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Characterization of pH and Ion Regulatory Proteins in Larval Mosquitoes

Permanent Link: http://ufdc.ufl.edu/UFE0024189/00001

Material Information

Title: Characterization of pH and Ion Regulatory Proteins in Larval Mosquitoes
Physical Description: 1 online resource (172 p.)
Language: english
Creator: Smith, Kristin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: anhydrase, gambiae, ion, mosquito, ph, rectum, rnai
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Mosquitoes have long been a major cause of human morbidity and mortality, and as a result, worldwide efforts are underway to control these disease vectors. Evidence suggests that targeting the larval stages of the mosquito lifecycle in addition to the adult stage may facilitate vector control. In order to specifically and safely target mosquito larvae using control methods, a thorough understanding of the biological systems crucial for larval survival is necessary. Here I characterize proteins that influence larvae s ability to tightly control the pH within their alimentary canal, as well as proteins involved in their ability to adapt to varying environmental conditions. Uncovering the methods of pH and ion regulation employed by larvae in both of these processes could lead to the discovery of novel targets and new and improved larvicides. First, I report the cloning and characterization of novel carbonic anhydrase transcripts from the larval Anopheles gambiae alimentary canal. Mosquito larvae generate a highly alkaline pH in a restricted area of the alimentary canal, and carbonic anhydrase is a crucial enzyme for the generation and maintenance of this pH. Characterization of mRNA expression and protein distribution in the case of one CA, AgCA9, reveal an alternate pathway for bicarbonate/carbonate transport into the lumen of the alimentary canal, updating the current model for larval pH regulation. Additionally, RNA interference work in an An. gambiae larval cell line demonstrates that AgCA9 is a protein capable of being manipulated by RNAi and suggests that this technique can be used to silence a CA in live mosquito larvae. Finally, antibody immunolocalization of AgCA9 reveals a novel subset of cells in the rectum of anopheline larvae. I characterize the cells of this rectum using immunohistochemistry, physiology, and pharmacology and use this data to develop a putative model of ion regulation in the anopheline rectum. This model supports my current hypothesis that anopheline larvae shift protein distribution to change the primary function of the rectum from absorption, in larvae reared in fresh water, to secretion, in larvae reared in saline water. Additionally, this work emphasizes the differences in rectal structure and protein regulation between anophelines and culicines, and suggests that these two subfamilies may be employ very different adaptive and regulatory strategies.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kristin Smith.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Linser, Paul J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024189:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024189/00001

Material Information

Title: Characterization of pH and Ion Regulatory Proteins in Larval Mosquitoes
Physical Description: 1 online resource (172 p.)
Language: english
Creator: Smith, Kristin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: anhydrase, gambiae, ion, mosquito, ph, rectum, rnai
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Mosquitoes have long been a major cause of human morbidity and mortality, and as a result, worldwide efforts are underway to control these disease vectors. Evidence suggests that targeting the larval stages of the mosquito lifecycle in addition to the adult stage may facilitate vector control. In order to specifically and safely target mosquito larvae using control methods, a thorough understanding of the biological systems crucial for larval survival is necessary. Here I characterize proteins that influence larvae s ability to tightly control the pH within their alimentary canal, as well as proteins involved in their ability to adapt to varying environmental conditions. Uncovering the methods of pH and ion regulation employed by larvae in both of these processes could lead to the discovery of novel targets and new and improved larvicides. First, I report the cloning and characterization of novel carbonic anhydrase transcripts from the larval Anopheles gambiae alimentary canal. Mosquito larvae generate a highly alkaline pH in a restricted area of the alimentary canal, and carbonic anhydrase is a crucial enzyme for the generation and maintenance of this pH. Characterization of mRNA expression and protein distribution in the case of one CA, AgCA9, reveal an alternate pathway for bicarbonate/carbonate transport into the lumen of the alimentary canal, updating the current model for larval pH regulation. Additionally, RNA interference work in an An. gambiae larval cell line demonstrates that AgCA9 is a protein capable of being manipulated by RNAi and suggests that this technique can be used to silence a CA in live mosquito larvae. Finally, antibody immunolocalization of AgCA9 reveals a novel subset of cells in the rectum of anopheline larvae. I characterize the cells of this rectum using immunohistochemistry, physiology, and pharmacology and use this data to develop a putative model of ion regulation in the anopheline rectum. This model supports my current hypothesis that anopheline larvae shift protein distribution to change the primary function of the rectum from absorption, in larvae reared in fresh water, to secretion, in larvae reared in saline water. Additionally, this work emphasizes the differences in rectal structure and protein regulation between anophelines and culicines, and suggests that these two subfamilies may be employ very different adaptive and regulatory strategies.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kristin Smith.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Linser, Paul J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024189:00001


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CHARACTERIZATION OF PH AND ION REGULATORY PROTEINS IN LARVAL MOSQUITOES By KRISTIN ELIZABETH SMITH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

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2009 Kristin E. Smith 2

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To my loving and supportive family 3

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ACKNOWLEDGMENTS I thank the family and friends who have s upported and encouraged me throughout the last five years. I thank my mother and father for always believing in me and never letting me forget it. I could not have done wit hout their encouragement and advi ce. I thank my other half, Robbie, for being an ear to talk to, a shoulder to cry on, and a fr iend with whom to celebrate my accomplishments and lament my failures. I than k the members of my committee as well as the faculty, staff and students of Wh itney for sharing their time, expertise, and opinions, thereby helping me accomplish much of what is written he rein. Finally, I thank my advisor, Prof. Paul Linser for his support, both financial and emoti onal, and for allowing me the freedom to expand my science in the directions which fit my interests. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................10 LIST OF ABBREVIATIONS........................................................................................................12 ABSTRACT...................................................................................................................................14 CHAPTER 1 INTRODUCTION................................................................................................................. .16 Significance................................................................................................................... .........16 pH Regulation in the Larval Alimentary Canal......................................................................17 Structure of the Larval Alimentary Canal..............................................................................18 Current Model of Alimenta ry Canal Alkalization..................................................................20 Ion Regulation in Mosquito Larvae........................................................................................21 Specific Aims..........................................................................................................................22 2 MATERIALS AND METHODS...........................................................................................26 Artificial Sea Water (ASW)....................................................................................................2 6 Experimental Insects........................................................................................................... ....26 Ag55 Cell Line.......................................................................................................................27 Isolation of RNA............................................................................................................... ......27 Isolation of Protein........................................................................................................... ......29 Cloning of AgCAs and AgAEs...............................................................................................30 Generation of cDNA Collections....................................................................................30 Cloning of Internal Fragments.........................................................................................31 Rapid Amplification of cDNA Ends (RACE).................................................................31 Cloning of Full-Length Gene Product.............................................................................32 Protein Alignments............................................................................................................. ....32 Calculation of Percent Identity...............................................................................................3 2 Detection of CA Activity........................................................................................................33 Quantitative PCR (QPCR)......................................................................................................33 Generation of First-Strand cDNA...................................................................................33 QPCR Reaction...............................................................................................................34 In situ Hybridization...............................................................................................................34 Generation of Digoxigenin Labeled Probes....................................................................34 Larval Preparation...........................................................................................................34 RNA Detection................................................................................................................35 Antibody Production...............................................................................................................36 5

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Peptide Blocking Assay..........................................................................................................37 Immunolocalization............................................................................................................. ...37 Phylogenetic Analysis............................................................................................................39 RT-PCR..................................................................................................................................39 Transcription of RNA.............................................................................................................39 Creation of dsDNA Flanked by T7 Promoter..................................................................40 Transcription of Sense and Antisense Strands into ssRNA.............................................40 Annealing of ssRNA........................................................................................................41 RNA Interference....................................................................................................................41 Northern Blot..........................................................................................................................41 Western Blot...........................................................................................................................42 Quantification of Protein.................................................................................................43 Acute Freshwater/Saline Challenges......................................................................................43 Quantification of Na+/K+-ATPase Signal Intensity................................................................44 Physiology..............................................................................................................................45 3 CLONING AND CHARACTERIZATION OF CARBONIC ANHYDRASE FROM ANOPHELES GAMBIAE GILES SENSU STRICTO LARVAE...........................................54 Introduction................................................................................................................... ..........54 Results.....................................................................................................................................56 Uniform CA Naming System..........................................................................................56 Detection of CA Activity in the Alimentary Canal of An. gambiae ...............................57 Cloning of Full-Length AgCAs from the Alimentary Canal of An. gambiae .................57 AgCA-RP2...............................................................................................................58 AgCA6.....................................................................................................................58 AgCA9.....................................................................................................................58 AgCAb.....................................................................................................................59 Detection of RNA Expression in Alimentary Canal Regions.........................................59 Verification of AgCA9 Antibody Specificity..................................................................60 Detection of AgCA9 Protein in An. gambiae Larvae......................................................61 Ectoperitrophic fluid................................................................................................61 Transitional region...................................................................................................62 Malpighian tubules...................................................................................................62 Rectum.....................................................................................................................63 Phylogenetic Analysis.....................................................................................................63 Discussion...............................................................................................................................64 CA Activity in An. gambiae Larvae................................................................................64 AgCA6, AgCA9, and AgCAb mRNA Localization.......................................................65 AgCA9 Protein Distribution............................................................................................66 Ectoperitrophic fluid................................................................................................66 Transitional region...................................................................................................68 Malpighian tubules...................................................................................................69 DAR cells.................................................................................................................70 Phylogeny.................................................................................................................72 4 SILENCING OF AGCA9 IN AN AN. GAMBIAE LARVAL CELL LINE, AG55...............90 6

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Introduction................................................................................................................... ..........90 Results.....................................................................................................................................91 AgAGO2 in Ag55............................................................................................................91 CA Isoforms in Ag55 Cells.............................................................................................92 AgCA9 mRNA Silencing................................................................................................92 AgCA9 Protein Down-Regulation..................................................................................93 Discussion...............................................................................................................................94 5 CHARACTERIZATION OF THE DAR AND NON-DAR CELL OF ANOPHELINE RECTA: PROTEIN LOCALIZATION................................................................................100 Introduction................................................................................................................... ........100 Results...................................................................................................................................102 Anopheline Rectal Structure..........................................................................................103 Ae. aegypti : Obligate Freshwater Culicine....................................................................104 An. gambiae : Obligate Freshwater Anopheline.............................................................104 Oc. taeniorhynchus: Saline-Tolerant Culicine..............................................................105 An. albimanus : Saline-Tolerant Anopheline.................................................................106 CA Isoforms in the Anopheline Rectum.......................................................................107 Discussion.............................................................................................................................108 Freshwater-Reared Larvae: V-ATPase and Na+/K+-ATPase Localization...................109 Saline Water-Reared Larvae: V-ATPase and Na+/K+-ATPase Localization................111 CA9 is Involved in CO2 Excretion................................................................................114 6 CHARACTERIZATION OF THE DAR AND NON-DAR CELLS OF ANOPHELINE RECTA: PHYSIOLO GICAL FUNCTIONS........................................................................126 Introduction................................................................................................................... ........126 Results...................................................................................................................................128 H+ Flux in Rectum.........................................................................................................128 Pharmacology................................................................................................................128 Discussion.............................................................................................................................129 H+ Fluxes are Likely Due to Many Transport Systems.................................................130 Pharmacology................................................................................................................131 Concanamycin A: V-ATPase inhibitor..................................................................131 Methazolamide: carbonic anhydrase inhibitor.......................................................133 DIDS: anion exchange inhibitor.............................................................................134 Putative Model for Ion Transpor t in the Anopheline Rectum.......................................135 7 CONCLUSIONS AND FUTURE DIRECTIONS...............................................................141 pH Regulation in the Larval Alimentary Canal....................................................................141 Ion Regulation in the Larval Rectum....................................................................................143 Final Thoughts......................................................................................................................144 APPENDIX 7

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CLONING AND CHARACTERIZATION OF MEMBERS OF THE SLC-4 BICARBONATE TRANSPORTER FAMILY FROM ANOPHELES GAMBIAE SENSU STRICTO LARVAE...............................................................................................146 Introduction................................................................................................................... ........146 Results...................................................................................................................................149 Phylogenetic Analysis...................................................................................................149 Cloning and Characterization of Full-Length AgAE3 cDNA from An. gambiae Larvae........................................................................................................................149 AgAE mRNA Expression in Alimentary Canal Regions..............................................150 Discussion.............................................................................................................................151 LIST OF REFERENCES.............................................................................................................161 BIOGRAPHICAL SKETCH.......................................................................................................172 8

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LIST OF TABLES Table page 2-1 Sequences of primers used in the generation of cDNA collections and RACE reactions.............................................................................................................................49 2-2 Sequences of forward and reverse primer s used to amplify CA and AE full-length gene products.....................................................................................................................50 2-3 Specifics of the primers used to amp lify full-length and partial AgAGO2 and CA sequences including primer sequences, anne aling temperatures, PCR cycles run, and expected length of products for each primer set................................................................51 2-4 Specifics of the primers used to gene rate AgCA9 or GFP dsDNA flanked by the T7 primer sequence on either the 5 or 3 extreme.................................................................52 2-5 Sequences of forward and reverse primer s used to detect CA and AE mRNA in qPCR reactions.....................................................................................................................53 3-1 An. gambiae carbonic anhydrase (CA) new naming convention.......................................74 9

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LIST OF FIGURES Figure page 1-1 Mosquito life cycle........................................................................................................ ....24 1-2 Structure of the larval alimentary canal.............................................................................25 3-1 Hanssons histochemical detection of CA activity in newly cut frozen sections of fourth-instar An. gambiae larvae........................................................................................75 3-2 Alignment of cloned An. gambiae CAs AgCA-RP2, AgCA6, and AgCA9 with H. sapiens CAII (HCAII) and D. melanogastor CAI (DrosCAI)..........................................76 3-3 Alignment of the predicted An. gambiae CA (AgCAb) with CAs from C. elegans and E. coli.............................................................................................................77 3-4 mRNA expression of cloned Ag CAs in the alimentary canal of An. gambiae larvae as determined by qPCR......................................................................................................78 3-5 mRNA expression of AgCA9 in An. gambiae larvae as determined by in situ hybridization......................................................................................................................79 3-6 Verification of the specificity of antibody 5563 in longitudinal sections of An. gambiae larvae...................................................................................................................80 3-7 Western blot used to determ ine AgCA9 protein localization in An. gambiae larval alimentary canal regions....................................................................................................82 3-8 Immunohistochemistry to detect AgCA9 and Na+K+-ATPase protein in longitudinal sections of whole An. gambiae larvae................................................................................83 3-9 Immunohistochemistry of AgCA9 in An. gambiae Malpighian tubule sections...............85 3-10 AgCA9 and Na+K+-ATPase distribution in An. gambiae recta.........................................86 3-11 Phylogenetic analysis of members of the -CA family in H. sapiens D. melanogaster Ae. aegypti and An. gambiae genomes......................................................87 3-12 Simplified model for the role of CA in larval alimentary canal pH regulation.................89 4-1 RT-PCR analysis of the expression of An. gambiae argonaut AgAGO2 in Ag55 cells and each of the eleven An. gambiae CAs and one CA in Ag55 cells and whole An. gambiae larvae.............................................................................................................97 4-2 Analyses of AgCA9 mRNA expression in unt reated Ag55 cells or those treated with AgCA9 or GFP dsRNA.....................................................................................................98 10

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4-3 Detection of AgCA9 protei n in untreated Ag55 cells or those treated with AgCA9 or GFP dsRNA.......................................................................................................................99 5-1 Na+K+-ATPase and V-ATPase protein distributio n in recta of various anopheline and culicine species reared in fresh and saline water.............................................................118 5-2 CA and Na+K+-ATPase protein distribution in recta of various anopheline and culicine species reared in fresh water..............................................................................120 5-3 Change in CA and Na+K+-ATPase protein distribution in recta of An. albimanus given acute fresh or sa line water challenges....................................................................121 5-4 RT-PCR analysis of the expression of each of the eleven An. gambiae CAs and the one CA in An. gambiae rectal tissue......................................................................123 5-5 Comparison of culicine and anopheline la rval rectal struct ure using confocal microscopy of whole mount immuno histochemical preparations...................................124 5-6 Comparison of the major protein localization patterns found in the recta of freshwater-reared culicines and anophelines to those in saline water-reared culicines and anophelines................................................................................................................125 6-1 Measure of H+ flux at the basal membrane of the DAR and non-DAR cells of An. albimanus larvae using SIET...........................................................................................138 6-2 Pharmacological inhibition of H+ flux at the basal membrane of 2% and 50% ASWreared An. albimanus recta...............................................................................................139 6-3 Putative model for ion trans port in the anopheline rectum of larvae reared in fresh or saline water......................................................................................................................140 A1 Function of H. sapiens SLC4 proteins.............................................................................154 A-2 Phylogenetic analysis of members of the SLC4 family in H. sapiens D. melanogaster Ae. aegypti and An. gambiae genomes....................................................155 A-3 Hydrophobicity plot of AgAE3.......................................................................................156 A-4 Protein alignment between An. gambiae AgAE3 and AgAE1, H. sapiens SLC4A1, and splice variants of H. sapiens SLC4A5, H. sapiens SLC4A8, and D. melanogaster NDae1..............................................................................................................................157 A-4 Continued.................................................................................................................. .......158 A-5 Protein alignment between An. gambiae AgAE1, AgAE2, and AgAE3.........................159 A-6 AgAE mRNA expression in the regions of An. gambiae alimentary canal ad determined by qPCR........................................................................................................160 11

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LIST OF ABBREVIATIONS AE Anion exchanger AMG Anterior midgut AP Alkaline phosphatase AR Anterior rectum ASW Artificial seawater CA Carbonic anhydrase CA-RP Carbonic anhydras e-related protein DAR Dorsal anterior rectal DEPC Diethylpyrocarbonate DIDS 4,4'-diisothiocyanostilbene-2,2'disulfonic acid ds Double-stranded FBS Fetal bovine serum FITC Fluorescein isothiocyanate GC Gastric caeca GPI Glycosylphosphatidylinositol HG Hindgut mg milligram ML multiple label MR4 Malaria Research and Reference Reagent Resource Center MT Malpighian tubule NAT Nutrient amino acid transporter NaP Sodium phosphate NBC Sodium/bicarbonate cotransporter NGS Normal goat serum 12

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NHE Sodium hydrogen exchanger Pen-strep Penicillin-streptomycin PM Peritrophic matrix PMG Posterior midgut PR Posterior rectum Pre-Inc Pre-incubation medium QPCR Quantitative PCR RACE Rapid amplification of cDNA ends RNAi RNA interference ROI Region of interest RT reverse transcription SIET Self-referencing ion-selective electrode technique siRNA Small interfering RNA SLC Solute carrier ss Single-stranded TBS Tris-buffered saline TEAHCL Triethanolamide hydrochloride TR Transitional region TRITC Tetramethyl Rhodamine Iso-Thiocyanate USDA United States Department of Agriculture 13

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF PH AND ION REGULATORY PROTEINS IN LARVAL MOSQUITOES By Kristin Elizabeth Smith May 2009 Chair: Paul Linser Major: Medical SciencesMolecular Cell Biology Mosquitoes have long been a major cause of human morbidity and mortality, and as a result, worldwide efforts are underway to control these disease vectors. Evidence suggests that targeting the larval stages of the mosquito lifecycle in addition to the adult stage may facilitate vector control. In order to sp ecifically and safely target mosq uito larvae using control methods, a thorough understanding of the biol ogical systems crucial for larval survival is necessary. Here I characterize proteins that influence larvaes ability to tightly contro l the pH within their alimentary canal, as well as proteins involved in their ability to adapt to varying environmental conditions. Uncovering the methods of pH and i on regulation employed by larvae in both of these processes could lead to the discovery of novel targets and new and improved larvicides. First, I report the cloning a nd characterization of novel carbon ic anhydrase transcripts from the larval Anopheles gambiae alimentary canal. Mosquito la rvae generate a highly alkaline pH in a restricted area of the alimentary canal, a nd carbonic anhydrase is a crucial enzyme for the generation and maintenance of this pH. Characterization of mRNA expression and protein distribution in the case of one CA, Ag CA9, reveal an alternate pathway for bicarbonate/carbonate transport in to the lumen of the alimenta ry canal, updating the current model for larval pH regulation. Additionally, RNA interference work in an An. gambiae larval 14

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cell line demonstrates that AgCA9 is a protein capable of being manipulated by RNAi and suggests that this technique can be used to silence a CA in live mosquito larvae. Finally, antibody immunolocaliza tion of AgCA9 reveals a nove l subset of cells in the rectum of anopheline larvae. I ch aracterize the cells of this r ectum using immunohistochemistry, physiology, and pharmacology and use this data to develop a putative model of ion regulation in the anopheline rectum. This model supports my current hypothesis that anopheline larvae shift protein distribution to change the primary function of the rect um from absorption, in larvae reared in fresh water, to secre tion, in larvae reared in saline water. Additionally, this work emphasizes the differences in rectal structur e and protein regulation between anophelines and culicines, and suggests that these two subfamilies may be employ very different adaptive and regulatory strategies. 15

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CHAPTER 1 INTRODUCTION Significance Mosquitoes (Diptera: Culicidae) are hosts to a number of pathogens including parasites, bacteria, viruses, and fungi. The Culicidae incl udes two subfamilies which comprise relevant disease vectors: the A nophelinae (including the Anopheles genus), and the Culicinae (including Aedes, Culex and Ochlerotatus genera). Members of both subfamilies spread pathogens through the infectious bite of the adult female and were responsible for more human death and disease in the 17th through 20th centuries than all other causes comb ined (Gubler, 1991). Malaria is the worst of all vector-borne diseas es and one of the top three ki llers among communicable diseases, infecting 500 million people worldwide, and killi ng at least two million people each year (The World Health Report, 1996; Sachs and Malane y, 2002). For these reasons, mosquitoes have been named the greatest menace of all diseasetransmitting insects (The World Health Report, 1996), and worldwide efforts to control mosquito populations have been either implemented or proposed. Mosquito control. Most mosquito control methods ta rget the adult stage; however, evidence suggests that targeting la rval stages in concert may va stly improve the efficacy of control strategies (Killeen et al., 2002a; Killeen et al., 2002b). Mosqu ito larvae develop from eggs laid in standing water or on sites that will be flooded, and hatch within a few days to a week. The juvenile mosquito pa sses through four aquatic larval instars, distinguishable by size, before pupating and emerging as a winged adult (C lements, 1992) (Figure 1-1). At any given time, the vast majority of mosquitoes on Earth are in the larval stage, an important phase in the insects life cycle. Development during this period is critical for the emergence of healthy adults capable of reproducing and spreading disease. It is also during the larval stages that mosquitoes 16

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are perhaps the most susceptible to control. Unlike adults that can avoid intervention by changing their location (i.e. flying away), larvae are st rictly aquatic and confined to the body of water in which they were hatched (Killeen et al., 2002a). For these reasons, the larval stages of the mosquito lifecycle are ideal targets for cont rol methods. Indeed, evidence supports the idea that targeting the larval stages in a rigorous, well managed manner may faci litate vector control (Killeen et al., 2002a; Killeen et al., 2002b). Currently, effective larvacides are available; however, the increasing occurrence of vector resistance to insecticides necessitates a constant search for new control methods. An ideal larvacide targets a protein or pathway that is both essential for larval mosquito viability and unique to mosquitoes so as not to affect other wildlife. To find su ch putative targets, a detailed understanding of larval biology is n eeded. To date, very little rese arch is dedicated to the biology and physiology of larval mosquitoes, or to the biol ogical processes necessary for larval survival. Two particularly important processes for larval survival are the generation and maintenance of a luminal pH gradient, and the regulation of hemolymph osmolarity in unstable ionic environments. pH Regulation in the Larval Alimentary Canal One of the most intriguing aspects of mosquito biology is the ability of larvae to generate and maintain a luminal pH in excess of 10.5 in a specific region of the alimentary canal (Dadd, 1975; Zhuang et al., 1999). This is one of the highest pHs known to exist in a biological system; the use of this high pH to initiate digestion sh arply contrasts most othe r organisms which initiate digestion in conditions that are more acidic, and can reach a pH equivalent to battery acid (~ pH 1.0) in certain situations. Becau se of the relative uniqueness of this mosquito characteristic, proteins responsible for the regulation of this pH may serve as targets for specific larvacides. 17

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The larval alimentary canal is an epithelial t ube one cell thick and composed of seven main regions listed here in order from anterior to posterior: the foregut, ga stric caeca (GC), anterior midgut (AMG), posterior midgut (PMG), Malpighi an tubules (MT), ileum and rectum (Figure 12). The pH of the alimentary canal lumen ranges from pH 6.5 in the rect um (Clark et al., 2007) to 7.5 in the GC and PMG to 10.5 in the AMG (Dadd, 1975) (see Figure 1-2). The highly alkaline pH is restricted to the AMG and exists in the abse nce of any morphological barriers between it and the near neutral pH values of the GC and PMG lumina. The alkaline AMG may have a role in the dissociati on of tannin-protein complexes that are found in the phytophagous diet of insect larvae (Berenbaum, 1980). Whereas larvae maintain a luminal pH of up to 10.5, they regulate th eir hemolymph to a constant pH of approximately 7.5 (Clark et al., 2004). Mosqu itoes have an open circulatory system, common to arthropods, which allows the hemolymph to circulate freely throughout the insect and bathe the alimentary canal epithelial cells directly on their basal si de. It is still largely a mystery how the cells of the AMG can be expose d to a nearly neutral pH in the hemolymph on their basal side while maintaining a pH as high as 10.5 on their apical side. The alimentary canal cells must exhibit a tightly regulated system of ion balance in order to function in such an extreme environment. Structure of the Larval Alimentary Canal Each alimentary canal region has a structure and gene expression pattern specialized for certain aspects of larval digestion as well as for biological processe s including pH and ion regulation (Neira et al., 2008). Th e GC are eight blind expansions of the alimentary canal and are composed of four cell types: (1) resorbing/ secreting cells, (2) ion-transporting cells, (3) caecal membrane-secreting cells, an d (4) imaginal cells. These cell types can be found in distinct regions of the GC or intermixed, depending on the species (Volkmann and Peters, 1989). 18

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Additionally, the cells of the GC secrete a fine granular substance, referred to as the caecal membrane, which separates the GC lumen from th e rest of the alimentary canal (Clements, 1992). Recent evidence suggests a major role for the GC in prot ein metabolism (Neira et al., 2008). The AMG cells possess short apical microvilli with abundant mitochondria in the apical cytoplasm and an extensive basal labyrinth which extends throughout most of the cell (Clements, 1992). The presence of small microvilli suggest s that this region is not involved in major transport processes, and in support of this th e AMG appears to express very few transcripts associated with transport compared to the other regions of the alimentary canal (Neira et al., 2008; K. E. Smith, unpublished data). However, mi croarray-based evidence indicates a role for this region in lipid metabolism and absorption (Neira et al., 2008). Additionally, the AMG lumen is highly alkaline (pH>10), suggesting an involvement of the cells in the generation and maintenance of this pH, and will be discussed in detail in the next section. The AMG is separated from the food bolus by a multilayered matrix com posed mostly of chitin and carbohydrate. This peritrophic matrix extends from the foregut through the rectum, thus creating the endoperitrophic space, or the area within the peritrophic matrix, and the ectoperitrophic space, or the area between the peritrophic matrix and the epithe lial cells (see Figure 1-2; Clements, 1992). The AMG is separated from the PMG by a brief transitional region. Although little information is currently availabl e regarding functions of this re gion, the transitional cells exhibit a distinct response to the Bacillus thuringiensis isrealensis CryVB toxin (Clark et al., 1999), suggesting that this region is di stinct from either the AMG or PMG. The PMG cells possess long apical microvilli and abundant mitochondria in th e apical cytoplasm with a less developed basal 19

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labyrinth than the AMG (Clements, 1992). This region plays a major ro le in digestion and absorption of both protein and carbo hydrates (Neira et al., 2008). Five MTs open into the alimentary canal a nd comprise two cell types, primary cells and stellate cells. The MTs functi on similar to human kidneys by pr oducing a primary urine. This urine is then passed posteriorly through the ileum, a muscular tube composed mostly of squamous cells, and into the rectum. The rectum modifies the primary urine, having a major role in ion regulation. This role is supported by the st ructure of the rectal cells, which possess tightly packed apical lamellae and an extensive basa l labyrinth (Meredith and Phillips, 1973). The various cells of the alimentary canal work together to maintain larval health in a constantly changing environment. Presently, a de tailed understanding of the mechanisms driving digestion and pH regulation in th e regions of the alimentary cana l is lacking; however, evidence supports roles for members of the carbonic anhydr ase family (CAs) (Corena et al., 2002; Seron et al., 2004; Strange and Phillip s, 1984; T. J. Seron, personal communication), and for the membrane-bound Cl-/HCO3 anion exchanger family (AEs; Strange and Phillips, 1984; Strange et al., 1984; Boudko et al., 2001a). Current Model of Alimentary Canal Alkalization The current model for larval alimentary canal alkalization was modified from that of Manduca sexta the tobacco hornworm. Like mosquito larvae, caterpillars generate a highly alkaline pH in their alimentary can al, in some cases exceeding pH 12. M. sexta generates this pH by the action of an H+ V-ATPase, which hyperpolarizes th e membranes of specialized goblet cells (Harvey et al., 1983), thereby driving the transport of luminal H+ for cellular K+ (Wieczorek et al., 1991; Azuma et al., 1995). This system is sufficient to support a pH of 11 with CO3 2(carbonate) as the counterion (Azuma et al., 1995). 20

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Similarly, larval mosquitoes energize ion tran sport in the alimentary canal by the action of an H+ V-ATPase (Filippova et al., 1998; Zhuang et al., 1999; Boudko et al., 2001b), which uses ATP to generate a proton gradient across a memb rane. In the GC and PMG, V-ATPase localizes to the apical membrane of the epithelial cells (Zhuang et al., 1999), hyperpolarizing the membrane and providing a supply of H+ ions to the lumen. The alkaline pH in the AMG of mosquito larvae is expected to be buffered, like M. sexta by CO3 2-. Indeed, CA in the cytoplasm of the epithelial cells (Corena et al., 2002) catalyzes the c onversion of carbon dioxide (CO2) to bicarbonate (HCO3 -) and a proton (H+). Plasma membrane anion exchangers on the apical membrane may facilitate the electroneutral transport of HCO3into the lumen in exchange for Cl-. HCO3 has a pKa of 6.3 and is sufficient to buffer the near neutral pH of the GC and AMG. By contrast, in the AMG, the V-ATPase localiz es to the basal membrane (Zhuang et al., 1999), and generates a net flux of protons out of the lumen (Boudko et al., 2001b). This may result in luminal HCO3losing an H+, thereby forming CO3 2which has a pKa exceeding 10.0. When paired with a strong cation such as K+ or Na+, CO3 2could buffer the highly alkaline AMG lumen. Ion Regulation in Mosquito Larvae A second biological process which is crucial for larval survival, but which has been largely overlooked in many species, is ion regulation. Larval mosquitoes are aquatic organisms that live in habitats of varying salinities ranging from rain pools and streams to salt marshes and hypersaline lakes. The majority of mosquito species live in fresh wa ter, having variable tolerance for low levels of salinity, whereas appr oximately 5% live in ei ther brackish or saline water (OMeara, 1976; Clements, 1992). A major ch allenge faced by all mosquito larvae is the tendency for these habitats to fluctuate widely in salinity due to such occurrences as rainfall or evaporation (Clements, 1992). For most larvae to survive in wate rs which are at times greatly 21

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hyperor hypo-osmotic to their hemolymph, they must possess highly developed systems for regulating the absorption a nd excretion of ions. Larval rectum: A key organ responsible for ion regula tion in larval mosquitoes is the rectum. The rectum modifies urine produced by the MT prior to excretion based on the ionic needs of the larva, reso rbing essential nutrients and excreting excess salts and waste products. Because larvae depend on ion regulation for survival, the recta of several culicine genera have been studied in detail (Ramsay, 1950; Asakura, 1970; Meredith and Phillips, 1973; Bradley and Phillips, 1975, Bradley and Phillips, 1977). Howeve r, little data has been published describing the recta of anophelines. Because anopheline mosquitoes account for 100% of the worlds human malaria vectors and are vectors for many other deadly diseases it is critical to understand the processes necessary for their survival (including ion regulation and adaptation). This knowledge can foster the development of novel techniques to reduce th e population of these vectors both specifically and safely. Specific Aims I predict that proteins intimate ly involved in both pHand ionregulation could be targeted in the production of specific and efficient larvacides. Based on th is prediction, the goals of this project were to (1) expand the current physiologi cal model of alimentary canal alkalization in mosquito larvae, and (2) devel op a better understanding of ion re gulation in anopheline larvae. The objectives for each of these goals are outlined in the following specific aims: 1. Clone, characterize, and localize th e expression of members of the Cl-/HCO3 transporter gene family in the larval mosquito alim entary canal (addressed in Appendix A) 2. Clone, characterize, and localize the expr ession of members of the carbonic anhydrase gene family in larval mosquito alim entary canal (addressed in Chapter 3) 3. Determine the specific role of members of the carbonic anhydrase family on larval alkalization using RNA interfer ence (addressed in Chapter 4) 22

