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Oostatic and trypsin modulating effects of AEDES-TMOF peptide and peptide-mimics on the mosquito, Aedes aegypti Linnaeus and the stable fly, Stomoxys calcitrans Linnaeus

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Oostatic and trypsin modulating effects of AEDES-TMOF peptide and peptide-mimics on the mosquito, Aedes aegypti Linnaeus and the stable fly, Stomoxys calcitrans Linnaeus
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Okedi, Loyce Martha Alungat, 1959-
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English
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xii, 139 leaves : ill. ; 29 cm.

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Animal feeding behavior ( jstor )
Blood ( jstor )
Digestion ( jstor )
Eggs ( jstor )
Enzymes ( jstor )
Female animals ( jstor )
Insects ( jstor )
Midgut ( jstor )
Oocytes ( jstor )
Ovaries ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF ( lcsh )
Entomology and Nematology thesis, Ph.D ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 2000.
Bibliography:
Includes bibliographical references (leaves 127-137).
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Printout.
General Note:
Vita.
Statement of Responsibility:
by Loyce Martha Alungat Okedi.

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OOSTATIC AND TRYPSIN MODULATING EFFECTS OF AEDES-TMOF PEPTIDE AND PEPTIDE-MIMICS ON THE MOSQUITO, AEDES AEGYPTI LINNAEUS AND THE STABLE FLY, STOMOXYS CALCITRANS LINNAEUS By LOYCE MARTHA ALUNGAT OKEDI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF 2000 FLORIDA

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I dedicate my dissertation to my parents, Samuel and Esther and my children Brian Samuel and Esther Grace.

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ACKNOWLEDGMENTS I thank all the people in Gainesville's Department of Entomology and Nematology and the USDA Center for Medical, Agricultural, and Veterinary Entomology who assisted me during the course of my studies. I greatly appreciate the supervision of my committee chairman. Dr. David A. Carlson and also Dr. Jerry Hogsette I especially thank them and Dr. Steve Valles for allowing me to use laboratory facilities and for their kind advice during the study. I thank Michelle Hossack, Frank Washington, Tok Fukuda, Marlene Faulkner, Charles Strong, and James Thomas for their assistance during my research. I thank Dr. Don Barnard and the administrative staff of the Mosquito and fly Research Unit for making my stay a pleasant lasting experience. I thank Dr. J. Nation, Dr. G. Smart and Dr. J. Capinera for all the guidance and for looking out for me during difficult times. I thank my parents and family for providing me with love and encouragement during the course of these studies. Last but not least, I thank the Director-General of the National Agricultural Research Organization, Entebbe, Uganda, Prof. Joseph Mukiibi for having provided me with a Uganda Government scholarship that enabled me to enroll fo doctoral training here at the University of Florida. Ui

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS iii LIST OF TABLES vii LIST OF FIGURES viii ABSTRACT xi CHAPTERS 1. GENERAL INTRODUCTION 1 Digestive physiology in Aedes aegypti 1 Egg Development in Aedes aegypti 5 Hormonal Control of Insect Reproduction in Aedes aegpyti 8 Hormonal Control of Reproduction in Stomoxys calcitrans 12 Relationship Between Digestive Enzymes and Egg Development .... 14 Rationale and Objectives 14 2. OOSTATIC ACTIVITY OF SYNTHETIC PEPTIDES AND PEPTIDE MIMICS ON THE MOSQUITO, AEDES AEGYPTI LINNAEUS AND THE STABLE FLY, STOMOXYS CALCITRANS LINNAEUS 18 Introduction 18 Specific Objective 22 Materials and Methods 22 Experimental Insects 22 Measurement of Oostatic Activity 25 Dose Response Effect of Treatments on A. aegypti 27 Effect of Treatments on the Stable Fly, S. calcitrans 27 Results 29 Effect of Ae-TMOF Peptides on A. aegypti 29 Effect of Pyrene-derived and Other Aedes-TMOF on A. aegypti 32 Dose Response Effect of Various Treatments on A. aegypti 42 iv

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Effect of Ae-TMOF Peptides on S. calcitrans 43 Discussion ^'^ 3. TRYPSIN MODULATING PROPERTIES OF SYNTHETIC PEPTIDES AND PEPTIDE MIMICS ON THE MOSQUITO AND THE STABLE FLY 53 Introduction 53 Specific objective 58 Materials and Methods 58 Experimental insects 58 Preparation of Insect Midguts for BApNA Assays 60 Protein Estimation Assays 61 Spectrophotometric Determination of Protein 62 Preparation of BApNA calibration curves 63 Preparation of insect midguts for [1,3^H] DFP assays ... 64 Determination of [1,3^H] DIP-TLE Products 64 Calibration if [1,3^H] DIP-TLE derivatives 67 , Polyacrylamide Gel Electrophoresis and Flourography 69 Results 72 The effect of Treatments on Blood Digestion (BApNA) 72 Comparison of Serine Proteases Produced in A. aegypti Females Treated with Ae-TMOF Peptides and Peptide Mimics 7 6 PAGE and fluorography of TLE and chymo-TLEs 80 Discussion 90 4. EVIDENCE OF OOSTATIC AND TRYPSIN MODULATING ACTIVITY IN OVARY-DERIVED EXTRACTS OF THE STABLE FLY, S. CALCITRANS Introduction 96 Materials and Methods 98 Stable fly rearing 98 Preparation of Stable Fly Ovary-Derived Factor (SFOV) .... 99 Characterization of the SFOV 99 Results 100 Preparation of Stable Fly Ovary-Derived Factor (SFOV) ... 100 Evaluation of Oostatic Activity 102 Characterization of the SFOV 102 Evaluation of Trypsin Modulating Properties 105 : MALDI-TOF-Mass Spectra of SFOV Sample 112 i Discussion 113 5. SUMMARY AND DISCUSSIONS 116 REFERENCES 121 V

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BIOGRAPHICAL SKETCH SIGNATURES

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LIST OF TABLES Table page 2-1 Egg development stages in the mosquito, A. aegpyti 26 2-2 Egg development stages in the stable fly S. calcitrans 26 2-3 Effect of TMOF peptides on egg development in A. aegpyti 30 2-4 Dose response of peptide mimics injected into A. aegypti 33 2-5 Response to pyrene peptide-mimics by A. aegypti 33 2-6 Dose response effects of Ae-TMOF peptide and mimics on egg development in A. aegypti 44 2-7 Effects of injected Ae-TMOF peptides and mimics on the stable fly, S. calcitrans 48 28 Effects of topical treatments of Ae-TMOF peptides and mimics on egg development in the stable fly, S. calcitrans 49 31 Trend of production of [1,3^H] DIP-TLE derivatives in the assays 70 3-2 Effect of TMOF peptides on trypsin activity in the female A. aegypti 73 3-3 Effect of TMOF peptides on trypsin activity in the female S. calcitrans 75 34 Effect of TMOF peptides on production of [1,3^H] DIP-TLE derivatives in A. aegypti with and without TPCK 80 41 A comparison of the effect of peak fractions of SFOV on ovarian development in A. aegypti 106 4-2 A comparison of the effect of SFOV on trypsin activity in female A. aegypti Ill vii

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LIST OF FIGURES Figure Paqe 2-1 Structures of synthetic TMOF peptide and peptide mimics used in the study 23 2-2 Structures of synthetic TMOF peptide and peptide mimics used in the study 24 2-3 Micro-capillary pullman used to pull capillary tubes to make glass needles 28 2-4 Peritrophic membrane forming around a freshly ingested blood meal in A. aegypti (1-2 hpbm) 35 2-5 Digested blood meal and developing eggs, 4 8 hpbm 35 -j 2-6 Developing eggs, 30 hpbm 36 2-7 Oviposited eggs, 72 hpbm 36 2-8 Appearance of gut and ovaries of TMOF-A treated mosquito, 30 hpbm 37 2-9 Appearance of gut and ovaries of B2 treated mosquito, 30 hpbm 37 2-10 Survival and growth inhibition effects of pyrene Ae-TMOF derived peptide-mimics on A. aegpyti 39 2-11 Appearance of gut and ovaries of A5 treated mosquito, 30 hpbm 40 2-12 Appearance of gut and ovaries of IPba (OtBu) -NH2 treated mosquito, 30 hpbm 40 2-13 Appearance of gut and ovaries of 9Fla-0H treated s mosquito, 30 hpbm 41 2-14 Appearance of gut and ovaries of PPPPPPNH2 treated mosquito, 30 hpbm 41 viii

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2-15 Growth and inhibition of A. aegypti oocytes following injection with AE-TMOF peptide and peptide mimics 4 5 216 Survival of injected A. aegypti mosquitoes after injection with Ae-TMOF peptide and peptidemimics 4 6 31 Charge relay system for a chymotrypsin molecule 57 3-2 Irreversible deactivation of TLEs by DFP .". 59 3-3 Spectra for standard trypsin degradation of BApNA by measurement of the free p-nitroaniline 66 3-4 Trend of radioactivity retained in standard incubations 71 3-5 Trend of radioactivity retained in incubations involving A. aegypti mosquitoes treated with various peptides 77 3-6 Effect of Ae-TMOF peptides and peptide mimics on tritiated DIP-TLE (with / without TPCK) 81 3-7 PAGE Fluorography of tritiated DlP-trypsinchymotrypsi^ like isozymes of female A. aegypti 83 3-8 PAGE Fluorography of tritiated DlP-trypsinchymotrypsin-like isozymes of female A. aegypti 85 3-9 PAGE Fluorography of tritiated DlP-trypsinchymotrypsinlike isozymes of female A. aegypti 88 3-10 PAGE Fluorography of tritiated DlP-trypsinchymotrypsin-like isozymes of female A. aegypti (with TPCK) 89 311 PAGE Fluorography of tritiated DlP-trypsinchymotrypsin-like isozymes of female S. calcitrans 91 41 Reverse phase HPLC gradient 101 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy OOSTATIC AND TRYPSIN-MODULATING EFFECTS OF AEDES-TMOF PEPTIDE AND PEPTIDE-MIMICS ON THE MOSQUITO AEDES AEGYPTI LINNAEUS AND THE STABLE FLY, STOMOXYS CALCITRANS LINNAEUS By Loyce Martha Alungat Okedi May 2000 Chairperson: D. A. Carlson J. Nation, J. Hogsette and E. Greiner Major Department: Entomology and Nematology The decapeptide trypsin modulating oostatic factor (TMOF) is oostatic when introduced too early in a gonotrophic cycle of the female mosquito, Aedes aegypti. The possibility that Aedes-TMOF-derived synthetic peptides and peptide mimics may affect egg development through modulating trypsin biosynthesis was evaluated in the mosquito, A. aegypti and on the stable fly, S. calcitrans . Additionally, a search for an ovary-derived factor similar in properties to the Aedes-TMOF was conducted. Oostatic activity in Aedes-TMOF peptides and peptide mimics varied from being oostatic to toxic to no effect. Synthetic peptides A, A5, A7 , B, and B2 were oostatic to mosquitoes, and A7 and A5 were slightly oostatic to stable flies. xi

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Peptides A4 and A6 were toxic to mosquitoes and also to stable flies. Peptide mimics with pyrene-butyric acid and carboranylalanine amidated to the N-terminal of the TMOF-A were oostatic to mosquitoes. A direct relationship was observed between the degree of oostatic activity and trypsin-modulating properties of these compounds. A stable fly ovary-derived factor was also discovered that had oostatic and trypsin-modulating properties when introduced to mosquitoes. Oostatic activity with the Aedes-TMOF synthetic peptides was inconsistent in stable flies, trypsin modulation was not regulated as their trypsin-like enzyme levels remain more or less constant through 5-7 days, over the five blood meals required to mature an egg batch. xii

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CHAPTER 1 GENERAL INTRODUCTION Physiology of Blood Digestion in Aedes aegyptl Female mosquitoes, Aedes aegypti, require a blood meal from a vertebrate host to obtain nutrients required for oogenesis. Ingestion of a blood meal triggers numerous morphological and biochemical changes in the midgut that make it competent to digest a blood meal. Electron microscopic studies of the midgut of the mosquito. A, aegypti show that it has three types of cells, digestive cells (Hecker et al. 1971), endocrine cells (Brown et al . 1985) and regenerative cells (Hecker et al. 1971). The midgut undergoes changes before and after ingestion of a blood meal whereby in unfed females, the digestive cells of the midgut contain no secretory granules (Hecker et al . 1971). In blood-fed mosquitoes, the surface of the rough endoplasmic reticulum greatly increases by loosening its tight whorls, a condition that exists in starved and sugarfed mosquitoes (Bauer et al . 1977). The sectretory granules appear in increasing number 8 and 24 hours post-blood meal (hpbm) (Rudin & Hecker 1979) . Free and membrane-bound 1

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2 ribosomes increase up to 24 and 36 hpbm, reach a peak and decline by 96 hpbm (Gander et al . 1980). The increase in amount of endoplasmic reticulum after a blood meal is due to the production of trypsin-like enzymes (Rudin & Hecker 1979) and the peritrophic membrane (Graf et al. 1986) . This was further supported by the fact that there was a large increase in trypsin enzyme activity (Briegel & Lea 1985) which was absent in midguts from un-fed A. aegypti. Immediately following the blood meal, two trypsins of molecular weight 32 and 36kDa are present in the midgut. These early forms of trypsin are replaced a few hours later with a 30 kDa trypsin, the late and major proteolytic enzyme involved in blood meal digestion (Barillas-Mury et al . 1991; Graf & Briegel 1989) . Tryptic activity and trypsin mRNA was only detectable 5 hpbm (Graf & Briegel, 1989) . Felix et al. (1993) established that trypsin synthesis occurred in two phases in female A. aegypti. The first phase of trypsin synthesis begins within a few hours of blood feeding when trypsin is synthesized (translated) directly from preformed mRNA. Subsequently, the peptides resulting from the initial phase of digestion, stimulate production of further mRNA from which additional trypsin is produced 8-10 hpbm. The second phase of trypsin synthesis starts about 810 hpbm, when trypsin is synthesized (transcribed) from

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3 newly formed mRNA and utilizes amino acids derived directly from the current blood meal (Felix et al . 1993) . Food ingested by insects is mostly macromolecular , in form of polysaccharides, proteins and lipids. While small molecules can pass into tissues directly, larger molecules must be broken down into smaller components before absorption through the peritrophic membrane and midgut epithelium can occur. Proteins, the predominant constituent of blood are usually found with carbohydrate and lipid moieties that are released during digestion and are digested by carbohydrases and esterases. Blood protein digestion involves two steps and two classes of enzymes. Foremost, proteinases or endopeptidases cleave large proteins into large peptides and then peptidases or exopeptidases progressively shorten the large peptides by removal of one or two amino acids at a time all the way to individual amino acid fragments. Endopeptidases hydrolyze peptide bonds at specific internal positions in the protein chain. The positions attacked are recognized by the nature of amino acids adjacent to the dipeptide bonds that are attacked. The principal endoproteases in the majority of insects are the serine proteases, trypsin and chymotrypsin . Trypsin and chymotrypsin belong to a family of endopeptidases that bear catalytically active serine and histidine residues at their

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4 active center. Trypsin and chymotrypsin recognize and hydrolyze peptide bonds involving the carboxyl group of the amino acids arginine or lysine in protein molecules. Chymotrypsin is less specific as cleaving of bonds involve carboxy groups of tyrosine, phenyl alanine and tryptophan and bonds involving other amino acids more slowly. Purification of trypsins extracted from A. aegypti 24 hpbm followed by SDS-PAGE at pH 8.4, yielded 3-6 active bands all with molecular masses in the range of 25-33 kDa . Analysis of amino acid sequences confirmed they belonged to the subclass of serine proteases and there was homology between mosquito trypsin and invertebrate and vertebrate trypsins. Hence the insect trypsin usually being referred to as trypsin-like enzymes (TLEs) (Gooding 1973, Briegel & Lea 1975, de Loof 1987) ) . Borovsky (1986) proposed a system for blood digestion in mosquitoes and proposed the following theory: "A signal is sent to midgut epithelial cells to initiate trypsin synthesis. Trypsin is secreted into the ectoperitrophic space, traverses the pores in the peritrophic membrane and begins to digest the clot around its periphery. Peptides, amino acids and polypeptides smaller than 23,000 daltons diffuse out through the peritrophic membrane into the ectoperitrophic space. Polypeptides and peptides are then further digested into free amino acids and get transported

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5 through the midgut epithelial cells into the hemolymph. The free amino acids are the used by the fat body to synthesize vitellogennin (egg yolk protein)". This theory is supported by studies on A. aegypti that show that 60% of total proteolytic activity occurs around the peritrophic area, and the rest of the activity is inside the midgut epithelium (Graf and Briegel 1982) . Egg Development in Aedes aegypti The female reproductive system in most insects is composed of a pair of ovaries connected to a pair of lateral oviducts. The lateral oviducts join to form the median oviduct that opens posteriorly into a genital chamber, the uterus (Chapman 1998). Opening laterally into the genital chamber is a spermatheca, a sac for storing sperm, and frequently a pair of accessory glands. Ovaries consist of ovariole compartments within which oocytes develop. Among the Diptera, the ovarioles open together into an expansion of the oviduct called the calyx. Each ovariole consists of a distal germarium in which oocytes stem from the oogonia and a more proximal vitellarium where yolk deposition into the oocytes takes place. The germarium and vitellarium reflect two phases of oocyte growth: the previtellogenic phase when the germarium

