MSU RETURNING MATERIALS: Place in book drop to remove this checkout from LIBRARIES —3——. your record. FINES will be charged if book is returned after the date stamped below. 5L 1-": ‘“ 5 A4! ESTERASE VARIATION AND CLASSIFICATION IN FOLSOMIA CANDIDA (Willem); AND ASSESSMENT OF THE SELECTIVE ADVANTAGE OF DIAZINONrRESISTANT ISOZYMES by Karin A. Grimnes A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology I982 ABSTRACT ESTERASE VARIATION AND CLASSIFICATION IN FOLSOMIA CANDIDA (Willem); AND ASSESSMENT OF THE SELECTIVE ADVANTAGE OF DIAZINON-RESISTANT ISOZYMES by Karin A. Grimnes Esterase enzymes of the parthenogenetic collembolan Folsomia candida (Willem) were investigated using starch and polyacrylamide electrophoresis. Ten strains were examined and four distinct estrerase zymograms were identified. All esterases were produced during all life stages. Esterases were classified as carboxyl (CBE) or choline esterases (CHE) based on substrate specificity and response to inhibitors. Two groups of CBEs were differentiated: slow migrating esterases found in both head and trunk, which might be involved in digestion, and fast migrating CBE found in trunk and hemolymph. Because of their substrate and inhibition characteristics, fast CBEs may regulate juvenile hormone titer in this organism. Choline esterases (CHE) of E, candida consisted of one group of isozymes with moderate electrophoretic mobility. Several CHEs were located in the head, and were thought to be esterases of the "brain"; other CHEs were found in the trunk, possibly functioning in the ventral nerve cord. Degree of inhibition of CHE activity was similar for all enzymes of this class except for esterase 5 in strain C, which required 10-1000 times the inhibitor concentration to cause 50% inhibition for esterase 5 of all other strains tested. The relationship between in vitro resistance of esterase 5 and in vivo resistance of strain C was investigated using the organophosphate Diazinon. Ten strains of E. candida were fed Diazinon-contaminated yeast (10-2000 ppm) over a 21 day treatment period. Diazinon treatment resulted in changes in behavior, and treated animals avoided food, did not consume exuvia, and had longer instar durations than control animals. Effects of Diazinon on F. candida were strain-dependent. Strain C, however, consistently showed optimal values for reproductive success in treated and control populations. The possible selective advantage for strain C over other strains investigated in this study was correlated with in vitro esterase resistance to Diazinon, but no causal relationship could be established. ACKNOWLEDGEMENTS I wish to thank the members of my committee: Dr. Jamers Miller, Department of Entomology, Dr. Ralph Pax, Department of Zoology and especially Dr. Renate Snider and Dr. Richard Snider, Chairman, for their aid in the preparation of this dissertation. Special thanks are extended to Dr. James Butcher, former Chairan of the Department of Zoology, for temporarily serving on my committee. I would also like to thank Dr. John Gill for help in statistical model design. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION MATERIALS AND METHODS CHAPTER 1: GENETIC AND PHYSIOLOGICAL VARIATION INTRODUCTION RESULTS DISCUSSION CHAPTER 2: CLASSIFICATION OF ESTERASES IN F. CANDIDA AND SUBSTANTIATION OF THE ROLE OF EACH CLASS INTRODUCTION RESULTS DISCUSSION CHAPTER 3: ORAL TOXICITY OF DIAZINON TO E. CANDIDA STRAINS POSSESSING IN VITRO DIAZINON;SUSCEPTIBLE AND -RESISTANT ISOZYMES INTRODUCTION RESULTS DISCUSSION CONCLUSION SUMMARY IMPROVEMENTS AND FUTURE WORK LITERATURE CITED iii Page viii 19 21 29 34 37 60 69 74 104 110 113 116 117 APPENDICIES I. Esterase Classification and Commercially Prepared Enzyme Sources Used for Inhibition Studies. 11. Naphthyl Esters Used in Substrate Studies. III. List of Pesticides Used in Inhibition Studies. Structure of Pesticides Used in Inhibition Studies. IV. Cross Classified Factorial Model with Double Nesting (after Gill, 1978). V. Migration Distance (mm) of Esterase Isozymes of Four Strains of F, candida in Polyacrylamide Gels. VI. Number (out of 32/samp1e) and Z Strain A‘Er candida with Detectable Esterase S and 6 Activity. Number (out of 32/sample) and Z Strain D.§. candida with Detectable Esterase S and 6 Activity. VII. Average Exuvia per jar in Strains of E. candida in Control and Diazinon Treated Jars (1000 ppm) (n=20 jars/ treated S jars/control, 5 animals/jar). VIII. Mortality (as number dead/total number) for Diazinon Treated and Control Individuals after 21 days. iv Page 125 126 127 128 129 130 131 132 133 138 LIST OF TABLES Table Page 1. Molt Cycle Phases of F. candida (after Palevody and Grimal, 1976). 17 2. Origin of E, candida Strains. 17 3. Summary of Enzyme Experiments used in This Study 18 4. Rf Values for Major ESterase Enzymes of E. candida on Polyacrylamide Gels. 24 5. Relative Distribution of Esterase Activity in Head and Trunk of E, candida, Expressed as Z of Control (=100%, using whole-body homogenates). 39 6. Differentiation of Esterase Classes Based on Inhibition Data from the Literature. 44 7. Inhibition of Commercially Prepared Esterases and Major Esterases of E. candida on Starch Gel Preparations. 44 8. Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 1 47 9 Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 3 48 10 Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 4 49 ll. 12. I3. 14. 15. 16. 17. 18. 19. LIST OF TABLES (Cont.) Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 5 Concentrations of Inhibitors (loglo) Needed to Reach 50% (ISO) Esterase Inhibition: Polyacrylamide Band 6 Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 7 Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 10 Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 11 Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 12 Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 13 and 14 Classification and Special Properties of Esterases in E, candida Based on Inhibition Studies Effect of Diazinon on Number of Days Spent Fasting (out of 21) by E: candida (N320 individuals/strain). vi Page 50 52 53 55 56 57 58 59 78 20. 21. 22. 23. 24. 25. 26. LIST OF TABLES (Cont.) Page Analysis of Variance of the Effects of Diazinon on Fasting Behavior of 7 Strains of E. candida (n=20 individuals/Strain) 79 Effects of Diazinon on Persistence of Exuvia in Days (n=2 exuvia per each of 20 individuals per strain) 95 Analysis of Variance of the Effects of Diazinon on Persistence of Exuvia in 7 Strains of F, candida (U= 2 Exuvia/Individual, E - 20 Individuals/Strain) 96 Effects of Diazinon on Instar Duration in E, candida (n-2 Observations on each of 20 Individuals/Strain) 98 Analysis of Variance of the Effects of Diazinon on Instar Duration in 7 Strains of F. candida (U82 Observations/Individual, E - 20 Individuals/Strain) 99 Effect of Diazinon on Oviposition Frequency in F, candida (in percent, based on the number of females out of 20/strain which oviposited at least once during the 21 day period) 101 Total Cumulative Mortality (Z) in F, candida after 21 days of Treatment with 1000 ppm Diazinon (Abbott's Corrected Mortality) 102 vii LIST OF FIGURES Figure l. 6. 7. Starch Gel Electrophoretic Patterns of Esterases in F3 candida. (Pattern D also found in Strains E and F) Polyacrylamide Gel Electrophoretic Pattern of Esterases in F, candida. (Pattern D also found in strains E and F, Pattern B also found in strains G, H, I and J). Cyclic Esterase Bands 5 and 6 in Strains A and D of E: candida. Each data point represents the 2 (out of 32 individuals) with detectable activity (after Grimnes, 1981). Effect of Alpha Naphthyl Ester Chain Length on Esterase Activity in Four Strains of E, candida. (X axis a chain length, Y axis - intensity of staining, where 0 - no staining, 3 - 1002 of normal staining reaction with ANA as a control) Effect of Beta Naphthyl Ester Chain Length on Esterase Activity in Four Strains of E. candida (X axis - chain length in Carbons, Y axis - intensity of staining where 0 - no staining, 3 - 1001 of normal staining reaction with ANA as a control) Effect of Diazinon on Mean Numbers (:LSE) of Exuvia per jar in Strain A of F, candida (n - 20 jars/treated, 5 jars/ control, 5 animals/jar). Effect of Diazinon on Mean Numbers (:_SE) of Exuvia per jar in Strain B of F. candida (n a 20 jars/treated, 5 jars/ control, 5 animals/jar). viii 22 23 28 40 41 81 82 LIST OF FIGURES (Cont.) Figure 8 10 11 12 12 13 14 Effect of Diazinon on Mean Numbers jar in Strain C of F. candida (n - control, 5 animals/jar). Effect of Diazinon on Mean Numbers jar in Strain D of F. candida (n - control, 5 animals/jar). Effect of Diazinon on Mean Numbers jar in Strain E of E. candida (n - control, 5 animals/jar). Effect of Diazinon on Mean Numbers jar in Strain G of F. candida (n - control, 5 animals/jar). Effect of Diazinon on Mean Numbers jar in Strain H of F. candida (n - control, 5 animals/jar). Effect of Diazinon on Mean Numbers jar in Strain I of F. candida (n - control, 5 animals/jar). Effect of Diazinon on Mean Numbers jar in Strain J of F, candida (n - control, 5 animals/jar). Effect of Diazinon on Mean Numbers (1 SE) of Exuvia 20 jars/treated, SE) of Exuvia jars/treated, SE) of Exuvia jars/treated, SE) of Exuvia jars/treated, SE) of Exuvia jars/treated, SE) of Exuvia jars/treated, (: SE) of Exuvia 20 jars/treated, (: SE) of Exuvia per 5 jars/ per 5 jars/ per 5 jars/ per 5 jars/ per 5 jars/ per 5 jars/ per 5 jars/ per jar in Strain D and E 0f.§' candida (n - 20 jars/treated, 5 jars/control, 5 animals/jar). ix Page 83 84 85 86 87 88 89 90 LIST OF FIGURES (Cont.) Figure Page 16 Effect of Diazinon on Mean Numbers (: SE) of Exuvia per jar in Strains B, G, and H of F. candida (n = 20 jars/ treated, 5 jars/control, 5 animals/jar). 91 17 Effect of Diazinon on Mean thbers (:_SE) of Exuvia per jar in Strains B, I and J of F. candida (n = 20 jars/ treated, 5 jars/control, 5 animals/jar). 92 18 Effect of Diazinon on Mean Numbers (2 SE) of Exuvia per jar in Strains A, B, C and D of F, candida (n = 20 jars/ treated, 5 jars/control, 5 animals/jar). 93 INTRODUCTION The isozymes of many insect enzyme systems have been extensively investigated (Wagner and Selander, 1974). Isozymes are potentially useful as biochemical markers to locate and follow populations, individuals or cells, and have also proven valuable in the study of cellular processes, metamorphosis and development. Alterations in enzyme titer of activity or shifts between isozymic forms provide a window through which the changing nature of the cellular environment can be investigated. Variation in function and behavior between isozymes can be used to predict the role of each, and the consequences of possessing such enzymes. An important tool in the study of isozymes and their significance is electrophoresis, the separation of proteins based on their shape, size and net charge. Improved isozyme detection techniques (Brewer and Sing, 1970) and use of polyacrylamide gel electrOphoresis (Ornstein, 1964; Davis, 1964) have increased enzyme systems available to researchers. The widespread use of electrOphoresis for random population surveys has produced large amounts of data concerning the natural variability of many major enzyme systems. In random surveys, however, individuals of unknown age, physiological or developmental state are analyzed. Thus, variation within each individual and variation among individuals and 1 2 populations are frequently combined. Most authors agree that mere compilation of electrophoretic variation from randomly assayed individuals results in little useful information about enzymes and other proteins (Mayr, 1976; Stephen, 1973). Approaches dealing with the significance of isozyme variation, with Special reference to the possible selective advantage of isozymic forms, are considered more fruitful (Dobzhansky, 1970; Lewontin, 1974). Ideally, an investigation of the isozymes comprising an enzyme system might involve the following questions: 1. What variation is present at the species, population, individual, tissue and cellular level? 2. What changes in isozymes occur throughout the life history of the individual (development, growth, seasonal variation)? 3. What roles do these isozymes fulfill in the organism? 4. What is the biological significance of the variation between isozymes and the consequences to the organism possessing a given complement of isozymes? The biological significance of enzymatic polymorphism can best be examined using an enzyme system which possesses both genetic variability and known physiological roles. The esterase system meets both criteria. Esterases are a widely distributed, highly polymorphic group of enzymes found in almost every form of life (Pearse, 1972); they catalyse ester hydrolysis. The general reaction is: Rl- C - OR2+ H20 > R1 - C-OH + R2 - OH 3 where R1 and R2 can be aromatic or aliphatic. Phosphate and thiophosphate esters may also be degraded (Pearse, 1972). Esterases have been studied extensively, and have received special attention in the insects, because of their great variability and important role in the cleavage and inactivation of juvenile hormone, acetylcholine and ester pesticides. Although these esterase functions are not unique to insects, in the insects they may be studied simultaneously. Acetylcholine is the major neurotransmitter in many insect central nervous systems (Chapman, 1971), and its cleavage is essential for proper neural function. A specific enzyme, acetylcholine esterase is responsible for the cleavage of acetylcholine (Ahmad, 1976). Another function of esterases is the cleavage of juvenile hormone (JH). Juvenile hormone, a methyl ester, is degraded to the non-active JH-acid form by esterase action (Ajami and Riddiford, 1973), and its inactivation is essential for normal metamorphosis (de Kort and Granger, 1981). The third role of esterases in insects is inactivation of esters entering the insect from the environment (Pearse, 1972), most notably the ester pesticides (organOphosphates and carbamates). Ester pesticides may interfere with esterase function by forming a stable enzyme-inhibitor complex, in which the enzyme becomes trapped (Plapp, 1976). Both juvenile hormone and acetylcholine cleavage are thereby reduced; the consequences may be death due to improper metamorphosis or lack of neural function (Gerorghiou, 1972). The relationship established between isozyme properties and pesticide resistance in insect populations in the field (Welling, 4 1977; Plapp et al., 1979) has indicated that esterases can be used to study the biological significance of isozymic variation. Genetic variation affecting esterase function can reduce pesticide effectiveness in several ways: acetylcholine esterase isozymes may be less susceptible to organophosphate inhibition; variation in non-target esterase isozymes may decrease the stability of enzyme-pesticide complexes, or increase the efficiency of cleavage, reducing pesticide levels. Examples of pesticide resistance via each of these mechanisms have been summarized by Plapp et a1. (1979). Most research on esterases has been confined to the pterygote orders, with only a few reports on esterases in apterygote species. Esterase research should be extended into the apterygote groups for several reasons. First, there is very little information on enzymes of any kind for apterygote species; for example, no acetylcholine esterase has been reported in these animals. Secondly, indiscriminate pesticide spraying eliminates beneficial species as well as harmful ones. Information on pesticide resistance of esterases may indicate which pesticides result in less damage to beneficial populations. A third reason for studying apterygote esterases is their role in melting and development. Apterygote orders are ametabolous; they show little change between hatching and attainment of sexual maturity. Wigglesworth (1964) suggested that apterygote forms were analogous to larval pterygotes. This is supported by isolation of JH in one thysanuran species, Thermobia domestics (Packard)(Madhavan et al., 1981), and of JH-like substances in other apterygote species. If JH is generally present in apterygotes, esterases may function in 5 regulation of JR titer. Also, since apterygote species tend to molt throughout their adult life (in contrast to pterygotes), apterygotes are ideal for the study of hormone and cellular interactions as a model for pterygote molting processes. A final reason for esterase study in apterygotes is that they are considered ”primitive" with respect to pterygotes on the basis of several developmental and morphological characteristics. Few comparisons of physiologial or cellular processes have been made between apterygotes and pterygote species, and this area is certainly worth investigating from both an evolutionary and phylogenetic standpoint. In the apterygotes, preliminary reports on esterase variation exist for only one collembolan species, Folsomia candida (Willem). Hart and Allamong (1979) surveyed this species for taxonomically relevant variation, and examined age-related changes in isozymes. Asher and Snider (1975) studied esterase variation related to molt cycle stage. Grimnes and Snider (1981) examined egg-laying behavior for several strains of F, candida which were differentiatied on the basis of esterase isozymes. F, candida is a useful model organism for the study of esterases in apterygote species because of the wealth of data concerning its various life processes. Physiology (hormone levels, activity of glands, digestion and molting), reproduction (maturation, provision and production of eggs) and susceptibility to pesticides have been studied. In addition, the biology of the species under a variety of controlled conditions has been extensively investigated (Snider, 1971; Snider and Butcher, 1973). 6 Another advantage for using F: candida is its parthenogenetic mode of reproduction. No breeding (and subsequent potential change in isozyme complement) is necessary to maintain pure culture lines. Since parthenogenesis in F. candida is accomplished by inihibition of meiosis II (Palevody, 1973), all offspring, barring mutation, should be homozygous. Combination of parthenogenesis with a rapid reproductive rate makes it extremely easy to raise large numbers of genetically identical F, candida for experimentation. In view of the limited knowledge of esterases in apterygotes, and the interesting preliminary data on F, candida, the esterase system of this species was chosen for investigation. The present study was designed to meet the following objectives; 1. To record genetic variation in esterases in E. candida populations and to determine the contribution of age, molt cycle stage and body tissue to the variation detected by electrophoresis, 2. To characterize the esterases 0f.