Identification of bacteria associated with malaria mosquitoes –Their characterisation and potential use Jenny Lindh Department of Genetics, Microbiology and Toxicology Stockholm University Bilden på framsidan är ritad av Emil 4år ©Jenny Lindh, Stockholm 2007 ISBN 91-7155-399-1 Printed in Sweden by US-AB, Stockholm 2007 Till min familj Abstract The use of transformed bacteria to stop or kill disease-causing agents in the gut of vector insects is called paratransgenics. Two of the major steps in creating a paratransgenic Anopheles mosquito, unable to spread the Plasmo- dium parasites that cause malaria, are to find a bacterium suitable for the purpose and a way to introduce the transformed bacterium into mosquitoes in the field. In this project, bacteria associated with malaria mosquitoes have been identified by phylogenetic analysis of their 16S rRNA genes. First, the midgut flora of field-caught Anopheles mosquitoes was examined using two pathways, one culture dependent and one culture independent. Second, six bacterial species from an An. gambiae laboratory colony, and third, ten iso- lates from Anopheles oviposition sites have been identified. Altogether, 32 bacterial species, representing 16 families, seven classes and four phyla were identified. Interestingly, several of them are related to bacteria known to be symbionts in other insects. Two possible ways of introducing bacteria into mosquitoes in the field in a paratransgenic approach were investigated in a laboratory setting. It was shown that sugar solutions with or without bacteria are equally attractive to An. gambiae mosquitoes and that the mosquitoes were able to take up bacte- ria from the water they emerged from. These results show that it may be possible to use sugar-baits and oviposition sites for distribution of geneti- cally modified bacteria in the field. To facilitate the distribution of the modified bacteria mosquito attractants should be used. We investigated whether the bacterial isolates identified in this project produce attractants affecting mosquito sugar-feeding or oviposi- tion site selection. To examine the sugar-feeding responses a dual-choice method based on coloured sugar solutions were developed. While no re- sponses were observed from the mosquitoes towards bacteria-containing sugar solutions, seven of the 19 isolates examined mediated positive oviposi- tion responses. The volatiles emitted from the bacterial isolates were sam- pled by Solid-Phase Microextraction (SPME) and identified by Gas- Chromatography coupled to Mass-Spectrometry (GC-MS). In total, 13 puta- tive oviposition attractants were identified among the volatiles emitted by the attractive bacteria List of publications This thesis is based on the following papers, which will be referred to in the text by their Roman numerals. Paper I and Paper II were reprinted with kind permission from American Society of Microbiology and Elsevier, respec- tively. I Lindh J.M., Terenius O. and Faye I. 16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new spe- cies related to known insect symbionts. Applied and Environ- mental Biology. 2005, 71: 7217–23. II Lindh J.M., Terenius O., Eriksson-Gonzales K., Knols B.G.J. and Faye I. Re-introducing bacteria in mosquitoes–A method for determination of mosquito feeding preferences based on coloured sugar solutions. Acta Tropica. 2006, 99:173-83. III Lindh J.M., Borg-Karlson A.-K. and Faye I. Investigation of transstadial and horizontal transfer of bacteria within an Anophe- les gambiae (Diptera: Culicidae) laboratory colony and oviposi- tion response of An. gambiae to bacteria-containing water. Manuscript. IV Lindh J.M., Kännaste, A., Knols B.G.J., Faye I. and Borg- Karlson A.-K. Identification of volatiles and oviposition re- sponses of Anopheles gambiae s.s. (Diptera: Culicidae) mosqui- toes to solutions containing bacteria previously isolated from An. gambiae s.l. midguts or oviposition sites. Manuscript. Additional papers not included in the thesis • Kämpfer P., Lindh J.M., Terenius O., Haghdoost S., Falsen E., Busse H-J. and Faye I. Thorsellia anophelis gen. nov., sp. nov., a new member of the Gammaproteobacteria. International Journal of Systematic and Evolutionary Microbiology. 2006, 56:335-338. • Kämpfer P., Terenius O., Lindh J.M. and Faye I. Janibacter anophelis sp. nov., isolated from the midgut of Anopheles arabien- sis. International Journal of Systematic and Evolutionary Microbiol- ogy. 2006, 56:389-392. Contents Introduction .....................................................................................................9 Malaria.............................................................................................................................9 The disease................................................................................................................9 Plasmodium parasites -The causative agent...........................................................10 Anopheles mosquitoes -The vectors .......................................................................11 Paratransgenics.............................................................................................................12 Paratransgenics in mosquitoes................................................................................13 Bacteria and mosquitoes...............................................................................................14 Bacteria as larval food .............................................................................................14 Transstadial transfer of bacteria ..............................................................................14 Bacteria in adult midguts..........................................................................................15 Midgut bacteria and the effect on Plasmodium parasites and the mosquito host...20 Bacteria as a source of mosquito semiochemicals..................................................21 Present investigation.....................................................................................24 Aims of this thesis..........................................................................................................24 Identification of bacteria associated with Anopheles mosquitoes.................................24 Methods ...................................................................................................................24 Results .....................................................................................................................26 Complementary discussion......................................................................................27 Laboratory investigations of two possible ways of introducing bacteria to mosquitoes in the field in a paratransgenic approach ..........................................................................37 Results .....................................................................................................................37 Complementary discussion......................................................................................39 Identification of putative semiochemicals for Anopheles mosquitoes with bacterial origin..............................................................................................................................41 Methods ...................................................................................................................41 Results .....................................................................................................................42 Complementary discussion......................................................................................45 Conclusions...................................................................................................................47 Acknowledgement.........................................................................................48 References....................................................................................................51 Abbreviations CCUG Culture Collection, University of Gothenburg GC-MS Gas-Chromatograph coupled to Mass-Spectrometry GFP Green Fluorescent Protein ICIPE International Centre of Insect Physiology and Ecology kb Kilobases KTH Kungliga Tekniska Högskolan (Royal Institute of Technology) LA Luria-Bertaini agar LB Luria-Bertaini broth ON Over-night PCA Principal Component Analysis PCR Polymerase Chain Reaction RDP II Ribosomal Database Project II rDNA ribosomal DNA rRNA ribosomal RNA s.l. sensu lato SPME Solid Phase Microextraction s.s. sensu stricto SU Stockholm University TTGE Temporal Temperature Gradient gel Electrophoresis Introduction Malaria The disease Malaria is by far the most significant parasitic disease that causes morbidity and mortality in humans. It was estimated that 300-500 million clinical cases and more than 1 million deaths due to malaria occur annually (WHO 2005). The majority of the fatal cases are in children under five years of age (Breman 2001). Malaria is endemic in over 100 countries (Fig.1). However, most of the deths (more than 80%) occur in sub Saharan Africa (Snow et al. 2005, WHO 2005). The problems with malaria are getting worse despite many campaigns against the disease, mostly due to resistance of parasites against drugs, mosquitoes against insecticides, the weak health systems and widespread poverty in Africa (Hargreaves et al. 2000, Hargreaves et al. 2003, White 2004, WHO 2005). Furthermore, global warming is anticipated