Overend, Gayle (2010) Drosophila as a model for the Anopheles Malpighian tubule. PhD thesis, University of Glasgow. http://theses.gla.ac.uk/1604/
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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
3
Abstract
Anopheles gambiae Plasmodium Anopheles Drosophila melanogaster Anopheles
Anopheles Drosophila AGAP097752 CG15406, AGAP012251 Picot, AGAP009005 ZnT35C, AGAP002587 CG8028 Anopheles Drosophila Anopheles Drosophila
AGAP007752 CG15406 Drosophila CG15406 4 CG15406 AGAP012251 Picot Anopheles Drosophila AGAP012251 Picot Picot Drosophila AGAP012251 Picot AGAP009005 ZnT35C ZnT35C Drosophila Picot CG15406 ZnT35C AGAP002587 CG8028 AGAP002587 CG8028 CG8028 Drosophila
Anopheles Drosophila Drosophila Drosophila Anopheles 5 Table of Contents
Abstract ...... 3 Index of Figures...... 12 Index of Tables...... 15 Abbreviations...... 16 Acknowledgements...... 19 Chapter 1 Introduction ...... 20 Anopheles gambiae Anopheles Anopheles 1.4 Drosophila melanogaster Drosophila Drosophila Drosophila Anopheles 1.8 Drosophila Anopheles Anopheles Drosophila 6 Drosophila Anopheles Chapter 2 Methods and Materials...... 49 2.1 Drosophila melanogaster Drosophila Drosophila 2.2 Anopheles gambiae Anopheles Anopheles 7 Drosophila Drosophila 2.11 In Situ Drosophila In Situ Anopheles In Situ Drosophila melanogaster Drosophila Drosophila Drosophila Drosophila 8 Chapter 3 Validation and Analysis of the Anopheles Malpighian Tubule Microarray ...... 85 Anopheles Anopheles Chapter 4 AGAP007752 and CG15406 as Insecticide Targets...... 114 Anopheles Drosophila Anopheles Drosophila AGAP007752 CG15406 AGAP007752 Anopheles 9 CG15406 Drosophila CG15406 CG15406 AGAP007752 CG15406 AGAP007752 CG15406 AGAP007752 CG15406 Chapter 5 AGAP012251 and Picot as Insecticide Targets...... 150 AGAP012251 Picot AGAP012251 Picot Picot AGAP012251 Picot 10 AGAP012251 Picot AGAP012251 Picot Chapter 6 AGAP009005 and ZnT35C as Insecticide Targets ...... 180 AGAP009005 ZnT35C AGAP009005 ZnT35C ZnT35C AGAP009005 ZnT35C AGAP009005 ZnT35C AGAP009005 ZnT35C Chapter 7 AGAP002587 and CG8028 as Insecticide Targets ...... 205 AGAP002587 CG8028 AGAP002587 CG8028 11 CG8028 AGAP002587 CG8028 AGAP002587 CG8028 AGAP002587 CG8028 Chapter 8 Conclusions and Further Work...... 228 Drosophila Anopheles Appendix ...... 237 Bibliography ...... 244 12 Index of Figures
Anopheles gambiae Drosophila Drosophila Drosophila Anopheles AGAP002587 AGAP007495 Anopheles AGAP007752 CG15406 CG15406 AGAP007752 Anopheles AGAP007752 Anopheles AGAP007752 Anopheles in situ Anopheles CG15406 Drosophila CG15406 in situ Drosophila Drosophila CG15406 13 CG15406 CG15406 CG15406 AGAP012251 Picot Anopheles AGAP012251. AGAP012251 Anopheles AGAP012251 Anopheles in situ Anopheles Picot Drosophila Picot Drosophila in situ Drosophila Drosophila Picot Picot ZnT35C CG3994) AGAP009005 ZnT35C Drosophila AGAP009005 Anopheles AGAP009005 Anopheles 14 AGAP009005 Anopheles in situ ZnT35C Drosophila ZnT35C Drosophila in situ ZnT35C ZnT35C AGAP002587 Anopheles AGAP002587 Anopheles AGAP002587 Anopheles in situ CG8028 Drosophila CG8028 Drosophila in situ CG8028 CG8028
15 Index of Tables
Drosophila Drosophila Taq Drosophila Drosophila Anopheles Drosophila Anopheles Anopheles Anopheles Drosophila CG15406 Drosophila CG15046 CG15406 Picot Drosophila Picot. Picot ZnT35C Drosophila ZnT35C ZnT35C CG8028 Drosophila CG8028 16
Abbreviations 17
18 19 Acknowledgements
20
Chapter 1 - Introduction 21 1.1 Summary
Anopheles gambiae, Anopheles Drosophila melanogaster Drosophila Drosophila Anopheles Drosophila Drosophila Anopheles
22 1.2 Diptera as a Pest Species
Anopheles gambiae Aedes aegypti Culicoides et al.
1.2.1 Insecticide Resistance in Mosquitoes
et al. Chrysanthemum 23
Anopheles gambiae
Cyp6g1 Drosophila melanogaster et al. Cyp6g1 Drosophila et al. Cyp6g1 Musca domestica 24
25
Figure 1.1 Distribution of Permethrin resistance in Anopheles gambiae in West Africa (W.H.O., 2005). 26 1.3 Anopheles gambiae
Anopheles Anopheles Anopheles Plasmodium Anopheles gambiae sensu strictu Plasmodium falciparum
et al. Cochliomyia hominovorrax Glossina morsitans Ceratitis capitata Anopheles et al.
27
Figure 1.2 Map of the world showing the predominant malaria vector in each country (C.D.C., 2004). 28 1.3.1 The Search for Novel Insecticide Targets in Anopheles
Anopheles
Anopheles Drosophila melanogaster et al.
Anopheles et al.
29 Anopheles
1.3.2 Genetics
1.3.2.1 Spontaneous Mutations
Anopheles Anopheles Anopheles white et al.
1.3.2.2 Directed Mutations
Anopheles Drosophila Drosophila 30 Aedes aegypti Anopheles stephensi Anopheles gambiae et al. et al.
et al. et al. et al. et al. Anopheles et al.
1.3.3 As an Experimental Organism
Anopheles Anopheles Plasmodium berghei Anopheles Anopheles gambiae et al.
Anopheles 31 Anopheles Aedes aegypti Culex pipiens Anopheles Plasmodium
Anopheles Aedes et al. et al. Anopheles et al Aedes et al.
1.3.4 Models for Anopheles
Anopheles gambiae Anopheles Anopheles Anopheles stephensi, pMinEGFP et al. Anopheles stephensi Aedes aegypti Plasmodium gallinaceum Aedes Aedes et al. et al. 32 Aedes Anopheles Aedes Aedes aegypti Drosophila melanogaster Drosophila Anopheles Drosophila Drosophila Anopheles Drosophila et al.
1.4 Drosophila melanogaster
Drosophila Drosophila 33 et al. Drosophila
1.4.1 Drosophila Physiology
Drosophila Drosophila Drosophila Drosophila Drosophila et al. et al. et al. et al.
Drosophila in vivo 34 1.4.2 Drosophila Genetics
Drosophila Drosophila Drosophila
1.4.2.1 Spontaneous Mutation
Drosophila
1.4.2.2 Directed Mutation
Drosophila
Drosophila 35 Drosophila
1.4.2.3 P-elements
et al.
white
et al. LacZ et al. 36 1.4.2.4 The UAS/GAL4 system
Drosophila
Enhancer Trap UAS-Gene X GAL4
X
GAL4
Genomic Enhancer GAL4 Gene X UAS Tissue-specific expression of GAL4 Transcriptional activation of Gene X
Figure 1.3 The UAS/GAL4 Drosophila expression system. Flies expressing GAL4 in a tissue- specific pattern are crossed with flies containing a UAS-transgene. The UAS-transgene is therefore only expressed in the chosen cell types (Brand and Perrimon, 1993).
37 Drosophila
Drosophila white et al.
1.5 Renal transport
38 Dixippus morosus Rhodnius prolixus Manduca sexta Aedes aegypti Schistocerca gregaria Acheta domesticus Formica polycenta Drosophila melanogaster et al. et al. et al. et al.
1.6 The Drosophila Malpighian tubules
Drosophila et al. 39 1.6.1 Morphology
Drosophila µ Drosophila et al.
et al. et al et al. et al. et al.
40
A B
C D E
Figure 1.4 Drosophila Malpighian tubule morphology. A: classical morphology (Wessing, 1978). B: detailed morphology and cell counts. C: stained principal cells, D: stained stellate cells, and E: stained bar-shaped cells in the initial segment, from enhancer trap investigations (Sozen et al. , 1997).
1.6.2 Physiology
Drosophila et al. 41 et al. Drosophila et al. et al. et al. et al. et al.
et al. et al. et al. et al. et al.
et al. et al.
1.7 The Anopheles Malpighian tubules
Anopheles 42 Anopheles Drosophila Aedes aegypti
1.7.1 Morphology
Anopheles Drosophila et al. Aedes aegypti
Anopheles Drosophila Drosophila et al. Drosophila Anopheles Anopheles et al.
1.7.2 Physiology
Aedes Anopheles Drosophila Anopheles et 43 al. et al.
et al. et al. Anopheles et al.
1.8 Drosophila as a Model for Anopheles
Drosophila et al. et al. Drosophila
1.8.1 As a Physiological Model
Anopheles Drosophila Drosophila, 44 Drosophila Drosophila cinnabar et al. et al. Drosophila Drosophila ( , 2002)
Drosophila Anopheles et al Drosophila sex peptide receptor SPR et al. Drosophila Anopheles SPR
Drosophila Anopheles et al Drosophila Plasmodium gallinaceum et al., Plasmodium Anopheles Plasmodium berghei Plasmodium Anopheles Drosophila et al.
Drosophila Anopheles
1.8.2 As a Malpighian Tubule Model
Drosophila Drosophila Drosophila et al 45 et al. Drosophila Anopheles stephensii Aedes aegypti Glossina mortisans Schistocerca gregaria et al. et al Drosophila Anopheles et al ) Anopheles Anopheles Drosophila Anopheles Drosophila et al Drosophila Anopheles stephensi Aedes aegypti et al. Drosophila et al. Aedes aegypti Aedes et al. Anopheles Drosophila
1.9 Microarray Analysis of Anopheles and Drosophila
46 Drosophila Anopheles Plasmodium Anopheles
1.9.1 Microarrays in Drosophila
Drosophila Drosophila et al et al.
et al. Drosophila
1.9.2 Microarrays in Anopheles
Anopheles et al. 47
et al et al. et al. Anopheles et al et al.
et al. et al. et al. et al.
48 1.10 Aims of this Project
Anopheles Drosophila Anopheles Drosophila Anopheles Anopheles Drosophila Drosophila Anopheles
49
Chapter 2 - Methods and Materials 50 Anopheles gambiae Drosophila melanogaster
2.1 Drosophila melanogaster
Drosophila melanogaster Drosophila
2.1.1 Drosophila Stocks
Drosophila melanogaster Drosophila w1118
Table 2.1 – Drosophila stocks used during this study.
Strain Genotype Purpose Oregon R Wild type in situ w1118 w1118 ; +/+; +/+ c42 GAL4 w; +/+; c42-GAL4/c42-GAL4 et al. Ac tin -GAL4 w; Act5c-GAL4/CyO; +/+ (Stergiopoulos et Drosophila al., 2009) Uro -GAL4 Uro-GAL4; +/+; +/+ (Terhzaz et al., 2010) c724 GAL4 w; c724 -GAL4 /c724 -GAL4 ; et al. +/+ w; +/+; elav c155 - ELAV -GAL4 GAL4/elav c155 -GAL4 (Bloomington Stock Centre) 51
Table 2.2 – Drosophila transgenic fly-lines created and used during this study.