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4. Characterize the rectal cell types in the larval anopheline mosquito (addressed in Chapters 5 and 6) 23

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Figure 1-1. Mosquito life cycle. Mosquitoes hatch from eggs and pass through four aquatic larval instar stages before pupating and emerging as a winged adult. 24

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Figure 1-2. Structure of the larval alimentary canal. The larval alimentary canal consists of the forgut, gastric caeca (GC), anterior midgut (A MG), transitional region (TR), posterior midgut (PMG), Malpighian tubules (MT), il eum and rectum. The rectum is further divided into DAR and non-DAR cells. The alimentary canal is lined with the peritrophic matrix (PM) which separates th e lumen into the ectoperitrophic space (EcS) and endoperitrophic spac e (EnS). The pH within the alimentary canal ranges from approximately 6.5 (yellow) in the rect um to 7.5 (orange) in the GC and PMG to 10.5 (red) in the AMG. 25

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CHAPTER 2 MATERIALS AND METHODS Artificial Sea Water (ASW) 100% artificial sea wa ter (ASW): 420 mmol l-1 NaCl; 9 mmol l-1 KCl; 12 mmol l-1 CaCl2.H2O; 23 mmol l-1 MgCl2.6H2O; 26 mmol l-1 MgSO4.7H2O; and 2 mmol l-1 NaHCO3 in milli-Q water (Millipore, Billerica, MA, USA), pH 8.1, osmolarity: 860 mosmol l-1 as measured using a 5500 vapor pressure osmometer (Wescor, Logan, UT, USA). All dilutions of the 100% ASW stock were made using milli-Q water. Experimental Insects Anopheles albimanus (STECLA), Anopheles gambiae (SS G3), Anopheles farauti (FAR1), and Anopheles stephensi (STE2) were hatched from eggs supplied by MR4 (The Malaria Research and Reference Reagents Resource Center) at the Centers for Disease Control and Prevention in Atlanta, GA, USA ( http://www.malaria.atcc.org ) and maintained as described in the supplier manual ( www2.ncid.cdc.gov/vector/vector.html ). Ochlerotatus taeniorhynchus, Aedes aegypti, Aedes albopictus and Anopheles quadrimaculatus were hatched from eggs supplied by the USDA (United States Department of Agriculture) in Gainesville, FL. An. aquasalis were hatched from eggs and reared to fourth-instar in 10% artificial ASW by Dr. Luciano Moreira at The Oswaldo Cruz Institute in Rio de Janeiro, Brazil. Unless otherwise stated, all larvae were reared in identical fresh water conditions (milli-Q water) at a density of approximately 100 larvae per 200 ml water. Additionally, certain species were hatched and reared in dilutions of ASW: An. albimanus (50% ASW), An. gambiae (10%, 20%, 30%, and 40% ASW, and acclimated to 60% ASW from milli-Q water by increasing the salinity by 10% each day beginning day 1 post hatch), Oc. taeniorhynchus (100% ASW), and Ae. 26

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aegypti (40% ASW). Unless otherwise stated, all la rvae were used at the early fourth-instar stage. Anopheline larvae were fed every othe r day with a dusting of ground TetraMin fish flakes (Tetra; Melle, Germany). Culicine larv ae were fed every other day with a mixture of brewer's yeast and liver powde r (2:3) (MP Biomedicals Inc., Solon, OH, USA). I evaluated the fresh-water species Ae. aegypti (n=2 egg batches) and An. gambiae (n=3 egg batches), for larval size and mortality rates wh en reared in fresh water, 30% and 40% ASW. Mortality rates were determined by isolating 100 ne wly hatched first-instar larvae into a separate container and counting the surviving larvae daily. This continued until larvae reached fourthinstar ( An. gambiae ) or until the first pupa was observed ( Ae. aegypti ). Ag55 Cell Line The Ag55 cell line (Pudney et. al., 1979) was obtained from Kimberly Keene (Colorado State University). The cells were grown in 75 cm2 flasks (Fisher Scientific, Pittsburgh, PA, USA) at 28C in Leibovitz's L-15 media (Sigma-A ldrich, St. Louis, MO, USA) with 10% fetal bovine serum (FBS, Atlantic Biologicals, Norc ross, GA, USA) and 1% Penicillin-Streptomycin solution (pen-strep; Sigma-aldrich). The ce ll media were changed every other day. Isolation of RNA To isolate RNA from mosquito tissues or cells, one of two reagents were used: TRI reagent RNA / DNA / Protein isolation reagen t (Molecular Research Ce nter, Inc; Cincinnati, OH, USA) and RNAqueous Kit (Applied Biosystems/Ambion, Austin, TX, USA). For TRI reagent samples, 50-100 mg of tissue, or cells grown on a culture dish <10 cm2, were homogenized in one ml of TRI REAGENT and incubated at room temperature for five minutes to permit complete dissociation of nucle oprotein complexes. The homogenate was then supplemented with 0.2 ml chloroform, vortexed fo r 15 seconds, incubated at room temperature for 15 minutes, and centrifuged 12,000g for 15 minutes at 4C. The aqueous phase contained the 27

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RNA and was transferred to a se parate tube. The DNA and protei n remained in the interface and organic phases and were stored at 4C for subsequent isolation of protein (see next section). The RNA was precipitated by combining with 0.5 ml is opropanol, incubated at room temperature for ten minutes, and centrifuged at 12,000g for eight mi nutes at 4C. The supernatant was removed and the RNA pellet washed with one ml 75% ethanol for five minutes at 4C. The supernatant again was removed and the RNA was air-dried for ten minutes under a fume-hood. RNA was resuspended in 0.5% SDS and incubated for 15 minutes at 55C. For RNAqueous samples, 1-75 mg, or cells grown on a culture dish < 60 cm2 were homogenized in lysis buffer (10 l mg-1 tissue). An equal volume of 64% ethanol was add to the lysate and mixed gently. The lysate/ethanol mi xture was applied to a Filter Cartridge assembled in a Collection Tube and centrifuged at 15,000g fo r one minute. The RNA was washed three times by applying 700 l Wash Solution #1 once, and 500 l Wash Solution #2/3 two successive times, centrifuging at 15,000g for one minute between each wash solution. The RNA was centrifuged an additional minute at 500g to ensu re all wash solution was removed before the RNA was eluted twice with 50 l Elution Solution preheated to 70C. RNA was then precipitated with one-half volum e LiCl Precipitation Solution, incubated for 30 minutes at -20C, and centrifuged for 30 minutes at top spee d. The supernatant was removed and the pellet was washed with cold 70% ethanol in diet hylpyrocarbonate (DEPC) treated water and centrifuged for 30 minutes at top speed. The supe rnatant was removed and the pellet air-dried for ten minutes under a fume-hood. The pellet wa s resuspended in DEPC treated water. All RNA was treated with DNase following ex traction to ensure removal of genomic DNA. DNase I (10 Units/ml RNA; New Engla nd Biolabs, Ipswich, MA, USA) and 10x DNase reaction buffer (1/10 final reaction volume) wa s combined with RNA and the mixture was 28

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incubated at 37C for 30 minutes. To inactivate DNase I, EDTA was added to a final concentration of 5 mmol l-1 and the reaction was incubated at 70C for ten minutes. RNA was stored at -20C until use. Isolation of Protein Protein was isolated from samples using TR I reagent RNA / DNA / Protein isolation reagent (Molecular Research Center, Inc.). Prot ocol was followed for RNA isolation (see above section) for 50-100 mg of tissue, or cells grown on a culture dish <10 cm2, until the step involving separation of aqueous and organic phases. The aque ous phase was removed and the organic phase was saved for isol ation of protein. First the DNA was precipitated by mixing the organic phase with 0.3 mls of 100% ethanol, incu bating at room temperature for three minutes, and pelleting the DNA at 2,000g for five minutes at 4C. The phenol-ethanol supernatant was removed and aliquoted 0.5 mls per tube. Three volumes of acetone were added to each aliquot; the samples were incubated for ten minutes at room temperature and centrifuged at 12,000g for ten minutes at 4C. The following was performe d three consecutive times: the protein pellet was dispersed in 0.5 ml of 0.3 mol l-1 guanidine hydrochloride in 95% ethanol + 2.5 % glycerol (V:V) using a small conical Teflon pestle (F isher K749515-0000) attached to a mechanical stirrer (~30 sec @ 800-1000 RPM). Following disper sal, another 0.5 ml aliquot of the guanidine hydrochloride/ethanol/glycerol wash solution was added and the sample was incubated for ten minutes at room temperature. The protein wa s pelleted again at 8,000g for five minutes. The protein was washed one final time in one ml of ethanol containing 2.5 % glycerol (V:V), incubated at room temperature for ten minutes, and pelleted at 8,000g for five minutes. The alcohol was decanted, and the tube was inverted to dry for 7-10 min at room temperature. The pellet was resuspended in a solution of 1% SDS. 29

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Cloning of AgCAs and AgAEs Generation of cDNA Collections Extracted RNA was precipitated by combining with 8 mol l-1 lithium chloride (1/10 final reaction volume) and 100% ethanol (2.5x final re action volume) and incubating at -20C for 30 minutes, centrifuging at top speed for 30 minutes, removing supernatant, and washing with 70% ethanol in DEPC-treated water. The RNA was then centrifuged for 20 minutes at top speed, the supernatant was removed and RNA was air-dri ed under a fume hood for 20 minutes. The RNA was then resuspended in 6 l DEPC-treated water and run (1.0 l) on a 1% agarose gel to ensure RNA quality. cDNA collections were generate d as described in Matz (2003). See Table 2-1 for all primer names referred to in this section and their corresponding sequences. The first strand synthesis reaction was prepared by combining 5.0 l RNA with 1.0 l TRSa primer (10 mol l-1) and heating to 65C for three minutes. To this was added 2.0 l 5x first-strand buffer (Superscript II RT, Invitrogen, Carlsbad, CA, USA), 1.0 l DTT (0.1 mol l-1), 1.0 l TS-oligo primer (5 mol l-1), 0.5 l dNTPs (10 mmol l-1 each dNTP), and 1.0 l superscript II reverse transcriptase (Invitrogen). This was incubated for one hour at 42C followed by three minutes at 65C and quick-cooled on ice. cDNA was amplified by combining 1.5 l of a 1:5 dilution of first-strand DNA, 3.0 l 10x advantage2 PCR buffer (Clontec h, Mountain View, CA, USA), 1.0 l dNTPs (10 mmol l-1 each dNTP), 1.5 l TRSa primer (2 mol l-1), 1.5 l TS-PCR primer (2 mol l-1), 1 l advantage II polymerase (Clontech), and 20 l DEPC treated water. The mixt ure was cycled 17 times at 94C for 40 seconds, 65C for one minute, and 72C for five minutes. 30

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Cloning of Internal Fragments Primers were designed to an internal regi on of each gene as predicted by Ensembl (www.ensembl.org). These primers were used in a PCR reaction to amplify the message from cDNA collections made to include the entire larval alimentary canal. Once amplified, the PCR product was sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the re action products were analyzed on an ABI Prism 310 Genetic Analyzer. This known sequence was us ed to design gene specific primers for the rapid amplification of cDNA ends (RACE). Rapid Amplification of cDNA Ends (RACE) RACE (Zhang and Frohman, 1997; modified by Ma tz et al., 1999) was used to determine the 3 and 5 ends using cDNA collections whic h were generated as described above. For all primer names listed in this sec tion and corresponding sequences see Table 2-1. First, specific primers (5_1, 3_1, 5_2, 3_2) were designed for each gene of interest. RACE was performed in two stages; three reactions were set up in the firs t stage: 5 RACE, 3 RACE, and a control. To each tube 2 l 10x advantage PCR buffer (Clontech), 0.5 l dNTP mix (10 mmol l-1 of each dNTP), 1.0 l first strand cDNA (diluted 1:5 in water), 0.5 l advantage DNA polymerase (Clontech), and 16 l sterile water were added. To the 5 RACE tube 1.0 l 5_1 (2 mol l-1) and 1.0 l 5_prox (2 mol l-1) were added. To the 3 RACE tube 1.0 l 3_1 (2 mol l-1) and 1.0 l 3_prox (2 mol l-1) were added. To the control tube 1.0 l 5_1 (2 mol l-1) and 1.0 l 3_1 (2 mol l-1) were added. PCR program: 94C for 40 seconds, XC (specific to the primer) for three minutes, 72C for two minutes. This program was run for 22-25 cycles. The second stage of RACE consisted of a nest ed PCR and required four tubes: 3 Nested, 3 control, 5 Nested, 5 control. A master mix was made consisting of 8.0 l 10x advantage buffer (Clontech), 2.0 l dNTP mix (10 mmol l-1 each dNTP), 4.0 l Udist (2 mol l-1), 2 l 31

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advantage DNA polymerase (Clontech), and 60 l sterile water. This master mix was dispensed into two tubes, 38 l each. To one tube was added 2 l of the first stage 3 RACE cDNA diluted 1:50. To the second tube was added 2 l of the first stage 5 RACE diluted 1:50. Each of these two mixtures was divided into two tubes, 19 l each. One of the resul ting mixtures contained the Udist primer alone and served as a negative control. To the other, 1 l of the corresponding RACE primer (2 mol l-1; 3_2 primer to the 3 Nested PCR tube, 5_2 primer to the 5 Nested PCR tube). PCR program: 12-20 cycles of 94C fo r 40 seconds, XC (specific to the primer) for three minutes, and 72C for two minutes. Cloning of Full-Length Gene Product Once the full sequence for each gene was determined, gene specific primers were designed to amplify the full length cDNA product (Table 22). The full length gene was ligated into the pCR4-TOPO vector (Invitrogen) and transformed into Top10 chemically-competent bacterial cells (Invitrogen) for sequence conf irmation as described above. Protein Alignments To generate a protein alignment, sequences were combined in a notebook document in FastA format. The sequences were aligned in Clus talX (Larkin et al., 2007) and visualized using Genedoc (Nicholas et al., 1997). Similarity gr oups were enabled using the default scoring matrix, Blosum 62 matrix. Regions with high si milarity are highlighted in black (100%). Regions of lesser similarity are highlighted in grey (>80%) and light grey (>60%). Calculation of Percent Identity To calculate the percent iden tity between AgAE3 and other AgAEs, a protein alignment was made between AgAE3 and either AgAE1 or AgAE2 as described above. The number of identical residues was then divided by the numbe r of residues in the longest sequence. The resulting fraction was multiplied by 100 to give a final percentage. 32

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Detection of CA Activity CA activity was detected in the alim entary canal of ear ly fourth-instar An. gambiae larvae using Hanssons method (Hansson, 1967) as desc ribed by Brown (Brown, 1980) (Figure 3-1). Briefly, early fourth-instar An. gambiae larvae were fixed by injecti on into the haemocoel of a 2.5% gluteraldehyde solution and immersed in the same solution for 30 minutes at room temperature. Twelve m sections were cut at -20C usi ng a Leica Cryostat CM 3050S (Leica, Bannockburn, IL, USA) and mounted on gelatin-coated slides. Using a slide basket, slides were immersed in incubation solution (1.75 mmol l-1 CoSO4, 11.7 mmol l-1 KH2PO4, 157.0 mmol l-1 NaHCO3, and 53.0 mmol l-1 H2SO4 in milli-Q water) briefly and then removed to permit adequate aeration of sections. This was repeated for approxi mately ten minutes, allowing 20-30 seconds to elapse between immersions. The s lides were then rinsed in milli-Q water and immersed in blackening reagent (0.5% ammonium su lfide) for five minutes. The slides were again rinsed in milli-Q water and mounted in 60% glycerol in PBS. The addition of the carbonic anhydrase inhibitor, methazolamide (10-3 mol l-1), to the incubation solution served as a control. Quantitative PCR (QPCR) Generation of First-Strand cDNA Superscript III Reverse Transcri ptase kit (Invitrogen) was us ed to generate cDNA from RNA using manufacturers instructions. Briefly, the following were combined in a single tube: <5.0 g total RNA, 1 l random hexamers (50 ng l-1), 1.0 l dNTP mix (10 mmol l-1 each dNTP), and DEPC-treated water to 10.0 l. Reactions were incubate d at 65C for five minutes and then quick cooled on ice for one minute. To each reaction were added the following: 2.0 l 10x RT buffer, 4.0 l MgCl2 (25 mmol l-1), 2.0 l DTT (0.1 mol l-1), 1.0 l RNaseOUT (40 U/ l), and SuperScript III RT (200 U/ l). The reactions were incubated at 25C for ten minutes, followed by 50C for 50 minutes, and terminated at 85C for five minutes. To eliminate residual 33

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RNA, 1.0 l of RNase H was added to each reaction and incubated at 37C for 20 minutes. cDNA was stored at 4C until use. QPCR Reaction Primers were designed to both the gene of in terest and an 18s ribosomal RNA endogenous control (specific to the species being examined ) using ABI primer express software (Applied Biosystems). The quantitative PCR (QPCR) reaction included 0.5 l cDNA (1-500 ng), 12.5 l SYBR green dye (Applied Biosystems), forw ard and reverse primers (50-300 nmol l-1 each), and water to a total of 25.0 l to wells of a 96 well plate (Applied Biosystems). Each reaction was run in triplicate. QPCR was performed using Applied Biosystems 7000 Sequence Detection system and data were analyzed using the method described in Pfaffl, (2001). In situ Hybridization Generation of Digoxigenin Labeled Probes Digoxigenin (DIG)-labeled RNA probes (both sense and antisense) were designed to the full length gene of interest us ing the DIG RNA labeling kit (R oche; Nutely, NJ, USA). The DNA template was a pCR4-TOPO vector cont aining the gene of interest. To 1.0 g of DNA were added 2.0 l 10x NTP labeling mixture, 2.0 l 10x transcription buffer, 1.0 l protector RNase Inhibitor, and 2.0 l RNA polymerase T3 or T7. The RNA polymerase was chosen based on desired strand of RNA (sense or antisense). The reaction was mixed gently and incubated for two hours at 37C. To remove template DNA, 2.0 l DNase I was added and the reaction was incubated for 15.0 minutes at 37C. The reaction was stopped by adding 2.0 l 0.2 mo1 l-1 EDTA (pH 8.0). Probes were stored at -20C until use. Larval Preparation Ten early fourth-instar An. gambiae larvae were dissected to expose the alimentary canal, five for sense and five for antisense detection. The heads of the cold-i mmobilized larvae were 34

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pinned down using fine st ainless-steel pins to a Sylgard layer at the bottom of a Petri dish containing 4% paraformaldehyde in PBS. The anal segment and the saddle papillae were removed using ulta-fine scissors and forceps and an incision wa s made longitudinally along the thorax. The carcass, including the fat body, centr al nervous system, trachea, and muscle, was separated from the alimentary canal. RNA was detected according to the in situ hybridization protocol outlined in Meles hkevitch et al (2006). RNA Detection Preparations were fixed for three hours in 4% paraformaldehyde in PBS and washed three times with PTW (1x PBS, 0.1 % Tween 20) for five minutes each. Preparations were incubated in a 3:1 PTW/MetOH followed by 1:1 PTW/Met OH, 1:3 PTW/MetOH and finally 100% MetOH for ten minutes each to permeablize the tissue. The tissue was rehydrated in 1:3 PTW/MetOH, 1:1 PTW/MetOH, 3:1 PTW/MetOH and finally PTW alone for ten minutes each. The tissue was then incubated with 0.3% TritonX100 in PBS for ten minutes, washed with PTW alone for five minutes and incubated in Proteinase K (10 g ml-1) in PTW at room temperature for ten minutes. The tissue was fixed a second time in 4% parafo rmaldehye in PBS at 4C for 20 minutes and washed twice with each of th e following: glycine (2.0 mg ml-1) in PTW, PTW, 0.1 mol l-1 triethanolamine hydrochloride pH 8.0 (TE AHCL), and acetic anhydr ide in TEAHCL (2.5 l ml1) for five minutes each. Finally, the tissue was washed three ti mes with PTW and prehybridized in hybridization buffer [50% formamide, 5.0 mmol l-1 EDTA, 5x SSC, 1x Denhardt solution (0.02% ficoll, 0.02 % polyvinylpirroli don, 0.02 % BSA), 0.1% Tween 20, 0.5 mg ml-1 yeast tRNA (Gibco BRL)] 6-8 hours at 50C. The tissue was hybridized with either sense or antisense probe (1.0 g ml-1) in fresh hybridization buffer at 50C for 12-14 hours. After hybridization, the tissue was incubated for 30 minutes each in: 50% formamide/ 5x SSC/ 1% SDS at 60C, 50% formamide/ 2x SSC/ 1% SDS at 60C, and twice with 0.2x SSC at 35

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55C. The tissue was then washed three times in PBT (1x PBS, 0.1% Triton X100, 2.0 mg ml-1 BSA) for five minutes each, and incubated in 10% normal goat serum (NGS) in PBT at 4C for one hour. To detect the probes, the tissue was incubated in a 1:2000 dilution of alkaline phosphatase-conjugated anti-DIG anti bodies in 1% NGS in PBT at 4 C and rocked for 24 hours. The tissue was washed three times with PBT for 20 minutes each and incubated twice in detection buffer (100 mmol l-1 NaCl, 50 mmol l-1 MgCl2, 0.1% Tween 20, 1 mmol l-1 levamisol, 100 mmol l-1 Tris-Cl, pH 9.5). Finally, the ti ssue was incubated in NBT/BCIP (20 l ml-1) in detection buffer at 4C in complete darkness unt il desired color results. The tissue was then washed with 4% paraformaldehyde in methanol at 4C for one hour, twice with 100% ethanol, and preserved in a 3:1 glycerol: PBS solution. Antibody Production CA9 chicken antibody was generated by Aves Labs, Inc. (Tigard, OR, USA) against the An. gambiae BSA-conjugated peptide: KEPIEVSHEQ LELFREMRC and was affinity-purified using the immunogen peptide (S mith, K. E. et al., 2007). Na+K+-ATPase monoclonal antibodies 5 that had been raised against the subunit of avian Na+K+-ATPase in mice were obtained from the Developmental Studies Hybridoma Bank in the form of hybridoma tissue culture supernatant (Lebov itz et al., 1989). A polyclonal antiserum raised agains t the B subunit of the V-type H+-ATPase of Culex quinquefasciatus (Filippova et al., 1998) was obtained from Professor Sarjeet Gill at the University of California, Riverside. For all experiments, the CA9 and V-ATPase an tibodies were used at a dilution of 1:1000, and the Na+K+-ATPase antibody was used at a dilution of 1:10. 36

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Peptide Blocking Assay To determine specificity, antibody was pre-incubated with peptide pr ior to detection of protein. First, the amount of antibody to be used in milligrams (mg) was calculated based on a 1:1000 dilution. Peptide was reconstituted in milli-Q water to 1.0 mg ml-1 and was added in 20% excess by weight. This was calculated by mu ltiplying mg of antibody by 0.4. The reconstituted peptide was combined with diluted antibody and inc ubated for 45 minutes at 37C prior to use. Immunolocalization Paraffin sectioned preparations: Primary fixation was achieved by injection into the larval haemocoel of a 4% formaldehyde soluti on diluted from ultrapure 16% formaldehyde (PolySciences, Inc., Warrington, PA, USA) with Tris-buffered saline (TBS), and larvae were immersed in 4% formaldehyde overnight at 4C. Larvae were transferred to Carnoys solution (60% ethanol, 30% chloroform, 10% glacial acetic acid) for 90 minutes on ice and washed twice for 30 minutes each with 100% ethanol. Larvae were then cleared with aniline: methylsalicylate (1:1) overnight followed by 100% methylsalicyla te overnight and embe dded in paraffin. Sections were cut six microns thick using a mi crotome and mounted on gelatin-coated slides. Sections were deparaffinized through successive five minute incubations in 100% xylene (2x) and xylene:ethanol (1:1), rehydrated through a series of five minute washes of graded ethanols: 100% (2x), 95% (2x), 80%, 70%, 50%, and finally washed three times in TBS. The slides were then blocked in pre-incubation buffer (pre-inc : TBS, 1% bovine serum albumin, 1% normal goat serum, 0.1% TritonX100) for 1-2 hours at room te mperature and incubated in primary antibodies in pre-inc for one hour at 37C. The slides were washed three times in TBS and incubated with the appropriate secondary anti bodies (Cy5or FITC-conjugated donkey-anti-chicken [to detect CA9], TRITC-conjugated goat anti-mouse [to detect Na+K+-ATPase], or FITC-conjugated goatanti-rabbit [to detect V-ATPase], Jackson Immu noResearch, West Grove, PA USA) at a dilution 37

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of 1:250 in pre-inc for one hour at 37C. The slides were again rinsed three times in TBS and mounted in 60% glycerol in TB S with phenylenediamine (Sigma-Aldrich Corp.) to diminish fluorescence quenching. Whole mount preparations: Larvae were dissected to se parate the gut from the rest of the larva. The dissected tissue was fixed in a 1:1 solution of hemolymph substitute solution (Clark et al., 1999; in mmol l-1: 42.5 NaCl, 3.0 KCl, 0.6 MgSO4, 5.0 CaCl2, 5.0 NaHCO3, 5.0 succinic acid, 5.0 malic acid, 5.0 L-proline, 9.1 Lglutamine, 8.7 L-histidine, 3.3 L-arginine, 10.0 dextrose, 25.0 Hepes, pH was adjusted to 7.0 with NaOH) and 4% formaldehyde overnight at 4C. The tissue was then washed twice with TB S for 30 minutes each at room temperature and incubated in pre-inc for 1-2 hour s at room temperature. The tissue was incubated with primary antibodies in pre-inc at 4C overnight. The fo llowing day the tissue was washed approximately ten times in pre-inc for 30 minutes each at room temperature. Secondary antibodies (FITCconjugated donkey-anti-chicken [t o detect CA9], TRITC-conjugate d goat anti-mouse [to detect Na+K+-ATPase], or FITC-conjugated goat-anti-rabbit [to detect V-ATPase]) were diluted 1:250 in pre-inc and incubated with the appropriate tissu e(s) overnight at 4C. The tissue was rinsed twice with pre-inc and once with TBS at room temperature for 30 minutes each, and then mounted in 60% glycerol in TB S with phenylenediamine (Sigma-Aldrich Corp.) to diminish fluorescence quenching. In all cases, multiple primary or secondary an tibodies were added simultaneously rather than sequentially. All secondary antibodies purch ased were specifically ML grade (multiple label; Jackson ImmunoResearch). This grade is affinity purifi ed and tested for minimal crossspecies immunoreactivity. For each image, an n>10 larval sections were observed. 38

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All images were captured using a Leica LSCM SP2 laser scanning confocal microscope (Leica Microsystems, Bannockburn, IL, USA). Fo r each signal in each preparation, laser intensity and detector sensitivity were set to captu re the full dynamic range of the fluorescence. Phylogenetic Analysis Predicted CA and AE sequences were obtained from the Ensembl database (www.ensembl.org) February 2007 release. Al ignments were created using ClustalX and trimmed and visualized using Genedoc. The phylogeny was prepared using MrBayes with the JTT amino acid substitution model and 1.5 milli on iterations. Trees were visualized using Treeview. All nodes were supported with >.95 posterior probability. RT-PCR cDNA was used as a template for PCR reactions with primers specif ic to the gene of interest (Table 2-3). The PCR mix consis ted of 1.0 l forward primer (10.0 mol l-1, 1.0 l reverse primer (10.0 mol l-1), 1.0 l cDNA template, and 22.0 l PCR SuperMix (Invitrogen). This mixture was heated to 95C for 2.0 minutes, followed by X cycles of 95C for 30 seconds, YC for 30 seconds and 72C for tw o minutes, and finally one cycle at 72C for five minutes for a final extension. The cycles run (X) for each reaction and the annealing temperatures (Y) can be found in Table 2-3. The pr oduct was run on a 1% agarose ge l, the DNA was extracted (Gelextraction kit, Qiagen, Valencia, CA, USA), a nd the product was ligated into the pCR4-TOPO vector (Invitrogen). The product was sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems). The reac tion products were analyzed on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Transcription of RNA Transcription was performed in three step s: creation of double st randed (ds)DNA flanked by the T7 promoter sequence on either the 5 or 3 end, transcription of both sense and antisense 39

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strands into single stranded ( ss)RNA, and finally, annealing of the two single strands into dsRNA. Creation of dsDNA Flanked by T7 Promoter First, primers were designed to amplify either AgCA9 or GFP DNA and to add a T7 promoter sequence at either the 5 or 3 end (Table 2-4). The template used was either AgCA9 cDNA in the pCR4-TOPO vector, or eGFP cDNA in the pGEMTEZ vector (Promega Corporation, Madison, WI). I received the eGFP from Lyric Bartholomay (University of Wisconsin). The reactions were cycled at 95C for five minutes, followed by X cycles of 95C for 30 seconds, YC for 30 seconds, and 72C for two minutes, and finally one more cycle at 72C for five minutes. The cycles run (X) for each reaction and the annealing temperatures (Y) are listed in Table 2-4. Genera tion of a single DNA product was verified by running the reaction on a 1% agarose gel. The product was purified using the PCR purification kit (Qiagen). Transcription of Sense and Antisense Strands into ssRNA Sense and antisense RNA were transcribed using the MEGAscript T7 transcription kit (Applied Biosystems/Ambion). Briefly, the fo llowing reagents were combined: 2.0 l ATP solution, 2.0 l GTP solution, 2.0 l CTP solu tion, 2.0 l UTP solution, 2.0 l 10x reaction buffer, 2.0 l T7 enzyme mix, 1.0 g DNA, and water to 20.0 l. The reaction was incubated for four hours at 37C. To remove template DNA, 1.0 l TURBO DNase (Applied Biosystems/Ambion) was added and the reacti on was incubated 15 minutes more at 37C. ssRNA was purified using MEGACle ar Kit (Applied Biosystems/A mbion), eluted in 100 l, and precipitated with 0.5 mol l-1 Ammonium Acetate. RNA was resuspended in DEPC-treated water. To verify the correct RNA size, 1.0 l of ssRNA was denatured by adding an equal amount of glyoxyl loading dye (Applied Biosystems/Ambi on) and heated to 50C for 30 minutes. The 40

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RNA was then run next to a 0.5-10 kb RNA la dder (Applied Biosystems/Ambion) on a 1% agarose gel made with sodium phosphate (NaP) running buffer. Annealing of ssRNA Each ssRNA strand was then adjusted to e qual concentrations for annealing. Equal amounts of sense and antisense ssRNA were combin ed in a tube and incubated in a water bath heated to 95C for five minutes. The reaction tubes remained in the water bath while it cooled to room temperature overnight. 1.0 l of ssRNA was denatured (as described above) and run next to 1.0 l of native dsRNA on 1% agarose gel in NaP buffer to verify annealing. The dsRNA ran slightly higher than the ssRNA. RNA Interference Ag55 cells were grown (see above section on Ag55 cell growth conditions for more information) to 70% confluency in six well plates (Fisher Scientific) fo r experimental (AgCA9 dsRNA treated) and two control groups (GFP dsRNA treated and untreated). The untreated group did not receive dsRNA, but was subjected to the same conditions as the other two groups. To treat the cells with dsRNA, media was replaced with 800 l L-15 media lacking FBS and 36.0 g dsRNA was added (AgCA9 dsRNA or GFP dsRNA). The plate was rocked at room temperature for 30 minutes and then 800 l L-15 media plus 20% FBS was added. Cells were incubated for 24, 48, 72 or 96 hours after which th ey were washed twice with two mls L-15 media lacking FBS and resuspended in 0.4 mL Tr izol reagent (Molecular Research Center) to extract RNA and protein (see above). Northern Blot To generate a radioactive northern probe, a pCR4-TOPO vector containing full length AgCA9 sequence was linearized by restriction enzy me digest. In a PCR tube the following was combined: 1.0 g linearized plasmid, 2.0 l 10x reaction buffer, 1.0 l dATP (10 mmol l-1), 1.0 41

