Susceptibility of Agriotes spp. larvae (Coleoptera: Elateridae) to "stress- 22 and-kill" strategies using spinosad and the entomopathogenic fungus 23 Metarhizium brunneum 24 Abstract 25 Wireworms (Agriotes obscurus, Agriotes lineatus, Agriotes sordidus) are 26 major pests of arable crops. Damage caused by these subterranean pests has 27 increased following the withdrawal of many traditional synthetic 28 insecticides. Wireworms are susceptible to entomopathogenic fungi 29 belonging to the genus

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... etarhizium. The aim of this study was to determine 30 if exposure of different wireworm species to sublethal doses of the 31 insecticide spinosad altered their susceptibility to different strains of 32 Metarhizium brunneum. Of the three wireworm species studied, A. obscurus 33 was the most susceptible species to the isolates of M. brunneum tested, 34 while A. lineatus and A. sordidus were not affected by the entomopathogen 35 on its own. However, combination of spinosad and M. brunneum could 36 cause significant mortality and, depending on the treatment combination, 37 some interactions were determined as being either antagonistic, additive or 38 synergistic. The efficacy of the stress-and-kill strategy was dependent on 39 the fungal strain, insect species, dose of stressing agent and time. M. 40 brunneum strain F52 and ART2825 combined with low doses of spinosad 41 resulted in a synergistic reaction. Scanning electron microscopy shows that 42 without spinosad, poor germination was seen on the wireworm cuticle, 43 indicating that it could be fungistatic. However, the increased susceptibility 44 2 of the wireworms to the stress-and-kill seem to indicate physiological 45 changes due to spinosad, facilitating germination and penetration of the 46 fungi. Our results show that stress and kill can provide wireworm control; 47 yet, the right combination between the fungi, stressing agent and insect 48 species is mandatory. 49