Fly -line Genotype Purpose CG15406.c1.l 1 w; +/+; CG15406.c1.l1/T M3,sb CG15406 CG15406.c1.l 4 w; +/+; CG15406.c1.l4/TM3,sb CG15406 CG15406.c1.l 9 w; CG15406.c1.l9/CyO; +/+ CG15406 CG15406.c2.l 4 w; +/+; CG15406.c2.l4/TM3,sb CG15406 CG15406.c2.l 7 w; CG15406.c2.l7/CyO; +/+ CG15406 CG15406.c2.l 8 w; CG15406.c2.l8/CyO; +/+ CG15406 CG15406.c3.l 3 w; CG15406.c3.l3/CyO; +/+ CG15406 CG15406.c3.l 4 w; +/+; CG15406.c3.l4/TM3,sb CG15406 CG15406.c3.l 5 w; CG15406.c3.l5/CyO; +/+ CG15406 Picot.c1.l 3 w; Picot.c1.l 3/CyO; +/+ Picot Picot.c1.l 5 w; +/+; Picot.c1.l 5/TM3,sb Picot Picot.c1.l 6 w; +/+; Picot.c1.l 6/TM3,sb Picot Picot.c2.l 3 w; Picot.c2.l 3/CyO; +/+ Picot Picot.c2.l 5 w; Picot.c2.l 5/CyO; +/+ Picot Picot.c2.l 6 w; +/+; Picot.c2.l 6/TM3,sb Picot Picot.c3.l 2 w; +/+; Picot.c3.l 2/TM3,sb Picot Picot.c3.l 3 w; Picot.c3.l 3/CyO; +/+ Picot Picot.c3.l 5 w; +/+; Picot.c3.l 5/TM3,sb Picot CG3994.c1.l 1 w; +/+; CG3994.c1.l 1/TM3,sb CG3994 CG3994.c1.l 3 w; CG3994.c1.l 3/CyO; +/+ CG3994 CG3 994.c1.l 4 w; CG3994.c1.l 4/CyO; +/+ CG3994 CG3994.c2.l 1 w; CG3994.c2.l 1/CyO; +/+ CG3994 CG3994.c2.l 2 w; CG3994.c2.l 2/CyO; +/+ CG3994 CG3994.c2.l 8 w; +/+; CG3994. c2.l 8/TM3,sb CG3994 CG3994.c3.l 1 w; CG3994.c3.l 1/CyO; +/+ CG3994 CG3994.c3.l 2 w; CG3994.c3.l 2/CyO; +/+ CG3994 CG3994.c3.l 4 w; +/+; CG3994.c3.l 4/TM3,sb CG3994 CG8028.c1.l1 w; +/+; CG8028.c1.l1 /TM3,sb CG8028 CG8028.c1.l3 w; +/+; CG8028.c1.l3 /TM3,sb CG8028 CG8028.c1.l4 w; CG8028.c1.l4 /CyO; +/+ CG8028 CG8028.c2.l1 w; CG8028.c2.l1 /CyO; +/+ CG8028 CG8028.c2.l2 w; CG8028.c2.l2 /CyO; +/+ CG8028 CG8028.c2.l4 w; +/+; CG8028.c2.l4 /TM3,sb CG8028 CG8028.c3.l 1 w; +/+; CG8028.c3.l1 /TM3,sb CG8028 CG8028.c3.l4 w; CG8028.c3.l4 /CyO; +/+ CG8028 CG8028.c3.l6 w; CG8028.c3.l6 /CyO; +/+ CG8028 52 2.1.2 Drosophila Rearing
Drosophila ° Drosophila
2.2 Anopheles gambiae
Anopheles in situ
2.2.1 Anopheles stocks
Anopheles gambiae
2.2.2 Anopheles rearing
Anopheles 53 2.3 Affymetrix Microarrays
et al Drosophila et al. Anopheles Anopheles
2.3.1 Sample Preparation
Anopheles Anopheles Anopheles Plasmodium Anopheles
Drosophila et al. Drosophila 54 2.3.2 Data Analysis
2.4 Nucleic Acid Isolation and Quantification
in situ
2.4.1 Genomic DNA Isolation
g g
2.4.2 Messenger RNA Extraction
55 µ
2.4.3 Complementary DNA Preparation
2.4.4 Plasmid DNA Isolation
2.4.5 Quantification of Nucleic Acids
56
2.5 Oligonucleotide Synthesis
°
2.6 Polymerase Chain Reaction
2.6.1 Standard PCR
Taq µ Taq 57
Table 2.3 Typical cycling procedure for standard DNA amplification using Taq DNA polymerase.
Step Temperature Time
°
°
°
°
°
2.6.2 Pfu PCR
Pfu Pyrococcus furiosus → →
Pfu µ Pfu µ µ µ
µ µ Pfu µ
58 Pfu Taq
2.6.3 Quantitative PCR
Drosophila Anopheles
µ Tbr
µ µ µ
µ rp49 s7 Drosophila Anopheles
59
Table 2.4 Typical cycling conditions for qPCR.
Step Temperature Time
°
°
° °
°
°
° °
rp49 s7 Drosophila Anopheles
Anopheles Drosophila Drosophila t 60 2.6.4 Direct Colony Screening by PCR
2.7 Cloning Procedures
2.7.1 Restriction Digests
° µ µ
µ
2.7.2 Agarose Gel Electrophoresis of DNA
61
2.7.3 Purification of DNA from Agarose Gels
2.7.4 DNA Ligations into pUAST Plasmids
2.7.5 Cloning PCR Products into TOPO Vectors
in situ 62 Anopheles Drosophila
2.7.6 Plasmids
Table 2.5 - Plasmids used during this study
Plasmid Purpose

 in situ Drosophila Picot
Drosophila CG3994
Drosophila CG15406
Drosophila CG8028
Drosophila Drosophila Drosophila CG15406, Picot, CG3994 CG8028 Drosophila 63
Figure 2.1 The Drosophila pWIZ UAS-RNAi vector. The pWIZ vector contains an intron from the white gene, flanked by two multiple-cloning sites. Insertion of an RNAi inverted repeat sequence of ~600 bp on each side of the white intron allows expression of a loopless hairpin RNA after splicing. Expression is activated by a GAL4 driver (Lee and Carthew, 2003).
2.7.7 Competent Cell Strains
Table 2.6 – Competent cell strains used during this study
Strain Genotype Purpose
mcr ∆ mrr-hsd mcr in situ φ lac ∆ ∆lac rec deo ara ∆ ara-leu gal gal rps end nup
φ lac ∆ ∆( lac arg deo rec end hsd pho sup thi λ gyr rel
64 2.7.8 Transformation of DH5 α Competent Cells
° °
2.7.9 Transformation of TOP10 Competent Cells
E. coli ° °
2.7.10 Plasmid Selection
µ µ 65
° °
2.7.11 Storage of Bacterial Cultures
°
2.7.12 Electrophoresis of RNA
66 2.7.13 Details of DNA Constructs
Table 2.7 - Generation of constructs used in this study
Construct Method of cloning PCR primer pairs
2.8 Automated DNA Sequencing
67
2.9 Germline Transformation
w1118 Drosophila µ
2.9.1 Determining the Chromosome of Insertion
2.10 Transgenic Drosophila crosses
68 2.10.1 Crossing Strategy
2.10.2 Emergence Counts of Drosophila Crosses
Picot Actin-GAL4 Picot Actin-GAL4
Picot Picot Actin-GAL4 Actin-GAL4 Actin-GAL4 Actin-GAL4 69
2.11 In Situ Hybridization of the Malpighian Tubules
In situ Drosophila Anopheles
2.11.1 Probe Preparation
in situ Anopheles Drosophila 
in vitro g g 70
2.11.2 Drosophila In Situ Hybridisation
71
In situ
100 mM phosphate buffer, pH 7.0 PBT buffer Hybridisation buffer Postfix buffer 20 X SSC, pH 7.0
2.11.3 Anopheles In Situ Hybridisation
72
2.12 Antibodies
Anopheles Drosophila
73
Table 2.8 Antibodies used during this study
Antibody and Source Dilution and Purpose
2.12.1 Design of Peptide Sequences
AGAP007752, AGAP012251, CG15406 Picot Anopheles Drosophila et al. et al.
74
Table 2.9 Antigenic sequences used for antibody production in rabbit.
Antibody Peptide sequence
2.12.2 Isolation of IgG Fraction from Immune Serum
2.12.3 Preparation of Affinity Columns
75
2.12.4 Affinity Purification of Antibodies
2.13 Protein Extraction
76 g
2.13.1 Bradford Assays
2.14 Electrophoresis and Western Blotting
2.14.1 Protein Electrophoresis
77
2.14.2 Western Blotting
2.14.3 Western Hybridisation
78
2.14.4 Western Signal Detection
2.15 Immunocytochemistry of Malpighian Tubules
Drosophila
2.15.1 Drosophila melanogaster
79
2.16 Confocal Microscopy
2.17 Fluid Secretion Assays
Drosophila 80 2.17.1 Fluid Secretion Assays in Drosophila
Drosophila et al. Drosophila
Figure 2.2 Diagram of the fluid secretion assay
2.18 Transport Assays
81 2.18.1 Transport Assays in Drosophila
Drosophila
82
Table 2.10 - Concentration of salts and amino acids used in the Drosophila salines. The minimal DS described (Linton and O'Donnell, 1999) was made without trehalose, and with the concentration of glucose varied during experimental optimisation. Schneider’s medium as previously described (Ashburner, 1989; Dow et al. , 1994).
Schneider’s DS Minimal DS medium (mM) (mM) (mM)
83 2.19 Drosophila Crosses on Sugar-Modified Food
CG15406 Drosophila
2.19.1 Gene Expression on Sugar-Modified Food
Table 2.11 Components of normal and sugar-modified Drosophila food. The quantities shown are for 1 l of food.
84 2.19.2 Drosophila Survival on Sugar-Modified Food
85
Chapter 3 - Validation and Analysis of the Anopheles Malpighian Tubule Microarray
86 3.1 Summary
Drosophila Anopheles Drosophila Anopheles Anopheles Anopheles Drosophila et al. Anopheles Drosophila
87 3.2 Introduction
Plasmodium/Anopheles Drosophila Mus musculus, Rattus norvegicus Arabidopsis thaliana, Caenorhabditis elegans Escherichia coli
3.2.1 Microarray Technology
in-situ 88
3.2.2 Microarrays Utilized in this Study
Anopheles gambiae et al Plasmodium Anopheles et al. Drosophila
Table 3.1 - Features of the Anopheles and Drosophila GeneChips used and referenced in this study (data as provided by Affymetrix).
Probe pairs per Predicted number of GeneChip Probe number probe set transcripts
Plasmodium/ 5,407 ( Plasmodium ) ~4,700 ( Plasmodium ) 11 Anopheles 16,941 ( Anopheles) ~16,000 ( Anopheles )
Drosophila 13,500 14 ~12,100
3.2.3 The Anopheles Malpighian Tubule Microarray
Anopheles gambiae
Anopheles 89
Table 3.2 - Transcriptomes compared in this study (The sample name for each group is used throughout this thesis).
Sample Gender of Life-Stage of Whole or Blood-fed (B) name mosquitoes mosquitoes tubules or sugar-fed (S)
Whole males & females adult whole sugar-fed
late-stage Larval males & females tubules fed on fish food larvae
MS males only adult tubules sugar-fed
FS females only adult tubules sugar-fed
sampled 3 h after FB females only adult tubules a blood meal
Plasmodium Plasmodium Plasmodium
Anopheles
90
Table 3.3 Interesting comparisons from the Anopheles microarray data-set.
Comparison Data of interest
91 3.3 Microarray Analysis
3.3.1 Initial microarray analysis
t
3.3.2 Validation of the Microarray
92 et al. Anopheles
93 3.4 Results
Anopheles Anopheles Anopheles Drosophila
3.4.1 Microarray Validation by Quantitative PCR
Anopheles Anopheles AGAP001858, AGAP010892, AGAP007495 AGAP008386 - AGAP007752, AGAP012251, AGAP009005, AGAP002587 94
A 6 Microarray 5 qPCR 4
3
2
Expression ratio Expression 1 (compared to(compared whole) 0
e l a MS FS FB ol rv a Wh L B 30 Microarray 25 qPCR 20
15
10
Expression ratio Expression 5 (compared (compared to whole) 0
e S B l M FS F ho rval W La C 50 Microarray 40 qPCR
30
20
Expression ratio ratio Expression 10 (compared to(compared whole) (compared to(compared whole) 0
le o MS FS FB Wh Larval D 35 Microarray 30 qPCR 25 20 15 10 Expression ratio Expression 5 (compared to(compared whole) 0
le o MS FS FB Larval Wh Figure 3.1 Comparison of Anopheles microarray and qPCR gene expression ratios for four genes. A: AGAP002858 , B: AGAP010892 , C: AGAP007495 and D: AGAP008386 (N=4, mean ± S.E.M.).
95 Anopheles Anopheles
3.4.2 The Usefulness of the Data
Anopheles et al Anopheles AGAP002587 AGAP007495 Anopheles gambiae et al. Anopheles 96
A) B)
C) D) 1250 60
1000 50 40 750 30 500 20 Signal strength Signal 250strength 10
0 0 S S s F hr hr hr hr hr e al S B M 4 8 2 MS F F arval day hol rv L FB 3 W La FB 2 FB 4 FB 7 FB 96 FB 15
Figure 3.2 Signal strength comparison of AGAP002587 and AGAP007495 in whole-mosquito and Malpighian tubule microarrays. Expression of AGAP002587 in A: whole mosquito and B: the Anopheles Malpighian tubules. Expression of AGAP007495 in C: whole mosquito and D: the Anopheles Malpighian tubules (Dissanayake et al. , 2006). Signal strength was calculated as explained in Section 2.3. (mean ± S.E.M., N=4 )
AGAP002587 AGAP002587 AGAP002587 et al AGAP002587 97 AGAP007495 et al Drosophila, et al.