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l dCTP (2 mmol l-1), 1.0 l dGTP (10 mmol l-1), 5.0 l 32P-labeled dUTP (800 Ci (mmol l-1)-1, 20.0 mCi ml-1; GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA), 2.0 l T7 RNA polymerase and water to 20.0 l. This reaction was incubated at 37C for one hour. The NorthernMax kit protocol (Applied Biosystems /Ambion) was used to run and analyze the Northern blot (Figure 4-2B). The RNA loaded for all treatment samples within a time point was equivalent, but differed betw een time points: 48 hours-10.0 g RNA, 72 hours-6.3 g RNA and 96 hours-8.0 g RNA. RNA extracted from cells at th e 24 hour time point was too little (less than 1.0 g) to be visualized on the Northern blot. Western Blot Protein samples were prepared and run on a NuPage 4-12% Bis-Tris gel, 1.0 mm x 12 well (Invitrogen). The protein was transferred to ni trocellulose in transfer buffer (5% 20x NuPAGE transfer buffer, 0.1% NuPAGE antioxidant and 10% MetOH in milliQ water) at 24 volts for 4 hours. The nitrocellulose was stained with fa st green (40 mls acetic acid, 100mls MetOH, 0.24 grams fast green, 100 mls water) for approximately 15 minutes, destained (5:1:5 MetOH, HOAc, H2O) for 15 minutes and rinsed with deionized wa ter. The individual la nes were separated and blocked with blotto (400 ml s TBS, 10 grams dry milk, 800 l Tween20) for 1 hour at room temperature. The primary antibody against AgCA9 was added at a 1:1000 dilution in blotto and rocked at 37C for 1 hour. The blot was washed three times in TBS at room temperature for five minutes each. The secondary antibody (Alkal ine phosphatase [AP]-conjugated donkey antichicken; Jackson ImmunoResearch) was added at a 1:250 dilution in blotto and incubated at 37C for one hour. The blot was again washed th ree times with TBS at room temperature for five minutes each. The labeled bands were detected using an AP conjuga te substrate kit (BioRad; Hercules, CA, USA) according to manufacturers instructions. 42

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Quantification of Protein The presence of a second protein which cr oss-reacted with the antibody was used as a loading control for each sample; this protein was distinguishable from AgCA9 by its slightly higher molecular weight. All bands were quantified using Image Quant 5.0 (Molecular Dynamics, Sunnyvale CA, USA) and analyzed usi ng Microsoft Excel. Values for each AgCA9 band were normalized by dividing by the value of the corresponding load ing control band. The percent down-regulation of AgCA9 protein was determined at each time point by the following equation: 100*1 B A where A is the normalized AgCA9 band in the AgCA9 dsRNA treated cells lane (lane 1 at each time point) and B is the averaged normalized valu es from both GFP dsRNA cells lane (lane 2 at each time point) and vehicle treated cells lane (lane 3 at each time point). Acute Freshwater/Saline Challenges To determine protein localization in larval rectal cells after acute exposures to fresh or dilute saline water (Figure 5-3), An. albimanus larvae were hatched in either fresh water or 25% ASW and reared individually in one ml of fresh water or 25 % ASW, respectively, in 24 well plates (Fisher Scientific). Larvae were caref ully monitored every 24 hours for molting. Newly molted larvae (within 24 hours) at the second-, th ird-, or fourth-instar stage were transferred from either fresh water to 25% ASW or from 25% ASW to fresh water. After 24, 48, and 72 hours larvae were removed and prepared for i mmunohistochemistry. Fourth-instar larvae could not be observed 72 hours post media transfer due to pupation. A minimal concentration of ASW was used which was shown to elicit Na+/K+-ATPase shift (25% ASW instead of 50% ASW). N>5 larva for each experimental group. Each experiment was performed in triplicate. 43

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Quantification of Na+/K+-ATPase Signal Intensity Because sources of variability exist in the imag ing system as well as in preparation of the samples, I was unable to quant itatively compare rectal Na+/K+-ATPase signal intensity between different samples. I was, however, able to co mpare signal between rectal cell types (dorsal anterior rectal [DAR] an d non-DAR, or anterior rectum [AR] and posterior rectum [PR]) in the same sample (Figures 5-1D, H, L and 5-3H). I therefore reported the change in protein distribution as a change in the ra tio of peak pixel intensity betw een the two rectal cell types in each sample. Because rectal cell type distribution of CA and VATPase did not change between larvae reared in fresh water and those reared in saline water, I only reported the pixel intensity ratios of Na+/K+-ATPase. To quantitate the immunostaining intensity between DAR and non-DAR cells (or AR and PR), the ROI (region of interest) function of th e Leica Confocal Quantify software (Leica Microsystems) was used to separately define all cel ls of either type in a given tissue section. Once I determined that no pixel intensities were beyond the dynamic range of the 8-bit grey scale, the peak pixel intensity of each ROI was calculated and us ed as the basis of comparison between DAR and non-DAR cells (or AR and PR). Three representative recta were quantified fr om each group (including the recta presented in Figure 5-1). For each rectum, the DAR/non-DAR (or AR/PR) peak Na+/K+-ATPase pixel intensity ratio was determined by dividing the p eak pixel intensity of the DAR cells (or AR) by the peak pixel intensity of the non-DAR cells (or PR). The DAR/non-DAR (AR/PR) ratios of the three recta from each group were then av eraged. Finally, the averaged DAR/non-DAR (AR/PR) ratios of the fresh water-reared larvae were plotted next to those of the saline waterreared larvae in each species us ing Graphpad Prism 3.0 graphing so ftware (La Jolla, CA, USA). Standard deviation between the three recta fr om each group was indicated. For each graph, a 44

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value of indicates that the peak pixel inte nsity was equal in the tw o cell types. A value greater than indicates greater peak pixel in tensity in the DAR cells (or AR) whereas a value less than indicates greater peak pixel intens ity in the non-DAR cells (or PR). Statistical significance was determined in Micr osoft Excel using a paired t-te st and reported as two-tailed P-values. Asterisks indicate a significant diffe rence between the DAR/non-DAR (AR/PR) ratios of larvae reared in fresh water a nd the ratios of those reared in saline water. P-Value > 0.05; ** P-Value > 0.005. Physiology Preparation of samples: Late third-instar An. albimanus larvae were anesthetized on ice for 30 minutes prior to dissection in artificial hemolymph solution (modified from Clark et al., 1999; in mmol l-1: 42.5 NaCl, 3.0 KCl, 0.6 MgSO4, 5.0 CaCl2, 5.0 NaHCO3, 5.0 succinic acid, 5.0 malic acid, 5.0 L-proline, 9.1 L-glutamine, 8. 7 L-histidine, 3.3 L-arginine, 10.0 dextrose, 5.0 HEPES in milli-Q water, pH 7.1; os molarity adjusted to 300 mosmol l-1 with D-mannitol). Larvae were pinned to a sylgard coated petr i dish through the head and the exoskeleton surrounding the ileum, Malpighian tubules and r ectum were removed. Exoskeleton surrounding the anterior extreme of the larva was left intact. Malpighian tubules we re either removed or pinned away from the rectum. The rectum wa s immobilized by adheri ng one side to a polylysine-coated coverslip which had been immersed in a 2.0 mg ml-1 poly-lysine solution in milliQ water for 30 minutes. Measurement of H+ flux at basal membrane of DAR and non-DAR cells: H+ gradients were measured at the basal membrane of An. albimanus recta using the self-referencing, ionselective microelectrodes technique (SIET; Smith et al., 1999; reviewed in Messerli et al., 2006; Smith, P. J. et al., 2007). Microelectrodes were pulled to a tip diameter of approximately 2-3 m from 1.5 mm diameter borosilicate thin-wall glass capillary tubes (TW1506, WPI, Sarasota, FL, 45

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USA) using a Sutter P-97 micropipette puller (Sutter instruments Inc., Novato, CA, USA) and salinized prior to use as described in Smith et al (1999). Microelectrodes were backfilled with 100 mmol l-1 KCl, 10.0 mmol l-1 HEPES, pH 7.4 and tip filled with a 50-200 m column of H+ ionophore B (Fluka, Buchs SG, Switzerland). H+ flux was measured at a distance approximately 5-10 m from the membrane beginning at the rectum-ileum junction and stopping when the end of the rectum was reached. Measurements were taken in 50 m increments at distinct positions along the anterior-posterior axis of the rectum. Reported values for each pos ition reflect an average of recordings taken over five minutes at 1000 samples per second with th e first 30% sampled before and after probe excursion being discarded (Figure 6-1B). H+ flux was considered outwardly directed, or an efflux, if the probe detected higher H+ activity closest to the membrane. By contrast, an inwardly directed H+ flux, or influx, was considered if the probe detected higher H+ activity furthest from the membrane. Seven bi ological replicates were performed. Because the DAR cells are not visibly discernable from non-DAR cells using a dissecting microscope, preparations were post-stained with rhodamine 123 (Molecula r Probes, Invitrogen, Carlsbad, California, USA) following H+ flux measurements and imaged using an Axio Observer Z1 microscope (Carl Zeiss In c., Thornwood, NY, USA) and an AxioCam MRc camera (Carl Zeiss Inc.). The fluorescent staining allowed for the iden tification of the DAR/non-DAR boundary (Figure 6-1A). Data was analyzed in Micros oft Excel and graphs were ge nerated using Graphpad Prism 3.0 graphing software. To determ ine the average DAR and non-DAR H+ fluxes (Figure 6-1C), all flux measurements taken from the DAR cells or non-DAR cells of a single prep were first averaged. Averaged values for all like meas urements were then averaged (all DAR cell 46

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measurements or all non-DAR cell measurements) a nd used as raw data for statistical analysis. Statistical significance was determined for all data in Microsoft Excel using a paired t-test and reported as two-tailed P-values. Pharmacology: To determine the effects of pha rmacological inhibitors on H+ flux, a single point of stable H+ efflux in the DAR cells, or influx in the non-DAR cells, was chosen for each preparation. The flux was considered stable if it did not significantly vary over a ten minute period. Measurements were recorded as describe d above for a period of ten minutes prior to, and ten minutes following addition of a pharmacological inhibitor. Concanamycin A (10-3 mol l-1; A.G. Scientific Inc., San Diego, CA, USA) and DIDS (103 mol l-1; Sigma-Aldrich Corp.) were pr epared in DMSO and added to the preparation at a final concentration of 10-6 mol l-1. Methazolamide (0.1 mol l-1 Sigma-Aldrich Corp.) was prepared in DMSO, diluted with artificial hemolymph to 10-3 mol l-1, and added to the preparation at a final concentration of 10-4 mol l-1. All reported results were an average of n 5 preparations. Results were reported as a ratio of flux after treatment, to flux before treatment with a pharmacological inhibitor (Figure 6-2). Data was analyzed in Micros oft Excel and graphs were ge nerated using Graphpad Prism 3.0 graphing software. For each graph, a value of for DAR cell flux, or -1, for non-DAR cell flux, indicates no change in H+ flux following addition of inhibi tor. A value greater than for DAR cell flux indicates an increase in H+ efflux, whereas a value less than -1 for non-DAR cell flux indicates an increase in H+ influx. A value less than for DAR cell flux indicates a decrease in H+ efflux, whereas a value greater than -1 for non-DAR cell flux indicates a decrease in H+ influx. Statistical significance was determined for all data in Microsoft Excel using a paired t-test and reporte d as two-tailed P-values. 47

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Flux (J) Calculations: Ion fluxes (J) were calculated using the following equation: x C C DJav slope dV av 10* Where D is the H+ diffusion coefficient (9.31*10-5 cm2 s-1), Cav is the background hemolymph [H+] concentration (mol cm-2), dV is the voltage difference between the near and far positions (mV), x is the probe ex cursion distance (2*10-3 cm2), and slope is the experimentally determined slope for each probe. To correct for the buffering capacity of wate r, bicarbonate, and histidine found in the artificial hemolymph, the following equation was used: )...1(*n i Hmeasured Htotalxx JJ The correction factor, xi, is the ratio of H+ bound buffer flux to free H+ flux and is calculated using the following equation for each buffer (B) present: 2][ ** HKa Ka B D D xH B i Where [B] is the buffer concentration, [H+] is the H+ concentration, and Ka is the acid constant of the buffer. 48

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Table 2-1. Sequences of primers used in the generation of cDNA collections and RACE reactions. Primer Sequence TRSa CGCAGTCGGTAC (T)13 TS-PCR AAGCAGTGGTATCAACGCAGAGT TS-oligo AAGCAGTGGTATCAACGCAGAGTACGCrGrGrG AgCA-RP2 3_1 GGTACCAGTTCGAGGAGATCT AgCA-RP2 3_2 GCACGGTCTGGATTATCCTCAATA AgCA-RP2 5_1 AGAAGATCCTTTGTACAGGACTTT AgCA-RP2 5_2 GTAGGGATCGAACAGTAGCTTCT AgCA6 3_1 TTTCACTGGGGCGACAAAAA AgCA6 3_2 GGATCGATCTGACGCGCTTCT AgCA6 5_1 TCGCCGACGGATTTGTACTTT AgCA6 5_2 GGGCCCGGAAGGAGATTGTT AgCA9 3_1 AGCATACGGTGGATGGTGAAT AgCA9 3_2 CGGCAAGGTCATTAACAACTTT AgCA9 5_1 GGAAGCAAACGAGCGATAATAT AgCA9 5_2 CTTCTGGGGTCCGTTCATTT AgCAb 3_1 GTCGTGAACAACATCAAGCATAT AgCAb 3_2 TAAGCCGCTCATCTTCTCGTCCGAAA AgAE3 3_1 GTGCGCTTTCTGTTTCTTCT AgAE3 3_2 ACTACTTCGATTGGATTGCTAGAA AgAE3 5_1 CCGTGTTGAGTCCATCCATAA AgAE3 5_2 GGTGTAGGATTCGTCGTTGAT 5_prox CGACGTGGACTATCCATGAACGCAAAGCAGTGGTATCAACGCAGA GT 3_prox CGACGTGGACTATCCA TGAACGCACGCAGTCGGTAC Udist TCGAGCGGCCGCCCGGGCAGGTC GACGTGGACTATCCATGAACGCA 49

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Table 2-2. Sequences of forward and reverse primers used to amplify CA and AE full-length gene products. CA Forward primer Reverse primer AgCA-RP2 CGAGCCATGTCAGTCAT CTATGGAAGCGAATGGAAC AgCA6 ATGCAGTTGTCGATTGGATC CTAGTACACCCAGTCCCATTTG AgCA9 TTAGTAGCTATCGACTTCACGCAG ATGTCTGTCACTTGGGGATACAC AgCAb TCACGAATAGTATCGCCGTAC ATGGAGCGTATATTGCGAG AeCA4 ATGTCGACCTCTTGGGGATAC TCAGTGTCCTCCGAATTCGC AgAE3 ATGGCTCATTTCCTCGATATTC TTAGGCAGGAATGGGAGCTT 50

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Table 2-3. Specifics of the primers used to amplify full-length and partial AgAGO2 and CA sequences including primer se quences, annealing temperatures, PCR cycles run, and expected length of products for each primer set. CA ENSEMBL ID Forward primer Reverse primer Length (bp) Annealing temp (C) # cyclesAgCA1 AGAP002360 ATGAGTGCGCTGTG TTTG ATCCAGTTGACCG TCTCC 800 57 35 AgCARP2 AGAP000715 CGAGCCATGTCAGT CAT CTATGGAAGCGA ATGGAAC 1032 55 35 AgCA3 AGAP000401 CACTACGTGGGCCA CTGGGAC CTAAAGTTTGGCG CGCCG 750 55 35 AgCA4 AGAP007550 AATAGCTTCAACCA CCAAACC GTGTCAGTGGGT GTCCTTC 913 57 35 AgCARP5 AGAP000688 CCGACAGTCACCCG TCA GGACCACCCTGC ATTAAC 657 63 40 AgCA6 AGAP003289 ATGCAGTTGTCGAT TGGATC CTAGTACACCCA GTCCCATTTG 957 60 35 AgCA7 AGAP002359 AGTTCACTTACCGC ATCTCG TGGAACTGATCG AACAGGTAC 904 57 40 AgCA8 AGAP010844 TCGTATGGTCACTG GCAC GACACTGCACTTC GTCCTC 930 58 35 AgCA9 AGAP010052 ATGTCTGTCACTTG GGGATACAC TTAGTAGCTATCG ACTTCACGCAG 831 60 35 AgCA10 AGAP004895 ATGAAAAGTTTCAC TTTATTGCTCTG TCAGTGGTAACTC AATCTGGATG 960 60 35 AgCA11 AGAP005066 ATGGCATCAAAAAC AACAAAGAAC TTACAGCTTCGAA AGCACAACGG 774 63 40 AgCAb AGAP002992 ATGGAGCGTATATT GCGAG TCACGAATAGTAT CGCCGTAC 768 60 35 AgAGO2 AGAP011537 CGCCCATACCTAAA CGTG GGCACTTGTTGTA CTGGATC 646 57 30 51

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Table 2-4. Specifics of the primers used to generate AgCA9 or GFP dsDNA flanked by the T7 primer sequence on either the 5 or 3 extreme. Gene T7 position Forward primer Reverse primer Annealing temp (C) Cycles run Length (bp)AgCA9 5' TAATACGACTCACTATAGG GATGTCTGTCACTTGGGGA TACAC TTAGTAGCTATCGAC TTCACGCAG 60 30 871 AgCA9 3' ATGTCTGTCACTTGGGGAT ACAC TAATACGACTCACTA TAGGGTTAGTAGCTA TCGACTTCACGCAG 60 30 871 GFP 5' TAATACGACTCACTATAGG GGAGGTGAAGTTCGAGGGC TCCATGCCGAGAGTG ATC 55 30 411 GFP 3' GAGGTGAAGTTCGAGGGC TAATACGACTCACTA TAGGGTCCATGCCGA GAGTGATC 55 30 411 52

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Table 2-5. Sequences of forward and reverse primers used to detect CA and AE mRNA in qPCR reactions. Gene Forward primer Reverse primer 18s GCGACCTCGTCGGTCAAG AGAGTTCCCGGGCACCAT AgCA6 TCC ATTACACAACACCCCATGT CGCGATGTTCCCTTCTCAAC AgCA9 CAACGGTAGGAGTATGGTCGATCT ACGTACCGCGCTTTGCA AgCAb AGCCTGGCCAAGTTTCAAA GACGAGAAGATGAGCGGCTTA AgAE2 CTGCGCGCCTGATTGTC ACACGCGCTCGATTTCG AgAE3 GTGTGTACGTGCGAATGTTCACT GCGTGATTAGGGCGGAAA 53

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CHAPTER 3 CLONING AND CHARACTERIZATION OF CARBONIC ANHYDRASE FROM ANOPHELES GAMBIAE GILES SENSU STRICTO LARVAE Introduction Members of the CA family have been implicat ed in the generation and maintenance of the larval mosquito AMG alkalization (Corena et al., 2002; Seron et al., 2004; T. J. Seron, personal communication). A current view of larval AMG alkalization proposes that CA in the epithelial cells of the alimentary canal catalyzes the formation of HCO3 -. HCO3 is then translocated into the lumen by a HCO3 transporter and deprotonated to CO3 2(pKa2 10.32) (Dow, 1984). Together with a strong cation such as K+ or Na+, CO3 2could serve as the buffer in the highly alkaline AMG lumen. Although ther e are still unanswered questions regarding this model, it is clear that CA is a major player in the regulati on of pH gradients within the larval mosquito alimentary canal (Corena et al., 2002; Seron et al., 2004; Strange and Phillips, 1984; T. J. Seron, personal communication). Multiple CAs have been cloned fr om, and immunolocalized to, the An. gambiae (Seron et al., 2004; Smith, K. E. et al., 2007; T. J. Se ron, personal communication; K. E. Smith, unpublished data) and Ae. aegypti (Corena et al., 2002; Seron et al., 2004; K. E. Smith, unpublished data) alimentary canals and it has been shown that inhibition of CA in mosquito larvae blocks AMG alkalization (Boudko et al., 2001a; Corena et al., 2002). CA catalyzes the reversible reaction: 3 2 2HCOH COOHCA and can generate HCO3 within alimentary canal epithelial cells. Living larvae are thought to actively excrete HCO3 -, resulting in a net alkalization of their surrounding media (Stobbart, 54

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1971). Moreover, addition of the global CA inhib itor, methazolamide, inhibits this alkalization (Corena et al., 2002). These data implicate CA as a key player in AMG alkalization. There are at least five genetically unrelated families of CA isoforms, , and (Alber and Ferry, 1996; Cronk et al., 2001; So et al., 2004; Lane et al ., 2005). Presently, members of the and families are not predicted to be present in any mosquito genome, and therefore they will not be discussed in this manuscript. Both and -CAs are found in invertebrates, while only the -CAs are present in vertebrates. -CAs are zinc metalloenzymes approximately 29 kDa in molecular weight. The active site of these CAs includes three histidines which are essential for coordina ting the zinc ion (Li ljas et al., 1972). -CA activity is specifically inhibited by certa in sulfonamide compounds (Mann and Keilin, 1940) including acetazolamide and methazolamide. The structures of several members of the -CA have been determined (Cronk et al., 2000; Kimber and Pai, 2000; Mitsuhashi et al., 2000). Although this class can hydrate CO2 with comparable efficiency to -CAs, the -CA active site differs in conserved amino acid residues from that of -CAs, and they are much le ss sensitive to the classic sulfonamide CA inhibitors. -CAs are predominant in the leaf tissues of vascular plants, as well as in bacteria and algae (Chegwidden and Carter, 2000); howev er, at least one -CA gene is predicted to be pr esent in both the An. gambiae and Ae. aegypti genomes (AGAP002992 and AAEL000816, respectively) as well as in the Drosophila melanogaster genome (FBgn0037646; www. ensembl.org ). In Homo sapiens, there are at least 14 -CAs which can be placed into five distinct groups based on their subcellular loca lization: (1) cytopl asmic CAs; (2) membrane-bound CAs, which are attached to the membrane via a glycosylphosphatidylinositol (GPI) anchor; (3) mitochondrial CAs; (4) secreted CAs; and (5) transmembrane CAs, which span the membrane once and have an 55

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extracellular N-terminal region and an intracellular C-terminal region (Chegwidden and Carter, 2000; Tashian et al., 2000). Additionally, three of the 14 H. sapiens CAs are CA-related proteins (CA-RPs), which have lost at leas t one essential histidine and are no longer active (Tashian et al., 2000). There are eleven CAs, and one CA predicted to be present in the An. gambiae genome ( www.ensembl.org), including cytoplasmic, membrane-bound, secreted, and transmembrane forms. Here I report the cloning and characterization of a cytosolic-like -CA (GenBank accession # DQ518576), henceforth referred to as AgCA9, from the alimentary canal of An. gambiae mosquito larvae. I also report the cloning of a CA-RP (AgCA-RP2; GenBank accession # FJ612599), a secreted-like CA (AgCA6; GenBank accession # DQ518577) and a CA (AgCAb; GenBank accession # EF065522) from the An. gambiae alimentary canal, as well as the mRNA characterizati on of AgCA6 and AgCAb. I repor t the phylogenetic analysis of members of the CA family from the H. sapiens D. melanogaster, Ae. aegypti and An. gambiae genomes and show that AgCA9 clusters with H. sapiens cytoplasmic CAs. I also suggest a uniform naming system for newly cloned An. gambiae CAs which does not currently exist. Results Uniform CA Naming System Table 3-1 lists the ensembl (www.ensembl.org) protein (ENSANGP) identifiers based on the July 2008 release, as well as the new suggested name convention of the twelve An. gambiae CAs (Smith, K. E. et al., 2007). Each name begins with a species identifier, Ag, followed by a protein abbreviation, CA, or CA-RP in the case of the CA-related proteins (CA-RPs). ENSANGP numbers were listed in ascending numerical order and -CAs were named sequentially beginning with AgCA1. The CA was named AgCAb. Also recorded are the 56

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accession numbers of those CAs which members of the Linser laboratory have fully cloned and sequence confirmed. Detection of CA Activity in the Alimentary Canal of An. gambiae Hanssons histochemical stain was used to detect CA activity in freshly cut frozen sections of early fourth-instar An. gambiae larvae (Figure 3-1). CA activit y was detected in the ventral nerve cord (Figure 3-1B) and in the cells of the GC and AMG (F igure 3-1C), the MTs (Figure 31D, E), and the dorsal anterior r ectal (DAR) region of the rectum (Figure 3-1D, E). CA activity was inhibited by the addition of methazolamide to the incubation solution in all regions except the MTs. Cloning of Full-Length AgCAs from the Alimentary Canal of An. gambiae Internal fragments of AgCA-RP2, AgCA6, AgCA9 and AgCAb were cloned from the An. gambiae larval alimentary canal using primers de signed according to the partial cDNA sequence predicted by ensembl. Using gene-specific sequ ences from these fragments (Table 2-1), rapid amplification of cDNA e nds (RACE) was used to amplify and sequence the 3 and 5 ends. The cDNA sequences spanned both the start and stop codons of each gene and were determined to be full length by the presence of a stop codon in the 5 UTR and the presence of a 3 stop codon followed by a poly-A tail in the 3 UTR. An alignment of the full lengt h protein sequences of the -CAs AgCA-RP2, AgCA6, and AgCA9, along with H. sapiens CAII (HCAII) and D. melanogaster CAI (DrosCAI) demonstrat ed their relative homology (Figure 3-2). The three histidines essential for CA catalytic activity, His-94, His-96, and His-119 (numbering is relative to HCAII), are indicated wi th asterisks. An alignment of the full length protein sequence of the CA, AgCAb, along with CA sequences from Caenorhabditis elegans (GenBank accession# NP_741809.1) and Escherichia coli (GenBank accession# AAC73237) indicated the relative homology of AgCAb to other CA sequences (Figure 3-3). The four amino acid 57

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residues which are conserved in all CA sequences and thought to coordinate the active-site zinc, Cys-42, Asp-44, His-98, and Cys-101 (numbering is relative to E. coli ; Cronk et al., 2001), are indicated with asterisks. AgCA-RP2 The full length AgCA-RP2 transcript was dete rmined to be 1,026 basepairs, corresponding to a protein product of 341 amino acids, with a molecular mass of 39.22 kDa as determined by The Sequence Manipulation Suite (Stothard, 2000). The sequence was submitted to the NCBI database and allocated the accession number FJ612599. The 5 extreme of the transcript encoded two in-frame methionine residues which were separated by only two amino acid residues. I currently do not know which methi onine acts as the star t codon in AgCA-RP2 translation. AgCA-RP2 is a pr edicted CA-RP and lacks two of the essential histidines for CA activity (Figure 3-2). Th e gene for AgCA-RP2 is located on chromosome X ( www.vectorbase.org ). AgCA6 The full length AgCA6 transcript was determin ed to be 957 basepairs, corresponding to a protein product of 318 amino acids, with a molecular mass of 36.02 kDa. The sequence was submitted to the NCBI database and allocated the accession number DQ518577. AgCA6 belongs to the CA family and is located on chromosome 2R ( www.vectorbase.org ). AgCA6 is predicted to be a secreted prot ein by the presence of a 32 amino acid signal peptide at the 5 extreme of the protein (Figure 3-2). AgCA9 The full length AgCA9 transcript was determin ed to be 831 basepairs, corresponding to a protein product of 276 amino acids, with a mo lecular mass of 31.5 kDa. The sequence was submitted to the NCBI database and allocated the accession number DQ518576. AgCA9 58

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belongs to the -CA family and is located on chromosome 3R ( www.vectorbase.org ). The protein is predicted to be cytoplasmically dist ributed based on the lack of signal peptide or transmembrane sequences. AgCAb The full length AgCAb transcript was determin ed to be 768 basepairs, corresponding to a protein product of 255 amino acids, with a molecu lar mass of 29.92 kDa. The gene is located on chromosome 2R ( www.vectorbase.org). The AgCAb sequence wa s submitted to the NCBI database and allocated the accession nu mber EF065522. AgCAb belongs to the CA family and shows poor homology with CAs (K. E. Smith, unpublished data), but shows high homology with other CA proteins (Figure 3-3) Specifically, AgCAb c ontains the four amino acid residues which are thought to be required for CA catalytic activity (Figure 3-3 asterisks). Detection of RNA Expression in Alimentary Canal Regions Quantitative PCR (qPCR) and in situ hybridization were used to detect CA mRNA within the mosquito larval alimentary canal. AgCA6, AgCA9, and AgCA b mRNA expression was evaluated using qPCR from cDNA derived from either the whole larval alimentary canal, GC, AMG, PMG, MT or rectum. The results were normalized to the An. gambiae 18s ribosomal RNA gene and reported relative to whole larval alimen tary canal (Figure 3-4). qPCR detected mRNA expression of all three genes in every re gion tested; however, each CA was expressed at a different level in each region. A specific CA was considered to be expressed significantly in a particular region if it was expresse d at a statistically si gnificant level (P-Value > 0.01) above that of whole larval alimentary canal samples. Ag CA6 and AgCA9 were expressed significantly in the rectum, and AgCAb was expressed significantly in the AMG and rectum. In situ hybridization was performed using full length DIG-labeled RNA probes to detect AgCA9 RNA in early four th-instar whole-mounted An. gambiae larval alimentary canals and 59

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carcasses (Figure 3-5). The most intense AgCA9 specific staining of the alimentary canal was seen throughout the cytoplasm of the cells of the GC, transitional region between the AMG and PMG (TR), and rectum, with weaker staining seen in the cells of the PMG. Currently, there is no method of morphologically discriminating the TR in the dissection of alimentary canal regions without electron microscopy or immunohistochemistry. Therefore, RNA from this region cannot be distinguished using qPCR. Th e most abundant staining of the carcass was seen in the fat body and muscle. No staining was seen in the alimentary canal or carcass using the sense probe. Verification of AgCA9 Antibody Specificity Two antibodies were generated by Aves Labs, Inc (Tigard, OR, USA) for use in Western blotting and immunohistochemistry. The first antibody (from hen 5340), wa s generated against the BSA-conjugated peptide: CZELGNRQLRE VDSY. The second antibody (from hen 5563) was generated against the BSA-conjugated pe ptide: KEPIEVSHEQLELF REMRC and affinity purified. Figure 3-2 illustrates the antigenic sites on the AgCA9 protein against which these antibodies were generated. Both antibodies sp ecifically recognized AgCA9 on a western blot (e.g. Figure 3-6A); however, the antibody from hen 5340 recognized a second protein as well (see Figure 4-3A). Only result s that were consiste nt between the two an tibodies were reported here. Western blot results for antibody 5563 are shown in Figure 3-6A. The blot was performed using protein extracted from whole An. gambiae fourth-instar larvae. The antibody detected a single specific band at 31.5 kDa representing AgCA9. The identity of the band was then verified by blocking the antibody with reconstituted peptide. When blocked, the antibody failed to detect any bands, further confirming that the anti body recognized AgCA9 in a specific manner. Specificity of protein localization seen in longitudinal sections (as discussed in the next section) was also verified using a preadsorption blocking assay. When the antibody was blocked with reconstituted peptide, it failed to detect AgCA 9 protein (Figure 3-6B-D ; compare 3-8A with 360

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6B and C, and 3-6D with 3-6E). Apparent e xpression in the exoskelet on is nonspecific (Figure 3-6B and C). Detection of AgCA9 Protein in An. gambiae Larvae Once the specificity of the affinity purified antibody 5563 was verified a Western blot was used to determine the presence of AgCA9 protei n in the individual regions of the alimentary canal including GC, AMG, PMG, MT, and rect um (Figure 3-7). Ag CA9 protein was most abundant in the GC and rectum, followed by the MT. AgCA9 detection in the AMG and PMG was relatively weak. Both whole mount larval preparations and l ongitudinal sections of whole larvae embedded in paraffin were used to detect AgCA9 protei n with antibodies 5563 and 5340. Specific protein was detected in the GC, ectoperitrophic fluid and in the cells of the TR, MT, and rectum as will be discussed in more detail below (Figur e 3-8A) (Smith, K. E. et al., 2007). Ectoperitrophic fluid While detection of AgCA9 protein on a Western blot indicated significan t protein levels in the GC, immunohistochemical results within the cells of the GC were variable. In most preparations AgCA9 was detected in the proteinace ous matrix that occupied the lumen of the GC and lined the alimentary canal (from the GC through the PMG) w ithin the ectoperi trophic space but was not detected in the f ood bolus (Figure 3-8A). AgCA9 protein is indeed within the ectoperitrophic space and not in the peritrophi c matrix (PM). The proteinaceous matrix expressing AgCA9 was m easured to be 10-27 m thick in both longitudinal and cross-sections of whole larvae embedded in paraffi n (e.g. Figure 3-8A), making it 10-30 times thicker than the PM in Ae. aegypti (Clements, 1992). This proteinaceous matrix is clearly non -cellular and most likely comprises the ectope ritrophic fluid, which is known to ha ve an important role in insect digestion (Terra and Ferreira, 1981; Terra et al., 1979). Both vitally and as a result of fixation, 61

