Qualitative differences occur in phenoloxidase activity in Anopheles mosquitoes refractory to Plasmodium.

In insects, the enzyme phenoloxidase (PO; EC 1.14.18.1) forms an integral part of the cellular and humoral defence system in response to foreign organisms. This enzyme has been shown to have at least two functions. Firstly, to synthesise melanin which encapsulates the invaders and, secondly, as a recognition system for generating other components of the host defence system. In a study with European black flies Simulium damnosum s.I., infected with Onchocerca spp., it was demonstrated that haemolymph phenoloxidase activities were lower than in traumatised specimens of Simulium (1). It was proposed that the PO system of black flies was not directly involved in immune reactions contrary to other dipterans. Recent studies on the encapsulation response of Anopheline mosquitoes to simian or monkey malaria, Plasmodium cynomolgi, in a selected line of Anopheles gambiae mosquitoes (2), showed that the ookinetes or early oocysts were encapsulated followed by melanisation. It was also shown that in the refractory mosquitoes oocysts of avian, rodent, monkey and human malaria were also encapsulated. These encapsulated or melanised bodies were described as a rare event in the Anopheles gambiae G3 (2), and it was concluded that this system of refractoriness is interesting as a biological model of insect immunity and host parasite interaction. The present study was focused on the infection of Anopheles gambiae mosquitoes with the rodent malaria Plasmodium yoelii nigeriensis. As no quantitative data has previously been available, a major part of the study has been to develop a sensitive microtitre plate phenoloxidase enzyme assay to reliably measure enzyme activity in haemolymph samples from individual mosquitoes. The system has been used to examine, in detail, temporal enzyme activities in the selected strains and in response to parasite infection. Four different strains of Anopheles gambiae S.S. were selected for susceptibility (KIL and Zands S) and refractoriness (REFMA and Zands R) to rodent malaria. The parasite, N67 strain of Plasmodium yoelii nigeriensis, was used from a large stock of infected mouse blood cryopreserved in liquid nitrogen. Stock material was prepared as previously described (3). The phenoloxidase activity was determined by a modification of a previously reported method