Hemolin expression during Cecropia development and its effect on malaria parasites

Hemolin is a lepidopteran member of the immunoglobulin superfamily, initially isolated from the giant silkmoth Hyalophora cecropia. Hemolin is also induced by stimulation with microbial cell wall components and was recently shown to be strongly upregulated by baculovirus and double stranded RNA. An interesting characteristic of the protein is that it is not only highly expressed during infection but also during development. The work presented in this thesis investigated the expression of hemolin during oogenesis and embryogenesis in H. cecropia. Vitellogenic follicles from ovaries were analysed for the presence of the protein by immunohistochemistry in whole-mount preparations and in cryosections. PCR was used to show the presence of Hemolin transcripts throughout vitellogenesis and choriogenesis and in fertilized and unfertilized mature eggs and Western blots showed the protein in unfertilized eggs, yolk cells and embryo. Injection of the moulting hormone 20-hydroxyecdysone (20E) into hibernating diapausing pupae (low metabolic state), upregulates Hemolin. When diapausing pupae were treated with 20E and the protein synthesis inhibitor cycloheximide, its expression stayed low. This shows that the hormone indirectly regulates Hemolin by some factor(s) induced by 20E. When both bacteria and 20E were injected into diapausing pupae, an enhanced induction of hemolin gene expression occurred. Despite the seemingly indirect 20E regulation, several putative hormone responsive elements were found in the upstream region of the Hemolin (HRE-IR, HRE-M and MRE). When these elements were analysed by gel electrophoresis mobility shift assays (EMSA) to investigate their binding to nuclear factors, all the sites resulted in specific retarded bands. The HRE-IR binding factor was clearly increased by ecdysone. Last but not least we have investigated the effect of Hemolin on development of the malaria parasite Plasmodium falciparum in the midgut of the Anopheles mosquitoes. Hemolin completely inhibits the development of the parasite into its final transmission stage, the sporozoite. A future goal is to generate para-transgenic mosquitoes, enforced by hemolin, to stop malaria transmission. Importantly, hemolin did not affect the mosquito fecundity when fed to the mosquito. We are currently constructing truncated forms of hemolin to gain insight into which parts are important for its effect on the parasite