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there are areas along the alimentary canal where th e PM lies very close to the epithelial cells. This results in a very thin ectoperitrophic spac e where the ectoperitrophic fluid is immediately adjacent to the apical membrane of the epithel ial cells. Based on nume rous immunolocalization experiments I do not believe that AgCA9 protein is located on the apical membrane of any alimentary canal epithelial cells, but is c onfined to the ectoperitrophic fluid. Transitional region The cells of the TR clearly expres s AgCA9 mRNA as demonstrated by in situ hybridization; however, protein detection was variable. In many preparations AgCA9 protein was observed in association with the periphery of the nucleus (Figure 3-8B). Another protein, Na+K+-ATPase, was found by others to have regionally specific membrane distribution in Ae. aegypti with a switch in polarity from the apical membrane in the AMG to the basal membrane in the PMG (Patrick et al., 2006). This polarity switch is also evident in An. gambiae and can be used to identify the AMG and PMG regions. The beginning of the TR is marked by the switch in localization of Na+K+-ATPase to the basal membrane (Figure 3-8C, arrowhead). The AgCA9 protein was restricted to the nuclei of the cells of the TR and did not ex tend into the AMG. No biochemical marker for the end of the TR and beginning of the PMG is known at this time. Counterstaining with the nuclear stain DRAQ-5 confirmed that CA protein was confined to the periphery of the nucleus, but was absent from the center region of the stru cture (Figure 3-8D). Malpighian tubules AgCA9 protein was detected in a punctate pa ttern within the principal cells of the MT, with stellate cells appearing devoid of protein (Figure 3-9). Th e protein appeared to associate with cytoplasmic inclusions throughout the MT a nd did not localize to either the membrane or nucleus. 62

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Rectum A patch of epithelial cells c overing roughly one quarter of th e rectum and positioned on the dorsal side of the apical extreme of this alimen tary canal region showed very intense and specific AgCA9 staining. These cells will henceforth be re ferred to as dorsal anterior rectal cells, or DAR cells, whereas the cells of the remainder of the rectum will be referred to as non-DAR cells. This localization pattern was recognized first by the anti body 5340 in whole mount larval alimentary canal preparations (Figure 3-10A), and specificity to AgCA 9 was verified using affinity purified antibody 5563 in paraffin sections (Figure 3-10B). To comp are and contrast this staining pattern a monoclonal antibody to Na+K+-ATPase was used, which was found by others working in parallel to produce a negative staining pattern in this region relative to the AgCA9 in whole mount larval preparations (Rheault et al., 2007). Indeed, this basal membrane protein produced a reversed image of the AgCA9 pattern (Figure 3-10C), localizing solely to the nonDAR cells. Localization in paraffin sections of the rectum showed AgCA9 protein to be clearly cytoplasmic whereas the Na+K+-ATPase localized to the extensive basal infoldings as described by Patrick et al., (2006). The unusual banding pattern seen in the DAR cells was investigated by counterstaining with TRITC-Phalloidin to reveal circumferential muscles surrounding the rectum (Figure 3-10D). Phylogenetic Analysis A phylogenetic tree comparing predicte d and cloned CA transcripts from H. sapiens D. melanogaster, Ae. aegypti and An. gambiae showed a general segrega tion of human and insect CAs (Figure 3-11). This separati on was not seen in the case of the CA-RPs. Interestingly, AgCA9 was the sole An. gambiae CA that clustered with H. sapiens CAs, along with its D. melanogaster and Ae. aegypti orthologues. Within the H. sapiens CAs, clustering occured between proteins with the same or similar subcellular localizations. There was a distinct 63

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separation between the secreted, membrane-bou nd, and transmembrane CAs. Cytosolic and mitochondrial CAs formed a fourth cluster. Discussion The ensembl database predicts a total of eleven -CA (two of which are CA-RPs), and one CA genes to be present in the An. gambiae genome, six of which have been fully cloned by the members of the Linser laboratory (e.g. Table 3-1) (Linser et al ., 2003; Smith, K. E. et al., 2007; T. J. Seron, personal communicatio n; K. E. Smith, unpublished data). Here I report the cloning of four of these six CAs: AgCA-RP2 a CA-RP, AgCA6, a secreted-like CA, AgCA9, a cytoplasmic -like CA, and AgCAb, a CA from the alimentary canal of the larval malaria mosquito, An. gambiae. Additionally, I report th e mRNA characterizatio n of AgCA6, AgCA9, and AgCAb, and protein characterization of AgCA9. CA Activity in An. gambiae Larvae CA activity was detected by Hanssons histochemical method in the ventral nerve cord and in the cells of the GC, AMG, MT, and rectum. A ll staining except that in the MT was inhibitable by the -CA inhibitor, methazolamide. These results differ from those reported by Corena et al., (2002) who reported CA activity only in the cells of the GC and PMG of Ae. aegypti using a similar method. This discrepancy could be attr ibuted to a difference in preparations; besides using a different mosquito species, Corena et al., (2002) detected CA activity in whole mount preparations compared to frozen sections whic h were used in this study. Hanssons method involves incubating tissue in media containing HCO3 PO4 2 and cobalt at pH 6. As described by Maren (1979), the method depends on the loss of CO2 from the surface of the tissue which displaces the CA reaction equili bria towards the production of OH-. The resulting increase in alkalization near sites of enzyma tic activity produces a cobalt precipitate. The use of whole mount preparations may have prevented proper CO2 loss from within the cells and resulted in 64

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false negative results. Additionally, Corena et al., (2002) did not report the pharmacological inhibition of CA to verify specificity of the stai ning. The CA staining patterns observed in this study support the mRNA localiza tion patterns identified for the three characterized CAs which will be discussed below. Inability to inhibit CA staini ng in the MTs has been previous ly reported (Palatroni et al., 1981) and could be due to several causes. It is possible that a non-specific precipitate is being formed in the concretion bodies of the MT (described in later text), and th at this precipitate is independent of CA activity. Alternatively, residu al staining could be due to an extraordinarily high concentration of the CA en zyme, in which case, a higher concentration of inhibitor may eliminate staining. However, increased inhibito r could result in the formation of an inhibitorcobalt complex rather than in the inhibition of CA directly (Muther, 196 6). Finally, MT CA activity could be mediated by AgCAb, a CA, which would be resistant to methazolamide inhibition. However, qPCR results indicated a lower level of AgCAb in the MT than most other regions of the alimentary canal. AgCA6, AgCA9, and AgCAb mRNA Localization AgCA6, AgCA9, and AgCAb displayed unique expression profiles when regional mRNA levels were compared to those in the whole a limentary canal, suggesting that individual cell types may employ specific CAs over others. The GC, PMG and MT did not significantly express AgCA6, AgCA9 or AgCAb, whereas the AMG significantly ex pressed only AgCAb when compared to whole alimentary canal. The rectum was the single region tested which showed a significant abundance of all three AgCAs above whole alimentary canal levels. This suggests that the rectum is heav ily involved in pH regulation. Indeed, CA in the rectum is implicated in both pHand ion-regulation, as discussed in more detail in Chapter 5, and consequently, multiple isoforms of the enzyme may be required by this region. The CA mRNA 65

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localization patterns were supported by microarray analysis performed using mRNA from the various regions of the An. gambiae alimentary canal (GC, AMG, PMG, hindgut (HG)) (Neira et al., 2008). Separate data were not available fo r MT and rectum, but were combined into one sample labeled HG. The microarray compared expression levels of approximately 14,000 An. gambiae genes (Affymetrix GeneChip Plasmodium / Anopheles Genome Array, Affymetrix Inc; Santa Clara, CA, USA) in each region of the a limentary canal as compared to whole insect. AgCA9 Protein Distribution Antibody probes against AgCA9 were used to determine protein dist ribution within the alimentary canal. AgCA9 was detected in the proteinaceous matrix that occupies the GC lumina as well as the length of the alimentary canal in the ectoperitrophic spac e. Additionally, AgCA9 protein was found in the cells of the TR, MT and in the cytoplasm of th e DAR cells within the rectum. There are obvious discrepancies between the measured AgCA9 mRNA and protein distribution. These can be explained simply by recalling that protei n localization does not necessarily mirror mRNA transcription. For exam ple, MT were found to have very little AgCA9 mRNA; however, I consistently saw protein in MTs of larval paraffin sections. A developmental shift in mRNA and protein production could account for these differences. All experiments were performed using early fourth-instar larvae. It is possible that AgCA9 mR NA is expressed in the MT of earlier larval stages and then wanes as the larvae nears pupation; if AgCA9 has a slow protein turnover rate, this would lead to the presence of prot ein in the absence of mRNA expression. Ectoperitrophic fluid According to the current model of pH regulat ion in the mosquito alimentary canal, CA catalyzes the production of HCO3 in the cells of the alimentary canal which is then translocated 66

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into the lumen by a plasma membrane anion ex changer. Our failure to localize an anion exchanger protein in the alimentary canal led members of the Linser la boratory to seek other possible means for HCO3 transport into the lumen. The detection of AgCA9 protein in the ectoperitrophic space obviated the need for an anion exchanger and offered an alternative mechanism for the production of HCO3 in the lumen (Figure 3-12). Mosquito alimentary canal epithelial cel ls are metabolically active, producing CO2. CO2 can diffuse freely across the cellular membrane a nd into the ectoperitrophic space where AgCA9 can catalyze its conversion to HCO3 -. HCO3 can buffer the lumina of the GC and PMG to a pH around 8.0. In the AMG epithelium, a basally located V-ATPase is positioned to move H+ vectorally out of the cells (Zhuang et al., 1999), re sulting in a transepithel ial potential that is lumen-negative (Clark et al., 1999) This likely results from th e combined activities of the VATPase and a K+/2H+ antiporter (Lepier et al., 1994) that produce a net movement of protons into the hemolymph in the AMG. A K+/2H+ antiporter has yet to be localized to the AMG, therefore this protein has been left out of the put ative model (Figure 3-12). This loss of luminal H+ should result in the deprotonation of HCO3 -, forming CO3 2-. This, combined with a strong cation such as K+ or Na+, would then provide the basis for buffering at a pH near 10.5. In caterpillars, the ectoperitrophic space was found by Gringorten et al., (1993) to be equal in pH to the endoperitrophic space. Thus, the same pH grad ient exists in both compartments of the lumen and HCO3 -/ CO3 2in the ectoperitrophic space could lead to a buffering of the entire lumen. How does a cytoplasmic-like protein, one that is not predicted to have a signal sequence, end up in the extracellular ectoper itrophic fluid? I suggest that AgCA9 protein is produced in the cells of the GC (recall that the GC was found to be enriched in AgCA9 mRNA) and secreted into the ectoperitrophic space by a type of exocytosis. There are several methods of secretion that do 67

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not require a signal sequence and are often utilized to export digestive enzymes in the cells of insect alimentary canals. Merocrine secretion is a classic form of exocytosis, occurring when membrane bound vesicles containing soluble proteins open onto the surface of the cell, allowing the proteins to be secreted in to the lumen (Hung et al., 2000). Alternativel y, apocrine release occurs when a portion of the plasma membrane buds off the cell, containing the proteins ( ibid ). Notably, there was an unexpected lack of accumulation of AgCA9 protein within the food bolus. The peritrophic matrix of An. gambiae larvae is permeable to 148 kDa particles (Edwards and Jacobs-Lorena, 2000) and would be expect ed to allow 31.5 kDa AgCA9 to freely diffuse into the food bolus. However, the native en zyme conformation of AgCA9 is unknown at this time and could be oligomeric. Additionally, the enzyme may be complexed with other enzymes, making it larger, or may associate with the PM and accumulate at this junction without passing through to the food bolus. Transitional region AgCA9 mRNA clearly localized to the TR using in situ hybridization; however, the protein localization was not as cl ear, appearing to localize to the periphery of the nuclei of the cells in this area. The unusual localization of AgCA9 to the nuclei is not unprecedented. There are many manuscripts that have noted CA in the nucleus us ing both immunoand enzymehistochemical methods (Hansson, 1967; Lutjen-Drecoll and Lonnerholm, 1981; Anderson et al., 1982; Brown et al., 1983; Brown and Kumpulainen, 1985; Toyosawa et al., 1996). These papers make no claims as to the validity of these fi ndings and most relate enzyme histochemical detection to the fact that the nuclei act as crystallization cente rs for the reaction products of Hanssons cobalt precipitation method (Ridde rstrle, 1991). I observed AgCA9 nuclear localization using immunohistoche mical analyses which are not based on enzymatic activity and hence nuclear CA remains a physiological mystery. 68

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The TR is described as a unique region in the alimentary canal which was previously thought to be a part of the PMG. This region is described as having a continuous morphological change between the AMG and PMG in contrast to a mixture of cells from each region (Clark et al., 2005). Clark et al., (2005) found the cells of this region to have long microvilli and a high density of mitochondria, suggesting a transport role involving a high rate of ATPase activity in Ae aegypti A distinct band of GPI-linked CA mRNA has previously been described in the anterior end of the PMG as well as an incr eased mRNA expression of an anion exchanger, AgAE1, in this region (Seron et al., 2004; T. J. Seron, personal communication). This localization to the nuclei of the TR identifies a di fferential quality of these cells compared to the rest of the alimentary canal. The beginning of the TR demarks the area at which the highly alkaline pH of the AMG drops to near neutral values within the lu men of the alimentary canal. I predict that these cells have an important role in the de-alkalization of the alimentary canal. At this time, no explanation is evident for the association of AgCA9 with the nuclei of these cells. Malpighian tubules MTs are important components of the mos quito osmoregulatory system, maintaining hemolymph volume and composition (Bradley, 1987b). An. gambiae larvae are equipped with five MT which are composed of two cell types: principal cells and stellate cells. The larger principal cells contained AgCA9 protein associated with membra nes of vesicular cytoplasmic inclusions, while no such protein wa s detected in the smaller stellate cells. Palatroni et al. (1981) localized CA activity to vesicular cytoplasmic inclusions within the MT of Culex pipiens using Hanssons method for histochemical localization. The authors noted that CA activity was only evident at the level of the membranes of these inclusions and was lacking on the cellular membrane, nucleus and other cytoplasmic structures The cytoplasm of prin cipal cells is densely packed with membrane-limited vesicles c ontaining concretion bodies, metallo-organic 69

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aggregates of Ca2+, Mg2+ and K+ that have roles in metal ion storage as well as transepithelial transport. Stellate cells not ably lack these concretion bod ies (Beyenbach, 2003; Clements, 1992). AgCA9 protein detection mirrored the en zymatic pattern shown by Palatroni et al., (1981), and possibly represents a ssociation with concretion bodies. I suggest AgCA9 may play a role in concretion formation and ion transpor t within the MT. In support of this, HCO3 formed by the CA-catalyzed reaction is an esse ntial component of concretion bodies in D. melanogaster and inhibition of CA inhibits concretion formation (Wessing and Zierold, 1997). Additionally, intracellular CA has an impor tant role in providing H+ and HCO3 to transport systems within D. melanogaster MTs (Wessing et al., 1999). DAR cells Meredith and Phillips analyzed the ultrastruc ture of freshwater vs. saltwater mosquito larvae, using Ae. aegypti and Aedes campestris respectively, as representative mosquitoes. They noted that whereas salt water mosquito larvae po ssess recta that are divided into two regions, the recta of freshwater mosquito larvae is uniform in structure and function, similar to the anterior rectum of salt water breeders (Meredith and Ph illips, 1973). This has been supported by light microscopy of two other species of mosquito, Aedes albopictus (Asakura, 1970) and Aedes detritus (Ramsay, 1950) as well as with hemipteran larvae, Hydrometra stagnorum and Halosalada lateralis (Goodchild, 1969) Meredith and Phillips hypothesized that the posterior region of the rectum is unique to saltwater breed ers and generates a hyperosmotic urine as a way of regulating the high salt intake. The anterior po rtion of the rectum is thought to selectively resorb ions, water and metabolites produced by the MTs (Meredith and Phillips, 1973). As primarily a fresh water breeder, An. gambiae larvae would be expected to have a uniform rectum. However, AgCA9 clearly localized solely to a subset of cells in the anteri or region of the rectum, 70

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suggesting distinctive functions of these cells within the rectum. The protein was not detected in the entire anterior rectum, but localized to cells on the dorsal side (DAR cells). The DAR cells also differentially expressed othe r pHand ionregulator y proteins such as Na+/K+-ATPase, V-ATPase (Okech et al 2008; Smith et al., 2008), and an An. gambiae cytoplasmic CA (AgCA11, accessi on # AY280613) (T. J. Seron, pe rsonal communication). This suggests a highly regulated system of pH and i on regulation within the rectum. Clearly the rectum of this particular fres hwater breeder is not uniform as originally thought; however the specific roles of the DAR cells remain to be determined. Most of the research regarding the larval mosquito rectum has been performed on members of the subfamily Culicinae, with no information available on the rect um of Anophelinae larvae, including An. gambiae. The DAR cells are likely unique to the larvae of anophelin e mosquitoes, as further investigated and discussed in Chapter 5. My data suggests that the DAR cells of the An. gambiae rectum may have a role similar to the anterior rectum of salt water culicines. The rectum is an important site of pH and HCO3 regulation in the larvae of Aedes dorsalis a salt-water mosquito capable of inhabiting hypersaline lakes (HCO3 and CO3 2concentrations as high as 1.0 2.4 mol-1 and pH values exceeding 10.0) (Strange et al., 1982). Using mi croperfused recta prepar ations, it was reported that the anterior rectum is a site of CO2 secretion in the form of a Cl--HCO3 exchange. Additionally, the authors pr edicted the involvement of a basolateral 1:1 Cl--HCO3 exchanger and a CA, based on data obtained using inhibito ry compounds (Strange et al., 1984). My data suggests that the DAR cells of An. gambiae may behave in a similar manner. AgCA9 within these cells can catalyze the conversion of CO2 to HCO3 which can then be transported into the lumen by an anion exchanger and excreted. Supporting this hypothesis, it is known that 71

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mosquito larvae alkalize their rearing media by ex creting bicarbonate (Stobbart, 1971) and it has been shown that this alkalizati on can be blocked using global CA inhibitors (Corena et al., 2002). Possible functions of both DAR and non-DAR cells wi ll be discussed further in Chapters 5 and 6. Phylogeny A phylogenetic analysis was performed to ex amine the relationship between CAs in the H. sapiens, D. melanogaster Ae. aegypti and An. gambiae genomes. Alignments were made between known and predicted protein sequences as reported by the Ensembl February 2007 release and a tree was generated. A distinct se paration was seen between insect and human CAs. However, CA-RPs showed no such distinction an d clustered together. This suggests that the CA-RPs are conserved between protostomes and de uterostomes and that active CA amplification took place after their divergence. Although CA-RPs lack CA activity, they are highly conserved proteins, indicating that they serve a critic al cellular function (T ashian et al., 2000). Interestingly, AgCA9 and its Drosophila and Aedes orthologs were the sole insect CAs that clustered with H. sapiens CAs. This association suggests that AgCA9 is closer to the primal protein from which other CAs branched. In fact AgCA9 was closely associated with CAVII, the H. sapiens CA which is the most highly conserved of the active CA isozymes and thought to most closely resemble the ances tral state (Hewett-Emmett and Tashian, 1996; Chegwidden and Carter, 2000). This implies that AgCA9 is a highly conserved protei n and likely to play a role in similarly conserved functionalities. Conclusions: Histochemical detection of CA activity in the larval alimentary canal challenges previous data and, along with the cloning and characterization of several CAs, suggests an alternative model for CA expression and activity in larval mosquitoes. The cloning of AgCA-RP2, and the cloning a nd characterization of secreted -like AgCA6, cytoplasmic-like 72

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AgCA9, and AgCAb, expands the current knowledge of AgCAs distribution in the alimentary canal of An. gambiae. These data suggest that each region of the alimentary canal expresses a different set of AgCAs, and that certain AgCAs may be optimally suited for certain cell types. Additionally, the detection of si gnificant and abundant levels of AgCA6, AgCA9 and AgCAb in the rectum supports curren t literature that reports heavy involvement of the larval rectum in pHand ion-regulation. The protein localization of the cytosolic-like -CA, AgCA9, to the ectoperitrophic fluid suggests that HCO3 production in the lumen is mediated by CO2 diffusion followed by the action of an extracellular CA. If AgCA9 actively catal yzes the production of HCO3 within the alimentary canal lumen, it would suggest that this CA is a key player in AMG alkalization, and therefore in larval survival. The protein was also detected in the cells of the TR, MT and DAR, a previously undescribed region of the rectum. I have shown differential localization of the ionregulatory protein Na+K+-ATPase in addition to AgCA9 in this region. The fact that DAR cells differentially express a number of pHand ionregulatory proteins sugg ests an overlooked and important role for these cells in larval pH regulation in anopheline mosquitoes. Finally, a phylogenetic analysis re vealed AgCA9 to cluster with H. sapiens CAs and in close association with CAVII, the most basal H. sapiens CA, implying a highly conserved, and thus important physiological role. Further studies should be performed to investigate the function of AgCA9, as well as the functions of AgCA-RP2, AgCA6, and AgCAb, specifically focusing on their role in pHand ion-regulation in each region of the alimentary canal. 73

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Table 3-1. An. gambiae carbonic anhydrase (CA) new naming convention. Table 3-1 lists the CA family (column 1), ensembl identifier (AGAP; column 2), and the suggested naming convention (column 3) for the 11 predicted -CAs, including the two predicted CA-related proteins (CA-RPs), and the single -CA encoded in the genome. Also noted are the accession numbers of those CAs which members of the Linser laboratory have fully cloned and sequence confirmed. The ensembl identifiers are based on the July 2008 release. CA family AGAP0 New convention Accession # -CA 02360 AgCA1 -CA-RP 00715 AgCA-RP2 FJ612599 -CA 00401 AgCA3 -CA 07550 AgCA4 -CA-RP 00688 AgCA-RP5 -CA 03289 AgCA6 DQ518577 -CA 02359 AgCA7 -CA 10844 AgCA8 -CA 10052 AgCA9 DQ518576 -CA 04895 AgCA10 AY280612 -CA 05066 AgCA11 AY280613 -CA 02992 AgCAb EF065522 74

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Figure 3-1. Hanssons histochemi cal detection of CA activity in newly cut frozen sections of fourth-instar An. gambiae larvae. A) Whole larval alimentary canal. Activity was detected in B) the ventral nerve cord, C) w ithin the cells of the GC and AMG, and D, E) within the cells of the MT and rectum. F, G) Treatment of incubation media with methazolamide inhibited CA activity in al l regions except for the MT. Abbreviations: AMG, anterior midgut; DAR, dorsal ante rior rectum; GC, gastric ceaca; MT, Malpighian tubules; PMG, posterior midgu t; SG, salivary glands. Scale bars: 250m (A), 125m (B, D, F), 200m (C, E, G). 75

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AgCA-RP2 : -----MSVMHPRIFLNVCFLLALGHEIEASWEEWW T Y DGISGPAFW GLINP QWNMCNKG R RQSPINVEPEKLLFDPYL RS : 75 AgCA6 : MQLSIGSRCCRSVSSLLFALLLIGGRYDGADGHRFGYSKPD -QRRW SKAHQSC----AG AHQSPIA I HSHRAVPLYMPAI : 75 AgCA9 : ------------------------------MSVTWGYTQMNGPQKW PEMFP QA----RG Q RQSPVDIVTSKTQNSGDL QE : 46 HCAII : ------------------------------MSHHWGYGKHNGPEHW HKDFP IA----KG E RQSPVDIDTHTAKYDPSL K: 45 DrosCAI : ------------------------------MSHHWGYTEENGPAHW AKEYP QA----SG H RQSPVDITPSSAKKGSEL NV : 46 AgCA-RP2 : LHI DKHKI SG---TLH N T G QSLVFRV EKDTKQH---VNI SSGPLAY-RY QFEEIYFHYGTDN NQGSEHH I HGYSF PGEIQ : 148 AgCA6 : ELV GYNNL LPGPMTIH N N G HSVSL S I PKTDPTSGKHPYI L GGK L EN-EY E LEGLH F HWGDKN NRG A EHV LNDIRY PLEMH : 154 AgCA9 : NPL RWTYV PENTRSLV N P G YCWKV D V NGKG------SML T GGPLQKEQF I LEQF H C HWGCSD SRGSEHT VDGESF AGELH : 120 HCAII : -PL SVSYDQATSLRI L N N G HAFNV EFDDSQDK----AVL K GGPLDG-TY R L I Q F H F HWGSLD GQGSEHT VDKKKY AAELH : 119 DrosCAI : APL KWKYV PEHTKSLV N P G YCWRV D V NGAD------SEL T GGPLGDQIF K LEQF H C HWGCTD SKGSEHT VDGVSY SGELH : 120 AgCA-RP2 : L YGFNKELY HNMSEAQHKAQGIVGI S L M L Q IGDTPNPEL RII TSAFNKV LYKGSSTPI RH-ISLRSLLP--NTEQY M TYE : 225 AgCA6 : IIHR N -KKYKSVGEALGYSDGLT VLGF F YQV TEQDAPSI NTL VRSFGQI VDYDQTLQL NHTFTLQSLIDGIDLTRFYTYK : 233 AgCA9 : LVHWNQSKYKSFAEAAGLPDGLA VLGVFLK VGKPHP-EL DII ARLLPFI THKGDRVTL NKPLDPARLLP--EGKAYWTYL : 197 HCAII : LVHWN-TKYGDFGKA VQQPDGLA VLGIFLK VGSAKP-GL QKV VDVLDSI KTKGKSADFTN-FDPRGLLP--ESLDYWTYP : 194 DrosCAI : LVHWNTTKYKSFGEAAAAPDGLA VLGVFLKAG NHHA-EL DKV TSLLQFV LHKGDRVTL PQGCDPGQLLP--DVHTYWTYE : 197 AgCA-RP2 : GST T H P G C W E STVWIILNKPIY ITKQELYALR K L MQGSEQTPKAPLGNNARPLQALHHRTVRTNIDFANTGV SKTQSCPT : 305 AgCA6 : GSLTTPPCS E A V T WVVFPDLL E LSVNQMKRFR T L DTGIHGSPMVDNYRALQPIGNRRV---------FVRKVNSRYTGLD : 304 AgCA9 : GSLTTPPCS E S V T WILFKEPIE VSHEQLELFR E M RCYDAAEECPCDETLNKQFD-------------YGKVINNFRPPLE : 264 HCAII : GSLTTPPLLE C V T WIVLKEPIS VSSEQVLKFR K L NFNGEGEPE-------------------------ELMVDNWRPAQP : 249 DrosCAI : GSLTTPPCS E S V I WIVFKTPIE VSDDQLNAMR N L NAYDVKEECPCNEFN-------------------GKVINNFRPPLP : 258 AgCA-RP2 : M YKDMYYKANRWTSEISARGRERGLPDIESVFHSLP : 341 AgCA6 : V FEGRHHSKWDWVY---------------------: 318 AgCA9 : L GNRQLREVDSY-----------------------: 276 HCAII : L KNRQIKASFK------------------------: 260 DrosCAI : L GKRELREIGGH-----------------------: 270 Figure 3-2. Alignment of cloned An. gambiae CAs AgCA-RP2, AgCA6, and AgCA9 with H. sapiens CAII (HCAII) and D. melanogastor CAI (DrosCAI). Regions with high similarity are highlighted in black (100% ). Regions of lesser similarity are highlighted in dark grey (>80% ) and light grey (>60%). The three histidines essential for CA activity (HCAII his-94, his-96, his-119 ) are indicted with asterisks. The signal peptide in AgCA6 is highlighted in red, and the antigenic sites against which the AgCA9 antibodies (5340 and 5563) were ge nerated are identified by blue bars. 76

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* An.gambiae : ---MERILRGVMRYRHTTREQMVQEFRKVRDNPQPKAVFFTCMDSRMIPTRFTETHVGDMFVVRNAGNLVPHAEH---FQ : 74 C.elegans : ---MNKILRGVIQFRNTIRKDLVKQFEEIKNNPSPTAVMFTCMDSRMLPTRFTQSQVGDMFVVRNAGNMIPDAPNYGAFS : 77 E.coli : MKDIDTLISNNALWSKMLVEEDPGFFEKLAQAQKPRFLWIGCSDSRVPAERLTGLEPGELFVHRNVANLVIHTDLN---: 76 An.gambiae : DEYFSCEPAALELGCVVNNIKHIIVCGHSDCKAMNLLYKLKDPEFASLDNRRISPLRAWLCEHANTSLAKFQNLKEIGLD : 154 C.elegans : EVSVNTEPAALELAVKRGGIRHIVVCGHSDCKAINTLYGLHQ---CPKNFDVTSPMDHWVRRNGFASVKRLNERLHRGPS : 154 E.coli : ------CLSVVQYAVDVLEVEHIIICGHYGCGGVQ----------AAVENPELGLINNWLLHIRDIWFKHS--------: 131 An.gambiae : KPLIFSSETPLRKFVAYIDPENNFAIEDKLSQVNTLQQIENVASYGFLKRRLESH-DLHIHALWFDIYTGDIYFFSRNSK : 233 C.elegans : SMKFESEVAPSQSFDAIIDPMDTLAMEDKLSQINVLQQLINICSHEFLKEYLESG-RLHIHGMWFDIYKGEDYLFSKDKK : 233 E.coli : ---------------SLLGEMPQERRLDTLCELNVMEQVYNLGHSTIMQSAWKRGQKVTIHGWAYGIHDGLLRDLDVTAT : 196 An.gambiae : RFIAIDESSIDRLLDEVRRYYS--------------: 255 C.elegans : RFVVIDEKTVTDLLAELNARYPVPEDQDGPVAFAKSN : 270 E.coli : NRETLEQR-YRHGISNLKLKHANHK-----------: 220 Figure 3-3. Alignment of the predicted An. gambiae CA (AgCAb) with CAs from C. elegans and E. coli. Regions with high similarity are highlighted in black (100%). Regions of lesser similarity are highlighted in dark grey (>80%) and light grey (>60%). The four amino acid residues essential for CA activity (E. coli cys-42, asp-44, his-98, and cys-101) are indicted with asterisks. 77

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Figure 3-4. mRNA expression of cloned AgCAs in the alimentary canal of An. gambiae larvae as determined by qPCR. qPCR was used to measure expression of AgCA6, AgCA9 and AgCAb mRNA in alimentary canal regi ons, gastric caeca (GC), anterior midgut (AMG), posterior midgut (PMG), Malpighian tubules (MT) and rectum, relative to whole alimentary canal. All values we re normalized to an 18s RNA endogenous control. Whole alimentary canal sample was normalized to a value of and is indicated by a dashed line. Error bars indicate standard deviation between three biological replicates. **: P-Value < 0.01; ***: P-Value < 0.001. 78

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Figure 3-5. mRNA expression of AgCA9 in An. gambiae larvae as determined by in situ hybridization. In situ hybridization was used to dete ct AgCA9 mRNA in whole larval alimentary canals and carcass. Antisense probe generated intense staining in the A) GC, transitional region between the anteri or and posterior midguts (indicated by arrow), and rectum of the larval alimentary canal with weaker staining seen in the cells of the PMG. C) Carcass showed the most intense staining in the muscle fibers and fat body. Sense probe did not significantl y stain any areas of the B) alimentary canal or D) carcass. For abbreviations see Figure 3-1 legend. Scale bars: 400 m. 79

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Figure 3-6. Verification of the specificity of antibody 5563 in longitu dinal sections of An. gambiae larvae. The specificity of the affi nity purified antibody 5563 was verified using a A) Western blot a nd B-E) immunohistochemistry. Image A shows the results of an antibody adsorption assay. The left pa nel of the image is th e fast green staining of total protein to ensure equal loading. Antibody was pre-incubated with peptide prior to detection of protein. The Western blot (right panel) illustrates the detection of AgCA9 protein with pre-immune IgY (l ane 1), affinity purified antibody 5563 IgY (lane 2), and peptide blocked IgY (lane 3) (A). Images B-E illustrate immunohistochemistry results obtained us ing affinity purified antibody 5563 IgY (E) and peptide blocked IgY (B, C, D) in long itudinal sections of whole larvae embedded in paraffin. The images were generated on a laser confocal microscope (Leica SP2). B and C are images of the same section of larval alimentary canal (without the rectum) with two different fluorochromes (FITC = green = AgCA9; TRITC = red = 80

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Na+K+-ATPase). D and E are images of the rectum positioned such that the anterior end is to the left. Na+K+-ATPase immunostaining was us ed as a counter stain to better visualize the regions of the alimenta ry canal in images C, D and E. The preadsorbed antibody (B, C, D) failed to dete ct AgCA9 protein compared with antibody not treated with peptide (E, also compare with Figure 3-8). A pparent exoskeleton staining is non-specific (B, C; arrows). For abbreviations see Figure 3-1 legend. Scale bars: 150 m (B, C); 75 m (D, E). 81

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Figure 3-7. Western blot used to determine AgCA9 protein localization in An. gambiae larval alimentary canal regions including GC, AMG, PMG, MT, and rectum. The left panel of Figure 3-7 is the fast green staining of to tal protein to ensure equal loading. The right panel shows protein detection usi ng an AgCA9 specific antibody, 5563. AgCA9 protein band is indicated by an arrow. For abbreviations see Figure 3-1 legend. 82

