Overend, Gayle (2010) Drosophila as a model for the Anopheles Malpighian tubule. PhD thesis, University of Glasgow. http://theses.gla.ac.uk/1604/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Drosophila as a model for the Anopheles Malpighian tubule A thesis submitted for the degree of Doctor of Philosophy at the University of Glasgow Gayle Overend Integrative and Systems Biology Faculty of Biomedical and Life Sciences University of Glasgow Glasgow G11 6NU UK August 2009 2 The research reported within this thesis is my own work except where otherwise stated, and has not been submitted for any other degree. Gayle Overend 3 Abstract The insect Malpighian tubule is involved in osmoregulation, detoxification and immune function, physiological processes which are essential for insect development and survival. As the Malpighian tubules contain many ion channels and transporters, they could be an effective tissue for targeting with novel pesticides to control populations of Diptera. Many of the insecticide compounds used to control insect pest species are no longer suited to their task, and so new means of control must be found. The malarial mosquito, Anopheles gambiae , spreads the Plasmodium parasite which is responsible for over one million deaths each year, and is one of the species on which many current insecticides are no longer effective. Anopheles is notoriously difficult to study due to a lack of natural mutation stocks and transgenic capabilities, as well as the difficulties involved with maintaining a colony. The fruit-fly Drosophila melanogaster is a useful model organism for Anopheles , and previous studies suggest that the mechanisms of Malpighian tubule function are well conserved between the two species. Following microarray investigations to identify genes which were highly enriched in both the Anopheles and Drosophila Malpighian tubules, four homologous gene- pairs were selected, AGAP097752 and CG15406, AGAP012251 and Picot, AGAP009005 and ZnT35C, and AGAP002587 and CG8028 . Analysis of the Anopheles Malpighian tubule microarray data-set showed ion channels and transporters to be highly expressed in the tubules, although similarly to Drosophila , very few of the renal up-regulated genes have been characterised. The gene-pairs chosen were all novel, but putatively predicted to be involved in sugar transport, phosphate transport, zinc transport and monocarboxylate transport respectively. These are functions which are likely to be essential, but so far remain unstudied in the insect renal system. The gene-pairs were chosen with two main purposes; to determine how closely expression of the genes was conserved between Anopheles and Drosophila , and also to determine which of the genes were essential, and could therefore be effective insecticide targets. The homologous gene-pair AGAP007752 and CG15406 have well-conserved expression in the Malpighian tubules, suggesting that they are functionally important genes. This was shown in Drosophila , where knockdown of CG15406 4 expression was lethal to the fly. A direct role in tubule fluid secretion was not found, and experiments to determine the sugars transported by CG15406 were inconclusive, possibly due to an abundance of highly-expressed sugar transporters in the tubules. The inorganic phosphate co-transporters AGAP012251 and Picot also show conservation of expression in the Malpighian tubules, and are likely to be involved in the transport of inorganic phosphate into the tubules for incorporation into metallo-organic concretions. In the Anopheles tubules the concretions are found in the main segment, in the Drosophila tubules they are located in the distal initial and transitional segments, where AGAP012251 and Picot are expressed. Picot is essential for Drosophila development through to adulthood, and for survival as an adult, although the transporter does not appear to be directly involved in fluid secretion. Expression of neither AGAP012251 nor Picot is confined to the tubules. The putative zinc transporters AGAP009005 and ZnT35C show a highly conserved expression pattern, and appear to be involved in the secretion of excess zinc from the Malpighian tubules. ZnT35C is essential early-on in Drosophila development, and for survival in the adult fly. Similarly to Picot and CG15406 , there is no direct role for ZnT35C in fluid secretion from the tubules under normal zinc conditions. The putative monocarboxylate transporters AGAP002587 and CG8028 are not as well conserved, as AGAP002587 is highly up- regulated in the tubules of female mosquitoes both before and after a blood- feed, whereas CG8028 has no sex-specific up-regulation. CG8028 is not essential for Drosophila development or survival, and plays no discernable role in fluid secretion. The data collected during this investigation suggests that in general there is a high level of conservation of expression between homologous transport genes in the Anopheles and Drosophila Malpighian tubules. The three gene-pairs which show the greatest conservation of expression are also essential for development and survival in Drosophila . This suggests that cross-species studies are an effective way of finding essential and important genes. The data collected also suggests that Drosophila is a reliable model for Anopheles , and could be used as a high-throughput system of finding genes which could be effective insecticide targets in Diptera. 5 Table of Contents Abstract ................................................................................ 3 Index of Figures...........................................................................12 Index of Tables............................................................................15 Abbreviations..............................................................................16 Acknowledgements.......................................................................19 Chapter 1 Introduction ...............................................................20 1.1 Summary ......................................................................... 21 1.2 Diptera as a Pest Species ...................................................... 22 1.2.1 Insecticide Resistance in Mosquitoes ................................... 22 1.3 Anopheles gambiae ............................................................. 26 1.3.1 The Search for Novel Insecticide Targets in Anopheles ............. 28 1.3.2 Genetics .................................................................... 29 1.3.2.1 Spontaneous Mutations.............................................. 29 1.3.2.2 Directed Mutations .................................................. 29 1.3.3 As an Experimental Organism ........................................... 30 1.3.4 Models for Anopheles ..................................................... 31 1.4 Drosophila melanogaster ...................................................... 32 1.4.1 Drosophila Physiology .................................................... 33 1.4.2 Drosophila Genetics....................................................... 34 1.4.2.1 Spontaneous Mutation............................................... 34 1.4.2.2 Directed Mutation.................................................... 34 1.4.2.3 P-elements............................................................ 35 1.4.2.4 The UAS/GAL4 system............................................... 36 1.5 Renal transport ................................................................. 37 1.6 The Drosophila Malpighian tubules .......................................... 38 1.6.1 Morphology ................................................................. 39 1.6.2 Physiology .................................................................. 40 1.7 The Anopheles Malpighian tubules ........................................... 41 1.7.1 Morphology ................................................................. 42 1.7.2 Physiology .................................................................. 42 1.8 Drosophila as a Model for Anopheles ........................................ 43 1.8.1 As a Physiological Model ................................................. 43 1.8.2 As a Malpighian Tubule Model ........................................... 44 1.9 Microarray Analysis of Anopheles and Drosophila .......................... 45 6 1.9.1 Microarrays in Drosophila ................................................ 46 1.9.2 Microarrays in Anopheles ................................................. 46 1.10 Aims of this Project ............................................................ 48 Chapter 2 Methods and Materials...................................................49 2.1 Drosophila melanogaster ...................................................... 50 2.1.1 Drosophila Stocks ......................................................... 50 2.1.2 Drosophila Rearing ........................................................ 52 2.2 Anopheles gambiae ............................................................