Developing transgenic models to induce late-life mortality in the malaria vector Anopheles gambiae Silke Fuchs A Thesis submitted in fulfilment of requirements for the degree of Doctor of Philosophy of Imperial College London Imperial College London Natural Sciences Imperial College Road, South Kensington London, SW7 2AZ 1 Declaration of Originality I declare that the intellectual content of this thesis is the product of my own research work. Any ideas or quotations from the work of other people are fully acknowledged in accordance with the standard referencing practices. 2 Abstract A key factor that limits the transmission of malaria is the age of its vector, Anopheles gambiae. This is due to the long extrinsic incubation period (EIP) of the parasite which can last a high proportion of the adult mosquito‟s life. Therefore, only a small percentage of a mosquito population is able to transmit malaria. This bottleneck could be of use in developing novel vector control strategies which are based on artificially shortening the adult life span of mosquitoes through the introduction of suitably tailored transgenic constructs. One strategy was to induce a toxic late-acting amino acid metabolism disease in mosquitoes that kills females after their amino acid rich blood meal. Phenylpyruvate, the toxic accumulation product in human Phenylketonuria disease, was found to cause increased death when fed to mosquitoes. GC-MS analysis showed that similar to PKU patients, phenylpyruvate had been converted to another toxic metabolite phenyllactate. Using RNAi, two enzymes PAH and PPO9 of the phenylalanine pathway were knocked down and their effect on survival, behaviour and melanisation immune response was investigated. While knockdown of one enzyme caused a reduced melanisation in response to parasite infection, reduced activity of the other led to a shortened adult life span but no change in melanisation. This led to the conclusion that both enzymes are regulating different processes and that melanisation is not necessary for mosquito survival and possibly mosquito immunity. Another strategy was to express long stretches of polyglutamines that are responsible for the toxicity of several neurodegenerative diseases, including Huntington's disease. Because uniform CAG repeats containing huntingtin exon 1 were highly instable in vitro and in vivo, different N-terminal huntingtin fragments with alternating CAG/CAA repeats fused to EGFP were inserted by attP/attB integration system in the model system Drosophila melanogaster and in Anopheles gambiae to characterise polyglutamine length dependent aggregation formation in the optic lobe and its effect on survival and behaviour. In both species, the polyglutamine fragments of putative toxic length tended to form aggregates in the photoreceptor cells. This led to a significant reduced adult life span in flies but not in mosquitoes. The latter showed a change in their chromatic vision ability. Alternative promoters were characterised for future HD mosquito models and could help to understand the observed difference between flies and mosquitoes. Both strategies have a potential as promising late-life acting vector control tools that merit further exploration. 3 Acknowledgements This thesis would not have been possible without the help of many people, some of whom are gratefully acknowledged below. I would like to thank Andrea for giving me the opportunity to work in this exciting field. I am heartily thankful to Tony for his support, guidance and encouragement throughout the years. Moreover, this thesis could not have been put together without the expertise and input of Volker Behrends, Holger Apitz, Sang Chan and Tibebu Habtewold. I am very grateful to have worked with such great lab members and friends. Niki, Kalle and Phi, I miss those lunch breaks and Frisbee sessions. Dan, I hope we will stay in touch although you turned your back onto Science. Miriam, Ann and Fede thanks for our nice chats inside the insectary. Lorenzo, those grapes kept me alive during the last days of writing. Roberto and Alekos, I wish you all the best making those transgenic lines. A big thanks also to my Mum and my brother who always supported me. And last not least, I want to thank my husband Robert for always being there for me, even when we lived apart. 4 Table of Contents Abstract ...................................................................................................................................... 3 Acknowledgements .................................................................................................................... 4 Table of Contents ....................................................................................................................... 5 1. Chapter: Introduction ................................................................................................... 17 1.1. Malaria: situation and trends ..................................................................................... 17 1.2. The biology of human malaria .................................................................................. 18 1.2.1. The parasite life cycle ........................................................................................ 18 1.2.2. The vector species .............................................................................................. 19 1.2.3. Importance of vector life span for malaria transmission ................................... 20 1.3. Malaria control strategies .......................................................................................... 22 1.3.1. Anti-malarial drugs ............................................................................................ 23 1.3.2. Vaccines ............................................................................................................. 24 1.4. Vector control ............................................................................................................ 25 1.4.1. Environmental management .............................................................................. 26 1.4.2. Chemical control by ITN and ITS ..................................................................... 26 1.4.3. Biological control............................................................................................... 28 1.5. Transgenic technologies as a new tool for vector control ......................................... 29 1.5.1. Population suppression ...................................................................................... 29 1.5.2. Population replacement ...................................................................................... 30 1.5.2.1. Population replacement using gene drive systems ..................................... 31 1.5.2.2. Manipulating the vectorial capacity ........................................................... 31 1.5.2.2.1. Interfering with mosquito tissue recognition by the parasite................... 32 1.5.2.2.2. Immune response effectors ...................................................................... 32 1.5.2.2.3. Manipulation of the vectors‟ feeding preference ..................................... 33 1.5.2.2.4. Inducing late-life mortality ...................................................................... 33 1.5.2.2.4.1. Inducing a blood-meal responsive amino acid disease ..................... 35 1.5.2.2.4.1.1. Phenylalanine/ tyrosine metabolism ........................................... 35 5 1.5.2.2.4.2. Introducing a polyglutamine disease in Anopheles gambiae mosquitoes ...........................................................................................................38 1.6. Aim............................................................................................................................ 41 2. Chapter: Material and Methods ................................................................................... 42 2.1. Bacterial Cultures ...................................................................................................... 42 2.2. Isolation of plasmid DNA (Miniprep, Maxiprep) ..................................................... 42 2.3. Restriction digestion of DNA .................................................................................... 42 2.4. Dephosphorylation of cut plasmids ........................................................................... 42 2.5. Extraction of DNA from Agarose gels ...................................................................... 43 2.6. Ligation of plasmid DNA .......................................................................................... 43 2.7. Transformation of E. coli with plasmid DNA ........................................................... 43 2.8. Polymerase- chain reaction of plasmid DNA ............................................................ 44 2.9. Generation of the httex1pQ transformation vectors .................................................. 44 2.10. Generation of the 3xP3 httNQEGFP transformation vectors ................................ 45 2.11. Generation of the ELAV httNQEGFP transformation vectors ............................... 46 2.12. Phenol/ Chloroform extraction .............................................................................. 46 2.13. Synthesis of capped integrase RNA ...................................................................... 46 2.14. Mosquito strains .................................................................................................... 47 2.15. Mosquito rearing...................................................................................................