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Figure 3-8. Immunohistochemistry to detect AgCA9 and Na+K+-ATPase protein in longitudinal sections of whole An. gambiae larvae. A) Whole alimentary canal; B, C, D) higher magnification of AMG, TR, and PMG. The images were generated on a laser confocal microscope (Leica SP2). The anteri or end of the larvae is towards the left. B, C, and D are images of the same section with the three different fluorochromes (FITC = green = AgCA9; TRITC = red = Na+K+-ATPase; Cy5 = blue= DRAQ-5 nuclear stain). AgCA9 protein detection was variable in th e epithelial cells of the GC but was consistently seen in the proteinaceous matrix that fills the GC lumen and lines the alimentary canal within the ectoperi trophic space (ectoperitr ophic fluid) (A). Na+K+-ATPase was used as a counter stain to identify the regions of the alimentary canal (A, C, D). Na+K+-ATPase is localized to the apical membrane in the cells of the AMG (C, hollow arrow) and to the basal membrane in the cells of the PMG (C, arrow). In the vicinity of the switch from apical to basal locali zation is the beginning of the TR (A, arrow; C, arrowhead). AgCA9 detection in the transitional region reveals an association with th e periphery of the nucleus (B, D; e.g. arrows). Nuclear 83

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localization was confirmed by counterstaini ng with the nuclear stain DRAQ-5 (D, blue). For abbreviations see Fi gure 3-1 legend. Scale bars: 150 m (A); 172.2 m (B, C, D). 84

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Figure 3-9. Immunohistochemistry of AgCA9 in An. gambiae Malpighian tubule sections. The confocal image demonstrates punctate AgCA9 protein localization within the principal cells of the MT (green). Stellate cells (e.g. arrows) do not appear to contain AgCA9 protein. The nuclei were detected using nuclear stain DRAQ-5 (blue). For abbreviation see Figure 3-1 le gend. Scale bar: 47.62 m. 85

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Figure 3-10. AgCA9 and Na+K+-ATPase distribution in An. gambiae recta. A, C, D) AgCA9 protein distribution in whole mount larvae and B) longitudinal paraffin sections of larvae. The Figure 3-10 shows laser confocal microscope images of An. gambiae larval rectum immuno-stained with chic ken antibodies 5340 (green; A, D) and 5563 (green; B), and mouse monoc lonal antibodies against Na+K+-ATPase (red; B, C). The ileum is indicated at the anterior end of the rectum (A, C, D). AgCA9 protein localized to the DAR cells exclusively (A, B, D arrow). The banding pattern seen in the DAR cells of whole mounts of the rectum is due to the circumferential muscles of the rectum. This is better seen in (D ) which shows muscle stained with TRITCPhalloidin (red). Na+K+-ATPase localized to the non-DAR cells (B, C). DAR cells are indicated with an arrow in each image. Scale bars: 150 m (A, C); 75 m (B, D). 86

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Figure 3-11. Phylogenetic analysis of members of the -CA family in H. sapiens (black), D. melanogaster (red), Ae. aegypti (blue), and An. gambiae (green) genomes. Phylogeny was prepared using MrBayes with the J TT amino acid substitution model and 1.5 million iterations. Interestingly, AgCA9 (starred) and its Drosophila and Aedes 87

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orthologs were the sole insect CA that cl usters with human CAs, indicating that it most closely resembles the primal CA from which other CAs branched. 88

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Figure 3-12. Simplified model for the role of CA in larval alimentary canal pH regulation. Metabolic CO2 can be converted to H+ and HCO3 either intracellularly, for use in ion transport processes, or extracellularly, by diffusing into the lumen. Whereas the gastric caeca and posterior midgut express an apical V-ATPase, translocating protons into the lumen, the anterior midgut e xpresses a basal V-ATPase, vectorally translocating protons out of the lumen. This results in the formation of luminal CO3 2-. HCO3 and CO3 2in the lumen can buffer the highly alkaline anterior midgut and the more neutral gastric caecae and pos terior midguts, respectively. 89

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CHAPTER 4 SILENCING OF AGCA9 IN AN AN. GAMBIAE LARVAL CELL LINE, AG55 Introduction Mosquito larvae can generate a highly alkaline pH (~10.5) in a restricted area of their alimentary canal (the AMG) (Dadd, 1975), de spite the absence of morphological barriers between the AMG and adjacent, more neutral, re gions of the alimentary canal. The alkaline environment is thought to aid in digestion and is crucial for larval surviv al (Corena et al., 2004). However, little is known about the mechanisms responsible for the generation and maintenance of the pH gradient. If well unde rstood, these mechanisms could be exploited for the discovery of novel targets for new and improved larvacides. One group of proteins with a role in AMG alkalization is the CA family (Boudko et al., 2001a; Corena et al., 2002). There are a pr edicted twelve genes belonging to the An. gambiae CA family ( www.ensembl.org February 2007 release), six of which have been cloned by members of the Linser laboratory (see Table 31 for accession numbers). The enzymes catalyze the conversion of H2O and CO2 to H+ and HCO3 and can generate HCO3 within alimentary canal epithelial cells. Larvae are thought to activ ely excrete HCO3 -, resulting in a net alkalization of their surrounding media (Stobbart, 1971). Moreove r, addition of the global CA inhibitor, methazolamide, inhibits this alkalization (C orena et al., 2002). These data implicate CA as a driving force for AMG alkalization. HCO3 generated by CA can be deprotonated to CO3 2which has a pKa in excess of 10.0. T ogether with a strong cation such as a K+ or Na+, CO3 2could serve as a buffer in the AMG lumen. The Linser research group has demonstrated the presence of CA within the epithelial cells of the mosquito larval alimentary canal (in both An. gambiae and Ae. aegypti ) using various 90

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methods. We have detected num erous CA mRNA transcripts using qPCR (Seron et al., 2004; Smith, K. E. et al., 2007; K. E. Smith, unpublished data), microarray analysis (Neira et al., 2008), and in situ hybridization (Corena et al., 2002; Seron et al., 2004; Smith, K. E. et al., 2007). We have shown the presence of CA proteins using immunohistochemistry (S eron et al., 2004; Smith, K. E. et al., 2007), and demonstrated CA protein activity in various mosquito species (Corena et al, 2002; Corena et al, 2004; K. E. Smith, unpublished data). In addition, we established that CA is a necessary enzyme for larval mosquito alimentary canal alkalization and survival (Corena et al, 2004). Although we have shown the CA family to be crucial for AMG alkalization, the specific roles of each member are unknown. A first step in determining if any one CA or combination of CAs is responsible for pH regulation is to silence them either individually or in concert. RNA interference (RNAi) is a powerful tool to manipulate mRNA levels in many organisms. This technique has recently been adapted for use in adult mosquitoes (Blandin et al., 2002) but is not well established for mosquito larvae. Therefore, I seek to first demonstrat e the silencing of a CA in an An. gambiae larval cell line, Ag55, prio r to attempting RNAi in liv e larvae. Here I report the mRNA silencing of an abundant alimentary canal CA, AgCA9 (GenBank accession # DQ518576), in Ag55 cells and the resulting down-regulation of the protein product. Results AgAGO2 in Ag55 One protein crucial for the RNAi pathway is argonaut, and the presence of this gene has been used to indicate active RNAi machinery (Hoa et al., 2003; Keene et al., 2004). To verify that argonaut was expressed in Ag55 cells, I performed PCR using Ag55 cDNA as a template and primers specific to a 646 ba sepair region of AgAGO2, the An. gambiae argonaute 2 gene (Table 2-3). The cDNA product (Figure 4-1A, arrow) showed 100% sequence identity to the 91

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AgAGO2 sequence, indicating that argonaut was present in the Ag55 cell line and that this cell line would be appropriate fo r RNAi experiments. CA Isoforms in Ag55 Cells To determine which CA genes were expressed in the Ag55 cell line, I used primers to the twelve CA sequences (11 CAs and one CA; Table 2-3) in i ndividual PCR reactions. Primers were designed to amplify either the full length mRNA, in the case of those fully cloned and sequenced genes, or a portion of the mRNA predicted by the An. gambiae genome ( www.ensembl.org). I determined the expression of CA ge nes in the Ag55 cell line as well as in whole larvae cDNA (Figure 4-1B). Whole larvae cDNA was used as a control to ensure that the primers detected the CA against which they we re designed. I detect ed all but AgCA1 and AgCA7 in the whole larvae controls using the pr imers indicated. In the Ag55 cells, I found only AgCA3, AgCA9 and AgCAb. AgCA 9 is abundant in the alimentary canal and thought to have a role in pH regulation (Smith, K. E. et al., 2007); therefore I chose to test if this CA could be silenced in an An. gambiae cell line using RNAi. AgCA9 mRNA Silencing Ag55 cells were treated with either full length AgCA9 doubl e-stranded (ds) RNA or GFP dsRNA (negative control). A second negative control group was exposed to the same conditions as the other two groups, but received no dsRNA. RNA was isolated from cells 24, 48, 72, or 96 hours after treatment and reverse transcribed into cDNA for use in qPCR assays to measure the expression of endogenous AgCA9 mRNA in each sample. Primers used to detect AgCA9 were gene specific and designed against the 3 UTR to elimin ate detection of exogenous RNA (dsRNA which was added to each culture). Three bi ological replicates of this experiment were performed and each resulted in a consistent knock-down of AgCA9 mRNA in the AgCA9 dsRNA treated cells compared to the GFP dsRNA or untreated cells. Statistically significant 92

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down regulation (P-Value < 0.05) of AgCA9 mRNA was seen after 24 hours and persisted throughout 96 hours, resulting in a down regulation of 88% after 96 hours (Figure 4-2A). In addition to qPCR, a Northern blot was pe rformed using RNA extracted from untreated cells, or those treated with AgCA9 dsRNA or GF P dsRNA for 48, 72, or 96 hours (Figure 4-2B). An equal amount of RNA was loaded for all trea tment groups within a time point, but differed between the four time points. AgCA9 mRNA wa s detected using a full length radioactive P32 RNA probe. The Northern blot showed a di stinct AgCA9 mRNA band at the approximate expected size (~ 1.5 kb with unt ranslated regions) in the contro l lanes (Figure 4-2B, lanes 2 and 3 at each time point, arrow) which is absent in the lanes corresponding to cells treated with AgCA9 dsRNA (lane 1 at each time point). Furthermore, siRNAs and cleaved endogenous AgCA9 mRNA were visible as a lower molecula r weight smear in the AgCA9 dsRNA treated lanes (Figure 4-2B, arrowheads). AgCA9 Protein Down-Regulation Antibody specificity: The antibody 5340 recognized AgCA9 on a western blot at the predicted molecular weight of 31.5 kDa as well as a second protein of a slightly higher molecular weight (Smith, K. E. et al., 2007). When anti body was incubated with the peptide antigen, neither band was recogni zed (Figure 4-3A). Western blot analysis: A Western blot was used to determine the presence of AgCA9 protein in untreated Ag55 cells or those trea ted with AgCA9 dsRNA or GFP dsRNA for 24, 48, 72, or 96 hours (Figure 4-3B). The presence of a second protein which cross-reacted with the antibody was used as a loading control; this pr otein was distinguishable from AgCA9 by its slightly higher molecular weight. The an tibody 5340 recognized AgCA9 at the expected molecular weight of 31.5 kDa in all control lanes (Figure 4-3B, la nes 2 and 3 at each time point). 93

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However, AgCA9 protein levels in cells treate d with AgCA9 dsRNA were considerably reduced (90% after 96 hours) compared to the control samp les (Figure 4-3B lane 1 at each time point). Discussion I used an An. gambiae larval cell line, Ag55, in which to silence AgCA9, a cytoplasmic CA cloned by members of the Linser laboratory and thought to have a role in pH regulation (Smith, K. E. et al., 2007). CA is an important component of larval alim entary canal alkalization and a crucial enzyme for larval survival. Numerous CA genes have been cloned and characterized to some extent by the Linser labor atory but we have yet to determine the specific functions of each member of the CA family in the alkalization process. The first step in uncovering the roles of each CA is to demonstrate that the mRNA can be stably silenced and that this silencing leads to a robust down-regulation of the corresponding protein. The Ag55 cell line is an An. gambiae neonatal 1st instar larval cell line which others have used for successful RNAi silenc ing (Konet et al., 2007). Additio nally, I verified that AgAGO2, an important gene in the RNAi pa thway, is expressed in this cell line. To determine which CA to silence, I identified those CA genes which were expressed in the Ag55 cell line. Because the cells were derived from first-instar larvae, I did not expect them to express the same complement of CA genes as fourth-instar larvae, the cDNA of which I used as a control. However, I was able to detect three CAs, AgCA3, AgCA9 and AgCAb in Ag55 cDNA. I chose to silence AgCA9; it is a good candidate for having a role in alimentary canal alkalization due to its abundant protein expression in the GC and ectoperitrophic space (S mith, K. E. et al., 2007). Additionally, AgCA9 was abundant in the Ag55 cell line, and therefore likely to have an important role in pH regulation. I was not able to detect two of the twelve CAs, AgCA1 and AgCA7 in the control template (whole fourth-instar la rvae cDNA). Either the predicte d sequences were incorrect and 94

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the primers did not anneal to the mRNA, or the CA s were expressed at a level too low to detect using my methods. The literature suggests that RNAi silencing can occur in as li ttle as 24 hours (Blitzer et al., 2005) and last up to at least ten days (Keene et al., 2004). I followed the expression of AgCA9 mRNA and protein daily from 24 to 96 hours due to the space constraint of the 6 well plates; after 96 hours, the cells began to overgrow and die. At each time point, I measured the relative abundance of AgCA9 mRNA and protein in each sa mple using qPCR and Northern and Western blots. Both qPCR and Northern blot indicated that cells trea ted with AgCA9 dsRNA expressed considerably less endogenous AgCA 9 mRNA than those treated with GFP dsRNA or those that were untreated. Likewise, the Western blot show ed that AgCA9 protein levels diminished in those cells treated with AgCA9 dsRNA. Similar to the cited literature, I found that this response was detectible within 24 hours of treatment and persisted throughout the 96 hours surveyed. These data demonstrate that dsRNA can be us ed to silence AgCA9 mRNA, as well as downregulate the protein prod uct, in the larval An. gambiae cell line, Ag55 cells. When using RNAi to investigate protein function, the half-life of the protein must be considered. If the protein is st able, with a half-life that exceeds the length of the experiment, protein levels will remain unchanged even if mRNA expression is decreased. These results indicate that AgCA9 is a protei n capable of being manipulated by RNAi and suggests that this technique can be used to silence a CA in live mosquito larvae. By down-regulating CA protein in live larvae, pH changes can be assessed and relative contribu tions of each CA to AMG pH regulation can be determined. There are several methods by which RNAi ha s been used to knock down genes in adult mosquitoes: direct injection of dsRNA (Roy et al., 2007; Hansen et al., 2007; Boisson et al., 95

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2006), introduction of a transgene which produces the RNA of interest in a hairpin form (Franz et al., 2006), or infection with a virus that pr oduces the dsRNA of interest (Adelman et al., 2001). To date there are only two reports of su ccessful RNAi in mosquito larvae, one in Ae. aegypti using injection of small interfering RNAs (siRNAs) (Blitzer et al., 2005) and one using transgenic An. stephensi larvae (Brown et al., 2003). My Ag55 cell line studies suggest that AgCA9 silencing in An. gambiae larvae is possible. Further work in live An. gambiae larvae will yield a greater understanding of this important family of gene s in the crucial process of pH regulation. 96

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Figure 4-1. RT-PCR analys is of the expression of An. gambiae argonaut AgAGO2 in Ag55 cells and each of the eleven An. gambiae CAs (AgCA1-11) and one CA (AgCAb) in Ag55 cells and whole An. gambiae larvae. A) Specific primers were used to detect a 646 bp portion of the AgAGO2 gene in Ag55 cells. B) Ag55 cDNA was used in a PCR reaction with primers specific to either the whole CA transcript or the partial predicted sequence. Whole An. gambiae fourth-instar larvae cDNA was used as a primer control (shown by a C above the la ne) in addition to the Ag55 experimental template (indicated by a A above the lane). See Table 2-3 for primer sequences and expected length of products. M: 1 kb DNA ladder (invitrogen). 97

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Figure 4-2. Analyses of AgCA 9 mRNA expression in untreated A g55 cells or those treated with AgCA9 or GFP dsRNA. AgCA9 mRNA was measured at 24, 48, 72, or 96 hours post-treatment using A) qPCR and at 48, 72, or 96 hours post-treatment using B) a northern blot. For the northern blot, a consistent amount of RNA was run for each sample from each time point: 48 hours (10 g), 72 hours (6.3 g), and 96 hours (8.0 g). Lanes 1: cells treated with AgCA9 dsRNA; lanes 2: cells treated with GFP dsRNA; lanes 3: untreated cells. Arrow: AgCA9; arrowheads: degraded endogenous mRNA and siRNAs. *: P-Value < 0.05, **: P-Value < 0.005. 98

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Figure 4-3. Detection of AgCA9 protein in untreated Ag55 cells or those treated with AgCA9 or GFP dsRNA. A) a blocking assay was used to verify antibody 5340 specificity. Equal amounts of whole An. gambiae fourth-instar protein were run in each lane. Lane 1 (pre): pre-immune IgY at a 1:1000 dilution. Lane 2 (IgY): immune IgY at a 1:1000 dilution. Lane 3 (block): immune IgY at a 1:1000 dilution blocked with peptide. AgCA9 is indicated with an arrow. The upper band represents a second protein specifically recognized by the antibody. B) a we stern blot was performed using protein from Ag55 cells which had been treated with either AgCA9 dsRNA (lanes 1), GFP dsRNA (lanes 2), or left untre ated (lanes 3) for 24, 48, 72, or 96 hours. AgCA9 protein was detected using a 1: 1000 dilution of antibody 5340. AgCA9 is indicated with an arrow. 99

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CHAPTER 5 CHARACTERIZATION OF THE DAR AND NON-DAR CELL OF ANOPHELINE RECTA: PROTEIN LOCALIZATION Chapters 3 and 4 have focused on larval pH regulation and the role of the CA family on this important process. I will focus Chapters 5 and 6 on another biological system which is important for larval adaptation and survival, ion regulation. Introduction Larval mosquitoes live in aquatic habitats that have the propensity to change drastically in both salinity and ionic compositi on. To survive in such dynamic environments, larvae must regulate their hemolymph osmolarity (~ 300 mosmol l-1) accordingly. In freshwater, larvae tend to gain water by osmosis and lose salts by diffusion. In saline water, larvae tend to lose water to their environment and gain salts (Clements, 1992). In fresh or dilute water, all larvae hyperregulate their hemolymph osmolarity by resorbing ions and nutrient s to produce a dilute urine. By contrast, larvae in saline water respond in one of three ways, depending on the species: obligate freshwater species are restricted to waters that ar e isoor hypo-osmotic to their hemolymph and cannot survive in higher osmolarities; saline-tolerant osmoregulators regulate their osmotic and ion concentrations by secr eting a hyper-osmotic urine to maintain a hemolymph osmolarity of approximately 350 mosmol l-1 over a species-specific range of external osmolarities (Bradley 1994); and saline-tolerant osmo conformers increase hemolymph osmolarity with increasing external osmo larity (in water exceeding 300 mosmol l-1) by producing organic compounds such as proline an d trehalose and accumulating them as non-toxic osmolytes in the hemolymph (Bradley, 1994). As saline-tolerant osmoconforming larvae use an osmoregulatory strategy which differs from the majority of saline-toler ant species (Bradley, 1987b), they will not be considered in the present study. 100

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A key organ responsible for ion regulation in la rval mosquitoes is the rectum. As larvae depend on ion regulation for survival, the rect a of several culicine genera (including Aedes and Culex) have been studied in detail. Saline-tolerant osmoregulating culicine species have a structurally distinct rectum co mpared to freshwater species (M eredith and Phillips, 1973). The recta of freshwater culicines are structurally unif orm and selectively resorb ions, water and nutrients from the primary urine produced by the MTs. Conversely, the r ecta of saline-tolerant culicines are structurally divided into distinct anterior and post erior regions. The structure and function of the anterior rectum (A R) is similar to the recta of freshwater culicines, whereas the function of the posterior rectum (PR) is to secrete a hyper-osmotic ur ine when larvae inhabit saline water (Meredith a nd Phillips, 1973; Bradley and Phillips 1977). The idea that the larval recta of freshwater and saline-tolerant larvae are distinct in structure a nd function is supported by data from various culicine species (Ramsay, 1950; Asakura, 1970; Bradley and Phillips, 1975, Bradley and Phillips, 1977). To my knowledge previous work on the mosquito larval rectum was restricted to the culicine subfamily, with little data published describing the recta of anophelines. I recently discovered a subset of cells on th e dorsal anterior rectum (DAR cells) of a freshwater anopheline, An. gambiae which were distinct from the rest of the rectum (non-DAR cells) with respect to localization of carboni c anhydrase (AgCA9, Smith, K. E. et al., 2007; Smith et al., 2008), V-ATPase, and Na+K+-ATPase (Okech et al., 2008 ; Smith et al., 2008). I observed similar protein localiza tion patterns in both freshwater and saline tolerant anophelines (Smith et al., 2008). This unique type of two-part rectum was originally detected in the salinetolerant Anopheles salbaii by light microscopy (Bradley, 1987a ; Bradley, 1994). These data led 101

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me to hypothesize that anopheline larvae are distinct from cu licine larvae in re ctal structure and ultimately in methods of ion regulation. V-ATPase and Na+K+-ATPase are well known membrane energizers important for ion regulation (e. g. Wieczorek, 1992; Skou, 1990) and recent work has established the presence of one or both of these proteins in the recta of culicines (Patrick et al., 2006; Smith et al., 2008) and anophelines (Okech et al., 2008; Smith et al., 200 8). Additionally, CA plays a major role in HCO3 secretion in the larval rectum (Strange and Phillips, 1984; Corena et al., 2002) and a specific CA isoform, CA9, localizes to the DAR cells of all anophelines examined to date including An. gambiae, Anopheles albimanus Anopheles farauti Anopheles quadrimaculatus Anopheles aquasalis and Anopheles stephensi (Smith et al., 2008), making it an excellent marker for these cells. In the present study, I compare the distribut ion of three ion-regul atory proteins (CA9, Na+K+-ATPase, and V-ATPase) in the recta of a freshwater culicine Aedes aegypti a freshwater anopheline An. gambiae a saline-tolerant culcine Ochlerotatus taeniorhynchus and a salinetolerant anopheline An. albimanus reared in fresh water and saline water. Additionally, I determine protein distribution in larvae either rear ed in freshwater and exposed to saline water, or reared in saline water and exposed to fres hwater, for 24, 48, and 72 hours to determine the effects of short term exposure to either of these conditions. From these analyses, I conclude that anophelines differ from culicines in larval rectal structure as well as in the regulation of protein expression. Additionally, I suggest putative functions for the DAR and non-DAR cells of anopheline larvae under both freshwater and saline water conditions. Results I compared the distribution of three proteins with known roles in ion regulation in the larval rectum: a specific carbonic anhydrase (CA9, Smith, K. E. et al., 2007), Na+/K+-ATPase, 102

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and V-ATPase. The proteins were localized in longitudinal paraffin sections of recta from freshwater and saline-tolerant anophelines and culicines reared in varying osmotic conditions; these conditions included both fr esh water and specific dilutions of ASW. The dilutions used were based on the published upper tolerance limit of each species (Bailey et al., 1981; Mosha and Subra, 1983; Mullenbach et al., 2004). I compared the freshwater mosquito species Ae aegypti (culicine) and An. gambiae (anopheline), with the saline-tolerant mosquito species Oc taeniorhynchus (culicine) and An albimanus (anopheline). Additionally, I evaluated the change in Na+/K+-ATPase distribution in An. gambiae, Oc. taeniorhynchus and An. albimanus as a ratio of Na+/K+-ATPase labeling intensity between the two rectal cell types in each species (either DAR cells versus non-DAR cells or AR versus PR). The ultrastructure of both freshwater and saline-tolerant culicines is characterized by highly infolded apical lamellae and extensive ba sal infoldings (Meredith and Phillips, 1973). Preliminary electron micrographs indicate that the membranes of the DAR and non-DAR cells of anophelines reared in fresh water are folded in a similar way. For the present study, the cells of all recta examined are therefore assumed to possess apical lamellae and basal infoldings. Anopheline Rectal Structure The immunolocalization patterns of CA9 and Na+/K+-ATPase were used to identify anopheline DAR and non-DAR cells, re spectively, in larvae reared in fresh water. No obvious differences in protein distribu tion were observed between the recta of freshwater anophelines ( An. gambiae, An. stephensi and An. quadrimaculatus) and saline-tolerant anophelines ( An. albimanus An. farauti and An. aquasalis ). In all larvae, CA9 local ized to DAR cells and Na+/K+ATPase localized to non-DAR cells (i.e. Figures 5-1B, 5-1J, 5-2B, 5-2D, and 5-4C) as described for An. gambiae (Smith, K. E. et al., 2007). This sim ilarity suggests that all anopheline larvae, regardless of saline-tolerance, have DAR and non-DAR cells. 103

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Ae. aegypti : Obligate Freshwater Culicine Ae. aegypti developed to fourth-insta r in concentrations of AS W up to a maximum of 40% (~ 350 mosmol l-1). After seven days post-hatch, 53% of larvae survived in 40% ASW, 82% survived in 30% ASW, and 63% survived in fr esh water. Protein localization did not differ between larvae reared in freshwater versus 40% ASW: Na+/K+-ATPase was present on the extensive basal infoldings, whereas V-ATPase localized to the apical lamellae (Figure 5-1A). CA9 was not detectible in the non-segmented rectum of Ae aegypti (Figure 5-2A). An. gambiae : Obligate Freshwater Anopheline An. gambiae developed to fourth-instar in ASW c oncentrations up to 40% but could not survive in higher salinities. After six days pos t-hatch 16% of larvae survived 40% ASW, but were developing much slower than those in lo wer salinities, 87% survived in 30% ASW, and 74% survived in freshwater. However, the larvae could be acclimated from freshwater to higher ASW concentrations by slowly increasing the ASW concentration by 10% ASW each day (up to 60% ASW for a maximum of 24 hours). The distribution of Na+/K+-ATPase, V-ATPase, and CA9 in An. gambiae reared in freshwater has been described previously (Smith K. E. et al., 2007; Rheault et al., 2007; Okech et al., 2008). Na+/K+-ATPase is restricted to the basal in foldings of the non-DAR cells (Figures 5-1B, 5-2B) whereas CA9 protein is restricted to the cytoplasm of the DAR cells (Figure 5-2B). V-ATPase localizes to the apical lamellae of the non-DAR cells, and appears to be cytoplasmic in the DAR cells (Figure 5-1B). The distribution pattern of all three proteins was identical in another obligate freshwater anopheline species, An. stephensi when reared in freshwater (results not shown). Distribution patterns of the thre e proteins did not change in An. gambiae larvae reared in 10% or 20% ASW. However, the distribution of Na+/K+-ATPase and V-ATPase showed subtle 104

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changes in the recta of larvae reared in 30% AS W, or acclimated to 60% ASW, compared with the recta of larvae rear ed in freshwater. Na+/K+-ATPase shifted from being undetectable in the DAR cells (Figure 5-1B) to being detectible on the basal infoldings of both DAR and non-DAR cells (Figure 5-1C). This change can be seen graphically in Figure 5-1D. When reared in freshwater, the DAR cells have significantly less Na+/K+-ATPase peak pixel intensity than the non-DAR cells. When acclimated to 60% ASW, there is no significant Na+/K+-ATPase difference between the DAR and non-DAR cells. In many larvae, this signal appeared reduced compared with that of those reared in freshwater. Additionally, V-ATPase appeared to localize to the cytoplasm of the non-DAR cells in addition to the apical lamellae (Figure 5-1C). Distribution of V-ATPase and CA9 in the DAR cells did not change. Oc. taeniorhynchus : Saline-Tolerant Culicine The rectum of Oc. taeniorhynchus is composed of regionalized anterior (AR) and posterior (PR) segments in contrast to DAR a nd non-DAR cells, and protein localization in these regions did not appear to change drastically between larvae reared in freshwater and those reared in 100% ASW. In both cases Na+/K+-ATPase and CA9 localized to the AR, Na+/K+-ATPase localized to the basal infoldings (Figures 5-1F, G, 5-2C) and CA9 localized to the cytoplasm (52C). The consistency of Na+/K+-ATPase distribution can be seen graphically in Figure 5-1H. There is significantly more Na+/K+-ATPase in the AR than th e PR when reared in both freshwater and 100% ASW, with no significant differences in Na+/K+-ATPase between the two rearing conditions. Conversely, V-ATPase localized mainly to the apical lamellae of the PR, and appeared absent from the AR (Figures 5-1F, G). However, many larvae reared in freshwater, but not 100% ASW, exhibited a low level of V-ATPase on the apical lamellae of the AR (Figure 51E asterisks). In all cases this signal was less intense than that in the PR. 105

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An. albimanus : Saline-Tolerant Anopheline Protein distribution in the recta of An. albimanus was identical to that in An. gambiae when larvae were reared in freshwater: Na+/K+-ATPase appeared to be restri cted to the basal infoldings of the non-DAR cells (Figures 5-1J, J inset, 5-2D); CA9 protein was evident only in the cytoplasm of the DAR cells (Fig 5-2D); V-ATPase localized to the apical infoldings of the nonDAR cells and the cytoplasm of the DAR cells (Figure 5-1J). This apparent cytoplasmic localization is better seen at a higher magnification in Figure 5-1I. The localization pattern of all three proteins was identical in anot her saline-tolerant anopheline species, An. farauti when reared in freshwater (results not shown). The distributions of CA9 and V-ATPase protei ns appeared unchanged in larvae reared in 50% ASW compared with those reared in freshwater, but Na+/K+-ATPase underwent a dramatic shift and appeared to localize mainly to the basa l infoldings of the DAR cells (Figure 5-1K and K inset), presenting a drastic increase of this pr otein in the DAR cells a nd a contrasting reduction in the non-DAR cells (compare Figures 5-1J inset and 5-1K inset) The change in Na+/K+ATPase distribution is shown gr aphically in Figure 5-1L. When reared in freshwater, Na+/K+ATPase peak pixel intensity was significantly gr eater in the non-DAR cells whereas when reared in 50% ASW, it was significantly greater in the DAR cells. To determine if the Na+/K+-ATPase protein shift is a reversib le event, larvae were reared in either freshwater or 25% ASW to second-, third-, or fourth-insta r and were transferred to 25% ASW or freshwater, respectively for 24, 48 or 72 hours. For each image in Figure 5-3, the data are presented graphically as the ratio of Na+/K+-ATPase peak pixel intensity in the DAR cells versus the non-DAR cells (Figure 5-3H). Lower case bar labels in Figure 5-3H correspond to the experimental group indicated by the uppercase letters (i.e. Figure 5-3A corresponds to bar a in Figure 5-3H). When larvae were reared in freshw ater and exposed briefly (for 24 to 48 hours) to 106

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25% ASW, the ability for larvae to shift rectal Na+/K+-ATPase distribution depended on the larval stage at which the exposure occurred. If exposed to ASW during the secondor thirdinstar stages, a change in Na+/K+-ATPase peak signal intensity from the non-DAR cells to the DAR cells was evident within 24 hours (Figure 53A, B, H bars a, b). Fourth-instar larvae exposed only for 24 hours expressed Na+/K+-ATPase in both DAR and non-DAR cells, as if in an intermediate stage (Figure 5-3C, H bar c). However, a change in Na+/K+-ATPase localization from the non-DAR cells to DAR cells was eviden t after 48 hours (Figure 5-3D, H bar d). In most cases, Na+/K+-ATPase shift was most dramatic in larvae exposed during the second larval stage. Most thirdand fourth-instar larvae retained some Na+/K+-ATPase signal in the non-DAR after exposure to 25% ASW. Slightly different results were found for larvae reared in 25% ASW and exposed to freshwater (for 24 to 72 hours). Wh ile second-instar larvae shifted Na+/K+-ATPase localization from DAR to non-DAR cells within 24 hours (Figure 5-3E, H bar e), third-in star (Figure 5-3F, H bar f) and fourth-instar (Figure 5-3G, H bar g) larvae did not fully shift Na+/K+-ATPase localization after 72 hours or 48 hours, respectively, and expr essed the protein in both DAR and non-DAR cells. Although fourth-instar larvae exhi bited very little difference in pixel intensity between the DAR and non-DAR cells after 48 hours, this difference was statistically significant. CA Isoforms in the Anopheline Rectum To determine if CA9 is the sole CA expressed in the rectum, the CA profile of the An. gambiae rectum was determined. Primers to the 12 CA sequences (11 CAs and one CA; Table 2-3) were used in individu al PCR reactions. Primers were designed to amplify either the full length mRNA, in the case of those fully clon ed and sequenced genes, or a portion of the mRNA predicted by the An. gambiae genome ( www.ensembl.org ). The expression of CA genes in An. gambiae rectal tissue was determined as well as expression of those in whole larvae 107

