Phospholipase A₂ activity is required for immune defense of European (Apis mellifera) and Asian (Apis cerana) honeybees against American foulbrood pathogen, Paenibacillus larvae

Honeybees require an efficient immune system to defend against microbial pathogens. The American foulbrood pathogen, Paenibacillus larvae, is lethal to honeybees and one of the main causes of colony collapse. This study investigated the immune responses of Apis mellifera and Apis cerana honeybees against the bacterial pathogen P. larvae. Both species of honeybee larvae exhibited significant mortality even at 10(2) 10(3) cfu/mL of P. larvae by diet-feeding, although A. mellifera appeared to be more tolerant to the bacterial pathogen than A. cerana. Upon bacterial infection, the two honeybee species expressed both cellular and humoral immune responses. Hemocytes of both species exhibited characteristic spreading behaviors, accompanied by cytoskeletal extension along with F-actin growth, and formed nodules. Larvae of both species also expressed an antimicrobial peptide called apolipophorin III (ApoLpIII) in response to bacterial infection. However, these immune responses were significantly suppressed by a specific inhibitor to phospholipase A(2) (PLA(2)). Each honeybee genome encodes four PLA(2) genes (PLA(2)A similar to PLA(2)D), representing four orthologous combinations between the two species. In response to P. larvae infection, both species significantly up-regulated PLA(2) enzyme activities and the expression of all four PLA(2) genes. To determine the roles of the four PLA(2)s in the immune responses, RNA interference (RNAi) was performed by injecting gene-specific double stranded RNAs (dsRNAs). All four RNAi treatments significantly suppressed the immune responses, and specific inhibition of the two secretory PLA(2)s (PLA(2)A and PLA(2)B) potently suppressed nodule formation and ApoLpIII expression. These results demonstrate the cellular and humoral immune responses of A. mellifera and A. cerana against P. larvae. This study suggests that eicosanoids play a crucial role in mediating common immune responses in two closely related honeybees.