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"""""' 5"“ '5‘“ ‘I'. 2'" I“ ””222 : - .2'-— I 22I THY-5‘s This is to certify that the thesis entitled COMPARATIVE PHYSIOLOGICAL STUDIES OF SCHISTOSOMA JAPONICUM AND SCHISTOSOMA MANSONI presented by Timothy C. Martin has been accepted towards fulfillment of the requirements for Ph. D. dggree in 200109,), Major professor __ (7, Date /MW 1) 04639 ”mum Michigan state University MSU LIBRARIES .25.!!!— iv RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped beIow. ‘leoww COMPARATIVE PHYSIOLOGICAL STUDIES OF SCHISTOSOMA JAPONICUM AND SCHISTOSOMA MANSONI By Timothy C. Martin A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology l982 ABSTRACT COMPARATIVE PHYSIOLOGICAL STUDIES OF SCHISTOSOMA JAPONICUM AND SCHISTOSOMA MANSONI By Timothy C. Martin Physiological comparisons of Schistosoma japonicum and Schistosoma 5 mansoni reveal several important differences. Ouabain (lO' M), lithium substitution and elevated K+ all increase the muscle tension of §, mansoni, but have little or no effect on the muscle tension of §, japonicum. In contrast, praziquantel (10'6 M) and low temperature (5°C) produce an equal increase in muscle tension in both species. §, japonicum and §, mansoni differ in their dependence of external Ca++ for the mediation of longitudinal muscle contraction. Muscle con- traction induced in §, mansoni by elevated K+, praziquantel, ouabain and low temperature are all attenuated by a 5 minute preincubation in media containing zero Ca++ (plus l0"4 M EGTA). Similar pretreatment of s, japonicum has no effect on praziquantel or low temperature induced contractions. The mechanical threshold for longitudinal muscle contraction is higher in §, japonicum than in §, mansoni. Microelectrode recordings showed that elevated K+ (60 mM) caused depolarization of the muscle in both parasites, but produced an increase in muscle tension only in §, mansoni. This difference in mechanical threshold appears to be due to a Timothy C. Martin specific difference in calcium permeability, as 45Ca++ accumulation in §, mansoni is nearly twice that in §, japonicum while uptake of 42K+ is the same in both species. Microelectrode recordings show that elevated K and Li+ substitution both caused muscle depolarization in _s_. M- gum_and §, mansoni, indicating that lithium and potassium ions are able to penetrate through the tegument to the level of the muscle in both parasites. The tegument appears to be a greater permeability barrier to cal- cium ions in §, japonicum than in §, mansoni. Upon removal of the tegu- ment with triton X-lOO, exposure of §, japonicum to elevated K+ produces a large muscle contraction that is similar to that observed in control §, mansoni. This contraction is dependent on the presence of Ca++ in the bathing medium. The reduced movement of Ca++ through the tegument of §, japonicum may be caused by fewer voltage sensitive calcium chan- nels or more active extrusion of Ca++ by a Ca++~Mg++ ATPase. §, £39931; cum_were less responsive to the muscle relaxing effects induced by the voltage-sensitive Ca++ channel blocker, 0-600. Enzyme analysis reveals a higher level of Ca++-Mg++ ATPase in the tegument of §, japonicum. While both parasites have an active Na+-K+ transport system, ouabain or lithium induced inhibition produced a greater degree of muscle depolarization and a larger increase in muscle tension in s, mansoni. To My Mother and Father 1'1 ACKNOWLEDGEMENTS I wish to express my appreciation to Dr. Ralph A. Pax for his guidance, helpful criticism and advice and patience during the preparation of this dissertation. I also want to thank Drs. Charles Tweedle and Gerard Gebber for serving on my guidance committee. Special thanks goes to Dr. James L. Bennett for his support and encouragement during the course of this project. I also wish to thank my colleagues in the laboratory: David P. Thompson, David R. Semeyn, Connie S. Bricker and Carla C. Siefker, for their personal advice and companionship. TABLE OF CONTENTS Page DEDICATION _______________________________________________________ ii ACKNOWLEDGEMENTS _________________________________________________ T l 1° LIST OF TABLES --------------------------------------------------- vi LIST OF FIGURES .................................................. vii INTRODUCTION ..................................................... 1 Pathology --------------------------------------------------- 3 Morphology .................................................. 3 General Anatomy ........................................ 3 Tegument ----------------------------------------------- 4 Muscle ................................................. 7 Biochemistry ................................................ 12 Pharmacology ................................................ 14 Hycanthone ............................................. 15 Metrifonate ............................................ 15 Oxamniquine ............................................ 15 Antimony ............................................... 15 Roll-3l28 .............................................. 15 Objectives .................................................. 15 MATERIALS AND METHODS -------------------------------------------- l8 Source and Maintenance of Animals --------------------------- l8 Recording Media ............................................. l8 Hank's Balanced Salt Solution -------------------------- 18 Potassium ---------------------------------------------- l9 Lithium ------------------------------------------------ 19 Calcium ................................................ 19 Mechanical Recordings --------------------------------------- l9 Microelectrode Recordings ----------------------------------- 23 Pharmacological Agents -------------------------------------- 24 Ouabain ................................................ 24 Praziquantel ------------------------------------------- 24 D-6OO -------------------------------------------------- 25 iv TABLE OF CONTENTS (continued) Page Temperature ................................................. 25 Triton X-lOO ................................................ 25 Ion Flux Studies ............................................ 26 Potassium .............................................. 25 Calcium ------------------------------------------------ 27 ATPase Assay ------------------------------------------------ 27 Statistical Procedure --------------------------------------- 28 RESULTS ---------------------------------------------------------- 29 Normal Activity --------------------------------------------- 29 Mechanical Activity ------------------------------------ 29 Electrical Activity ------------------------------------ 29 Effect of Altered Ion Concentration ------------------------- 34 Potassium .............................................. 34 Lithium ------------------------------------------------ 42 Low Temperature ............................................. 51 Pharmacological Agents -------------------------------------- 57 Ouabain ................................................ 57 D-6OO -------------------------------------------------- 67 Praziquantel ------------------------------------------- 57 Ion Accumulation -------------------------------------------- 67 Potassium ---------------------------------------------- 67 Calcium ................................................ 72 Triton Treatment ............................................ 72 Mechanical Activity ------------------------------------ 80 Electrical Activity ------------------------------------ 80 Potassium .............................................. 80 Calcium ................................................ 88 Ouabain ------------------------------------------------ 88 Lithium ------------------------------------------------ 88 Ca++-Mg++ ATPase -------------------------------------------- 95 DISCUSSION ------------------------------------------------------- 99 Calcium Permeability ---------------------------------------- 99 Regulation of Calcium --------------------------------------- l03 Active Transport -------------------------------------------- lOS Significance of the Physiological Differences --------------- lO7 SUMMARY .......................................................... 109 BIBLIOGRAPHY ----------------------------------------------------- 111 Table LIST OF TABLES The effects of elevated K+ concentrations on the mechanical and electrical activity of S, japonicum and S, mansoni ----------------------------------------- A comparison of the effects of LiCl HBS on the mecha- nical and electrical activity of S, japonicum and S, mansoni ------------------------------------------------ A comparison of the effects on ouabain, low temperature D-600, zero Ca++ and praziquantel on the mechanical activity of S, japonicum and S, mansoni ---------------- A comparison of ion uptake in S, japonicum and S, mansoni ................................................ A comparison of normal and triton-treated S. japonicum; the effects of ouabain, LiCl H35 and 60 mill—"K+ on mechanical activity ------------------------------------ A comparison of Ca++ and/or Mg++ dependent enzymes in S, japonicum and S, mansoni ---------------------------- vi Page 35 54 60 79 83 98 Figure 10 ll 12 l3 l4 LIST OF FIGURES Scanning electron micrograph of paired S. japonicum and S. mansoni ------------------------------ ' .. """ . . .. ..... Scanning electron microscopy comparing the tegumental surface 0f S. japonicum and S. mansoni ----------------- Transmission electron microscopy of tegument, muscle and extracellular space of male S. mansoni injected with HRP ----------------------------------------------- Schematic diagram of apparatus used to make mechanical recordings from male S, japonicum and S, mansoni ------- Sample chart recordings of spontaneous contractile activity from schistosomes ............................. Potential profile obtained while penetrating into the dorsal surface of an adult male S. japonicum with a microelectrode ----------------------------------------- Chart recordings comparing the effects of elevated K+ on the muscle tension of S, japonicum and S, mansoni--- The effects of 60 mM K+ on the muscle tension of schistosomes ........................................... . . + The effects of various concentrations of K on muscle tension ................................................ The effects of various concentrations of K+ on E1 and E2 of schistosomes ..................................... Time course of effect of 60 mM K+ on E1 and E2 in S. japonicum .............................................. Chart recordings comparing the effects of LiCl HBS on muscle tension of S, japonicum and S, mansoni ---------- The effects of l38 mM Li+ on the muscle tension of schistosomes ........................................... Time course of effect of 138 mM Li+ on E1 and E2 in japonicum ........................................... Page 10 20 3O 32 36 38 4O 43 45 47 49 52 LIST OF FIGURES (continued) Figure 15 l6 l7 l8 T9 20 2T 22 23 24 25 26 27 28 29 30 The effect of lowered temperature on the muscle tension of schistosomes ........................................ The effect of zero Ca++ on the low temperature induced muscle contracture in schistosomes ..................... Chart recordings comparing the effects of ouabain on muscle tension of S. japonicum and S mansoni ---------- The effects of ouabain on the muscle tension of schistosomes ........................................... Time course of the effect of ouabain on El and Ezin japonicum ........................................... The effect of 0-600 on the muscle tension of schisto- somes .................................................. The effect of zero Ca++ on the praziquantel induced muscle contraction in schistosomes ..................... 42K+ by male schistosomes over one hour ------ 42K+ by male schistosomes over two hours ----- Uptake 0f 45Ca ++ by male schistOSOmes .................. Uptake of Uptake of Scanning electron microscopy showing the effect of triton X- lOO treatment on the tegument of S. japonicum- Chart recordings showing the effect of 60 mM K on the muscle tension of normal and triton- treated S. japo- nicum -------------------------------------------------- The effect of 60 mM K+ on the muscle tension of normal and triton- treated S. japonicum ........................ Chart recording of the effect of zero Ca+ +HBS, 60 mM K+ H88 and l. 4 mM Ca++ on the muscle tension of a triton- treated S. japonicum ---------------------------- Chart recording showing the effect of l4 mM Ca ++ on the K+ contracture of triton- treated S. japonicum ---------- The effect of ouabain on the muscle tension of normal and triton- treated S. japonicum ........................ viii Page 55 58 61 63 65 68 7O 73 75 77 BI 84 86 89 91 93 LIST OF FIGURES (continued) Figure Page 31 The effect of Li+ on the muscle tension of normal and triton-treated S, japonicum ---------------------------- 96 32 Mechanical threshold for the activation of muscle contraction in S, japonicum and S, mansoni ------------- lOl ix INTRODUCTION Schistosoma japonicum and S, mansoni are two medially important trematode parasites of man. It is estimated that these parasites infect well over 500 million people. S, japonicum is endemic to Southeast Asia, China, Japan and the Phillipines, while S, mansoni is distributed throughout most of Africa and parts of South and Central America. S, japonicum and S, mansoni belong to the same genus and are simi- lar in several respects. Superficially adults appear to be similar though S, japonicum is somewhat larger and is devoid of the tegumental spines characteristic of S, mansoni. The complicated life cycle of the two parasites is almost identical. Free swimming cercariae pene- trate the skin of the human host and migrate through the body. Both parasites finally develop to maturity in the mesenteric veins and portal system. Females then lay large numbers of eggs, some of which penetrate the intestinal wall to be expelled in the feces (Noble and Noble, l976). In general, S, japonicum causes more pathology than S, mansoni. While the parasites appear to be quite similar in some respects, there are several important differences between them. The host- parasite interface, i.e. the tegumental surface, is quite different in the two worms. The tegument of S, mansoni has spines and bosses while 2 the tegument of S, japonicum lacks both of these structures and in- stead is composed of a multifolded membrane which gives its surface a sponge-like appearance (Sakamoto and Ishii, 1977). Both parasites depend primarily on anaerobic respiration and both utilize many of the same sugars and amino acids for energy, but recent evidence indi- cates that the TCA cycle and electron transport may be more important in the metabolism of S, japonicum than in S, mansoni (Huang, l980). S, japonicum is resistant to several antischistosomal drugs which are quite effective against S, mansoni. Three of these drugs, hycan- thone, metrifonate and Roll-3l28 are thought to act by interfering with normal nerve or muscle function. While this would suggest that the two parasites may differ with respect to nerve or muscle function, virtually no work has been undertaken to determine the underlying physiological cause or causes for these differences in drug sensiti- vity. There have been no previous attempts to compare the basic physio- logy of these two schistosomes. This is due in part to the prevalent belief that an understanding of the physiology of S, japonicum can be inferred from studies of other species of human schistosomes. It was also only recently that techniques were developed which could quanti- tatively measure mechanical and electrical activity in schistosomes. Using these techniques, I have undertaken studies comparing S, 332931; ggm_and S, mansoni in the hope that these studies might help to clarify the underlying causes for the differential sensitivity of these two parasites to various antischistosomal drugs. Pathology Most of the pathology associated with schistosomiasis is caused by the deposition of parasite eggs in the host's tissue and the subse- quent immune response. Female worms release eggs into the fine mesen- teric vessels of the small and large intestine. Approximately half of the eggs penetrate the intestinal wall to be passed in the feces (Kang and Fan, l973). Under proper conditions these eggs hatch into miracidia and penetrate the skin of an aquatic snail, thus maintain- ing the parasite's life cycle. Eggs which are not expelled in the feces are washed into the liver, or become lodged in the intestinal wall, forming granulomas. It is this pathology which is responsible for the symptoms of schistosomiasis; i.e., diarrhea, abdominal pain, anemia and spleen and liver enlargement. While the etiology and symptomology of S, mansoni and S, jgpggj; ggm_infections are the same, there is a difference in the severity of disease caused by the two parasites. Infections caused by S, Sgpggj; 93m are more destructive than those caused by S, mansoni. There are two reasons for this. First, each female S, japonicum can lay ten times the number of eggs laid by a female S, mansoni (Kang and Fan, 1973). Second, the tissue reaction to the ova of S, japonicum is more acute than the reaction to S, mansoni eggs. The immune response to S, japonicum eggs occurs more rapidly and appears to be a general- ized reaction. S, mansoni eggs cause a delayed but more specific immune response (Smither, l972). Morphology- General Anatomy. Adult male S, japonicum average 15 mm in length, while the adult male S, mansoni average l0 mm in length. Both 4 parasites are about