§° candida, identify esterase class, and use data from these studies to support the role traditionally assigned to each esterase class, 3. To ascertain the biological significance (and possible selective advantage) of isozymic forms by analyzing the relationship between isozymic prOperties and organismal response to environmental stress imposed by an organophosphate pesticide. 7 MATERIALS AND METHODS Experimental Design To satisfy the objectivesof this study, two distinct types of experiments were performed on F, candida: electrophoretic assays to detect and characterize esterase activity, and oral toxicity studies on whole animals to quantify the differences in biology (behavior and physiology) among strains. Enzyme Studies Esterase enzymes were investigated in strains currently in culture to detect the extent of esterase variation available for further research. Animals in various stages of the molt cycle were analyzed to characterize the changes in isozyme titir or activity associated with ecdysis. Esterases in the embryonic and early post-embryonic period (from newly laid eggs up to small juveniles) were followed to record changes in enzymes during development. The results of these studies are discussed in Chapter 1. Esterases were characterized by testing for substrate specificity and sensitivity to inhibitors and ester pesticides. The relative distribution of esterase isozymes between head and trunk of F, candida was investigated. Results were used to classify esterases and to estimate functional differences between esterases of the same class. Substrate specificity, body location, inhibition and molt cycle variation data were used to substantiate the role for each esterase classs in F, candida. These studies are dealt with in Chapter 2. Toxicity Studies Oral toxicity studies with Diazinon were conducted to relate 8 observed i3_!i££2 pesticide resistance of esterase enzymes to pesticide resistance of the whole animal. Changes in biology (egg laying, molting feeding) due to pesticide treatment were investigated. This work is discussed in Chapter 3. Rearing Methods Culture methods utilized in this study were those of Snider (1973). Clear plastic jars with snap-on lids (2.5 x 3.5 cm for rearing individuals, 3.7 x 5.4 cm for stock cultures) were filled to a depth of 0.5 cm with a watery slurry of 1:1 plaster-charcoal mixture. The charcoal was Darco G60 (Sargent-Welch Scientific Company). After the plaster-charcoal substrate had hardened, the jars were closed, and set aside until needed. When animals needed to be transferred, they were tapped gently into jars; eggs were moved with a needle. Distilled water was added periodically to maintain relative humidity close to 100%. All cultures were maintained at 15.5°C in a constant temperature cabinet, and exposed to light only for obseration. Powdered Brewers yeast was used as food and replaced when necessary. Isolation and Identification of Strains Cultures of females were obtained from various laboratories or geographic locations. Isogenic clone lines were started with parthenogenetic females taken from each culture, and clone populations were designated as ”strains". Strains were maintained as stock cultures; individuals were removed as needed for experimentation. Individual animals of each strain were electrophoretically assayed to identify the complement of esterase enzymes present in that strain. 9 Esterases in E, candida are known to vary during the process of ecdysis (Asher and Snider, 1975). In order to avoid indiscriminate mixing of molt cycle variation with strain variation in esterases, a standard time during the molt cycle was chosen for electrophoretic assay. Palevody and Grimal (1976) divided the molt cycles between ovipositions into five repeating phases which relate molt cycle stage to egg production and feeding behavior (Table 1). Preliminary studies indicated that the maximum number of esterases were present on the second day of Phase 3 (P3 +2). Animals from many strains were cultured individually and frozen (-20°C) on day P3+2. Electrophoretic analysis of these animals produced a "standard” esterase isozyme pattern for each strain. The standard pattern of each strain was used as a control, against which all tissue, age of molt cycle variation studies were compared. Several strains were selected for further study. Locality of original source, collector and data of initial culture are presented in Table 2. Two strains were studied from the localities of Kellogg Biological Station, Michigan (Strains E and F), Belgium (B and H) and Coldwater Cave, Iowa (I and J). Methods for Production of Enzyme Source Material Enzyme experiments were designed to answer questions concerning esterase variability, behavior, location and role in F, candida and required the use of four different enzyme sources. Whole F, candida staged to P3+2 were used as the enzyme source for substrate-inhibitor and pesticide studies. Large numbers of F. candida females of various strains were isolated and their biology (egglaying, molting, fasting) was followed to insure they were representative for the 10 whole species. Animals were frozen at -20°C on day P3+2. Storage of up to six months at -20°C had no effect on esterase activity. Whole F, candida, which were staged to a variety of times in the molt cycle, were used to study molt cycle variation in esterases. Animals from strains A and D were isolated and observed daily for two weeks to establish molt cycle phase. After that, the animals were observed every six hours for entry into Phase 2 (the first six hour period in which gut contents were absent). Phase 2, which lasts about two days was divided into nine intervals of six hours (0-6 hours into Phase 2, 6-12 hrs, 12-18 hrs, 180-24 hrs, 24-30 hrs, 30-36 hrs, 36-42 hrs, 42-48 hrs, 48-54 hrs into Phase 2). F3 candida were killed by freezing as they entered these intervals until 32 individuals of each strain (A and D) had been collected for each interval in Phase 2. Eggs and juveniles of strain D were used to study changes in esterases during early development. Females of strain D were isolated and allowed to reproduce. Eggs were pooled and samples removed from the culture jars every two days. At 15.5°C, development takes approximately 17-19 days (Snider, 1971). Samples from day 0 of life (eggs) to day 35 (juveniles) were collected, frozen at -20°C and assayed for esterase activity. Head and trunk samples of each strain were used in tissue localization studies. These samples were produced by removing the head from the remainder of the body (trunk), using a pair of minuten needles and slicing across the neck segment. Animals were anesthetized with C02 prior to dissection. Heads were pooled separately from trunk sections and frozen at -20°C until assayed. 11 Controls for Enzyme Experiments For the inhibition and pesticide studies, one portion of the gel was incubated with distilled water without additional chemicals as a control. After incubation, all slices were stained with alpha naphthyl acetate. In substrate studies, gels stained with other ester substrates were compared to an identical gel stained with alpha naphthyl acetate. For all other studies, controls consisted of origins filled with whole body homogenates (P3+2) of the apprOpriate F, candida strains. Effectiveness of specific inhibitors against esterase classes was verified by testing the same inhibitors against commercially prepared esterase enzymes. Esterase source and enzyme commission identification number of the purchased enzymes may be found in Appendix 1. Horizontal Starch Gel Electrophoresis Starch slab gels were prepared by dissolving 28 gm starch (Electrostarch, Electrostarch Company, Madison, Wisconsin) in 250 mls gel buffer of 0.005 M histidine pH 8.9. The gel solution was stirred, boiled until viscous, degassed, and poured into a 14.0 x 9.0 x 1.0 cm high plexiglass tray with removable ends. One or two slotmakers were inserted into the slab gel to produce up to 20 origins. The slab gels were refrigerated two hours and slotmakers removed. Whole body homogenates were pipetted into the slots, for a final concentration of 80% (by volume) of the total homogenate of one adult_§. candida at each origin. Tray buffers consisted of 0.41 M_Sodium citrate, pH 8.0 and 10% NaCl (in the electrode chamber). Bridging between buffer chambers 12 and gel surface was accomplished with Whatman # 1 filter paper. Electrophoresis was carried out at 4°C, 15 mA, for 5 hours. During electrohoresis, a gradual rise in gel resistance resulted in a loss of amperage. This was corrected by using a Bio Rad 400 power source, set to constant current mode. Direction of migration was toward the positive pole. After electrophoresis and prior to subsequent treatment, the gel was removed from the tray and sliced horizontally into four identical slices with a wire gel slicer. Polyacrylamide Slab Gel ElectrOphoresis Polyacrylamide slab gels (7.5 2 Acrylamide) containing 0.2 Z N,N methylene-bis-acrylamide (BIS), 1.0 ul/ml N,N,N',N'-tetra -methy1ethylenediamine (TEMED), and 1.0 mg/ml ammonium persulfate, were made with a gel buffer of 0.01! Tris-0.077M Glycine pH 8.9 (modified from Davis, 1964). Sodium dodecyl sulfate (SDS) was omitted to retain enzyme structure and activity. This method separates proteins on the basis of their size, shape and overall charge (Ornstein, 1964). Slab gels (15.5 x 11.0 cm x 1.5 mm thick) were poured between glass plates, and a slotmaker containing 20 origins was inserted. Polymerization at room temperature took one hour. The enzyme source was either whole animals, portioned animals or eggs; crushed in gel buffer contianing 202 glycerol and 0.052 Brompophenol blue as an electrophoretic marker. Each origin contained approximately 80% (by volume) of the total homogenate of one adult_§. candida. Slab gels were electrophoresed using either a Hoefer Scientific Slab Gel Electrophoresis Unit (Model SE 500) or a model constructed 13 from plexiglass (Price, 1968). Power was supplied by Heathkit regulated power supply (Model IP-17) on constant current mode. Gel buffer filled both upper and lower chambers. Electrophoresis at 4°C, 15 mA per side was carried out until the dye marker reached the end of the glass plates (2.5 - 3.0 hours). Direction of migration was toward the positive pole. Replicates of the electrophoretic patterns were produced by repeating the order of the enzyme sources sequentially along the twenty origins. These "strips" were cut apart after electrOphoresis was completed and subjected to further treatment. One strip of the electrophoretic gel from each slab gel run was used as the control, and stained under optimal conditions to locate all esterase enzymes (method below). A representative gel was measured and migration distance for esterase enzymes recorded. The Rf values were calcualted as follows: Rfsdistance of esterase band from origin/distance of dye marker from origin. Enzyme Staining Esterase enzymes were located in the gel (starch or polyacrylamide) by a substrate-diazo dye coupling reaction. The substrate was alpha naphthyl acetate and the diazo-coupling salt was Fast Blue RR. This particular salt was chosen because its presence inhibits staining reactions of acid phosphatases (Cook and Forgash, 1965) which might obscure identification of esterase enzymes. Gel slices were incubated in a staining mixture containing 1.0 mg/ml Fast Blue RR in 0.04M Trizma base pH 6.25. Alpha naphthyl acetate was dissolved in acetone and added to the staining mixture. The final concentration of substrate was 0.2 mg/ml (1.07 mM) while 14 acetone final concentration was 22 (v/v). In substrate studies, alpha naphthyl acetate was replaced by alpha or beta esters of increasing chain length. Esters of 2-14 carbon lengths were used (Appendix II). Final concentration of substrate was maintained at 1.074 mM. Replicate gel was stained with alpha naphthyl acetate for use as a control in the substrate studies. Inhibitor Studies Two types of inhibitor studies were performed in the course of this study. Specific inhibitors were used to differentiate among esterase classes, in both starch and polyacrylamide slab gels. Degree of inhibition and the structural components essential for inhibition of esterase activity were estimated through the use of a number of high grade ester pesticides (90-99% pure, from the Environmental Protection Agency). Structures and specific nomenclature for trade name pesticides and other inhibitors may be found in Appendix IV. Specific inhibitors or ester pesticides were dissolved in acetone and added to distilled water to give a final concentration of 10’3,10’4, 10"5 or 10‘7'M_inhibitor. Whenever acetone was an unsuitable solvent, methanol or hexane was substituted. Control gels were treated with distilled water containing 22 v/v) acetone, methanol or hexane. After incubation for one hour at room temperature, the gel slices were rinsed and stained with alpha naphthyl acetate. After staining, the gels were rinsed in distilled water and scored for presence or absence of each esterase enzyme band. 15 Staining intensity of treated enzymes was estimated by comparison to control gel slices and scored on a scale of 0-3. A score of 0 indicated complete absence of staining (0.0%), 1 indicated 50% intensity and 3 represented a staining reaction equal to control gel slices (100%). The concentration of pesticide which caused 50% reduction in staining intensity was designated as the 150 inhibitor level. Summary of Enzyme Experiments in F. candida All enzyme experiments conducted during this study are summarized in Table 3. This table includes the methods, enzyme sources and controls used for each experiment. Originally, the term "isozyme” was used only for electrophoretic variants from the same structural gene. Many reasearchers now use "isozyme” in reference to all electrophoretic variants in an enzyme system. Since no attempt was made to locate the structural genes for esterases in.F, candida, ”isozyme" was applied in its broadest sense. The terms ”isozyme" or "enzyme band“ were used interchangeably for any esterase enzyme in F, candida. ”Esterase pattern" or ”zymogram" were used to describe the characteristic appearance of the electrophoretic slab gel after staining with alpha naphthyl acetate. An esterase pattern contained a number of individual isozymes, each with slightly different mobility. Toxicity Studies Diazinon was chosen for the toxicity studies because preliminary investigation indicated that it was an effective esterase inhibitor. Diazinon (992 pure, EPA) was mixed in acetone and added to dry Brewer's yeast forming a slurry. Control yeast was treated with l6 acetone. All slurries were dried by hand stirring. Resulting levels of contamination were 10,50,100,1000 and 2000 ppm diazinon, with 0 pm diazinon in the control yeast. Yeast was stored at -20°C, and small amounts were removed as needed. Animals used in toxicity experiments were F, candida of the following age categories: a. random adults from stock cultures, unknown age, approximately equal size. b. randomly selected adults from cultures of known age. c. first instar juveniles, hatched from eggs retrieved from stock cultures. Replicate experiments were performed for each esterase strain with animals of each age category. Observations of adult feeding behavior, accumulation of exuvia, egglaying and mortality were made daily. For juveniles, only mortality was recorded. Yeast was replaced every third day and dead animals removed daily. An animal was considered dead if no movement occurred even when it was prodded. Mortality was expressed as Abbott's corrected mortality (Abbott, 1925): Mortality - 100 x (Z live control - 1 live treated)/(Z live control). Statistical Methods Sublethal reactions of F. candida to pesticide exposure were analyzed using a two factor cross-classified model for analysis of variance, where strain of F. candida and treatment level were considered as two independent factors. Models for data analysis were constructed using methods suggested by Gill (1978) and are included in Appendix V. 17 Table 1. Molt Cycle Phases of E. candida (after Palevody and Grimal, 1976). 1 Intermolt 1 - Feeding 1 2 Fasting 1 M1 Exuviation 1 3 Intermolt 2 - Feeding 2 4 Fasting 2 M2 Exuviation 2 5 Egglaying Recognition gut dark (full) gut clear (empty) exuvia produced gut dark gut clear exuvia produced gut dark or clear Duration (15.5°C) 4-5 days 48-54 hours 5 days 48-54 hours 24 hours ending with oviposition new Phase 1 begins~~~~ Table 2. Origin of_§. candida Strains. Strain Locality/Collector/Initial culture date A MSU, lab culture, E. Lansing, Michigan R. J. Snider, 1965 B Ball Park, MSU Campus, E. Lansing, Michigan, R. M. Snider, 1976 C Iowa, lab culture, K. Christiansen, 1976 D Woldumar Nature Center, Lansing, Michigan, R. J. Snider, 1970 E Kellogg Biological Station, Kalamazoo Co. F S. J. Loring, 1979 G Belgium, lab culture, C. Gregorie-Wibo, 1980 H I Iowa, Coldwater Cave, K. Christiansen, 1980 ...Qom¢oomoamm we xwoaocaauou wcwewu mauxo uaoa mnu ou muomou ~+mm «s 18 .mvfivcmu .m we mmumammoso: mean oaons u mm: s m T2 3 TS BORN mCOHUQHUGUUfiOU movaoqummm house an cowownweaa m>onm mm m>onm mm aom< m>onm mm mmmumumm wsficamum1onm mm N+mm.:m3 aom< ovwamahuomxaom mamas mmmuoumm nouanancfi oc mace wcacamum1¢z< wcwCHmuml5fi vcm mwwm o samuum some owwfiuoam um cmuouw wcacqmumndz< ca oucmummmmm N+mm.mm3 mmaaco>an was ammo o oedemaxuumhaoa mmmumumm magnuum Ham mxcsuu coaooa wcucfimumlmz< mommumumm mo N+mm.:m3 no mess: voaoom Ham ovaemamuommaoa coaumooa meow a.< mcwmuum mm :H uon mafia u: o N+ma.=m3 camauoam ecum.mm2 a.< madcasumuaze coaumaus> noumum sackcluao: mcamuum dam «sswaacfimumlu=m .N+mm.:m3 ¢«~+mm.