in A. gossypii will potentially increase assay accuracy and decrease test cost. INTRODUCTION In Australian cotton systems, the emergence of the aphid vectored disease ‘cotton bunchy top’ in the 1998-1999 growing season, led to a reduction in aphid tolerance and more targeted insecticide applications for aphid control. During the subsequent growing seasons, resistance levels in A. gossypii increased to dimethoate and pirimicarb causing control failures in many Australian cotton growing regions (Herron et al. 2001). Currently, aphid control relies on insecticides from several modes of action; however, the major group used for aphid control in Australian cotton is the neonicotiniods primarily as a seed dressing (Maas 2011). Neonicotinoid resistance in A. gossypii was first detected in season 2007-2008 from where it quickly increased in both level and abundance particularly causing control issues with the foliar neonicotinoids used against A. gossypii such as acetamiprid and clothianidin (Herron and Wilson 2011). Here I present recent resistance data showing a marked change in neonicotinoid resistance in A. gossypii and show the practical consequence of neonicotinoid resistance on A. gossypii control plus introduce a new molecular based method to characterise pirimicarb resistance. METHODS Aphids were collected from commercial cotton fields or cotton plants in the vicinity of commercial crops. They were sent to the Elizabeth McArthur Agricultural Institute (EMAI) and cultured separately on pesticide-free cotton at 25 ± 4 o C under natural light (Herron et al. 2001). Strain integrity was assured by maintaining aphid populations in purpose built insect proof cages. Aphid bioassay tests required placing them in a 35 mm Petri dish on an excised cotton plant leaf disc fixed in agar (Herron et al. 2001). Briefly, batches of ten apterous adult female aphids per leaf disc were then sprayed with insecticide with the aid of a Potter spray tower. All tests were replicated and included a water-only sprayed control. After spraying, clear plastic film was used to cover the Petri dishes, which were then maintained at 25  0.1 o C in 16:8 L:D for 24 h after which mortality was assessed. Pirimicarb resistance was previously detected via bioassay and then an established DNA based method of M c Loon and Herron (2009) but we are concurrently trialling a quantitative real-time PCR (qPCR) method to estimate the resistance allele frequency from pooled DNA. Conventional PCR detects resistance alleles in individual aphids and typically 20-50 individual aphids are needed to accurately estimate the resistance allele frequency per strain (sampling site). Our new method for estimating the resistance allele frequency is based on Taqman probes for resistant and susceptible alleles in one PCR reaction with DNA extracted from pooled (hundreds) aphids. The resistance allele frequency can be accurately estimated based on the ratio of increased fluorescence intensity between the resistance probe and susceptible probe at each PCR cycle. The practical consequence of neonicotinoid resistance was tested using a randomised complete block glasshouse trial where neonicotinoid seed dressings were challenged with neonicotinoid resistance aphids. Resistant aphids were challenged against a thiamethoxam seed treatment (Cruiser 350 SF) at consecutive weekly intervals post germination until product failure relative to an untreated control. RESULTS Although testing for season 2011-2012 is still incomplete it is obvious there is a significant reduction in neonicotinoid resistance from the previous season high of 94% to 22% (Figure 1). Pirimicarb resistance testing is also still under way