l mm wide. Female S. japonicum and S, mansoni are longer, 20 mm and 16 mm respectively, and thinner, approximately 0.2 mm wide. The males of both species have a groove, the gynecophoral canal, along most of their body length. The female usually lies within this groove (Figure l). Much of the general anatomy is the same for the two male schisto- somes. Both species are covered with a tegumental epithelium. The tegument covers the parasite's outer circular and inner longitudinal muscle layer. Beneath the muscle layers, the nephridia, testes and intestinal cecae are suspended in a parenchyma matrix. The nervous system of both species consists mainly of two paired circumesopha- geal ganglia and four lateral nerve trunks. Small fibers from the lateral nerve trunks branch to innervate muscle bundles and tegumental sensory receptors (Silk and Spence, 1969). Tegument. The body surfaces of both S, japonicum and S, mansoni are covered by an anuclear tegument. The dorsal tegument of male worms is usually from 3 to 5 microns in thickness, while the ventral tegument is relatively thin, measuring only l to 3 microns. The tegu- ment is composed of many dense secretion granules, vacuoles of varying size and a few mitochondria, which originate in underlying nucleated epithelial cells. Protoplasmic channels connect the tegument with the nucleated epithelial cells which are located beneath the muscle layers. In contrast to the tegument, the epithelial cells of both schistosomes, contain many mitochondria (Morris and Threadgold, 1968; Inatomi e£_gl,, 1970). S, japonicum and S, mansoni have small (1x4 um) crystalloid spines embedded in their tegument. These spines are distributed over the Figure 1. Scanning electron micrograph of paired S, japonicum and S, mansoni. Female is lying within the gynecophoric canal of the male. OS, oral sucker; VS, ventral sucker; GC, gynecophoric canal; F, female; M, Male. Calibration, 0.6 mm. Kindly supplied by D. P. Thompson, Dept. of Zoology, Michigan State University. S. japonicum " ' A S. mansoni Figure 1 7 entire tegumental surface of S, mansoni, while the spines are located only near the ventral sucker and in the gynecophoral canal of S, japonicum. The dorsal tegument of S, mansoni is also invested with numerous tegumentary bosses, which are not found on S, japonicum. Scanning electron micrographs (4000X, kindly supplied by C. S. Bricker) show that except for the spines, bosses and what appear to be small sensory bulbs, the tegument of S, mansoni is smooth (Figure 2). S, japonicum also has the small sensory bulbs, but the actual tegumental surface is quite different from that of S, mansoni. S, japonicum's tegumental surface is composed of many complicated ridges and depressions, giving it a sponge-like appearance. The complexity of the tegumental surface of S, japonicum actually increases its surface area, relative to S, mansoni (Sakamoto and Ishii, 1977; Voge et_gl,, 1978; Ma and He, 1981). Recent work by Fetterer £3.21: (1981a) and Bricker gt_gl, (1982) has shown that a distinct electrical potential can be recorded across the outer tegumental membrane of S, mansoni. This potential has a value of -35:7.4 mv across the ventral tegument (Fetterer g§_§l, 1981) and -51:O.6 mv across the dorsal tegument (Bricker et_§l,, l982). Iontophoretic injection of horseradish peroxidase (HRP) when these large potentials are recorded, show that the electrical potential originates from the tegument and associated structures; i.e., tegu- mental cytons and cytoplasmic channels (Panel A, Figure 3). Mggglg, The muscle of S, japonicum and S, mansoni appears to be smooth muscle, having no striations. The arrangement of this muscle, i.e. circular, longitudinal and dorsal-ventral, is the same in the two parasites. Circular muscle is located beneath the tegument with the Figure 2. Scanning electron microscopy comparing the tegumental surface of S, japonicum and S, mansoni. A and B show low magnifica- tion (6OOX) of the tegument of S. ja onicum and S. mansoni, respec- tively. C and D show high magnification (4000X)_bf the tegument of S, japonicum and S, mansoni, respectively. SB, sensory bulbs; S, spine; 8, bosses. Calibration bar for A and B, 13 uM; calibration bar for C and D, 2.5 uM. Kindly supplied by C. S. Bricker, Dept. of Zoology, University of Vermont. Figure 2 10 Figure 3. Transmission electron microsc0py of tegument, muscle and extracellular space of male S, mansoni injected with HRP. A, injec- tion of HRP into dorsal tegument and associated structure (E1 = -51:O.6 mv). B, longitudinal muscle and C, circular muscle injected with HRP (E2 = -10+0.5 mv). T, tegument; C, tegumental cyton; LM, longitudinal muscle; CM, circular muscle, ES, extracellular space. Calibration, 8 microns. Kindly supplied by C. S. Bricker. ll Figure 3 12 fibers arranged perpendicular to the long axis of the body. Longitu- dinal muscle lies below the circular muscle and is oriented in an anterior-posterior direction. The dorsal-ventral muscle extends through the body of the worm in a dorsal-ventral fashion. The muscles are surrounded by fine collagen-like fibers of connective tissue. Detailed information about muscle structure is available only for S, mansoni (Silk gt_gl,, 1969). In this parasite, the cell body of the muscle fiber is located below the fibers at the same level as the tegumental cytons. The muscle cell body connects to the muscle fiber by cytoplasmic processes. While there is a poorly defined sarcoplasmic reticulum in the muscle of S, mansoni, no one has demon- strated that a sarcoplasmic reticulum is present in the muscle of S, japonicum. As previously described, when a microelectrode is advanced into S, mansoni, the large tegumental (E1) potential is encountered. Further advancement of the microelectrode shows that another, less negative potential (E2) lies below the tegument (Bricker §£_gl,, 1982). This potential has a value of -28:D.6 mv. Iontophoretic injection of HRP shows that this potential originates from longi- tudinal and circular muscle (Panel B and C, Figure 3). Biochemistry Adult schistosomes have a functional digestive tract, the surface of which is modified for absorption. It is generally believed that the adult parasite ingests erythrocytes along with small organic com- pounds circulating through the host's portal system (Read, 1972). Uptake of these smaller nutrient molecules can also take place through 13 the tegument. In S, mansoni, trans-tegumental uptake of a variety of sugars and amino acids had been demonstrated (Cornford and Oldendorf, 1979). Indirect evidence for the tegumental uptake of sugars and amino acids in S, japonicum comes from experiments which show that these compounds are metabolized to C02 at rates that approximately equal to that in S, mansoni (Bruce 3} S1,, 1972, 1974). Metabolism of carbohydrates is the major source of energy for adult S, mansoni and S, japonicum (Read, 1972; Bruce et_§l,, 1974). While both parasites metabolize glucose, fructose, mannose and glucosamine, there are species differences in the amount of catabolism of each of these sugars. For example, male S, japonicum will produce approximately equal amounts of CO2 from glucose, fructose and mannose, while male S, mansoni produces most C02 from glucose, and lesser amounts from mannose and fructose. Less CO2 is produced from glucosamine than any of the other sugars, and the amount of glucosamine utilized by S, mansoni and S, japonicum is approximately the same. Amino acids are used by both adult S, mansoni and S, japonicum for energy production, but this energy source appears to be secondary to carbohydrate metabolism. Bruce §£_213 (1972) found that the amino acids alanine, arginine, histidine, glutamine and aspartic acid are all meta- bolized to CO2 by both schistosome species. The amino acid proline, however, was metabolized to CO2 only by S, mansoni (Bruce e§_§l,, 1972). Energy metabolism in adult schistosomes appears to be primarily anaerobic. Both parasites have active pyruvate kinase and lactic de- hydrogenase (Bueding and Saz, 1968). In the case of S, mansoni, carbo- hydrate is converted to lactic acid which is then excreted (Read, 1972). 