«:m3 Ham .uommaoa\noumum coaumdum> Houucoo oouaom maxucm magmuum muonuoz ucoaaummxm zvsum mans ca vow: mucmewumaxm mahwam mo humeaam .m magma 19 Chapter 1: Genetic and Physiological Variation INTRODUCTION Esterases have been investigated in a number of insect species. Esterase variability has been used to calculate genetic distances within and between populations (Wagner and Selander, 1974; Hudson and Jui, 1976), and to provide marker enzymes to study the phenomena of reproductive isolation (Halliday, 1979), drift and founder effect (Rockwood-Sluss et al., 1973; Krimbas and Tsakas, 1971) and dispersal (Sell et al., 1974). Esterase variation has also become an important tool in systematics (Stephen and Cheldelin, 1970) especially in LepidOptera (Hudson and Jui, 1976; Sell et al. 1974). Polymorphism in alpha and beta esterases were used by Throckmorton (1977) to develop phylogenies of Drosophila species. Hart and Allamong (1979) studied esterases of several species of Collembola, in an attempt to substantiate taxonomic designations previously established in this order. In general, much information has been gained from biochemical taxonomy, but it has become apparent that no fixed number of isozymic differences exist between taxa (Wagner and Selander, 1974). Extensive knowledge of enzyme variability is essential if biochemical taxonomy is to contribute to systematics (Avise, 1974; Staney, 1981). Within individual insects, esterases may vary between tissues, as in the tissue specific esterases of the cockroach (Cook and Forgash, 1965). Esterases also vary with time, with enzyme titer or activity changing during development. Examples have been cited for DrosoPhila species (Pantelouris and Downer, 1969) and other Diptera (Ahmad, 1976), Lepidoptera (Clements, 1967) and Hemiptera 20 (Nemec, 1972). Esterase variation associated with metamorphosis and control of JH titer has frequently been reported, and has been reviewed by de Kort and Granger (1981). Enzyme variation through time (due to developmental, metamorphic or physiological state of the animal) is often confounded with estimates of genetic variability in large population surveys using randomly assayed individuals. It is obvious that some knowledge of enzyme variability is essential before changes in esterases through time can be quantified. Esterase variability contributed by changes within individual animals can then be removed from population survey data. Preliminary investigations of esterases in.§, candida have uncovered age-related variation (Hart and Allamong, 1979) and changes in esterases associated with molt cycle stage (Asher and Snider, 1975). Molt cycle variation consisted of the disappearance of two esterase isozymes at some time prior to molting and their reappearance when exuviation was complete. Based on these data, a more complete study of esterases ian. candida was conducted to quantify changes in enzyme pattern which were associated with life processes. In the present study, esterase variability in laboratory populations of F. candida was investigated with starch and plyacrylamide electrophoresis. A survey of all obtainable strains was conducted. Changes in esterase enzymes during development and molting were examined by assaying individuals staged to specific time periods in these processes. 21 RESULTS Genetic Variation in Strains of F. candida Starch gel electrophoresis of individuals from strains maintained in the laboratory produced the esterase zymograms of Figure 1. Four zymogram patterns were identified: Pattern A (from strain A—MSU lab culture), Pattern B (from strain B-MSU Ball Park), Pattern C (from strain C-Iowa) and Pattern D (strain D from Woldumar Nature Center, strains E and F from Kellogg Biological Station). Although each esterase pattern contained enzymes of different mobility, there were also similarities between patterns. Each strain possessed two analogous groups of esterase enzymes: one collection of slow migrating enzymes (4-5 bands of activity) and a faster migrating group of esterases (typified by bands 5 and 6 of strains A and D). Staining at the origin also occurred in all strains, indicating that some additional enzymes did not enter the gel material. Although starch gels were technically easy and inexpensive, the degree of resolution was insufficient to adequately discriminate between closely migrating esterase bands. Polyacrylamide gel electrophoresis allowed increased resolution between esterase bands, and produced superior zymograms. Consequently, polyacrylamide electrophoresis was used for substrate and inhibitor studies (Chapter 2). Typical esterase patterns from polyacrylamide gels were recorded for each strain available in culture (Figure 2). Migration distance was measured (Appendix V) and Rf values were calculated for the major bands of staining activity (Table 4). Esterase enzymes were referred to by number (based on polyacrylamide assay, Figure 2) 6A —_ - c so I?! + O- — S /\ ;- - IIIIIII 3 - C 3 —--— g —- m- — > E -— _ _ g — A B C D STRAINS Figure 1. Starch Gel Electrophoretic Patterns of Esterases in §.candida. (Pattern D also found in strains E and F) 23 15—— Is 14---- l4 _+. o— “— n.— '*— n— A u “ --"" 'Illllllll IIIIII. u_ w .- w_ — a 30— lo— : . q 0 s——- s (E, 1 z 1 1 L h 0 w- u— 0 I— c a ‘- '_ ’- ~ a) .. «o- o “— u_ «- 3 3- '— “z ’— a— —- a u C) m 1 1 l 2. A B C D Figure 2. Polyacrylamide Gel Electrophoretic Patterns of Esterases in §,candida (Pattern D also found in strains E and F, Pattern B also found in strains G,H,I and J). 24 TABLE 4. Rf Values for Major Esterase Enzymes of F. candida on Polyacrylamide Gels. Strain Isozyme A B C D band ‘_-““ 1 0.006 0.006 0.006 0.006 3 0.067 0.067 0.054 0.067 4 0.081 0.081 0.087 0.081 5 0.094 0.101 0.107 0.094 6.0 0.115 - - 0.115 6.5 ~ 0.135 0.135 - 7 0.235 0.235 0.235 0.235 10 0.336 0.323 0.309 0.323 11 0.370 0.350 0.336 0.377 12 0.444 - , 0.404 0.444 13 0.485 - - 0.485 14 - 0.511 0.511 - 25 throughout the course of this study. Even with the increased resolving power of polyacrylamide gels over starch gels, no differentiation could be made betwen strains D (from Woldumar Nature Center), E and F (Kellogg Biological Station). All appeared to contain the esterase complement of Pattern D. Pattern B was recovered from strain B (Ball Park, MSU), G and H (Belgium) and I and J (Iowa) cultures. Similarities in esterase patterns existed for all strains investigated in this study, both in number of esterase enzymes, intensity of staining reaction, and migration distance. Because of these similarities, esterases separated by polyacrylamide electrophoresis could be divided into three ”sets” of enzymes (Figure 2). These sets were similar to the groups of esterases identified from starch gel electrophoresis, with the addition of a group of low migrating enzymes which remained close to the origin. Set 1 consisted of esterases l and 2, the lowest migrating bands. Both appeared to have identical Rf values in all four patterns. Esterase l (Rf value of 0.006) was always present, but esterase 2 was difficult to score consistently, because of its weak staining intensity. For this reason, data on esterase 2 were omitted in further studies. Set 2 esterases had Rf values of 0.054 to 0.235 and consisted of three to four major bands (3-6) and two minor ones (6.5, 7). This group contained the strongest and most rapidly staining esterases in the polyacrylamide gels. Set 3 esterases were the fastest migrating bands (8-15), with Rf values from 0.309 to 0.511. The major bands in this group were 26 more variable among strains than the esterases of set 2. Several intensely staining esterases (bands 10, ll, 12) were located between the minor esterases of set 3. Minor bands included the slowest migrating esterases of this set (8 and 9) as well as the fastest migrating enzymes on the gel (13-15). Esterases 8 and 9 were difficult to score in later studies and were omitted from data summaries. Individuals which had been damaged during transfer from culture jars to assay tubes leaked hemolymph onto the absorptive charcoal surface, and later showed a substantial reduction in set 3 esterase activity. From this observation, it was concluded that a major portion of the set 3 activity in F. candida was located in the hemolymph. An extensive survey of the esterases within the species ( F: candida) was not attempted. Only populations readily obtainable were examined, primarily to characterize strains suitable for further study. Esterases in Development of F. candida Samples of eggs and juveniles of strain D were frozen on day 0 (newly laid eggs) to day 35 (small juveniles). These samples were electrophoresed in starch gels to document changes in esterases during embryonic and early post—embryonic development. The six esterase isozymes which are present in strain D adults (Figure 1) were detected in eggs just after oviposition. No change in the number of esterase isozymes occurred during development (0-35 days after oviposition). Many eggs or young were pooled for these enzyme assays, since the esterase activity contained in a single egg of juvenile was below 27 the resolution limits for the staining reaction. Use of pooled samples meant that small alterations in specific esterase levels during development could not be detected. Molt Cycle Variation in Adults Molt cycle variation in esterases was followed in detail by dividing Phase 2 (Palevody and Grimal, 1976), normally lasting 48-54 hours, into 9 six-hour intervals. A number of strain A and D animals were analyzed by starch gel electrOphoresis upon entering each of the intervals (32 animals per interval per strain). These data have been discussed elsewhere (Grimnes, 1981) and are presented here in brief. Esterase bands 1-4 remained constant throughout Phase 2, while bands 5 and 6 showed variation in titer correlated with intervals in Phase 2 (Figure 3). Band 6 was first to disappear, reaching minimal levels in both populations by 6-12 hours into Phase 2, but reappeared rapidly between 18-30 hours into Phase 2. By 42-48 hours, band 6 was present in 1002 of the assayed populations. Band 5 in both strains disappeared and reappeared at a later time. Minimal levls of band 5 in both populations existed between 12 and 30 hours. Total recovery (100%) of band 5 activity was not reached during the course of Phase 2 in either strain. Differences between strains A and D in the loss and return of esterase activity appeared minor. 28 . 8 $ 5 A B Ecendide with detectable esterase activity (1») osi'zi'szksbs'sls Hours into Phase 2 Figure 3. Cyclic Esterase Bands 5 and 6 in Strains A and D of §.candida. Each data point represents the 2 (out of 32 individuals) with detectable activity (after Grimnes,1981). U 485'4 29 DISCUSSION In general, direct comparison between starch and polyacrylamide zymograms is not always possible because changes in the buffer system and gel material often produce substantial differences in results. However, 3, candida zymograms produced by both techniques were similar enough to tentatively identify esterase bands 1-4 on starch as set 2 enzymes (bands 3-6) on polyacrylamide gels, starch band 5 as polyacrylamide band 10, and starch bands 63 and 6F as polyacrylamide bands 11 and 12, respectively. Cycling esterases first observed in starch gel electrophoresis are probably esterases 10-12 on polyacrylamide, and were located in the hemolymph. Additional information concerning the behavior of each isozyme is needed to confirm these designations. Comparison between esterase zymograms of the present study and the results of Hart and Allamong (1979) indicated similarities between the F, candida populations used in both investigations. Hart and Allamong did not discuss individual zymograms, but divided the polyacrylamide gel into "windows” of Rf values and recorded the frequency with which esterase bands appeared in the windows. The major esterases of Hart and Allamong had Rf value ranges (Rf - 0.26-0.33, 0.34-0.44, 0.45-0.50) which corresponded closely to the fast migrating bands in the strains analyzed here (strain A: 0.336, 0.370, 0.444; strain B: 0.323, 0.350; Strain C: 0.309, 0.336, 0.404; strain D: 0.323, 0.377, 0.444). In contrast to the fast migrating esterases, Hart and Allamong found very little esterase activity in the Rf values corresponding to Set 2 esterase activity in Strains A through D (Rf - 0.67 to 0.235). 30 In F, candida, developmental studies indicated that all esterases were present from the egg stage onwards. In contrast, Hart and Allamong (1979) reported a gradual increase in the number of esterase bands with increasing size of juveniles (individual assays), until the adult pattern was reached. These discrepancies can be explained by differences in technique. During electrophoresis of single individuals of small size, minor bands may not contain enough enzyme to show a visible staining reaction. Theoretically, the most intensely staining bands will be the first to become visible, either by virtue of their increased activity or their titer, in assays of single small animals. This was observed by Hart and Allamong, since the report that esterases appeared during development in an order corresponding to their intensity. In the present study, several juveniles were used for each assay. Therefore, any esterase possessed by them had a greater chance of being detected. The disadvantage of this method was that estimation of enzyme levels in individuals assayed by this method was not possible. Periodic cycling of esterase activity correlated to molt cycle phase in F, candida was observed for esterase bands 5 and 6 (starch gels). Cycling esterases disappeared during fasting (Phase 2) and reappeared later in the cycle (end of Phase 2 or phase 3). Three possible hypotheses could explain this phenomenon. Esterase cycling may be: a.) unrelated to molting, b.) correlated to it for indirect reasons, or c.) involved in molt cycle control. With respect to the first hypothesis, it is difficult, if not impossible, to support the idea that esterase cycling is totally random, or free running with respect to the molt cycle. Variation in 31 esterase staining has been observed whenever animals of Phase 2 or 4 are analyzed. The second hypotheses, i.e., indirect correlation to exuviation, would involve cycling esterase participation in processes related to molting, but not in control of molting. Some examples of molt-related processes are elimination of midgut epithelium and gut flora, which are essential for complete replacement of midgut cells (mesenteron), and resumption of feeding in Phase 3. If cycling esterase activity were digestive in nature and restricted to mesenteron cells, loss of activity could result from elimination of mesenteron lining. Mesenteron is shed during the fasting period in Collembola (Palevody, 1974; Thibaud, 1968). New mesenteron, however, forms beneath the old mesenteron layer and is already present when old mesenteron is discarded. In order to produce the cycling effect seen during Phase 2 of F. candida (esterase 6 returning before esterase 5), activation of esterase enzymes in the new mesenteron would have to be staggered, with esterase 6 activated first. Enzyme loss could be attributed to loss of gut flora, which are eliminated along with mesenteron cells. RepOpulation of gut flora occurs by feeding on fecal material (Richards, 1974) and could account for the gradual increase in esterase activity. However, there are two problems with this idea of gut flora esterase activity: a.) Since esterase 6 cycles faster than esterase 5, selective uptake of various gut flora (containing this enzyme) from fecal matter would have to occur, and b.) F. candida do not begin feeding until Phase 2 is completed. Therefore, repopulation of gut flora could not occur 32 before the end of Phase 2, which contradicts the observed reappearance of esterase 6 early in Phase 2. Neither proposal for indirect involvement of esterases in molting (either mesenteron of gut flora loss), involves hemolymph esterases. In fact, they preclude esterase location in the hemolymph. Since cycling esterase activity has been found primarily in the hemolymph in F. candida, it appears likely that the esterases fulfill some function other than in digestion or association with or in gut flora. The third hypothesis, that cycling esterase activity in ER candida is involved in molt cycle control, is substantiated by the present data. Esterases exert control over the molt cycle by cleavage and regulation of juvenile hormone levels (Ajami, 1976). JH esterases are located in the hemolymph of most insect species (Ajami, 1976; de Wilde, 1981), consistent with location of cycling esterases in F, candida. Furthermore, the cyclic changes during Phase 2 of the molt cycle in E. candida are consistent with the behavior of esterases involved in molt cycle control in pterygotes. Pterygote larval stages, especially last larval instars, show a rise in JH and ecdysone levels prior to each molt (Basic and Hsaio, 1977). JR esterases are present during each pterygote larval instar, but their titer falls just before molting occurs (Fain and Riddiford, 1975). Apterygotes exhibit concerted JH and ecdysone fluctuation, at least in the two groups investigated (Thysanura: Rohdendorf and Watson, 1969; Rohendorf and Sehnal, 1973; Collembola: Palevody and Grimal, 1976; Palevody et al., 1977). In apterygotes, as in larval pterygotes, JH esterase activity should therefore be high between 33 molts and fall prior to molting. This matches the behavior observed for cycling esterases (starch bands 5 and 6) during the molt cycle in F, candida. Attempts to utilize esterase pattern as a correlating factor in taxonomic studies in E. candida have met with varying degrees of success. The species of F. candida was divided into two taxonomic types (K. Christiansen, personal communication), based on morphological characters but no relationship between type and esterase pattern was observed for the strain used in this study. On the other hand, Hart and Allamong (1979) were able to demonstrate a substantial difference in esterase zymograms between F. candida populations and other species of Collembola. No consistent correlation could be drawn between esterase pattern and locality of collection. Pattern B was found in populations from Michigan (Strain B), Iowa (Strains I and J) and Belgium (Strains G and H). In contrast, esterase pattern D (from strains D, E and F) appeared limited to Michigan. Only one strain containing pattern A (Strain A, Michigan) and pattern C (Strain C, Iowa) have been observed. The only physiological parameter in F. candida which has been investigated with reference to esterase pattern is egglaying behavior (Grimnes and Snider, 1981). Strains E, F and D (all esterase pattern D) were more similar to each other in egglaying behavior and fecundity than to any other strain tested (A and C Strains). Since egg production depends on many enzymes and hormones, this correlation can not be directly attributed to esterase pattern without further research. 