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cDNA (Figure 5-4). Whole larvae cDNA was used as a control to ensu re that the primers detected the CA against which they were de signed. All CAs but AgCA1 and AgCA7 were detected in the whole larvae controls using the pr imers indicated. In the rectal tissue, AgCA3, AgCA6, AgCA9 and AgCAb were detected. Discussion Ion regulation by the rectum helps mosquito la rvae to survive, and adapt to, a constantly changing environment. A wealth of literature ha s focused on the structure and function of the larval culicine rectum and the distinction between its role in freshwater and saline-tolerant species (e. g. Wigglesworth, 1972; Meredith and Phillips, 1973). However, the discovery that the anopheline rectum is struct urally distinct from any culic ine species described (Bradley, 1987a; Bradley, 1994; Smith, K. E. et al., 2007) suggested that anophelines may utilize a unique method of ion regulation. To test this hypothesis, I compared th e localization patterns of three proteins involved in ion regulation (CA9, Na+/K+-ATPase, and V-ATPase) in the recta of anopheline and culicine larv ae (including freshwater larvae and saline-tolerant larvae) reared in fresh versus saline water. Three key points regarding the comparison of anopheline and culicine recta emerge from these data: (1) In contrast to obligate freshwater and salinetolerant culicines which have structurally distinct recta, all anophelines examined (regardle ss of saline-tolerance) possess a similarly structured rectum, consisting of DA R and non-DAR cells (Figur e 5-5). (2) Anopheline larvae undergo a dramatic shift in rectal Na+/K+-ATPase localization when reared in freshwater versus saline water. This shift is not seen in any culicine larvae examined. (3) With the exception of Ae. aegypti the freshwater culicine examined, CA 9 consistently localized to an anterior region of the rectum in both culicine and anopheline larvae regard less of the salinity of the rearing water. The first two key points, along with the localization patterns of Na+/K+108

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ATPase and V-ATPase (discussed below), will be used to suggest putative functions for the anopheline rectal regions. Distribution of CA9 will be discussed in a separate section. Figures 55 and 5-6 summarize my results; Figure 5-5 i llustrates the difference s between anopheline and culicine recta and Figure 5-6 summar izes protein distribut ion in anopheline and culicine recta in a schematic form. FreshwaterReared Larvae: V-ATPase and Na+/K+-ATPase Localization When reared in freshwater, th e protein localization patterns in An. gambiae and An. albimanus recta were identical: non-DAR cells cont ained an apical VATPase and basal Na+/K+ATPase whereas the DAR cells were enriched in cytoplasmic V-ATPase. The protein localization pattern of the non-DAR cells in An. gambiae and An. albimanus was similar to that of the freshwater culicine, Ae. aegypti which also expressed an apical V-ATPase and basal Na+/K+-ATPase in its non-segmented rectum The detection of V-ATPase in Ae. aegypti larval rectum confirms the finding of Filippova et al., (1998) but conflicts with the findings of Patrick et al. (2006) who detected V-ATPase mRNA in the rectum, but did not detect pr otein when using the same antibody against the B subunit which was us ed in this study. However, ultrastructural studies of the larval Ae. aegypti rectum identified a particulate coat on the apical lamellae (Meredith and Phillips, 1973) s uggesting the presence of portasomes (Clements, 1992) which correspond to the V1 portion of the V-ATPase (Harvey, 1992; Radermacher et al., 1999; Zhuang et al., 1999). Ae. aegypti is a freshwater culicine that can a cclimate to 50% ASW by osmoconforming in water hyper-osmotic to their hemolymph (average hemolymph of Ae. aegypti reared in freshwater ~ 250 mosmol l-1; Edwards, 1982; Clements, 1992). Larvae possess a non-segmented rectum which selectively resorbs ions from the primary urine. Like Ae. aegypti anophelines reared in freshwater actively resorb ions from the environment via their rectum to maintain 109

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constant ionic and osmotic hemolymph concentr ations (Bradley, 1994). The combination of basal Na+/K+-ATPase and apical V-ATPase in the rectum is ideally suited for this task. The polarity is similar to that in frog skin (Ehren feld and Klein, 1997), in which the V-ATPase hyperpolarizes the apical membrane, driving Na+ into the cell from the Na+ deficient environment. The Na+/K+-ATPase then transports the Na+ across the basal membrane into the hemolymph, replenishing ions which are lost to the aquatic environment, as first suggested by Koefoed-Johnsen and Ussing (1958). One example of V-ATPase mediated Na+ transport into mosquito cells occurs in the alimentary canal of many species: Na+ is driven into the cell by the electrical coupling of a VATPase and a Na+: amino acid nutrient amino acid transpor ter (NAT) (Boudko et al., 2005). Hyperpolarization of the apical membrane by V-ATPase drives electrophoretic Na+: amino acid symport into the cells. The efflux of H+ and influx of Na+ driven by the coupling of these two proteins constitutes a Na+/H+ exchanger (NHE), NHEV-NAT (Okech et al., 2008). In addition to driving Na+, V-ATPase hyperpolarization of the apical membrane ma y drive the absorption of numerous other essential ions. In Oc. taeniorhynchus reared in freshwater, a similar synergy between V-ATPase (apical) and Na+/K+-ATPase (basal) is evident in the AR, a regi on that has a resorptiv e function (Bradley and Phillips, 1977). This finding provides further support that the physiological coupling between these ATPases is involved in resorbi ng essential ions from the primary urine. Importantly, when Oc. taeniorhynchus are reared in 100% ASW, the apical V-ATPase appears to localize to the cytoplasm, breaking the coupling with Na+/K+-ATPase, which may indicate that these cells decrease their resorptive function in the presence of high salinity. 110

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The cytoplasmic localization of V-ATPase, a me mbrane protein, in the DAR cells of both An. gambiae and An. albimanus, as well as in the AR of saline-reared Oc. taeniorhynchus was unexpected, although there are severa l possible explanations. The rectal ultrastruc ture of the DAR cells has not been extensivel y studied, but the AR of saline-tolerant culicines exhibits a highly infolded basal membrane which constitutes the major elaboration of the surface area (Meredith and Phillips, 1973). The apparent cyt oplasmic V-ATPase may in stead be localizing to these basal infoldings. Alternatively, this lo calization could represent V-ATPase protein on the membrane of vacuoles within the cells (Harvey, 1992) or subun its that have dissociated from their membrane-bound V0 anchors. Dissociation of V1 from V0 occurs during molting in caterpillars (Sumner et al., 1995) and during glucose deprivation in yeast (Kane and Parra, 2000), and indicates inactivatio n of the protein. Saline Water-Reared Larvae: V-ATPase and Na+/K+-ATPase Localization When anopheline larvae were reared in sa line water, I observed a shift in Na+/K+-ATPase localization to the DAR cells (this protein was not abundant in the DAR cells of freshwaterreared larvae). Whereas in An. albimanus this shift was drastic, accompanied by a decrease in Na+/K+-ATPase in the non-DAR cells, in An. gambiae Na+/K+-ATPase was present in both DAR and non-DAR cells, suggesting an intermediate condition. In An. gambiae I also noted a marked reduction in the overall Na+/K+-ATPase signal as well as a no ticeable increase in V-ATPase signal in the cytoplasm of all re ctal cells. This may indicate a breakdown in the ion-transporting functions of the cells. As an obligate freshwater larva, An. gambiae lacks the ability to secrete a hyperosmotic urine and in fact cannot survive more than 24 h ours when acclimated to 60% ASW. It is possible that the stress incurred by exposure to saline water causes a breakdown in the cellular components of the ion-regulator y organs, which would lead to death. 111

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In saline-tolerant larvae such as An. albimanus I did not observe any decrease in Na+/K+ATPase protein signal or any indi cations of protein degradation. An. albimanus larvae can survive to pupation in up to 75% ASW, with about 35% of the first-instar larvae reaching pupation (Hurlbut, 1943). Th e dramatic shift in Na+/K+-ATPase (upregulation in the DAR cells and downregulation in the non-DAR cells) appears to disrupt the phys iological coupling between Na+/K+-ATPase and V-ATPase which I predict to be responsible for Na+ resorption in freshwater conditions. This shift renders the non-DAR cells similar in protei n localization to the PR of the saline-tolerant culicine Oc. taeniorhynchus and is likely to result in the ability of the non-DAR cells of An. albimanus to secrete a hyper-osmotic urine in a fashion similar to Oc. taeniorhynchus as discussed below. Oc. taeniorhynchus larvae are highly saline-tolerant, able to survive up to 300% ASW by regulating their hemolymph to maintain a constant osmolarity of about 350 mosmol l-1 (Nayar and Sauerman, 1974; Bradley, 1994). The rectum of this species differs from that of the freshwater Ae. aegypti by the presence of an additional segment, the PR, which secretes a hyperosmotic urine and rids the hemolymph of excess ions from a saline environment. The PR of Oc. taeniorhynchus expressed high levels of V-ATPase on th e apical lamellae but did not appear to express Na+/K+-ATPase. The presence of an apical V-ATPase is supported by ultrastructural studies of other saline-tolerant culicines which described a partic ulate coat made up of spherical subunits on the cytoplasmic surf ace of the apical lamellae (M eredith and Phillips, 1973), indicating V-ATPase containing portasomes. As mentioned above and discussed in greater detail in Harvey (1992), the hyperpolarizing actio n of V-ATPase can be used to drive many ion transporting processes against an otherwise unfavorable concentr ation gradient. I hypothesize that the V-ATPase on the apical lamellae of the PR in Oc. taeniorhynchus and in the non-DAR 112

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cells in An. albimanus is energizing one or more transporte rs also present on this membrane to translocate ions from the cells to the lumen in or der to excrete excess ions from the hemolymph. The ability for An. albimanus larvae to shift Na+/K+-ATPase protein distribution in response to salinity, as well as th e reversibility of this event, may be dependent on the larval stage at which exposure occurs. Younger larvae (s econdand third-instars) reared in freshwater and exposed to 25% ASW exhibited a shift in Na+/K+-ATPase distribution from non-DAR to DAR cells in as little as 24 hours. More matu re larvae (fourth-instars) exposed to 25% ASW for 24 hours expressed Na+/K+-ATPase in both non-DAR cells and DAR cells as if in an intermediate state between freshwater and saline water protein expression. After 48 hours, the majority of Na+/K+-ATPase was expressed in the DAR cells of fourth-instar larvae. This could indicate that early instar larvae are far more plastic in terms of regulating gene and/or protein expression than late larvae. Slightly different results we re obtained when rearing la rvae in 25% ASW and exposing them to freshwater. Whereas second-instar larvae shifted Na+/K+-ATPase distribution from the DAR to non-DAR cells within 24 hours of exposur e to freshwater, third-and fourth-instar larvae consistently expressed the pr otein in both DAR and non-DAR ce lls even after 72 and 48 hours, respectively. This may indicate that at the second-instar larval stages, the Na+/K+-ATPase protein shift is fully reversible, wherea s more mature larvae cannot downregulate Na+/K+ATPase expression in the DAR cells within the pe riod of time observed. It is possible that the concentration of ASW was too low to cause a co mplete shift in distribution in more mature larvae, and that larvae would respond differently if reared in 50% ASW versus 25% ASW. It is interesting that a protein shift is seen at all in 25% ASW (osmolarity = 250 mosmol l-1), as the osmolarity is less than that of the An. albimanus hemolymph (osmolarity ~ 300 mosmol l-1). 113

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This may be due to the abruptness of the salinity change, with larvae being transferred directly from freshwater to 25% ASW in the present stud y. Additionally, it could indicate that some event signals the rectum to shift Na+/K+-ATPase protein before it is actually necessary to produce a hyper-osmotic urine, as if in preparation. CA9 is Involved in CO2 Excretion CA9 consistently localized to the DAR cells of all anopheline mosquitoes examined (including An. gambiae, An. albimanus An. farauti An. quadrimaculatus An. aquasalis and An. stephensi ), as well as to the AR of Oc. taeniorhynchus regardless of rearing water salinity. Members of the CA family are implicated in both ion and pH regul ation in a number of organisms (e.g. Henry, 1984; Corena et al., 2004; Schewe et al., 2008) and catalyze the hydration of CO2 to H2CO3 which instantaneously dissociates into H+ and HCO3 -. CA within the epithelial cells can convert cellular CO2 to HCO3 which can then be excreted by the action of a Cl-/HCO3 exchanger (Smith, K. E. et al., 2007). Because the localization of CA9 did not change in any species in response to being rear ed in saline water compared w ith freshwater, I hypothesize that those rectal cells abundant in CA9 protein have at least one role th at is independent of external water salinity. One possible role for CA expressing cells is the transport of HCO3 -, from blood to lumen (in preparation for excretion), or from lumen to blood (absorption). In this way, the cells may use metabolic CO2 to regulate hemolymph HCO3 levels. Determining the presence and polarity of Cl-/HCO3 exchangers in these cells would re veal the direction in which HCO3 is being transported. Alternatively, cells that are enri ched in CA protein could be more metabolically active than neighboring cells which produce less CA. Cells with a robust metabolism would produce higher levels of CO2 that in turn, could induce synthesi s of high levels of CA protein to convert the toxic CO2 to HCO3 prior to excretion. In suppor t of this suggestion, there is 114

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evidence that the AR of Aedes dorsalis is involved in HCO3 secretion (Strange et al., 1984) which is mediated by a rectal CA (Strange and Phillips, 1984). Although I was unable to dete ct CA9 in the recta of Ae. aegypti larvae, this does not indicate a lack of CA activity in the rectum of this species. There are 13 predicted CA genes in the Ae. aegypti genome ( www.ensembl.org ), any of which could cat alyze the conversion of CO2 to HCO3 in the rectal cells. In support of CA activity in Ae. aegypti recta, alkalization of the rearing medium by starved Ae. aegypti larvae was reported by Stobbart (1971) who suggested that they may be excreting K+ HCO3 -. A similar alkalization wa s reported by Corena et al., (2002), who noted that the alkalization is blocke d by global CA inhibitors. However, Clark et al., (2007) reported that the rectal lumen of Ae. aegypti is acidic (pH<6.2), and not alkaline which would be expected for a larva excreting either K+ or Na+ and HCO3 -. It is possible that HCO3 secreted into the lumen associates with H+ ions provided by an apical V-ATPase to form H2CO3, which would dissociate into H+ and HCO3 when excreted from the rectum. However, as Clark et al., (2007) di scuss, the pKa of H2CO3/HCO3 is 6.4 and in an environment more acidic than this pH, HCO3 would exist primarily in the form of CO2. Another possibility is that CA is not present in the recta of Ae. aegypti Neither Stobbart (1971) nor Co rena et al., (2002) directly measured the pH of rectal excretions, and it is possible that the larvae alkalized their media by some other manner (i.e. gaseous respiration via the tracheal system). The role of CA in Ae. aegypti recta, as well as in other anopheline and cu licine species, can be further examined by measuring the precise pH and the concentration of HCO3 directly from the rectal contents. There is evidence for the expression of more than one CA in the recta of anophelines. Three additional CA isoforms are present in the recta of An. gambiae, AgCA3, AgCA6, and AgCAb. Whereas AgCA3 is similar to AgCA9 in that it is a predicted cytoplasmic CA, AgCA6 115

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is a predicted secreted CA and AgCAb is a predicted -CA. It is likely that each of these CAs has a distinct role in the rectum. At present, it is impossible to dis tinguish DAR from non-DAR using a dissecting microscope, and therefore I cannot determine the mRNA expression of each CA within the rectum using q-pcr, but because of the specific nature of AgCA9 localization to the DAR cells, I hypothesize that AgCA3, AgCA6 and AgCAb are likewise differentially localized in the rectum, each perfor ming a specific role. It is possi ble that all three isoforms are localized to the DAR cells, as is suggested by CA enzyme histochemistry (see Chapter 3) which detects an abundance of CA activity in the DAR cells compared with the non-DAR cells. It is likely that orthologs of these CA isoforms are also present in the recta of various culicine species. The generation of specific antibodies to each of these CAs will re veal their localization patterns within the rectal cells and can offer insi ght into the roles of each rectal region in CArelated functions. Conclusions: Based on the comparison of ion-regulat ory protein localization in culicine and anopheline larval recta I hypo thesize that saline-tolerant a nophelines secrete a hyper-osmotic urine by the same rectal cells th at are present in freshwater anophelines; this is in contrast to saline-tolerant culicines, which have a separate rectal region to secrete a hyper-osmotic urine. When reared in fresh water, both saline-tolerant and freshwater anophelin e larvae actively resorb water and nutrients from the primary urine without excreting salt. In s upport of this idea, the protein localization patterns of anopheline nonDAR cells resemble those of the freshwater culicine rectal cells wh ich are known to be activ e in resorbing ions. When exposed to saline water, saline-tolerant anophelines activate a regi on of the rectum (non-DAR cells) to secrete a hyper-osmotic urine by shifting protein localizati on of certain membrane energizing proteins such as Na+/K+-ATPase. This shift breaks the system of ion resorption in the non-DAR cells and 116

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the rectum functions in a way that is similar to th at of a salt water culici ne rectum, with the DAR cells performing the task of th e AR (resorption) and the non-DAR cells performing the task of the PR (excretion). These data suggest that two subfamilies of mosquitoes, anophelines and culicines, differ greatly in rectal structur e. These data also suggest that a nophelines regulate protein expression differently than do culicines when reared in sali ne water. The present study demonstrates that data obtained from one species of mosquito cannot necessarily be applied to all species. The majority of currently available information con cerning rectal structure an d function, as well as ion regulation (a system crucial for larval surv ival), pertains to culicine species. The data presented here, along with ultrastructural and physiological research currently underway, will expand that information to include the e qually important anopheline subfamily. 117

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Figure 5-1. Na+K+-ATPase and V-ATPase protein distribut ion in recta of various anopheline and culicine species reared in fresh and saline water. Na+K+-ATPase (red) and VATPase (green) protein distribution was determined in longitudinal sections of the recta of A) Ae. aegypti ; B and C) An. gambiae; E, F, and G) Oc. taeniorhynchus ; and I, J, and K) An. albimanus reared in fresh or saline wate r. D, H, and L) Distribution of Na+K+-ATPase is indicted as a ratio of Na+K+-ATPase peak pixel intensity in the DAR cells (or AR) versus the non-DAR cells (or PR). Arrowheads demark the junction between DAR and non-DAR cells in anophelines and arrows demark the junction between AR and PR in culicines. The inset images in panels J and K are identical to the corresponding panel, but lack V-ATPase staining signal, thereby giving a clearer view of Na+K+-ATPase distribution. The apparent cytoplasmic localization of V-ATPase in An. albimanus is shown in higher magnification in (I). Asterisks in panel E demark weak V-ATPa se signal on the apical lamellae of fresh water-reared Oc. taeniorhynchus Asterisks in panels D, H, and L indicate a significant difference in the Na+K+-ATPase peak pixel inte nsities between the DAR 118

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and non-DAR cells (or AR and PR). PValue > 0.05; ** P-Value > 0.005. AR: anterior rectum, ASW: artificial sea water, DAR: dorsal anterior rectum, L: lumen, PR: posterior rectum. Scale bars: 150 m (A ), 75 m (B, J, J inset), 86.13 m (C), 149.36 m (E, F), 99.32 m (G, K, K inset), 12 m (I). 119

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Figure 5-2. CA and Na+K+-ATPase protein distribution in recta of various anopheline and culicine species reared in fresh water. A) Ae. aegypti B) An. gambiae C) Oc. taeniorhynchus, and D) An. albimanus Na+K+-ATPase (red) was used as a counterstain. CA (blue) dist ribution did not change in larv ae reared in saline water; therefore only images of freshwater reared larvae are shown. Arrowheads indicate the junction between DAR and non-DAR cells. Arrows indicate junction between the AR and PR. AR: anterior rectum, DAR: dorsal anterior rectum, L: lumen, PR: posterior rectum. Scale bars: 150 m (A), 75 m (B), 149.36 m(C), 99.32 m (D). 120

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Figure 5-3. Change in CA and Na+K+-ATPase protein distribution in recta of An. albimanus given acute fresh or saline water challenges. Immunohistochemical detection of Na+/K+-ATPase (red) and CA9 (green) in An. albimanus larvae. Larvae reared in freshwater and transferred to 25% ASW during A) the second-instar stage for 24 hours, B) during the third-instar stage for 24 hours, and dur ing the fourth-instar stage for C) 24 hours or D) 48 hours. Also s hown are larvae reared in 25% ASW and transferred to freshwater dur ing the E) second-instar stag e for 24 hours, F) during the third-instar stage for 72 hour s and G) during the fourth-ins tar stage for 48 hours. H) Distribution of Na+K+-ATPase in each sample is indicted as a ratio of Na+K+-ATPase peak pixel intensity in the DAR cells versus the non-DAR cells. Lowercase bar 121

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labels in H correspond to the uppercase letters in Figure 5-3 panels (i.e. Figure 5-3A corresponds to Figure 5-3H bar a). Distribu tion of CA9 is indicative of DAR cells. Na+K+-ATPase and CA9 colocalization is indicated in yellow. In each set of images, the top panel shows both Na+K+-ATPase and CA9 localization, whereas the bottom panel is the same picture showing only Na+K+-ATPase localization. Asterisks in panel H indicate a statistically significant difference in Na+K+-ATPase peak pixel intensities between the DAR and non-DAR cells. P-Value > 0.05; ** P-Value > 0.005; *** P-Value >.001. L: lumen. Scale bars: 75 m (A, B, C, E), 150 m (D, G), 65.17 m (F). 122

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Figure 5-4. RT-PCR analysis of the expr ession of each of the eleven An. gambiae CAs (AgCA1-11) and the one CA (AgCAb) in An. gambiae rectal tissue. Rectal cDNA was used in a PCR reaction with primers speci fic to either the whole CA transcript or the partial predicted sequence. Whole An. gambiae fourth-instar larvae cDNA was used as a primer control (shown by a C above the lane) in addition to the rectal cDNA experimental template (i ndicated by a R above the lane). See Table 2-3 for primer sequences and expected length of products. All bands shown were the expected size for each gene amplified. M: 1 kb DNA ladder (invitrogen). 123

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Figure 5-5. Comparison of culic ine and anopheline larval rect al structure using confocal microscopy of whole mount im munohistochemical preparatio ns. Structure of A) the freshwater culicine, Ae. aegypti B) the saline-tolerant culicine, Oc. taeniorhynchus, and C) the freshwater anopheline, An. gambiae. Data similar to these have also been reported elsewhere (Patrick and Gill, 2003; Smith, K. E. et al, 2007; Okech et al., 2008). Protein localizations are shown for vi sual distinction of rectal segments in each species. Immunolocalizati ons indicate distribution of Na+/K+-ATPase (red), VATPase (green), and CA9 (blue). AR: ante rior rectum, DAR: dorsa l anterior rectum, PR: posterior rectum. Scale ba rs: 150 m (A, C), 186 m (B). 124

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125 Figure 5-6. Comparison of th e major protein localization pa tterns found in the recta of freshwater-reared culicines and anophelines to those in saline water-reared culicines and anophelines. Figure 5-6 allows an easy comparison of the proteins in the anopheline and culicine recta. In all cases CA9 localizes to the cytoplasm, Na+K+ATPase (NaK) localizes to th e basal membrane, and V-ATPase localizes to the apical membrane. The non-DAR cells of freshwat er-reared anophelines, both freshwater and saline-tolerant, resembled the recta of freshwater culicines in protein localization; both expressed Na+K+-ATPase and V-ATPase. When reared in saline water, the anopheline rectum shifted Na+K+-ATPase localization and resembled the saline-water culicine rectum in protein localization; anopheline DAR cells and culicine AR possessed CA9 and Na+K+-ATPase, whereas anopheline non-DAR cells and culicine PR possessed V-ATPase.

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CHAPTER 6 CHARACTERIZATION OF THE DAR AND NON-DAR CELLS OF ANOPHELINE RECTA: PHYSIOLOGICAL FUNCTIONS Introduction Mosquito larvae, like all aquatic organisms, must adapt to an environment which can change drastically in ionic composition and sali nity. Adaptation to these varying conditions requires a highly developed system for the regula tion of ions, and in mosquitoes, a major organ responsible for this regulation is the rectum. Although, to date, the majority of work on larval ion regulation has focused on the culicine subfamily I and others have shown that the recta of anopheline larvae differ from that of both freshw ater and saline-tolerant culicine species in structure (Bradley, 1987a; Bradley 1987b; Smith, K. E. et al., 2007) and in regulation of protein expression (Smith et al., 2008). The recta of anopheline larvae are similarly stru ctured regardless of saline tolerance, but can adapt to saline environments by shifting th e distribution of at l east one ion-regulatory protein, Na+/K+-ATPase. As a result, different salinities elicit different patterns of protein expression in the rectal regions, and may result in a change in overall function of the rectum. In this way, larvae can survive in freshwater by resorbing essential ions and nutrients, and in saline water (in the case of saline-tolerant species) by generating a hyperosmotic urine, while using the same rectal cell types. I hypothesize that anopheline larvae shift protein distributi on patterns in their rectal regions to activate the non-DAR cells, which comprise approximately 75% of the rectum, to either absorb or secrete ions based on envi ronmental ion load. By expressing different combinations of ion-regulatory proteins, the rectal cells may fa vor the function of one protein over another. Many ion-regulatory transporte rs can activate other proteins by generating favorable electrochemical gradients across a memb rane. Often, these gradients are generated by 126

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the uneven distribution of H+ across a membrane, and indeed many proteins facilitate the transport of H+ across insect membranes including V-ATPase (Wieczorek et al., 1991), Na+/H+exchangers (Pullikuth et al., 2006), Na+/H+ antiporters (Padan et al., 2001; Rheault et al., 2007), and K+/H+ exchangers (Harvey and Nedergaard, 1964). The self-referencing ion-selective microelectrode technique (SIET) was developed to measure ion-specific flux across a membrane at the single cell level while virtually eliminating the artifact of voltage drift (Kht reiber and Jaffe, 1990; Kochian et al., 1992; Smith et al., 1999). The selectivity of the electrode is based on the characteristics of commercially available ionophores suspended in a liquid membrane at the tip of a microelectrode (Smith et al., 1994). The electrode self-references by re cording voltage values at two points within an ion gradient, a point near the ion source and a point a know n distance away from the ion source. By subtracting the voltage measured at the far point from that at the near point, drift and background signals, as well as any contaminating voltages can be minimized (Smith, P. J. et al., 2007). Here I report H+ flux at the basal membrane of the DAR and non-DAR cells of freshwater and saline water-reared An. albimanus larvae using SIET, and the effects of pharmacological inhibition of that flux. My da ta indicate there are distinct differences between the DAR and nonDAR cells both in the direction of net H+ flux and in the proteins mediating this flux. Additionally, there are distinct differences in the roles of several of these proteins in H+ flux depending on the salinity of the larval rearing media. I use these results, together with previous immunohistochemical data, to generate a putative model for ion control in the larval anopheline rectum and discuss how this model relates to my previous hypothesis rega rding the role of the anopheline rectum in ion regulation. 127

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Results H+ Flux in Rectum H+ flux was determined at the basal membrane of the DAR and non-DAR cells of An. albimanus larvae reared in either 2% or 50% ASW using SIET (Figure 6-1). Because DAR and non-DAR cell types cannot be distinguished using a dissecting microscope, measurements were taken along the anterior-posterior axis of one side of the rectum and preparations were poststained with rhodamine 123 to visualize DAR and non-DAR cell types (Figure 6-1A). H+ flux was measured at points approximately 5-10 m from the membrane in 50 m increments along the axis of the rectum (Figure 6-1B). In all larvae a significant H+ efflux was observed averaging 1.3 x 10-7 mol cm-2 s-1 in the area of the DAR cells. Although this efflux was consistent among all larvae, the magnitude of the flux varied greatly between larvae ranging from 1.38 x 10-8 to 3.27 x 10-7 mol cm-2 s-1. As the probe neared the DAR/non-DAR boundary, the H+ flux decreased significantly and fluctuated between an influx and efflux, averaging 9.6 x 10-9 mol cm-2 s-1. Again the magnitude of this flux varied between larvae, but in each individual larva, a trend was noted in which DAR cells exhibited a large H+ efflux which decreased consid erably at the DAR/non-DAR cell boundary, and often reversed direction in the non-DAR cells (Figure 6-1C). Pharmacology To determine the proteins involved in H+ flux at the basal membrane of the DAR and nonDAR cells, the effects of concanamycin A, methazolamide and DIDS were evaluated at a single point of H+ efflux at the DAR cells and influx at the non-DAR cells (Figure 6-2). Concanamycin A is a specific inhibitor of the H+ pump, V-ATPase; methazolamide is a specific inhibitor of CA, a known marker for DAR cells and an enzy me which catalyzes the production of H+ and HCO3 within a cell; DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid Disodium salt) is a 128

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specific inhibitor of anion exchange and is eff ective against bicarbonate transporters, proteins which frequently work in synergy with CAs (M cMurtrie et al., 2004). For each inhibitor, n 5 larvae were examined. Concanamycin A at a concentration of 10-6 mol l-1 significantly decreased efflux at the DAR cells of larvae reared in both 2% (P-val ue < 0.01) and 50% (P-v alue < 0.05) ASW and significantly decreased n on-DAR cell influx in larvae reared in 50% ASW (P-value < 0.05). Concanamycin A had no significant effect on non-DAR cell influx in 2% ASW reared larvae at the concentration used. Methazaolamide at a concentration of 10-4 mol l-1 significantly decreased H+ efflux in the DAR cells of larvae reared in both 2% (P-value < 0.05) and 50% (P-value < 0.01) ASW, whereas it significantly increased non-DAR cell influx in larvae reared in both 2% (P-value < 0.001) and 50% (P-value < 0.01) ASW. DIDS at a concentration of 10-6 mol l-1 significantly decreased H+ efflux in the DAR cells of larvae reared in 2% ASW (P-value < 0.01) and significantly increased DAR cell H+ efflux in larvae reared in 50% ASW (P-value < 0.05). DIDS significantly incr eased non-DAR cell influx in larvae reared in both 2% (P-value < 0.01) and 50% (P-value < 0.05) ASW. No significant effects were detected in either cell type of la rvae reared in either 2% or 50% ASW using 0.1% DMSO. In at least one case, each inhibitor (con canamycin A, methazolamide, DIDS) caused the reversal of flux, in which treatment resulted in a H+ efflux becoming an influx. No instances were observed where treatment resulted in a H+ influx becoming an efflux. Discussion I previously hypothesized that the primary f unction of the rectum of saline-tolerant anophelines changes based on the ionic needs of the larva, an d that this change is due to a shift in 129

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the distribution of ion-regulatory proteins (Smith et al., 2008). Here I gain insight into the functional characteristics of the anophe line DAR and non-DAR cells by measuring H+ flux at the basal membrane of 2% and 50% ASW-reared An. albimanus recta using SIET. Because H+ is coupled to many transport processes, H+ flux at a membrane can be indicative of transport across that membrane. A large H+ efflux was measured from the DAR cells into the hemolymph; this contrasted with the flux measured in the non-DAR cells which diminished drastically, often becoming an influx. The H+ flux in both DAR and non-DAR cells was shown to be independent of rearing salinity and mediated in part by the actions of V-ATPase, CA and anion exchange. Here I have combined data from H+ flux measurements and pharmacological inhibition in each region of the rectum to generate a putative model of ion regulation which will be discussed in more detail in the following sections (Figure 6-3). H+ Fluxes are Likely Due to Many Transport Systems Treatment with pharmacological inhibitors strongly suggests that the measured H+ fluxes, large effluxes at the DAR cell basal membra ne and influxes at the non-DAR cell basal membrane, are the collective result of many different H+ transport systems. Several cases were noted in which treatment of an H+ efflux at the DAR cell membrane with an inhibitor resulted in a measured H+ influx. These results support the idea that the measured fluxes are not the products of a single H+ source, but the cumulative effects of many H+ transporting proteins, likely generating numerous H+ effluxes and influxes at the basal membrane. The large fluxes measured at the DAR cell membrane are likely the result of an excess of H+ efflux into the hemolymph over H+ influx into the cell. No instances were observed in which treatment with an inhibitor resulted in an H+ influx at the non-DAR cells becoming an efflux. This ma y represent a functional difference between the DAR and non-DAR cells; however, the major ity of the inhibitors increased the H+ influx in the 130

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non-DAR cells, and therefore would not be expected to reverse the flux. It is likely that the low effluxes/influxes measured in the non-DAR cells are the result of relativ ely equal numbers of H+ being translocated into the hemolymph as there were into these cells. This observation also affects the interpretati on of pharmacological data; it is impossible to discern an increase in efflux from a decrease in influx and vice versa. Here I represent changes in H+ flux due to pharmacological inhi bition as a ratio of flux with and without, treatment with an inhibitor. However, I have not reported the percent flux inhibition, as it would be an irrelevant value. The H+ flux measurements represent a mixture of influxes and effluxes, and I cannot distinguish which are affected by the inhibitor. Therefore, speculations on inhibitor effects will be based on known and predicted protein distribution patterns and the classical view of the specific effects of the pharmacological experiments. Pharmacology Although H+ flux at the basal membrane of the r ectum did not appear to differ between larvae reared in 2% and 50% ASW, the effects of specific pharmacological inhibitors on that flux, specifically the effects of concanamycin A on the non-DAR cells and of DIDS on the DAR cells, indicate functional differences between the recta of larvae reared in different salinities (Figure 6-2). Concanamycin A: V-ATPase inhibitor Concanamycin A is a membrane permeable macro lide antibiotic that specifically inhibits V-type H+-ATPase-mediated H+ movement (Huss et al., 2002). V-ATPase is an H+ pump and well known membrane energizer important fo r ion regulation (e. g. Wieczorek, 1992). Treatment of DAR cells with concanamycin A resulted in a significant decrease in H+ efflux in larvae reared in both 2% and 50% ASW (Figure 6-2). This suggest s the presence of a V-ATPase on the basal membrane of the DAR cells pumping H+ into the unstirred layer of the hemolymph. 131