14 Additional evidence for anaerobic metabolism comes from studies which demonstrate that the activity of TCA cycle enzymes is very low in both species (Smith and Brown, 1977a). More recent evidence by Huang (1980) shows that S, japonicum has an active TCA cycle and several electron transport proteins. Huang provides two explanations for this discrepancy. First, he suggests that electron transport and oxi- dative phosphorylation may be more important for energy production in adult S, japonicum than in adult S, mansoni. A more likely explana- tion, according to Huang, is that both species have an active electron transport and oxidative phosphorylation system, jg_yjyg, During worm collection and subsequent enzyme determination, other investigators failed to maintain appropriate oxygen tension in the incubation media, and thus caused the parasites to shift from the normal aerobic to anaerobic metabolism. Pharmacology Many of the comparative studies of S, japonicum and S, mansoni have involved the parasite's sensitivity or resistance to anthelmintic drugs. Both parasites reside in the mesenteric veins, and much of their general biology is the same. It is therefore not surprising that both parasites are equally sensitive to several antischistosomal drugs; e.g., tubercidin, praziquantel and several cyanide compounds. What is surprising, is the fact that S, japonicum is resistant to several drugs which are quite effective against S, mansoni. These com- pounds include hycanthone, metrifonate, oxamniquine, the complex anti- monials and the benzodiazepine, Roll-3128. 15 Hycanthone. The mechanism of hycanthone's action has not been clearly shown, but it may interfere with either the serotonin (5-HT) system (Chou e§_gl,, 1973) or the cholinergic system (Hillman g§_al,, 1977). The drug has also been shown to irreversibly damage the tegu- ment of S, mansoni. The tegument of S, japonicum is not affected (Hillman e£_gl,, 1977). Clinical trials have shown hycanthone to be effective in the treatment of S, mansoni infections (Goodman and Gilman, 1976). In contrast, the drug is ineffective in treatment of S, Sggggj; ggm_infestations (Yarinsky, 1972). Recent evidence that the drug may be mutagenic (Meadow et_§l,, 1973) has caused a decline in its wide- spread use. Metrifonate. This drug is an organophosphate and as such is thought to work by inactivating the acetylcholinesterases. It is the drug of choice for treatment of infections caused by S, haematobium (Blair, 1977), another human schistosome which lives in the blood vessels of the urinary bladder. Metrifonate is also effective, al- though to a lesser degree, against S, mansoni (James and Webber, 1972). It does not, however, affect S, japonicum (James and Webbe, 1974). Oxamniquine. The mechanism of action for oxamniquine is unknown. In general, S, mansoni are susceptible to oxamniquine, although differ- ent strains of this species do exhibit varying degrees of drug resis- tance. S, japonicum, however, seems to be totally resistant to oxamniquine. Antimony. The antimony compounds are thought to inhibit phospho- fructokinase, the enzyme that catalyzes the rate-limiting step in the glycolytic pathway (Goodman and Gilman, l976). Antimonial compounds were the first effective antischistosomal drugs used for the treainnent 16 of schistosomiasis. Antimony potassium tartrate and antimony sodium tartrate are effective in treatment of both S, japonicum and S, mansoni infections. Both drugs are also toxic and are responsible for a variety of side effects. More recently, a complex antimony compound has been developed which is less toxic, antimony sodium dimercapto- succinate. This new drug will cure diseases caused by both species, but is less effective in treatment of S, japonicum infections. Roll-3128. The mechanism of action for Roll-3128 is unknown. Clinical studies have shown that it is effective against S, mansoni but of little use against S, japonicum (Stohler, 1978). The jg_vitro response of the two schistosomes is also different. S, mansoni typi- cally reacts to the drug with a large muscle contracture, while S, japonicum do not contract (Pax §£_§1,, 1978). The use of this drug therapeutically is questionable as like some other benzodiazepines, it causes sedation at effective antischistosomal doses. Objectives Schistosoma japonicum and S, mansoni are two medically important trematode parasites of man. While comparative studies of the two animals have shown that they differ with respect to drug sensitivity, i.e. S, japonicum is resistant, while S, mansoni is sensitive, to cer- tain antischistosomal drugs, virtually no work has been undertaken to determine the underlying physiological cause or causes for these differences. A knowledge of the basic physiology of S, japonicum and S, mansoni, which may be responsible for the variability in drug re- sponse, is essential, not only for describing the mechanism of action 17 of known antischistosomals, but also for the rational development of new effective drugs. I have undertaken this study to determine if S, japonicum and S, mansoni differ in some basic physiological way, and if these differ- ences may be related to variability in drug response. The objectives of this study are: (1) To determine what physiological differences exist between S, japonicum and S, mansoni, with respecto to motility. (2) To determine the effect of environmental conditions and drugs, which alter motility, on the electrophysiology of the parasite's muscle. MATERIALS AND METHODS Source and Maintenance of Animals Female white mice (Mus musculus) infected with Schistosoma japg: nicum (Formosa, Chinese and Philippine strain) were obtained from the laboratory of Dr. Y. S. Liang, University of Lowell, Lowell, MA. Female white mice infected with Schistosoma mansoni (Puerto Rican strain) were obtained from the laboratory of Dr. J. L. Bennett, Michi- gan State University, East Lansing, MI. All parasites were collected from the mesenteric and portal veins, 45 to 60 days post-infection and were maintained at 37°C in RPMI/1640 (Grand Island Biological Company) for mechanical and electrical studies. For biochemical or uptake studies, parasites were collected in Eagle's medium (Grand Island Biological Company) containing 0.5% pentobarbital to permit separation of male and female worms. All experiments were carried out on adult male schistosomes within 12 hours of their removal from the mice. Recording Media Hank's Balanced Salt Solution. All experiments were carried out in Hank's Balanced Salt Solution (HBS) or a modified HBS. The concen- tration of constituents of H85 are: 138 mM NaCl, 5.4 mM KCl, 0.5 mM M9504, 1.4 mM CaCl 0.5 mM KH2PO4, 0.25 mM NaZHPO4, 20 mM Hepes (N-2- 23 hydroxyethylpiperazine-N-Z-ethanesulfonic acid) and 0.1% glucose. The pH was adjusted to 7.4 using 6 N NaOH and osmolality was 310 mOSM. 18 19 Potassium. The effect of elevated K+ HBS on mechanical and elec- trical activity of the schistosomes was tested. HBS with 60 mM or 177 mM K+ was made by increasing KCl concentration while decreasing NaCl concentration. All other constituents in the medium were un- changed. The modified KCl and NaCl concentrations were as follows: 60 mM K+ HBS, 60 mM KCl and 87 mM NaCl; 177 m M K+ HBS, 177 mM KCl and zero NaCl. Lithium. In order to determine the effect of Li+ on mechanical and electrical activity of the parasites, a modified HBS, in which LiCl was completely substituted for NaCl, was used. Final concentra- tion of LiCl was 138 mM. Calcium. The effects of elevated Ca++ and zero Ca++ on the muscle tension of the two parasites were tested. Zero Ca++ HBS was prepared by omitting CaCl2 from the HBS and adding 10"4 M ethylene glycol bis(B-aminoethyl ether) N,N'-tetracetic acid (EGTA). Elevated Ca++ HBS was made by raising CaCl2 to 14 mM and omitting phosphate and sulfate (M9504, KH2P04 and NaZHP04) to prevent precipitation of Ca++. Zero Ca++-60 mM K+ HBS was prepared as zero Ca++ HBS with 60 mM KCl and 87 mM NaCl. Mechanical Recordings Changes in the muscle tension of S, japonicum and S, mansoni were measured using a suction pipette-balance arm system as described by Fetterer gt_gl, (1977, 1978). Figure 4 shows a schematic diagram of this system. The two suction pipettes were made from polyethylene tubing (i.d. 0.3 mm, o.d. 1.0 mm) which was drawn out to give an inside 20 Figure 4. Schematic diagram of apparatus used to make mechanical recordings from male Schistosoma japonicum and S. mansoni. 21 Stationary Pipette Flexible Pipette Shutter li——2X—-|—x_{ ' Figure 4 22 diameter of 0.1 mm. One pipette was 2.5 mm long and inflexible. The other pipette was constructed to make it as flexible as possible. First, the length of tubing from which it was drawn was longer (5.5 mm) and second, it was drawn out so that a second constriction was placed about 1.0 cm above its tip. A 0.25 mm stainless steel wire, 5 cm long was connected to the end of the flexible pipette. The end of this wire rested on the balance arm, which was also made of 0.25 mm stainless steel wire and was 11 cm long. The fulcrum for the balance arm was placed 6 cm from the contact to the flexible pipette. The other end of the balance arm was attached to a blackened acetate strip which served as a shutter for the phototube in a modified "A" myo- graph (Narco Biosystems, Inc., Houstin, TX). The output of this trans- ducer was connected to a type 7173 transducer coupler and amplified by a type 7000 amplifier (both by Narco Biosystems, Inc.). Output from the amplifier was displayed on a physiograph (Narco Biosystems, Inc.) as a pen deflection. At the beginning of each experiment, 2.5 ml of buffered medium was placed in the polyethylene dish, which served as the recording chamber. The temperature of the recording chamber was maintained at 37°C, except in the low temperature experiment. The recording appara- tus was calibrated by adding a 4 mg weight to the shutter end of the balance arm and observing the magnitude of the pen deflection on the physiograph. The tension exerted on the flexible pipette by the schistosome could later be expressed in terms of force necessary to cause an equivalent displacement of the shutter. 