34 Chapter 2: Classification of Esterases and Substantiation of their Roles in E, candida INTRODUCTION An important tool for comparison and identification of the complicated assortment of esterases uncovered by electrophoresis is the division of esterases into ”classes”. Esterases are classifed according to their ability to cleave substrates of various kinds, and the degree of inhibition of their staining activity by certain specific compounds. Aldridge (1953) proposed the first differentiation between esterases based on the degree of Paraoxon inhibition. A more complex scheme was suggested by Augustinson (1961) and improved by Holmes and Masters (1967). Pearse (1972) reviewed these methods for general histo- logical work. Although the classification system was described using vertebrate species, application to invertebrates has been relatively successful. This is the classification scheme followed here. There are five generally accepted esterase classes. These are the carboxyl esterases (CBE, B-esterases, aliesterases) which preferentially hydrolyze aliphatic esters; arylesterases (A-esterases) more active toward aromatic than aliphatic esters, but inhibited by sulphydryl reagents; acetyl esterases (C-esterases) also active toward aromatic esters but not inhibited by sulphydryl reagents; and the choline esterases (CHE), which hydrolyze choline esters and are inhibited by eserine. The choline esterases are frequently divided into acetylcholine esterases (ACE) which work on short chain choline esters, and butylcholine esterases (BCE), which show greater activity toward butylcholine than acetylcholine. This 35 division has been based primarily on mammalian choline esterases, and may not be valid throughout the insects (V033 and Matsumura, 1964). Lipases are sometimes included as an esterase class but are readily distinguished from other esterases because of their preference for substrates with ester chains of five carbons or more (Pearse, 1972). A number of roles have been attributed to esterases within insects; several to specific classes. Three three major functions of esterases have been reported in the literature: cleavage and inactivation of acetylcholine, of juvenile hormone and of ester pesticides. Esterases are also thought to participate in degradation of esters which are taken up in the diet. Acetylcholine esterases (ACE) are responsible for the cleavage of acetylcholine in most vertebrates and in many insect nervous systems. Esterases are essential, therefore, in the termination of a normal neural impulse. Choline esterases of insect brain are generally assumed to be acetylcholine esterases, even when their ester chain length specificity has not been determined (Ahmad, 1976). ACE enzymes may also play an important part in insect development and diapause (Smallman and Mansingh, 1969). Appearance of specific choline esterases has been asociated with diapause termination in crickets (Jameson et al., 1976) and silkworms (Kai and Nishi, 1976). The role of esterases in the regulation of juvenile hormone has been reviewed by de Kort and Granger (1981). Degradation of the methyl ester of JH by esterases during specific developmental time periods has been documented in Lepidoptera (Weirich and Wren, 1973; 36 Sandburg et al., 1975; Hwang-Hsu et al., 1979) and Coleoptera (Kramer and de Kort, 1976). Generally, peaks of esterase activity occur prior to stages in which low JH titer is essential to normal development, i.e., prior to the larval-pupal molt or the pupal-adult molt. In apterygotes, presence of JH has been established in only one thysanuran species, Thermobia domestics (Madhavan et al., 1981) and theorized for one collembolan species, Folsomia candida (Palevody and Grimal, 1976). Data on esterases exist for only one of these species, with three reports for F. candida (Asher and Snider, 1975; Hart and Allamong, 1979; Grimnes, 1981). Grimnes (1981) followed changes in esterase levels associated with the molt cycle of E, candida; results have been discussed above. Esterases function in the detoxification of ester pesticides, both carbamates and organophosphates (Matsumura and Sakai, 1968; Pasteur and Sinegre, 1975). Organophosphates may interfere with esterase function, especially acetylcholine esterases, by forming enzyme-inhibitor complexes and delaying cleavage (Moriarty, 1969). The normal ester cleavage reaction may or may not proceed to the acetylation step, or may stop at the final deacylation of enzyme (Stegwee, 1960; Pratt, 1975). Failure of the esterase to complete the cleavage reaction and release the ester chain as an alcohol results in inhibition. Inhibition is either competitive or irreversible (Pearse, 1972; Plapp, 1976), with degree of esterase inhibition being highly species-specific for each compound (Moriarty, 1969). Intensive studies of inhibition at the enzyme level have been performed on insect esterases (Hammock et al., 1977) but these studies are rare. 37 Esterases are also involved in the process of digestion; they break down natural esters occurring in the diet. Marcuzzi and LaFisca (1977) have suggested that lipases (long chain esterases) of litter invertebrates participate in the degradation of higher oils, waxes and cutine present in leaf litter. In their estimation, the contribution of invertebrate lipase to litter breakdown may be as important as microfloral activity. In the present study, esterases in F} candida were tested with a number of specific inhibitors and ester pesticides to determine the class of each isozyme. Data from additional studies on tissue localization (head vs. trunk) and substrate specificity were used (along with esterase class identity) to substantiate the role for the isozymes of each esterase class in F3 candida. RESULTS Tissue Localization of Esterases Localization of esterase enzymes within the body of F. candida was only partially successful. Mid-gut sections were removed from the thorax (after the head was severed) but were inseparable from fat body cells. Mid-gut and fat body cells contained insufficient enzyme activity to produce identifiable esterase bands after polyacrylamide electrophoresis. In addition, loss of hemolymph and, subsequently, of esterase 3 activity could not be prevented once the thorax had been teased Open. Simple division between head and trunk proved more feasible; when the head was severed on a wax dissection plate, little hemolymph was lost. Trunk segments which leaked a large quantity of hemolymph were not assayed. Quantity of biological material was adjusted by the addition of heads and trunks until staining was as 38 intense as the control. Whole body homogenates of individual E, candida of the appropriate strain were used as control material. Esterases were not equally distributed throughout the body of F, candida. Estimation of the proportion of each esterase in head and trunk homogenates is given in Table 5. A11 strains (A-J) produced equivalent results when assayed in this manner. Slower migrating esterases (bands 3-4) with the exception of band I, appeared predominantly in head homogenates. Esterases 5, 6 and 7 showed an increasingly trunk-based distribution. The fastest migrating esterases (bands 10-14) were confined to the trunk and were not present in detectable quantities in head homogenates. Alpha and Beta Naphthyl Ester Substrate studies Esterase enzymes of F3 candida hydrolyzed a number of different substrates. Assessment of specific cleavage abilities for each esterase band was achieved by using of alpha and beta naphthyl esters of increasing chain length. Although these esters may never be encountered in the field, their structures were useful for colorimetric identification (by diazo-coupling) of esterase activity. Other ester substrates, such as acetylcholine and JH, could not be identified directly, since no color reaction could be used and therefore, were not tested. Chain length of esters and intensity of the staining reaction for esterases of strains A, B, C and D are presented in Figures 4 and 5. Alpha naphthyl acetate (ANA) was used as the control substrate; intensity of staining with other esters was compared to ANA staining and scored from 0 (no staining) to 3 (dark as control). As a 39 TABLE 5. Relative Distribution of Esterase Activity in Head and Trunk of_F. candida, Expressed as Z of Control (=100%, using whole-body homogenates). Band number Head Trunk Polyacrylamide -----_- * Origin 50 50 l 50 50 3 100 0 4 100 0 5 75 25 6.0, 6.5 50 50 7 25 75 10 0 100 ll 0 100 12 0 100 13, 14 0 100 3 13 z 1 1343‘ we 3 11 I I 34‘ a 11 a 3 n s 8 I '6...” ‘IS .- U vi C 3 “NC I 3136000 1 t “M. h 3| r tldfb HF“ f 154‘600 F Louise‘nfl H". I 8.8416 '1' 1 II“. l ‘86 i 1:l.L|""'-£‘l"-T'fi‘ )e.‘ 0 81mm A h. an. .0 a m P 3 fl 1 ‘L—Irnr 13* 6 Hub aansseeou 2 40 but! 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'0 C 't asst I zen: 1348' I 3 ,1 Z 2 11 Z 1 1 z 456 1.349 73“? 1 g 1 g 1 i 1 1 1 1 a '45 $345 1345 c ’1”: D 6mm: A B Honimdhmmluwmmmmmvitymmmof - z.m,orma-mmmm.rm-mamm.m mma-mammmmamuumon. 42 consequence, all staining intensity results for the alpha ester of two carbons (Figure 4) were recorded as 3, since that was alpha naphthyl acetate. The bar graphs for esterases in strains A-D (Figure 4-5) were spaced in the figures according to the electrophoretic mobility of the esterase band. The results for fast migrating esterase were placed at the bottom of the figure, slow migrating esterases at the top. This facilitated comparisons between reactions of esterases of similar and different mobilities. Although no two esterases were identical in their ability to cleave esters used in this study, there were a number of similarities among the isozymes. Based on substrate specificity, three sets of esterases could be differentiated. These sets were analogous to enzyme groups previously established by electrophoretic mobility and staining intensity criteria (Chapter 1). Slow migrating esterases (l and 2), Set 1, were active only on alpha esters of short length. Beta esters were not hydrolyzed. A second set of esterases (bands 3-8) appeared to hydrolyze either alpha or beta esters with chain lengths of up to 5 carbons (valerate), after which activity declined as ester chain length increased. An exception to this behavior was noted for esterase 6.5, found in strains B and C. This enzyme was not able to hydrolyze beta esters of more than two carbons. Esterases 9-14 (Set 3) were only slightly active toward either alpha or beta esters; their activity generally declined after chain lengths of two carbons and it was negligible against esters of 4 carbons or greater in length. All esterase enzymes in_E, candida showed a decrease in activity when carbon chain length of the substrate was increased. 43 This was an indication that none of the isozymes were lipases. Lipases preferentially hydrolyze very long chain esters, and although they could possibly stain with the methods employed in this study (Pearse, 1972), an increase in activity with chain length would be expected. Specific Inhibitor and Pesticide Studies Differentiation between esterase classes through the use of specific inhibitors has been attempted by a number of researchers, whose results are summarized in Table 6. Most of these inhibitors were used in the present study. However, Paraoxon and DP? (diisoprOpylfluorophosphate), usually used in classification schemes to inhibit ACE, BCE and CBE enzymes, were extremely toxic and difficult to obtain. Sigma Chemical Company suggested PMSF as a substitute for Paraoxon and DFP, based on the work of Fahrney and Gold (1963), who reported that PMSF inhibited rat brain and other mammalian ACE but not electric eel ACE; inhibition probably is species-specific. A number of organophosphates and carbamate pesticides were also employed; these substances inhibit choline and carboxyl esterases. Pesticides, their structures and their chemical names are listed in Appendix III. The class differentiation system was tested by using commercially prepared esterases of each class to verify their sensitivity to each inhibitor. The results, given in Table 7, indicated that each purified esterase behaved as expected for its class. ACE and BCEs were strongly inhibited by PMSF but not eserine sulfate. Inhibitors of the classification system were applied to 44 TABLE 6. Differentiation of Esterase Classes Based on Inhibition Data from the Literature. Inhibitors (Concentrations ARE as loglo) >3 Eserine (-5) O Eserine Sulfate (-5) 0 CMPS (-3) +1- PMSF (-5) 0 EDTA {-3) -H- LaNO3 ++ Paraoxon 0 Acetyl choline iodide (-3) 0 Atropine Sulfate (-3) 0 References: l. Clements, 1967 2. Holmes and Masters, 1967 ”-Q-‘ ‘somm- Esterase Class CBE AE ACE BCE References "B" "C“ O 0 ++ ++ 1,2 0 0 ++ ++ 4,2 0 0 0 0 4 sp 0 sp sp 5 0 0 0 0 1,3 0 0 0 0 1 ++ 0 ++ ++ 1,3,4,2 0 0 ++ 0 2 0 0 ++ 0 3 3. Stephen and Cheldelin, 1970 4. Hooper, 1976 5. Fahrney and Gold, 1963 Key to Symbols: 0 no inhibition ++ severe inhibition sp inhibition appears to be species-dependent TABLE 7. Inhibition of Commercially Prepared Esterases and Major Esterases of E. candida on Starch Gel Preparations. Esterase Class - Inhibitor Substance (Concentration as log10 or Band Eserine Sulfate CMPS PMSF EDTA LaNO3 -5 -4 -7 -3 -4 -3 -3 -3 Carboxylesterase "B" 0 0 0 0 + ++ 0 0 Acetylesterase ”C” 0 0 0 0 0 0 O 0 Acetylcholine esterase (ACE) ++ ++- 0 O + ++ 0 0 Butyl choline esterase (BCE) ++ ++ 0 0 + ++ 0 0 Starch gel band F. candida ‘7 2 + ++ 0 0 ++ ++ 0 0 3 + ++ 0 0 ++ ++ 0 0 5 0 0 0 0 0 + 0 0 6 0 0 0 0 0 + 0 0 Key to Symbols: 0 no inhibition '+ partial inhibition -++ severe inhibition 45 F. candida esterases after starch gel electrophoresis (Table 7). Enzymes 2 and 3 (starch) were completely inhibited by PMSF and eserine sulfate, and were either acetyl or butyl choline esterases. Bands 5 and 6 (starch) were partially inhibited by PMSF, but not by eserine sulfate, and therefore, were conclusively identified as carboxyl esterases. All four esterases were affected by at least one inhibitor, so they could not be acetyl esterases. No esterases were sensitive to CMPS, EDTA or lanthanum ions, therefore, none were aryl esterases. Additional information on the esterases of F. candida was obtained by repeating this classification research on enzymes separated by polyacrylamide electrophoresis. Polyacrylamide gel electrophoresis was superior to starch in terms of resolution and detection of enzyme inhibition because enzyme bands were sharper and easier to differentiate. The esterase numbering system follows that portrayed in Figure 2. The inhibitor and pesticide concentrations needed to reduce alpha naphthylacetate staining activity to 50% of the control staining activity (150 value), were recorded for each esterase enzyme in strains A, B, C and D (Tables 8-17). The 150 values were expressed as loglo concentration for easier comparison of values. In Tables 8-17, the symbol + indicated that a 10 ‘3M_solution resulted in a reduction (but not 502) in esterase staining. The symbol 0 was used to indicate that even a 10‘3 M solution, the highest concentration available for testing due to solubility limitations, had no effect on esterase staining activity. Several pesticides had no effect on any esterase of F, candida: i.e., Guthion (Azinphosmethyl), Abate (Temephos), Chlordimeform, 46 Dimethoate (Cygon), Atrazine and Paraquat. Concentrations of up to 10'?! EDTA or LaNO3 had no effect on esterase activity. These substances were not included in Tables 8-17. The origin of the gel always exhibited esterase activity, indicating that some enzymes were not able to penetrate the gel surface. Eserine sulfate partially inhibited these enzymes, proving some activity was due to choline esterases. Since staining activity at the gel origin appeared to result from a mixture of esterases, their classification was not attempted. Esterase enzyme 1 (Table 8) behaved like a carboxyl esterase. It was not inhibited by a sulphydryl reagent (CMPS), by EDTA or LaNO3, therefore, it was not an aryl esterase. Eserine sulfate was without effect, so this enzyme was not a choline esterase. Although esterase l was not severely inhibited by organOphosphate pesticides, it showed a slight susceptibility toward them, confirming its identification as a carboxyl esterase. The third, fourth and fifth esterase enzymes (Table 9, 10 and 11) were choline esterases. Resistance to CMPS, EDTA and LaN03, and inhibition by 10'5 M eserine sulfate, was consistent with choline esterase behavior. All three esterases were inhibited by PMSF. Staining activity was severely depressed after incubation with a number of the pesticides used in this study. This was expected, since choline esterases are the target enzymes for organophosphate and carbamate pesticides, which interfere with normal esterase function. Esterase 5 (Table 11) was especially interesting because its behavior differed in one of the stains used in this study. Esterase 47 TABLE 8. Concentrations of Inhibitors (loglo) Needed to Reach 502 (ISO) Esterase Inhibition: Polyacrylamide Band 1. ‘a-------.—..—m”--a’ Inhibitor A B C D Organophosphates Dipterex (Trichlorfon) 0 0 0 0 Naled -3 -3 -4 -4 Dichlovos -3 -3 -3 -3 Malathion + + + + Malaoxon O O 0 O Dursban (Ch10pyrifos) -3 + + + Re ldan -3 -3 -3 -3 Diazinon + + + + Dyfonate + + + + Dyfonate (Ox Analog) + + + + Bromophos methyl -3 -3 -3 -3 Ronnel. -3 -3 -3 -3 Methyl Parathion + + + + Ethyl Parathion + + + + Fenthrothion 0 0 0 0 Carbamates Mexacarbate (Zectran) 0 0 0 0 Aminocarb 0 0 0 0 Carbaryl (Sevin) + + + 0 Carbofuran 0 0 0 0 Specific Inhibitors Atropine Sulfate 0 0 0 0 Eserine Sulfate 0 0 0 0 CMPS 0 0 0 0 PMSF na na na na Key to symbols: 0 no effect on esterase activity from a 10"3 M solution of inhibi + slight effect on esterase activity from a 10T3 M solution of inhibitor na data not available for this inhibitor tor TABLE 9. Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 3. Inhibitor A B C D Organophosphates Dipterex_(Trichlorfon) -5 -5 -3.3 -3.3 Naled -5 -6 -6 -6 Dichlovos -5 na -4.3 -4 Malathion + + + + Malaoxon -3 + + -3 Dursban (Chlopyrifos) -6 -6 -6 -5 Re ldan -3 -3 -3 -3 Diazinon -5 -6 -5 -5 Dyfonate -4 na na -4 Dyfonate (Ox Analog) -6 na -6 -6 Bromophos methyl -3 -3 -3 -3 Ronnel. -3 -3 -3 -3 Methyl Parathion -4 0 -3 -3 Ethyl Parathion -5 na -4 -6 Fenthrothion -3 na na -3.3 Carbamates Mexacarbate (Zectran) -4.3 -4.3 -5 -4 Aminocarb -4 -4 -4 -4 Carbaryl (Sevin) -5 -3.3 -5 -5 Carbofuran -4 -4 -4 -4 Specific Inhibitors Atropine Sulfate + + + + Eserine Sulfate -5 -5 -5 -5 CMPS 0 0 0 0 PMSF -3 -3 -3 -3 Key to symbols: 0 no effect on esterase activity from a 10'3 M solution of inhibitor '+ slight effect on esterase activity from a 10F3 M solution of inhibitor ‘ na data not available for this inhibitor 49 TABLE 10. Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 4. Inhibitor A B C D OrganOphosphates--- Dipterex (Trichlorfon) -5 -5 -4 -4.3 Naled -5 -6 -6 -6 Dichlovos -6 -7 -5 -4 Malathion + O 0 + Malaoxon -3 -3 -3 -3 Dursban (Chlopyrifos) -5 -6 -6 -5 Re ldan -3 -3 -3 -3 Diazinon -5 -5 -5 -5 Dyfonate -3.3 -4.3 -3.3 -3 Dyfonate (Ox Analog) -6 -6 -6 -6 BromOphos methyl -3 -3 -3 -3 Ronnel -3 -3 -3 -3 Methyl Parathion -5 -4.3 + -3 Ethyl Parathion -6 -6 -6 -6 Fenthrothion -4 -4 -4 -4 Carbamates Mexacarbate (Zectran) -4.3 -4.3 -4.3 -4.3 Aminocarb -4 -4 -4 -4 Carbaryl (Sevin) -5 -5 -4 -5 Carbofuran -4 -4 -4 -4 Specific Inhibitors Atropine Sulfate + -3 -3 + Eserine Sulfate -5 -5 -5 -5 CMPS 0 0 0 0 PMSF -3 -3 -3 -3 Key to symbols: 0 no effect on esterase activity from a 10‘3 M solution of inhibitor + slight effect on esterase activity from a 10‘3 M solution of inhibitor " na data not available for this inhibitor 50 TABLE II. Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 5. Inhibitor A B C D orgaaaanagsh‘aigg“ ' °" ‘ ' ' ...._.-.._.....