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6 is regulated by the oocyte genome, and the trophic phase is when the larger vitellarium is regulated by genes outside the oocyte also involved in embryonic growth. Throughout the holometabolous orders of insects, each oocyte leaves the germarium portion of the ovariole associated with one or more nurse cells. In higher Diptera the nurse cells and oocyte occupy the same follicle with the oocyte always occupying the proximal position. As oocyte development progresses, mRNAs, ribonucleoprotein, ribosomes and other cytoplasmic constituents are transferred from the trophocytes to the oocyte to add to the oocyte euplasm (nuclear material) . Gradually the trophocytes collapse and the oocyte progresses in development up to metaphase of the first meiotic maturation division until ovulation as in Drosophila (Foe et al. 1993), or in Culex by sperm entry and syngamy (Jost 1971). Meiosis is not complete until after fertilization . The oocyte euplasm makes up less than 10% of the total oocyte content. The remaining 90% or more is yolk, 40% of which is largely triacylglycerols and the rest is three to five proteins ranging in size from 44,000 to 51,000 kDa (Kawooya & Law 1988) . The accumulation of yolk in the oocytes occurs in the vitellarium and results in a very rapid increase in the size of the oocyte. Cyclic yolk accumulation, which results in a gonotrophic cycle, is often

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dependent on nutrition, which in mosquitoes and specifically A. aegypti, depends primarily on the intake of blood. The single batch of eggs produced after each blood meal and the number of eggs and frequency of oviposition all depend directly on the availability and size of the blood meal. Newly emerged A. aegypti female mosquitoes become reproductively competent after a chain of events that commence with the release of juvenile hormones (JH) from the corpora allata. Juvenile hormones stimulate the growth of the primary follicles of newly emerged females to grow in size to a pre-vitellogenic stage (Lea 1963; Gwadz & Spielman 1975) . In A. aegypti, JH levels are high during the first two days post-emergence, and thereafter gradually decline. This could be due to a feedback inhibition by the resting ovaries themselves (Feinsoid & Spielman 1980 a&b, Rossignol et al., 1981, Shapiro et al . , 1986). Once the proximal follicle length reaches 50-75 ^m, no further follicle growth is possible in the absence of a blood meal. Clements (1956) and Gillet (1956) suggested the presence of a brain-derived humoral factor responsible for mosquito egg development. As the accumulation of yolk progresses within the oocyte, the surrounding follicular epithelium develops extensive intercellular spaces between follicle cells. The intercellular spaces permit direct access of the hemolymph to the oocyte surface. Selective uptake of vitellogenin by

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8 endocytosis, from the hemolymph, occurs through the help of specific receptors on the plasma membrane of the oocyte (Raikhel & Dhadialla 1992, Ferenz 1993) . Follicle epithelium cells may also produce yolk proteins in smaller amounts that are directly contributed to the oocyte. The uptake of proteins from the hemolymph is regulated by juvenile hormone and produces changes in the surface of the oocyte (Yin et al. 1989). The fat body synthesizes yolk proteins, lipids and proenzymes that are involved in yolk metabolism during embryogenesis . Yolk transportation to the oocyte is also receptor-mediated and conducted by low-density recyclable lipophorins (Troy et al 1975, Gondim et al. 1989). Hormonal Control of Insect Reproduction in Aedes aagypti The trophic phase of ovarian development begins with the release of ovarian ecdysteroidogenic hormone (EH) formerly referred to as the "egg development neurosecretory hormone" that stimulates the ovaries to synthesize 20hydroxyecdysone (Lea & Brown 1990) . Midgut wall distention, from uptake of blood, stimulates the median neurosecretory cells of the brain to secrete ecdysteroidogenic hormone into the hemolymph (Meola & Lea 1972) . In A. aegypti, EH is released twice: immediately after

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9 blood meal uptake and later, 8 hours post-blood meal. The release of juvenile hormone prepares primary ovarian follicles to become receptive to EH (Shapiro & Hagedorn 1982) and the fat body to become receptive to 20hydroxyecdysone (Hagedorn 1977). Ecdysone produced in the ovaries stimulates the fat bodies to produce vitellogenin, the egg yolk protein precursors (Hagedorn et al. 1975). About 20 hours post-blood meal, secondary ovarian follicles separate from primary follicles. This probably results from a rise in the level of 20-hydroxyecdysone (Beckenmeyer & Lea 1980). The vitelline envelope synthesis, initiated and maintained by 20-hydroxyecdysone, reaches its peak in the hemolymph at this time (Raikhel & Lea 1991). The follicle epithelial cells begin to produce the proteins of both the vitelline envelope and chorion as yolk accumulation nears its end. Normally vitellogenin synthesis stops about 30 to 45 hours post-blood meal. The vitelline envelope is essentially a proteinaceous layer surrounding the oocyte, while the chorion is a proteinaceous complex that later also contributes to the darkening and hardening of the eggs. Chorion secretion occurs in two phases resulting in the formation of two layers of the chorion, the endochorion and exochorion. Chorionic genes, 15a-l and 15a-2, share 68% identity and appear to code for endochorionic proteins in A. aegypti (Lin et al. 1993) . The proteins deduced from the

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10 sequences of the 15-a genes shared a highly conserved hydrophobic sequence with the Drosophila proteins. Northern analysis of RNA extracted from vitelline membrane epithelia revealed a 650 base pair RNA transcript only in ovaries of blood-fed females. The RNA transcript was detectable at 5 hours, abundant at 20-30 hours and declined at 40-50 hours post-blood meal. Furthermore, a tritiated transcript used to probe ovary sections frozen at 20 hours post-blood meal hybridized only to the ovarian follicle cells. This showed a more pronounced expression of the 15a-l gene in follicles adjacent to the oocyte than in follicles adjacent to the nurse cells. This suggested that the local environment around and within the follicle cell modulated gene expression (Lin et al . 1993). Raikhel and Lea (1982 & 1991) suggests that chorionic proteins, too, are synthesized by follicle cells adjacent to the oocyte and not by those adjacent to the nurse cells. While proximal follicles develop to maturity after uptake of the first blood meal, the secondary follicles separate from their germaria and grow to the previtellogenic resting stage (-50 |im) . Treatment with 20-hydroxyecdysone can cause penultimate follicles to separate from germaria and then if females are blood-fed later, yolk deposition occurs simultaneously in the ultimate and penultimate follicles (Beckenmeyer & Lea 1980) . Females that retained a

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11 full complement of mature oocytes, and took a second blood meal, could not develop any of their penultimate eggs further beyond the resting stage. Borovsky (1988) and Borovsky et al. (1990, 1991a, 1996) put forward an explanation for termination of vitellogenin synthesis, involving negative feedback arising from an ovary-derived peptide secreted by the follicular epithelial cells in the mosquito, A. aegypti. The decapeptide sequence is tyrosineaspartic acid-proline-alanine-proline (6) -COOH (Mr 1047.6 Da). Injection of the peptide into normal blood-fed females inhibited blood meal digestion and resulted in retarded ovarian development. Therefore, the ovary-derived peptide was described as "trypsin modulating oostatic factor-A" (TMOF-A) secreted by ovaries 24 to 48 hours after a blood meal. It is released into the hemolymph and binds to midgut receptors where it blocks further production of trypsin-like enzymes and indirectly inhibits vitellogenesis in autogenous females (Borovsky et al . 1993). That trypsin synthesis continued longer than normal in ovariectomized blood-fed females supports the fact that TMOFs have a functional role in the termination of trypsin synthesis. Lin et al. (1993) provide evidence that ovarian follicular epithelial cells of the mosquito, A. aegypti synthesize a chorionic protein encoded by gene 15a-2 with an open reading frame at the N-terminal coding for TMOF-B.

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12 TMOF-B only differs from TMOF-A in the order of the first two amino acid residues (Borovsky et al. 1990a) . It is possible that, in evolution, peptide (s) synthesized and processed during ovarian development acquired a role in regulating the secretion of trypsin in hematophagous insects. A factor was found to inhibit bloodmeal digestion and inevitably block ovarian development, if applied too early to young females. Hormonal Control of Reproduction in Stanioxys caJLcltians Unlike nematoceran blood-feeders like mosquitoes and black flies (Diptera: Culicidae and Simuliidae respectively) , blood-feeding Diptera in the sub-order Cyclorrapha, such as stable flies (Diptera: Muscidae) do not exhibit gonotrophic discordance i.e. producing one batch of eggs per full blood meal. Stable flies fed from 48 hours post-emergence need multiple blood meals to produce mature eggs with three to six blood meals required to mature the first batch of eggs (Kuzina 1942). Chia et al . (1982) reported that five blood meals are needed for the first egg batch and three to mature the second batch. Moobola & Cupp (1978) demonstrated that stable fly ovaries enter a juvenile hormone-mediated resting stage following eclosion.

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13 Sutcliffe et al. (1993) suspected that the bloodfeeding act results in the release of hormones that stimulate the resumption of ovarian development. Houseman & Morrison (1986) showed that in stable flies, the relationship between the fat body and egg development was uncertain because the fat body neither produces vitellogenin nor any female-specific protein during vitellogenesis . However, Sutcliffe et al . , (1993) noted while fat body growth proceeded similarly with the first two blood meals in controls. However, fat body growth was halted in those females that were offered fewer than three blood meals during the period they were expected to have matured the first egg batch. Hence it was established that blood-feeding history in stable flies affected survival and more so, the reproductive process. Besides gonotrophic discordance, stable flies also differ from mosquitoes in the way they manage their blood meal. Mosquitoes digest a single full-sized blood meal as a bolus with digestion proceeding over the entire surface of the meal. Digestion products of the bolus are made available immediately to mature an egg batch. Stable flies, on the other hand, mature an egg batch from several blood meals and digestion is organized into a production line that involves food storage, digestion and absorption happening at different positions along the length of the gut. Stable

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14 flies are described as continuous-digesters as opposed to mosquitoes that are batch-digesters (Lehane et al . 1995). The batch-digesters undergo considerably more (and dramatic) changes in the levels of midgut digestive enzymes, which suggests more regulation control during the digestive cycle (Blakemore et al. 1995) . Hence the trend of regulation control of enzyme processes that are involved in blood meal digestion, as described in the mosquito, A. aegypti, is less dramatic in the stable fly. While A. aegypti has an early trypsin mRNA transcript transcribed to form trypsin-like enzyme within the course of some 24 hours, Scalcitrans contain an inactivated trypsin zymogen that is converted to a fully active trypsin-like enzymes (Moffatt et al. 1995). The Relation Between Digestive Enzymes and Egg Development Because of the role of hematophagous arthropods as vectors, the digestive physiology of these arthropods has been intensely studied. Much of the attention has centered on demonstrating the presence of the enzymes by using an invitro assay or crude homogenates of some portion of the gut such as the midgut, or whole insects. Since proteins are the most obvious nutrient in blood, attention has been directed to studying proteinases in hematophagous arthropods including insects. The proteinases have been classified as

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15 cathepsins if the optimum pH is acidic, or trypsin-like if the optimum pH of activity is alkaline. Studies have also involved the state of the enzyme prior to and after ingestion of a blood meal; what initiates a rise in enzyme activity and what terminates the rise (Gooding 1966) . In A. aegypti, there is a transitory decline in midgut proteinase activity immediately after blood feeding which Fisk & Shambaugh (1952) suspected to be due to substrate depletion of enzymes or inhibition by specific proteinase inhibitors in blood serum. On the contrary, in S. calcitrans , blood feeding neither caused an immediate rise nor a transitory decline in midgut proteinase activity. A steady rise was noted in proteinase content in the midgut that was suggested to be due to activation of pre-existing material or de novo synthesis of the enzyme (Fisk 1950, Champlain & Fisk 1956). Lehane (1976, 1977) proposed that a secretagogue mechanism involving intake of a blood meal operates during the digestive process of hematophagous insects. Gooding (1969) used BApNA to determine levels of trypsin present in guts based on moles of BApNA hydrolyzed per minute per gut. He characterized and determined that synthesis of mRNA and protein occur after ingestion of a blood meal.

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16 Rationale and Objectives Initial studies on mosquito TMOFs centered on the effects of TMOFs on ovarian development and digestive enzyme activity in A. aegypti. In subsequent studies, attempts were made to modify the original TMOF peptide sequences and/or design substituents to create novel bioactive analogs (mimics) . The small native decapeptides , TMOF-A and B, are highly water soluble and therefore unlikely to pass readily through the insect cuticle. The mimics though still hydrophilic compounds, are synthesized with the potential to remain unaffected by natural enzyme degrading systems that can sequester or remove TMOF circulating in the insect hemolymph. Design of hydrophobic side chains might confer prolonged activity of chemically modified peptides as with Pheromone Biosynthesis Activation Neuropeptide (PBAN). The research objective is to establish the effects of these synthetic Aedes-TMOF peptides and peptide-mimics as oostatic and trypsin biosynthesis modulators for A. aegypti and S. calcitrans . Specific reversible substrates for trypsin-like enzymes such as BApNA was used to determine whether trypsin levels were affected in mosquitoes and stable flies treated with Aedes-TMOF peptide and peptidemimics. Actual levels of trypsin-like enzymes present were

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17 using an irreversible substrate such as tritiated DFP to determine enzyme levels by scintillation counting and PAGE gel fluorography to define isozyme patterns. Isozyme patterns were studied to observe discrepancies from the saline control treated insects. In A. aegypti an early trypsin transcript would be seen to be converted {or not converted) to the late trypsin form by the pattern of the TLE isozymes. While newly emerged female mosquitoes need one blood meal to mature an egg batch, the female stable fly requires up to five sequential blood meals to mature the first egg-batch. Borovsky (1991b) observed that A. aegypti ovary-derived TMOF (Ae-TMOF) retarded trypsin biosynthesis and activity in several hematophagous insects, including stable flies, in which up to 48% inhibition of trypsin activity occurred. This suggests cross-bioactivity of Aedes-TMOF in hematophagous insects that have primarily trypsin-like enzymes (TLEs) for protein digestion. A second objective was to investigate the possible occurrence of Aedes-TMOF-like peptides in the ovaries of stable flies. If present, to determine whether it can modulate blood protein digestion and affect egg development in either the mosquito, A. aegypti or the stable fly, S. calcitrans .

