Supplementary Material for: The Role of Hemocytes in <b><i>Anopheles gambiae</i></b> 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 <i>Toll</i> signaling in hemocytes on <i>Plasmodium </i>survival was investigated by transferring hemocytes or cell-free hemolymph from donor mosquitoes in which the suppressor <i>cactus </i>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 <i>Toll</i> pathway. A comparative analysis of hemocyte populations between susceptible G3 and the refractory L3-5 <i>Anopheles gambiae</i> mosquito strains did not reveal significant differences under basal conditions or in response to <i>Plasmodium berghei</i> infection. The response of susceptible mosquitoes to different <i>Plasmodium</i> species revealed similar kinetics following infection with <i>P. berghei,</i><i>P. yoelii</i> or <i>P. falciparum,</i> but the strength of the priming response was stronger in less compatible mosquito-parasite pairs. The <i>Toll, Imd,</i><i>STAT</i> or <i>JNK</i> signaling cascades were not essential for the production of the hemocyte differentiation factor (HDF) in response to <i>P. berghei </i>infection, but disruption of <i>Toll, STAT</i> or <i>JNK </i>abolished hemocyte differentiation in response to HDF. We conclude that hemocytes are key mediators of <i>A. gambiae</i> antiplasmodial responses.