-- 7" Dipterex (Tkichlorfon) -6 -5 -3 -4 Naled -5 -6 -3.3 -5 Dichlovos -7 -7 -3.3 -7 Malathion -3 + 0 + Malaoxon -4.3 -3 -3 -3.3 Dursban (Chlopyrifos) -6 -6 -3 -6 Reldan. -3 -3 O -3 Diazinon -6 -6 -3 -5 Dyfonate -4.3 -6 -3 -5 Dyfonate (0x Analog) -6 -7 -6 -7 Bromophos methyl -3 -3 0 -3 Ronnel -3 -3 0 -3 Methyl Parathion -4.3 -4.3 + -5 Ethyl Parathion -7 -6 + -5 Fenthrothion -6 -4 -4 -4.3 Carbamates Mexacarbate (Zectran) -4.3 -4.3 -4.3 -4.3 Aminocarb -4 -4 -4 -4 Carbaryl (Sevin) -6 -4 -4.3 -4.3 Carbofuran -5 -5 -4 -5 Specific Inhibitors Atropine Sulfate + + 0 + Eserine Sulfate -4 -4 -4 -4 CMPS 0 0 0 0 PMSF -3 -3 -3 -3 Key to symbols: 0 no effect on esterase activity from a 10'"3 M solution of inhibitor + slight effect on esterase activity from a 10‘3 M solution of inhibitor " na data not available for this inhibitor 51 5 in strain C was consistently more resistant to 0P compounds than esterase 5 of strains A, B and D. This reduced sensitivity was apparent not only in the 150 values, but also in the degree of sensitivity to OP levels of 10'3M. Esterase 5 in strain C (esterase 5C) showed no change in activity with several compounds listed in Table 10 (0), while the same enzyme in other strains showed some inhibition, if not 50% reduction in staining activity. Pesticides which produced this degree of differential staining activity between strains were Malathion, Reldan, Bromophos methyl, Ronnel and the specific inhibitor, atropine sulfate. Comparison of 150 values between esterase 5C and esterase 5 of the other strains ranged from 1 (10 times the concentration; Parathion, Carbofuran) to 3 (1000 times the concentration; Dichlorovos, Dursban, Dipterex and Diazinon). In general, esterase SC was equally or more resistant to all inhibitors used in this study. Esterase 6.0 (Table 12) was found only in strains A and D. It was resistant to CMPS, EDTA and LaNO3, but susceptible to eserine sulfate, therefore, it was a choline esterase. Since PMSF did not inhibit esterase 6.0 to the same degree as other choline esterases in F, candida, esterase 6.0 was considered a PMSF-resistant choline esterase. Esterase 7 (Table 13) was similar in mobility in all four strains, and appeared to be a choline esterase. It was not sensitive to CMPS, EDTA or LaNO3, but was very susceptible to PMSF (IO-RM). In contrast to esterases 3-6, enzyme 7 was frequently resistant to OPs or carbamates. Enzymes 10-14 (Tables 14-17) were carboxyl esterases. They were 52 TABLE 12. Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 6. Inhibitor A B C D eigenstates """ '“ Dipterex (Trichlorfon) -6 -6 Naled -6 -5 Dichlovos -7 -7 Malathion + -5 Malaoxon -4 -4.3 Dursban (Chlopyrifos) -6 -6 Reldan na na Diazinon -6 -5 Dyfonate -4 -4 Dyfonate (Ox Analog) -6 -6 Bromophos methyl na na Ronnel na na Methyl Parathion -4 -4 Ethyl Parathion -6 -5 Fenthrothion -4.3 -5 Carbamates Mexacarbate (Zectran) -4.3 -4 Aminocarb -4 -4 Carbaryl (Sevin) -6 -4.3 Carbofuran -4 -3.3 Specific Inhibitors Atropine Sulfate + + Eserine Sulfate -5 -5 CMPS 0 0 PMSF -5 + Key to symbols: 0 no effect on esterase activity from a 10"3 M solution of inhibitor + slight effect on esterase activity from a 10‘3 M solution of inhibitor '- na data not available for this inhibitor 53 TABLE I3. Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 7. Inhibitor A B C D Ofganophosphates Dipterex (Trichlorfon) -4 -4 -4 -4 Naled -6 -6 -6 -6 Dichlovos -7 -7 -7 -7 Malathion 0 0 0 0 Malaoxon 0 0 0 0 Dursban (Chlopyrifos) -6 -4.3 -4.3 -4.3 Reldan na na na na Diazinon -6 -4.3 -4.3 -6 Dyfonate O O 0 0 Dyfonate (Ox Analog) 0 0 0 0 Bromophos methyl na na na na Ronnel na na na na Methyl Parathion -3 -3 + -3 Ethyl Parathion 0 -3 -4 0 Fenthrothion O + + 0 Carbamates Mexacarbate (Zectran) + + 0 0 Aminocarb 0 0 O O Carbaryl (Sevin) -4.3 -5 -3 -4.3 Carbofuran 0 0 0 0 Specific Inhibitors Atropine Sulfate na na na na Eserine Sulfate na na na na CMPS 0 0 0 0 PMSF -5 -5 -5 -5 Key to symbols: 0 no effect on esterase activity from a 10"3 M solution of inhibitor + slight effect on esterase activity from a 10F3 M solution of inhibitor " na data not available for this inhibitor 54 not affected by CMPS, EDTA, LaNO3, eserine sulfate or PSMF, and were either resistant to many pesticides or able to hydrolyze them. Most carboxyl esterases are especially susceptible to Dipterex and Dichlorvos (Clements, 1967), and this was true for E. candida CBEs. According to the degree of inhibition caused by pesticides, esterases 10-14 were very similar among strains; the greatest difference between them was their electrophoretic mobility. Esterase 10 was probably the same enzyme as esterase 5 on starch gels, while esterases 11 and 12 were starch bands 68 and 6F respectively. Therefore, these three carboxyl esterases (10-12) would be expected to show the same cycle of activity during molt cycle phases that was first detected with starch gel electrophoresis in the course of this study. A summary of the identity of the esterases of F. candida is given in Table 18. All isozymes were classified as choline (Set 2) or carboxyl esterases (Set 1 and 3). l Acetyl or aryl esterases or lipases were not apparent in F: candida. 55 TABLE 14. Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 10. Inhibitor A B C D Organophosphates ....... Dipterex (TriEhIorfon) -4.3 -4 -4 -4.3 Naled -5 -4.3 na -5 Dichlovos -4.3 -4.3 -4.3 -4.3 Malathion O 0 O O Malaoxon 0 0 0 0 Dursban (Chlopyrifos) + + + + Reldan 0 0 O 0 Diazinon + O 0 + Dyfonate 0 O 0 0 Dyfonate (Ox Analog) 0 O 0 0 Bromophos methyl 0 0 0 0 Ronnel 0 0 0 0 Methyl Parathion + 0 0 0 Ethyl Parathion O 0 0 0 Fenthrothion 0 0 0 O Carbamates Mexacarbate (Zectran) + 0 0 + Aminocarb 0 0 0 0 Carbaryl (Sevin) + + + + Carbofuran + 0 + + Specific Inhibitors Atr0pine Sulfate 0 O 0 0 Eserine Sulfate 0 0 0 0 CMPS 0 0 0 0 PMSF 0 0 0 O Key to symbols: 0 no effect on esterase activity from a 10'3 M solution of inhibitor + slight effect on esterase activity from a 10‘3 M solution of inhibitor ' na data not available for this inhibitor 56 TABLE 15. Concentrations of Inhibitors (loglo) Needed to reach 50% (150) Esterase Inhibition: Polyacrylamide Band 11. Inhibitor A B C D Organophosphates Dipterex (Trichlorfon) -4.3 -4.3 -4 -4.3 Naled -6 -5 -5 -5 Dichlovos -3.3 -3.3 -3.3 -3.3 Malathion 0 0 0 0 Malaoxon 0 0 0 0 Dursban (Chlopyrifos) + O + + Reldan 0 0 0 0 Diazinon 0 0 0 0 Dyfonate 0 0 0 O Dyfonate (0x Analog) 0 0 0 0 Bromophos methyl 0 0 0 O Ronnel 0 0 0 0 Methyl Parathion 0 0 0 0 Ethyl Parathion 0 0 0 0 Fenthrothion 0 0 0 0 Carbamates Mexacarbate (Zectran) + 0 0 + Aminocarb 0 0 0 0 Carbaryl (Sevin) + 0 0 + Carbofuran + 0 0 + Specific Inhibigors Atropine Sulfate ’“ o o o o Eserine Sulfate 0 O 0 0 CMPS 0 0 0 0 PMSF 0 0 0 0 Key to symbols: 0 no effect on esterase activity from a 10'3 M solution of inhibitor + slight effect on esterase activity from a 1053 M solution of inhibitor " na data not available for this inhibitor 57 TABLE 16. Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 12. Inhibitor 6F§5n°6§fi5§phates Dipterex (Trichlorfon) Naled Dichlovos Malathion Malaoxon Dursban (Chlopyrifos) Reldan Diazinon Dyfonate Dyfonate (Ox Analog) Bromophos methyl Ronnel Methyl Parathion Ethyl Parathion Fenthrothion I U1 I i D-an& O Cauaua it but 0 on Ii #9» O was»: c>c>c>c>c>C>c>C>c>+-C>C> c>C>c>c>c>c>c>C>c>+-C>C> c>c>c>c>c>c>c>c>c>+ c>c> Carbamates Mexacarbate (Zectran) Aminocarb Carbaryl (Sevin) Carbofuran 0000 0000 0000 Specific Inhibitors Atropine Squate Eserine Sulfate CMPS PMSF 0000 0000 0000 ‘ -‘~-4-'. ’“~."-‘ Key to symbols: 0 no effect on esterase activity from a 10'3 M solution of inhibitor + slight effect on esterase activity from a 1033 M solution of inhibitor " na data not available for this inhibitor 58 TABLE 17. Concentrations of Inhibitors (loglo) Needed to Reach 50% (150) Esterase Inhibition: Polyacrylamide Band 13 and 14. .Q---O----~--mm-“-~-“-. Inhibitor A B C D Band 13 14 14 13 Organophosphates Dipterex (Trichlorfon) -4.3 -4.3 -6 -6 Naled -4.3 us -4.3 -4.3 Dichlovos na -3.3 -3.3 na Malathion O 0 O O Malaoxon 0 0 0 0 Dursban (Chlopyrifos) + + + + Reldan 0 -3 -3 0 Diazinon 0 0 0 0 Dyfonate 0 O 0 0 Dyfonate (Ox Analog) na na na na Bromophos methyl 0 -3 -3 0 Ronnel O -3 0 0 Methyl Parathion 0 0 0 0 Ethyl Parathion O 0 0 O Fenthrothion 0 0 0 0 Carbamates Mexacarbate (Zectran) 0 0 0 0 Aminocarb 0 0 0 0 Carbaryl (Sevin) 0 O 0 0 Carbofuran 0 0 0 0 Specific Inhibitors Atropine Sulfate 0 O 0 0 Eserine Sulfate O 0 O 0 CMPS 0 0 0 0 PMSF 0 0 0 0 ‘-:O~.---- Key to symbols: 0 no effect on esterase activity from a 10‘3 M solution of inhibitor + slight effect on esterase activity from a 1533 M solution of inhibitor - na data not available for this inhibitor 59 TABLE 18. Classification and Special PrOperties of Esterases in E. candida Based on Inhibition Studies. Isozyme band Esterase Class Special PrOperties Carboxyl esterase Choline esterase Choline esterase Choline esterase Choline esterase Choline esterase Carboxyl esterase Carboxyl esterase Carboxyl esterase Carboxyl esterase Carboxyl esterase -a--.--- susceptible to GP resistant to OP in strain C PMSF resistant OP and carbamate resistant 60 DISCUSSION Major esterases in F. candida homogenates were identified as choline esterases or carboxyl esterases on the basis of inhibition, substrate and tissue localization data. These two classes of esterses will be discussed separately below. Choline esterases (acetyl or butyl) of F. candida were identified as such because of their sensitivity to most organophosphate pesticides (including PMSF) and eserine. In addition, choline esterases were resistant to CMPS, EDTA and lanthanum ions. This behavior was also observed in acetylcholine esterase and butylcholine esterase purified from mammalian sources. Choline esterases in F, candida consisted of several intense bands (esterases 3-5) and two minor bands (esterases 6.0 and 7). Comparison of inhibition data from starch and polyacrylamide gels confirms the homology between starch esterases 1-4 and polyacrylamide bands 3-6. All four strains (A-D) contained several choline esterase isozymes of similar mobility. With the exception of esterase 5 in strain C, all of these enzymes showed similar sensitivity to inhibitors and pesticides used in this study. Choline esterases in E. candida could not be divided into acetylcholine (short chain) and butylcholine (long chain) esterases on the basis of substrate and inhibitor data. All choline esterases cleaved substrate esters of up to 5 carbons in length, and both alpha and beta isomers were hydrolyzed. ACE-specific inhibitors (atropine sulfate and acetylthiocholine iodide), originally suggested for use on vertebrate esterases (Pearse, 1972), failed to differentiate between acetyl and butyl choline esterases. A11 choline esterases 61 were slightly inhibited by 10‘3'M_atropine sulfate and acetylthiocholine. Data for F, candida indicated that they possessed only one group of choline esterases, which were all very similar in behavior. These results are consistent with the data of V033 and Matsumura (1965) who reported that, unlike vertebrates, insect choline esterases were not restricted by substrate length, forming one group of esterase activity. Since the division between ACE and BCE appears more obscure in the insects than in vertebrates, further research is needed to validate the existence of two choline classes within insect species. Due to this lack of discrimination the choline esterase of_§. candida were considered as one enzyme class. The role of acetylcholine esterase in the cleavage of the neurotransmitter acetylcholine has already been demonstated (Pearse, 1972). Because of the difficulty in identifying acetylcholine esterase in insects, all choline esterases have been assumed to participate in neurotransmission (H00per, 1975). Cook and Forgash (1965) reported that most choline esterase activity in the cockroach was confined to the nervous system. In F. candida, choline esterases were located in both head and trunk sections of the body, but not in the hemolymph. Esterases 3 and 4 were restricted to head homogenates, and were responsible for most of the esterase activity of the head. It is possible that these esterases are the major esterases of the sub- and supra- esophageal ganglia, and that they function in the cleavage of acetylcholine. The remaining choline esterases (bands 5-7) were increasingly prevalent in trunk homogenates (25% band 5, 50% band 6, 75% band 7), and were tentatively associated with the ventral nerve cord. Predominance of 62 choline esterase activity in the head of F. candida was consistent with data from Diptera where 50% of head homogenate and 35% of trunk homogenate esterase activity was due to choline esterase isozymes (Ho and Sudderuddin, 1976). Comparison of choline esterases of this study with the results of Hart and Allamong (1979) for E, candida indicated several discrepancies between the populations. In Hart and Allamong's populations, no choline esterases were discovered; isozyme bands (with the exception of origin esterase activity) were not sensitive to eserine sulfate. In addition, their polyacrylamide gels lacked any major staining activity in the Rf range of 0.054-0.23 (the range of choline esterases in this study). Hart and Allamong suggested tight compartmentalization or membrane attachment for these enzymes in E} candida, thereby rendering them incapable of entering the gel material. In the present study, choline esterase activity was recorded for both origin and gel-located isozyme bands. The difference between these two studies may be due to choice of technique. Samples for electrophoresis were prepared by crushing animals in strong buffer solution containing 5% sucrose. In the present study, a dilute gel buffer was used; strong solutions gave unsatisfactory results in preliminary studies and are known to interfere with enzyme extraction (Beckindorf and Stephen, 1970). Analysis of Hart and Allamong's F. candida with dilute buffer would probably result in choline esterase activity corresponding to that found in this study. Similar behavior among strains was recorded for all choline esterases except for band 5 of strain C (esterase CS). This isozyme was more resistant to inhibition by almost every organophosphate 63 used. 150 values for esterase SC indicated that it was resistant to 10-1000 times the concentration of pesticides needed to inhibit band 5 in other strains (A, B and D). Two explanations exist for the resistance: a.) C5 is able to cleave OP compounds at a faster rate than do other choline esterases, or b.) the active site of C5 is not as compatible with most organOphosphate structures compared to other choline estereases, so that an enzyme-inhibitor complex does not form. Both explanations postulate a change in the active site as the cause of resistance. If the resistance to pesticides shown by C5 also occurs ig_yiyg, and if the isozyme is an important contributor to neural activity in E, candida, possession of C5 may give strain C an advantage when encountering organophosphates in the environment. Carboxyl esterases (CBE) in F, candida were identified by their characteristic behavior during inhibition studies. CBEs were resistant to eserine and CMPs, but susceptible to some organophosphates. Two groups of CBE activity were found in E, candida homogenates: esterase l, the slowest migrating isozyme, and a group of fast migrating esterases (10-14). These groups of CBE activity were analogous to sets 1 and 3, respectively, which have been identified on the basis of mobility and staining intensity (polyacrylamide gels) in Chapter 1. Comparison between the results of inhibition studies on starch and polyacrylamide gels confirmed that starch bands 5, 6S and 6F were polyacrylamide enzymes 10, 11 and 12, respectively. The slowest migrating carboxyl esterase, esterase 1, was equally distributed between head and body homogenates of F, candida. Since staining intensity was not affected by damage to the animal (and 64 loss of fluids), esterase l was not located in the hemolymph. CBE activity in the head has been attributed to salivary gland enzymes (and digestion ) in Diptera (Ho and Sudderruddin, 1976). Esterase 1 could be a salivary gland esterase in F3 candida. Unlike those of many insect species, the salivary glands in Collembola extend into the thorax (Schaller, 1970), which would explain the distribution of esterase 1. The enzyme would function in cleavage of esters taken up in the diet. Hart and Allamong (1979) did not report any slow migrating carboxyl esterases in their populations of F. candida (Rf value < 0.20). The remaining carboxyl esterases in E3 candida were the fastest esterases in the polyacrylamide gel (Rf value = 0.30 to 0.51). Each strain (A-J) possessed several intensely staining enzymes in this region, as did the population of F. candida studied by Hart and Allamong (1979). These isozymes (Set 3) were restricted to the thorax and abdomen, and were also present in the hemolymph. A11 CBEs were generally resistant to organophosphate pesticides, consistent with their ability to cleave esters entering the cell from the external environment (Pearse, 1972), and their known function in pesticide hydrolysis (Plapp, 1976). Cycling esterase (10-12) participation in the cleavage of juvenile hormone has already been suggested on the basis of their correlation with the molt cycle of F. candida (Chapter 1). Additional information gained from esterase body location, substrate specificity, and inhibition studies can be used to support their action in JH cleavage. Esterases 10-12 were located in the trunk and hemolymph of F. Candida. Fat body and hemolymph are the major sources of JH esterases in many insect 65 species (de Wilde, 1981; Vince and Gilbert, 1977; de Kort and Granger, 1981). Since homogenization releases stored proteins, (Beckindorf and Stephen, 1970), JH esterases from the fat body would be expected in trunk homogenates. Studies on the substrate specificity of esterases 10-12 in F. candida demonstrated that esterase activity declines as ester chain length increased. A three-carbon ester was less likely to be hydrolyzed than the control two-carbon ester. Methyl pesticides were more effective than ethyl pesticides in causing inhibition of esterase 10-12 activity. Both of these results indicated that very short chain esters are preferentially recognized by cycling esterases of E: candida. Hammock et a1. (1977) found that chain length was the critical feature of the JH molecule necessary for cleavage by JH esterase; their ability to hydrolyze JH fell rapidly as the JH methyl ester was replaced by longer chains. This relationship has been verified by Sandburg et a1. (1977) and Kramer and de Kort (1976). Isomeric ester chains, such as isopropyl JH, are also generally not hydrolyzed by JH esterases (Weirich and Wren, 1973). In conclusion, JH esterases are generally much more active toward methyl esters than longer chains, and this was