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CHAPTER 2 OOSTATIC ACTIVITY OF SYNTHETIC PEPTIDES AND PEPTIDE MIMICS ON THE MOSQUITO, AEDES AEGYPTI, LINNAEUS AND THE STABLE FLY, STOMOXYS CALCITRANS, LINNEAUS Introduction The existence of substances inhibiting egg follicle growth has been suspected since early studies on a number of insects including the cockroaches, Blatella germanica and Blatta orientalis (Iwavnov & Mescherskaya 1935), the eye gnat, Hippelates collusor (Adams and Mulla 1967) and the house fly, Musca domestica (Adams and Nelson 1968 and Kelly et al. 1984) . In mosquitoes, Meola and Lea (1972) and Else and Judson (1972) separately observed that during oogenesis the ovary secretes a humoral factor that inhibits yolk deposition into less developed follicles. Such ecstatic humoral factors were found to be peptide-like in the triatomine bug, Rhodnius prolixus (Davey & Kunster 1981) and the mosquito, A. aegypti (Borovsky 1982) . As a gonotrophic cycle progresses, humoral responses closely related to protein deposition into the oocytes invoke a feed back mechanism that results in the release of peptide fragments into the hemolymph. These are recognized 18

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19 on the surfaces of midgut epithelial cells as signals to reduce or halt trypsin production altogether. Borovsky et al., {1991b) described an ovary-derived peptide originating from vitellogenic A. aegypti female mosquitoes with oostatic and trypsin modulating properties and named it a trypsin modulating oostatic factor [TMOF] . TMOF is secreted from the follicular epithelium of mature oocytes in ovaries of A. aegypti mosquitoes, then transported and bound to the midgut cells where it inhibits synthesis of trypsin-like enzymes. The peptide could be a by-product of yolk proteins cleaved off and liberated during the editing process of yolk protein precursors on the vitellin membrane. Amino acid sequences for mosquito-derived TMOF (Ae-TMOF) peptides are: NH2Tyrosine-Aspartic acid-Proline-Alanine(Proline) e-COOH for TMOF-A; NH2-Aspartic acid-Tyrosine-Proline-Alanine(Proline) 6-COOH for analog B and NH2-Proline-Alanine(Proline) 6-COOH for analog C (Borovsky & Carlson 1989, Carlson et al., 1993, 1994). Borovsky et al., (1991b) established that Ae-TMOF is not species specific in activity because it also modulates synthesis of trypsin-like enzymes and egg development in other mosquitoes like Culex nigripalpus , Cx. quiquefasciatus , Anopheles quadrimaculatus and A. albimanus . When injected into S. calcitrans , TMOF-A inhibited trypsin biosynthesis up to 46% at 4.8 nanomoles, a quantity that causes total inhibition of trypsin

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20 biosynthesis in A. aegypti (Borovsky et al . 1991a) . This treatment effectively deprives a treated female of the end products of blood protein digestion and hence limits availability of yolk protein precursors to be used by the fat body for yolk protein synthesis (Borovsky, t al. 1990). Recently, Bylemans et al. (1994, 1995) isolated two small peptides with f olliculostatic and trypsin modulating activity in the gray flesh fly, Neobelliera bullata. One acts like TMOF, while the second factor, Neb-colloostatin o Neb-TMOF, inhibits yolk uptake by pre-vitellogenic oocytes and decreases the concentrations of vitellogenin present in the hemolymph. However, these TMOFs are structurally very different from Ae-TMOF-A and/or B. Studies on mosquito TMOF first centered on its effects on ovarian development and midgut digestive processes in A. aegypti mosquitoes. Attempts have involved shortening, or modifying original peptide sequences and designing substituents to create novel analogs that were assayed for both oostatic and trypsin modulating activity. Small native peptides containing just ten amino acids as found in the Ae TMOF are highly water soluble and therefore unlikely to readily pass through the insect cuticle. A potentially useful route for bioactive peptides involves structural modification or chemical derivativization of the TMOF-A sequence, HaN-Tyrosine-Aspartic acid-Proline-Alanine-

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21 (Proline) eCOOH (see Figure 2-1). Modifications to increase binding, potency and life span could involve blocking tyrosine OH via esterif ication with substituents to inhibit lysis; replacement of the internal proline with hydroxyproline or anthranilyl ; and by exchanging, endblocking and derivatizing various amino acids. The peptidemimics can also be hydrophilic compounds, but synthesized with modifications that theoretically will remain unaffected by natural enzyme degrading systems that sequester or remove TMOF circulating in the insect hemolymph. Modifications could include addition of hydrophobic side chains at the Nterminal of TMOF-A, replacing the proline in the third position with anthraninyl in peptide A4 ; replacing the proline in the third position with hydroxyproline in A5; adding a phenoxyacetyl group to the first amino acid tyrosine in A6, replacing all the 1-amino acid composition in TMOF-A with d-amino acids to form the retro-inverso analog of TMOF-A in A7 ; modification of TMOF-B by replacing the aspartic acid with a succinyl group at the N-terminal in B2 . Other modifications could involve the presence of pyrene substituents to the peptide to form: or pyrene-butyric acid to form IPba-Y (OtBu) DPAPPPPPPNH2 or Carboranylalanine to form CbAla-ARYDPAPPPPPPNH2, 9-f luoreneacetic acid to form 9Fla-ARYDPAPPPPPP0H and 9Fla-ARYDPAPPPPPPNH2 . Such alterations could prolong the activity of chemically

PAGE 33

22 modified peptides by providing means for transportation of bioactive substances across the insect cuticle. Specific Objective The research objective therefore is to establish the effects of the various synthetic TMOF peptides and peptidemimics as egg development arrestants for biting flies such as the mosquito, A. aegypti and the stable fly, S. calcitrans . Hence, a bioassay was developed to evaluate the effect of Aedes-TMOF derived peptides and peptide-mimics on egg development in the study insects. Materials and Methods Experimental Insects USDA Insectary reared A. aegypti (L) larvae were fed on liver powder/yeast (1:1) until they pupated. Maintenance conditions were 28°C and 80% relative humidity. The adults eclosed and were held in cages until they were 5 days old.

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23 Figure 2-1. Structures of synthetic TMOF peptide and peptide-mimics used in the study.

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Figure 2-2. Structures of synthetic TMOF peptide-miitiics with pyrene, 9-Fluoroacetic acid and caboranylalanine added to Aedes-TMOF.

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25 and maintained on 10% w/v sugar water soaked cotton-wool pads. The adults were offered a guinea-pig blood meal and after three hours, the females that fully engorged were used in the bioassays with the various treatments. Insectaryreared stable fly adults, S. calcitrans (L) , obtained from pupae from the USDA-CMAVE insectaries were maintained at 28°C and 80% relative humidity. A normal gonotrophic cycle development takes about three days in the mosquito, A. aegypti and seven to ten days in stable fly, S. calcitrans (see Table 2-1 and 2-2, respectively) . Measurement of Oosta'tic Activi'ty Dose-response treatments involved injecting peptides and peptide-mimics dissolved in Ringer's saline at various concentrations ranging from 1-40 |ig per female. The structures of peptides used in the study are described in Figures 2-1 and 2-2. The injection treatments were applied between the third and fourth pleura with glass needles pulled out of microcapillary with a Pullman (Figure 2-3) . A capillary tube is placed within a filament loop of the Pullman, screws of the rotor tightened and the switch set to heat the filament. As the filament became red hot, the capillary softens around the filament area and the pulling action on the two sides of

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26 Table 2-1. Egg development stages in the A. aegypti maintained at 27 °C onrp> ^Phri «?toDhers 1963) . mosquito, and blood fed Stage Follicle Characteristics Length (fim) of stage Time post eclosion 2 3 4 sn no blood meal taken 200 blood meal ^ digested 450 blood meal digested 600-800 Oviposition, ready to take another blood meal 0-3/5 days 24 hpbm 52 hpbm 72-96 hpbm Thereafter, oviposition every 72 hpbm. Table 2-2. Egg development stages in the stable fly, S. calcitrans maintained at 24 °C and blood-fed daily (Moboola & Cupp 1978) . Stage Follicle Characteristics Days post length {\m) of stage eclosion N 70-160 Ovary with 16 undifferentiated cells tightly enclosed by tracheal system; no yolk 0 I 160-300 Oocyte occupying 20% of follicle Trace of yolk 3 II 300-680 Oocyte occupying 40% of follicle follicle elongated 4-5 III 680-820 Oocyte occupying >50% of follicle nurse cells reach maximum devt 5-6 IV 800-900 Oocyte occupying 70-90% of follicle Nurse cells degenerate 6-8 V 900-1200 Oocyte occupying 100% of follicle nurse cells in-apparent hatching pleat present chorion and surrounding entire follicle thickened 7-10 Thereafter, oviposition every 2-3 days.

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27 the capillary pulls the capillary into a fine "needle-like" filament. The treated insects were examined at various hours post blood meal under a dissecting microscope to determine stages of egg development. Dose Response Effect of Various Treatments on A. asgyptl Following initial treatments at 5 \iq per female, the mosquitoes were injected with various doses of the peptides to determine dose-response effects. As mosquitoes became available, doses between 1 and 40 |J.g per insect were injected into each female and occasionally males. Evaluation of ecstatic activity was done the same way as above with 5 |J.g treatments. Meanwhile some guts were also pulled and stored in 50 mM TRIS-HCl buffer, pH 7.9 or 8.4 for various trypsin assays described in Chapter 3. The Effect of Treatments on the Stable Fly, S. calcltreuis Two-day old adults were offered a meal of bovine bloodsoaked cotton-wool pads. Three hours later, they were used in the bioassays with various treatments either by injection or topical application. After seven days from day of treatment, both the saline treated control and those that had received various peptide and peptide-mimics were

PAGE 39

Figure 2-3. Micro-capillary Pullman used to pull capillary tubes to make glass needles.

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29 dissected and egg follicle length in ultimate follicles determined . By this time, egg follicle length in control flies had reached 1200 jam and the eggs had or were being oviposited. The proportion that had retarded egg follicle development by day 7 was averaged and scored against the proportion that had normal development and the dead. Samples of gut were retained for trypsin analysis (see Chapter 3). Results The Effect of Ae-TMOF Peptides on A. aegypti Female A. aegypti injected with 5 |xg of oostatic hormone, TMOF-A, and peptides A5, A7 and A8 had ovaries with ultimate eggs developed about half the normal follicle length (263.5 + 12.7; 184.2 + 72.7; 250 + 24.7 and 223.3 + 56.3, respectively) compared to controls at 4 60 + 28.1 [im at 48 hpbm. The trend at 72 hours, when controls had oviposited eggs of 673.5 + 32 ^m, revealed retarded development of egg follicles in the treated females with TMOF-A, A5, A7, A8 and B (Table 2-3), respectively. Yolk deposition into the ultimate oocytes was inhibited. However females injected

PAGE 41

30 c: c * £i W r\ IM U J u_ 1 1 H Li, vj 1 r\ CO 1 t rTi 1— ' X, or\ n ) /— « U *j 1 u / n3 rn fA CM m ni / — ^ t — 1 V — ' 00 lO 00 n\ CN] CNJ 1 — 1 1 — I CNJ 1 — 1 1—1 c ^ n n -P 1 1 CN CO cn lT) to m (11 CO u •H * CO /]\ , 1 U/ ' 1 I—* ID ) t — 1 ID o o LT) •H lU 1 \ cu Cn CO H \ — 1 T3 U' ( ' J CN] V-H CO Tj QJ ' C CO ( — 1 O o ^1 (11 CM Q) o o o o in LD £ •H -P .H II eg cs] (\1 rH cn CNJ rH p U Cu Ol, *W -H c (U o nH ^^ cn CN r-H CO u CIj CNJ CNJ CTi ["t [ [ 1 ti 1 M— 1 UJ CNJ f— ( CNJ lD CO 4-) (0 Zl c CD + 1 c 1 1 1 1 + 1+1 + 1 + 1 + 1 + 1 E-I Q) CJ" T3 1 1 ll 1 CO S-i Tj CM • CO " 0) -P (J u; vU o to fO o o CO rH to 1 — 1 u 00 (D CO o LO CN tl) -P U •H ^ CsJ TJ CM \ N ll M U -H P 0) rH M-l O "O W M-l \
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31 with saline developed their eggs to > 650 ^im which they oviposited at 72 hpbm (Table 2-3) . Hence a delay in egg development to oviposition over several days resulted in some treated females (as with A5) which had oocytes measuring 453.8 + 52.9 ^m at 96 hours, 24 hours after control females had oviposited. ' . ; , Figures 2-4 to 2-6 are light microscope photomicrographs of stages of blood-meal digestion and egg development at different times after the first blood meal. Figure 2-4 is the midgut of a normal blood-fed female mosquito, 3 hpbm. The blood is bright red in color with the peritrophic membrane just beginning to form around the blood. The oocytes are rather transparent, about 50 |am long, and have not developed beyond resting stage. By 48 hpbm, the blood has been digested and is brownish in color (Figure 25) . The oocytes have increased in size to over 280 ^im in length and are opaque in appearance. Figures 2-6, and 2-9 to 2-14 shows ovaries of treated insects 30 hpbm, mostly with translucent oocytes and some evidence of still bright red blood in the gut, indicating occurrence of little protein digestion. Oostatic activity of treatments was confirmed if measurements of ultimate oocytes lengths at various hpbm were less than those in the controls .

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32 Figure 2-6 shows ovaries of saline-treated blood-fed mosquitoes, 30 hpbm, in which eggs are clearly opaque and about 200 (im long. This constituted the standard oocyte size to which development in other treated insects was compared. Figure 2-7 shows recently oviposited eggs 72 hpbm, with maximum attainable egg follicle lengths of -650 ^m by which time eggs had been oviposited. Figures 2-8 and 2-9 show TMOF-A and B2 treatments with guts where the blood meal ingested 30 hours earlier was shrunken in size but still bright red in color, indicating little or no blood digestion. The reduction in size of the blood meal implied that other normal processes, such as peristalisis and diuresis, that accompany the digestion of a blood meal, were uninterrupted. Figure 2-11 shows rather translucent eggs in ovaries of A5 treated mosquitoes and egg follicle length was <150 [tm indicating retarded growth compared to controls. The Effect of Pyrene -Derived and other Aedes-TMOF Mimics on A. aegyptl Tables 2-4 and 2-5 show the effect of injecting bloodfed females with pyrene, carboraynl and 9-Fluroacetic acid derivatives of Ae-TMOF peptides. The results indicated oostatic activity in IPba (OtBu) DPAPPPPPP-NH2 where 70% of treated insects were retarded with doses of 4 nM and 80% were retarded with doses of 2nM.

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33 Table 2-4. Dose response of peptide-mimics injected into A. aegypti showing follicle lengths reached by the no. of treated females (nm) . Ultimate oocytes follicle length Treatment Dose/fern in nM <100 \irti 100-200 ^im >200 ^m Dead Ipba (OtBu) -* 2 8 0 0 2 4 7 0 0 3 PPPPPP-NH2 2.3 1 9 0 0 4 . 6 1 5 0 4 CbAla-* 7.0 4 • Z '' 0 4 11.3 0 0 10 0 9Fla-*0H 4.7 0 0 10 0 9.4 0 0 0 10 9Fla-* 2.0 0 7 3 0 3.6 0 5 0 5 7.3 0 0 10 0 Control sal 0 0 10 0 NOTE: Females were injected 3 hpbm and examined 30 hpbm. (See Fig. 2-2 for formulae) . *=ARYDPAPPPPPP-NH2 Table 2-5 Response to pyrene peptide mimics by A. aegypti showing follicle lengths reached by the number of treated females. Treatment Dose/ Ultimate oocyte length female by number attaining stage (nM) <300 |J,m 300-499 |im >500 ^im Dead pyl 40 0 0 0 20 **pyl 40 0 0 0 20 pyl 4 3 0 15 2 py3 40 0 0 20 0 py5 40 0 1 4 0 control sal 0 0 20 0 NOTE: Females injected 3 hpbm and examined 72 hpbm. (See figure 2-2 for formulae) ** topical application

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34 CbAla-ARYDPAPPPPPP-NH2 injection of 7.0 nM revealed 40% of insects with retarded egg development. 9Fla-ARYDPAPPPPPP-NH2 showed moderate activity as 50% and 70% had egg sizes of less than 200 ^im with 3.6 and 2.0 nM of 640 [(1)3] respectively; PPPPPP-NH2 showed ecstatic activity as 50% and 90% of insects had egg sizes below normal. Figure 2-3 shows responses with the pba derivatives, clearly indicating ecstatic activity with IPba (OtBu) DPAPPPPPP-NH2 and PPPPPPNH2 fragment. Overall mortality was observed particularly with 9Fla-ARYDPAPPPPPP-NH2 at a dosage of 9.4 nM while at 4.7 nM the mimic was neither toxic nor ecstatic . Responses with pyrene mimics were noted only 72 hpbm whereby 15% treated with pyl (obtained one purification step before PPPPPP-NH2) at 4nM had retarded growth indicated by under developed egg size. Pyl was toxic at 40 nM when applied by injection or topically. Py3 (obtained one purification step before IPba (OtBu) DPAPPPPPP-NH2) and Py5 (obtained one purification step before 5 81-Pba [(|)5] ) at the dose of 40nM were neither ecstatic nor toxic. Hence Pyl had oostatic activity at lower concentration but was toxic at higher concentrations. Figures 2-12 through 2-14 shew ovaries of mosguitoes treated with pba TMOF peptide mimics IPba (OtBu) DPAPPPPPP-NH2) , 9Fla-DPAPPPPPP-NH2, 9FlaDPAPPPPPP-OH, and PPPPPP-NH2 (Table 2-4).

PAGE 46

Figure 2-5. Digested blood meal and developing eggs ~280 |im (48 hpbm) .

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36 Figure 2-7: Oviposited eggs 72 hpbm (>650 long) .

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37 Figure 2-8 : Appearance of ovaries and gut and of TMOF-A treated mosquito, 30hpbm. Eggs underdeveloped beyond 50 |am long and blood still bright red in color Figure 2-9: Appearance of gut and ovaries of B2 treated mosquito, 30hpbm. Blood still red, eggs are not developed beyond 100 (im long.

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38 Oostatic activity was evident since the egg size was below normal and the eggs were transparent indicating little yolk deposition into the oocytes at 30 hpbm. Figure 2-12 shows a few partly developed eggs (-200 |im) in a mass of under developed translucent eggs (<100 (xm) in the ovary dissected from female mosquito treated with IPba (OtBu) DPAPPPPPP-NH2 . A similar trend of the presence of some portion of a rather translucent ovary with opaque oocyte plaques is evident in Figure 2-12. These imply the presence of a few eggs developed to -200 \im within a mass of underdeveloped eggs with treatment, 9Fla-ARYDPAPPPPPP-0H . Figure 2-14 a translucent mass of tiny underdeveloped eggs <150 |im long with more obvious opaque appearance with treatment, PPPPPP-OH. The activity with the mimics, IPba (OtBu) DPAPPPPPP-NH2, PPPPPP-NH2, 9Fla-ARYDPAPPPPPP-0H, 9Fla-ARYDPAPPPPPP-NH2. CbAla-ARYDPAPPPPPP-OH and even A5, B2 revealed that the ovaries appeared to have some resources tc distribute into the yolk granules of the eggs but there may not have been enough to achieve completely normal egg development in the ultimate egg follicles in the ovaries.