23 After the system was calibrated, a worm was placed in the re- cording chamber. The inflexible pipette was attached to the dorsal surface, about 1 mm from the posterior end of the parasite. The flexible pipette was then placed on the parasite anterior to the in- flexible pipette. The worm was then stretched to produce an overall tension of 8 to 16 mg and a distance of 1.25 to 1.75 mm between pipettes. The 4 mg load was then applied to the shutter end of the balance arm. After placement of the suction pipettes, a minimum of 10 minutes of equilibration time was allowed before recordings were begun. The force exerted by the schistosome is dependent upon the length of the worm over which the measurements were taken. In this way, tension changes can be expressed in terms of milligrams per millimeter of worm, as compared to baseline tension; i.e., the tension level just prior to drug or altered ion treatment. Microelectrode Recordings The microelectrode recordings were made using the procedure de- scribed by Fetterer g§_gl, (1981) and Bricker §t_gl, (1982). Micro- electrodes were pulled with a horizontal electrode puller (Narashige Instruments) from 1.5 mm capillary tubing (WPI, New Haven, CT). These microelectrodes had resistances between 20 and 40 MOhms. The micro- electrodes were filled with 3 M KCl and connected to a preamplifier (M-4A, WPI) via a Ag-AgCl wire. The output of the preamplifier was displayed on an oscilloscope (Tektronix 5118) and recorded on a chart recorder (Gould Model 220). Fluid in the recording chamber was grounded with a 3 M KCl-Agar bridge with an Ag-AgCl wire. 24 Parasites were immobilized in 50 mg% pentobarbital—HBS to permit removal of the female from the gynocophoral canal. Males were then pinned to the sylgard with minuten insect pins with their ventral side down. After securing the parasite, the chamber was washed three times with drug-free HBS. Parasites were incubated in fresh HBS at 37°C for an additional 10 minutes before any measurements were taken. Microelectrode penetrations were made on the mid-dorsal surface lateral to the gut and medial to the edge of the worm. Recordings were made from electrical compartments (E1 and E2) at approximately one minute intervals. Electrical potentials were monitored for five minutes before addition of drugs or ions, and for another 10 to 20 minutes afterward. Advancement of the microelectrodes were controlled by a Leitz micromanipulator. Pharmacological Agents Ouabain. The effect of the cardiac glycoside, ouabain, on the mechanical and electrical activity of the parasites was also tested. Ouabain (Sigma) was dissolved in a 50-50 mixture of dimethylsulfoxide (DMSO).and distilled water (dH20). A subsequent dilution (1:10) was made with dHZO. Twenty-five ul of this solution was then added to 2.5 m1 of bathing fluid, bringing the final concentration of ouabain to -5 10 M. Praziquantel. Praziquantel (kindly supplied by Drs. P. Andrews and H. Thomas of the Bayer Institute of Chemotherapy. Wuppertal, West 2 Germany) was initially dissolved in DMSO to a concentration of 10' M. Two subsequent dilutions (1:10) were made with dH20. Twenty-five pl 25 4 of this solution (10' M) was then added to the bathing fluid, bringing 6 the final concentration of praziquantel to 10‘ M. D-600. The D-600 (Knoll A.G., Ludwigshafen am Rhein) was dis- solved in DMSO at a concentration of 10'2M. In the experiments measur- ing the effect of D-600 on muscle tension, 25 ul of this concentration was added to 2.5 ml of bathing fluid, bringing the final concentration 4 of D-600 to 10- M. Temperature The effect of lowered temperature (5°C) on the muscle tension of the two parasites was determined. The temperature of the incubation chamber was lowered by circulating ice-water, rather than warm water (37°C), through the temperature regulating system. After 30 minutes, the incubation temperature was returned to 37°C. Triton X-100 Triton X-100 (Research Products International) was used to remove the tegument of S, japonicum and S, mansoni according to the technique of Oaks g§_§l, (1981). Parasites were separated and males were incu- bated in 0.2% triton X-100 at 4°C for 10 minutes. The worms were gently shaken with a vortex mixer (Vortex Genie, Scientific Products, Inc.) for 30 seconds). The worms were then allowed to settle, and the supernatant containing the tegumental fragments was removed and dis- carded. Parasites were then washed (7X) with cold (4°C) HBS and after- ward, incubated for at least one hour in fresh HBS at 37°C, before any experiments were performed. 26 Ion Flux Studies 42K+ uptake by schistosomes were performed Potassium. Studies on by placing male S, mansoni (20 per vial) in each of 8 vials and male S, japonicum (15 per vial) in each of 8 vials containing 2 m1 of HBS. Parasites were then pre-incubated at 37°C for at least one hour. Half 5 of the parasites from each group, were then incubated in 10' M ouabain for 10 minutes. After this incubation period, 2.0 m1 of HBS containing 42K+ (courtesy of the Nuclear Reactor Lab, Michigan State Uni- 8 uCI of versity) was exchanged for normal HBS in the 4 control vials of each group while the 4 vials of each group containing HBS with ouabain was exchanged for HBS containing both ouabain (10'5M) and 42K“. The above procedure was repeated for each time point tested (i.e., 2, 5, 10, 30, 60 and 120 minutes). The parasites were then separated from the incu- bation medium by filtration over a 25 mm glass fiber filter (Whatman GF/B) placed under vacuum on a millipore filtration apparatus (Milli- pore Co., Model XX10-024-00). The trapped parasites were then washed three times in 5 ml ice-cold HBS, weighed, placed in a vial containing 0.5 ml tissue solubilizer (NCS, Amersham Copr., Arlington Hts., IL) and incubated at 50°C for 60 minutes. Acetic acid (150 pl) was added to all samples. The solubilized parasites were then placed in a scin- tillation vial containing 5 ml of scintillation fluid (ACS, Amersham Corp.) and counted with a scintillation spectrometer (Beckman, Model 7000). Prior to the filtration process, a 20 ul aliquot was collected from each vial and the activity in it counted. In this way 42 it was possible to express K+ accumulation as mM K+ per kilogram wet weight. 27 45 ++ Ca accumu- Calcium. Parasites were prepared for studies of lation by first incubating worms (15 per vial) in 2.0 m1 HBS for a minimum of one hour (37°C). The HBS was then removed and replaced 45Ca++ (New England Nuclear, 15.1 with 2.0 ml of HBS containing 4 uCi mCi/mg). The worms were incubated in label containing media (37°C) for 15, 30 or 60 minute intervals. At the end of the incubation period, the parasites were filtered using glass fiber filters (Whatman GB/F) on a Millipore filter apparatus (Millipore Inc.) and rinsed three times with 5 ml of ice-cold HBS. After the parasites were trapped on the GB/F filters, they were treated as described above for the 42K+ 45 experiments. The activity of Ca++ was expressed as counts per minute per milligram wet weight. ATPase Assay_ Ca++- and Mg++-dependent ATPases (i.e., Ca++-Mg++ ATPase, Ca++- ATPase and Mg++-ATPase) were measured according to the procedure de- scribed by Sulakhe e}_gl, (1973). Parasites (40 per vial) were rinsed (3X) in appropriate buffer and then homogenized at 4°C. The concen- tration of the constituents in the buffered mediums were as follows: for Ca++-Mg++ ATPase buffer - 50 mM tris-HCl, 5 mM MgCl and 40 uM CaClz (pH=7.0); for Ca++-ATPase buffer - 50 mM tris-HCl, 5 mM CaClZ, 0.5 mM EDTA (pH=7.0); and for Mg++-ATPase buffer - 50 mM tris-HCl, 5 mM MgCl and 0.5 mM EGTA (pH=7.5). 