observed for esterases 10-12 in E, candida. Degree of inhibition observed for cycling esterases 10-12 is consistent with inhibition levels reported for JH esterases in other insect species. JH esterases in the cockroach were resistant (less than 50% reduction in staining with a 10‘4 M solution) to Carbaryl, Aldicarb and PMSF (Hammock et al., 1977). F, candida esterases 10-12 were even more resistant to these substances; a 66 10’3 M solution generally caused little change in staining activity. In contrast, F, candida cycling esterases were inhibited by Dichlorvos, Dipterex and Naled. JH esterases in the locust (Pratt, 1975) and Manduca sexta (Sandburg et al., 1975) were also sensitive to these inhibitors. In the majority of insect species, JH esterases have been reported as a subclass of carboxyl esterases (de Kort and Granger, 1981). In Coleoptera (Kramer and de Kort, 1976), Lepidoptera (Whitmore et al., 1972), OrthOptera (Pratt, 1975) and Diptera (Ahmad, 1976), they were susceptible to Paraoxon and DFP as well as other CBE inhibitors. However, mosquito JH esterase was identified as an aryl eterase (Hooper, 1975) becausemof its sensitivity to CMPS (10-4 ‘M); this appears to be a rare phenomenon (de Kort and Granger, 1981), since JH esterases, in general are carboxyl esterases. In view of their cycling nature, inhibition sensitivity, substrate specificity and class, esterases 10-12 can be identified as JH esterases in F, candida. Three esterase classes were not identified ion, candida during this study: acetyl esterases, aryl esterases and lipases. Acetyl esterases (C-esterases) are found primarily in higher plants, especially citrus fruit (Pearse, 1972). They are not sensitive to any of the inhibitors used here; this was verified with purified orange peel acetyl esterase (Table 7). Since all F. candida esterases were sensitive to l or more organophosphates, none of them were acetyl esterases. Aryl esterases (A-esterases) are characterized by their resistance to organophosphates and concurrent sensitivity to CMPS, 67 EDTA and lanthanum ions. Although several of the fast migrating isozymes in F, candida were relatively OP resistant, none were also inhibited by CMPS, EDTA or lanthanum nitrate, and therefore, were not aryl esterases. In contrast, Hart and Allamong's (1979) F: candida population contained several aryl esterases, high mobility isozymes sensitive to CMPS. However, considering all esterase isozymes discovered in the species up to this point, aryl esterases occur relatively infrequently when compared to choline or carboxyl esterases in F, cagdida, which is also true of most pterygote insects (Ho and Sudderuddin, 1976; Ahmad, 1976). No lipases were identified in F. candida with the staining methods used in this study. Although esters of 14 carbons were not hydrolyzed at any appreciable rate by any esterase in E, candida, studies with longer esters (i.e., trigylycerides) are necessary before lipase activity can be completely ruled out. Lipases are common in litter invertebrates who participate in the chemical breakdown of litter (Marcuzzi and LaFisca, 1977). Since Collembola are thought to be involved in physical (rather than chemical) modification of litter preparatory to microfloral activity (Richards, 1974), lipase enzymes may be relatively rare in species of this group. Minor bands (2, 6, 5, 8 and 9) could not be classified because they were too faint to score during inhibition studies. On a volume comparison, with one animal per slot, these minor bands made very small contributions to overall esterase activity. Attempts to increase staining intensity by loading extra material in the gel wells resulted in overloading of major bands, causing spreading, 68 streaking and interference with both enzyme migration and staining. Because of the low activity of minor esterases, it is suggested here that they were enzymes of the mesenteron, gut flora, or fungi consumed in the diet. 69 Chapter 3: Ifl_vivo Effects of Diazinon on Strains of F, candida Possessing £2.V1tr° Diazinon-Susceptible and-Resistant Esterase Isozymes. INTRODUCTION OrganOphosphates and carbamate pesticides exert their toxic effects on insects by inhibiting choline esterase activity in the nerve cord, thereby interfering with neural transmission (Casida, 1955; Moriarity, 1969; Plapp et al., 1979). Carboxyl esterases (CBE) are inhibited to a lesser extent than choline esterases (CHE) and are able to cleave ester pesticide molecules. This rapid hydrolysis by CBEs results in an overall reduction of level of pesticide in the insect (Welling, 1977), thereby reducing its toxicity. Insect populations resistant to ester pesticides have been documented. Correlation of pesticide resistance i3 vivo with altered esterase behavior £3 :iggg has revealed several mechanisms for pesticide ineffectiveness: A). presence of a mutant CBE or CHE enzyme, b). increase in CBE titer or activity, c). decrease in CBE titer or activity, or d.) a factor completely independent of esterase action. These mechanisms will be considered independently. Pesticide resistance has been correlated to the presence of mutant esterase enzymes in Diptera (Chuo and Sherman, 1971; Plapp et al., 1979), Acarina (V033 and Matsumura, 1965) and Coleoptera (Sudderuddin and Lin, 1978). When esterases are no longer impaired by organophosphates or carbamates, toxic effects of these chemicals are reduced. Altered enzymes may be substrate-specific, conferring resistance to only one compound (Krueger and Casida, 1961; Matsumura 70 and Voss, 1964). In mosquitos, for instance, resistance to Malathion did not extend to closely related forms of the molecule (Oppenorth, 1965). General, or cross resistance, is more commonly encountered (Welling, 1977) and results from a loss of structural specificity at the active site of the enzyme (V033 and Matsumura, 1965; Plapp, 1976). The most frequently reported mechanism of pesticide resistance is an increase in carboxyl esterase activity or titer. Examples have been found in Lepidoptera (Riskallah et al., 1979), Diptera (Matsumura and Brown, 1961; Forgash, Cook and Riley, 1962; Matsumura and Hogendijk, 1964; Sudderuddin, 1973; Hooper, 1976). HomOptera (Sawiki et al., 1977; Devonshire, 1977) and Acarina (Matsumura and Voss, 1964). In all of these cases, increases in enzyme amount or activity reduced the severity of organophosphate poisoning by enhancing pesticide degradation rates. Pesticide resistance has also been correlated to a reduction in esterase activity or amount (Plapp and Bigley, 1961; van Asperen and Mazijk, 1965; Ahmad, 1974), without definitive explanation. Plapp (1976) has suggested, however, that the apparent decrease in esterase action was a result of the technique used to estimate it. Since activity was measured by colorimetric assay, the observed reduction could have been due to reduced substrate cleavage rates by esterase enzymes. Plapp (1976) concluded that mutant CBE enzymes capable of rapid pesticide hydrolysis might not cleave colorimetric substances as efficiently as predicted, thereby resulting in an underestimate of esterase activity. Finally, resistance to ester pesticides may be the result of a 71 mechanism unrelated to esterase action. For instance, possession of reduced cuticular absorption rates for Malathion and Parathion resulted in a mosquito strain resistant to these compounds (Matsumura and Brown, 1963). Plapp (1976) has reviewed this mechanism and its function in resistance to organophosphate and carbamate pesticides; he also discussed the contributory role of mixed-function oxidase and transferase enzyme systems and concluded that in many insect species, more than one enzyme system is involved in pesticide resistance. Reports of Apterygota resistant to ester pesticides are rare. The relationship between degree of resistance and esterase properties has never been investigated. Only in a few Collembolan species, most particularly F, candida, has any pesticide toxicity been examined. Low doses of topically applied insecticides were very toxic to F. candida_(Thompson and Core, 1972), organophosphates more so than carbamates; this was confirmed in soil contact studies (Tomlin, 1975). Insect mortality has traditionally been the only parameter recorded during tepical application and soil contact studies. These experiments are designed to measure 24 or 48 hour toxicity and are concerned only with immediate effects (i.e., knockdown and kill rates). Sub-lethal effects are known to appear in advance of mortality (Welling, 1977) and may contribute to overall pesticide effects in field populations. Prolonged uptake of pesticide may curtail reproduction (Georghiou, 1972) or affect behavior (Moriarty, 1969). Sub-lethal effects are generally examined in extremely low dose studies or in oral toxicity studies using contaminated food sources 0 72 Few oral toxicity studies have been performed with Collembola (Eijsackers, 1975). Extremely high doses of pesticides and herbicides are often reached before there is appreciable mortality. Carbofuran (250-2000 ppm) caused reduced egg production and 10-100% mortality in F. candida after a 40 day exposure (Gregorie-Wibo, 1978). Yeast contaminated with the herbicides Atrazine and Paraquat reduced fecundity and caused longer instar durations in F. candida, but very little cumulative mortality, even after 22 weeks (Subagja and Snider, 1981). Guthion-contaminated yeast resulted in 7 day mortalities of 1% (10 ppm), 37% (1000 ppm) and 53% (3000 ppm), but no sub-lethal effects were reported (R. M. Snider, personal communication). DDT-contaminated yeast (100-150,000 ppm) stimulated egg production in E. candida, but did not cause any mortality (Butcher and Snider, 1975). All of the chemicals previously used in oral toxicity studies on ‘F. candida, with the exception of DDT, were tested for i2_!i552 inhibition of esterase activity (Chapter 2). Carbofuran was the only chemical which also produced substantial in_!i££2_esterase inhibition, with 50% inhibition of choline esterase activity at 10’?! and complete inhibition (100%) at 10"3 M, Atrazine, Paraquat and Guthion had no effect on esterase activity. Therefore, based on the data available in the literature, only one organOphosphate (Guthion) and one carbamate (Carbofuran) caused severe mortality in F, candida, but in the present study, only Carbofuran affected esterase activity in vitro. Several other pesticides, however, caused both in yingmortality in topical application and soil contact studies (Thompson and Gore, 73 1972); and £2.X£££2 inhibition of esterases. Oral toxicity of sub-lethal effects of these pesticides on F3 candida have never been reported. One example is the organOphosphate pesticide Diazinon, which, therefore is suitable for oral toxicity studies. Diazinon (P-S) is toxified to Diazoxon (P-0) and detoxified by cleavage of either one of its two ethyl esters (Eijsackers, 1975; Oppenoorth, 1965). Diazinon has been shown to readily penetrate the nerve sheath in Diptera (Lord et al., 1963), and its oxygen analog establishes an equilibrium concentration between the CNS and hemolymph (Welling, 1977). Thompson and Core (1972) have shown that E. candida was susceptible to Diazinon (100% kill at 0.1% topically applied, or 0.5 ppm in soil). In the present study (Chapter 2), concentrations of 10"5 M Diazinon generally inhibited choline esterase (bands 3-7) activity by 50%. Esterase 5 of strain C was more resistant (than in strains A, B and D) to inhibition by Diazinon since a 10“3{M_solution was necessary for 50% inhibition of esterase activity. Esterase 5 was equally distributed between head and trunk homogenates and was identified as a choline esterase involved in ventral nerve cord function. Other major esterases (carboxyl esterases 10-14) were unaffected by Diazinon. Given that strain-specific differences in ig_yi££g_esterase behavior were known to exist, feeding experiments on F. candida strains A-J were conducted with Diazinon-containinated yeast to ascertain; a.) the sub-lethal effects and mortality caused by exposure to Diazinon, b.) the possible existence of_ig'vivo strain-specific reSponses to Diazinon, c.) the selective advantage 74 (if any) conferred to strain C by esterase 5 (Diazinon-resistant isozyme). RESULTS Behavioral differences between treated and control individuals were observed for a number of normal activities and are qualitatively described below. Other parameters which could be quantified, included: fasting behavior, consumption of exuvia, instar duration, oviposition frequency and mortality. Strains A-J were fed Diazinon-contaminated yeast (10, 50, 100, 1000, and 2000 ppm) for 21 consecutive days. Contamination levels of 10, 50 and 100 ppm caused neither noticeable sub-lethal effects nor mortality in F. candida adults, while a dosage of 2000 ppm resulted in active avoidance of food. Therefore, most of the observations reported below are based on animals exposed to 1000 ppm Diazinon. In 1000 and 2000 ppm treatments, sub-lethal effects increased in frequency or became more obvious as the treatment period (21 days) progressed. Sensing F, candida lack discernible eyes and normally sense the substrate by touching it with the tips of the antennae several times each second, often while running rapidly. In the present study, treated animals touched the surface with ther antennae more often than control animals, usually at such a high frequency that their terminal antennal segments were difficut to observe. Treated animals also used a larger portion of their antennae for sensing, holding them flat against the substrate surface, while simultaneously touching the substrate with their mouthparts, and often were found 75 dead in this position. At high concentrations of Diazinon (2000 ppm), animals wiped their antennae and mouthparts across the substrate, as if to clean them. Treated individuals, if they survived, had only brown-tipped stubs of one segment long, instead of normal four-segmented antennae. Locomotion Walking and springing activity were affected by Diazinon treatment, which caused loss of coordination. When animals attempted to walk, the furcula dragged behind them. Repeated attempts to draw the furcula up to its normal position under the abdomen and to hold it with the retinaculum were rarely successful. After a few steps, the furcula slipped loose again 31d trailed behind. Unlike control animals, treated individuals did not land on their feet after springing, but rather on their sides. Unable to coordinate leg and body movements, animals wriggled ineffectively and only regained their footing after a great deal of effort. If a jump landed them in water dr0plets formed by condensation on the side of the jar (normally no severe hazard) they became trapped by the water's surface tension and were unable to escape. Treated individuals which fell into a small hole in the substrate had difficulty finding their way out. Grooming and‘Bodydngement Grooming activity in treated animals was reduced or absent. As a result, their tarsi became charcoal covered and fungal hyphae which had invaded the integument were not removed. Unusual body movements consisted of stretching the abdomen from side to side, or arching it forward, toward the head. The head was frequently bent toward 76 the abdomen several times in quick succession, while the animal was either standing still or walking. Feeding Throughout these experiments treated animals consumed less food than control animals, an effect which was particularly evident in jars containing 5 individuals apiece. Controls, in groups of 5, routinely consumed all of their food in the time between feedings. In contrast, treated animals appeared to avoid the food, so that a substantial portion of contaminated yeast remained when treated cultures were refed. An additional indication of avoidance behavior was found in the pattern of fecal deposition. F. candida in culture indiscriminately deposit fecal matter on all areas of the substrate, which results in a darkening of the food source. Fecal depostion was observed on control yeast, but not on treated yeast, indicating that treated animals spent less time in the immediate vicinity of their food. Differences in degree of fecal deposition were most pronounced at 2000 ppm Diazinon. Fasting Behavior Since F. candida routinely consume charcoal substrate along with food, a gut outline can be detected through the integument when the animal is feeding. In contrast, fasting is indicated when no gut outline can be discerned. F. candida normally fast for 24-48 hours before exuviation takes place (Palevody, 1973; Grimnes, 1981). Because food avoidance was suggested by observations on fecal deposition and food consumption the effect of Diazinon on fasting was quantified. Using visual estimation of gut contents, the time spend fasting 77 was assessed in treated and control individuals from 8 strains (20 animals/strain, except strain E in which replication was reduced to 12 due to mortality). For each individual, the total number of days Spent fasting (8 duration of fasting), out of 21 observation days, was recorded, and means/strain were calculated (Table 19). These values, therefore, reflect the overall extent of fasting, and not its continuity (or molt-related cyclicity). Control means for fasting duration were significantly lower than Diazinon-treatment means (Table 19) with the exception of strain E. In this context, significance was defined as the extent to which differences between control and treatment means exceeded the sum of the standard errors for both means. Comparison among strains of esterase pattern B (strains B,G,H,I and J) and within pattern D (strains D and E) did not indicate any correlation between fasting behavior and esterase pattern. Fasting duration in control individuals was lowest for strains C and H; strain C also exhibited the lowest fasting duration of all strains when subjected to Diazinon. A two factor cross classified model, with pesticide concentration and strain as cross classified fixed effects, was used for an analysis of variance of these data (Appendix IV). Strain E was not included because its replication was not equal to that of other strains. Each treatment-strain combination contained 20 data points. Results are given in Table 20. The F-ratios for treatment and strain effects indicated that these two factors were significant (p<0.001) without interaction between them. 78 TABLE 19. Effect of Diazinon on Number of Days Spent Fasting (out of 21) by F, candida (n - 20 individuals/strain). Control 0 ppm Diazinon 1000 ppm Strain mean 1 SE mean -_i-_ SE B 12.05 .51 14.95 .47 C 7.65 .56 10.00 .39 D 10.50 .60 12.80 .56 E* 11.83 .75 10.92 .87 G 9.25 .46 12.40 .55 H 7.65 .54 11.50 .67 I 12.15 .65 13.60 .59 J 10.05 .60 11.20 .50 -.—.--“-.-. *Based on 12 observations/mean, not included in the analysis of variance. 