Synergistic effects of neonicotinoids in combination with entomopathogenic fungi on the insecticidal activity against the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae) and their effects on the host immune system

The combined use of entomopathogenic fungi and neonicotinoids was examined. A combination of six types of neonicotinoids, acetamiprid, imidacloprid, clothianidin, nitenpyram, dinotefuran, and thiacloprid, and two fungal species, Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae) and Metarhizium sp. (Hypocreales: Clavicipitaceae), were administered to adult red flour beetles, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). The mortality of test beetles caused by the combined treatment was compared to those caused solely by neonicotinoids or a solvent control. Significantly enhanced mortality was observed when B. bassiana was used in combination with imidacloprid, thiacloprid, or clothianidin. Combination index (CI) analyses revealed that the enhanced insecticidal effects were synergistic and stronger for imidacloprid and thiacloprid. The effect of neonicotinoids on the beetle's humoral as well as cellular innate immune responses were investigated. The mRNA levels of the Toll pathway-dependent antimicrobial peptide (AMP) gene, cecropin3, due to exposure to B. bassiana were slightly suppressed by the concurrent application of imidacloprid and thiacloprid. Encapsulation responses with melanin deposition against abiotic material tended to be suppressed by neonicotinoid treatment. These results collectively suggest that the synergistic effects of certain neonicotinoids co-used with B. bassiana might be associated with adverse impact of the neonicotinoids on the beetle's antifungal immune defense. In addition, RNA interference-mediated silencing of crucial genes for the normal Toll pathway signaling rendered the knockdown beetles more susceptible to B. bassiana infection.