3.4.3 Genes which are Highly Expressed in the Tubules
et al. Drosophila et al. Anopheles et al.
Anopheles Drosophila Anopheles in vivo Anopheles Anopheles et al. 98
Table 3.4 – Largest gene signal strengths in the Anopheles Malpighian tubule microarray at each life-stage. (N=4, mean ± S.E.M.). (Genes which appear in more than one category are colour-coded, function predicted by Ensembl (Hubbard et al. , 2009)).
Gene Signal Function Strength Whole Anopheles
AGAP010134 22239 ± 1371 Arrestin-like (signal transduction) AGAP009863 21601 ± 1988 ATP dependent RNA helicase-like AGAP009360 21382 ± 2922 Unknown AGAP003768 21310 ± 1151. Ribosomal subunit-like AGAP001164 21015 ± 1117 Opsin-like (G-coupled protein receptor)
Larval tubules
AGAP011061 25267 ± 1260 Unknown AGAP012881 21986 ± 1285 Metallopeptidase-like AGAP010469 20518 ± 934 Unknown AGAP002599 20263 ± 1803 Ubiquitn-like AGAP009441 20050 ± 125 Translation elongation factor-like
Male tubules
AGAP010364 22392 ± 1652 Unknown *ENSANGT00000011142 21624 ± 1424 No longer mapped AGAP003757 21305 ± 1758 Unknown AGAP008219 20794 ± 1761 Cytochrome P450 AGAP000604 20206 ± 2450 Unknown
Female tubules
AGAP010364 28314 ± 4680 Unknown AGAP005802 24616 ± 3846 60s Ribosomal protein AGAP004422 24386 ± 2632 Ribosomal protein-like *ENSANGT00000011142 23622 ± 3288 No longer mapped AGAP004462 22691 ± 2634 Unknown
Female blood-fed tubules
AGAP000604 27465 ± 973 Unknown AGAP010364 24658 ± 654 Unknown *ENSANGT00000011142 20052 ± 691 No longer mapped AGAP005802 19912 ± 790 60s Ribosomal protein AGAP004422 19891 ± 917 Ribosomal protein-like 99 et al. ENSANGT00000011142 et al. ENSANGT00000011142 et al.
3.4.4 Genes which are Highly Enriched in the Tubules
et al.
100
Table 3.5 – Genes highly enriched in the Anopheles Malpighian tubules in comparison to whole mosquito as determined by microarray (N=4, mean ± S.E.M.). (Genes which appear more than once are colour-coded, function predicted by Ensembl (Hubbard et al. , 2009)).
Gene Enrichment Signal strength Putative Function (± S.E.M.) (± S.E.M.)
Larval tubules
AGAP011682 4110 ± 768 14541 ± 1302 Unknown AGAP010469 4034 ± 1628 20518 ± 934 Unknown AGAP010361 3016 ± 1490 14338 ± 536 Unknown AGAP005671 2720 ± 969 11096 ± 3123 Unknown AGAP012881 2201 ± 915 1160 ± 291 Unknown
Male tubules AGAP000603 239 ± 75 4370 ± 635 Unknown AGAP008731 116 ± 23 7244 ± 852 laccase-like AGAP008593 89 ± 23 4552 ± 582 Unknown AGAP004133 84 ± 8 8084 ± 757 Unknown AGAP003758 62 ± 14 2209 ± 400 Unknown
Female tubules AGAP000603 393 ± 162 5119 ± 587 Unknown *ENSANGT00000020967 68 ± 16 679 ± 216 No longer mapped AGAP010365 58 ± 11 6716 ± 1417 Unknown AGAP006196 50 ± 5 965 ± 346 Unknown AGAP008731 46 ± 9 2888 ± 496 laccase-like
Female blood -fed tubules AGAP004350 542 ± 142 16347 ± 1180 MFS-like AGAP000603 1067 ± 480 12983 ± 502 Unknown *ENSANGT00000020967 624 ± 190 5098 ± 244 No longer mapped AGAP008731 200 ± 38 13340 ± 262 laccase-like AGAP003205 167 ± 21 503 ± 8 Unknown
AGAP010469, 101
A) B)
Figure 3.3 Expression of transcripts in the tubules in comparison to whole mosquito. Number of transcripts which are A) down-regulated or B) up-regulated in each life-style.
Anopheles 102
3.4.5 Comparison of the Malpighian Tubule Transcriptome During Different Life-stages
t
Anopheles
103
a) b)
c) d)
e) f)
Figure 3.4 A comparison of the number of transcripts significantly up- or down-regulated in the Anopheles Malpighian tubules at the four life-stages assayed. (Statistical significance determined by Student’s t-test, p<0.05). 104 3.4.6 Genes for Further Investigation
Anopheles
• Anopheles
• Anopheles
•
• Anopheles
Anopheles Drosophila
3.4.6.1 The Gene Selection Process
Anopheles 105
Drosophila Drosophila Drosophila et al. Anopheles Drosophila et al. Drosophila et al. Drosophila Drosophila
• Anopheles Drosophila
• Drosophila
• 106 Drosophila et al.
3.4.6.2 Genes Selected for Study
Anopheles Drosophila Anopheles Drosophila
Table 3.6 The four homologous gene-pairs chosen from the Anopheles and Drosophila Malpighian tubule microarrays for further study (Benson et al. , 2009; Hubbard et al. , 2009).
Anopheles gene Drosophila gene Putative function
AGAP007752 CG15406 Sugar transporter
AGAP012251 Picot (CG8098) Inorganic phosphate co-transporter
AGAP009005 ZnT35C (CG3994) Zinc transporter
AGAP002587 CG8028 Monocarboxylate transporter
Anopheles Drosophila Picot 107
3.4.7 Additional Genes of Interest from the Anopheles Microarray
AGAP008731, AGAP010725 AGAP004005
AGAP008731 AGAP003738 Anopheles et al AGAP008731 AGAP003738 et al. Drosophila CG3759 et al. et al. et al. et al. Drosophila et al. et al.
Anopheles AGAP010725 Drosophila AGAP010725 Prestin, Prestin et al. Prestin Drosophila 108 et al. AGAP010275 Prestin Prestin Prestin
AGAP004050 Anopheles Drosophila Doublesex (dsx).Dsx Drosophila dsx Drosophila Anopheles et al. dsx Drosophila et al. dsx dsx
109 3.5 Discussion
Anopheles Anopheles Drosophila
3.5.1 Microarray Analysis and Validation
Anopheles Anopheles Drosophila in vivo Anopheles, Aedes aegypti et al. et al. Anopheles Anopheles Plasmodium
Anopheles Anopheles 110
Figure 3.5 Comparison of the average microarray and qPCR tubule enrichment ratios for the four genes from the Anopheles microarray validated in this chapter. The line shown is y=x.
Anopheles Drosophila et al. Anopheles Drosophila 111
et al. et al.
3.5.2 Gene Selection
Anopheles Drosophila Drosophila Drosophila
Anopheles Drosophila 112
Drosophila Anopheles Drosophila Anopheles
113 3.6 Conclusions
Anopheles Anopheles Drosophila Anopheles Drosophila Anopheles Drosophila AGAP007752 CG15406, AGAP012251 Picot, AGAP009005 ZnT35C, AGAP002587 CG8028. Anopheles Drosophila
114
Chapter 4 - AGAP007752 and CG15406 as Insecticide Targets
115 4.1 Summary
Anopheles Anopheles Drosophila Anopheles AGAP007752 Drosophila CG15406 Anopheles Drosophila CG15406 CG15406 CG15406 CG15406 CG15406 CG15406 CG15406 Anopheles Drosophila AGAP007752 CG15406 AGAP007752 CG15406
116 4.2 Introduction
AGAP007752 CG15406 Anopheles Drosophila AGAP007752 CG15406
4.2.1 The Importance of Sugars
117 4.2.2 Sugar Uptake in Anopheles and Drosophila
Anopheles Anopheles Drosophila Drosophila Anopheles Drosophila et al. et al. et al.
4.2.3 Sugar Transporters in Anopheles and Drosophila
Anopheles Drosophila Drosophila sut1, sut2, sut3, Glut1 CG7882 et al. Sut1 sut2 Glut1 Sut3 CG7882 et al. GLUT1, GLUT2, GLUT3 GLUT4 CG1213, CG10960, CG6484, CG30035, CG8234, sut4 CG1208 et al et al. GLUT6 GLUT8
Drosophila CG15406 CG15408, CG3285, CG14606, CG14605, CG33281 CG33282 et al 118 et al. Anopheles Aedes aegypti
*
*
Figure 4.1 Gene tree image of AGAP007752 and CG15406 and their homologues. (Tree generated by Ensembl, see Section 3.4.6.2 (Hubbard et al. , 2009; Vilella, 2009)). 119 4.2.4 Sugar Transport in Mammals
et al. GLUT1-4 GLUT5, GLUT7, GLUT9 GLUT11 GLUT5 et al. GLUT7 GLUT9 et al. GLUT6, GLUT8, GLUT10, GLUT12 HMIT GLUT 8, GLUT10 GLUT12 et al. et al. et al. HMIT et al.
GLUT1 GLUT5 GLUT1 et al. 120 4.2.5 The Putative Sugar Transporters AGAP007752 and CG15406
AGAP007752 CG15406 et al. AGAP007752 CG15406
CG15406 AGAP007752 CG15406 CG15406 Anopheles Drosophila CG15406 AGAP007752
121
A
B
C
PEPPQ ETKG GRK EX(6)RG DRYGRK EX(6)R COOH NH 2
Figure 4.2 The predicted structures of the mammalian GLUT family and CG15406 . The structures of A: Class I and Class II and B: Class III members of the human GLUT family (Uldry and Thorens, 2004); C: the predicted structure of CG15406 showing conservation of human GLUT signature sequences . CG15406 is predicted to have a similar 12 TM domain structure to the human GLUTs, with the carboxy- and amino-terminal ends on the cytosolic side of the membrane. All of the motifs found in the intracellular loops are at least partially conserved between the human GLUTs and CG15406 . CG15406 structure predicted using protein prediction tools provided by the Centre for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/TMHMM/) and ExPASy Proteomics Tools (http://www.expasy.ch/tools/). 122 CG15406 Drosophila CG15406 AGAP007752 Anopheles AGAP007752 CG15406 in vitro in vivo
Table 4.1 CG15406 expression in larval and adult Drosophila (Chintapalli et al. , 2007).
Tissue mRNA Signal Pre sent Call Enrichment Affy Call Brain 3 ± 1 1 of 4 0 Down Head 6 ± 0 0 of 4 0 Down Thoracicoabdominal 5 ± 1 0 of 4 0 Down ganglion Salivary gland 15 ± 2 0 of 4 0.09 Down Crop 2 ± 0 0 of 4 0 Down Midgut 19 ± 1 4 of 4 0.1 Down Tubule 4941 ± 253 4 of 4 29.8 Up Hindgut 8 ± 1 0 of 4 0 Down Heart 11 ± 6 3 of 4 0.07 Down Fat body 29 ± 29 3 of 4 0.18 Down Ovary 3 ± 0 0 of 4 0 Down Testis 10 ± 0 4 of 4 0.1 Down Male accessory 8 ± 2 0 of 4 0.1 Down glands Virgin spermatheca 26 ± 17 3 of 4 0.16 Down Mated spermatheca 12 ± 1 3 of 4 0.07 Down Adult carcass 18 ± 4 2 of 4 0.1 Down Larval CNS 3 ± 2 0 of 4 0.02 Down Larval Salivary gland 3 ± 0 0 of 4 0.02 Down Larval midgut 9 ± 2 1 of 4 0.06 Down Larval tubule 4193 ± 108 4 of 4 25.3 Up Larval hindgut 63 ± 3 4 of 4 0.39 Down Larval fat body 14 ± 3 0 of 4 0.1 Down S2 cells (growing) 3 ± 0 0 of 4 0.02 Down Whole fly 165 ± 22 4 of 4 123 4.3 Results
AGAP007752 CG15406 AGAP007752 CG15406 in situ CG15406 Drosophila CG15406 Drosophila CG15406 AGAP007752
4.3.1 AGAP007752 Expression in the Anopheles Tubules
AGAP007752 Anopheles in situ
4.3.1.1 Expression of the Transcript
AGAP007752 Anopheles
AGAP007752 AGAP007752 AGAP007752 124 AGAP007752
12000 FB FS MS Larval Whole *** *** *** - 10000 Larval *** *** *** MS - - 8000 FS -
6000
4000
Signal strength 2000
0
e S F rval MS FB a Whol L
Figure 4.3 Microarray data for AGAP007752 expression in the Anopheles tubules at different stages in the life-cycle. The attached table shows the statistical significance between life-stages. (N=4, mean ± S.E.M.) One-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.