PAGE 50

39 TS Q) T3 U e (0 (0 -P 0 (U c U m V c (0 o o o in i : 1 1 — J 0) c H (0 04 On 04 Cu CM I 2 CTl (0 < U a: o CQ o Oi M 3 -H

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Figure 2-11: Appearance of gut and ovaries of A5 treated mosquito, 30hpbm. Eggs are not developed beyond 150 fJ.m long. Figure 2-12: Appearance of gut and ovaries of iPba (OtBu) DPAPPPPPP-OH treated mosquito, 30hpbm, Eggs translucent & <150 |im long.

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41 Figure 2-13: Appearance of gut and ovaries of 9FlaARYDPAPPPPPP-OH treated mosquito, 30hpbm. Eggs translucent but few developed beyond to 200 [im long. Appear fuzzy in this picture. Figure 2-1 A: Appearance of gut and ovaries of PPPPPP-NH2 treated mosquito, SOhpbm. Eggs translucent & < 150 |J.m long.

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42 Dose Response Effect of Various Treatments on A. aagypti Table 2-6 and Figures 2-15 to 2-16 show results of exposing the females to serial doses of the various peptides and peptide-mimics . Besides TMOF-A and B that had oostatic activity, A5, and B2 were over 70% of the females had underdeveloped eggs at 10 ^g and A7 at 5 Jig per insect. Increasing the dosages to >10^g per insect usually resulted in increased mortality, as with B2 . Decreasing doses led to instances where a few eggs could be found developing at an apparently normal pace scattered within a mass of underdeveloped and rather transparent eggs. In such cases, it was not easy to establish oostatic activity with mimics shown in Figure 2-8 through 2-14. However guts of treated insects that had responded to 10 ng were pulled for trypsin assays. A positive correlation was seen between TMOF-A and B and peptides A5, A7 and B2 for oostatic properties, while toxicity was evident in peptide-mimics A4 and A6. Figure 2-15 and 2-16 illustrate oostatic activity and toxic properties of the various compounds. Data in Figure 2-15 and 2-16 indicate that uniform doses of the compounds could not yield uniform results. However dosage/mortality curves could not be easily derived under the conditions of these tests at the dosage rates tested.

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43 The Effect of Treatments on the SteJsle Fly, S. calcltxans As usual the oostatic effects were more definite with TMOF-A, with eggs retarded in 60% insects, eggs developing normally in 32.5% and 10% of the insects dead. TMOF-B retarded eggs in 31.1% of the insects, eggs developed normally in 48.0% of the insects and 10.4% of the insects died. Peptide-mimic A4 resulted in 5.8% of the insects with eggs retarded, 24.5% of the insects with eggs developing normally and 62.5% of the insects died. A6 had similar trends with 5.0% of the insects with eggs retarded, 19.4% of the insects with eggs developing normally and 70.8% of the insects died. A4 (with anthraninyl) and A6 (with phenoxyacetyl ) appeared more toxic than oostatic to the stable flies. A5 caused 41.8% of the insects to have eggs retarded, and 10.5% of the insects with eggs normally developed, and 28,7% insects died. B2 had 30.8% of insects with eggs retarded, 60% of insects with eggs developing normally, and 17.5% of the insects died. These two peptides, A5 (with hydroxyproline) and B2 (with succynyl) appeared oostatic and less toxic to stable fly females. The dead flies (by day 7) survived the initial injection and appeared un-damaged by injection 24 hours after treatment.

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44 Table 2-6. Dose-response effects of Ae-TMOF peptides and mimics on egg development in A. aegypti showing follicle lengths reached by the number of treated females (|i.m) . Ultimate egg follicle length reached Treatment Dose/fem (number attaining stage) (^g) <100 nm 100-200 nm >200 ^im Dead O CI X XiXC. n 0 20 0 7i 1 3 2 5 0 A 0 0 0 A 15 20 0 0 0 A4 0 0 0 20 A4 9.6 0 0 0 20 A4 19.3 0 0 0 20 AS 2 0 g w 0 A5 5 8 4 3 5 A5 10 13 4 2 1 A5 20 5 0 0 15 A6 0 0 0 ?0 A6 4 4 0 2 14 A6 5 0 0 0 20 A6 8 2 0 2 16 A7 1 0 0 10 10 A7 5 13 2 2 3 B2 10 7 1 2 10 B2 21 0 0 0 20 B2 42 10 1 2 7 B 5 0 0 20 0 B 10 8 2 0 0 B 20 3 0 5 12 Females fed blood, treated 3 hpbm and dissected 30 hpbm.

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45 O Eh m < Si 4J •H C o •H P O (1) -o C •H c o o CO 0) -u o o o u m CO o o c ^ O (0 -H 4-> H C c ITS •H CO (U -p 3 O Q4 i-i (U in I
PAGE 57

46 •O -O T3 -O TJ nj (tj fTJ (tJ n] d) 01 0) (U XI H H > -P a Q) CO a • 1 CM 0) U d -H

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47 in Table 2-8 and show that A4 has oostatic properties when applied topically. Egg follicles were retarded in 56.2% and 72.2% of the insects when A4 was applied to back and legs of stable flies, respectively. A6 retarded egg growth in 31.4% and 0% when applied to back and legs respectively. Retardation was obvious with applications to the back as 85.7% of the insects had retarded egg development from peptide TMOF-A and 68% with TMOF-B. It was not possible to keep all of the peptide treatment on the insects because the peptides were dissolved in DMSO and the treatment droplets tended to fall off; thus there was little control on how much peptide was taken up by legs. The DMSO solvent used in this study was not in itself toxic to the stable flies as treated flies continued to develop normally in all processes to seven days. Discussion Small peptides in the Ae-TMOF family are highly watersoluble and therefore less likely to readily pass through the insect cuticle. The developed mimics are hydrophilic compounds designed and synthesized with a potential to remain unaffected by the insect's body system, that can sequester or remove TMOF from the insect. Most modifications

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48 Table 2-7. Effects of injected Ae-TMOF peptides and mimics on the stable fly, S. calcitrans showing follicle length category reached and the percentage of treated females. Ultimate egg length reached Peptide (% + SD attaining stage by day 7) 5ug <800nm >800nm Dead A 60 + 27 .3 32. 5 + 3. 5 10 + 14 1 A4 5.8 + 2. 7 24 . 5 + 17 .8 62 5 + 43 6 A5 41. 8 + 17 . 6 10. 5 + 0. 7 28 7 + 12 8 A6 5.0 + 2. 0 19. 4 + 13 .7 70. 8 + 31 8 A7 28 . 1 + 10 .8 32. 6 + 5. 6 20 5 + 9.9 B 31. 1 + 13 .5 48. 0 + 3. 4 10 4 + 1.3 B2 30. 8 + 17 .0 60 + 0 17 5 + 12 9 CONTROL 0 100 0 Stable flies fed blood, treated 3-6 hpbm, dissected on day 7

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49 Table 2-8. Effects of topical treatments of Ae-TMOF peptides and peptide mimics on egg development in S. calcitrans showing follicle length category and the % of treated females. Ultimate egg follicle length Peptide Treatment (% attaining stage by day 7) 5ug Site <800nm >800nm Dead A back 85.7 14 .3 0 A leg 25. 6 74.4 0 A4 back 56.2 43.8 0 A4 leg 72.2 27.8 0 A6 back 31.8 68.2 0 A6 leg 0 100 0 A7 back 70 30 0 A7 leg 43.8 56.3 0 B back 68 32 0 B leg 4,5 95.5 0 CONTROL back 0 100 0 Leg 0 100 0 stable flies fed blood, treated 3-6 hpbm, dissected on day 7

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50 in the peptides were at the N-terminus of the original TMOFA. Addition of a phenoxyacetyl group to the tyrosine in the chain of A6 and the replacement of aspartic acid in position 2 with an anthraninyl group in A4 proved more toxic than oostatic to the insects. However, the replacement of a third position proline with the hydroxyproline group in A5 and the addition of a succinyl group to aspartic acid at the N-terminus of the TMOF-B in B2 sequence all seemed to retain oostatic properties. A7, the TMOF retroinverso analog proved oostatic to mosquitoes with no difference between its effect and TMOF-A and B. The carboranylalanine and pyrenebutyric acid mimics of TMOF and the hexaproline fragment proved oostatic to mosquitoes. The hexaproline fragment affected egg development mosquito insect with normal development and 90% were less than normal. It is possible the insects also are affected by polyproline fragments floating in the hemolymph. This is interesting since many polyproline fragments are found in the 15-a gene of yolk proteins (Lin et al., 1993 and Marten, 1996) . The effect of adding a pyrene-butyric acid to the TMOF peptide was that the derivative, Ipba (OtBu) DPAPPPPPP-NH2, was oostatic at low but toxic at higher dosages to mosquitoes. The f luoroneacetic acid mimics, 9Fla-ARYDPAPPPPPP-0H and 9Fla-ARYDPAPPPPPP-NH2 had

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51 no oostatic activity on mosquitoes. 9Fla-ARYDPAPPPPPP-0H however appeared to be toxic at lower concentrations. The fact that a more dilute sample was more toxic could be the reverse as addition of saline as a diluent may have released more quantities of the compounds bound to the walls of the container, thus effectively increasing the concentration. Increased bioactivity shown with CbAla and IPba derived peptide-mimics could well be due to enhanced or prolonged binding of the peptide-mimics to the trypsin biosynthesis receptors located in the midgut. Alternatively, increased bioactivity could have been due to the inability of the insect body system to degrade and sequester circulating peptide mimic in the hemolymph. Hence the mode of action which is unknown could be then due to the insect's gut continuing to receive a signal to negatively affect or inhibit trypsin production process. It is also possible that the treated insects were stressed, producing little TLEs to manage the blood meal. This would lead to scarce yolk protein resources absorbed into the hemolymph, from the midgut, and hence such insects could only develop a few eggs probably by re-distributing available yolk protein precursors. This was seen with TMOF-A at 5 ^g where 25% of the insects appeared to have normal ultimate oocyte development (Table 2-6) .

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52 The scenario was also seen with TMOF-B at 5 Hg; A5 at 2.5, 5 and 10 Hg; A7 at 5 \ig; and also B2 at both 10 and 42 |4.g. This could have led to a few to many normal looking (opaque and normal size) eggs among smaller (translucent) ones. In such cases, it would be difficult to determine rea oostatic activity of the compound in question.

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CHAPTER 3 TRYPSIN MODULATING PROPERTIES OF SYNTHETIC PEPTIDES AND PEPTI DE-MIMICS ON THE MOSQUITO, AEDES AEGYPTI, LINNAEUS AND THE STABLE FLY, STOMOXYS CALCITRANS LINNAEUS Introduction An adult female mosquito requires a blood meal to obtain protein nutrients for egg-yolk protein synthesis { vitellogenesis ) . The uptake of a blood meal stimulates the midgut cells to synthesize and secrete various proteindigesting enzymes such as trypsin, chymotrypsin, and amino and carboxy-peptidases that make the midgut competent to digest a blood meal. In A. aegypti, post-feeding induction of trypsin activity is separable into early and late phases (Felix et al. 1991), and each phase is characterized by the presence of a specific group of trypsin isozymes (Graf & Briegel 1989) . The early phase begins immediately after feeding (Rudin & Hecker 1979) and involves the translation of mRNA that is already present in the midgut epithelial cells before feeding (Felix et al . 1991). Translation of early trypsin mRNA occurs only after ingestion of a blood or a protein meal (Noriega et al , 1996a, b) . The early trypsin mRNA levels decrease rapidly during the first 24 53

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54 hours following feeding and rise again at the end of blood digestion, about 60 hours after feeding (Noriega et al. 1996b, 1997) . Early trypsin plays an essential role in the activation of late trypsin transcription, resulting in the major endoprotease involved in blood meal digestion. Late trypsin isozyme levels peak about 24 hours after uptake of a blood meal and most of the blood meal will have been digested at this time (Graf & Briegel 1985) . Midgut protease levels enable efficient utilization of blood, because blood digestion has a direct correlation with vitellogenesis as digested blood proteins supply the amino acids for yolk production. However, any negative feedback response to the midgut can affect the turnover rate of early trypsin transcript into late trypsin, leading to reduced trypsin availability. This leads to retarded blood digestion in the gut and hence, reduced availability of amino acids as precursors for egg protein synthesis (Borovsky et al. 1989). In mosquitoes, Meola and Lea (1972) and Else and Judson (1972) observed that during oogenesis the ovary secreted a humoral factor that inhibited yolk deposition in less developed follicles. A factor named TMOF was eventually isolated, purified, identified and synthesized from A. aegypti ovaries. The mosquito derived TMOF (Ae-TMOF) amino acid sequences for

PAGE 66

55 TMOF-A and TMOF-B are provided in figure 2-1 (Borovsky & Carlson 1989, Carlson et al. 1993, 1994). Borovsky et al. (1990) noted that the Ae-TMOF is a non species specific modulator of trypsin synthesis and egg development in various mosquito species (e.g., Culex nigripalpus, Cx. quiquefasciatus , Anopheles quadrimaculatus and A. alhimanus) . TMOF-A also inhibited trypsin biosynthesis in the stable fly, Stomoxys calcitrans, up to 46% at 4.8 nM, a quantity that causes total inhibition of egg development in A. aegypti (Borovsky et al, 1991). Recently, Bylemans et al. (1994, 1995) isolated two small peptides with f olliculostatic and trypsin modulating activity from the gray flesh fly Neobelliera (Sarcophaga) bullata. The first factor is TMOF-like, while the second one, Neb-colloostatin, inhibits yolk uptake into oocytes and decreases vitellogenin concentrations in the hemolymph. Trypsin belongs to a family of serine proteases, protein digesting enzymes that have a serine amino acid residue at the active site. Mammalian alpha-chymotrypsin has three peptide chains interconnected by two disulfide bonds. The most critical charges that play a role in enzyme activity of these proteases are serine 195, histidine 57 and aspartic acid 102. Serine at position 195 is a strong nucleophile and plays an active role in the reactivity of

PAGE 67

56 both trypsin and chymotrypsin . These serine-bearing proteases hydrolyze peptide bonds by attacking the carbonyl group of the peptide bond through the oxygen atom of the hydroxyl group of serine 195. The active site is later regenerated by addition of water resulting in proton removal and ionic attack of the carbonyl carbon of the acyl group (Figure 3-1). Trypsin-like enzyme activity has been reported in most insects examined with exceptions in the order Hemiptera, and the Cucujiformia in the order Coleoptera (Terra et al. 1996). Protease activity is frequently assayed by its esterase activity on specific synthetic (chromogenic) substrates. Trypsin and trypsinlike enzymes preferentially cleave protein chains on the carboxyl side of peptide bonds bearing either of two basic L-amino acids, lysine and arginine . Trypsin activity can be assayed spectrophotometrically with a soluble chromogenic substrate such as alpha Nbenzoyl-DL-arginine-p-nitroanilide (BApNA) . BApNA bears the amino acid arginine on the carboxyl end which trypsin and trypsin-like enzymes recognize with specifity. The colorimetric assays spectrophotometrically follow the degradation of BApNA to yield p-nitroanilide, a distinct yellow product, exclusively diagnostic of the presence of

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57 Asp 102 — C-O H — N/^NH — O — Sor 195 His 57 Asp 102 — C—0—H H"f^N—HO — Set 195 B His 57 Figure 3-1. Charge relay system for a chymotrypsin molecule. A = normal charge distribution; B = Conversion o the molecule into powerful nucleophile by the removal of one proton by aspartate 102 from serine 195 via histidine 57 results in the conversion of serine 195 into a powerful nucleophile (Stryer 1995) .

