Anti-parasitic and Anti-viral Immune Responses in Insects Olle Terenius Department of Genetics, Microbiology and Toxicology Stockholm University 2004 Doctoral thesis from the Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Abstract Insects encounter many microorganisms in nature and to survive they have developed counter measures against the invading pathogens. In Drosophila melanogaster research on insect immunity has mainly been focused on infections by bacteria and fungi. We have explored the immune response against natural infections of the parasite Octosporea muscaedomesticae and the Drosophila C virus as compared to natural infections of bacteria and fungi. By using Affymetrix Drosophila GeneChips, we were able to obtain 48 genes uniquely induced after parasitic infection. It was also clearly shown that natural infections led to different results than when injecting the pathogens. In order to search for the ultimate role of the lepidopteran protein hemolin, we used RNA interference (RNAi). We could show that injection of double stranded RNA (dsRNA) of Hemolin in pupae of Hyalophora cecropia led to embryonic malformation and lethality and that there was a sex specific difference. We continued the RNAi investigation of hemolin in another lepidopteran species, Antheraea pernyi, and discovered that hemolin was induced by dsRNA per se. A similar induction of hemolin was seen after infection with baculovirus and we therefore performed in vivo experiments on baculovirus infected pupae. We could show that a low dose of dsHemolin prolonged the period before the A. pernyi pupae showed any symptoms of infection, while a high dose led to a more rapid onset of symptoms. By performing in silico analysis of the hemolin sequence from A. pernyi in comparison with other Hemolin sequences, it was possible to select a number of sites that either by being strongly conserved or variable could be important targets for future studies of hemolin function. © Olle Terenius, 2004 ISBN 91-7265-930-0 pp. 1-68 PrintCenter Stockholms Universitet, Stockholm 2004 2 Table of contents List of papers included in the thesis 5 Abbreviations 6 Introduction 8 Aims of this thesis 12 Parasites in Drosophila 13 Comparative analysis of reports on anti-parasitic immune responses in Drosophila melanogaster 16 Virus in Drosophila 21 RNA interference in insects 24 RNAi – a defence against intruding RNA 25 The RNA interference mechanism 25 Baculovirus and Hemolin in Lepidoptera 27 Background 27 Physiological protection against baculovirus infection 31 Developmental resistance 31 Developmental expression of Hemolin 36 Immunological protection against baculovirus infection 39 The importance of haemocytes in the response to baculovirus infection 40 The importance of phenoloxidase in the response to baculovirus infection 42 3 The involvement of hemolin in immune response to baculovirus 45 A model for anti-viral response in Lepidoptera with hemolin as a key player 48 Phylogenetic aspects on hemolin and baculovirus in Lepidoptera 50 Summary of the results 54 Acknowledgement 55 References 59 4 List of papers included in the thesis This thesis is based on the following papers#, which will be referred to in the text by roman numerals. In addition some previously unpublished data are presented. I. Roxström-Lindquist K*, Terenius O*, Faye I (2004) Parasite-specific immune response in adult Drosophila melanogaster: a genomic study. EMBO Reports 5: 207-212. * These authors contributed equally to the work. II. Bettencourt R, Terenius O, Faye I (2002) Hemolin gene silencing by ds-RNA injected into Cecropia pupae is lethal to next generation embryos. Insect Molecular Biology 11: 267-271. III. Hirai M, Terenius O, Li W, Faye I (2004) Baculovirus and dsRNA induce Hemolin, but no antibacterial activity, in Antheraea pernyi. Insect Molecular Biology 13: 399-405. IV. Li W*, Terenius O*, Hirai M, Nilsson AS, Faye I (2004) Molecular characterization, immunological and phylogenetic analysis of Hemolin, in the Chinese oak silkmoth, Antheraea pernyi. Manuscript. * These authors contributed equally to the work. #The published papers were reproduced with the permission from the copyright holders. 5 Abbreviations 20E 20-hydroxy-ecdysone AcMNPV Autographa californica multinucleocapsid nucleopolyhedrovirus ALP alkaline phosphatase ApNPV Antheraea pernyi nucleopolyhedrovirus BmNPV Bombyx mori nucleopolyhedrovirus BV budded virion DCV Drosophila C virus dsHemolin double stranded RNA of Hemolin dsRNA double stranded RNA EcR ecdysone receptor egt ecdysteroid UDP-glycosyltransferase FHV flock house virus HPLC high performance liquid chromatography LD (LD; lethal dose) the amount of a material, given all 50 at once, which causes the death of 50% of a group of test animals LdMNPV Lymantria dispar nucleopolyhedrovirus MALDI-TOF Matrix-assisted laser desorption/ionisation-time of flight MNPV multinucleocapsid nucleopolyhedrovirus mRNA messenger RNA ODV occlusion derived virion 6 PCR polymerase chain reaction per os by mouth phk pherokine PO phenoloxidase qRT-PCR quantitative reverse transcriptase PCR RNAi RNA interference S2 Schneider’s cell line 2 siRNA short interfering RNA SNPV single nucleocapsid nucleopolyhedrovirus USP ultraspiracle protein 7 Introduction Insecta is the most successful group of terrestrial animals, both by number of individuals and number of species. Insects outnumber the rest of the terrestrial animals by several orders of magnitude, for example are the species of vertebrates (fish, amphibians, reptiles, birds and mammals together) only as many as the coleopteran family Curculionidae. However, one has to take into consideration that in the vertebrate classes most species are described, which is not the case for insects. It is only in the best-known insect families where the number of newly described species (per year and total number of species in the family) is as low as in vertebrates (O. Terenius and S. Firle, unpublished). Some insects can appear in gargantuan numbers as in locust swarms, consisting of many billion individuals that eat several tons of plant material each day. These swarms have been reported since historic times and were one of the punishments for Pharaoh not letting Moses and his people leave Egypt “and there remained not any green thing in the trees, or in the herbs of the field, through all the land of Egypt” (Exodus, 10:15). While I am writing this, locust swarms strike sub-Saharan Africa leading to devastating consequences. Another important torment for humans caused by the act of insects is plague, which is spread by fleas and in medieval times made such impact on humanity that it caused the ever-growing world population to temporally decrease. Today, the plague is feared, but very rare. This is however, not the case for the vector- 8 borne (insect-transmitted) disease malaria that is spread by mosquitoes and ends a human life every 20 second. In contrast, insects also may have a direct beneficial impact on human life by producing honey and silk. In all these cases, their impact is linked to the fact that they are so numerous. How have the insects become so successful? Could their rapid development and massive production of progeny suffice as explanations, or are there other things that contribute to the success? A short-lived species could be expected to invest more in reproductive fitness than in life prolonging traits. However, many insects do live for long periods of time. In the Palaearctic and Nearctic regions, hibernation is necessary and lasts over several months. For some species, like the currently reproducing 17-year cicada (Boyce, 2004), the life prolonging traits are undoubtedly crucial. One of the important traits that make the insects survive for long enough periods of time to produce their progeny is their defence system against invading microbes. In the last three decades, since the inducible immune response against bacteria was first discovered in Drosophila melanogaster by Boman and co-workers (1972), the field of insect immunity has expanded tremendously including also anti-fungal response in insects (for reviews, see Hultmark, 2003; Hoffman, 2003; Steiner, 2004). Moreover, this knowledge has been integrated with the innate immune response in mammals and today there are a number of interesting reviews comparing the innate immune responses in mammals and insects (for recent examples, see Boman, 2003; Beutler, 2004). The anti-parasitic responses have been studied 9