AGAP007752
et al Anopheles et al. et al. et al. AGAP007752 125 AGAP007752 AGAP007752
A 25 Microarray 20 qPCR
15
10
Expression ratio Expression 5 (compared to to (compared whole) 0
le rval MS FS FB ho a W L
B 1.25 **
1.00
0.75
0.50
Expression ratio ratio Expression 0.25 (compared (compared to FS) (compared to(compared FS)
0.00 r h FS FB 4 +2 B F
Figure 4.4 qPCR validation of AGAP007752 expression in Anopheles . A: ratio of expression as calculated from microarray and qPCR data. B: ratio of expression in female tubules 3 h and 24 h after a blood-feed (N ≥4, mean ± S.E.M.). One-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.
AGAP007752 Drosophila et al. 126 In situ AGAP007752 Anopheles
A midgut B
proximal
proximal
midgut
distal
distal
C midgut D
distal end F
E distal
distal end proximal
midgut
G
distal end
Figure 4.5 Expression of AGAP007752 in the adult Anopheles tubules as determined by in situ hybridisation . A: low power view of the male tubules showing expression throughout. B: low power view of the female tubules showing expression throughout. C: high power view showing expression in the female lower tubule where it attaches to the gut. D: high power view of the distal end of the female tubules showing strong expression in the principal cells. E: main section of the male tubule showing expression in the principal cells. F: low power view of female tubule stained with sense probe as a control, showing no expression. G: high power view of the female distal end stained with sense probe as a control, showing no expression. 127 AGAP007752 AGAP007752 AGAP007752
4.3.1.2 Expression of the Protein
AGAP007752 in situ in situ et al. Anopheles Anopheles
MT H W ~82 kDa
~64 kDa
~49 kDa AGAP007752
~37 kDa
Alpha tubulin
Figure 4.6 Expression of the AGAP007752 protein in Anopheles as determined by Western blotting. A band the correct size for the AGAP007752 protein (~50 kDa) can be seen in protein extracted from the Malpighian tubules (MT), head (H) and whole-body (W). Alpha tubulin was also blotted for as a control for protein integrity. 128 Anopheles
4.3.2 CG15406 Expression in the Drosophila Tubules
CG15406 Drosophila in situ
4.3.2.1 Expression of the Transcript
CG15406 AGAP007752 Drosophila et al. CG15406 CG15406 129
A B
50 1.25 *** 40 1.00
30 0.75 ***
20 0.50 Expression ratio Expression ratio 10 *** 0.25 (Compared to whole) (Compared to Male MT) 0 0.00 Whole Larval MT Adult MT Male MT Female MT
Figure 4.7 Expression of CG15406 in the Drosophila Malpighian tubules as determined by qPCR. A: expression in the larval and adult Malpighian tubules in comparison to whole-fly. B: expression in the female tubules in comparison to the male tubules (N=4, mean ± S.E.M.). Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.
CG15406 Drosophila et al.
In situ Drosophila 130
A B
IS
MS MS IS
MS LS
U LS MS LS U
C D E F IS MS MS U
IS IS LS U MS
Figure 4.8 Expression of CG15406 in the adult tubules as determined by In situ hybridisation. Low power views showing expression in the ureter (U), lower segment (LS), main segment (MS) and initial segment (IS) in the male (A) and female (B) tubules. C: high power shot of the posterior distal tubule showing staining in the principal cells. D: high power shot of the anterior distal tubule showing faint staining in the initial segment. E: and F: sense controls showing no staining in the tubules.
CG15406 Drosophila AGAP007752
4.3.2.2 Expression of the Protein
131
d d a a T T e e F F M M h h W W d d d d d d 2 7 2 7 2 7 ~115 kDa ~82 kDa ~64 kDa ~49 kDa ~52 kDa
~37 kDa
~26 kDa
Alpha tubulin
Figure 4.9 Expression of CG15406 in Drosophila as determined by Western blotting. A protein band the correct size for CG15406 (~52 kDa) can be seen in the Malpighian tubules of 2- day old (2d MT) and 7-day old (7d-MT) flies, the head of 2-day old (2d head) and 7-day old (7d head) flies, and whole-flies aged to 2-days (2d WF) and 7-days old (7d WF). Alpha tubulin was probed as a control for protein integrity.
CG15406
132
Figure 4.10 Localisation of CG15406 in adult Drosophila tubules by immunocytochemistry. A: and B: Initial segment (IS) and main segment (MS) showing binding at the basolateral membrane (20x magnification). C: Antibody binding at the basolateral membrane of the main segment showing binding in the principal cells, but not the stellate cells (40x magnification). D: Control tubules treated with pre-immune serum and Texas Red (40x magnification). Left hand panel – Texas Red staining of CG15406 (red); middle panel – DAPI nuclear staining (blue); right hand panel – Texas Red and DAPI merged.
133 CG15406 in situ CG15406
4.3.3 RNAi Knockdown of CG15406
Drosophila CG15406 CG15406 w1118 CyO ,Sb CG15406
4.3.3.1 Effect on Fly Survival
134
Table 4.2 Pattern of expression of GAL4 drivers used to drive UAS-RNAi fly-lines.
Driver line Pattern of expression Actin -GAL4
C42-GAL4 Uro -GAL4
C724-GAL4
Table 4.3 Effect of CG15406 RNAi knockdown on fly survival (cross N=3). (Raw data is presented in Appendix 2).
Fly -line Actin -GAL4 C42 -GAL4 Uro -GAL4 C724 -GAL4
CG15406.c1.l1 lethal semi -lethal CG15406.c1.l4 lethal semi -lethal CG15406.c1.l9 lethal CG15406.c2.l4 semi -lethal CG15406.c2.l7 CG15406.c2.l8 CG15406.c3.l3 lethal CG15406.c 3.l4 lethal CG15406.c3.l5 semi -lethal
Actin-GAL4 CG15406 135 c42-GAL4 Actin-GAL4
CG15406 CG15406 CG15406 Actin-GAL4 CG15406 Actin-GAL4 c42-GAL4 CG3285, CG15406
CG15406 t c42-GAL4 c42-GAL4 in situ
CG3285 CG3285 Drosophila CG15406 CG15406 136
A 1.25 *** ** *** 1.00
0.75
0.50
Expression ratio ratio Expression 0.25 (compared (compared to parental) C42 (compared (compared to parental) C42 0.00 4 .l1 l .l9 .l9 C42 1 1 1 c .c1. c .c 6 6 0 0 4 4 5 5 15406. 1 15406. 1 G G CG15406.c1.l1 CG C CG > 2 4 B C42>C C42>CG15406.c1.l4C 1.25 *** 1.00 ***
0.75 **
0.50
Expression ratio Expression 0.25
0.00 (compared (compared to parental) ACT 4 8 .l .l7 .l7 l 2 2 .c .c2.l4 c .c2. .c2.l8 6 6 6 6 0 0 0 0 4 4 4 4 5406.c2 5406. Actin-Gal4 1 G15 G1 G15 C CG C C T>CG15 T> C A AC ACT>CG15 C 1.50 * *** 1.25 *** 1.00
0.75
0.50
Expression ratio Expression 0.25
(compared (compared to parental) C42 0.00 4 .l3 l .l5 .l5 C42 3 3 3 c .c3. c .c 6 6 0 0 4 4 5 5 15406. 1 15406. 1 G G CG15406.c3.l3 CG C CG > 2 4 C42>C C42>CG15406.c3.l4C
D 1.75 * 1.50 1.25 1.00 0.75 0.50 Expression ratio Expression 0.25
(compared to(compared parentals) 0.00
CG15406.c1.l4 CG15406.c2.l8
c42>CG15406.c1.l4 ACT>CG15406.c2.l8 Figure 4.11 qPCR verification of CG15406 knockdown in the tubules. CG15406 expression in A: RNAi lines from construct 1, B: RNAi lines from construct 2, and C: RNAi lines from construct 3. D: Quantification of CG3285 expression in driven RNAi lines (N=3, mean ± S.E.M.) (Student’s t- test, *p<0.05, **p<0.01, ***p<0.001). 137
1 2 3 4 5 6 ~82 kDa Lane Fly-line ~64 kDa 1 c42>CG15406.c1.l10 ~49 kDa ~52 kDa 2 CG15406.c1.l10 ~37 kDa 3 ACT>CG15406.c2.l4 ~26 kDa 4 CG15406.c2.l4 5 c42>CG15406.c3.l4 Alpha tubulin 6 CG15406.c3.l4
Figure 4.12 Protein expression of CG15406 in three RNAi fly-lines. Three RNAi lines driven by Actin-GAL4 or c42-GAL4 , and their respective parental lines, were blotted for CG15406 expression in the tubules. Alpha tubulin was blotted as a control for protein integrity.
4.3.3.2 Effect on Fluid Secretion from the Tubules
CG15406 et al.
CG15406 138 CG15406
A 2.0
-1) C42 CG15406.c1.l10 1.5 C42>CG15406.c1.l10
1.0
0.5
Secretion Secretion raterate (nl(nl min min drosokinin 0.0 0 10 20 30 40 50 60 70 80 Time (min) B 2.0
-1) c42 CG15406.c2.l4 1.5 ACT>CG15406.c2.l4
1.0
0.5
Secretion raterate (nl(nl min min drosokinin 0.0 0 10 20 30 40 50 60 70 80 Time (min) C 2.0
-1) c42 CG15406.c3.l4 1.5 c42>CG15406.c3.l4
1.0
0.5
Secretion rate (nl min drosokinin 0.0 0 10 20 30 40 50 60 70 80 Time (min)
Figure 4.13 Effect of CG15406 knockdown on the rate of fluid secretion from the tubules. Typical experimental results, showing the response of three driven RNAi lines and the appropriate parental lines to Drosokinin after 30 min of steady secretion (mean ± S.E.M., N ≥10). 139 4.3.4 Sugars Transported by CG15406
CG15406 CG15406, Drosophila Drosophila
4.3.4.1 Fly Survival on Glucose, Fructose and Sucrose
CG15406 CG15406
2.0 *** *** 1.5 ***
1.0
0.5 Expression ratio Expression (compared to(compared Normal) 0.0
Normal Glucose Fructose Sucrose
Figure 4.14 CG15406 expression in the tubules of flies raised on fly-food with modified sugar content. Expression is compared in flies raised on normal, glucose-rich, fructose-rich, and sucrose-rich food (N=4, mean ± S.E.M.). Student’s t-test comparing normal food to modified food, *p<0.05, **p<0.01, ***p<0.001.
CG15406 CG15406 140
CG15406 Actin-GAL4 c42-GAL4
A B 0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2 Proportion of of Proportion of 0.1 0.1 c42>RNAi eclosures c42>RNAi eclosures ACT>RNAi eclosures 0.0 0.0 al e os orm N Normal Gluc Sucrose Glucose Fructose Sucrose Fructose C D 0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2 Proportion of Proportion of of 0.1 0.1 c42>RNAi eclosures ACT>RNAi eclosures ACT>RNAi eclosures 0.0 0.0 e e al se se se se s m o mal o o r co r ros o u cto ct N u No ru Gl r Sucr Gluc F Suc F
Figure 4.15 Survival of RNAi fly-lines on sugar-modified food. A: and B: CG15406.c2.l4 driven by Actin-GAL4 and c42-GAL4 . C and D: CG15406.c3.l4 driven by Actin-GAL4 and c42-GAL4 . (N=4, mean ± S.E.M.). Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.
CG15406.c2.l4 CG15406.c3.l4 141 CG15406
4.3.4.2 Glucose Transport by the Tubules
CG15406
0.4
0.3
0.2
0.1 Transport Transport ratio
0.0 9 9 4 R l .l4 l4 n c1. c2.l c3. c3.l4 rego O
CG15406.c1.lCG15406.CG15406.c2CG15406.CG15406.CG15406. 42> C ACT> C42>
Figure 4.16 Transport ratio of 3H-labelled glucose in wild-type and RNAi fly-lines. (mean ± S.E.M., N>12 ).