79 TABLE 20. Analysis of Variance of the Effects of Diazinon on Fasting Behavior of 7 Strains of F. candida (n - 20 individuals/ strain). Effect SS Treatment (A) 423.63 1 422.63 69.82 ** Strain (B) 677.37 6 112.90 17.3 ** Interaction (AB) 55.17 6 9.19 1.41 -. .--.---‘--‘-‘-M----‘-“‘-------‘ **p < .001 80 Consumption of Exuvia F3 candida normally consume their exuvia within a few days after molting, sooner if food is lacking. Studies with 2000 ppm Diazinon indicated that treated individuals were less likely to eat their exuvia than control animals. Cast skins clearly accumulated in treatment jars, were generally avoided by the animals, and were eaten only sporadically. This phenomenon was quantified by making daily counts of the number of exuvia present in jars containing 5 animals (treated at 1000 ppm versus control), from day 7 to day 21 of the treatment period. Nine strains of F. candida were used (20 treated and 5 control jars per strain), and pertinent means :LS.E. are plotted in Figures 6-14. Means (without S.E.) for several strains possessing one esterase pattern are plotted together in Figures 15-18 for within-pattern comparison. Numerical means and standard errors used in the production of these graphs are given in Appendix VII. All strains fed contaminated yeast showed an increase in the mean number of exuvia present. Strain A (Figure 6) accumulated the highest number of exuvia per day (reaching a maximum of 9 exuvia per jar), while the corresponding control peaked at slightly over 4 and then declined. Strain D (Figure 9) showed the next highest number of exuvia during the treatment period. Strains B, G, H, I and J (Figures 7, 11 12, 13 and 14) exhibited only a moderate accumulation of exuvia per jar, followed by strain C (figure 8), for which the lowest treatment means were recorded. 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M NO m :fimuum CH umh uma mH>sxm mo Ammfiv amassz :mmz co cocHNmfia mo uummwm .OH muswwm EUCZ UXD>HC 0..th WCZ 86 .Auwh\mameficm m .Houuaou\mumn m .cmummuu\mumn omucv pzwzhcwmh mo w>¢c mu mg #— _ Nu cu 41P+++_ \\ \ \ \Q \ Q o my..-r--+--+. w .5528 o SEBoEo 8:85: o zEEmcla a: mvfivcmu.m.wo o :Hmuum SH umm Hum mw>axm mo Ammfiv umnasz saw: so aoawumwn mo uommmm .HH muswfim ISHJGZZZ MJ><:3)>*‘CE O.hJQ: 'DCEO: 87 NN .AHMn\mHmEHcm m .Houucou\mumn m .vmummuu\mumfi Omuav mvwwcmo.m.wo : cwmuum ca umw uma mfi>5xm mo Ammfiv umneaz cam: co cocqnmfio mo uumwmm .NH muawfim hzwzhcumh mo wrca ON m— mu Va I w ‘ s? “I Nu c— o _ _ - .5528 x 335?; 852: z zEEmI on :wcz UXD>HCE LINK WCK 88 .Aumm\mHmEH:m m .Houuaoo\mumh m .wmummuu\mumfl om ucv mvwvcmu.m.wo H :«muum cw umh uma mfi>=xm mo Ammflv mumnasz cmmz so confinmwa mo uummwm .MH whamfim hzwxhcwxh mo m>¢o NN on mu m— z 3 cu m m b _ _ p _ _ _ + + 1,, +...+.-.+---+..-.,...+---+....¢ o + l N K c 7 m 1 v w. c ~ .9 m x u a 40529.. a z—cEm?--o I o m awhcumh H zEthI : a: 89 . Han mHmEHcm m .Houucou mumm m .vmummuu mumh om us A \ \ \ V mwfiwcmo.m.mo h cwwuum cw mwpbxm mo Ammflv “@3532 cwa :0 confinmwa mo uummwm .QH muswfim hzwzhcumh no wrco NN ON Du m~ vfl N— cu m m .1: P b _ P — _ + _ D + lilo \\+ II I N 1 V I m Jomhzou _... zucmbmbuouo I m cupcwmh .1 zucthI o" ZINC: UXD>H¢ (LING: "JCK 90 NN ON r .Auwn\mamafiam m .Houucou\mumh m .umuwwuu\mumh ON ucv mo m vcm a mcfimuum a“ paw awn wH>=xm mo Ammflv quE:z ammz co cocanmwo mo uumwmm .mH muswfim mu _ pzuzhcumh mo m>mo mu _ v— _ Nu n: . o _ — — cwhcwmp w zucmhmofié owhcumh o z—cmhmI mvwvchnM cu IIUUCIflZL u1><:::>-c:: lfl.hJO£ '3‘!!! 91 NN ON b .Aumn\mameficm m .Houucou\mumn m .Omumwuu\mumn ON uav OO O wcm O.m mCHmuum :H van uma mH>axm mo AMmHv umnasz ammz so :ocfinmfia mo wommmm .OH musmam mg _ pzwzhmwmh ....O m>¢O mg _ «a _ N— Ou O O _ _ 82$: 2 zafimfi 8:85 6 235359 8:55 o EESI m3 2:3 .M Ow IZMJOIZI UJ><233>F‘G: Q.hJO: '7CICK 92 NN ON _ .Aumn\mameficm m .Houucoo\mumm m .Omummuu\mumn ON "CO «0 O tam H.m mcwmuum a“ umw pma mH>sxm mo Ammflv umnesz saw: no cocfiumfin mo uummmm .NH musmfim o— _ hzuzhcwmp no mrco On _ «— _ Nu O“ . O _ — _ OM25»: .a z~¢¢.—w$:i¢ OPEOE. O z—czhmo---o cub—gum» O zuczhmI mcwvcmuam O~ IIMJOEIL hJ><:D>’**OE 0.MJO: 'Ddflfl: 93 .Aumn\mHmEficm m .Houucou\mumn m .vmummuu\mumh ON usv mvfiuamunm mo O vcm o.m.< mcwmuum cw pmh umm mw>zxm mo AMmHv “mnesz saw: so confinmfln mo uumwwm .OH muawfim pzuthcuzh mo w>OO Nw ON O" m: I N— Ow O O _\ b[ p p _ P _ O ‘9 ‘1 \ss \’0 \ I‘ll s \ {*\\-/ \\. 1T..£\ “INVOB 1 N . /0 I‘ll“\\ \\0llll.l|||°llll‘\\\\ \\\ ( \o.....-0\ h a... 1 0‘39. \m..\m..!m...:ma.. w "l...“‘. \ \\ R \\ \ I m ....R /w\\m 8.531.— O zuczhmuim .532: u 235mg}... 835: m 2335021. I O Owhcuzh a 232.51 O— ZUCZ mx=>~¢ 1:.qu 'JCBK 94 (B,G,H,I and J) showed similar but not identical trends in number of exuvia during Diazinon exposure (Figures 16-18). In conclusion, Diazinon treatment increased the number of exuvia present in each jar. This increase could have been due to decreased consumption of exuvia (avoidance) or to increased molting frequency and correspondingly shorter instar duration. In order to discriminate between these two possibilities, observations of molting instar duration and exuvia persistence (number of days a cast or skin could be identified) were made on individual animals. Data on isolated individuals indicated that accumulation of exuvia was the result of reduced consumption rates and not shorter instar duration (discussed below). Exuvia persistence (in days) was recorded for two exuvia in each of 20 jars, and means (based on 40 observations) for control and treated individuals of each strain are given in Table 21. Means for strain E were based on 12 jars (24 observations) instead of 20 jars. On a scale from longest to shortest mean persistence of cast skins, treated strains could be arrayed as: D>E>B>I>G>H>J>C. Mean values for control individuals were significantly lower than treatment means for all strains. Again, means of both treated and control individuals were lowest for strain C. In addition, strain C also showed the smallest increase in exuvia persistence (difference, in days, between treated and control means) due to Diazinon exposure. Analysis of variance was performed in the data using a model summarized in Appendix IV, using pesticide and strain as cross classified fixed effects (Table 22). Strain E was not included 95 TABLE 21: Effects of Diazinon on Persistence of Exuvia (in days) (n-2 exuvia per each of 20 individuals per strain) Control 0 ppm Diazinon 1000 ppm Strain Mean :_ SE Mean :. SE B 5.88 .58 8.28 .74 C 1.85 .20 3.45 .43 D 5.60 .59 9.18 .83 E* 2.17 .34 8.83 1.17 C 2.28 .31 5.95 .66 H 2.25 .37 5.63 .11 I 4.28 .47 6.25 .66 J 1.93 .27 4.48 .51 - -0-----.-.--W-~.Q~.--‘-“ * Based on 24 observations/mean, not included in the analysis of variance. 96 TABLE 22: Analysis of Variance of the Effects of Diazinon on Persistence of Exuvia in 7 Strains of E. candida (U=2 exuvia/individual, E=20 individuals/strain) EFFECT SS df MS F-ratio Treatment (A) 1050.24 1 1050.24 80.9 ** Strain (8) 1639.53 6 273.25 21.05** Intraction (AB) 79.55 6 13.26 1.02 Individuals (E) 3452.36 266 12.98 3.68* Observations (U) 989.13 280 3.53 --- -‘C--—‘“ * .001A>C, D>J>B, E, F, H and I. The results did not indicate the presence of any esterase pattern-related mortality; strains D, E and F (pattern D) and strains B, G, H, I and J were not similar. In every strain, juveniles which survived 1000 ppm treatment were smaller than control juveniles, a result directly related to food avoidance behavior (i.e., reduced food intake). Effects of Diazinon on adult survival were measured in three trials (Table 26). Trial 1 consisted of animals of unknown age (isolated from crowded stock cultrues in which competition for food was severe) and treated imnediately. Trial 2 was conducted on animals selected for equal size and fed uncontaminated yeast for 4 days prior to treatnent. Trial 3 animals were raised from isolated eggs, and were fed regularly until they attained the size of aimals used in trials 1 and 2 (30 days). Although adults used in these trials were not identical in age, the most important factor determining survival seemed to be the nutritional stress level, i.e., degree of feeding prior to treatment. Adults fed contaminated yeast immediately after isolation (Trial 1) were more likely to die than animals which had been fed with untreated yeast for 30 days (Trial 3) or even for 4 days (Trial 2) prior to treatment initiation (Table 26). Thus mortality in strains A (43%), D (312) and C (22%) was high for stressed individuals, but 104 drOpped to 02 for strain D and 5% for strain C when treatment followed 30 days of regular feeding. In general, all strains showed greater survival potential if fed four days prior to treatment, even more so if fed for 30 days prior to treatment. Strain E was the only strain in which increased feeding did not result in decreased mortality. Animals in Trial 1 were nutritionally stressed, and, as a result, were probably hungry. When fed treated yeast, they consumed it, and the associated pesticide at a greater rate per unit time in trial 1 (than in later trials). Mortality, therefore, was much higher in trial 1, and probably was directly related to degree of stress. Differences in stress levels are also known to affect survival during topical application and soil contact studies (Thompson and Core, 1972). No clear relationship between mortality in adults and esterase pattern was established in this study. Strains B, G, H, I and J (all esterase pattern B) did exhibit relatively low mortality in both stressed and non-stressed (fed) adults, but strains D and E (both esterase pattern D) did not appear to be similar. Strain C, which possessed an in vitro Diazinon-resistant isozyme (Chapter 2), was not immune to the toxic effects of the pesticide. Although feeding prior to treatment reduced mortality from 22% to 5%, strain C was the only strain in which survival was not 100% in trial 3. DISCUSSION Many factors determine the oral toxicity of a pesticide in a given species (Winteringham, 1969). Gustatory preferences or 105 avoidance behavior influence the amount of pesticide taken up by the animal (Georghiou, 1972). Processes of toxication and detoxification by gut flora and rate of pesticide transport through the gut wall tissues, modify pesticide levele in the hemolymph (Eijsackers and Van der Drift, 1977). Large differences between the effects of oral and topical applications, for the same pesticide and species, often reflect the relative importance of factors limiting pesticide action (Matsumura and Boush, 1966). Detection of oral toxicity in laboratory populations of Collembola often requires a high concentration of pesticide in the food source (Subagja and Snider, 1981; Gregorie-Wibo, 1978). Eijsackers (1975) has suggested that survival at low doses was due either to high detoxification rates by gut flora or to pesticide absorption to charcoal which had been ingested by the animals. In the present study, as well, high concentrations of Diazinon were needed to cause mortality; Diazinon-contaminated yeast at 2000 ppm killed a significant proportion of the population over the three week treatment period. Avoidance of food at this dosage probably contributed to the overall variablity in mortality results, both with in and among strains. At 1000 ppm, avoidance of comtaminated yeast was reduced but not completely eliminated. This dose, only moderately toxic to the animals, proved adequate for examination of sub-lethal effects of Diazinon in strains of E. candida. Biological changes in E. candida due to Diazinon (sub-lethal effects and mortality) were divided into two groups: effects which were primarily related to Diazinon's mode of action in the nervous system (direct effects), and effects which were a consequence of food 106 avoidance (indirect effects). Diazinon directly affected sensing activity, coordination, locomotion, oviposition and survival. Diazinon, or Diazoxon, is known to inhibit choline esterase activity of the brain and ventral nerve cord (Plapp, 1976). This was verified in the present study since Diazinon severly inhibited in_vitro staining activity of E. candida choline esterases (Chapter 2). Rapid antennal vibration, tremors in body parts, and uncoordinated movements confirmed in 1112.5hat Diazinon affected neural transmission in E. candida. Since neural impulse blocking in insects does not interfere with breathing (unlike vertebrates), temporary immobilization without death can occur; this was occasionally observed in E. candida. Welling (1977) has reported that Diazinon attacks esterases of the thoracic ganglia first, and then, eventually, brain esterases. Abnormal locomotion in E, candida is best ascribed to Diazinon's effect on the thoracic ganglia, while death could have been the result of inhibition of brain esterases. Another direct effect of Diazinon was the persistence and accumulation of uneaten exuvia in jars of treated animals. Since Collembola lack Malpighian tubules, wastes are stored by conversion to insoluble forms and deposition in fat body cells or in the integument (Schaller, 1970). In the course of Diazinon treatment, pesticide may have been stored in the integument, causing the animals to avoid their exuvia as they avoided contaminated food. In many insect species (Moriarty, 1969) Diazinon causes a reduction in egg viability and numbers of fertile females, and retards follicular development. Reduced frequency of oviposition in all strains of E, ggpdida was, most likely, a direct effect of 107 Diazinon. Indirectly, the pesticides impact on E. candida was aggravated by poor nutritional staate of test animals. Thus, as a result of avoidance of contaminated food, a) adults may have lacked sufficient reserves for egg provisioning, b) treated juveniles were smaller than control juveniles, and c) instar duration was prolonged. Mortality is only one measure of the deleterious effect Diazinon can have on a population 0f.E'.EEEQ£QE' If sub-lethal effects are extrapolated to field populations, one can easily imagine that impairment of springing, walking and sensing would increase predation and desiccation rates, and keep animals from escaping sprayed areas or contaminated food sources. Poor nutrition as a result of food avoidance leads to fewer molts, reduced chance of egglaying and slower growth rates; in turn, all of these are reinforced by an increased susceptibility to Diazinon due to nutritional stress. In conclusion, mortality figures alone tend to severely underestimate the potential damage to E3 candida_populations exposed to a pesticide like Diazinon. Investigation of Diazinon's effects was performed on a number of strains of E, candida. Analysis of the data for instar duration, persistence of exuvia and fasting duration indicated that strain was a significant contributory factor to observed variation (p<0.001); both control and treated means were strain dependent. Pesticide effect was also significant (p<0.001) but interaction between strain and treatment was not. This lack of interaction strongly indicated that the pesticide affected all strains similarly, although actual mean values differed among strains. Knowing that strain and 108 pesticide both had significant effects, the data were analyzed with respect to esterase pattern. A relationship between the animal's behavior and the enzymes it possessed could not be conclusively demonstrated. Neither pairs from the same geographic locality, nor strains of the same esterase pattern showed clear or consistent similarities in their biological performance. The discovery of an in li££2.Diazinon-resistant esterase isozyme in strain C (esterase 5) raised the question whether an altered choline esterase would be less affected by Diazinon in_!izg_as well, resulting in reduced mortality and/or sub-lethal effects, and thus provide a selective advantage for strain C. Strain C esterase 5 was more resistant to organophosphates than esterase 5 in other starins. Furthermore, all other esterases of strain C were equally or more resistant to inhibitors and pesticides when compared to esterases of strains A, B and D. Therefore, in. zi££2_responses of Strain C esterases to Diazinon were examined to determdne the potential for strain C to maintain or increase its contribution to the next generation (a selective advantage), relative to other strains. Strain C had the shortest instar duration in both treated and control individuals. Faster molting results in more oviposition opportunities (every other instar), and a potentially faster growth and recruitment rate. Strain C spent less time fasting, and consumed cast skins rapidly under both treated and control conditions. This indicated that strain C could be accumulating nutrients at a faster rate than other strains which showed a greater degree of avoidance. A greater percentage of females of strain C 109 laid eggs under treated and control conditions than any other strain. In addition, a previous study showed that strain C produced more eggs in the first two ovipositions than strains A, D, E and F (Grimnes and Snider, 1981), and were larger in size by the first oviposition. Therefore, in terms of egg production, frequency of oviposition, instar duration and feeding, strain C seemed to be at an advantage. In contrast, strain C exhibited high mortality (22%) when treated with Diazinon immediately after isolation from stock cultures. When well-fed for 30 days prior to treatment, mortality dropped to 5%, but this value was the highest of any strain under these conditions. Also, Grimnes and Snider (1981) reported that strain C egg production was reduced in later instars, and that egg viability was slightly lower than for strains A, D, E and F. In conclusion, a slight selective advantage was observed for strain C when treated with Diazinon-contaminated yeast (1000 ppm). Slightly higher mortality when compared to other strains may be offset by increased feeding, faster molting and more oviposition opportunities. Strain C is known to lay more eggs and grow at a faster rate in early instars (Grimnes and Snider, 1981). Since juveniles showed only moderate mortality (62), it is possible that eggs laid by strain C may hatch and grow to maturity faster and with more success than other strains. Overall, faster generation time and high fecundity during early instars would result in a selective advantage for strain C. 110 CONCLUSION The esterase system of the apterygote soil collembolan E. candida was investigated for four major reasons: 1. The general lack of data on enzymes in apterygotes, 2. The possible role of esterases in JH control during molting and reproduction, a role which has been proven in pterygotes and suggested in apterygotes, 3. The function of esterases in pesticide metabolism and resistance, and 4. the "primitive” status of apterygotes relative to pterygote species. The esterase system of_§. candida consisted