2 The homogenized tissue was then centrifuged at 3000 RPM in a Beck- man centrifuge (4°C) for 15 minutes. The supernatant was then removed and discarded and the tissue was resuspended in fresh buffer, and again 28 centrifuged. The tissue was washed using this procedure two additional times. After the washing, the vials containing the suspended tissue were incubated at 37°C. To start the reaction, ATP was added to each sample (final concentration, 5 mM). After a 10 minutes incubation period, 250 pl of trichloroacetic acid was added to each sample and the vial immediately removed from the warm water bath and placed in an ice bath. The vials were centrifuged at 3000 RPM for 15 minutes and an aliquot of the supernant removed. Enzymatic activity was deter- mined by measuring P04 released into the supernatant using a spectro- photometer (Spectronic 20, Bausch and Lomb). Tissue samples were taken before addition of ATP and protein content was determined by the method of Lowry e§_gl, (1951) with bovine serum albumin (Sigma) as the standard. All enzymatic activity was expressed as P04 released per hour per milligram of protein. Statistical Procedure Unless otherwise noted, results are expressed as the mean of a minimum of 6 :_one standard error of the mean. Standard two-tailed t-tests were used to determine statistical differences between means. RESULTS Normal Activity Mechanical Activity. The mechanical activity recorded from adult male Schistosoma japonicum normally consisted of spontaneous contractions of variable frequency (5-50 per minute) and amplitude (Figure 5). This normal motility was indistinguishable from that ob- served in S, mansoni (Fetterer gt_gl,, 1977). Both parasites showed, over time, a gradual relaxation in baseline tension. During the first ten minutes, after parasites were hooked up, the muscle tension of S, japonicum decreased by -0.73:0.26 mg (N=12) while that of S, mansoni decreased by -l.05:0.25 mg (N=12). This muscle relaxation of the two parasites was not significantly different (P30.2). Electrical Activity. The two species also possess similar elec- trical characteristics. As demonstrated by Fetterer e£_gl, (1981a) and Bricker e£_§l, (1982), S, mansoni contains three discrete electri- cal compartments. These electrical compartments have been anatomi- cally identified as originating from (1) tegument (E1), (2) muscle (E2) and (3) extracellular space (E3). Figure 6 shows the potential changes observed as a microelectrode is advanced into S, japonicum. The most negative potential, E], was the first potential encountered. It had a value of -60.2:2.1 mv. This compares to the values I recorded for S, mansoni of -53.3:1.4 mv. 29 30 Figure 5. Sample chart recordings of spontaneous contractile acti- vity from schistosomes. The top trace represents activity recorded from S. japonicum. The bottom trace represents activity recorded fromS _. manson1. 31 "WMWNMMW s. W... '2 m9 30sec I Figure 5 32 Figure 6. Potential profile obtained while penetrating into the dorsal surface of an adult male S, japonicum with a microelectrode. The sharp vertical drop represents penetration of E1. The first upward potential change represents E2. The second upward potential change represents E3. 34 The next potential recorded was E2. It had a value of -27.8:j.4 mv in S, japonicum and -23.5:1.2 mv in S, mansoni. E3 was also recorded from S, japonicum but no quantifiable data concerning this potential was collected. Table 1 compares average values recorded for E1 and E2 in S, japonicum and S, mansoni. Though the values recorded for E1 and E2 were significantly more negative in S, japonicum than in S, mansoni (P§,01) it seems clear that these potentials in S, japonicum represent the same potentials as identified in S, mansoni as tegument and muscle. Effect of Altered Ion Concentration Potassium. Fetterer e§_§l, (1978) demonstrated that exposure of S, mansoni to an elevated K+ HBS (60 mM) incubation medium caused a rapid, sustained increase in the muscle tension. I have measured a tension increase of 2.9:0.6 mg at two minutes in this parasite. The muscle tension continued to increase and by the end of the 20 minute test period reached a value of 3.9:0.6 mg. In contrast, when 60 mM K+ was applied to S, japonicum, only a small increase in tension was ob- served (Figures 7 and 8). Even after 20 minutes, the tension was in— creased by only l.5:1.1 mg; after 60 minutes by only 2.1:1.3 mg. The tension at 60 minutes was still significantly less than that achieved by S, mansoni at 20 minutes (P:.01). Tension changes in response to 177 mM K+ was also examined (Figure 9). HBS with 177 mM K+ caused a large, sustained muscle con- traction in both parasites. The large tension increase induced by 177 mM K+ was not significantly different in the two schistosomes 35 TABLE 1 The Effects of Elevated K+ Concentrations on the Mechanical and Electrical Activity of S, japonicum and S, mansoni S. japonicum S. mansoni Mechanical Tension Change 50 mM K++at 20 min 0.92 _+_ 1.10 mg 3.80 i 0.50 mg: 177 mM K at 20 min 2.84 i 0.83 mg 3.24 i 0.85 mg 60 mM K+ at 60 min 2.08 :_1.30 mg Not Performed Electrical Electrical Potential‘ 1:1 (tegument) (11:24) -60.2 i 2.1 mv -53.3 i 1.4 mv: E2 (tegument) (N=24) -27.8 :_1.4 mv -23.5 :_l.2 mv Depolarization of Electrical Potential amuse in 60 mM K++at 5 min 28.2 i 7.0 mv 13.3 i 1.7 mv: Emusc in 177 mM K at 5 min 31.7 :_2 9 mv 21.7 :_3.0 mv N=6 unless otherwise noted. Statistical analysis compares S. japonicum and S, mansoni. °(P:.01) b(P:,20) 36 Figure 7. Chart recordings comparing the effects of elevated K+ on the muscle tension of S, japonicum and S, mansoni. At the arrow the medium (HBS) was replaced by 60 mM K+ HBS. Top trace, S, japonicum; bottom trace, S, mansoni. 37 WW 3,, S. mansoni F ' |2mg A 2min Figure 7 ' 38 Figure 8. The effects of 60 mM K+ on the muscle tension of schisto- somes. Values are means i one S.E.M. (N=6). At the arrow the medium was replaced with HBS containing 60 mM KT. Open circles, S, japoni- 93m; closed circles, S, mansoni. A TENSION (mg) 39 09'4’81'21'62'0 3'0 TIME (MIN) Figure 8 V 40 40 Figure 9. The effects of various concentration of K+ on muscle ten- sion of schistosomes. Values are means :_one S.E.M. (N=6). All measurements were taken f've minutes after medium was replaced with HBS containing elevated K . Open circles: S. japonicum; closed circles: S, mansoni. '— A TENSION(m9) (y 41 6'0 177 mM K+ Figure 9 42 (P3,20). Only at the concentration of 60 mM K+ was the tension in- crease significantly greater in S, mansoni than in S, japonicum (P5, 01 ) . The effect of elevated K+ concentrations on tegumental and muscle potentials in the two schistosomes is shown in Figure 10. It should be noted that 60 mM K+ HBS, while it had little effect on the muscle tension of S, japonicum, caused a large depolarization of both tegu- ment and muscle in this parasite. Even within the first minute after exposure to 60 mM K+, tegument of S, japonicum was depolarized from ~56.0:3.5 mv to -26.0:3.0 mv, while muscle was depolarized from -13.0:2.0 mv to +1.0:7.0 mv (Figure 11). The effect of elevated K+ medium on the mechanical and electrical activity of the two parasites is summarized in Table l. Lithium. When S, mansoni was exposed to an HBS in which LiCl had been substituted for NaCl, there is a gradual increase in tension (Fetterer gt_gl,, 1981a). Maximum tension reached in my experiments averaged 4.2:0.8 mg, and the half-time for response was six minutes. By contrast, when S, japonicum was exposed to the LiCl substituted HBS, there was a drop in maintained tension, so that after five minutes it reached a minimum of -1.34:0.30 mg below the control level. The half-time for this tension drop was three minutes. After this transient relaxation, the tension of S, japonicum gradually increased (Figures 12 and 13). If permitted to incubate in LiCl HBS for one hours, the tension in the musculature of S, japonicum eventually reached a tension level of 2.5:0.5 mg (Figure 13). This maximum in- crease in tension of S, japonicum which occurred between 50 and 60 43 Figure 10. The effects of various concentrations of K+ on E1 and E2 of schistosomes. Values are means :_one S.E.M. (N=6). All measure- ments were taken five minutes after medium was replaced with HBS containing elevated K+. E1 is shown by broken lines. E2 is shown by solid lines. Open circles: S, japonicum; closed circles: S, mansoni. 44, 177 10: 534 mM K“ Figure 10 45 Figure 11. Time course of effect of 60 mM K+ on E1 and E2 in S, japonicum. Values are means :_one S.E.M. (N=6). Animals were pre- 1ncubated in PB/HBS. At the arrow the medium was exchanged with HBS containing 60 mM KT. Open circles, E]; closed circles, E2. 46 TIME (MIN) .Figure 11 47 Figure 12. Chart recordings comparing the effects of LiCl HBS on muscle tension of S, japonicum and S, mansoni. At the arrow the medium was replaced by HBS containing 138 mM Li+ (a complete substi- tution of Li+ for NaT). Top trace, S, japonicum; bottom trace, S. mansoni. 