142
CG15406 Oregon R
CG15406 CG15406 143
A 1.25 30 m 1.00
0.75 3 hr 0.50
Scintillation ratio ratio Scintillation 0.25 (compared to 30 m sample) m 30 to (compared (compared to 30 m sample) m 30 to (compared 0.00 9 9 .l9 .l 1 1 .c1.l 6.c 0 06 4 5406.c 54 1 1 15 G G CG C C > 2 4 C C42>CG15406.c1.l9
B 1.25 30 m 1.00 3 hr
0.75
0.50
Scintillation ratio ratio Scintillation 0.25 (compared to 30 m sample) m 30 to (compared (compared to 30 m sample) m 30 to (compared 0.00
4 l4 l4 l4 .l . . . 2 .c .c2 6 6 0 40 406.c2 406.c2 54 5 5 5 1 G C CG1 CG1 CG1 T> T> AC AC
C 1.25 30 m 1.00 3 hr 0.75
0.50
Scintillation ratio Scintillation 0.25
(compared to 30 m sample) m 30 to (compared 0.00 4 .l .l4 3.l4 3 .c .c 6 6 6.c3 0 0 40 5 54 1 G G154 G15406.c3.l4 CG1 C C C > 2 4 C C42>
Figure 4.17 Uptake of 3H-labelled glucose by CG15406 RNAi lines. Uptake of glucose after 3 h by three different GAL4-driven UAS-RNAi lines and their parental fly-lines. (mean ± S.E.M., N>12) 144 CG15406 CG15406 CG15406
145 4.4 Discussion
AGAP007752 CG15406 Anopheles Drosophila CG15406 AGAP007752
4.4.1 AGAP007752 and CG15406 in the Tubules
AGAP007752 CG15406 AGAP007752 CG15406 In situ Anopheles Drosophila CG15406 CG15406
AGAP007752 AGAP007752 CG15406 146 Anopheles
in situ AGAP007752 CG15406 AGAP007752 CG15406 Anopheles Drosophila
CG15406 CG15406 Actin-GAL4 CG15406 CG15406 c42-GAL4 c42- GAL4 c42-GAL4 CG15406 147 CG15406
4.4.2 AGAP007752 and CG15406 as Sugar Transporters
AGAP007752 CG15406 Drosophila CG3285 et al. CG15406 CG3285 et al. Drosophila AGAP007752 CG15406
CG15406 CG15406 CG15406 148 CG15406
CG15406 CG15406 CG15406 CG15406 CG15406
4.4.3 AGAP007752 and CG15406 as Insecticide Targets
AGAP007752 CG15406 CG15406 Anopheles Drosophila AGAP007752 CG15406
149 4.5 Conclusion
AGAP007752 CG15406 Anopheles Drosophila Anopheles Drosophila CG15406 Xenopus AGAP007752 CG15406 CG15406 AGAP007752 CG15406 AGAP007752
150
Chapter 5 - AGAP012251 and Picot as Insecticide Targets
151 5.1 Summary
Anopheles Anopheles Drosophila et al. Anopheles AGAP012251 Drosophila Picot AGAP012251 Picot Anopheles Drosophila Drosophila Picot AGAP012251 Anopheles Picot Picot Picot Anopheles Drosophila AGAP012251 Picot AGAP012251 Picot
152 5.2 Introduction
5.2.1 Inorganic Phosphate
Aedes aegypti Drosophila et al. et al. Drosophila et al. 153
5.2.2 Inorganic Phosphate Co-transporters
Drosophila et al. Drosophila et al.
5.2.2.1 Mammalian Phosphate Transporters
et al. PiT-1 PiT-2 et al. PiT-1 PiT-2
et al. et al. 154 et al. et al. et al.
NaPi-IIa NaPi-IIb NaPi-IIc
et al. et al. et al.
et al. et al. et al. sialin et al. et al.
Anopheles Drosophila AGAP004257 CG7628 et al. CG7628 et al. Drosophila VGlut Anopheles AGAP007992 SLC17A6, SLC17A7 SLC17A8 VGlut et al. 155 AGAP012251 Picot et al. AGAP012251 Picot
Figure 5.1 Alignment tree of genes closely related to AGAP012251 and Picot . All of the highly homologous genes are from Anopheles , Aedes and Drosophila , suggesting a protein family which is highly insect-specific. Evolutionary tree generated by Ensembl, see Section 3.4.6.2 (Hubbard et al. , 2009; Vilella, 2009). 156 Anopheles Drosophila Aedes aegypti
5.2.3 Inorganic Phosphate in the Malpighian Tubules
Drosophila Rhodnius prolixus et al. Aedes aegypti Anopheles albimanus et al .
Drosophila
Drosophila Drosophila Rhodnius prolixus Drosophila et al. Rhodnius Drosophila et al. Anopheles albimanus, et al. Anopheles albimanus et al. 157
Drosophila Rhodnius Anopheles
Drosophila et al.
Drosophila hydei,
5.2.4 The Putative Inorganic Phosphate Co-transporters AGAP012251 and Picot
AGAP012251 Picot CG8098 , AGAP012251 Picot Picot Drosophila AGAP012251 Anopheles AGAP012251 Picot
Drosophila et al. Picot AGAP012251
Picot AGAP012251 Picot AMYREL Picot 158 et al.
Picot
Drosophila et al.
AGAP012251 Anopheles
Table 5.1 Picot expression in adult and larval Drosophila (Chintapalli et al. , 2007).
Tissue mRNA Signal Present Call Enrichment Affy Call Brain 835 ± 37 4 of 4 1.80 Up Head 682 ± 17 4 of 4 1.50 Up Thoracicoabdominal 1058 ± 13 4 of 4 2.30 Up ganglion Salivary gland 703 ± 26 4 of 4 1.50 Up Crop 101 ± 5 4 of 4 0.20 Down Midgut 1652 ± 92 4 of 4 3.50 Up Tubule 3621 ± 397 4 of 4 7.70 Up Hindgut 3336 ± 95 4 of 4 7.10 Up Heart 58 ± 18 4 of 4 0.68 Down Fat body 150 ± 6 4 of 4 0.12 Down Ovary 175 ± 5 4 of 4 0.40 Down Testis 150 ± 6 4 of 4 0.30 Down Male accessory glands 85 ± 7 4 of 4 0.20 Down Virgin spermetheca 573 ± 37 4 of 4 1.22 None Mated spermetheca 769 ± 92 4 of 4 1.64 Up Adult carcass 262 ± 16 4 of 4 0.60 Down Larval CNS 1012 ± 118 4 of 4 2.15 Up Larval salivary gland 285 ± 23 4 of 4 0.61 Down Larval midgut 1433 ± 52 4 of 4 3.05 Up Larval tubule 2982 ± 136 4 of 4 6.30 Up Larval hindgut 3329 ± 125 4 of 4 7.08 Up Larval fat body 7 ± 0 0 of 4 0.00 Down S2 cells (growing) 13 ± 1 4 of 4 0.03 Down Whole fly 470 ± 26 4 of 4 159 5.3 Results
AGAP0012251 Picot AGAP012251 Picot in situ Picot Drosophila Picot Drosophila
5.3.1 AGAP012251 in the Tubules
AGAP012251 Anopheles AGAP012251 in situ AGAP012251
5.3.1.1 Expression of the Transcript
AGAP012251 Anopheles 160
4000 FB FS MS Larval Whole *** *** *** - 3000 Larval * * ** MS - - FS - 2000
1000 Signal strength
0
S ole M FS FB Wh Larval
Figure 5.2 Anopheles Malpighian tubule microarray data for AGAP012251. The attached table shows the statistical significance between life-stages. (N=4, mean ± S.E.M.) One-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.
AGAP012251 AGAP012251 AGAP012251
AGAP012251 AGAP012251 161
A 12.5 Microarray 10.0 qPCR
7.5
5.0
Expression ratio Expression 2.5 (compared to(compared whole) 0.0 e S B ol rval M FS F Wh La
B ** 3.0 ** 2.5
2.0
1.5
1.0 Expression ratio ratio Expression (compared (compared to FS) (compared to(compared FS) 0.5
0.0 r B h FS F 4 +2 B F
Figure 5.3 qPCR validation of AGAP012251 expression in the Anopheles tubules . A: ratio of expression as calculated from microarray and qPCR data. B: ratio of expression in female tubules 3 h and 24 h after a blood-meal (N ≥4, mean ± S.E.M.). One-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.
AGAP012251
AGAP012251 162 Drosophila et al. In situ AGAP012251 Anopheles
A B midgut
midgut distal
proximal
proximal distal C distal end D
middle segment
midgut
E F G distal
proximal distal end
midgut distal end
Figure 5.4 Expression of AGAP012251 in the adult Anopheles tubules. A: low power view of the male tubules showing expression throughout. B: low power view of the female tubules showing expression throughout. C: low power view showing expression in the female lower tubule where it joins the gut. D: high power view of the middle of the female tubule showing expression. E: high power view of the distal end of the female tubule showing expression. F: low power view of the female tubules stained with sense probe as a control, showing no expression. G: high power view of the male distal end stained with sense probe as a control, showing no expression. 163 in situ AGAP012251 AGAP012251 Anopheles in situ AGAP012251
5.3.1.2 Expression of the Protein
In situ AGAP012251 in situ Anopheles AGAP012251 Anopheles
Anopheles AGAP012251 164
MT H W
~82 kDa
~64 kDa AGAP012251
~49 kDa
~37 kDa
Alpha tubulin
Figure 5.5 Protein expression of AGAP012251 in Anopheles as determined by Western blotting. A band the size of AGAP012251 (~58 kDa) can be seen in protein extracted from the Malpighian tubules (MT), head (H) and whole mosquito (W). Alpha tubulin was blotted as a control for protein integrity.
5.3.2 Picot in the Tubules
Picot Drosophila Picot Picot in situ
5.3.2.1 Expression of the Transcript
Picot AGAP012251 Drosophila et al. et al. Picot 165
A B 25 2.0 *** ** 20 1.5 15 1.0 10
0.5 Expression ratio ratio 5 Expression ratio (Compared to whole) (Compared to whole) * (Compared to Male MT) 0 0.0 Whole Larval MT Adult MT Male MT Female MT
Figure 5.6 Expression of Picot in the Drosophila Malpighian tubules as determined by qPCR. A: expression in the larval and adult Malpighian tubules in comparison to the average whole-fly expression, as determined by qPCR. B: expression in the female tubules in comparison to the male tubules as determined by qPCR (N=4, mean ± S.E.M.). Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.
Picot Picot Anopheles albimanus et al.
In situ Drosophila Picot
Picot in situ AGAP012251 Picot 166
A B LS IS
ureter LS MS LS
LS MS
MS
MS
IS
C E
MS IS IS
D LS MS ureter
MS
MS
Figure 5.7 Expression of Picot in the Drosophila adult tubules as determined by in situ hybridisation. Low power views showing expression in the ureter, lower segment (LS), main segment (MS) and initial segment (IS) in the anterior (A) and posterior (B) tubules. C: high power shot of the anterior initial segment showing staining. D: high power shot of the posterior main segment showing faint staining. E: sense probe control showing no staining in the tubules.
5.3.2.2 Expression of the Protein
167
d d a a T e F T e F M h W M h W d d d d d d 2 2 2 7 7 7 ~115 kDa ~115 kDa ~82 kDa ~82 kDa
~64 kDa ~64 kDa Picot ~49 kDa ~49 kDa ~32 kDa ~32 kDa
Alpha tubulin
Figure 5.8 Expression of Picot in Drosophila as determined by Western blotting. A strong protein band the correct size for Picot (~58 kDa) can be seen in the Malpighian tubule, head and whole-fly samples of 7-day old flies, but only a faint band of Picot can be seen in the Malpighian tubules of 2 day-old flies. Alpha tubulin was probed as a control for protein integrity.
Picot
Picot 168
MS MS MS
TS TS TS A
TS TS TS MS MS MS B
IS IS IS
basolateral basolateral basolateral
C
IS IS IS
TS TS TS
D
Figure 5.9 Localisation of Picot in the Drosophila adult tubules by immunocytochemistry. A: and B: antibody binding at the basolateral membrane of the transitional segment (TS) but not the main segment (MS) (20x magnification). C: antibody binding at the basolateral membrane of the initial segment (IS) (40x magnification). D: initial and transitional segment of control tubules treated with pre-immune serum and Texas Red (40x magnification). 169 in-situ Picot
Picot
5.3.3 RNAi Knockdown of Picot
Picot Drosophila Picot Picot w1118 CyO Sb Picot
5.3.3.1 Effect on Fly Survival
Picot et al. Picot Drosophila
170
Table 5.2 Pattern of expression of UAS/GAL4 drivers used to knock-down Picot.
Driver line Pattern of expression Actin -GAL4
C42-GAL4
C724-GAL4 ELAV -GAL4
Table 5.3 Effect of Picot RNAi knockdown on fly survival (cross N=3) (Raw data is presented in Appendix 2) .
