A method for assessing the virulence of entomopathogenic fungi (Hypocreales: Clavicipitaceae) on Atta laevigata (Hymenoptera: Formicidae)

ABSTRACT Leafcutter ants are social insects that cultivate a symbiotic fungus and cut leaves of nearly any plant, making them major generalist pests. Entomopathogenic fungi are promising biological control agents, particularly because they are easy to propagate and can effectively infect leafcutter ants. However, virulence studies have faced challenges in keeping leafcutter ants alive long enough to evaluate the effects of entomopathogenic fungi on their survival. This study describes a method for the collection, preparation, inoculation, and maintenance of leafcutter ants in bioassays using the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill., with Atta laevigata (Smith, 1858) as a model insect. We tested six liquid diets to assess their effects on ant longevity and detailed all bioassay preparation steps and procedures. The maltose diet allowed ants in the control group treated with sterilised water + 0.1% Tween 80 to exhibit a significantly higher mean survival time than those treated with a dose of 105 conidia per insect. The use of this liquid diet in the virulence bioassay enabled precise calculation of the median lethal dose (LD50 = 9.590 × 102) and the lethal dose for 95% of the population (LD95 = 3.912 × 105), estimated on the fifth day after fungal application. The low LD50 and LD95 values underscore the high virulence of B. bassiana against A. laevigata and support their potential use in biological control programmes. The method described is cost-effective, easily replicable, and provides accurate, consistent results for evaluating the effects of entomopathogenic fungi on leafcutter ants in laboratory bioassays.