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58 trypsin and trypsin-like enzymes. Actual trypsin levels can be established by using [^H] diiso-propylf luorophosphate (^H DFP) , a substrate that can selectively and irreversibly acylate the serine residue at the trypsin active site, to form [^H] diiso-propylphosphate (DIP) derivatives (see Figure 3-2) (Graf and Briegel 1985; Borovsky and Schlein 1988). The DIP derivatives can be visualized on native PAGE gels with f luorography . Specific Objective The objective of this work was to determine whether promising peptides and peptide-mimics affect protein digestion in the gut of A. aegypti and S. calcitrans by influencing levels of trypsin. Materials and Methods Experimental Insects USDA insectary-reared A. aegypti larvae were fed on liver powder/yeast (1:1) until they pupated. Maintenance

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59 ACTIVE ENZYME O (His 57 Ser 19S CH3 P " CH3 HC-O-P-O-CH CH3 O CH3 (His 57 Asp 102V I Vr\ Ser 195 F CH3 9 J H CH3 O CH3 _0-P-0-CH CH3 ASP 102k 1 r Asp 102. CH3 "-0^»^ P-O-CH HC ^ CH3 (His 57 Ser 195 O-H nOn -h CH3 CHS HC-O-P-O-CH CH3 O CH3 INACTIVE ENZYME Figure 3-2. Irreversible deactivation of trypsin-like enzymes by DFP,

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60 conditions were 28°C and 80% relative humidity. The adults eclosed and were held in cages until they were 5 day old, and maintained on 10%w/v sugar water soaked cotton pads. The treated female mosquitoes were examined 30 hours post blood meal by dissecting them with the aid of a dissecting microscope to establish the stages of egg development attained. Midguts were pulled out for trypsin assays with BApNA and tritiated DFP. Insectary reared stable fly S. calcitrans adults obtained as pupae from USDA-CMAVE insectaries were maintained at 28°C and 80% relative humidity. Newly emerged adults were fed on bovine blood soaked into cotton pads. Adults were injected with selected treatments. The treated stable flies were examined several days after a blood meal, ad lib, by dissection under a dissecting microscope to establish stages of egg development attained. The alimentary canal was pulled and placed in buffer for the trypsin assays as with mosquitoes. Preparation of Insect Midgut for BApNA Assays Female mosquitoes engorged in 30 minutes to one hour when exposed to a guinea-pig. Three hours later fully fed adults were easy to separate by appearance of the swollen, reddish abdomen for injection or for topical treatments.

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61 The guts were dissected and placed in 0.1 ml of 50 mM TRISHCl buffer with 0.1 M CaCl2, pH 7 . 9 per abdomen 30 hours post blood meal (hpbm) for later trypsin assays. Insectary reared stable fly adults engorged in 1-3 hours following exposure to bovine blood soaked cotton-wool pads. They were injected with selected treatments and examined several days later, after dissecting to establish stages of egg development. The guts were dissected and placed in 0.1 ml of 50 mM TRIS-HCl buffer with 0.1 M CaCl2, pH 7.9 per abdomen and homogenized in 100 |il of buffer. A portion of the gut homogenate was diluted 5x for protein estimation with Bradford's reagent (Bradford 1976). Midguts were then stored at -20°C. The procedure was repeated until 1-5 samples were available to run the BApNA assay, whereupon they were thawed to room temperature and homogenized with a glass homogenizer. The homogenates were centrifuged at 4°C at 14,000g for 20 minutes and supernates used for protein estimation and subsequent trypsin activity assays. Pro'tein Estima'tion Assays The Bio-Rad protein assay is based on the changing of the absorbance maximum of an acid solution of Coomassie

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62 Brilliant Blue G-250 shifting from 465 nm to 595 nm when binding to a protein occurs. The Coomassie Blue dye reagent was prepared by dissolving 0.25 g of Brilliant Blue G (Aldrich) in 125 ml of 95% ethanol to which was added 250 ml of 85% 0-phosphoric acid. The solution was shaken well, left to stand for 15 minutes and made to to 500 ml with water after 30 minutes and again after three hours. The stock solution was then filtered through two pieces of Whatman #1 filter paper and the filtrate collected and stored. Reagent concentration was obtained by diluting the stock solution five times. Spectrophotometric Determination of Protein Three ml of reagent was measured into three tubes and 97.5 ml of TRIS-HCl buffer with 0.1 M CaCl2, pH 8,5 was added to each tube. Crude homogenate (100 |il) was added and measurements of absorbance taken after 2 minutes on a spectrophotometer at 595 nm. Three readings were averaged and based on the standard curve with bovine serum albumin, and calculations of protein concentrations in mg/ml were made with every sample (Bradford 1976) . The calculations were extrapolated to determine mg of protein per midgut required for calculations of nm of BApNA per gut.

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63 Preparation of B^NA Calibration Curves A stock solution of 1 mg/ml of porcine pancreatic trypsin type IX (Sigma) was prepared in 0 . IM HCl and stored at -20°C to ensure stability. BApNA (43.5 mg) was dissolved in 1 ml of DMSO and made to 1 nM BApNA with 50mM TRIS-HCl buffer with 0.1 M CaCl2, pH 8.5. To determine whether treatments with the experimental peptides affected trypsin production, non-treated guts were also included in the assays as controls for experimentally treated samples. BApNA was prepared by incubating O.lM BApNA in 50 mM TRIS-HCl buffer with 0.1 M CaCl2, pH 8.5, with gut sample digestive enzymes and measuring rate of hydrolysis of BApNA to p-nitroaniline (pNA) . The quantity of enzyme that hydrolyzes one nmole of BApNA per minute was determined by measuring absorbency at 410 nm of 1 ml of the reaction mixture. The values obtained from the gradient, defining differences in absorbance values reflecting the rate of pnitroaniline formation per minute over two minutes, were used to calculate nmoles of BApNA degraded per minute per insect midgut. Standard curves were obtained with porcine pancreatic trypsin IX (Sigma) incubated with BApNA (Figure 3-3) .

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64 Preparation of Insect Midguts for DFP Assays Following eclosion, adults were held in cages until they had reached the appropriate stages after which they were allowed to feed on a guinea-pig for one hour. Three hours later, those that had fully engorged received treatments under cold anesthesia. The bioassay treatments involved injecting insects between the third and fourth abdominal pleura with a peptide or peptide-mimic solution in Ringer's saline with a micro-capillary pulled glass needle. After examining the mosquitoes under dissecting microscope for stages of egg development attained, the guts were dissected and transferred into 0.1 ml per gut of 50 mM TRIS-HCl buffer, pH 8 . 5 with 0 . 1 M CaCla. The samples were homogenized and centrifuged at 4°C at 14,000g for 20 minutes. The supernatants were stored at -20°C until use and the procedure was repeated when additional samples were available . .>'''' • ' • -f i , f. ' , ' Determination of [1,3-^H] DIP-TLE Products [1,3H] DIP-trypsin-like derivatives were prepared by incubating above supernates, with/without tosyl-Lphenylchloro ketone (TPCK) inhibitor, with 1 ^il of [1,3-^H]

PAGE 76

65 DFP (5 microCuries (nCi)) per ^1/ specific activity 35 |aCi /millimole (mM) , New England Nuclear) for 24 hours at 4°C. Likewise different dilutions (0-5 (xg) of porcine pancreas trypsin and controls containing 100 |il of TRIS-HCl buffer, 0.1 M CaCl2, pH 7.9, were also incubated with [1,3-^H] DFP as above, Supernatants were incubated with [1,3-^H] DFP in the presence or absence of TPCK. TPCK inhibits chymotrypsin activity. Five |al of lOmM TPCK per 50 |al of midgut homogenate inhibits up to 95% of [1,3-^H] DIP-chymotrypsin derivatives from forming (Borovsky and Schlein 1988). TPCK binds specifically with chymotrypsin and allows the formation and hence estimation of only the [1,3-^H] DIPtrypsin-like products. Aliquots (5-50 |J.l) of the different dilutions (0-5 [ig) were adsorbed onto filter paper (4X4 cm) . The papers were then washed for 15 minutes in 200 ml of 10% Trichloroacetic acid (TCA) , replaced twice with 5% TCA for 15 minutes and followed by a 15 minute wash in absolute alcohol. The papers were dried at 50°C and added to tubes containing 3 ml of scintillation fluid (Liquiflor) and radioactivity was counted in a liquid scintillation counter. The maximum range of radioactivity expected is around 610,000 counts per minute (cpm) of [1,3-^H] DIPtrypsin-like derivatives. Calibration curves with

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66 o VD o o o o o CO o in o (Nl o o o (0 T3 C O U 0) m 0) g +) C i 3 n (0 1 >i J3 < Z n o c o •H P <0 •o u (U •o c •H 0) & M (T3 • T3 Q) C XI -P -H uiu 01 1 aaueqjosqv CO M O M-l (0 -P u a 0) w o I u

PAGE 78

67 trypsin were constructed by plotting radioactivity in disintegrations per minute (dpm) of [1,3-'h] DIP derivati auto-corrected from cpm by the counter. The procedure wa: standardized against trypsin concentrations in ^g/0.1 ml concentrations of trypsin-like enzymes. Calibration of [1,3-^H] DIPtrypsin-like Derivatives Several [1,3-^H] DIP-trypsin-like derivatives were quantified by scintillation counts and visualized on PAGE gels after treatment with Ae-TMOF, peptides and peptide mimics dissolved in Ringer's saline. To establish a linear relationship between the conversion of [1,3-'^H] DFP to [1,3^H] DIP derivatives, blanks/standards, control and treated midgut homogenates were incubated in 0.1 ml of 50mM TRISHCl buffer, pH 7 . 9 containing lOmM CaCl2. Incubations were done for 24 hours at 4 °C in the presence of 5 ^Ci of [1,3^H] DFP and radioactivity (dpm) of [1,3-^H] DIP derivatives determined. With blanks, determinations were made of the number of washes and for which concentrations of TCA were needed to be above background noise, compared to controls (Table 3-1) . This assay proved that TCA washes at various

PAGE 79

68 concentrations were effective at removing any un-reacted DFP substrate radioactivity on filter papers. As all counts were below 100 dpm, it was decided to proceed with washes of 10% TCA replaced twice with 5% TCA washes, for 10 minutes in every case as in Borovsky & Schlein (1987) . The standards set up with porcine pancreatic trypsin revealed that [1,3-^H] DIP-TLE products remained bound to the filter paper after several washes as 58,045 dpm were in the range expected with 0 . 1 mg /ml of trypsin. Scintillation counts of 33,968 dpm for the reference and 229,994 dpm with 1.2 ^1 of [1,3-^H] DFP incubated alone, proved that the counter was actually detecting tritiated incubates (Table 3-1) . Figure 3-4 shows a linear relationship between the quantities of trypsin in incubates and dpm counts of tritiated DIP-TLE derivatives (r^=0.9993). The straight lines obtained were used to quantitatively determine the amounts of trypsin like enzymes in insects treated with the various treatments described in Chapter 2.

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69 Polyacrylamide Gel Electrophoresis (PAGE) and Fluorography Polyacrylamide Gel Electrophoresis (PAGE) of [1,3H] DIPtrypsin-like derivatives of midgut homogenates were run in a modified Laemli system (Laemli 1970) as described by Borovsky & Schlein (1988). A slab gel (1.5mm thick, 15 cm long), containing a stacking gel of 3% (w/v) (3 cm) polyacrylamide and 0.125 M TRIS-HCl, pH 6 . 8 and a separating gel of 10% (w/v) (12 cm) polyacrylamide and 0.375 M TRIS-HCl, pH 8.8 was used during electrophoresis. In each gel several lanes were run simultaneously. PAGE was run at 25 volts for 2 hour and at 35 volts for 1 hour. Gels were initially stained in 0.1% (w/v) Coomassie brilliant blue R-250 for 15 minutes. They were then de-stained in a mixture of acetic acid, methanol and water (10% glacial acetic acid: 25% methanol) for one hour, washed in 10% glacial acetic acid: 10% methanol for one hour and destained overnight (5% glacial acetic acid: 7% methanol) . The gels were then incubated with dimethylsulf oxide (DMSO) (80 ml) for one hour, the solution was replaced with 22% 2,5-diphenyl oxazole (PPO) in DMSO and incubated for one hour. After the incubations, gels were rinsed with distilled water and kept in water for 30 minutes to facilitate full precipitation of

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70 Table 3-1. Trend of production of [1,3^H] DIP-TLE derivatives in the assays. Trypsin levels [1,3-^H] DIP derivatives (dpm) Blank 43.11 TCA washes, 10: 10:5 54.06 TCA washes, 10: 10:10 62.50 TCA washes, 5:5 :5 55.94 TCA washes, 10: 10:5:5 49.64 TCA washes, 10: 10:10:5 47.65 TCA washes, 10: 10:10:10 53.71 Reference 33, 968 . 00 1.2 ^il [1,3-^H] DFP 229, 994 0.0 mg porcine pancreatic trypsin 678 .75 0.1 mg 58, 045. 6 1 . 0 mg 121, 046

PAGE 82

71

PAGE 83

72 PPO. The gels were dried at 60°C for several days under vacuum on a filter paper in a gel drier and dry gels were exposed to x-ray films at -70°C for 45-60 days and then developed in a dark room. Results The Effect of Treatments on Midgut Blood Digestion Injections of oostatic hormone into blood-fed female A. aegypti resulted in inhibition of oostatic activity (Table 2-3) and modulation of trypsin-like enzymes in the midgut of treated females at 30 hpbm. Persistently high protein levels in the midgut, above 200 mg/ml, confirmed that blood was actually retained in their guts during the whole treatment course. An indication of which component of the total protein was trypsin was done through observing an activity gradient from the rate of hydrolysis of BApNA with crude gut homogenates (Table 3-2). Trypsin activity recorded in guts of the control and saline control insects was 4.92 and 3.7 6 nmoles of BApNA/minute/gut , respectively. Treated insects had trypsin activity of 2.87 for TMOF-A, 2.04 for A5, and 2.00 for TMOF-B. The other peptides had

PAGE 84

73 • 0) U 3 n •0 0) 6 0) o o o o — O o o o ' o i-H rH rH rH n rH rH r« c P tjl C p Q) -H CNI en O CM rH O O iH > 0) e CD o <^ CM rH rH ro O ^i) o ro r-H p -P o cn e •H H U c -P 03 a, o to C OQ c n • 0) •H -H to .H a 0 CNJ m rH o a p, >i e CTi CD o CM m o t— o CO rH rH 0) >^ c t^ CM CM o rH rH CM O CM .H o o g 0) X! a o x: H e e e e e e e o c Cn CP CP c c c c c c 4-1 Q) -p H. O -H c u o o p (0 a; o o o o o o o (0 -P E e rH rH rH rH rH O CTi O 0) >i (0 -p Q) •H c > Cd -H Eh H -H -P o o o DC to S-l s-l 2 -p -p 1 c • c c rg -H -H o O "3 to 1 CO a u U CM OQ Q. to 1 +J >, 0) 0) c >, 0) 2 o (0 O H H o c c 1 1 rH Eh fH to 0 •H (t3 (0 (tJ e X3 iH rH rH rH rH rH X> II m ti) Q) o (0 CM >i 0^ E-i u CO < < < CQ Oi cr\ cn U rH *

PAGE 85

74 activity levels below 2.0, e.g., 0.26 for females that survived the toxic A6 treatment, 1.63 for A7, and 1.31 for B2. The pyrenebutyric acid-derivatives of TMOF-A (pba) variously affected trypsin activity as activity was 2.46 for 9Fla-NH2, 0.76 for pyl collected one step before lPba(0tBu)-NH2, 1.09 for 9Fla-0H, 1.36 for P,6), 0.16 for CblAla-NH2 and 0.19 for IPba (OtBu) -NH2 . In the previous chapter, various light photo-micrographs revealed a bright red color of blood, persistent for 24 hpbm, which strongly suggested the presence of undigested blood in the gut of treated insects, and even beyond one to two days post treatment. However, the gut of control mosquitoes had blood digestion ongoing and the blood had turned a characteristic darkish brown color, indicating protein-degradation by proteolytic enzymes. Trypsin activity recorded in the gut of the control and saline control stable flies was 3.82 and 2.24 nmoles of BApNA/minute/gut, respectively (Table 3-3). Treated insects had trypsin activity of 2.76 for TMOF-B, 3.72 for B2, and 1.255 for a stable fly ovary-derived peptide described in Chapter 4 .

