Supplementary Material for: The Role of Hemocytes in Anopheles gambiae Antiplasmodial Immunity

Hemocytes synthesize key components of the mosquito complement-like system, but their role in the activation of antiplasmodial responses has not been established. The effect of activating Toll signaling in hemocytes on Plasmodium survival was investigated by transferring hemocytes or cell-free hemolymph from donor mosquitoes in which the suppressor cactus was silenced. These transfers greatly enhanced antiplasmodial immunity, indicating that hemocytes are active players in the activation of the complement-like system, through an effector/effectors regulated by the Toll pathway. A comparative analysis of hemocyte populations between susceptible G3 and the refractory L3-5 Anopheles gambiae mosquito strains did not reveal significant differences under basal conditions or in response to Plasmodium berghei infection. The response of susceptible mosquitoes to different Plasmodium species revealed similar kinetics following infection with P. berghei,P. yoelii or P. falciparum, but the strength of the priming response was stronger in less compatible mosquito-parasite pairs. The Toll, Imd,STAT or JNK signaling cascades were not essential for the production of the hemocyte differentiation factor (HDF) in response to P. berghei infection, but disruption of Toll, STAT or JNK abolished hemocyte differentiation in response to HDF. We conclude that hemocytes are key mediators of A. gambiae antiplasmodial responses.