This proposal describes experiments investigating the intracellular melanization of filarial nematode larvae of Brugia malayi, B. pahangi and Dirofilaria immitis in the target tissues of mosquito species. We have available highly susceptible and highly refractory strains of Anopheles quadrimaculatus to both Brugia species. In the refractory strains of An. quadrimaculatus most of the developing larvae of both Brugia species are melanized intracellularly about 3 to 4 days after the infective blood meal. We will conduct investigations using electron microscopy (standard and cytochemical) to characterize the process of intracellular melanization of developing larvae and to locate phenoloxidase activity in the thoracic muscles of An. quadrimaculatus. We will further compare these processes with extracellular melanization of microfilariae in the abdominal hemocoel of the same species of mosquito. We will study the effects of different prophenoloxidase and phenoloxidase inhibitors to differentiate the processes of intracellular and extracellular melanization of Brugia larvae, both in vivo and in vitro. We will also measure soluble phenoloxidase in the thoracic muscles of An. quadrimaculatus. In addition we will investigate the process of intracellular melanization of developing larvae of B. malayi in the thoraces and of D. immitis in the Malpighian tubules of Aedes sollicitans maintained in organ culture. These studies will show, if extracellular melanization of microfilariae in the hemocoel and intracellular melanization of developing larvae in the target tissue of mosquitoes follow the same or different melanization processes and/or if different time intervals are involved before either melanization response occurs.