Fly -line Actin -GAL4 C42 -GAL4 C724 -GAL4 ELAV -GAL4
Picot.c1.l3 lethal semi -lethal Picot.c1.l5 lethal semi -lethal Picot.c1.l6 lethal Picot.c2.l3 semi -lethal Picot.c2.l5 semi -lethal Picot.c2.l6 lethal Picot.c3.l2 semi -lethal Picot.c3.l3 semi -lethal Picot.c3.l5 lethal
Actin-GAL4 Picot et al. et al. 171 Picot
c42-GAL4 C42-GAL4 Picot Picot
Picot c42-GAL4 Actin-GAL4 CG6978, Drosophila Picot
Picot t CG6978 Picot 172
A 1.25 ** * * 1.00
0.75
0.50
Expression ratio Expression 0.25
(compared to (compared parental) C42 0.00 3 3 5 6 6 .l .l C42 1.l 1.l 1.l .c t.c1 t.c1 .c .c o o icot cot P Pic Pic Picot Pi > > 42 42 C C42>Picot.c1.l5 C B 1.25 *** *** 1.00 ***
0.75
0.50
Expression ratio Expression 0.25
(compared to (compared parental) C42 0.00 3 3 5 6 6 .l .l C42 2.l 2.l 2.l .c t.c2 t.c2 .c .c o o icot cot P Pic Pic Picot Pi > > 42 42 C C42>Picot.c2.l5 C C 1.25 *** *** 1.00 ***
0.75
0.50
Expression ratio ratio Expression 0.25 (compared (compared to parental) C42 (compared (compared to parental) C42 0.00 2 3 4 .l2 .l3 .l .l5 3 3 C c3 t.c t.c3 t. t.c o o o o c ic Pi Pic P Picot.c3.l5 > 2>Pic 4 C42>Picot.c3.l2 C C42 D 1.25
1.00
0.75
0.50
Expression ratio ratio Expression 0.25 (compared to(compared parental) (compared (compared to parental) 0.00
.l3 l3 1 1. 3.l6 c t.c t.c t. o o o c ic ic pi p picot.c3.l6 > 2 2>p c4 c4
Figure 5.10 qPCR verification of Picot knockdown in RNAi fly-line tubules. Picot expression in A: RNAi lines from construct 1, B: RNAi lines from construct 2, and C: RNAi lines from construct 3. D: Quantification of CG6978 expression in driven RNAi lines (N=3, mean ± S.E.M.) (Student’s t- test, *p<0.05, **p<0.01, ***p<0.001). 173
1 2 3 4 5 6 ~82 kDa Lane Fly-line ~64 kDa ~58 kDa 1 c42>picot.c1.l6 ~49 kDa 2 picot.c1.l6 ~37 kDa 3 c42>picot.c2.l3 ~26 kDa 4 picot.c2.l3 5 c42>picot.c3.l3 Alpha tubulin 6 picot.c3.l3
Figure 5.11 Picot expression in three GAL4-driven UAS-RNAi fly-lines and their parental fly- lines as determined by Western blotting. Alpha tubulin was blotted as a control for protein integrity.
c42-GAL4 Picot
5.3.3.2 Effect on Fluid Secretion from the Tubules
Picot et al.
Picot Drosophila
Picot 174
A 2.0
-1) C42 picot.c1.l6 1.5 C42>picot.c1.l6
1.0
0.5 drosokinin Secretion rate (nl min 0.0 0 10 20 30 40 50 60 70 80 Time (min)
B 2.0
-1) C42 picot.c2.l3 1.5 c42>picot.c2.l3
1.0
0.5 drosokinin Secretion0.0 rate (nl min 0 10 20 30 40 50 60 70 80 Time (min)
C 2.0
-1) C42 picot.c3.l3 1.5 C42>picot.c3.l3
1.0
0.5
drosokinin Secretion rate (nl min 0.0 0 10 20 30 40 50 60 70 80 Time (min)
Figure 5.12 Effect of Picot RNAi knockdown on the rate of fluid secretion from the tubules as determined by secretion assays. Typical experimental results, showing the response of three driven RNAi lines and the appropriate RNAi parental lines to Drosokinin after 30 min of steady secretion (mean ± S.E.M., N ≥10).
175 5.4 Discussion
Drosophila AGAP012251 Picot Picot AGAP012251
5.4.1 AGAP012251 and Picot in the Tubules
AGAP012251 Picot AGAP012251 Anopheles Picot Drosophila AGAP012251 Troglophilus neglectus et al. Drosophila Anopheles gambiae Anopheles albimanus et al.
In situ AGAP012251 Anopheles 176 Anopheles Drosophila Picot Picot
Picot
Picot Picot Actin-GAL4 Picot et al. et al. Picot Picot Drosophila
Picot c42-GAL4 c42-GAL4 Picot Picot 177 Drosophila
in situ AGAP012251 Picot Anopheles Drosophila Picot AGAP012251
5.4.2 AGAP012251 and Picot as Inorganic Phosphate Co-transporters
AGAP012251 Picot et al. Anopheles Drosophila
AGAP012251 Picot Drosophila et al. AGAP012251 Picot
AGAP012251 Picot
Picot Picot c42-GAL4, Picot Picot 178 Picot Picot Picot
5.4.3 AGAP012251 and Picot as Insecticide Targets
AGAP012251 Picot Picot AGAP012251 Picot,
AGAP012251 Picot
179 5.5 Conclusion
AGAP012251 Picot Anopheles Drosophila Picot AGAP012251 Anopheles Drosophila AGAP012251 Anopheles Picot Drosophila Picot Drosophila Picot Picot
180
Chapter 6 - AGAP009005 and ZnT35C as Insecticide Targets
181 6.1 Summary
Anopheles Anopheles Drosophila et al. Anopheles AGAP009005 Drosophila ZnT35C AGAP009005 ZnT35C Anopheles Drosophila AGAP009005 ZnT35C ZnT35C ZnT35C ZnT35C AGAP009005 ZnT35C Anopheles Drosophila AGAP009005 ZnT35C
182 6.2 Introduction
AGAP009005 ZnT35C Anopheles Drosophila
6.2.1 The Importance of Zinc Regulation
et al. et al. et al.
6.2.2 Zinc Transport in Mammals
183 et al.
metal-responsive-element-binding transcription factor-1 MTF-1 MTF-1
6.2.3 Zinc Transport in Insects
Drosophila et al.
Drosophila 184 et al. CG10505 CG10505 CG10505 et al. GstD2 GstD5 S et al. ZnT35C
Drosophila et al. ZIP1, CG9430, CG7816 et al. CG13189, CG10006, CG4334, Zip3, CG2177 Drosophila fear of intimacy foi et al. et al. Foi et al. Drosophila Catsup, et al. Drosophila Zip3 et al. Anopheles et al.
Drosophila et al. et al. ZnT35C 185 Drosophila et al. Anopheles et al. AGAP009005 ZnT35C Anopheles
6.2.3.1 Zinc Transport in the Malpighian Tubules
Musca domestica et al. Drosophila hydei et al Drosophila et al Drosophila et al. 186 6.2.4 The Putative Zinc Transporters AGAP009005 and ZnT35C
AGAP009005 ZnT35C et al. ZnT35C CG3994 AGAP009005
Figure 6.1 Homology tree for ZnT35C ( CG3994) and AGAP009005 . The tree shows a close relationship between the Anopheles and Drosophila zinc transporters. (Tree generated by Ensembl, see Section 3.4.6.2 (Hubbard et al. , 2009; Vilella, 2009).
AGAP009005 ZnT35C ZnT35C AGAP009005 Anopheles
187
Table 6.1 ZnT35C expression in larval and adult Drosophila (Chintapalli et al. , 2007).
Tissue mRNA Signal Pre sent Call Enrichment Affy Call Brain 4 ± 1 1 of 4 0.00 Down Head 8 ± 0 1 of 4 0.00 Down Thoracicoabdominal 8 ± 1 0 of 4 0.00 Down ganglion Salivary gland 11 ± 4 0 of 4 0.05 Down Crop 5 ± 1 0 of 4 0.00 Down Midgut 9 ± 3 1 of 4 0.00 Down
Tubu le 4645 ± 86 4 of 4 22.10 Up Hindgut 59 ± 9 4 of 4 0.30 Down Heart 8 ± 1 2 of 4 0.04 Down Fat body 17 ± 9 4 of 4 0.08 Down Ovary 275 ± 3 4 of 4 1.30 None Testis 477 ± 22 4 of 4 2.30 Up Male accessory glands 6 ± 1 0 of 4 0.00 Down Virgin spermatheca 15 ± 1 2 of 4 0.07 Down Mated spermatheca 11 ± 1 2 of 4 0.05 Down Adult carcass 30 ± 3 3 of 4 0.10 Down Larval CNS 6 ± 1 2 of 4 0.03 Down Larval Salivary gland 7 ± 0 1 of 4 0.04 Down Larval midgut 7 ± 1 0 of 4 0.04 Down Larval tubule 1960 ± 123 4 of 4 9.30 Up Larval hindgut 13 ± 1 4 of 4 0.07 Down
Larval fat body 50 ± 19 3 of 4 0.20 Down S2 cells (growing) 6 ± 1 1 of 4 0.03 Down Whole fly 210 ± 24 4 of 4
ZnT35C et al Drosophila et al. ZnT35C Drosophila ZnT35C dMTF-1 ZnT35C ZnT35C Drosophila ZnT35C 188
A B
Figure 6.2 ZnT35C expression in whole-fly Drosophila . A: The transcript levels of ZnT35C in whole-fly measured after exposure to different foods: NF, normal food; Cd, 50 µM CdCl 2; Cu, 0.5 mM CuSO 4; Zn, 5 mM ZnCl 2. Results are shown for wild-type flies (MTF-1 wf) and flies in which dMTF-1 expression has been knocked down (MTF-1 KO). Representative gels are shown below the bars. B: Expression pattern of the ZnT35C-GFP fusion protein driven by the ZnT35C promoter in Drosophila larvae. A segment of Malpighian tubule from an animal fed with zinc shows strong green fluorescence on the apical membrane (Yepiskoposyan et al. , 2006).
ZnT35C ZnT35C Drosophila Anopheles ZnT35C AGAP009005, .
189 6.3 Results
AGAP009005 ZnT35C Anopheles Drosophila AGAP009005 ZnT35C in situ ZnT35C Drosophila ZnT35C Drosophila
6.3.1 AGAP009005 in the Tubules
AGAP009005 Anopheles Anopheles AGAP009005 Anopheles in situ
6.3.1.1 Expression of the Transcript
AGAP009005 Anopheles 190
17500 FB FS MS Larval 15000 Whole *** *** *** ** Larval ** *** *** 12500 MS *** *** 10000 FS * 7500 5000 Signal strength 2500 0
ole val MS FS FB h ar W L
Figure 6.3 Microarray data for AGAP009005 expression in the Anopheles tubules at different life-stages. The attached table shows the statistical significance between life-stages. (N=4, mean ± S.E.M.) One-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.
AGAP009005 AGAP009005
Anopheles AGAP009005 AGAP009005 191
A 25 Microarray 20 qPCR
15
10
Expression ratio Expression 5 (compared to(compared whole) 0 S M FS FB Whole Larval
B **
5 **
4
3
2
Expression ratio ratio Expression 1 (compared to(compared FS) (compared to(compared FS)
0 r B h FS F 4 +2 B F
Figure 6.4 qPCR validation of AGAP009005 expression in the Anopheles tubules. A: ratio of expression as calculated from microarray and qPCR data. B: ratio of expression in female tubules 3 h and 24 h after a blood-meal (N ≥4, mean ± S.E.M.). One-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.
AGAP009005 AGAP009005 AGAP009005 AGAP009005 192 AGAP009005
AGAP009005 In situ AGAP009005 Anopheles
A B proximal
distal midgut
distal
distal
distal
distal
C proximal DE
F distal end distal
distal end
Figure 6.5 Expression of AGAP009005 in the adult Anopheles tubules. A: low power view of the male tubules showing expression throughout. B: low power view of the female tubules showing expression throughout. C: high power view showing expression in the female tubule from the proximal segment to the distal end. D: high power view of the distal end of the female tubule showing strong expression in the principal cells, with lighter areas which may be stellate cells. E: low power view of male tubule stained with sense probe as a control, showing no expression. G: high power view of the female distal end stained with sense probe as a control, showing no expression. 193 in situ AGAP009005 in situ AGAP009005 AGAP009005
6.3.2 ZnT35C in the Tubules
ZnT35C Drosophila Drosophila et al. in situ
6.3.2.1 Expression of the Transcript
ZnT35C Drosophila AGAP009005 et al. et al. ZnT35C Drosophila 194
A B 20 *** 2.0 *** 15 1.5
10 1.0
5 0.5 Expression ratio Expression ratio
(Com pared to whole) ** (Com pared to Male MT) 0 0.0 Whole Larval MT Adult MT Male MT Female MT
Figure 6.6 Expression of ZnT35C in the Drosophila Malpighian tubules. A: expression in the larval and adult Malpighian tubules in comparison to the average whole-fly expression. B: expression in the female tubules in comparison to the male tubules (N=4, mean ± S.E.M.). Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.