48 S. iaponicum S. mansoni 2mg w 2min 4 Figure 12 49 Figure 13. The effects of 138 mM Li+ on the muscle tension of schistosomes. Values are means i_0ne S.E.M. (N=6 . At the arrow the medium was replaced with HBS containing 138 mM Li . Open circles, S, japonicum; closed circles, S, mansoni. SO A TENSION (mg) 0'4'8'1'21'520 3'0 4'0 5'0 6'0 1 TIME (MIN) Figure 13 51 minutes, was still significantly less than the tension increase of S, mansoni at 16 minutes (P§,Ol). LiCl HBS, though it induced no tension increase in S, japonicum until after 15 minutes, did cause depolarization of both the tegument and muscle at times considerably shorter than this. Even after only ten minutes, the tegument was depolarized from -60.0:3.0 mv to -28.0: 5.0 mv, while the muscle was depolarized from -20.0:3.0 mv to -12.0: 3.0 mv (Figure 14). This is similar to the Li+ effect on S, mansoni in which tegument and muscle are both depolarized (Bricker gt_§l,, 1982). The effects of LiCl substitution on mechanical and electrical activity of the two parasites is summarized in Table 2. Low Temperature To test the effects of lowered temperature on the muscle tension of the two parasites, the temperature of the circulating fluid in the bathing chamber was lowered from 37°C to 5°C. Both S, japonicum and S, mansoni responded to the low temperature with a maintained increase in muscle tension (Figure 15). In both, the increase in tension was slow in onset, reaching a value of only 0.4:0.2 mg for S, japonicum and 0.1:0.1 mg for S, mansoni at two minutes. Between two and ten minutes the tension increased considerably for both S, japonicum (2.9:0.3 mg) and S, mansoni (2.7:0.3 mg). After ten minutes the muscle tension continued to increase, but as a much slower rate. By 30 minutes, muscle tension for the two parasites was approximately 3.5 mg. When bathing temperature was returned to 37°C, both parasites relaxed at the same rate. Most of this relaxation occurred during the first five minutes; i.e., the muscle tension of S, japonicum dropped 52 Figure 14. Time course of effect of 138 mM Li+ on E1 and E2 in S, ja onicum. Values are means :_one S.E.M. (N=6). Animals were pre- 1ncubated in PB/HBS.+ At the arrow the medium was replaced with HBS containing 138 mM Li . Open circles, E]; closed circles, E2. 53 -4 -2 9 2 4 6 8 10 TIME (MIN) Figure 14 54 TABLE 2 A Comparison of the Effects of LiCl HBS on the Mechanical and Electrical Activity of S, japonicum and S, mansoni S. japonicum S. mansoni Mechanical Tension Change LiCl HBS for 20 min 0.40 :_0.73 mg 3.19 :_0.82 mg° LiCl HBS for 60 min 2.47 :_0.51 mg Not Performed Electrical Depolarization of Electrical Potential Ete -LiCl at 10 min 23.7 :_1.5 mv 193 E 9 -LiC1 at 10 min 8.1 :_1.0 mv 12 I'llUSC N=6 for all experiments. a(P301) bVa1ues determined by c. s. Bricker gt_§l, (1982) 11 to 15 minutes after Li+ substitution. 55 Figure 15. The effect of lowered temperature on the muscle tension of schistosomes. Values are means :_one S.E.M. (N=6). At the first arrow, the temperature of the bathing medium was lowered to 5°C. At the second arrow the temperature of the bathing medium was returned to 37°C. Open circles, S, japonicum; closed circles, S, mansoni. ATENSION (mg) 56 20 TIME (min) Figure 15 57 to O.8:0.5 mg while that of S, mansoni dropped to 1.4:0.5 mg. Fif- teen minutes after returning the temperature to 37°C, the muscle ten- sion of S, japonicum (0.4:0.3 mg) and S, mansoni (0.4:0.2 mg) was only slightly elevated above control levels. When the two parasites were preincubated in zero Ca++ plus EGTA, they responded differently to low temperature (Figure 16, Table 3). S, japonicum responded with a gradual increase in muscle tension, reaching 3.49:0.86 mg by 20 minutes. In contrast, incubation of S, mansoni in zero Ca++ rendered this parasite unresponsive to the tension inducing effects of low temperature. Addition of Ca++ back into the bath, (final concentration of Ca++ being 1.4 mM), produced no further increase in S, japonicum's muscle tension, but did cause an immediate increase in the muscle tension of S, mansoni (4.60:0.87 mg two minutes after addition of Ca++). Pharmacolggical Agents Ouabain. A sample chart recording comparing the response of S, japonicum and S, mansoni to ouabain (10'5M) is shown in Figure 17. In my experiments for S, mansoni, the maximum tension increase observed was 4.5:O.7 mg and the half-time for maximum contraction was six minutes. The responses of S, japonicum to ouabain was much different than that of S, mansoni. Tension did not begin to increase until after 15 minutes and even after 60 minutes the maximum tension in- crease was only 1.5:0.4 mg (Figure 18). Neither the tegumental or muscle potential of S, japonicum was affected by ouabain (Figure 19). This is in contrast to ouabain's effect on electrical potentials in S, mansoni (Bricker gt_gl,, l982) 58 Figure 16. The effect of zero Ca++ on the low temperature induced muscle contracture in schistosomes. Values are means + one S.E.M. (N36). Bathing medium (HBS) was exchanged for zero Ca1F+ HBS plus 10 4M EGTA, five minutes prior to lowering the temperature. At the first arrow, the temperature of the bathing medium was lowered to 5°C. At the second arrow CaCl was added to the bath, bringing the concentration of Ca++ to 1.4 m8. Open circles, S. japonicum; closed circles, S, mansoni. 7' A TENSION (mg) 59 TIME (MIN) Figure 16 60 TABLE 3 A Comparisgg of the Effects of Ouabain, Low Temperature, D-600 Zero Ca and Praziquantel on the Mechanical Activity of S, japonicum and S, mansoni S. japonicum S. mansoni Mechanical Tension Change 5 5 Ouabain (10' M) at 20 min Ouabain (10' M) at 60 min Low Temperature at 30 min Zero Ca++ and Low Tempera- ture at 20 min 0-500 (10‘4M) at 20 min Zero Ca++_gnd Praziquan- tel (10 M) at 5 min Electrical E in Ouabain at 10 min teg . . . Emusc 1n Ouaba1n at 10 m1n 0.31 :_0.82 mg 3.54 :_0.98 mga 1.46 :_O.44 mg Not Performed 3.57 :_0.38 mg 3.35 :_0.31 mg° 3.49 :_0.86 mg 0.13 :_0.59 mg° 0.10 :_0.60 mg -1 00 :_0.50 mgb 3.50 :_0.84 mg 0.73 :_1.35 mga Depolarization of Electrical Potential -1.8 :_1.8 mv 25 mvg 2.6 :_5.3 mv 12 mv N=6 for all experiments. a(P301) b(.01
0.2 (Figure 22). Only at the five
42 +
minute time point did S, mansoni accumulate significantly more K
42
than did S, japonicum (.055P5,10). In another experiment, K+ was
monitored over a two hour period. In this experiment also there was
no significant difference between S, japonicum and S, mansoni (Figure
42K+ attributed to active transport (via a Na+,K+-ATPase) was
5
23).
eliminated by preincubating some parasites in ouabain (10' M) for 10
minutes. This concentration of ouabain was also maintained in the
42
incubation fluid during the K+ uptake measurement. Ouabain caused
42
a significant reduction in K+ uptake in both worms (P§,01) (Figure
22).
45
Calcium. The data showing the accumulation of Ca++ over a
one hour time period are shown in Figure 24. At all three time points
measured (15, 30 and 60 minutes), S, mansoni accumulated a signifi-
1.45 ++
cantly greater amount 0 Ca than did S, jannicum (P<.01). The
rate of 45Ca++ accumulation was greater for S, mansoni than S, japoni-
g9m_during the first 30 minutes of incubation but after 30 minutes
42
the rate was the same. Table 4 summarizes the uptake of K+ and
45Ca++ by the two worms.
Triton Treatment
The tegument of S, mansoni can be effectively removed by incuba-
tion in 0.2% triton X-lOO (5°C) for ten minutes followed by 30 seconds
of gentle vortexing (Oaks gt_gl,, 1978). My results have shown that
this technique is equally effective in removing the tegument of
73
Figure 22. Uptake of 42K+ by male sahistosomes over one hour. Values
are means :_on S.E.M. )N=4). Total ZKT accumulation is shown by
solid lines. K+ accumulation in the presence of 10'5M ouabain is
shown by broken lines. Open circles, S, japonicum; closed circles,
S, mansoni.
' 4216‘ (mM/Kg)
74
TIME (MIN)
Figure 22
75
Figure 23. Uptake of 42K+ by male schistosomes over two hours. Values
are means :_one S.E.M. (N=4). Open circles, S, jagonicum; closed
circles, S, mansoni.
76
5'0
TIME (MIN)
Figure 23
120
77
Figure 24. Uptake of 45Ca++ in male schistosomes. Values are
means :_S.E.M. (N=8). Open circles, S, jannicum; closed circles,
S. mansoni.
3000
5
inf
C0 CPM/ mg wet wt.
45
78
1'5
3'0
TIME (MIN)
Figure 24
79
TABLE 4
A Comparison of Ion Uptake in S, jagonicum and S, mansoni
Amount of Isotope Accumulated
Ion
S. japonicum S. mansoni
42 + . _ + + b
K -60 m1n (N—4) 8.23 :_l.0 mM K /kg 9.35 :_1.9 mM K /kg
42I<"-120 min (N=4) 25.0 i 1.1 mM K+/kg 22.0 _+_ 0.9 mM K+/kga
45 ++
Ca -60 min (N=8) 1796 _+_ 172 CPM/mg wt 3000 _+_ 193 CPM/mg wta
a(Pfr,01). Statistical analysis compares S, japonicum and S, mansoni
va ues.
b(P_>_. 02)
80
S, japonicum. Scanning electron microscopy (kindly supplied by C. S.
Bricker) showed that the convoluted surface of normal S, jagonicum
was absent in the triton treated worms. These worms, instead ex-
hibited a striated surface resembling muscle fibers (Figure 25).
Mechanical Activity. Triton treated S, jannicum in HBS ex-
hibited less spontaneous activity than did untreated S, jagonicum. In
over half the parasites tested, there were no spontaneous contrac-
tions, whatsoever. Triton treated S, jagonicum showed a gradual re-
laxation in baseline muscle tension, similar to that observed in
untreated worms.