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Apparent cytoplasmic localization of a V-AT Pase was observed in the DAR cells of both An. gambiae and An. albimanus using immunohistochemistry (Sm ith et al., 2008) and it was suggested that the protein may in fact localize to the basal membrane. Preliminary evidence suggests that similar to the recta of freshwater culicines and the AR of saline-tole rant culicines (Meredith and Phillips, 1973), the ba sal membrane of DAR cells is elaborated into an extensive basal labyrinth which, because of an apparent lack of apical lamellae, extends throughout the majority of the cell (K. E. Smith, unpublished obs ervations). V-ATPase protein distributed on the basal membrane may appear cytoplasmic due to the nature of the membrane. Treatment of non-DAR cells with concanamyc in A elicited no significant effect on H+ influx in 2% ASW-reared larvae, but resulted in a significant decrease in H+ influx in 50% ASW-reared larvae (Figure 6-2). This suggests a difference in V-ATPase protein distribution or function between the non-DAR cells of larvae reared in 2% and 50% ASW; however, V-ATPase has been shown to localize to the apical membrane of the non-DAR cells in larvae reared in both conditions (Okech et al., 2008; Smith et al., 2008) This difference in pharmacological inhibition could be explained by the differe ntial distributio n of another H+ transporter such as a sodiumhydrogen exchanger (NHE). In most cases, insect NHEs mediate the el ectroneutral exchange of intracellular Na+ for extracellular H+ (Beyenbach, 1995; Maddrell and ODonnell 1992). However, there is evidence that they can also function in the opposite direction (Pullikuth et al., 2006), exchanging extracellular Na+ for intracellular H+, similar to mammalian NHEs (Orlowski and Grinstein 2004) Two NHEs, AeNHE3 and AeNHE8, have been cloned from Ae. aegypti and localized to the recta of larvae (AeNHE3; Pullikuth et al., 2006) and adults (AeNHE8; Kangethe et al., 2007). A similar NHE may provide another means of apical H+ transport into the lumen of An. 132

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gambiae non-DAR cells in the event of apical V-ATPase inhibition. If this NHE were downregulated in 50% ASW-reared larvae, concanamyc in A inhibition of the apical V-ATPase would impede H+ transport into the lumen, thereby backing up the basal H+ influx. In support of my hypothesis that the non-DAR cells of freshwater-re ared larvae absorb ions, NHE, in conjunction with a basally distributed Na+/K+-ATPase (Okech et al., 2007; Smith, K. E. et al., 2007; Smith et al., 2008), may mediate the uptake of sodium from the urine. The down-regulation of NHE and Na+/K+-ATPase proteins in the non-DAR cells of 50% ASWreared larvae could decrease sodium resorption a nd may reflect the diminished ionic needs of the larvae. Methazolamide: carbonic anhydrase inhibitor Methazolamide is a specific inhibitor of -CAs, one isoform of which (AgCA9) I have previously identified as a reliable marker for DA R cells (Smith, K. E. et al., 2007; Smith et al., 2008). Additionally, the mR NA of least two other -CA isoforms and one -CA isoform, are present in whole recta samples of freshwater-reared An. gambiae larvae: AgCA3, a cytoplasmic CA, AgCA6, a secreted CA, and AgCAb, a -CA against which methazolamide is ineffective (see Chapter 3). Both and -CA isoforms catalyze the conversion of cellular CO2 to HCO3 and H+. Treatment with methazolamide resulted in a significant decrease in DAR cell H+ efflux and a significant incr ease in non-DAR cell H+ influx in larvae reared in both 2% and 50% ASW (Figure 6-2). This data supports the known distribution of AgCA 9 in the DAR cells and suggests that CA(s) influence H+ flux in the non-DAR cells as well possibly through the presence of an extracellular or cytoplasmic CA is oform associated with the cells. Alternatively, CA activity in the DAR cells could indirectly mediate proteins involved in H+ flux in the non-DAR cells. Many systems exhibit colocalization of CA and a membrane V-ATPase which translocates H+ 133

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produced by the CA-mediated r eaction out of the cell (Breton et al., 1996; Breton et al., 1999; Breton, 2001; Paunescu et al., 2008 ). Therefore, inhibition of a cellular CA would decrease H+ available for V-ATPase transport, decreasing the V-ATPase-mediated flux. Likewise, the decrease in DAR cell H+ efflux following methazolamide treat ment supports the prediction of a basal V-ATPase in this cell type. By contrast, the non-DAR cells of An. albimanus larvae are known to express an apical V-ATPase when larv ae are reared in both fr esh and saline water (Smith et al., 2008). The methazolamide-related increase in non-DAR cell H+ influx may indicate that a cellular CA is the main source of H+ for the apical V-ATPase. Inhibition of this CA would deplete the V-ATPase of H+ needed to energize the membrane, and may lead to an upregulation of other H+ transport systems on the basal membrane to compensate for the loss of H+. Alternatively, a decrease in CA-mediated H+ generation could decr ease intracellular H+ concentration, altering the H+ gradient and leading to an increase in basal H+ influx down the concentration gradient. DIDS: anion exchange inhibitor DIDS is a specific inhibitor of cellular anion exchange and is effective against multiple proteins including Na+-driven and Na+-independent HCO3 -/Cltransporters and Na+/HCO3 cotransporter as well as Clchannels (Grassl & Aronson, 1986; Helbig et al., 1988; Boron, 2001; Matulef and Maduke, 2005). Treatment with DIDS significantly decreased DAR cell H+ efflux in 2% ASW-reared larvae, while significantly increasing DAR cell H+ efflux in 50% ASWreared larvae. This suggests differential distribu tion of protein activity me diating the transport of HCO3 or Clin the DAR cells of larvae reared in 2% versus 50% ASW. Treatment of non-DAR cells with DIDS significantly increased H+ influx in larvae reared in both 2% and 50% ASW, suggesting no significant differe nces in protein activity medi ating the transport of HCO3 or Clin this cell type. 134

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Three HCO3 transporting proteins belonging to the SLC4 family (Romero et al., 2004) are predicted to be expressed in the An. gambiae genome; the mRNA of only one of these proteins, AgAE1, is abundantly expressed in the hindgut (MTs and rectum) as measured by micro-array and quantitative-PCR (Neira et al., 2008; M. V. Neira, pe rsonal communication). HCO3 transporting proteins are often co-l ocalized with CAs to facilitate the transport out of the cell of HCO3 produced by the CA-catalyzed reaction (McMur trie et al., 2004; Sterling et al., 2001). As mosquito larvae are thought to excrete HCO3 from the rectum, alkaliz ing their media (Stobbart, 1971), I predict AgAE1 to localize to th e apical membrane, transporting HCO3 from the cells into the lumen of the rectum. Due to the wide range of proteins i nhibited by DIDS, I cannot draw any other specific conclusi ons regarding my model of ion tr ansport except to say that the results strongly suggest that DIDS-sensitive anio n exchange protein activity changes in the DAR cells, but not the non-DAR cells, depending on the salinity of rearing water. Putative Model for Ion Transport in the Anopheline Rectum I have generated a putative model for ion transport in the larval rectum of anopheline mosquitoes (Figure 6-3) based on current physio logical and pharmacologi cal data as well as previous immunohistochemical data (Rheault et al., 2007; Okech et al., 2008; Smith et al., 2008). This model supports my hypothesis that the prim ary function of the non-DAR cells, the cell type which comprises approximately 75% of the cells in the rectum, changes in response to the ionic needs of the larvae. Anophelines reared in fresh water actively resorb ions via their rectum to maintain constant ionic and osmotic hemolymph concentrations (Bradley, 1994). By contrast, anophelines reared in saline wate r must excrete the excess ions i ngested while feeding via rectal production of a hyperosmotic urine. A potential mechanism for Na+ resorption is evident in the non-DAR cells of 2% ASW-reared larvae, whereby Na+ is transported from the lumen into the cell via an NHE and is then transported into the hemolymph in exchange for a K+ via a Na+K+135

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ATPase. Basal Na+K+-ATPase is considerably down-regulated in the non-DAR cells of larvae reared in 50% ASW (Smith et al., 2008), and I predict that the apical NHE may also be downregulated in saline conditions. This change in protein distribution would interfere with Na+ resorption in the non-DAR cells and may indicate a shift in pr imary rectal function, from resorption in fresh water, to secretion in saline water. The DAR cells comprise approximately 25% of the cells in the rectum and are hypothesized to have a main role in HCO3 excretion (Smith et al., 2008). However, this hypothesis is based on the idea that CA excl usively localizes to the DAR cells. As pharmacological data now suggests that CA is present in both DAR and non-DAR cells, it is likely that DAR cells have a role in basal reso rption of essential ions and nutrients from the primary urine, similar to that suggested for the AR of culicine larvae (Meredith and Phillips, 1973; Bradley and Phillips, 1977). Cha nges in the response of DAR cell H+ flux to DIDS in larvae reared in 2% and 50% ASW cannot readil y be explained without further analysis of protein distribution. It is possible that the effects of DIDS are related to the absence or presence of a basal membrane Na+K+-ATPase in DAR cells of larvae reared in 2% and 50% ASW, respectively. The shift in localization of this protein may regulate the rate or type of ion resorption mediated by the DAR cells. Conclusions: I have further characterized the DAR and non-DAR cell types of the anopheline larval rectum using SIET. I have dem onstrated that the two ce ll types differ in basal membrane H+ flux and that V-ATPase, CA, and anion exchanger proteins have a major role in mediating H+ flux in both cell types. Additionall y, differences in the response of H+ flux to inhibitors in larvae reared in 2% versus 50% ASW indicate cha nges in protein function between the two rearing conditions. These data, al ong with previous imm unohistochemical results, 136

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support a model of rectal ion regulation in whic h the non-DAR cells have a resorptive function in freshwater-reared larvae and a se cretive function in saline water-reared larvae. In this way, anopheline larvae may adapt to varying salinities. Ion regulation is a biological pr ocess crucial to the survival of mosquito larvae. The ability to shift protein distribution, th ereby altering cell func tion, affords larvae the ability to survive and flourish in a wider range of ha bitats. This work has begun to characterize the function of the anopheline rectum as it relates to adaptati on and ion regulation. Only through a full understanding of the proteins involved in, and the mechanisms responsible for these adaptive abilities can we take advantage of this vital process for the development of vector control. 137

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Figure 6-1. Measure of H+ flux at the basal membrane of the DAR and non-DAR cells of An. albimanus larvae using SIET. A) Image illustra ting the larval rectum as viewed through a dissecting microscope during H+ flux measurements (top panel), and poststained with rhodamine 123 to visual ize the DAR/non-DAR boundary (arrows; bottom panel). B) H+ flux for seven individual prepar ations beginning at the rectumileum junction and stoping at the end of th e rectum, measured in 50m intervals. Each data point represents an average of continuous measurements taken over five minute periods. C) Averaged DAR and non-DAR H+ flux measurements. Error bars indicate standard error of the mean. Difference in H+ flux between the DAR and nonDAR cells was statistically significant (P < 0.01) as determined using a paired t-test and reported as a two-tailed P-value. Scale bar: 100m. e: electrode. 138

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Figure 6-2. Pharmacol ogical inhibition of H+ flux at the basal membra ne of 2% and 50% ASWreared An. albimanus recta. Inhibitors include a V-ATPase inhibitor, concanamycin A (C), a carbonic anhydrase inhibitor, meth azolamide (M), and an anion exchange inhibitor, DIDS, as well as a DMSO vehicle control. Asterisks indicate a significant difference between H+ flux with and without inhibitor. 139

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140 Figure 6-3. Putative model for ion transport in th e anopheline rectum of larvae reared in fresh or saline water. Proteins in color ar e those which have been previously immunolocalized, whereas proteins in black are those wh ich are predicted. This model suggest that the non-DAR cells of 2% ASW-reared larvae function in resorbing ions from the urin e as indicated by the NHE/Na+K+-ATPase-mediated sodium resorption pathway. This function likely changes to a secretive one in larvae reared in 50% ASW. AE: anion ex changer; CA: carbonic anhydrase; Na/K: sodium/potassium ATPase; NHE: sodium /hydrogen exchanger; V: V-ATPase.

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CHAPTER 7 CONCLUSIONS AND FUTURE DIRECTIONS pH Regulation in the Larval Alimentary Canal Conclusions: I have cloned the full-length transc ripts of four CA genes from the An. gambiae alimentary canal including two CAs predicted to translate to secreted (AgCA6) and cytoplasmic (AgCA9) proteins, one CA-RP (CA-RP2), and one CA (AgCAb). mRNA localization of three of these genes indicates a significant abunda nce of AgCAb in the AMG and AgCA6, AgCA9 and AgCAb in the rectum. Th rough protein localization I detected one CA, AgCA9, in the GC, transitional region, ectoperitr ophic space, MT and DAR cells of the rectum. Additionally, I have demonstrat ed AgCA9 mRNA silencing and protein down-regulation in an An. gambiae larval cell line, paving the way for functiona l studies in live larvae. This work has advanced the current model of AMG alkalization in mosquito larvae and has linked CA function with ion-regulation as well as pH-regulation. The previous model of midgut alkalization in mosquito larvae relied on plasma membrane HCO3 transporters to translocate cellular HCO3 into the lumen where it buffered the near neutral milieus of the GC and PMG. An api cally located V-ATPase in the AMG would cause HCO3 to be stripped of a H+ to form CO3 2which could buffer the highly alkaline AMG lumen. The inability to localize a HCO3 transporter to the apical membrane of the alimentary canal challenged the validity of this model. However, localization of a CA to the ectoperitrophic space modified the original model such that HCO3 transporters are no longer essential. The ectoperitrophic space is conti nuous in pH with the endoperitr ophic space and therefore can control pH across the whole widt h of the alimentary canal. A simplified version of the new model for luminal pH buffering of larval mosquito alimentary canal is illustrated in Figure 3-12. Epithelial cells are metabolically active and 141

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produce CO2. CO2 can diffuse through the epithelial cells and into the lumen where a luminal CA in the ectoperitrophic space catalyzes the conversion of CO2 to HCO3 -. CA catalyzes the same reaction within the epithelial cells, generating H+. In the GC and PMG, an apically located V-ATPase hyperpolarizes the membrane by pumping H+ from the cells into the lumen. By contrast, in the AMG epithelium, a basally located V-ATPase is positioned to move H+ ions vectorally out of the cells (Zhuang et al., 1999), re sulting in a transepithel ial potential that is lumen-negative (Clark et al., 1999). This results in the loss of H+ from HCO3 -, forming CO3 2-. In addition to revising the model for midgut alkalization, I have demonstrated the potential for RNA interference-mediated CA silenc ing. Although there has been little success in down-regulating protein expressi on in anopheline mosquito larvae, I have shown that it is possible in an An. gambiae larval cell line. Becaus e the half life of the protein of interest is an important factor in RNAi, not all proteins can be down-regulat ed using this mechanism. Through my cell culture work I have shown that at least one CA has the potential to be downregulated in live larvae using RNAi. Future directions: There remain at least six uncha racterized CA genes present by the An. gambiae genome. The cloning and char acterization of these genes will further complete the model for midgut alkalization in mosquito larvae. Additionally, although the localization of a CA in the ectoperitrophic space offers an alternative mechanism for HCO3 access to the lumen, localization of predicted anion ex changers is still a major goal of the laboratory. Some progress towards this goal is described in appendix A of this manuscript. Whereas demonstration of AgCA 9 mRNA silencing in the Ag55 cell line was a first step in identifying the role of this CA, similar experiment s in live larvae have no t been as successful. To investigate the reasons for poor RNAi succes s in larvae, I suggest silencing AgCA9 using 142

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RNAi in adult mosquitoes. This technique is used often and is widely successful in adults, and will indicate if there is an inherent problem with the gene being silenced, or with the larval stages of the mosquito life cycle, which makes RNAi s ilencing of AgCA9 in larvae a difficult endeavor. An alternative approach to RNAi silencing in mosquito larvae is the development of transgenic larvae. By microinjecting preblastoderm embryo s with a CA gene linked to a genetic drive system such as a transposable element, larvae can be genetically manipulated to express CAspecific inverted-repeat sequences which form dsR NA in the larvae (see Franz et al., 2006). Ion Regulation in the Larval Rectum Conclusions: Through AgCA9 protein localization, I revealed an unexpected staining pattern which identified a novel s ubset of cells in the larval a nopheline rectum. I discovered that the larval anopheline rectum differ s from that of the larval culici ne rectum in structure and in regulation of protein expression, suggesting that anophelines a nd culicines regulate hemolymph osmolarity in slightly different ways. Specifica lly, the larval anopheline r ectum appears to shift primary distribution of Na+K+-ATPase protein from the non-DAR cells in fresh water-reared larvae, to the DAR cells of saline water-reared larvae. This is in stark contrast to culicine larvae which do not appear to shift protein di stribution in response to salinity. Additionally, I demonstrated that the anopheline DAR cells differ functionally from the non-DAR cells in basal membrane H+ flux, as well as in the proteins mediating that flux. Finally, I showed that an anion exchange inhibitor, DIDS, and a V-ATPase inhibitor, concanamycin A, had different effects on the DAR and non-DAR cells, respectively, of larvae reared in fresh versus saline water, suggesti ng a change in protein f unction in response to salinity. Together my immunohistochemical, physiologi cal, and pharmacological data support the hypothesis that the major cell type of the a nopheline rectum, the non-DAR cells, changes its 143

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primary function from that of absorption in larvae reared in fresh water, to secretion in larvae reared in saline water. This suggests a remarkable method of adaptation which has not been described in mosquitoes to date. These data not only prompt the development of a new model for anopheline ion regulation in th e rectum (Figure 6-3), but also stress the differences between anopheline and culicine larvae. It is clear from this data that anophelines and culicines differ in very important ways, and that data generated in culicine species cannot nece ssarily be applied to anopheline species and vice versa. Importantly, these differences ma y affect larval response, or adaptation, to vector control strategies. Future directions: To test my hypothesis regard ing a change in anopheline rectal function, the absorptive and secretive prope rties of the DAR and non-DAR cells must be characterized. This can be done using in vitro rectal ligation studies monitoring the concentration and volume of fluid accumulated in each region over time (see Bradley and Phillips, 1977). However, the difficulty lies in identifying, and se parating the DAR and nonDAR cells using a dissecting microscope. Perhaps an easier approach to further characterizing the function of the larval anopheline rectal cell types is to perform a similar physiological study on the recta of culic ine larvae, the functions of which have already been determined. By comparing basal H+ flux and pharmacological inhibition of that flux of either the freshwater cu licine rectum, or the salinetolerant culicine AR and PR, to that of the anopheline DAR and non-DAR cells, predications regarding function can be made. Final Thoughts As I have stressed throughout this manuscrip t, a thorough and detailed understanding of mosquito biology and physiology can facilitate th e development of safe and effective control methods. The cloning and characte rization of several CAs from the larval mosquito alimentary 144

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canal have emphasized the global importance of this enzyme family to mosquito survival. CA is a central player in larval pH and ion regulation, two processes crucial for larval adaptation and survival. Additionally, CA ac tivity is important to adult disease transmission. Recent unpublished work in malaria transmission assays indicate that CA inhibitors may block Plasmodium infection of mosquitoes, thereby interferi ng with the spread of this deadly disease (M. P. Corena, P. J. Linser, personal communicati on). I am confident that by characterizing the remaining six An. gambiae CAs, we will uncover new roles for this enzyme in both larval regulation strategies and adultplasmodium interactions. My research has not only revealed methods of pH and ion regulation in mosquito larvae not previously known, but has also shown that previous models of these processes may have been inaccurate. Specifically, the newly characterized structure and function of of the larval anopheline rectum stresses the idea that tw o subfamilies of mosquitoes, culicines and anophelines, are in fact very different. Presently, much larval re search, including the majority of research on ion regulation, is performed on culicine mosquitoes; my work emphasizes the importance of conducting parallel research on anopheline species, the most important malaria vectors. The differences between these two subfamilies may have profound effects on larval adaptation and/or response to vector control methods, and these di fferences may translate to the adult stage as well. By not fully understand ing the biology, physiology, and pharmacology of anophelines, the sole vectors for human malaria, we will continue to fail at eradication of this and other vector born diseases. 145

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APPENDIX A CLONING AND CHARACTERIZATION OF ME MBERS OF THE SLC-4 BICARBONATE TRANSPORTER FAMILY FROM ANOPHELES GAMBIAE SENSU STRICTO LARVAE This appendix describes data generated towards aim one of my proposal: to clone, characterize, and localize the e xpression of members of the Cl-/HCO3 transporter gene family in the larval mosquito alimentary canal. The work reported here addresses several key issues in regards to this aim but must be expanded to produce a publishable story. Introduction The previous model for AMG alkalization in mosquito larvae proposed that HCO3 generated by cytoplasmic CA was translocated into the alimentary canal lumen by HCO3 transporters and then paired with a strong cation (X+) to buffer the lumen as either X2CO3 or XHCO3 (Boudko et al., 2001b). This model was recently updated based on data which suggested that a luminal CA in the alimentary canal obviates the need for plasma membrane HCO3 transporters in AMG alkalization (Smith, K. E. et al., 2007; Chapter 3 of this manuscript). However, CA is also expressed in the cells of the alimentary canal, particularly in the GC and rectum, catalyzing HCO3 production within those cells (Smith, K. E. et al., 2007; Chapter 3 of this manuscript). HCO3 is a membrane impermeant ion and requires a transport protein to cross the plasma membrane for absorption into the hemolymph or excretion into the lumen (McMurtrie et al., 2004). Base d on this requirement, I predict that plasma membrane HCO3 transporters localize to the membranes of epithelial cells to translocate HCO3 from the cells into the hemolymph or lumen. HCO3 transporters belong to the solute carrier 4 (SLC4) gene family and have been described in detail in H. sapiens (Romero et al., 2004). Many of the characterized H. sapiens SLC4 genes share considerable homology with genes predicted by the An. gambiae and Ae. aegypti genomes. 146

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As reviewed by Romero et al. (2004), the SLC4 family of HCO3 transporters consists of integral membrane proteins that transport bicar bonate or carbonate across the plasma membrane in exchange for at least one monoatomic ion. Proteins are characterized by 10-14 transmembrane domains which separate long N-terminal and shorter C-terminal hydrophilic domains, both of which are intracellular. Based on physiological characterization of H. sapiens SLC4 proteins, the family includes three functional subgroups: electroneutral Cl--HCO3 exchangers, electrogenic Na+/ HCO3 cotransporters and electroneutral Na+-coupled HCO3 transporters. Despite having structural and functional sim ilarities, the three SLC4 subgroups differ in their specific transport properties (F igure A-1). The electroneutral Cl--HCO3 exchangers transport Cland HCO3 across most mammalian membranes in a 1:1 electroneutral exchange (Sterling et al., 2001). In most cell types, these exchangers driv e the exchange of extracellular Clfor intracellular HCO3 -, but the reaction is bidirecti onal and direction depends on the transmembrane chemical gradients of the substr ates (Romero et al., 2004). The electrogenic Na+/ HCO3 cotransporters (NBCe) mediate moveme nt in the same direction of one Na+ and two or three HCO3 -. In most cases, the NBCes move one Na+ and two HCO3 into a cell, thereby generating a net negative charge within that cel l; however, there are notable exceptions to this rule (Romero et al, 2004). The electroneutral Na+-coupled HCO3 transporters mediate the movement of Na+ and HCO3 across the plasma membrane in one of three ways, defined by each of three H. sapiens proteins belonging to this group: (1) NBCn1 mediates the uptake of one Na+ and one HCO3 -, but no Cl-; (2) NDCBE mediates the uptake of one Na+ and two HCO3 -, coupled with the exit of one Cl-; and (3) NCBE was originally thought to be a Na+-driven Cl--HCO3 147

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transporter, although there is pr eliminary evidence against a Clrequirement (Romero et al., 2004). Boudko et al., (2001a) reported clear evidence of an electroneutral Cl--HCO3 exchanger in the AMG of Ae. aegypti larvae. They detected a strong Cl-/ HCO3 concentration difference across the AMG epithelium, with low levels of Clin the lumen and high levels in the hemolymph. By contrast, HCO3 had the inverse distribution. These results suggest that the lumen is being supplied with a source of HCO3 while simultaneously being depleted of Cl-. Additionally, Boudko et al., (2001a) reported a strong Clefflux from the epithelial cells into the hemolymph which was inhibited by the anion exchange blocker DIDS, thus providing further evidence for AE mediated Clmovement. They hypothesized that CA in the cytoplasm of the epithelial cells catalyzes the production of HCO3 -, which is transported into the lumen by a HCO3 transporter in exchange for a Clion. The Clion is then transported from the cytoplasm into the hemolymph via a nonspecific chlo ride channel (Boudko et al., 2001a). There are three SLC4 genes (AgAEs) predicted to be present in the An. gambiae genome. Members of the Linser laboratory have previously cloned one gene, AgAE1, from An gambiae cDNA (genbank accession # AY280611) (T. J. Seron, personal communication; M. V. Neira, personal communication). This gene is orthologous to the H. sapiens electroneutral Na+-coupled HCO3 transporters as well as to another insect gene, D. melanogaster Na+-coupled HCO3 transporter, NDAE1 (Romero et al., 2000), which exchanges Na+ and HCO3 for Cland H+. The remaining two An. gambiae SLC4 genes, AgAE2 and AgAE3, are predicted to be orthologous to the H. sapiens AE genes. Here I describe the cloning of AgAE3, and the mRNA characterization of AgAE2 and AgAE3, from the An. gambiae larval alimentary canal. Additionally, I suggest 148

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putative functions for AgAE1, AgAE2, and Ag AE3 based on a phylogenetic analysis comparing SLC4 proteins from H. sapiens D. melanogaster Ae. aegypti and An. gambiae genomes. Results Phylogenetic Analysis A phylogenetic tree was generated to compar e the predicted and cloned SLC4 proteins from H. sapiens D. melanogaster, Ae. aegypti and An. gambiae genomes (Figure A-2). The tree showed a distinct clustering of H. sapiens SLC4 proteins into three main groups: electroneutral Cl--HCO3 exchangers (SLC4A1-3), electrogenic Na+-HCO3 cotransporters (SLC4A4 and 5), and electroneutral Na+-coupled Cl--HCO3 transporters (SLC4A7, 8, and 10). SLC4A9, a protein with uncertain functi on, clustered with the electrogenic Na+-HCO3 cotransporters. SLC4A11, a protein with no functional data available, clustered away from all other proteins. Insect SLC4 proteins formed clusters which were distinct from those of the H. sapiens AgAE1 and its Ae. aegypti ortholog clustered with D. melanogaster NDAE1. Together these proteins clustered closer to the H. sapiens Na+-dependent HCO3 transporters, both the electrogenic Na+-HCO3 cotransporters a nd the elecroneutral Na+-coupled Cl-HCO3 transporters, than to the electroneutral Cl--HCO3 exchangers. AgAE2 and AgAE3 (starred) clustered with their uncharacterized Ae. aegypti and D. melanogaster orthologs, and this group of proteins clustered closely with the H. sapiens electroneutral Cl--HCO3 exchangers. Cloning and Characterization of Full-Length AgAE3 cDNA from An. gambiae Larvae An internal fragment of the AgAE3 cDNA was cloned from the An. gambiae larval alimentary canal using gene specific primers; th e primers were designed according to the partial cDNA sequence predicted by Ensembl. Using ge ne-specific sequences from this fragment, RACE was used to amplify and sequence the 3 and 5 ends of the gene (see Table 2-1 for 149

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AgAE3 RACE primers). The full-length cDNA (genbank accession # EU068741) is 2,983 basepairs and spans both the star t and stop codons. The cDNA was determined to be full length by the presence of a stop codon in the 5 UTR and the presence of a 3 po ly-A tail. AgAE3 is located on chromosome 3L ( www.vectorbase.org ). The full length AgAE3 cDNA is predicted to translate to a protein product of 993 amino acids with a molecular mass of 108 kDa ( http://bioinformatics.org/sms/ ). A hydrophobicity plot (Figure A-3; DNAMAN, version 3.2 Lynnon Biosoft, Quebec, Canada) indicated AgAE3 contains ten hydrophobic transmembrane domains (F igures A-3 and A-4). A protein alignment between AgAE3, AgAE1, H. sapiens SLC4A1 (electroneutral Cl--HCO3 exchanger), and splice variants of H. sapiens SLC4A5 (electrogenic Na+/ HCO3 cotransporter), H. sapiens SLC4A8 (electroneutral Na+-coupled HCO3 transporter), and D. melanogaster NDae1 demonstrated that AgAE3 shares considerable homology with other known SLC4 proteins (F igure A-4). A second protein alignment between the three predicted An. gambiae AgAEs, AgAE1, AgAE2, AgAE3 (Figure A-5), indicated that AgAE3 shares 27 % identity with AgAE1 and 59% identity with AgAE2. AgAE mRNA Expression in Alimentary Canal Regions Quantitative PCR (qPCR) was used to detect AgAE mRNA within the mosquito larval alimentary canal. AgAE3 and AgAE2 mRNA expression levels were evaluated from cDNA derived from either whole larval alimentary can al, GC, AMG, PMG, MT or rectum. The results were normalized to the An. gambiae 18s ribosomal RNA gene and reported relative to whole larval alimentary canal (Figure A-6). AgAE2 and AgAE3 mRNA were dete cted in every region tested; however, each region expressed the AgAE s at different levels. AgAE2 was evenly distributed in the GC, AMG, and MT, with less e xpression detected in th e PMG and rectum. No single region expressed AgAE2 si gnificantly above whole larval alimentary canal levels. By 150

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contrast, AgAE3 was significantly expressed by the AMG (P-Value < 0.001) and MT (P-Value < 0.01) above whole larval a limentary canal levels. Discussion AgAE3, a member of the SLC4 HCO3 transporter family, was cloned from, and mRNA expression was characterized in, the alimentary canal of An. gambiae larvae. The predicted protein consists of 993 amino acids and ten predicted hydrophobic transmembrane domains. AgAE3 shares 59% identity with another An. gambiae HCO3 transporter, AgAE2, and indeed the two AgAEs cluster together in a phylogenetic an alysis. AgAE3 shares less similarity with a previously cloned AgAE, AgAE1, and clusters aw ay from this AgAE. These data support the prediction that AgAE3 and AgAE 2 function as electroneutral Cl--HCO3 exchangers, whereas AgAE1 functions as a Na+-coupled HCO3 transporter. The phylogenetic analysis also id entified distinct groups of H. sapiens SLC4 proteins, and as expected, proteins known to have similar functions cluster together in one of three main groups: electroneutral Cl--HCO3 exchangers, electrogenic Na+-HCO3 cotransporters, and electroneutral Na+-coupled Cl--HCO3 transporters. An unexpected result was the placement of SLCA9, which clustered with the electrogenic Na+-HCO3 cotransporters. Although the function of SLC4A9 is still under question, Romero et al (2004) reported that its cDNA is most closely related to the electroneutral Na+-coupled Cl--HCO3 transporters. By contrast, functional studies suggest SLC4A9 behaves as an electroneutral Cl--HCO3 exchangers (Ko et al., 2002). At this time, there is no physiological evidence to suppo rt SLC4A9 clustering with the electrogenic Na+HCO3 cotransporters. One striking property of the H. sapiens and D. melanogaster SLC4 family members is the existence of numerous splice variants for each gene. In the H. sapiens genome, 29 individual variants exist for a total of ten genes. In the D. melanogaster genome, 13 individual variants 151