ZnT35C Drosophila Anopheles AGAP009005 ZnT35C
In situ Drosophila
in situ ZnT35C Drosophila ZnT35C in situ ZnT35C Drosophila 195
A B
ureter
ureter hindgut MS MS MS
MS
TS IS
CD E
ureter
MS MS
IS
Figure 6.7 Expression of ZnT35C in the adult Drosophila tubules as determined by in situ hybridisation. Low power views showing expression in the ureter, main segment (MS), transitional segment (TS) and initial segment (IS) in the (A) male and (B) female tubules. C: high power shot of the main segment showing staining. D: high power shot of the anterior distal tubule showing staining in the initial segment. E: sense probe control showing no staining in the tubules.
6.3.3 RNAi Knockdown of ZnT35C
ZnT35C Drosophila ZnT35C ZnT35C w1118 196 Sb ZnT35C
6.3.3.1 Effect on Fly Survival
Table 6.2 Pattern of expression of GAL4 drivers used to knock-down ZnT35C
Driver line Pattern of expression Actin -GAL4
C42-GAL4
Uro -GAL4
C724 -GAL4
197
Table 6.3 Effect of ZnT35C knockdown on fly survival (cross N=3, raw data is presented in Appendix 2).
Fly -line Actin -GAL4 C42 -GAL4 Uro -GAL4 C724 -GAL4
CG3994.c1.l1 CG3994.c1.l3 CG3994.c1.l4 CG3994.c2.l1 semi -lethal CG3994.c2.l2 lethal CG3994.c2.l8 lethal CG3994.c3.l1 CG3994.c3.l2 semi -lethal CG3994.c3.l4
Actin-GAL4 Actin-GAL4 ZnT35C
ZnT35C Actin-GAL4 Actin-GAL4 c42-GAL4 CG11163, ZnT35C 198
A 1.25 *** *** *** 1.00
0.75
0.50
Expression ratio Expression 0.25
0.00 (compared (compared to ACT parental) 4 3 4 l .l1 .l .l .l4 a 1 1 G .c c 4.c1 - -c1 n- 9 94 4 4 ti 9 99 99 Ac 3 3 3 G G G CG39 CG3994.c1.l1CG3994.c1.l3 C C > > T T C CT>C A A AC B 1.25 *** *** *** 1.00
0.75
0.50
Expression ratio Expression ratio 0.25 (compared to parental) C42 (compared (compared to parental) C42 0.00
l2 C42 2.l1 .c2.l1 c2.l8 4.c2.l8 94 94.c 94.c2. 94.c2.l2 9 94. 9 39 39 39 39 CG CG CG39
C C42>CG C42>CG3 C42>CG 1.25 ** *** *** 1.00
0.75
0.50
Expression ratio Expression 0.25
0.00 (compared (compared to ACT parental)
l4 l1 .l4 a 3. 3.l1 3.l2 3 .c c 4.c 4.c 94.c3.l4 99 994 9 99 Actin-G 3 3 3994.c3.l23 3 G G G3994. G G G C C C CT>C A ACT>C ACT>C D 1.5 *
1.0
0.5 Expresion ratio Expresion ratio (compared to parentals) (compared (compared to parentals) 0.0 2 .l3 .l3 .l 1 1 2 .c .c 4 4 9 9 9 9 G3994.c G3 G3 C CG3994.c2.l2 C T>C 2> C 4 A C
Figure 6.8 qPCR verification of ZnT35C knockdown in the tubules of driven RNAi lines. ZnT35C expression in A: RNAi lines from construct 1, B: RNAi lines from construct 2, and C: RNAi lines from construct 3. D: Quantification of CG11163 expression in driven RNAi lines (N=3, mean ± S.E.M.) (Student’s t-test, *p<0.05, **p<0.01, ***p<0.001). 199 ZnT35C t CG11163 ZnT35C
6.3.3.2 Effect on Fluid Secretion from the Tubules
ZnT35C et al.
ZnT35C CG3994.c1.l3 ZnT35C CG3994.c1.l3 ZnT35C ZnT35C 200
A 2.0 )
-1 Actin GAL4 CG3994.c1.l3 1.5 ACT>CG3994.c1.l3
1.0
0.5 drosokinin Secretion Secretion raterate (nl(nl min min 0.0 0 10 20 30 40 50 60 70 80 Time (min) B 2.0 )
-1 Actin GAL4 CG3994.c2.l1 1.5 C42>CG3994.c2.l1
1.0
0.5
drosokinin Secretion Secretion rate (nl min 0.0 0 10 20 30 40 50 60 70 80 Time (min) C 2.0 )
-1 Actin GAL4 CG3994.c3.l1 1.5 ACT>CG3994.c3.l1
1.0
0.5
Secretion Secretion raterate (nl(nl min min drosokinin 0.0 0 10 20 30 40 50 60 70 80 Time (min)
Figure 6.9 Effect of ZnT35C knockdown on the rate of fluid secretion from the tubules. Typical experimental results, showing the response of three GAL4-driven UAS-RNAi lines and the appropriate UAS-RNAi and GAL4 driver parental lines to Drosokinin after 30 min of steady secretion (mean ± S.E.M., N ≥10). 201 6.4 Discussion
Drosophila, Anopheles AGAP009005 ZnT35C Anopheles Drosophila ZnT35C AGAP009005
6.4.1 AGAP009005 and ZnT35C in the Tubules
AGAP009005 ZnT35C AGAP009005 Anopheles ZnT35C Anopheles Drosophila AGAP009005 ZnT35C
AGAP009005 et al. AGAP009005 et al. et al. et al. Anopheles Drosophila 202 AGAP009005
in situ AGAP009005 ZnT35C et al ZnT35C Drosophila et al. ZnT35C
ZnT35C ZnT35C Actin-GAL4 ZnT35C Drosophila ZnT35C ZnT35C c42-GAL4 UrO-GAL4
in situ AGAP009005 ZnT35C Anopheles Drosophila ZnT35C AGAP009005, Drosophila Anopheles
6.4.2 AGAP009005 and ZnT35C as Zinc Transporters
AGAP009005 ZnT35C Drosophila ZnT35C 203 AGAP009005 ZnT35C et al.
et al ZnT35C ZnT35C ZnT35C ZnT35C Drosophila
6.4.3 AGAP009005 and ZnT35C as Insecticide Targets
et al AGAP009005 ZnT35C ZnT35C Drosophila ZnT35C AGAP009005 ZnT35C Anopheles AGAP009005
AGAP009005 ZnT35C Anopheles Drosophila AGAP009005 ZnT35C
204 6.5 Conclusion
AGAP009005 ZnT35C AGAP009005 ZnT35C Anopheles Drosophila ZnT35C Drosophila ZnT35C et al. ZnT35C Drosophila ZnT35C in situ Anopheles Drosophila Xenopus AGAP009005 ZnT35C
205
Chapter 7 - AGAP002587 and CG8028 as Insecticide Targets
206 7.1 Summary
Anopheles Drosophila et al. Anopheles AGAP002587 Drosophila CG8028 Anopheles Drosophila In situ CG8028 CG8028 CG8028 Anopheles Drosophila AGAP002587 CG8028 AGAP002587 CG8028
207 7.2 Introduction
AGAP002587 CG8028 Anopheles Drosophila
7.2.1 The Importance of Monocarboxylic Acids
et al. 208
7.2.2 Monocarboxylate Transport in Mammals
MCT1-4 MCT6 MCT8 MCT10 et al. et al. et al.
SLC5A8 SLC5A12 et al. et al. SLC5A8 SLC5A12 et al.
7.2.3 Monocarboxylate Transport in Diptera
Drosophila Anopheles, Drosophila 209 Silnoon outsider
et al LKB1 Silnoon CG8271 et al. Silnoon Drosophila et al. Anopheles AGAP010881 (Hubbard et al., 2009) Silnoon Drosophila LKB1 Silnoon
Drosophila outsider CG8062 p53 et al. outsider outsider p53 outsider
7.2.4 The Putative Monocarboxylate Transporters AGAP002587 and CG8028
AGAP002587 CG8028 et al.
AGAP002587 CG8028 210 AGAP002587 CG8028 MCT6 MCT8
CG8468 CG8389 CG12286, Drosophila CG8028 et al. CG8028 Drosophila CG8028 AGAP002587 Anopheles
211
Table 7.1 CG8028 expression in larval and adult Drosophila (Chintapalli et al. , 2007).
Tissue mRNA Signal Pre sent Call Enrichment Affy Call Brain 6 ± 0 3 of 4 0.20 Down Head 5 ± 1 0 of 4 0.20 Down Thoracicoabdominal 4 ± 1 0 of 4 0.10 Down ganglion Salivary gland 19 ± 4 3 of 4 0.56 Down Crop 7 ± 2 0 of 4 0.20 Down Midgut 9 ± 2 3 of 4 0.30 Down
Tubu le 2008 ± 231 4 of 4 59.00 Up Hindgut 8 ± 1 2 of 4 0.30 Down Heart 5 ± 0 0 of 4 0.16 Down Fat body 14 ± 6 4 of 4 0.41 Down Ovary 4 ± 1 0 of 4 0.10 Down Testis 6 ± 0 0 of 4 0.20 Down Male accessory glands 10 ± 1 3 of 4 0.30 Down Virgin spermatheca 10 ± 1 3 of 4 0.33 Down Mated spermatheca 11 ± 2 4 of 4 0.30 Down Adult carcass 13 ± 5 2 of 4 0.40 Down Larval CNS 5 ± 0 4 of 4 0.16 Down Larval Salivary gland 6 ± 2 1 of 4 0.19 Down Larval midgut 10 ± 1 4 of 4 0.31 Down Larval tubule 626 ± 54 4 of 4 18.4 Up Larval hindgut 11 ± 2 4 of 4 0.35 Down
Larval fat body 9 ± 0 1 of 4 0.30 Down S2 cell (growing) 13 ± 1 4 of 4 0.39 Down Whole fly 34 ± 4 4 of 4
212 7.3 Results
AGAP002587 CG8028 Anopheles Drosophila AGAP002587 CG8028 in situ CG8028 Drosophila CG8028 CG8028 Drosophila
7.3.1 AGAP002587 in the Tubules
AGAP002587 Anopheles Anopheles in situ
7.3.1.1 Expression of the Transcript
AGAP002587 Anopheles 213
12500 FB FS MS Larval
10000 Whole *** * - - Larval *** * - 7500 MS *** * FS *** 5000
Signal strength 2500
0 l a rv MS FS FB Whole La
Figure 7.1 Microarray data for AGAP002587 expression in the Anopheles tubules at different life-stages . The attached table shows the statistical significance between life-stages. (N=4, mean ± S.E.M.). One-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.
AGAP002587 AGAP002587 Anopheles AGAP002587
AGAP002587 AGAP002587 214
A 140 Microarray 120 qPCR 100 80 60 40 Expression ratio Expression 20 (compared to(compared whole) 0 e al B rv MS FS F hol a W L
B *** 20 ***
15
10
5 Expression ratio ratio Expression (compared to(compared FS) (compared to(compared FS)
0
FS FB +24h FB
Figure 7.2 qPCR validation of AGAP002587 expression in Anopheles . A: ratio of expression as calculated from microarray and qPCR data. B: ratio of expression in female tubules 3 h and 24 h after a blood-meal (N ≥4, mean ± S.E.M.). One-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.
AGAP002587 AGAP002587 AGAP002587 215 AGAP002587 In situ AGAP002587 Anopheles
A B
proximal
proximal midgut
distal end
distal distal distal end
C DEmidgut proximal
proximal
proximal
proximal
distal end distal distal
Figure 7.3 Expression of AGAP002587 in the adult Anopheles tubules as determined by in situ hybridisation. A: low power view of the male tubules showing expression throughout. B: low power view of the female tubules showing expression throughout. C: high power view showing expression in the female distal end. D: high power view of the female tubule where it joins the midgut showing expression. E: low power view of male tubule stained with sense probe as a control, showing no expression.