Electrical Activity, Microelectrode recordings from S, jagoni-
g9g_after triton treatment, showed that two electrical potentials could
usually be measured. The first electrical potential encountered when
a microelectrode was advanced into the worm, had a value of -28.3:1.3
mv. This was not significantly different from the muscle values
recorded from untreated S, japonicum (-28.9:1.0 mv) (P3,2) (Table 5).
A large negative potential, similar in magnitude to tegument was not
present in S, jagonicum after tegument removal.
Potassium. A typical chart recording showing the effect of 60
mM K+ HBS on normal and triton-treated S, jagonicum is shown in Figure
26. As already described, normal parasites are affected little by
60 mM K'. However, after tegument removal, S, jagonicum responded
with a large rapid increase in muscle tension. Within 15 seconds, for
the six animals tested, the tension level was increased by an average
of 3.2:0.7 mg and the muscle always remained contracted for the dura-
tion of the 10 minutes test period (Figure 27). This tension increase
81
Figure 25. Scanning electron microscopy showing the effect of triton
X-lOO treatment on the tegument of S, japonicum. Upper micrograph:
low magnification (6OOX) of the tegument of normal (left) and triton
treated (right) S. japonicum. Lower micrograph: high magnification
(4000X) of the tEgument of normal (left) and triton treated (right)
S. japonicum. Calibration bar for A and B, 13 uM; calibration bar
for C and D, 2.5 uM. Kindly supplied by C. S. Bricker, Dept. of
Zoology. University of Vermont.
82
It}; '
O
‘11
Figure 25
83
TABLE 5
A Comparison of Normal and Triton Tregted S, jagonicum; The Effects
of Ouabain, LiCl HBS and 60 mM K on Mechanical Activity
S. japonicum S. japonicum
(normal) (triton)
Mechanical Tension Change
50 mM K+ at 10 min 0.92 :1.10 mg 2.77 i 0.89 mg:
Ouabain at 20 min 0.31 :_0.82 mg 0.39 :_O.31 mga
LiCl at 20 min 0 4O :_0 73 mg 2 43 :_O 34 mg
Electrical Electrical Potential
E -60.3 + 2.5 mv
teg - b
Emusc -28.9 :_1.0 mv -28.2 :_O.5 mv
N=6 for all experiments.
a(P301)
b(P.lO:P>.05)
C(P_>_,20)
84
Figure 26. Chart recordings showing the effect of 60 mM K+ on the
muscle tension of normal and triton-treated S, japonicum. At the
arrow the medium was exchanged with HBS containing 60 mM K+. Top
trace, normal S, jagonicum; bottom trace, S, japonicum after tegu-
ment removal.
4..
K
85
Control
Triton
|2_mg-
2min
Figure 26
86
Figure 27. The effect of 60 mM K+ on the muscle tension of normal
and triton-treated S. japonicum. Values are means + one S.E.M. (N=6).
At the arrow the medium was replaced with HBS contaTning 60 mM K+.
Open circles, normal worms; closed circles, triton-treated worms.
A TENSION (mg)
87
3.
1
2.
I L
T _0
0|
oKfidsi 2 5 1'0 "
TIME (MIN)
Figure 27
88
in triton-treated S, japonicum was not significantly different from
K+ induced tension increase in untreated S, mansoni (P3,2).
Calcium. Incubation of triton-treated S, japonicum in zero Ca++
plus 10'4
M EGTA HBS caused a decrease in muscle tension, reaching a
minimum of -3.74:0.71 mg by 10 minutes (N=6) (Figure 28). Exchange
with 60 mM K+-zero Ca++ media caused only a slight transient change in
muscle tension during a two minute test period but addition of 1.4 mM
++ . . . . .
, back 1nto the med1um, caused a large, rap1d increase 1n muscle
Ca
tension (4.72:0.79 mg after one minute).
In several types of muscle, elevated levels of Ca++ reduce the
size of the K+ induced contraction, presumably by stabilizing the
muscle membrane (Luttgau, 1963; Nasledov gtngl., 1966). This possibi-
lity was tested for S, japonicum by incubating triton-treated parasites
in 14 mM Ca++ and measuring the effect of 60 mM K+ on muscle tension.
In both 1.4 mM Ca++ and 14 mM Ca++, S, japonicum responded to 60 mM
K+ with a large, sustained muscle contraction of comparable magnitude
(Figure 29).
Ouabain. Tegumental removal using triton alters S, japonicum's
responses to both ouabain and LiCl. As described above, S, japonicum
responds to ouabain with only a gradual increase in tension. After
removal of the tegument, these parasites were even less responsive to
ouabain. After 20 minutes, triton-treated S, jagonicum reached a ten-
sion level of less than 0.4:0.3 mg, while the tension increase of
normal worms was 1.4:O.5 mg (Figure 30).
Lithium. As already described, normal S, jagonicum respond to
LiCl HBS with a transient decrease in muscle tension followed by an
89
Figure 28. Chart recording of the effect of zero Ca++ HBS, 60 mM K+
HBS and 1.4 mM Ca++ on the muscle tension of a triton-treated S,
japonicum. At the first arrow the medium was exchanged with HBS con-
ta1n1ng zero Ca++ plus 10'4M EGTA. At the second arrow, the zero Ca++
medium was replaced with zero Ca++-6O mM K+ medium. At the third arrow,
Ca++ was added back into the bath to bring the final Ca++ concentra—
tion to 1.4 mM.
9O
+4}
(7‘3! l(+' Ckf'+'
Figure 28
91
Figure 29. Chart recording showing the effect of 14 mM (:a++ on the
KT contracture of triton treated S. japonicum. Upper trace: K+ in-
duced contracture of parasite bathéd in 1.4 mM Ca++. Lower trace:
K+ induced contracture of parasite bathed in 14 mM Ca++.
92
t
K" .1
(\w—v— __fi1_~._fi:_
K
_IL.
4*
Figure 29
93
Figure 30. The effect of ouabain on the muscle tension of normal and
triton-treated S, japonicum. Values are means :_one S.E.M. (N=6).
At the arrow the med1um was replaced by HBS containing 10'5M ouabain.
Open circles, normal S, japonicum; closed circles, S, jagonicum after
tegument removal.
A TENSION (mg)
94
2.
1.
.. i
-1.
- 9 , 9 i 5 lb 26'
TIME (MIN)
Figure 30
95
increase in muscle tension. In contrast, triton-treated S, japonicum
exhibited an increase in tension throughout the 20 minute test
period. At 20 minutes the tension was significantly higher for these
parasites (2.4:O.3 mg) than for untreated S, japonicum (1.2:O.3 mg)
(P§,Ol). The transient relaxation seen in normal worms was not ob-
served in triton-treated ones (Figure 31). A comparison of the effects
of high K', ouabain and LiCl on the muscle tension of normal and
triton-treated S, jagonicum is given in Table 5.
Ca++-Mg++ ATPase
The amount of Ca++ and/or Mg++-activated ATPase activity in crude
homogenates of S, jagonicum and S, mansoni is given in Table 6. The
amount of total Ca++-Mg++ ATPase was similar in the two parasites but
S, japonicum contained significantly more Ca++-ATPase than did S,
mansoni. After tegumental removal, total Ca++-Mg++ ATPase activity
in S, jagonicum stayed the same as control, while the activity in
triton-treated S, mansoni increased when compared to its control.
96
Figure 31. The effect of Li+ on the muscle tension of normal and
triton-treated S, japonicum. Values are means :_one S.E.M. (N=6).
At the arrow the medium was replaced by HBS containing 138 mM Li .
Open circles, normal 5. japonicum; closed circles, S, japonicum
after tegument removaTI
A TENSION (mg)
97
+c
Nd
U11
10
TIME (MIN)
Figure 31
2'0
98
TABLE 6
A Comparison of Ca++ and/or Mg++ Dependent Enzymes in
S, japonicum and S, mansoni
S. japonicum S. mansoni
Enzymg_ Enzyme Activity Measured as mM
EQ4/mggprotein/hr
Total Ca++-Mg++ ATPase 595 i 17 730 : 10C
Ca++-ATPase 414 i 35 288 i 29a
Mg++-ATPase 550 i 11 504 : 15b
Total Ca++-Mg++ ATPase 558 _+_ 25 992 1 13°
in triton-treated worms
N=4. Statistical analysis compares S, janonicum and S, mansoni
values.
a(133.01)
b(.10