PAGE 86

75 Ti Q) M CO IB QJ — e — . , — . o o o o o rH CU rH U c (U CO c CTl CTl x: -H CO CM -p (U e • • o O • tji 4J \ CTl in • • c o 1 \ rH c rH •H -H 0 > e m • H CO 4-1 < U (D H 3 < M c C 03 -rH H > e CO rH 0 a o CNJ un (Ti O >1 6 00 CM CM C rH u c i c 4-) O .H rH -H -H O o > 4-) •H U -P 4-) 4-1 O C c u a -H O o (0 a u CJ u CO (U c >1 H 0^ iH o c c CO a: (0 n o H > a e rH O >i II Q) Q) o (0 CM Dm (J m OQ CO Eh -K

PAGE 87

76 Comparison of Total Serine Proteases Produced in A. Aogyptl Females Treated With Ae-TMOF Peptides and Peptide Mimics The production of total tritiated DIP-TLE products varied greatly in treated mosquitoes. The smallest amount of serine proteases was secreted by A. aegypti females that were un-fed 3 hours later and gave counts of 3,046 dpm. This was in comparison to 4,922, 39,432 and 93,850 dpm in blood-fed controls 3 hpbm injected with saline, 30hpbm injected with saline and 30hpbm touched (but not injected) , respectively (Table 3-1 and Figures 3-5 and 3-6) . In all cases with treatments dissolved in saline, and dpm counts were lower than saline controls at 39,4322 dpm (Figures 3-6 & 7) . The counts from various treatments were A, 10 ^g yielded 20,181 dpm; B, 10 ^ig yielded 10, 402 dpm; B2, 10 ^ig yielded 7, 044 dpm; A5, 10 ^ig yielded 3, 902 dpm while A7, 10 ^g yielded 16,325 dpm. Peptide treatments affected protease levels available in the guts of treated insects, and the results were that little or no digestion of blood occurred, with reduction in the end products available to the insect for egg development. Trypsin production was correlated with retarded egg development. In addition to an effect on normal trypsin production, a chymotrypsin inhibitor (TPCK)

PAGE 88

77 (0 g 0) t-l c H c I O U Q) O rH ^^ (0 e (U r~ <4-l u o > c H to O c iH o H II 4J (0 O i3 u • to -H (U T3 c H H +J CI "0 tl) 1 > •H > +J -H •H 3 u (0 TD o tu -H j-> TJ (0 (0 o o o o CM uidp in I n 0) M 3 D» -H

PAGE 89

78 was incorporated in some of the incubations and comparisons made to ascertain how much retardation would be achieved with and without chymotrypsin products included. In all cases the addition of TPCK into the incubates led to decreased amounts of [1,3^H] DIP-TLE products in the female A. aegypti mosquito. Table 3-4 and Figure 3-7 show the trend of production of [1,3^H] DIP-TLE derivatives in the presence or absence of TPCK, The results revealed dpm of 3,046 (blood-fed at 3 hours), 4,922 (blood fed), 39,432 (injected with saline at 30 hours) and 93,850 in saline only treated controls that were un-fed at 3 hours. Hence the saline injection alone affected protease enzyme production in the female mosquito because just pricking the insect cuticle results in some trauma. Consequently, the saline control at 30 hours (39,432 dpm) was taken as the control for the experiment in all cases. There was, however, less pronounced retardation with this control value as opposed to adopting the 93,850 dpm count (Table 3-4). Based on the 39,432 dpm count as control, reduction in production of tritiated DIP-TLE derivatives was up to 51% for A, 41% for A7, 64% for B, 57% for B2 and 100% for A5 . The trend with pba-mimics was 40% with IPba (OtBu) -NH2, 42% with 9Fla-0H, and 111% with 9FlaNH2 topically applied. Adding TPCK revealed that not all

PAGE 90

79 protease activity in the gut of these female mosquitoes was trypsin-like . There was reduced dpm counts with TPCK included in the incubations. Counts as low as 32% were scored in saline only controls at 3 hours as opposed to 13,186 dpm (100%) in saline only controls at 30 hours. With the 13,186 dpm count taken as control in gut samples incubated with TPCK, the counts were 26% for A, 61 % for A7, 41% for B, 70% for B2 and 23% for A5 . The trend with pba-mimics was up to 37% with IPba (OtBu) -NH2, 20% with 9FlaOH, and 122% with 9Fla-NH2 topically applied. Polyacrylaunide Gel Electrophoresis and Fluorography of Trypsin and Chymotrypsin-like Enzymes. Various aliquots of incubates were separated on PAGE gels and visualized by fluorography with X-ray films incubated for 45-60 days. Several isoenzyme bands were detected in treated adult females of A. aegypti (Figures 37 to 3-9) . Figure 3-7 reveals that nulliparous females never exposed to a blood meal had almost no bands of tritiated DIP-TLEs (lane B from 3,046 dpm) as compared to blood fed controls only 3 hours later in which three bands were evident (lane A from 5,462 dpm). Thirty hours post blood meal, up to six bands of trypsin isozymes were

PAGE 91

Table 3-4. Effect of TMOF peptides on production of [1,3^H] DIP-TLE derivatives in A. aegypti with and without TPCK. Treatment Time (hpbm) Levels DIP-TLE no TPCK of [1,3-^H] derivatives (dpm) % TPCK % Saline un-fed 3 3, 046 8 Saline 3 4, 922 12 2, 807 21 Saline control 30 0 _/ , *! 0 ^ 100 13, 186 inn -L u u A, 10|ag 30 20,181 51 13,279 101 B, lO^ig 30 25,281 64 5, 468 41 A5, lO^g 30 39, 606 100 3, 025 23 A7, lOng 30 16, 325 41 8, Oil 61 B2, lOfXg 30 22, 417 57 9,2 62 70 IPba (OtBu) -NH2, 4 nm 30 15, 968 40 4, 869 37 9Fla-0H, 4.6 nm 30 16, 445 42 2, 628 20 CbAla-NH2, wash 30 3, 805 29 9Fla-NH2, 7 nm top 30 43, 666 111 16, 042 120 Un-inj ected 30 93, 850 237 Blood fed taken as 0 hpbm; injected at 3 hpbm and dissected at 30 hpbm.

PAGE 92

81 c -H (0 sx >1 u 4-> -H 1 4-) Oi H a >l Q tj> a H 4-> X3 to c > (0 •H i-l CO i JS -P < H 0 3 e 0 >, M 0 4-1 u u J= <1) o 4J c -H <4-l (0 £ td sjunoo Ludp I ro
PAGE 93

82 visible in saline treated females (lane D from 39,432 dpm) . Guts from A. aegypti females treated with peptides B, B2, A, A7 and B were placed in lane E from 10,402 dpm, lane F from 7,044 dpm, lane G from 20,181 dpm, lane H from 16,325 dpm and lane I from 16,541 dpm counts respectively. The upper bands clearly present in lane D of the control appeared less pronounced in incubated guts (lanes E through I) from the treated mosquitoes. Apparently, the bands present in lane A with 3 hpbm saline control gut incubates were the only prominent bands seen in lanes E through I. This indicated little change in TLE profiles from 3 hpbm when the mosquitoes received treatment. It is possible that there was retarded conversion of early trypsin mRNA needed to trigger transcription of late trypsin prominently represented as the third dark band in the controls 30 hpbm. Figure 3-8 further shows that two clear bands were present in 3 hpbm blood-fed females (lane I) compared to 4 bands in the controls 30 hpbm (lane H from 13,186 dpm). Saline controls with less dpm of tritiated DIP-TLE still continued to reveal 4 distinct bands irrespective of low dpm counts. Other lanes (A, B, C, F and G) with treatments showed less of the bands found in the saline control (lane I) 30 hpbm. Lane A showing results from treatment with 10 ^g of B had

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83 BCD F G H I Figure 3-7. PAGE Fluorography of [1,3H] DlP-trypsinchymotrypsin like isozymes of females of A. aegypti. The Xray film was exposed to the gel for 60 days at -70°C. Each lane represents gut aliquots from one midgut equivalent. (A) (B) (C) (D) (E) (F) (G) (H) (I) Saline control 3hpbm Saline control, un-fed 3h Saline control 3hpbm Saline control B, B2, A, A7, B, 10)ag lO^g long lOMg lO^g (5, 4 62 dpm) ; later (3,04 6 dpm) ; (6, 541 dpm) ; (39, 432 dpm) ; (10, 402 dpm) ; (7, 044 dpm) ; (20, 181 dpm) ; (16, 325 dpm) ; (16, 541 dpm) . All trypsin levels at 30 hpbm, unless stated otherwise.

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84 2 distinct bands that were seen also in lanes F and G with guts from 10 [iq treatment with A5 . Lane B from mosquitoes treated with IPba (OtBu) -NH2 showed 2 diminished bands with little tritiated DIP-TLE products. Lane D shows 2 clear and two unclear bands present in guts from 9Fla-NH2 treatment with 87,038 dpm. There was no retardation of tritiated DIPTLE products in this case. Figure 3-9 is an over-exposed plate kept in the cold for 70 days before developing, but otherwise similar to Figure 3-10. The gel however was prepared with less quantities (12 ^1) of aliquots per lane as opposed to 30 jil per lane in the rest of the gels. It shows that saline treated controls (lane M) had several bands at 3 hpbm that were not present in lane L 30 hpbm. Not all the same bands continued to persist in treated females 30 hpbm (lane L) . Lane D is a blank lane. In the treated insects some isozymes which disappeared in the controls 30 hpbm continued to persist while also some isozymes that appeared in the 30 hpbm saline control showed up. But in all cases (except for the topical treatments), the dpm counts in this aliquots from treated insects continued to be lower than 39,850 taken as the control dpm count at 30 hpbm. Lanes G and H had treatments where IPba (OtBu) -NH2 and 9Fla-NH2 mimics were applied topically.

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85 I ABC D EF6HI Figure 3-8. PAGE Fluorography of [1,3^H] DlP-trypsinchymotrypsin like isozymes of females of A. aegypti. The Xray film was exposed to the gel for 45 days at -70°C. Each lane represents gut aliquots from one midgut equivalent. (A) B, lO^ig (25, 851 dpm) ; (B) IPba (OtBu) -NH2, 4nmol (15,968 dpm) ; (C) 9Fla-0H, 4.6nmol (16,445 dpm) ; (D) 9Fla-NH2, 2.0 nmol (87,038 dpm) ; (E) IPba (OtBu) -NH2, 4nmol. top, (136,126 dpm) ; (F) A5, lOiag (31,249 dpm) ; (G) A5, lO^g (56, 950 dpm) ; (H) Control, (13,186 dpm) ; (I) Control, 3 hpbm (4,922 dpm). All trypsin levels at 30 hpbm, unless stated otherwise.

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86 The dpm counts were 136,126 and 87,038 for the topical applications with IPba (OtBu) -NH2 and 9Fla-NH2, respectively, compared to injections with the same mimics that showed 15,968 and 25,851 dpm, respectively. Lane G showed much activity since probably the insect continued to synthesize its TLEs unaffected by the topical treatment. Figure 3-10 shows incubations of some guts done with TPCK, an inhibitor of the production or expression of [1,3^H] DIP-chymotrypsin-like enzyme products in the incubates. The dpm counts were much lower in all cases than saline controls at 3 and 30 hpbm, which gave 2,807 and 18,786 dpm, respectively. Dpm counts in treatments were 5,468; 13,279; 6,485; 8,011; 6,226 and 7,044 dpm for guts dissected out of females treated with B; A; A5; A7; 9Fla-0H and B2, respectively. The bands of tritiated DIP-TLE products were similar in lanes B and C and somewhat similar to lane D with saline treatments at 3 hpbm; B, 10 ^ig; and A, 10 ^ig, respectively. This showed that retarding production of chymotrypsin-like products leads to evidence that trypsinlike products present in controls were absent in guts from treatments B and A. Lane E with A5, 10 ^ig treatment was similar to controls at 30 hpbm. However, treatment A7, and 9Fla-0H had upper profile bands absent revealing that the second isozyme present in controls 30 hpbm was diminished.

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87 In all lanes B through H a middle isozyme band present in control lane A of 30 hpbm was absent. This revealed that three isozyme bands are affected differently by the treatments. They probably all have to be present in proportion to enable normal trypsin activity. Figure 3-11 gives data on incubates from stable fly females and males. Control un-fed female had 6,969 dpm compared to un-fed male with 5,332 dpm. Three later dpm counts were 4,486 and 5,360 for female and males, respectively. Dpm counts did not change much after feeding as females had 7,892 and 5,345 dpm 24 and 120 hpbm given blood ad lib, while guts from a female treated with A7 at 5 ^g had 4,138 dpm. Male dpm counts were 6,345 at 24 hpbm. The data showed a different trend of effect on protease availability in guts of the stable fly, as [1,3"^HjOIP-TLE product levels appeared stable or uniform in females from emergence to 120 hpbm. This supports the fact that stable fly TLEs are produced and present in the gut all the time and no dramatic episodes occur since they are not batch feeders.

PAGE 99

88 ABC DEFG HI J KLM Figure 3-9. PAGE Fluorography of [1,3^H] DlP-trypsinchymotrypsin like isozymes of females of A. aegypti. The Xray film was exposed to the gel for 60 days at -70°C. Each lane represents gut aliquots from one midgut equivalent. (A) B2, lO^g (22, 417 dpm) ; (B) B2, lOng (21,159 dpm) ; (C) Saline (13, 186 dpm) ; (D) Blank; (E) A5, lO^g (39, 606 dpm) ; (F) A5, lO^ig, (14, 046 dpm) ; (G) IPba (OtBu) -NH2 4nmol. top (136, 126 dpm) ; (H) 9Fla-NH2, 4nmol. Top. (87, 038 dpm) ; (I) 9Fla-0H, 4 . 6nmol (16,445 dpm) ; (J) IPba (OtBu) -NH2, 4nmol (15, 968 dpm) ; (K) 9Fla-NH2, 4nm (25, 851 dpm) ; (L) Saline (13, 186 dpm) ; (M) Saline 3hpbm (5, 462 dpm) ; All trypsin levels at 30 hpbm, unless stated otherwise.

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89 AB CD EF 6H Figure 3-10. PAGE Fluorography of [1,3^H] DIP-trypsin-like isozymes of females of A. aegypti. The X-ray film was exposed to the gel for 60 days at -70°C. Each lane represents gut aliquots from one midgut equivalent inhibited with TPCK. (A) Saline, (18,78 6 dpm) ; (B) Saline, 3hpbm (2, 807 dpm) ; (C) B, lO^ig (5, 4 68 dpm) ; (D) A, lOng (13,27 9 dpm) ; (E) A5, lO^ig (6, 485 dpm) ; (F) A7, lO^ig (8,011 dpm) ; (G) 9Fla-0H, 4 , 6nm (6, 22 6 dpm) ; (H) B2, lO^ig (7,04 4 dpm) All trypsin levels at 30 hpbm, unless stated otherwise

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90 Discussion Gooding et al. (1973) reported that following uptake of a blood meal de novo synthesis of trypsin-like enzymes was initiated in the midgut of female mosquitoes. Briegel & Lea (1975) suggested that the stimulation was due to globular proteins present in the blood meal. These enzymes provide for orderly digestion of blood that is necessary for successful egg development. The overall process of ovary and egg development is regulated by a complex series of hormonal interactions. The mechanism whereby Aedes-TMOF oostatic hormone and the derived peptide-mimics cause inhibition of proteolytic enzyme activity was followed by concentrations that would cause over 50% oostatic activity. A direct relationship between oostatic activity and lack of synthesis of proteolytic enzymes was explored by directly injecting oostatic hormone into female A. aegypti (Borovsky 1988) . About 75% of protease activity in the midgut of female A. aegypti is trypsin-like (Briegel & Lea 1975) . Saline control TLE activity was lower than that from sham colony control guts. Based on the saline control activity at 3.76 nmol BApNA per minute per gut, the lowest activity

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" ^ ' i 91 . A B CDE F 6HIJ Figure 3-11, PAGE Fluorography of [1,3^H] DlP-trypsinchymotrypsin like isozymes of females of S. calcltrans . The X-ray film was exposed to the gel for 60 days at -70°C. Each lane represents gut aliquots from one midgut equivalent. (A) Control Un-fed female (6,969 dpm) ; (B) Control un-fed male, (5, 332 dpm) ; (C) Control un-fed female, 3h later (4,486 dpm) ; (D) Control un-fed male, 3h later (5,360 dpm) (E) Vitellogenic control 60 h later (5, 345 dpm) ; (F) Control 24 hpbm female (7,892 dpm) ; (G) Control 24 hpbm female (5,313 dpm) ; (H) Control 24 hpbm male (6, 345 dpm) ; (I) Saline control 24 hpbm male (8, 244 dpm) ; (J) A7, 5 ng, 24 hpbm (4,138 dpm). All trypsin levels at 30 hpbm, unless stated otherwise

PAGE 103

92 was observed with A6, CblAla-NH2 and 9Fla-0H, hexaproline fragment, IPba (OtBu) -NH2 and PYl collected one purification step before IPba (OtBu) -NH2, with less than 1 nmol BApNA degraded. Moderate activity was observed with TMOF-A, A5, A7, TMOF-B, B2, and 9Fla-NH2. TLE activity could not be assessed with toxic samples that generally proved lethal to the insects between 3 and 30 hpbm for mosquitoes as in A4 and A6. However, Graf & Briegel (1985) and Borovsky & Schlein (1988) reported that several [1,3-^H] DIP-TLE isozymes are synthesized in the midgut of A. aegypti, sand flies, P. papatasi, house flies, M. domestica, stable flies, S. calcitrans and even lice, Pediculus humanus . Female mosquitoes injected with oostatic hormone synthesized 87% less of the [1,3-^H] DIP-TLEs i.e. 13,625: 99,167 cpm (Borovsky et al . 1989) . In these studies saline control readings of 93,850 dpm were obtained with fresh sham operated females, while saline controls placed in storage for months gave readings of 39,432 dpm. When compared to the 39,432 dpm reading, 50% reduction in TLEs levels was obtained (79% if the 93,432 dpm count is taken as standard) . Percentage retardations below 50% were attained with A7 at 10 and IPba (OtBu) -NH2. Retarded TLE levels were also obtained in insects that survived the toxic 9Fla-

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93 OH mimic. It is possible that toxic peptide-mimics cause the stressed (dying) insects to not perform physiological functions normally. Samples were shared between assays with BApNA and tritiated DFP such that in cases with A4 and A6, samples were not available for DFP assays. The two assays show reduced activity and levels of enzymes, especially so with the toxic peptides and moderate TLE activity and levels with ecstatic peptides and peptide-mimics. The fact that there were reduced levels of tritiated DIP-TLE products in all cases revealed that not all products in this mosquitoes were trypsin-like . TPCK inhibits production of chymotrypsin-like enzymes. Therefore even with retarded TLE activity in treated mosquitoes, chymotrypsin digests some amount of blood meal protein to avail the insect with some amino acids for yolk protein synthesis. This could also be another reason why some peptide and peptide-mimic treatments were only partially oostatic even if little TLE were being produced. Isozyme profiles in saline treated controls supported earlier studies by Noriega et al. (1996a & b) and Khalkok et al. (1993) that the trypsin profiles in A. aegypti change following uptake of a blood meal from an early trypsin to late trypsin form in about 24-36 hours. With bands present in 3 hpbm guts, it was interesting to see how

PAGE 105

94 they continued to persist in treated mosquito guts and the absence of the late trypsin prominent band seen in Figure 3-7 kept at -70°C for 60 days. This indicated that the conversion of early to late trypsin was being negatively affected by the treatments. Figure 3-9 also exposed at -70°C for 60 days showed some mosaics whereby both the early and late trypsin bands appeared. This revealed a situations whereby partial ecstatic activity was recorded and the incubates were from a homogenized group sample. Incubations with TPCK, a chymotrypsin inhibitor revealed, less banding at 3 hpbm and in all treatments at 30 hpbm. This also showed that the 3 hpbm band was present in TMOF-A, TMOF-B treated insects and the band present in 30 hpbm saline treated insects was clearly absent. This indicated inhibition of trypsin-like enzymes in the midgut of mosquitoes that were treated with TMOF-A and B. The peptide and peptide mimics treatments revealed interesting mosaics whereby some early trypsin bands and also the late trypsin band appeared slightly with A5 with a light late trypsin band and A7, B2 and 9Fla-0H with light early trypsin bands. This again showed that the normal process of conversion of early to late trypsin was inhibited with TMOF-A and B, and slightly affected with the peptide mimics.