of choline and carboxyl esterases. Within these classes, several isozymes were present and were similar to each other in most respects, except for their electrophoretic mobility. The behavior of the esterases of E. candida was like that of pterygote esterases. Choline esterases were located in the head, possibly in the “brain” and in the trunk, where they were tentatively associated with the ventral nerve cord. It was concluded that carboxyl esterases were probably located in the salivary glands, hemolymph and fat body, and functioned in digestion, pesticide hydrolysis and JH control. No clear case of pesticide resistance was uncovered in this study. A Diazinon-resistant choline esterase possessed by strain C was correlated to a slight selective advantage for strain C over other strains when exposed to Diazinon, but this enzyme did not increase the animal's chance of survival during treatment. Enzyme assays indicated that E. candida carboxyl esterases were resistant to a number of pesticides 12.31552) which is also true for pterygote carboxyl esterases. Several carboxyl esterases in E, candida exhibited cylic changes 111 in titer or activity correlated to molt cycle which suggested that these enzymes function in the control of JH levels. Class, substrate specificity and inhibition characteristics of these esterases were similar to those of esterases involved in the control of JH in pterygote species. The precise timing of esterase activity in E. candida supports the hypothesis that these esterases limit JH levels in apterygotes in the same manner as in pterygotes. Because apterygote species show no great morphological changes at the onset of reproduction, they have been considered ametabolous (showing no metamorphosis) and neotenous (reproducing in the juvenile state). Many apterygotes continue to molt and reproduce throughout their "adult" life, unlike pterygotes in wich metamorphosis to the adult state coincides with the last molt. In pterygotes, both ecdysone and JH surges are necessary for egg production (maturation of oocytes and provisioning), and these insects generally produce only one large egg batch. The surge in these two hormones, which normally cause a molt in the larval stage, have no effect on the adult cuticle, which has already undergone final differentiation. In apterygotes, by contrast, these hormones continue to have the same effect on cuticle as in younger stages, i.e., retention of the ”juvenile" culticle, and lack of wings. Since JH titer has never reached a critically low level during molting (at least according to the current theory on ametabolous deve10pment), it is possible that no permanent differentiation to the adult form has occurred. JH and ecdysone continue to fluctuate in the adult and provide signals for vitelogenesis and egg maturation. Sequential egg production could be a consequence of hormonal control of molting, or molting may be only 112 a side effect of hormonal control over reproduction. In any case, the processes of molting, reproduction and lack of dramatic metamorphosis are intimately related in E, candida. In conclusion, the esterase system in E, candida cannot be called ”primitive", either in terms of isozyme variability, properties, class or proposed function within this apterygote species. Apterygote esterases were comparable to pterygote esterases in every respect addressed in this study. Additional enzyme systems in apterygotes must be investigated, however, before comparisons of many cellular processes between apterygotes and pterygote species can be undertaken. 113 SUMMARY 1. Esterase enzymes were studied in ten strains of E. candida. Four distinctly different electrophoretic zymograms were uncovered and described. 2. Each zymogram consisted of at least ten esterase bands which belonged to three groups: slow-migrating carboxyl esterases, choline esterases of moderate mobility and fast-migrating carboxyl esterases. Choline esterases could not be divided into acetyl and butyl choline esterases. No aryl or acetyl esterases, or lipases, were identified in E, candida. 3. Isozymes within each of the three enzyme groups behaved similarly when treated with substrates, inhibitors and pesticides. Differential mobility of isozymes among esterase patterns was not correlated to variation in enzyme action. An important exception was noted for strain C, in which one choline esterase was resistant to 10-1000 times greater pesticide concentrations than corresponding esterases in other strains. 4. Esterases were present in the eggs of E. candida from time of oviposition onwards. Although amounts of each enzyme were probably lower in early stages, electrophoresis revealed that all esterase enzymes were present throughout embryonic development. 5. Esterases were non-randomly distributed between the head and the remainder of the body (trunk). Major choline esterase activity appeared in head homogenates, possibly in association with neural transmission in sub- and supra- esophageal ganglia. Several choline esterases apeared to be minor contributors to esterase activity in trunk homogenates. These were tentatively identified as 114 choline esterases of the ventral nerve cord. 6. Carboxyl esterases were primarily located in the trunk region and were theorized to function in digestion and cleavage of esters in the hemolymph. Consistent with the CBEs of othe insect species, CBEs in .E' candida were able to degrade ester pesticides i3 vitro. 7. Several fast-migrating carboxyl esterases showed changes in staining intensity correlated to molt cycle stage, i.e., high activity between molts, low activity immediately prior to molting. This phenomenon was termed physiological variation. All the properties determined for these esterases in E, candida were so similar to those of JH esterases in pterygotes that it was suggested that cycling esterases control JH levels in E, candida. A method by which esterases would regulate JH titer was discussed. 8. Treatment of E. candida strains with Diazinon-contaminated yeast indicated that mortality was dependent on degree of nutritional stress, i.e., amount of feeding prior to treatment initiation. Diazinon (1000 ppm) caused reduced oviposition, increased instar durations, reduction in feeding and accumulation and persistence of cast exuvia. At higher concentrations (2000 ppm), it caused food avoidance behavior, permanent antennal damage and high mortality. 9. Although pesticide effects were significant, as were strain effects (p<0.001) there was no interaction between them for the parameters analyzed. In general, pesticide treatment caused equivalent effects of all strains, although actual means differed between strains. Attempts to relate strains of similar esterase pattern to their reaction to pesticide treatment indicated that there was no over-all correlation between esterase pattern and strain 115 biology. 10. Strain C appeared to have a selective advantage when compared to other strains of g, candida. Increased oviposition and molting frequency and increased feeding gave individuals of strain C a better chance to increase population size, even during Diazinon treatment. Previous data on strain C's faster growth rate and greater egg production substantiated this advantage. The slightly higher mortality observed for Diazinon treated adults of Strain C would reduce adult numbers but would not affect the selective advantage conferred to strain C by a shorter generation time and higher reproductive rate. Strain C's advantage was correlated to its possession of a Diazinon resistant esterase isozyme, but a causal relationship could not be proven without further research. 116 IMPROVEMENTS AND FUTURE WORK This investigation dealt with esterase variation, isozyme properties and the contribution of these enzymes to the biology 0f.E° candida. More work is necessary to define the precise function of each esterase isozyme. Research on esterases in Collembola has indicated that these enzymes may be useful in resolving taxonomic problems. This study should be extended to other arthropods to elucidate biochemical relationships within and between groups, and to supplement current phylogenies based on morphological characteristics. Esterases have also been implicated in organOphosphate resistance shown by several insect species. Further investigations should examine the degree to which the complement and properties of isozymes within organisms can be used to predict the effectiveness of pesticide application on populations in the field. In the present study, Diazinon caused a delcine in survivorship, growth and oviposition frequency in_§. candida, effects which appeared to be strain - dependent. 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Enzyme Commission Common Name Source if purchased Designation* ............ from Sigma 3 Hydrolases 3.1 Acting on ester bonds 3.11 Carboxylic ester hydrolases 3.1.1.1 Carboxylesterase (B) Hog liver 3.1.1.2 Arylesterase (A) 3.1.1.3 Lipase 3.1.1.4 Phospholipase A 3.1.1.6 Acetylesterase (C) Orange peel 3.1.1.7 Acetylcholine esterae (ACE) Bovine 3.1.1.8 Pseudocholine esterase (BCE) Erythrocyte 3.1.1.10 Atropine sulfate esterases Horse serum 3.1.1.11 Pectinesterase 3.1.1.12 Vitamin A esterase 3.1.1.13 Cholesterol esterase 3.1.1.14 Chlorophyllase *Barman, 1969. APPENDIX II. 126 Naphthyl Esters used in Substrate Studies. length of Carbon Chain 2 3 10 12 14 Alpha Ester Beta Ester acetate acetate propionate propionate butyrate butyrate valerate valerate caproate caprylate caprate laurate myristrate APPENDIX III. Common or trade name -.--.---- . ----. Abate Aminocarb Atrazine Bromphos methyl (Nexion) Carbary1(Sevin) Carbofuran CMPS Chlordimeform Cygon(dimethoate) Diazinon Dichlorovos Dipterex (Trichlorfon) Dursban (Chlorpyrifos) Dyfonate (S) Dyfonate (O) Fenitrothion Guthion (Azinophosmethyl) Ma lao xon Malathion Mexacarbate (Zectran) Naled Paraquat Parathion,ethy1 Parathion, methyl PMSF Reldan (Chlorpyrifos,M) Ronnel 127 List of Pesticides Used in Inhibition Studies. 0,0,0,0'-tetramethy1 0,0'-(thio-di-p-phenylene) diphosphorothioate 4-dimethy1amino-m-toly1 methylcarbamate 2-chloro-4-(ethylamino)-6-(isopropy1amino)-1,3,5- triazine O-(4-bromo-2,5-dichloropheny1)0,0-dimethylphos- phorothioate 1 napthyl N-methylcarbamate 2,3-dihydro-2,2-dimethylbenzofuran-7yl methylcar- bamate chloromethyphenylsulfonic acid N'-(4-chloro-o-toly1)-N,N-dimethylformamidine 0,0-dimethy1 S-(N-methylcarbamoylmethyl) phospho- rodithioate 0,0-diethy1 0-(2-isopropyl-6-methy1-4-pyrimidinyl) phosphorothioate 2,2-cichloroviny1 dimethyl phosphate dimethy1(2,2,2-trichloro-1-hydroxyethyl) phosphate 0,0-diethy1 0-(3,5,6-trichloro-2-pyridy1) phospho-rothioate O-ethyl S-phenyl ethylphosphonodithioate O-ethyl S-phenyl ethylphosphonothioate 0,0-dimethy1 0-(4-nitro-m-toy1) phosphorothioate 0,0-dimethyl phosphorodithioate S-ester with 3-(mercaptomethyl)- 1,2,3-benzotriazin-4(3H)-one 0,0-dimethyl S-[1,2-di(ethoxycarbony1)ethy1] phosphorodithiate diethyl mercaptosuccinate, S-ester with 0,0-dimethy1 phosphoroditioate 4-dimethylamino-3,5-xy1y1 methylcarbamate 1,2-dibromo-2,2-dichloroethy1 dimethyl phosphate 1,1'-dimethy1-4,4'-bipyridium dichloride 0,0-diethyl-O-p-nitrophenyl phosphorothioate 0,0-dimethyl-O-p-nitrophenyl phosphorothioate phenylmethylsulfonyl flouride 0,0-dimethyl 0-(3,5,6-trichloro-2-pyridy1) phosphorothioate 0,0-dimethyl 0-92,4,S-trichlorophenyl) phosphorothioate APPENDIX III. 128 Structure of Pesticides Used in Inhibition Studies. a, J? Phosphates R /,P~Ra Thiophosphates 1 Name R1 R2 R3 ,ou Dipterex OCH3 OCH3 -CH-CC15 (trichlorfon) Br Naled 00113 00113 - 0-CH-CCiz3r Dichlovos OCH3 OCH3 -o—-CH=CC|02_ ,cug-Cflrcedr Ma lathion mm 001;, —s-— c— c: O~Cz“5 C513" RQO'CLHS' Malaoxon OCH3 OCH3 .— 5-é\c¢— 84.2.145- c> Dursban OC2H5 OCZHS _O%}.u_ CL Re ldan OCH3 OCH3 .0% Di 1 0c 3 cc 3 .. ~ ’ ‘ 82 non 2 5 2 5 0 CHCCHs); Dyfonate C2H5 OCZHS ’S‘C:> Dyfonate (ox) C2H5 OCZHS -54c:> Bromphos CH3 Ronnel Parathion (methyl) Parathion (ethyl) Fenthrothion Carbamates Mexacarbate (Zectran) Aminocarb Carbaryl (Sevin) Carbofuran C) i 0033 0053 PE? OCH3 OCH3 "0@-NOL OC2H5 OCZHS -0-@’“°z 0033 00235 -o 02 C"; a 9 CH3-N-C-R C“: 12‘: —o@'NCCH3)z “a as C“! 129 APPENDIX IV Cross classified Factorial model with Double Nesting (after Gill, 1978). The data for persistence of exuvia, instar duratin and fasting duration were analyzed using the model: Y = A1 + 31 + (AB)ij + E(ij)k + U(ijk)1 where: Y - the observed value Ai - the effect of the treatment Bj - the effect of the strain (AB)ij - the interaction between treatment and strain E(ij)k - the effect of the animal (nested in the strain treatment combination U(ijk)l - the repeated observation (nested in the animal-drops out without replication) For this model the F-ratios are calculated using the following mean squares: treatment effects F - (MS treatment)/(MS animal) strain effect F - (MS strains)/(MS animal) interaction F a (MS interaction)/(MS animal) error F - (MS anima1)/(MS observation) 130 APPENDIX V. Migration Distance (mm) of Esterase Isozymes of four Strains of E. candida in Polyacrylamide Gels. Isozyme STRAIN Band A B C D 1 .74 .74 .74 .74 3 7.4 7.4 5.9 7.4 4 8.9 8.9 9.6 8.9 5 10.4 11.1 11.8 10.4 6a 12.6 - - 12.6 6d - 14.8 14.8 - 7 25.9 25.9 25.9 25.9 10 37.0 35.5 34.0 35.5 11 40.7 28.5 37.0 41.4 12 48.8 - 44.4 48.8 13 53.3 - - 53.3 14 - 56.2 56.2 - Dye front 110.0 131 APPENDIX VI. Number (out of 32/samp1e) and 2 Strain A F, candida with Detectable Esterase 5 and 6 Activity. 6 hr Interval Esterase 5 Esterase 6 Phase 2 Number Percent Number Percent 0-6 9 28.1 11 34.4 6-12 2 6.3 0 0.0 12-18 0 0.0 2 6.3 18-24 0 0.0 20 62.5 24-30 0 0.0 23 71.9 30-36 1 3.1 29 90.6 36-42 5 15.6 31 96.9 42-48 21 65.6 32 100.0 48-54 22 68.7 32 100.0 flw‘-‘-‘--'.“ “‘ APPENDIX VI. 6 hr Interval Esterase 5 Phase 2 Nmnber 0-6 22 6-12 7 12-18 1 18-24 1 24-30 0 30-36 2 36-42 8 42-48 25 48-54 26 -~.--.‘ -- -- -‘+o.—«“d¢--«“‘*‘.“w-‘9 m“ 132 Percent ---. C-“-‘ Q—O‘-—«-.‘-. -- u. o - O O O O 68.8 21.9 3.1 3.1 0.0 6.3 25.0 78.1 81.3 with Detectable Esterase 5 and 6 Activity. Number 9 3 7 24 28 32 32 32 32 Esterase 6 Number (our of 32/sample) and Z Strain D_§, candida Percent O ~O—oflm-‘ -‘-- 28.1 9.4 21.9 75.0 87.5 100.0 100.0 100.0 100.0 133 APPENDIX VII. Average Exuvia per Jar in Strains of_§. candida in Control and Diazinon Treated Jars (1000 ppm) (n=20 jars/treated, 5 jars/control, 5 animals/jar). Day of Strain of F. cancica Treatment Strain I Strain J control 1000 ppm control 1000 ppm ..... mean SE mean SE mean Se mean SE 7 0.4 .22 1.2 .17 0.8 .34 1.0 .26 8 0.6 .22 2.1 .25 1.4 .46 1.2 .32 9 0.6 .36 2.4 .25 0.6 .54 1.2 .34 10 0.2 .18 2.3 .28 1.0 .40 1.6 .32 11 0.4 .22 2.3 .21 0.8 .34 2.3 .28 12 0.4 .36 2.0 .25 2.0 .56 2.6 .29 13 0.6 .36 1.8 .21 1.6 .46 3.5 .18 14 1.0 .49 2.5 .30 2.6 .46 3.4 .22 15 1.2 .71 3.4 .16 2.6 .36 2.8 .24 16 0.4 .36 3.0 .38 1.4 .78 3.1 .27 17 1.0 .40 3.2 .32 1.5 .58 3.4 .28 18 1.6 .46 3.3 .25 1.6 .36 3.6 .30 19 1.2 .52 4.2 .36 1.8 .33 4.1 .31 20 1.4 .54 4.2 .25 0.6 .36 4.1 .33 21 1.0 .49 7.0 .22 0.0 0.0 4.1 .33 «W---OQOOo-O-Cor--------------00---. -. 134 APPENDIX VII. Average Exuvia per Jar in Strains of F. candida in Control and Diazinon Treated Jars (1000 ppm) (n=20 jars/treated, 5 jars/control, 5 animals/jar) Day of Strain of E. candida Treatment Strain A Strain C control 1000 ppm control 1000 ppm ..._--_----J¥¥¥l SE mean SE .353? SE mean SE 7 3.6 .82 3.5 .21 1.0 .71 1.65 .28 8 3.8 .43 4.0 .17 0.4 .34 2.6 .34 9 2.6 .60 4.8 .14 0.2 .17 2.2 .35 10 2.6 .67 4.6 .15 0.4 .36 2.0 .32 11 1.8 .82 4.3 .21 0.2 .18 1.8 .30 12 2.8 .80 5.1 .24 0.6 .22 2.3 .28 13 3.2 .18 6.4 .27 0.8 .44 2.2 .22 14 3.8 .44 6.9 .22 1.4 .60 2.0 .26 15 4.4 .36 7.6 .37 0.6 .54 2.5 .32 16 2.2 .72 7.7 .41 0.0 0.0 3.1 .45 17 2.1 .42 8.3 .32 .1 .09 2.0 .44 18 2.0 .27 8.9 .26 0.2 .18 2.0 .44 19 2.4 .73 9.0 .26 0.0 0.0 2.1 .37 20 2.0 .28 9.1 .30 0.0 0.0 2.3 .34 21 3.4 .67 8.7 .43 0.2 .18 2.3 .29 -----‘----“-‘—.“‘---- 0 Q 0-.-- .‘-‘-.-¢.-----.‘-C-.- O O o----¢Qu.--‘-“--.““ 135 APPENDIX VII. Average Exuvia per Jar in STrains of F. candida in Control and Diazinon Treated Jars (1000 ppm) (n-20 jars/treated, 5 jars/control, 5 animals/jar). Day of Strain of E. candida Treatment Strain B Strain control 1000 ppm control 1000 ppm *--- mean--- ‘SE mean_y SE mean Se mean SE 7 0.80 .34 1.1 .19 8 1.4 .44 2.2 .25 9 2.2 .46 2.4 .24 10 2.8 .33 3.1 .22 11 2.8 .59 2.9 .29 12 2.2 .82 3.0- .29 13 0.8 .33 2.8 .27 14 1.6 .33 2.3 .27 15 1.8 .67 2.3 .27 16 1.8 .86 2.9 .30 17 1.7 .85 4.2 .33 18 1.6 .83 4.5 .35 19 1.8 .72 4.7 .41 20 1.4 .61 4.6 .46 21 1.2 .44 4.3 .44 136 APPENDIX VII. Average Exuvia per Jar in Strains of F, candida in Control and Diazinon Treated Jars (1000 ppm) (n=20 jars/treated, 5 jars/control, 5 animals/jar). Day of Strain of F. candida Treatment Strain G Strain H control 1000 ppm control 1000 ppm mean_ SE mean SE mean Se mean SE 7 0.8 .52 2.2 .29 1.0 .45 0.7 .33 8 0.4 .22 2.4 .28 2.6 .60 3.4 .29 9 0.8 .52 2.7 .37 2.6 .54 3.5 .28 10 0.2 .18 2.2 .34 2.0 .57 3.3 .21 11 0.2 .18 1.9 .33 1.4 .46 3.4 .23 12 0.2 .18 2.7 .31 0.4 .22 3.5 .29 13 0.0 0.0 4.0 .25 0.0 0.0 3.6 .31 14 1.6 .61 4.0 .33 0.0 0.0 3.7 .28 15 1.2 .72 3.4 .16 0.6 .75 4.0 .25 16 1.2 .66 4.3 .40 0.2 .18 3.4 .40 17 0.8 .50 4.2 .37 0.1 .09 3.8 .20 18 0.4 .36 4.0 .35 0.0 0.0 4.1 .30 19 0.4 .36 4.1 .37 0.4 .36 4.2 .36 20 0.0 0.0 5.3 .48 0.0 0.0 4.3 .41 21 0.2 .18 4.3 .41 0.2 .18 4.1 .38 M------¢----‘4C--.—.‘-‘-“‘-O —-‘----‘-&-‘ 137 APPENDIX VII. Average Exuvia per Jar in Strains of E. candida in Control and Diazinon Treated Jars (1000 ppm) (n=20 jars/treated, 5 jars/control, 5 animals/jar). Day of Strain of E. candida Treatment Strain D Strain E control 1000 ppm control 1000 ppm *------~--__mean SE mean EE--JT%¥1....J¥E--J¥¥¥1 SE 7 1.2 .44 2.2 .21 0.0 0.0 2.4 .27 8 2.4 .67 3.25 .28 1.2 .44 2.9 .24 9 2.0 .56 3.7 .25 0.2 .18 2.6 .26 10 2.0 .63 4.2 .19 0.8 .33 2.0 .20 11 1.2 .44 4.2 .22 2.2 .52 2.3 .26 12 1.4 .54 4.1 .21 2.2 .34 2.5 .28 13 1.0 .56 4.2 .27 2.8 .18 3.0 .16 14 2.8 .34 4.2 .29 2.2 .44 3.6 .23 15 2.6 .82 4.3 .25 2.6 .72 4.7 .35 16 2.2 .59 4.6 .38 1.4 .46 5.3 .44 17 1.8 .51 5.5 .39 1.0 .30 4.8 .42 18 1.4 .44 6.4 .40 0.6 .34 4.3 .41 19 2.0 .63 6.5 .38 1.0 .40 4.1 .37 20 2.2 .66 6.7 .41 0.8 .50 5.0 .39 21 1.8 .44 6.0 .40 0.8 .33 5.2 .36 APPENDIX VIII. 138 Mortality (as number dead/total number) for Diazinon Treated and Control Individuals after 21 days. JUVENILES STRAIN 28/121 11/104 9/82 7/116 27/107 15/118 71.147 12/178 17/110 14/195 2/24 6/21 1/20 0/19 5/21 5/30 8/23 3/37 3/23 5/53 Trial 1 43/100 3/100 22/100 31/100 29/100 na 7/100 6/100 15/100 5/100 treated control treated control ADULTS Trial 2 Trial 3 treated control treated_control 0/25 0/25 0/25 3/25 0/25 na 2/25 1/25 2/25 2/25 3/100 28/100 4/100 22/100 10/100 na 1/100 0/100 1/100 4/100 0/25 5/25 0/25 0/25 0/25 na 0/25 0/25 1/25 0/25 na 1/25 2/40 12/40 10/25 na 1/30 0/30 5/25 0/25 na 2/25 1/40 16/40 10/25 na 1/30 0/30 5/25 2/25