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exist for a total of two genes. Multip le splice variants may also exist for An. gambiae or Ae. aegypti SLC4 members; this prediction is supported by data indicating the presence of numerous forms of the previously cloned AgAE1, each which differ slightly at the 3 cytoplasmic tail (M. V. Neira, personal communication). Splice va riants of a single gene could represent amplification products of incompletely spliced pre-mRNA, as in the case of H. sapiens SLC4A5 (Romero et al., 2004). Alternativel y, splice variants could be f unctional transcripts of a single gene which differ in regions required for regulat ion, thereby allowing a tighter control over the expression, and subsequent activ ity, of certain genes. AgAE mRNA expression was examined in the fi ve main regions of the larval alimentary canal, the GC, AMG, PMG, MT, and rectum, us ing qPCR. An even distribution of AgAE2 mRNA was detected in the GC, AMG and MT with less expression in the PMG and rectum, although AgAE2 was not detected at significant levels in any region above whole alimentary canal levels. This is supported by data gene rated from microarray an alysis which detected slightly more AgAE2 mRNA in the GC and AMG than in the PMG or hindgut (MT and rectum combined) samples (Neira et al., 2008). By contrast, AgAE3 mRNA was more abundant than AgAE2 in all regions of the alimentary canal although only expression in the AMG and MT was considered statistically significant compared to whole alimentary canal levels. This expression pattern is not consistent with microarray data published by members of the Linser laboratory (Neira et al., 2008) which dete cted approximately equal concen trations of AgAE3 mRNA in all alimentary canal regions, none of which signifi cantly express AgAE3 above the whole larval insect sample. There are several possible explanations fo r the contradicting AgAE3 mRNA expression data. Each technique detects a sp ecific, but distinct sequence of AgAE3 cDNA. If, as predicted, 152

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there are several splice variants of AgAE3, the two techniques may have identified two individual forms of the mRNA. Additionally, the microarray analysis compared mRNA expression in each alimentary canal region to wh ole insect, whereas qPCR analysis compared mRNA expression in each region to whole alimentary canal. If there is significant AgAE3 mRNA expression in the larval carcass, these tw o quantitative techniques would report different results. Finally, the qPCR data is questionabl e in that the sum of mRNA expression in the alimentary canal parts exceeds that of the whole. This data represents an average of four biological replicates each of wh ich consistently indicated simila r expression patterns. Presently there is no explanation for such results and this is sue will need to be resolved prior to publishing AgAE mRNA characterization. Conclusions: In conclusion, I have cloned a full-length version of an An. gambiae SLC4 family member, AgAE3. Phylogenetic analysis suggests that AgAE3 is homologous to another An. gambiae SLC4 member, AgAE2, and that both AgAEs are most similar to the H. sapiens electroneutral Cl--HCO3 exchangers. QPCR analyses indicate that mRNA of both AgAEs is present throughout the alimentary canal, although at different levels in each region. Additionally, it is predicted that the two genes express multiple transcripts; numerous splice variants of an AgAE could potentially increase the mRNA in each region, and may also increase the level of regulation possible. These two AgAE genes, along with the previously cloned AgAE1 gene, are likely to be responsible for the transport of HCO3 from the epithelial cells into the lumen or hemolymph of the alimentary cana l. Identification of protein localization and demonstration of activity will shed new light on an important step in both pHand ionregulation in the larval mosquito alimentary canal. 153

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Figure A1. Function of H. sapiens SLC4 proteins. Electroneutral Cl--HCO3 exchangers transport Cland HCO3 across most mammalian membranes in a 1:1 electroneutral exchange, usually exchanging extracellular Clfor intracellular HCO3 -. Electrogenic Na+/ HCO3 cotransporters mediate movement in the same direction of one Na+ and two or three HCO3 -, usually transporting both ions into of the cell. Electroneutral Na+-coupled HCO3 transporters mediate the movement of Na+ and HCO3 across the plasma membrane by at least two known m echanisms. One mechanism includes the uptake of one Na+ and one HCO3 -, but no Cl-, transporting both ions into the cell. The second mechanism includes the uptake of one Na+ and two HCO3 into the cell, coupled with the exit of one Cl-. 154

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Figure A-2. Phylogenetic analysis of members of the SLC4 family in H. sapiens (black), D. melanogaster (red), Ae. aegypti (blue), and An. gambiae (green) genomes. Phylogeny was prepared using MrBayes with the J TT amino acid substitution model and 1.5 million iterations. AgAE2 and AgAE3 are starred. 155

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Figure A-3. Hydrophobicity plot of AgAE3. The graph plots th e predicted hydropathy (Y-axis) of a protein against the amino acid sequence (X-axis). AgAE3 is predicted to consist of ten transmembrane domains which are identified numerically. 156

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AgAE3 : -----------------------------------------------------------------MAHFLD--IPYADGSMRRKSSIASSVLARHYINNA : 33 AgAE1 : -----------MMDHG-----VVDEEAPIDPRLKNRTF---TADQDFEGHRAHTVFVGVHIPGSSRRHSQRRRHKHHQASRENGDKGSTGSEAERPVTPP : 81 Ndae1a : ---------------------MAEKNEYIELPWTMNSS---SGDDEAP------------------KDPRTGGEDFTQQFTEN----------DFEVTPP : 48 SLC4A1 : -----------------------------------------------------------------MEELQDDYEDMMEENLEQEEYEDPDIPESQMEEPA : 35 SLC4A5 : MKVKEEKAGVGKLDHTNHRRRFPDQKECPPIHIGLPVPTYPQRKTDQKGHLSGLQKVHWG------LRPDQPQQELTGPGSGASSQDSSMDLISRTRSPA : 94 SLC4A8 : MPAAGSNEPDGVLSYQ-----RPDEEAVVDQGGTSTILNIHYEKEELEGHRTLYVGVRMPLGRQSHRHHRTHGQKHRRRGRGKGASQGEEGLEALAHDTP : 95 AgAE3 : SRSINSSCAQINESDKAN--DTNHKVFVQLNELTGSGENQE--WKQTARWIKYEENLEEEADRWGRPHVAALSFHSLLNLRHCLETGVVLMSLEANDLSI : 129 AgAE1 : AQRVQFILGGEVGGEGDGTQHESHPLFSEMEELVK--EGDEMAWKETARWVKFEEDVEEGGNRWSKPHVATLSLHSLFELRSLLLNGTVTLDMEAVSLKQ : 179 Ndae1a : AQRVQFILGEDVDDG----THVSHPLFSEMGMLVK--EGDEIEWKETARWIKFEEDVEEGGNRWSKPHVATLSLHSLFELRRLLVNGSVMLDMEAQNLEV : 142 SLC4A1 : AHDTEATATDYHTTSHP----GTHKVYVELQELVMDEKNQELRWMEAARWVQLEENLGENG-AWGRPHLSHLTFWSLLELRRVFTKGTVLLDLQETSLAG : 130 SLC4A5 : AEQLQDILGEEDEAP-------NPTLFTEMDTLQH--DGDQMEWKESARWIKFEEKVEEGGERWSKPHVSTLSLHSLFELRTCLQTGTVLLDLDSGSLPQ : 185 SLC4A8 : SQRVQFILGTEEDEE-----HVPHELFTELDEICMK-EGEDAEWKETARWLKFEEDVEDGGERWSKPYVATLSLHSLFELRSCLINGTVLLDMHANSIEE : 189 AgAE3 : VAHRVVEQMVARELIHEDDKPVIARALLLRHRHVNENTHGGIFYGSRRRLSSFTSIRSVKNDSRKPRIIPVV---------------------------: 201 AgAE1 : IAELVCENMVNSGTLPVEARDKVIDALLKRHKHQHEFDSKKSRLPLIRSLADIGKNHSSSKNMPRSSTSKSISSANSFPMHVVSSAPSGMHEEGESALGP : 279 Ndae1a : MADLVCDHMVSAGTLPPGVKDKVKDALLRRHRHQHEY-AKKTRLPIIRSLADM-RNHSSSK--KKKSNSK-----------------------------: 208 SLC4A1 : VANQLLDRFIFEDQIRPQDREELLRALLLKHSHAGELEALG---GVKP------AVLTRSGDPSQP-LLP-----------------------------: 190 SLC4A5 : IIDDVIEKQIEDGLLRPELRERVSYVLLRRHRHQTKK-------PIHRSLADIGKSVSTTNRSPARSPGAG----------------------------: 249 SLC4A8 : ISDLILDQQELSSDLNDSMRVKVREALLKKHHHQNEK-KRNNLIPIVRSFAEVGKKQSDPHLMDKHGQTVS----------------------------: 259 AgAE3 : ------------------------------------------------------------------------EINGHGGQSEMRLNINDESYTSSQEDIK : 229 AgAE1 : VSPQTALLYGSKDQRGHYLALPTVEDQANTNSPPNSSSATSNGSNPNASGKPPTNGNSGSGKHLTVPGRAAGENMTQSPSNVSMPRNASSGELQNGEHKT : 379 Ndae1a : ----------------------------HSRPAQNLSSIT--------------------------------EDMVKSPSNQSMARPGSGTELSEQQHKG : 248 SLC4A1 : ------------------------------------------------------------------------------QHSSLETQLFCEQGDGGTEGHS : 212 SLC4A5 : --------------------------PSLHHSTEDLRMRQ---------------------------------SANYGRLCHAQSRSMNDISLTPNTDQR : 290 SLC4A8 : ---------------------------PQSVPTTNLEVKN-------------------------------GVNCEHSPVDLS---------------KV : 286 AgAE3 : MCTILKRIPEGAEATTVLVGAVDFLEQPTIAFVRLAEGIPMPSITEVPIPVRFLFLLLGPQKTDLDYHEVGRSIATLMSNEHFHDIAYTADDREELLSAI : 329 AgAE1 : NTHFMRKIPPGAEASNILVGEVDFLDKTLSAFLRLNTASVMGDLTEVPVPTRFIFILLGPPGSHGSFHEIGRAMATLMSDEIFHEVAYRAKRREHLLAGI : 479 Ndae1a : NTHFMRKIPPGAEASNILVGEVDFLERTLSCFIRLSQAVVLGDLTEVPVPTRFVFILLGPPGSQSNFHEIGRAMATLMSDEIFHEVAYRARKRDHLLSGV : 348 SLC4A1 : PSGILEKIPPDSEATLVLVGRADFLEQPVLGFVRLQEAAELEAV-ELPVPIRFLFVLLGPEAPHIDYTQLGRAAATLMSERVFRIDAYMAQSRGELLHSL : 311 SLC4A5 : KNKFMKKIPKDSEASNVLVGEVDFLDQPFIAFVRLIQSAMLGGVTEVPVPTRFLFILLGPSGRAKSYNEIGRAIATLMVDDLFSDVAYKARNREDLIAGI : 390 SLC4A8 : DLHFMKKIPTGAEASNVLVGEVDILDRPIVAFVRLSPAVLLSGLTEVPIPTRFLFILLGPVGKGQQYHEIGRSMATIMTDEIFHDVAYKAKERDDLLAGI : 386 AgAE3 : DDFLEDSIVLPPSKWE-RQGLLPFEELKARSDMIRLRKKKALDEMIKSKQPPLVTSEEEKKLLTVGGGGG---------------------------GGD : 401 AgAE1 : DEFLDAVTVLPPGEWDPSIRIEPPAAIPPQ---------------EVRKRPPEKNPKEEID--------------------------------------E : 526 Ndae1a : DEFLDAVTVLPPGEWDPTIRIEPPAAIPSQ---------------EVRKRPPE-LPKEEVD--------------------------------------E : 394 SLC4A1 : EGFLDCSLVLPPTDAPSEQALLSLVPVQR--ELLRRR----------YQSSPAKPDSSFYKGLDLNGG-------------------------------: 367 SLC4A5 : DEFLDEVIVLPPGEWDPNIRIEPPKKVPSA---------------DKRKSVFSLAELGQMNGSVGGGGGAPGGGNGGGGGGGSGGGAGSGGAGGTSSGDD : 475 SLC4A8 : DEFLDQVTVLPPGEWDPSIRIEPPKNVPSQ---------------EKRK-MPGVPNGNVCH--------------------------------------I : 432 AgAE3 : GGSDGKKPPNNPLEKTNRLWGGVINDIKRRYPMYKSDIMDGLNTEALAATIFMYFAALSTAITFGGLCSDKTDNLIGISESLLSDAIFGMVFHLLAGQPL : 501 AgAE1 : ELEEQRQREEAGLSRTGRLFGGLINDLKRKAPFYLSDFKDGLSMQCVASWIFLYFACLSPIIPFGGLLGTATGNNIAAMESLVSGFVCGIGYGFFSGQPL : 626 Ndae1a : EEEEARLREENGLSRTGRLFGGLINDIKRKAPWYISDYKDALSMQCVASWIFLYFACLSPIITFGGLLAEATGKHMAAMESLVSGFVCGMGYGFFSGQPL : 494 SLC4A1 : --------PDDPLQQTGQLFGGLVRDIRRRYPYYLSDITDAFSPQVLAAVIFIYFAALSPAITFGGLLGEKTRNQMGVSELLISTAVQGILFALLGAQPL : 459 SLC4A5 : GEMPAMHEIGEELIWTGRFFGGLCLDIKRKLPWFPSDFYDGFHIQSISAILFIYLGCITNAITFGGLLGDATDNYQGVMESFLGTAMAGSLFCLFSGQPL : 575 SLC4A8 : EQEPHGGHSGPELQRTGRLFGGLVLDIKRKAPWYWSDYRDALSLQCLASFLFLYCACMSPVITFGGLLGEATEGRISAIESLFGASMTGIAYSLFAGQAL : 532 AgAE3 : LIIGTTGPLVLFDEALNQFCISNDFNFLTVRVYVGCWLVVIALLVSAFEGSVYVRMFTRFTQEIFSALITLLFIFETSIKLVSVYVRHPLLSEYVYKNIT : 601 AgAE1 : TILGSTGPVLVFETIVYEFCQKVGWDYLTFRFWIGTWISIILVVLVAVDASALVCYITRFTEENFACLIAVIFIYKAIENVFHIGQEYPLNTA-----GG : 721 Ndae1a : TILGSTGPVLVFESIIYEFCLKMGWDYMTFRFWIGMWVAGICIVLTAIDASALVCYITRFTEENFATLIAFIFIYKAIENVMVIGKNFPVN-------QG : 587 SLC4A1 : LVVGFSGPLLVFEEAFFSFCETNGLEYIVGRVWIGFWLILLVVLVVAFEGSFLVRFISRYTQEIFSFLISLIFIYETFSKLIKIFQDHPLQKTYNYN--: 556 SLC4A5 : IILSSTGPILIFEKLLFDFSKGNGLDYMEFRLWIGLHSAVQCLILVATDASFIIKYITRFTEEGFSTLISFIFIYDAIKKMIGAFKYYPINMDFKPNFIT : 675 SLC4A8 : TILGSTGPVLVFEKILFKFCKDYALSYLSLRACIGLWTAFLCIVLVATDASSLVCYITRFTEEAFASLICIIFIYEAIEKLIHLAETYPIHMHSQLDHLS : 632 AgAE3 : VAPLVPTPCLEVSNGTT--------------SIGLLENNLTMVVNGTLGLVSNMDHLLIPEDARGPRNQPNTALFCTILMFGTFSLAYYLKLFRNSHFLG : 687 AgAE1 : KYDCACLPPAAHE--------------------LTPEAVHQWAQYDLRTCKTLNGTLTGADCDKPE-YVSDVFLMSIVLFLGTYIISVILKDFKNALFFP : 800 Ndae1a : IYDCVCTPPIG-----------------------SNASVIDYAKYNWDSCESYNGTLVGGDCGTP--PTENVFLMSVVLCAGTFLISTVLKEFKNALFFP : 662 SLC4A1 : -VLMVPKP-------------------------------------------------------QGP--LPNTALLSLVLMAGTFFFAMMLRKFKNSSYFP : 598 SLC4A5 : TYKCECVAPDTVNTTVFNASAPLAPDTNASLYNLLNLTALDWSLLSKKECLSYGGRLLGNSCKFIP----DLALMSFILFFGTYSMTLTLKKFKFSRYFP : 771 SLC4A8 : LYYCRCTLPENPNNHTL----------QYWKDHNIVTAEVHWANLTVSECQEMHGEFMGSACGHHGPYTPDVLFWSCILFFTTFILSSTLKTFKTSRYFP : 722 AgAE3 : RTARRALGDFGVPISIAIFVLVDYLTPQVYTEKLSVPEGLSPSDQTRRGWLIP----MGGVPSWLPFLASIPALLMYILIFMETHIAELIVDKPERGLKK : 783 AgAE1 : AVVRQFISDFSVTIAIFSMTLLDVFTR-IATPKLDVPSEFKRTI-PDRGWIIM--PFHESNPAWSSALAILPALLGTILIFMDQQITAVIINRKEHKLTK : 896 Ndae1a : SIVRQYISDFSVLIAIFAMSFFDYSLG-VPTQKLEVPNELKPTL-STRGWLIP--PFVEKNPWWSAIIAVFPALLGTILIFMDQQITAVIVNRKENKLKK : 758 SLC4A1 : GKLRRVIGDFGVPISILIMVLVDFFIQDTYTQKLSVPDGFKVSNSSARGWVIHPLGLRSEFPIWMMFASALPALLVFILIFLESQITTLIVSKPERKMVK : 698 SLC4A5 : TKVRALVADFSIVFSILMFCGIDACFG-LETPKLHVPSVIKPTR-PDRGWFVA--PFG-KNPWWVYPASILPALLVTILIFMDQQITAVIVNRKENKLKK : 866 SLC4A8 : TRVRSMVSDFAVFLTIFTMVIIDFLIG-VPSPKLQVPSVFKPTR-DDRGWIIN--PIG-PNPWWTVIAAIIPALLCTILIFMDQQITAVIINRKEHKLKK : 817 AgAE3 : GSGLHMDVVLLCVINTVCGFFGMPWHSAATVRSVSHVSAVTIMSRTHAPGDAPHIADVKEQRVSGFVVSVLVGLSVVMAPILRLIPMSVLFGVFLYLGIA : 883 AgAE1 : GCGYHLDLFVLACLIQICTMMGLPWFVAATVLSINHVNSLKKESETAAPGEKPQFIGVREQRVTHILIFLMIGCSVLLTPLLSHIPMPVLYGVFLYMGVS : 996 Ndae1a : GCGYHLDLFILSILIAICSMMGLPWFVAATVLSINHVNSLKLESECSAPGEKPQFLGVREQRVTHILIFLTIGVSVLLTPLLGNIPMPVLFGVFLYMGVA : 858 SLC4A1 : GSGFHLDLLLVVGMGGVAALFGMPWLSATTVRSVTHANALTVMGKASTPGAAAQIQEVKEQRISGLLVAVLVGLSILMEPILSRIPLAVLFGIFLYMGVT : 798 SLC4A5 : AAGYHLDLFWVGILMALCSFMGLPWYVAATVISIAHIDSLKMETETSAPGEQPQFLGVREQRVTGIIVFILTGISVFLAPILKILGLIIVRRLLDFI--F : 964 SLC4A8 : GCGYHLDLLMVAIMLGVCSIMGLPWFVAATVLSITHVNSLKLESECSAPGEQPKFLGIREQRVTGLMIFVLMGCSVFMTAILKFIPMPVLYGVFLYMGVS : 917 Figure A-4. Protein alignment between An. gambiae AgAE3 and AgAE1, H. sapiens SLC4A1, and splice variants of H. sapiens SLC4A5, H. sapiens SLC4A8, and D. melanogaster NDae1. Alignment demonstrates that AgAE 3 shares significant homology with other known SLC4 proteins. Regions with high similarity are highlighted in black (100%). Regions of lesser similarity are highlighted in grey (>80%) and light grey (>60%). Predicted transmembrane domains are indicated with red bars. AgAE3 : SMSGVQLFERLRLFLMPVKHHPQVLFVRRVRTWKMHLFTFIQILALAVMLAVKSSPFAMAFPSFLIFMVPIR-IQMEKFFSPLELRALDSSQPNEGAEDE : 982 AgAE1 : ALKGLQFFDRLLIMLMPAKYQPDYMFLRQVPIRRVHLFTMIQLACFAVLWLIKSLSITSILFPLMLVVMIGVRKSLDYIFTKRELKILDDIMPEMTKRAR : 1096 Ndae1a : SLKGLQFFDRILIMFMPAKYQPDYMFLRQVPIKRVHLFTMIQLACLIILWLIKSFSQTSILFPLMLVVMIGIRKALDLVFTRRELKILDDIMPEMTKRAA : 958 SLC4A1 : SLSGIQLFDRILLLFKPPKYHPDVPYVKRVKTWRMHLFTGIQIICLAVLWVVKSTPASLALPFVLILTVPLRRVLLPLIFRNVELQCLDADDAKATFDEE : 898 SLC4A5 : SQHDLAWIDN----ILPEKEKKETDKKRKR-KKGAHEDCDEEPQ----------FPPPSVIKIPMESVQSDPQNGIHCIARKRSSSWSYSL--------: 1040 SLC4A8 : SLQGIQFFDRLKLFGMPAKHQPDFIYLRHVPLRKVHLFTLIQLTCLVLLWVIK-ASPAAIVFPMMVLALVFVRKVMDLCFSKRELSWLDDLMPES-KKKK : 1015 AgAE3 : P--DFYEQAPIPA--------------------------------------------------------------------------------------: 993 157

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AgAE1 : ADDLHQLEDGEDSGRHSADNLQLPLENGTVNEKGKINISEEVNRTTIWKQVNNCNVLFTKKQTPVKHSQQQLSKLSETEKRLSTMREEDEVADEPSRQLS : 1196 Ndae1a : ADDLHKLDAEVG--------------------------------------------LLARIFPWGKGSR-------------------SRVVTKPP---: 991 SLC4A1 : EGRDEYDEVAMPV--------------------------------------------------------------------------------------: 911 SLC4A5 : ---------------------------------------------------------------------------------------------------: SLC4A8 : LDDAKKKAKEEE---------------------------------------------------------------------------------------: 1027 AgAE3 : ---------------------------------------------------------------: AgAE1 : HQKRTQKMKREFWMNSRSGSSNGRANSNNSASSTTAAAGGKIVCAEESAALSRGSSKSASETQV : 1260 Ndae1a : ------------------GLDDGIVGSGG------AVGAGLITCTTSNANEKEFEAQSSLLKK: 1030 SLC4A1 : ---------------------------------------------------------------: SLC4A5 : ---------------------------------------------------------------: SLC4A8 : -----------------------------------------VIVLAPTVYLGASNYRT-----: 1044 Figure A-4. Continued. 158

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AgAE1 : --MMDHGVVDEEAPIDPR LKN RTF TADQDFEGHRAHTVFVGVHIPGSSRRHS QRR RHKHHQASR ENGDKGS TGS E AERPVT PPA QRVQF I : 88 AgAE2 : RHHPHKSRKFSL QEYHP EWRRQ S G T EGGPTGR RISVQPEDATLQEADIDELT SHR S D DPR A L R R HKVSAQS GAS LVNINRKEAA PPLQH L : 90 AgAE3 : -----MAHFLDI PYAD GSMRRKSSIA SSVLAR --------------------HYINN ASR S I N---------SS C AQINE S DKA N----: 51 AgAE1 : L GGEVGGEGDGTQH ESH P LFS EME ELVKEG DEMAWKETARWVKFEEDVEEGG NRWS KPHVATLSL HSLFELRSLL LNG T V T LDMEAVSL K : 178 AgAE2 : L --PSSKYKKMYDHSP H E VFVQLDELTGSGEDR EWKETARWIKYEEDVEEGADRWGRPHVASLSFHSLLNLR R CLETGVVLMDLEEKDLP : 178 AgAE3 : -------------DT N H K VFVQLNELTGSGENQ EWKQTARWIKYEENLEEE ADRWGRPHVAA LSFHSLLNLRH CLETGVVLMS LEAN DLS : 128 AgAE1 : QIAELV C E N MVNSGTL PVEAR DKVID ALLK RHKHQHE FDSKKSRLPLIRSLADIG KNHSSSKNMPRS S TSKSISSANSFPMHVVSSAPSG : 268 AgAE2 : SVAY RVVEQMVVDELIHEDDKA VIMRALLLRHRHVNES S --------------HGGFSFGP KRKY SSYTSLQ VSRPGPQQPAHQPAKDQQ : 254 AgAE3 : IVAH RVVEQMVARELIHEDDKP VIA RALLLRHRHVNEN T --------------HGGIFYGSRRRL SSFTSIRSVK N-------------: 190 AgAE1 : MHEEGES ALGPVSPQTALLYGS KDQRGHYLALPT VEDQA NTNSPPNSSSATS NGSN PNASGKP P T NGN SGSG K H LTV PGRAAGE NMT Q S P : 358 AgAE2 : RHQRHAS GPHRCRRSGSQDSSS TATAAAAPVTLT TVTSA VGMRRNNSPLSLT SVDDKKPRIVPA TEINGHGGG H G E T RINISEETYTSSQ : 344 AgAE3 : -----------------------------------------------------D S RKPRIIPVVEINGHGG-QSEMRLNINDESYTSSQ : 225 AgAE1 : SNVS M PRNASS GELQNGEHKTNTHFMRKIPP GAEASN ILVGE VDFLDKTLSAFLRLNTASVM GDLTEVPVPT RFIFILLGPPGS HGSFHE : 448 AgAE2 : EDIKMR T QKES -------------ILKRIPEGAEATTVLVGAVDFLEQPTIAFVRLAEGIPMPSITEVPIPVRFLFILLGPQKT E LDYHE : 421 AgAE3 : EDIKMC T -----------------ILKRIPEGAEATTVLVGAVDFLEQPTIAFVRLAEGIPMPSITEVPIPVRFLFLLLGPQKT D LDYHE : 298 AgAE1 : IGRA MATLMSDEI FHE VAYRAKRREH LLAGIDEFLDAVTVLPPGE W DPSIRI EPPAAIPPQEV R K RPPEK NPKE E I DEELEE QRQREEAG : 538 AgAE2 : VGRSIATLMSNEHFHDIAYKADDRR QLLSAINEFLDDSIVLPPGKWERQA LLPFEELKAKSDMIRLRKKKALDEK IKSKQS QLLTSEEEK : 511 AgAE3 : VGRSIATLMSNEHFHDIAYT ADDREELLSAIDD FLE DSIVLPPS KWERQG LLPFEELKARSDMIRLRKKKALDEM IKSKQPPLVTSEEEK : 388 AgAE1 : -------------------------L S RTG RLFGGLINDLKRKA P F Y L SDFKDGLSMQ C VASWIFLYFAC LSPII P FGGLLGTAT G N N I A : 603 AgAE2 : KLLAAAGGA G D G G GGG DGKKPPK NPLEKTNRLWGGLINDIKRRYPMYKSDIMDGLNTE T LAATIFMYFAALSTAITFGGLS SDKTH NLIG : 601 AgAE3 : KLLTVGGGG G G G D GGS DGKKPPN NPLEKTNRLWGGVINDIKRRYPMYKSDIMDGLNTE A LAATIFMYFAALSTAITFGGLC SDKTD NLIG : 478 AgAE1 : AMESLVSGFV C GIG Y GFFSGQPLT ILGSTGPVLVFETIV Y EFCQKVGWDYLTF R F WIGT WIS IIL VVLV A VDAS ALV CYITRFTEEN F AC : 693 AgAE2 : ISETLVSASM V GLVFHLLAGQPLVIIGTTGPLLLFDEALNQFCISNDFS FLTVRVYVGCWLA VIALVVSAFEGSVYVRLFTRFTQEIFSA : 691 AgAE3 : ISESLLSDAI F GMVFHLLAGQPLLIIGTTGPLVLFDEALNQFCISNDFNFLTVRVYVGCWL V VIALLVSAFEGSVYVRMFTRFTQEIFSA : 568 AgAE1 : LIA VIFIYKAI ENV FHI GQEYPLN T AGGKYD CACLPPAAH--------------ELTPE A V HQWAQYDL RTCKTLNGTL TGAD CDKP EYV : 769 AgAE2 : LITLIYIV ETVMKLLSVYK RHPLLA EYLYKNVTEPQPLPM P Y LEEERNGTTP T S ELLA E S LTTAVNGTIA M AQD AANLLIPEDGTGPL N R : 781 AgAE3 : LITLLFIFETS IKLVSVYV RHPLLSEYVYKNITVAP L VPT P C LEVS-NGTTS IGLLENNLTMVVNGTLG L VSNMDH LLIPEDARGPR N Q : 656 AgAE1 : SD VFL MSIVLF LGTYI I SVILKD FKNALF FPAVVR QFI S DFS V T I A I FSMTLLDVFT -RI A T P KLD VPSEFKRT IPDR GWIIMPFHESNP : 858 AgAE2 : PNTALFCTILT LGTFSLAYYLKLFRNSHFLGRN ARRALGDFGVPISIALFVLVDYMI PQVYTEKLSVPEGLSPSDETRRGWIIPLG GVP : 870 AgAE3 : PNTALFCTILMFGTFSLAYYLKLFRNSHFLGRT ARRALGDFGVPISIAIFVLVDYLTPQVYTEKLSVPEGLSPSDQTRRGWLIPMG GVP : 745 AgAE1 : AW SSALAI LPALLGTILIFMDQQITA VIINRK E HKL T KGC G Y HLDLF VLACL IQICTMMGLPWFVAATVL SIN HVNSL KKES E T A APGEK : 948 AgAE2 : GWMPFVAG IPALLVYILIFMETHISELIVDKPERGLKKGSGLHMDIVLLC F LNTVCGFFGMPWHC AATVRSVTHVSAVTIMSRTHAPGDA : 960 AgAE3 : SWLPFLAS IPALLMYILIFMETHIA ELIVDKPERGLKKGSGLHMDVVLLCV INTVCGFFGMPWHS AATVRSVSHVSAVTIMSRTHAPGDA : 835 AgAE1 : P QFIGVREQRVTHILIF LMIGC SVLLT PLLSHIPMP VLYGVFLYMGVSAL K GLQFFD RLL I M LMPA K YQP DYMFLRQ VPIRRVHLFTM IQ : 1038 AgAE2 : PHIT DVKEQRISGFF VSLMVGLSVT MAPILRLIPMSVLFGVFLYMGIASMSGVQFFERLRLYLMPVKHHPQVP FVRRVPTWKMHLFTFVQ : 1050 AgAE3 : PHIA DVKEQRVSGFVVSVLVGLSVVMAPILRLIPMSVLFGVFLYLGIASMSGVQ L FERLRLFLMPVKHHPQVLFVRRVR TWKMHLFTFIQ : 925 AgAE1 : L ACFAVLWL IKSLSIT S I L FPLMLVVMIG VRKS L DYIFTKRELKI LDDIMP EMTKRARAD DLHQ LEDGEDSGRHSADNLQLPLENGTVNE : 1128 AgAE2 : ILALAMLWAVKSSPFSLAFPFFLIMMVP IRKQ L ESIFSPLELRA LDGSQP NEGAEDEPD FYEQ APIPA--------------------: 1118 AgAE3 : ILALAVML AVKSSPFA-MA----------------------------------------------------------------------: 943 AgAE1 : KGKINISEEVNRTTIWKQVNNCNVLFTKKQTPVKHSQQQLSKLSETEKRLSTMREEDEVADEPSRQLSHQKRTQKMKREFWMNSRSGSSN : 1218 AgAE2 : -----------------------------------------------------------------------------------------: AgAE3 : -----------------------------------------------------------------------------------------: AgAE1 : GRANSNNSASSTTAAAGGKIVCAEESAALSRGSSKSASETQV : 1260 AgAE2 : -----------------------------------------: AgAE3 : -----------------------------------------: Figure A-5. Protein alignment between An. gambiae AgAE1, AgAE2, and AgAE3. Alignment demonstrates that AgAE3 shares a higher sequence identity with AgAE2 (59%) than with AgAE1 (27%). Regions with high sim ilarity are highlighted in black (100%). Regions of lesser similarity are highlighted in grey (>80%) and light grey (>60%). 159

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160 Figure A-6. AgAE mRNA expr ession in the regions of An. gambiae alimentary canal ad determined by qPCR. Results were normalized to the An. gambiae 18s ribosomal RNA gene and reported relative to whole larval alimentary canal. Whole alimentary canal sample was normalized to a value of and is indicated by a dashed line. Error bars indicate standard error between three (AgAE2) or four (AgAE3) individual runs. Abbreviations: AMG, anterior midgut; GC, gastric caeca; MT, Malpighian tubules; PMG, posterior mi dgut. *: P-Value < 0.05; ***: P-Value < 0.001.

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BIOGRAPHICAL SKETCH Kristin E. Smith was born in Somers Point, NJ and raised in West Chester PA. She is the oldest of three half-sib lings, a sister and two brothers. Sh e received her Bachelor of Science degree from Pennsylvania State University in 200 4, graduating with a major in microbiology and a minor in biochemistry and molecular biology. It was during her undergraduate studies that she participated in the co-operative education program, working for a total of 18 months at GlaxoSmithKline Pharmaceuticals at the Collegev ille and King of Prussia, PA branches. At GlaxoSmithKline she developed a love for the industrial work se tting. Following graduation, Kristin moved to Gaivesville, FL to pursue an advanced degree at the University of Florida College of Medicine. A love for the smaller community at Whitney and isolated environment brought her to the Whitney Laboratory where she wo rked under the direction of Prof. Paul Linser characterizing various ionand phregulatory proteins in the larval mosquito alimentary canal. Upon completion of her doctorate degree, she has secured a post-doctoral position at Mayo Clinic in Jacksonville FL, invest igating the mechanisms of action of various psychotropic drugs. 172