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95 The trend of banding was not followed in stable flies in which dpm counts did not reveal dramatic differences between un-fed and saline treated controls from day zero to 60 hours provided blood available ad lib. The banding pattern of the isozymes showed remarkable tritiated DIP-TLE products with females at 24 and 60 from first blood meal. There were more bands formed in the 24 post first blood meal lanes when compared to the other lanes. This supported the idea that early in the adult life, mature trypsin results from cleavage of 2 kDa fragment of about 18 amino acids to produce a mature trypsin isozymes of 22,900 and 31,600 kDa. Stable flies have translational and not transcriptional control of trypsin biosynthesis (Moffatt & Lehane 1990) . While trypsin is stored as an inactive proenzyme, it is secreted into the gut lumen in a fully active form.

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CHAPTER 4 EVIDENCE OF OOSTATIC AND TRYPSIN MODULATING ACTIVITY IN EXTRACTS FROM OVARIES OF THE STABLE FLY, STOMOXYS CALCITRANS Introduction Since the report on the existence of an ovarian development inhibiting hormone present in the corpora lutea of ootheca-bearing cockroaches, Blatella germanica and Blatta orientalis , research has been geared towards the elucidation of mechanisms regulating oogenesis and egg production. Since then research has uncovered oostatic hormones in a variety of insects with a majority of them of ovarian origin (Iwavnov and Mescherskaya 1935). In Diptera, Adams et al., (1968) first demonstrated the existence of oostatic hormone (s) in the house fly, Musca domestica in when complete inhibition of a second gonotropic cycle occurred, mature eggs from the first ovarian cycle were retained. Furthermore, extracts from mature ovaries injected into newly emerged females inhibited progress into the vitellogenic phase of ovarian maturation. Meola and Lea (1972) demonstrated a similar mechanism in mosquitoes. 96

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97 involving an ovary produced humoral factor inhibiting yolk deposition in less developed egg follicles. In A. aegypti, Borovsky (1985) observed that ovaryderived TMOF (Aedes-TMOF) , injected into newly emerged females disrupts orderly blood digestion essential for successful egg development. Trypsin biosynthesis in several hematophagous insects, including stable flies, was retarded up to 48%. This suggested cross-reactivity of Aedes-TMOF among hematophagous insects that use trypsin-like enzymes for protein digestion. To date, three inhibitors of ovarian development from Diptera were characterized: These are Aedes-TMOF from A. aegypti (Borovsky et al . , 1990, 1993) and Neb-TMOF and Neb-Collostatin from Neobellaria bullata (Bylemans et al., 1994). It is possible that all three oostatins are cleaved products of yolk proteins. Cross reactivity of Aedes-TMOF across hematophagous insects that utilize trypsin-like-enzymes for protein digestion and the occurrence of such factors in both mosquitoes and flesh flies led to speculation that TMOF-like peptides occur in the ovaries of stable flies too. Such an ovary-derived hormone may function to modulate blood digestion and can therefore be assessed for bioactivity in the mosquito, A. aegypti and the stable fly, S. calcitrans . While A. aegypti needs one blood meal to mature an egg batch, female stable flies require up to five sequential

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98 blood meals to mature the first egg batch. Therefore I extracted, characterized, and evaluated an extract from vitellogenic ovaries of stable flies for a fly ovary derived factor (SFOV) with similar properties to Aedesand NebTMOF. Assays for oostatic and trypsin modulating properties were conducted on A. aegypti and S. calcitrans. Characterization of SFOV was done following the procedure used for Aedes-TMOF (Borovsky et al . , 1990). The evaluations for bioactivity were performed as described with Aedes-TMOF peptides and peptide-mimics on A. aegypti and S. calcitrans . Materials and Methods St£j3le Fly Rearing Stable flies reared in the fly rearing facilities at the USDA-CMAVE insectaries were used for this study. Maintenance conditions were as described in Chapter 3. Newly emerged adults at various ages provided ovaries for the search for a stable fly oostatic factor.

PAGE 110

99 Preparation of the Stable Fly Ovary-Derived Factor (SFOV) . SFOV was isolated from vitellogenic ovaries five days after female stable flies were fed blood ad lib. The ovaries were dissected out and placed in distilled water containing 1 mM phenyl methyl sulfonyl fluoride (PMSF) acidified to pH 4.5 with 1 M hydrochloric acid. Fifty ovary equivalents were homogenized and the homogenate was centrifuged for 20 minutes at 20,000g in an Eppendorf centrifuge at 4 °C and the supernatant harvested. The supernatant was heat treated at 50 °C in a water bath for 10 minutes and recentrif uged in the cold for 20 minutes. The fresh supernatant was collected, frozen and lyophilized on a Virtis lyophilizer. The freeze-dried sample was reconstituted in 0.1% trif luoroacetic acid (TFA) in HPLC water and placed on SepPak (35 cc) and eluted with increasing concentrations (10, 20, 30, 40, 50, 80%) of acetonitrile / 0.1% TFA (Borovsky, et al., 1990). The fractions were dried on a Speed-Vac at 40°C, lyophilized and reconstituted in saline for bioassays for both ovarian development and trypsin biosynthesis and activity. Characterization of the SFOV. A portion of the lyophilized 10% acetonitrile in 0.1% TFA fraction from Sep-Pak separation of vitellogenic ovaries

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100 was reconstituted with 0.1% TFA and further separated with Hewlett Packard High Pressure Liquid Chromatography (HPLC) on a reverse-phase Cis HPLC column. The HPLC parameters were as follows: two minutes pre-run with 100% water in 0.1% TFA, followed with a gradient run of 0 to 80% acetonitrile in 0.1% TFA over 30 minutes (Figure 4-1) at -800 millibars. Samples evaluated came from newly emerged females, 2-day old females, vitellogenic females, and 5-day old males. The methods of chapters 2 and 3 were adopted for evaluation of oostatic and trypsin modulating activity, respectively. Results Preparation of the Stable Fly Ovary-Derived Factor (SFOV) . The preparation process for the ovary-derived peptides placed strong emphasis on looking for a safe procedure that would not damage conformation and active site(s) of a probable SFOV. It was expected that an SFOV would be similar to Aedes-TMOF which is a deca-peptide with its active site for recognizing and interacting with the midgut receptors

PAGE 112

101

PAGE 113

102 responsible trypsin production center around the amino acid tyrosine found at the N-terminal . Evaluation of Oostatic Activity and Trypsin Properties Ovaries from 200-1000 females in a batch provided a starting tissue sample. Heat-treatment at 50 °C for exactly 10 minutes enabled larger peptides and yolk proteins that might be toxic to precipitate. Lyophilizing allowed removal of solvent from the sample without use of heat, which denatures proteins and peptides creating distortions in configuration of compounds and possibly affecting their bioactivity. The 0.1% TFA was the preferred solvent for HPLC runs both with Sep-Pak and regular chromatography on the Hewlett Packard Chromatograph . Characterization of the SFOV Data summarized in Figure 4-1 were obtained by taking readings of % acetonitrile in 0.1% TFA against time as the gradient was running. High peaks were consistently found highest at around 10 minutes, for five sets of samples were collected from 1997 to 1999. At times 0-4, 4-6, 6-9, 9-12, 12-15 minutes into the run, collection of fractions 1, 2, 3,

PAGE 114

103 4, and 5 were made up to twenty times, respectively. The acetonitrile in the collected fractions was removed from samples by using a Speed-vac and the remaining liquid frozen and lyophilized to remove the remaining liquid. A portion was reconstituted in Ringer's saline, and injected into 5day old mosquitoes at varying doses, and remaining portions that were bioactive were submitted to ICBR for Matrix Assisted Laser Desorption lonization-Time of Flight-Mass Spectroscopy (MALDI-TOF-MS ) . Evaluation of SFOV Oostatic Activity Bioactivity of the lyophilized Sep-Pak fractions reconstituted with Ix Ringer's saline from 10, 20, 30, to 40% acetonitrile varied when tested against A. aegypti. Dried weights obtained for the fractions were 8.5, 160, 80, 20, 13, 13, and 84 mg for fractions at 10, 20, 30, 40, 50, 60, and 80% acetonitrile in 0.1% TEA respectively. The dried fractions were reconstituted in 100 |al start solution and dilutions made to appropriate concentrations so that around 10 \iq was injected into each female treated. Usually mortality resulted at higher concentrations with no survivors at 20 and 40 \iq doses (not shown) . Females that survived at the dose of 10 |J,g per female revealed oostatic

PAGE 115

104 activity with the 10% acetonitrile fraction as egg follicle length was no more than 88.5 |im + 19.41 (21/0) at 30 hpbm. This was in comparison to follicle length measurements for the 20, 30, 40, 50% and saline control fractions of 166 ^m (12/8), 111 nm (9/12), 163 ^m (15/4), 100 ^im (1/19) and >200 [im (18/2) respectively, at 30 hpbm (Table 4-1). The 10% acetonitrile in 0.1% TFA Sep-Pak fractions were lyophilized and the dried sample reconstituted in 0.1% TFA. It was injected into the HPLC (Hewlett Packard) on reversephase Ci8 HPLC to attain further separation. The fractions were collected based on Fig 4-2a as a guide and portions were collected at: 1) 0-4, 2) 4-6, 3) 6-9, 4) 9-10, 5) 1012, and 6) 12-25 minutes and categorized as portions 1, 2, 3, 4, 5 and 6, respectively. Individual portions 1, 2, 3, 4, and 5 were re-analyzed under the same conditions (as above) on the reverse-phase Cis HPLC and gave consistently similar peaks with retention times similar to peaks that appeared in the original 10% ACN fraction with portions 4 and 5 (Figures 4-2b-d) . The collected portions 1, 2, 3, 4 and 5 were bioassayed on 5-day old A. aegypti females, 3 hpbm and ecstatic activity scored at 30 hpbm. The bioassays showed that portion 4 tested at 6.1 |J.g / female had oostatic activity and egg follicle length was 83.33 + 12.19 ^m (15/5), compared to 200 + 28.1 |am (18/2) for controls (see

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105 Table 4-1) . Portion 5 at 6.7 ^ig / female was toxic, with egg follicle length of 75 (im (1/9) compared to 200 + 28.1 fam (90%) for controls. Evaluation of Trypsin Modulating Activity Injections of SFOV Sep-Pak fractions and HPLC portions into blood-fed female A. aegypti resulted in inhibition of trypsin-like enzyme (TLB) activity in the midgut of treated females at 30 hpbm (Table 4-2) with fraction 10% acetonitrile in 0.1% TFA and specifically portion 4 collected off the HPLC at 9-10 minutes. The kinetics of trypsin-like enzymes were 2.37 6 nmoles BApNA/minute/gut with SFOV HPLC portion 4, 4.916 nmoles with colony control, and 3.764 nmoles with saline injected control females. The protein content measurement (total blood protein and enzyme protein) in all samples analyzed was above 250 mg/ml, which indicated that the insects were alive during the period of treatment. Analysis of excreta from treated insects revealed some trypsin activity in the excreta but very low protein content which in some cases could not be calculated (results not shown) .

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106 Table 4-1. A comparison of the effect of peak fractions of SFOV on ovarian development in A. aegypti showing follicle length achieved and number attaining length or number dead (%) % ACN in Dose per Follicle length Mortality 0.1% TEA female at 30 hpbm (Hg) (\im) (%) (%) Control 200 + 28. 1 (18) (10) 50% ACN 6.5 100 (1) (95) 40% ACN 10 163.33 + 39. 94 (15) (20) 30% ACN 8 111.11 + 22. 05 (9) (60) 20% ACN 8 166.67 + 32. 57 (12) (40) 10% ACN 8.5 88.46 + 19. 41 (21) (0) 4 of 10% ACN* 6.1 83.33 + 12. 19 (15) (25) 5 of 10% ACN* 6.7 75 (1) (95) Follicle length at 30 hpbm. * = portions 4 and 5 obtained 9-10 and 10-12 minutes into the HPLC run of the 10% acetonitrile Sep-Pak fraction.

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107 ure 4-2a. Reverse phase Cis HPLC Chromatogram of 6-day old, vitellogenic, S. calcitrans females (10% ACN/0.1% TFA Sep-Pak fraction 0-30 mins) a"S9saas }o ooi'oss t'oEz V on ;i

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108 Figure 4-2b. Reverse phase Cis HPLC Chromatogram of 6-day old, vitellogenic, S. calcitrans females (10% ACN/0.1% TFA Sep-Pak portion 4-6 mins)

PAGE 120

109 Figure 4-2c. Reverse phase Cis HPLC Chromatogram of 6-day old, vitellogenic, S. calcitrans females (10% ACN/0.1% TFA Sep-Pak portion 9-10 mins

PAGE 121

110 Reverse phase Cis HPLC Chromatogram of 6-day old, vitellogenic, S. calcitrans females (10% ACN/0.1% TFA Sep-Pak portion 10-12 mins)

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Ill Table 4-2. A comparison of the effect of SFOV on trypsin activity in female A. aegypti. Females Fed on pig Trypsin activity (nmol BApNA/min/gut ) Protein (mg/ml) Control 4 . 916 289.824 Saline control 3.764 269.567 A 2 .872 278.080 A5 lOug/female 2 .043 501.667 A6 lOug/female 0.264 349.239 A7 1.632 332.160 A8 lOug/female 1.309 407 . 102 B lOug/female 2.002 332.320 SFOV (HPLC portion) 2 .376 254 .27 Trypsin activity and levels at 30 hours post blood meal.

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112 Matrix Assisted Laser Desorption lonization-Time of FlightMass Spectra (MALDI-TOF-MS) of the SFOV Sanqple. MALDI-TOF analysis of 10% SFOV Sep-Pak fractions from newly emerged, two-day old, vitellogenic females and older males revealed the presence of more peptide compounds in the sample from vitellogenic females. MALDI-TOF-MS spectra showed the presence of a candidate peptide at m/z 908,8 in the bioactive fraction (Figure 4-3a) . This peptide was not seen in spectra of younger females or males of the same age as vitellogenic females. Other potential peptides were seen at in/z ~ 2, 935 in both young and vitellogenic females, and others ranging from 3,603 to 6,944 were present only in females with vitellogenic ovaries (Figure 4-3a & b) . Amino acid analysis of HPLC portion 4, originally from the 10% Acetonitrile in 0.1% TFA fraction, revealed a peptide of the following amino acids: Aspartic acid, Asparigine, Serine, Glutamine, Threonine, Glycine, Glutatamic acid, Histidine, Alanine, Arginine, Tyrosine, Proline, Methionine, Valine, Phenyl alanine, Isoleucine, Lysine and Leucine. The proportions of Proline, Arginine and Lysine were highest.

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113 Figure 4-3a. Mass spectra of HPLC fraction from 10% ACN in 0.1% TFA from vitellogenic ovaries of S. calcitrans (L) offered blood for four days.