In situ AGAP002587 Anopheles AGAP002587 AGAP002587 216
7.3.2 CG8028 in the Tubules
CG8028 Drosophila Drosophila et al. in situ
7.3.2.1 Expression of the Transcript
CG8028 Drosophila AGAP002587 et al. et al. CG8028 Drosophila CG8028
A B 50 *** 1.25
40 1.00
30 0.75
20 0.50
Expression10 ratio Expression0.25 ratioratio (com pared to whole) (Com pared to Male MT) ** (Com pared to Male MT) 0 0.00 Whole Larval MT Adult MT Male MT Female MT
Figure 7.4 Expression of CG8028 in the Drosophila Malpighian tubules as determined by qPCR. A: expression in the larval and adult Malpighian tubules in comparison to whole-fly. B: expression in the female tubules in comparison to the male tubules (N=4, mean ± S.E.M.). Student’s t-test, *p<0.05, **p<0.01, ***p<0.001. 217 CG8028 Anopheles CG8028 Drosophila
In situ Drosophila CG8028
A B TS TS MS IS ureter IS LS
MS MS LS
MS LS
LS TS MS MS
IS
TS
IS
C DE F
ureter TS
MS
MS MS
LS IS LS ureter LS LS
Figure 7.5 Expression of CG8028 in adult Drosophila tubules as determined by in situ hybridisation. Low power views showing expression in the ureter, lower segment (LS), main segment (MS), transitional segment (TS) and initial segment (IS) in the male (A) and female (B) tubules. C: high power view of the ureter and lower segment showing staining. D: high power view of the main segment showing staining. E: high power view of the anterior initial and transitional segments showing staining. F: sense control showing no staining in the tubules.
CG8028 Drosophila CG8028 218 CG8028 in situ CG8028 Drosophila
7.3.3 RNAi Knockdown of CG8028
CG8028 CG8028 w1118 Sb CG8028
7.3.3.1 Effect on Fly Survival
Table 7.2 Pattern of expression of GAL4 drivers used in this study.
GAL4 Driver line Pattern of expression Actin -GAL4
C42 ELAV -GAL4
C724-GAL4
219
Table 7.3 Effect of CG8028 knockdown on fly survival (cross N=3, raw data is presented in Appendix 2) .
Fly -line Actin -GAL4 C42 -GAL4 ELAV -GAL4 C724 -GAL4
CG8028.c1.l1 CG8028.c1.l3 CG8028.c1.l4 CG8028.c2.l1 CG8028.c2.l2 CG8028.c2.l4 CG8028.c3.l1 CG8028.c3.l4 CG8028.c3.l6
CG8028 CG8028 CG8028 Actin-GAL4 CG8034, CG8028 220
A 1.25 *** *** *** 1.00
0.75
0.50
Expression ratio Expression 0.25
0.00 (compared to ACT parental) ACT to (compared
l4 l1 .l4 l4 .l6 1. Ga .c1. .c1 .c .c1 8 8 in- 28 28 t 0 02 0 02 c 8028.c1.l1 A G G8 CG8 CG8 CG8028.c1.l6 T>CG8 ACT>C AC ACT>C B 1.25 *** *** *** 1.00
0.75
0.50
Expression ratio Expression 0.25
0.00 (compared to ACT parental) ACT to (compared
.l1 2.l4 -Gal4 .c2.l1 .c2 .c2.l2 .c2.l2 .c2.l4 .c 8 Actin 8028 8028 8028 CG CG802 CG >CG
ACT>CG8028 ACT ACT>CG8028 C ** 1.25 *** ** 1.00
0.75
0.50
Expression ratio Expression 0.25
0.00 (compared to ACT parental) ACT to (compared
al4 -G in 8.c3.l1 8.c3.l1 Act 8028.c3.l4 8028.c3.l4 8028.c3.l6 8028.c3.l6 CG802 CG CG CG >CG T>CG802 AC ACT ACT> D 1.5
1.0
0.5 Expression ratio Expression
(compared to parentals) to (compared 0.0
2.l1 .c1.l1 8 8.c1.l1 8.c 02 G8 CG802 C CG802 CT> A ACT>CG8028.c2.l1
Figure 7.6 qPCR verification of CG8028 RNAi knockdown in the tubules. CG8028 expression in A: RNAi lines from construct 1, B: RNAi lines from construct 2, and C: RNAi lines from construct 3. D: Quantification of CG8034 expression in driven RNAi lines (N=3, mean ± S.E.M.) (Student’s t- test, *p<0.05, **p<0.01, ***p<0.001). 221 CG8028 t CG8034 CG8028 CG8028 CG8028
7.3.3.2 Effect on Fluid Secretion from the Tubules
CG8028 et al.
CG8028 CG8028 CG8028 222
A 2.5
-1) Actin GAL4 2.0 CG8028.c1.l1 ACT>CG8028.c1.l1 1.5
1.0
0.5 drosokinin Secretion Secretion raterate (nl(nl min min 0.0 0 10 20 30 40 50 60 70 80 Time (min)
B 2.5
-1) Actin GAL4 2.0 CG8028.c2.l1 ACT>CG8028.c2.l1 1.5
1.0
0.5
Secretion Secretion raterate (nl(nl min min drosokinin 0.0 0 10 20 30 40 50 60 70 80 Time (min)
C 2.5
-1) Actin GAL4 2.0 CG8028.c3.l6 ACT 1.0 0.5 Secretion rate (nl min drosokinin 0.0 0 10 20 30 40 50 60 70 80 Time (min) Figure 7.7 Effect of CG8028 knockdown on the rate of fluid secretion from the tubules. Typical experimental results, showing the response of three driven RNAi lines and the appropriate RNAi parental lines to Drosokinin after 30 min of steady secretion (mean ± S.E.M., N ≥10). 223 7.4 Discussion Drosophila Anopheles Drosophila AGAP002587 CG8028 Anopheles Drosophila CG8028 AGAP002587 7.4.1 AGAP002587 and CG8028 in the Tubules AGAP002587 CG8028 Anopheles AGAP002587 AGAP002587 Drosophila CG8028 Anopheles, CG8028 Drosophila Anopheles Drosophila in situ AGAP002587 CG8028 Anopheles 224 AGAP002587 Drosophila CG8028 CG8028 CG8028 Actin-GAL4 CG8028 c42-GAL4 UrO-GAL4 CG8028 Drosophila CG8028 in situ CG8028 AGAP002587 AGAP002587 Drosophila Drosophila Anopheles Drosophila AGAP002587 7.4.2 AGAP002587 and CG8028 as Monocarboxylate Transporters AGAP002587 CG8028 Drosophila CG8028 225 AGAP002587 CG8028 Aedes aegypti et al. AGAP002587 CG8028 CG8028 CG8028 CG8028 AGAP002587 Xenopus 7.4.3 AGAP002587 and CG8028 as Insecticide Targets AGAP002587 CG8028 CG8028 Drosophila Drosophila AGAP002587 Anopheles AGAP002587 AGAP002587 CG8028 Anopheles Drosophila CG8028 AGAP002587 AGAP002587 Aedes aegypti 226 Aedes Anopheles Anopheles Anopheles Anopheles Aedes, et al. 227 7.5 Conclusion AGAP002587 CG8028 CG8028 Drosophila AGAP002587 CG8028 Anopheles Drosophila Anopheles Drosophila, Aedes aegypti AGAP002587 Anopheles Xenopus CG8028 AGAP002587 228 Chapter 8 - Conclusions and Further Work 229 8.1 Summary Anopheles Anopheles Drosophila Drosophila Anopheles 8.2 Conclusions Anopheles Aedes 8.2.1 Tissue-specific Microarrays are Advantageous for Tissue- specific Studies Anopheles Anopheles Anopheles et al et al. et al. Drosophila et al. et al. Drosophila 230 Table 8.1 Genes which are predominately expressed in unexpected tissues (Chintapalli et al. , 2007). Anopheles Anopheles 8.2.2 Transporters are Highly Expressed in the Malpighian Tubules of Dipterans Drosophila et al. Anopheles Drosophila Anopheles 231 8.2.3 Drosophila is a Good Model for the Anopheles Malpighian Tubules Drosophila Anopheles AGAP002587 CG8028 AGAP002587 CG8028 Anopheles Drosophila Drosophila 8.2.4 Cross-species Studies are an Effective Strategy for Identifying Essential Genes 232 Anopheles Anopheles Drosophila Drosophila Drosophila CG8028 Drosophila Drosophila 8.2.5 Limitations of the Study Drosophila Drosophila CG15406 Picot 233 Actin-GAL4 Drosophila Drosophila Anopheles Drosophila 234 CG15406 Picot Drosophila et al. et al. Xenopus oocyte Anopheles Anopheles Anopheles 235 8.3 Future Work Anopheles Drosophila Anopheles Drosophila Drosophila Anopheles Anopheles Anopheles Drosophila Xenopus laevis Anopheles Drosophila Anopheles 236 Drosophila Anopheles Drosophila Drosophila 237 Appendix 1 Drosophila melanogaster Primer Name Primer Purpose Primer Sequence Picot Picot CG3994 CG3994 CG15406 CG15406 CG8028 CG8028 CG4330 CG4330 CG11163 CG11163 CG8051 CG8051 CG15408 CG15408 In situ Drosophila melanogaster Primer name Primer purpose Primer sequence CG15406 in situ CG15406 in situ Picot in situ Picot in situ CG3994 in situ CG3994 in situ CG8028 situ CG8028 in situ 238 Anopheles gambiae Primer Name Primer Purpose Primer Sequence AGA P007752 AGAP007752 AGAP012251 AGAP012251 AGAP009005 AGAP009005 AGAP002587 AGAP002587 AGAP002858 AGAP002858 AGAP010892 AGAP010892 AGAP007495 AGAP007495 AGAP008386 AGAP008386 In situ Anopheles gambiae Primer name Primer purpose Primer sequence AGAP007752 in situ AGAP007752 in situ AGAP012251 in situ AGAP012251 in situ AGAP00900 5 in situ AGAP009005 in situ AGAP002587 in situ AGAP002587 in situ 239 Drosophila melanogaster Primer Name Primer Purpose Primer Sequence CG15406 CG15406 CG15406 CG15406 CG15406 CG15406 Picot Pic ot Picot Picot Picot Picot CG3994 CG3994 CG3994 CG3994 CG3994 CG3994 CG8028 CG8028 CG8028 CG8028 CG8028 CG8028 240 Appendix 2 CG15406 Chi Fly -line Actin -GAL4 C42 -GAL4 Uro -GAL4 C724 -GAL4 CG15406.c1.l1 CG15406.c1.l4 CG15406.c1.l9 CG15406.c2.l4 CG15406.c2.l7 CG15406.c2.l8 CG15406.c3.l3 CG15406.c3.l4 CG15406.c3.l5 1 3 241 Picot Chi Fly -line Actin -GAL4 C42 -GAL4 C724 -GAL4 ELAV -GAL4 Picot.c1.l3 Picot.c1.l5 Picot.c1.l6 Picot.c2.l3. Picot.c2.l5 Picot.c2.l6 Picot.c3.l2 Picot.c3.l3 Picot.c3.l5 1 3 242 CG3994 Chi Fly -line Actin -GAL4 C42 -GAL4 Uro -GAL4 C724 -GAL4 CG3994.c1.l1 CG3994.c1.l3 CG3994.c1.l4 CG3994.c2.l1 CG3994.c2.l2 CG3994.c2.l8 CG3994.c3.l1 CG3994.c3.l2 CG3994.c3.l4 1 3 243 CG8028 Chi Fly -line Actin -GAL4 C42 -GAL4 ELAV -GAL4 C724 -GAL4 CG8028.c1.l1 CG8028.c1.l3 CG8028.c1.l4 CG8028.c2.l1 CG8028.c2.l2 CG8028.c2.l4 CG8028.c3.l1 CG8028.c3.l4 CG8028.c3.l6 1 3 244 Bibliography , et al. 287 92 104 32 65 304 56 37 12 245 4 12 212 32 118 180 285 3 12 246 267 168 207 43 405 3 295 39 90 247 557 152 447 128 24 269 21 622 248 5 7 197 83 44 7 10 183 249 40 14 278 20 66 279 38 15 447 250 343 Pt 2 60 95 36 344 97 772 426 , et al. 298 8 , et al. 37 251 126 22 36 32 183 95 517 ( Pt 3) 27 8 252 49 18 276 81 9 463 8 30 135 253 34 456 202 38 24 14 207 157 254 : 6 15 37 , et al. 35 45 24 255 167 10 99 33 80 28 , et al. 316 204 199 256 198 36 132 43 253 94 60 37 114 207 257 206 207 511 14 288 20 103 2 200 8 258 1 28 8 94 392 3 153 17 259 37 275 10 4 (Epub ahead of print) 5 101 447 260 176 130 19 293 49 5 17 206 261 45 38 vol. 7 31 203 266 153 9 262 135 30 15 451 44 34 47 207 53