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114 Figure 4-3b. Mass spectra of HPLC fraction from 10% ACN in 0.1% TFA from ovaries of 3-day old S. calcitrans (L) offered blood for two days.

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115 Figure 4-4. Amino acid analysis of SFOV portion 4 of the 10% ACN in 0.1% TFA fraction. 3/15/99 6:06 PM t. i.3 3.Q 19 Incerpolaced baseline i>eajc NO RT Peajc ID Type Heigtac fm>l He 3 30 *»P c 320540 297.68 3 96 JUn G 546855 428.50 3 4 54 Ser c 540999 1013.11 4 4 76 Sin c 11524S 113.72 5 4 93 Thr c 290S34 395.50 6 5 15 Sly c 577'565 667.24 56 Giu 590359 564. SO a 6 03 His c 194022 396.76 9 5 92 Al* C 737412 792. OS 10 7 53 Krg c 1688897 9168.82 7 as 135209 1760.53 12 3 25 11645 0.00 13 S 62 Tyr c 71066 7S.78 14 9 03 31623 0.00 IS 9 27 26487 0.00 1« 9 S7 1005 0.00 » 90 12625 497.05 13 10 50 Pro c 757214S 9346.02 1» 13 98 Met c 236895 261.88 20 11 25 val c 957729 937 82 21 36 12752 3.00 22 12 33 ptncc c 272527 547.64 23 12 57 180576 0.00 24 13 30 469633 0.00 25 13 89 lie c 18990 26.55 16 14 19 Lya c 1224377 1122.05 27 14 38 35089 0.00 2i 15 43 3752 0.00 23 IS (2 3363 O.OO !0 16 06 422 0.00 31 IS SO 5543 7.00 32 17 2^ 3413 0.00 33 17 SO 414 56.77 34 17 90 10524 559.79 Appltad Biosyacavu Prociaa PROCISE-HT

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116 Discussion The ability of oostatic extracts to inhibit vitellogenesis has now been established in several species of Diptera including M. domestica (Adams et al. 1968), Aedes atropalpus, D. melanogaster (Kelly et al. 1984), A. aegypti (Borovsky 1985), and recently N. bullata (Bylemans et al. 1994). The existence of oostatic hormone was observed first in aqueous ethanol extracts of ovaries from M. domestica . The extracts inhibited ovarian development but were relatively toxic. Semi-purified extracts induced 50% inhibition of ovarian development at 25 \iq (Adams et al . 1968, 1970), while heat-treated aqueous extracts were oostatic at a dose of only 11.2 |J.g protein. Tobe (1980) cautioned that a general mechanism of action of such oostatins was difficult to establish since it would probably act on different levels of the endocrine hierarchy controlling reproduction in insects. Mechanisms may inhibit release of neurosecretory material from the median neurosecretory cells e.g. EDNH (Adams 1981); act directly on the as ovary inhibitors JH-induced follicle cell patency as in R. prolixus (Davey & Huebner 1974) or may inhibit ovarian ecdysteroid synthesis in A. atropalpus (Kelly et al. 1984) .

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117 The extraction procedure that was used to obtain AedesTMOF was adopted for the present search for the stable fly derived oostatic hormone. Oostatic hormone activity was first noticed with ovary extracts that were prepared from five-day old vitellogenic females. Vitellogenic ovary sample was compared to samples prepared the same way obtained from newly emerged, three-day old females exposed to two blood meals ad lib or even 5-day old male abdomens. Stable flies use the first two blood meals for general adult development and it is the last three blood meals that are directly used for vitellogenic growth Moobola & Cupp (1978) . Oostatic activity of extracts appears low during pre-vitellogenesis and increases during vitellogenic stages. Cross-reactivity between Ae-TMOF and NEB-TMOF has also been reported (Bylemans et al. 1994) . These data suggest that oostatins are widely distributed in insects that exhibit cyclic ovarian maturation, especially as Musca oostatins were recognized by Ae-TMOF antibody (Borovsky et al . 1994a) . However, Neb-collostatin has no sequence similarity with AeTMOF but appear to bear the same mode of action. Oostatic hormone may therefore be a family of small peptides synthesized and secreted in different tissues to inhibit the initiation of second and subsequent cycles of oocyte development while the already mature and developing first cycle oocytes are still present.

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118 Dissecting ovaries from females was done with at least 200-1000 females to enable describing the sample in terms of ovary equivalents later. Placing ovary tissue into 1 mM PMSF at pH 4.5 helped to preserve the bioactivity of any peptides when held under in acidic conditions. Heat treatment at no higher than 50 °C enabled larger peptides, yolk proteins and toxins to precipitate and not interfere with sample preparation later. From then onwards freezing temperatures were preferred as well as acid conditions to retain bioactivity . , Lyophilizing enabled drying of the sample without use of harsh heat which would have denatured the peptides, leading to distorted conformations and loss of bioactivity. TMOF-like peptides modulate trypsin bioactivity by recognizing a midgut receptor and interacting with it. Hence the emphasis on use of a procedure unlikely to damage an active site(s) suspected to reside on Aedes-TMOF is on the amino acid tyrosine found at the N-terminal of TMOF (Borovsky et al . 1994b). : Lyophilized samples were reconstituted in 0.1% TFA, which is acidic in nature, to preserve the peptides in a bioactive condition. TFA also promotes adsorption of peptides on the front of the Cis reverse phase columns both in the Sep-Pak and RP-HPLC columns. Adsorption to the column front is the basis of RP-HPLC that enables eluents run

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119 through the column at an increasing gradient of polar to non-polar solvents to separate compounds by their polarity. As the gradient progresses, and depending on its interaction with both the column and the peptides, the peptides are separated. Both the Sep-Pak and RP-HPLC use the same type of Ci8 reverse phase column packing. The Sep-Pak columns enabled rapid separation of the sample into fractions that were checked for bioactivity. After confirming that the 10% fraction was bioactive, a portion of the sample was reconstituted in 0.1% TFA for HPLC, and later for MALDI-TOF-MS spectra. Preparative HPLC of samples collected sequentially and processed at separate times between 1997 and 1999 continued to yield the same characteristic peaks seen in Figure 4-2a. Furthermore, the individual portions were collected on the RP-HPLC at different times by using an increasing acetonitrile concentration gradient over 30 minutes. All the portions collected at 0-4, 4-6, 6-9, 9-10 and 10-12 minutes continued to yield peaks at times very similar to the original 10% acetonitrile /0.1% Sep-Pak sample from which they were collected. This provided the confidence that HPLC was an excellent tool for separation of the peptide fractions. The individual portions were injected into A. aegypti females and fraction 4 with a near-perfect single peak was found to be ecstatic at 30 hpbm. The females were analyzed for

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120 evidence of egg development retardation and also gut bioactivity. Females injected with fraction 4 had reduced trypsin-like activity and produced less trypsin over the 30 hours until dissection. MALDI-TOF-MS of portion 4 included a candidate material, possibly a peptide, that could be responsible for biological activity. Females injected with portion 4 had little gut gut trypsin-like activity as it probably produced very little trypsin over the 30 hours until they were dissected. MALDI-TOF of 10% acetonitrile in 0.1% TFA showed a posible novel peptide responsible for biological activity seen here.

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CHAPTER 5 SUMMARY AND CONCLUSIONS Because of the role of hematophagous arthropods as vectors, the reproduction of these arthropods has attracted much attention. Adult hematophagous insects require a blood meal for egg production and midgut proteases play a major role in the digestion of the meal. Digestive enzymes in hematophagous insect vectors constitute one of the early barriers for any parasite ingested with a blood meal. Since proteins are the most obvious nutrient in blood, much effort has been placed on studying proteinases in hematophagous arthropods . The present study was designed to investigate the effect of different chemically formulated peptides and mimics derived from Aedes-TMOF on egg development and trypsin like enzymes were affected. Compounds with AedesTMOF activity could be possible oostatins for hematophagous insects. In A. aegypti, there is a transitory decline in midgut proteinase activity immediately after blood feeding which Fisk & Shambaugh (1952) suspected to be due to substrate depletion of enzymes or inhibition by specific proteinase inhibitors in blood serum, Khalkok et al . (1993) 121

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i • % \ ', ' : 122 confirmed that there was a transtition from an early trypsin to late trypsin. This study on various Aedes-TMOF peptide and peptide mimics revealed that the conversion of early to late trypsin can be delayed by certain treatments. The fact that autogenous Aedes atropalpus with mature or nearly mature eggs are incapable of normal digestion of a blood meal (Hudson 1970) and that decapitation of A. aegypti after feeding depresses proteinase levels suggested the possibility of hormonal involvement in digestion especially when females are gravid with eggs. The appearance of hormonal signals early in a gonotropic cycle leads to information being transmitted to the midgut to depress its level of TLE production. While A. aegypti has naturally occurring TMOF coming into play to moderate trypsin production, this study proved that altering the original natural peptide could achieve an oostatic effect or a toxic effect. Gooding (1973) used BApNA to evaluate levels of trypsin present in guts based on nmol BApNA hydrolyzed per minute per gut and was able to characterize and determine that in A. aegypti both mRNA and trypsin protein synthesis take place after ingestion of a blood meal. Isoenzyme profiles of tritiated DIP-TLE products in saline treated controls supported earlier studies by Noriega et al . , 1996a

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123 & b) that an early trypsin mRNA transcript appears and is prominent within 3 hours from uptake of a blood meal. It then disappears and is replaced by the mature late trypsin form that actively digests a blood meal by 24-36 hpbm. BApNA was also used in this studies to determine whether trypsin activity profiles were affected in mosquitoes and stable flies treated with Aedes-TMOF peptide and peptide-mimics . Injections with all the treatments affected trypsin activity. BApNA as a substrate was very useful because the rate of degradation to p-NA directly depended on how much active trypsin was available in the gut homogenate. Lower degradation rates implied that less enzyme was available to degrade (cleave) the substrate and release the colorimetric p-NA. In addition, more support to the amount of available active TLE was obtained by irreversibly binding available TLEs to tritiated DFP. Actual levels determined by scintillation counting revealed that peptide and peptidemimics treatments were also affecting trypsin levels. This case was not seen with topically applied peptides. Acetone used to dissolve them may not have been the best solvent for peptide-mimics. It was therefore not likely that much mimic could have stayed on the insect cuticle long enough to penetrate the cuticle. Levels of actual TLEs present were established by irreversibly binding TLEs to tritiated DFP

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124 and determine by scintillation counting and isoenzyme profile patterning detected by fluorography of the tritiated DIP-TLE products. " , Egg development closely follows blood digestion as primary egg follicles begin to grow in size beyond the resting stage after uptake of a blood meal. The primary follicles of newly emerged females are very small (below 50 [im long) and cannot be differentiated from the surrounding nurse cells. In treated mosquitoes with peptide and peptidemimic, the primary follicle growth is not so synchronized and does not occur in a normal pace when compared to saline treated controls that had reached over 200 fxm over the 30 hpbm period. This was correlated to the fact that with most peptide treatments, the early trypsin profiles continued to be seen long after this early transcript had disappeared from saline control guts. In some cases, there were both early and late trypsin transcripts present at 30 hpbm, which implied that some late trypsin biosynthesis was going on. However, in both situations, the persistence of an early trypsin transcript must have created a deficit in late trypsin supply to digest the blood to provide yolk protein precursors .

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125 In S. calcitrans , blood feeding neither causes an immediate rise nor a transitory decline in general midgut proteinase activity. A steady rise of TLE activity was instead noted in proteinase content in the midgut. This may be due to activation of pre-existing material or de novo synthesis of the enzyme (Champlain & Fisk 1956) . It was not conclusive whether TLE production was actually being affected by the treatments. It was concluded that Aedes-TMOF peptides were not active oostatins for stable flies. In the course of five blood meals required for maturing an egg batch, it appeared that the insect could recover and compensate for any retarded availability of blood mealderived yolk protein precursors. The search for natural TMOFs in stable flies revealed an ovary derived ecstatic factor (SFOV) from vitellogenic ovaries. SFOV was ecstatic and trypsin modulating in A. aegypti and had trypsin modulating properties but no observable ecstatic activity on the stable fly. It is possible that stable flies possess prolinespecific peptidases in their hemolymph that could actively sequester, destroy and thereby regulate any excess or unnatural peptides circulating in the hemolymph (Martensen et al., 1998) . While Neb-TMOF (sequence NPTNLH) that was hydrolyzed

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126 by this peptidase had only one proline in its sequence, a high proline content was observed in the amino acid composition of the SFOV portion 4 obtained at 9-10 minutes in the RP-HPLC run. . v--

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135 Marten, E. J. 1996. The vitelline membranes of A. aegypti and Drosophila melanogaster . Invert, Reproduction & Devel. 30: 255-264. Martensen, I., J. Koolman & R. Mentlein. 1998. Proline specific peptidase from the blue blow fly, Calliphora vicina hydrolyses in vitro the ecdysiostatic peptide trypsin modulating oostatic factor (Neb-TMOF) . Arch. Insect Biochem & Physiol. 37: 146-157. Meola, R. & A. 0. Lea. 1972. Humoral inhibition of egg development in mosquitoes. J. Med. Entomol . 9: 99-103. Moobola, S. M. & E. W. Cupp. 1978. Ovarian development in the stable fly, S. calcitrans in relation to diet and juvenile hormone control. Physiol. Entomol. 3: 317-321. Moffatt, M. R. & M. J. Lehane. 1990. Trypsin is stored as an inactive zymogen in the midgut of S. calcitrans . Insect Biochem. 20: 719-723. Noriega, F. G., J. E. Pennington, C. Barillas-Mury, X. Y. Wang & M. A. Wells. 1996a. Early trypsin, an Aedes aegypti female specific protease, is posttranscriptionally regulated by the blood meal. Insect Molec. Biol. 5: 25-29. Noriega, F. G., X. Y. Wang, J. E. Pennington, C. BarillasMury & M. A. Wells. 1996b. Early trypsin, a female specific midgut protease in A. aegypti: isolation, amino terminal sequence determination, and cloning and sequencing of the gene. Insect Biochem. Molec. Biol. 26: 119-126. Noriega, F. G., D. K. Shah & M. A. Wells. Juvenile hormone controls early trypsin gene transcription in the midgut of A. aegypti. Insect Mol. Biolo. 6 (1) : 63-66. Pennington, J. E., & F. G. Noriega. 1995. The expression of early trypsin in A. aegypti. J Cell Biochem. 21A (Suppl) : 211. Raikhel, A. S. & T. S. Dhadialla. 1992. Accumulation of yolk proteins in insect oocytes. Ann. Review Entomol. 37: 217-251.

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137 Yin, C. M., B. X. Lou, M. F. Li & J. G. Stoffolano. 1989. Precocene II treatment inhibits terminal oocyte development but not vitellogenin synthesis and release in the black blow fly, Phormia regina Meigen. J. Insect Physiol. 35: 465-474.

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138 BIOGRAPHICAL SKETCH Loyce Okedi was born on 27 October 1959 in Ngora, Uganda. She grew up in Scroti town where she attended Soroti Primary School up to 1971. She then proceeded to attend Gayaza High School, Kampala, where she attained both the East African Certificate of Education in 1975 and the Advanced East African Certificate of Education in 1977. She received her Bachelor of Science degree (Honors) in zoology and botany from Makerere University in 1981. In 1982 she joined the Uganda Trypanosomiasis and Malaria Research Organisation and worked in the malaria section. She was awarded a research training grant from the by WHO/TDR Special Programme for Research and Training in Tropical Diseases and enrolled for a master's degree at the University of Nairobi, Kenya, She obtained a Master of Science degree in medical entomology in 1990 under the supervision of Professor C. P. M. Khamala. Her research was on resistance to synthetic insecticides by malaria vectors of Anopheles gambiae larvae from rice schemes in Kenya and advocated for bacterial larvicides {Bacillus thuringiensis and B. sphaericus) . On returning to Uganda Trypanosomiasis Research Organisation at Tororo (now Livestock Health Research

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138 Institute) she joined the trypanosomiasis program and worked on the tsetse and trypanosomiasis epidemiology project on Lake Victoria Islands in Uganda prior to enrolling at the University of Florida, Gainesville.

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. David A. Carlson, Chair Associate Professor of Entomology and Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Phi'. Jerome Hogset'1 Assistant Professor oj Entomology and Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree ^^of^ Doctor of Philosophy. ^ames L, Nation, Professor of Entomology and Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards o^.,s£:±olarly presentation and is fully adequate, in scope'^^^f^d duality, as a dissertation for the degree oy'^ff^or jbfi^hi/osophy . Greiner Professor of Veterinary Medicine

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This dissertation was submitted to the Graduate Faculty of the College of Agricultural and Life Sciences and to the Graduate School and was accepted as partial fulfillment of the requirement^ for the degree of Doctor Philosophy. /V A / May 2000 Dean, College of Agricultural and Life Sciences Dean, Graduate School