DEVELOPMENTAL CHANGES IN PHYSIOLOGICAL RESPONSES TO DRUGS AND IMMUNE SUBSTANCES IN SCHISTOSDMA MANSONI By David Paul Thompson A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology and Neuroscience Program 1984 n”) Q o, ) n. .3 ABSTRACT Developmental Changes in Physiological Responses to Drugs and Immune Substances in Schistosoma mansoni by David Paul Thompson Immature post-transformational stages of Schistosoma mansoni (schistosomula) were studied using physiological and biochemical tech- niques to examine developmental changes in their responsiveness to drugs and immune substances. Electrical activity in schistosomula is highly sensitive to a num- ber of agents believed to affect the neuromuscular system, metabolism or the host/parasite interface of adult S, mansoni. Among the wide range of substances tested, measurable effects due to potassium cyanide and immune serum plus complement were restricted to freshly transformed schistosomula. Schistosomula, however, were less responsive than adults to the putative neurotransmitters SHT and dopamine, as well as pentobarbital-sodium and the anthelmintic, oltipraz. Skin- and mecha- nically-transformed schistosomula exhibited similar responses to all treatments. To examine more closely the conversion to a cyanide-insensitive state, immature and adult §, mansoni were studied electrophysiologi- cally and by monitoring the fate of exogenous [14Cngucose. Electrical 3 recordings revealed that the parasite becomes insensitive to lO' M David Paul Thompson cyanide by.24 hr after transformation. This finding may be explained on the basis of metabolic changes that occur between the 3 hr and 24 hr stages resulting in a 77% drop in CO2 evolution and an 84% increase in lactate excretion. The timecourse of this conversion was similar in skin- and mechanically-transformed schistosomula, and was unaffected by inhibition of protein synthesis. No Pasteur effect is detected in immature or adult schistosomes. Based on physiological recordings, 3 hr schistosomula are highly sensitive to 24 hr incubations in antiserum plus complement, while adult schistosomes are refractory to this treatment. Cytochalasin B (lo-SM) and colChicine (lo-4M) effect varying degrees of tegumental disruption in adult §, mansoni, but no measurable effects on tegument or muscle physiology. Furthermore, their effects, do not appear to be exacerbated by concurrent exposure to immune serum plus complement. to Linda, Trevor and Trent ii ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. Ralph A. Fax (Dept. of Zoology/Neuroscience Program), my major professor, for his guidance and constructive criticism throughout this investigation. A special thanks to Dr. James L. Bennett (Dept. of Pharmacology/Toxico- logy) for assistance in obtaining the NIH grant which funded work on the schistosomula, and for providing creative insights during these in- vestigations. Thanks also to Dr. Rudy Bernard (Dept. of Physiology), Dr. Richard Hill (Dept. of Zoology), Dr. Harold Miller (Dept. of Micro- biology/Public Health), and Dr. Charles Tweedle (Dept. of Anatomy/ Neuroscience Program) for helpfully serving as committee members and consultants. I would also like to thank Dr. Glenn Hatton (Dept. of Psychology, Director, Neuroscience Program) for enthusiastically loan- ing equipment as well as technical expertise on numerous occasions. Finally, thanks and best wishes to my colleagues in parasite research, Dan Morrison, Tim Martin, Dave Semeyn, Carla Siefker, Connie Bricker, Beth Van de Waa, Liz Nolde Mussie, Joe Depenbusch, Amy Abrahamsen, Helen Cirrito, Ivy Mao, and George Cheng. Their friendship has been the most rewarding aspect of my work. TABLE OF CONTENTS Page LIST OF TABLES --------------------------------------------------- vii LIST OF FIGURES .................................................. viii GENERAL INTRODUCTION --------------------------------------------- 1 Schistosoma mansoni: Parasite, life-cycle and disease ------ l Anatomical and physiological characteristics relevant to the present study ------------------------------------------ 5 Developmental changes in the parasite within the definitive host --------------------------------------------------- 15 Metabolic changes occurring in S, mansoni within the defini- tive host ---------------------------------------------- 23 OBJECTIVES ....................................................... 27 SECTION I ........................................................ 31 Schistosoma mansoni: A comparative study of schistosomula transformed mechanically and by skin penetration. Electro- physiological responses to a wide range of substances ------- Summary ................................................ Introduction ........................................... Materials and Methods .................................. Preparation and culture of schistosomula ---------- Apparatus and recording procedures ................ Experimental treatments ........................... Results ................................................ Characteristics of spontaneous electrical activity Ion SubStitution experiments ---------------------- Drug responses .................................... Responses to antischistosomulum antibody ---------- Discussion ............................................. iv 31 31 34 34 35 38 4O 4O 43 43 49 49 TABLE OF CONTENTS (continued) Page SECTION II ------------------------------------------------------- 55 Changes in glucose metabolism and cyanide-sensitivity in Schistosoma mansoni during development ---------------------- 55 Summary ------------------------------------------------ 55 Introduction ------------------------------------------- 56 Materials and Methods ---------------------------------- 58 Parasite preparations and incubation media -------- 58 Electrophysiological and mechanical recordings---- 60 Incubation of [ 4C]glucose-containing media and analysis of metabolites ---------------------- 62 Results ------------------------------------------------ 64 Characteristics of volume conducted electrical -potentials----e ----------------------------- 64 Developmental changes in substrate utilization---- 64 Developmental changes in physiological responses to metabolic inhibitors ---------------------- 67 Effects of metabolic inhibitors on substrate uti- lization ------------------------------------- 70 Discussion --------------------------------------------- 75 SECTION III ------------------------------------------------------ 79 Schistosoma mansoni: Physiological effects of concurrent in Vitro exposure to antiserum plus complement and drugs tfiat affect cytoskeletal components---------< --------------- 79 Summary ------------------------------------------------ 79 Introduction ------------------------------------------- 80 Materials and Methods--: ------------------------------- Bl Parasites, incubation media, drugs and immune sub- stances -------------------------------------- 8l Electrophysiological and mechanical recordings---- 82 Turnover of [ 2 IJ-labelled surface components---- 84 Scanning and transmission electron microscopy ----- 88 Results ------------------------------------------------ 89 Effects of drugs and immune substances on tegu- mental morphology ---------------------------- 89 Microelectrode and surface electrical recordings-- 93 Mechanical recggdings ----------------------------- lO3 Turnover of [ I]-labelled surface components---- lO3 Discussion --------------------------------------------- ll6 TABLE OF CONTENTS (continued) Page GENERAL DISCUSSION ---------------------------------------------------- 125 The electrophysiological assay ----------------------------------- 125 Implications of the study ---------------------------------------- l30 SUMMARY --------------------------------------------------------------- 136 APPENDIX -------------------------------------------------------------- l40 BIBLIOGRAPHT ---------------------------------------------------------- 141 vi Table LIST OF TABLES Page Changes in electrical activity and motility in mechani- cal- and skin-transformed schistosomula brought about by experimental substances ----------------------------- 44 Tegumental potentials recorded in adult male S. mansoni after 24 hr incubations in RPMI-l640 alone (A), with drugs (B), or concurrently with dru s and immune mouse serum plus guinea pig complement (C ------------------- 94 vii Figure «DOOM TO LIST OF FIGURES Page Life-cycle of S, mansoni ------------------------------- 2 Micrographs of cercaria (left) and adult S, mansoni---- 7 Schematic of tegument and muscle layers in S, mansoni-- lO Schematic of apparatus used for recording endogenous electrical transients from the surface of schistosomes- 36 Electrical activity recorded from the surface of me- chanically-transformed 3 hr schistosomUla under control conditions and after the addition of 50% ethyl alcohol to the recording medium -------------------------------- 4l Developmental changes in S. mansoni in the distribution -of metabolic endproducts after 4 hr incubations in D- [I4C]glucose-containing media under control conditions- 65 Frequency of endogenous electrical potentials recorded from developmental stages of S, mansoni under control conditions (hatched bars) or after 1 hr incubations in media containing lO'3M cyanide (open bars) or 10' M antimony (shaded bars) --------------------------------- 68 Effects of incubation for 1 hr (open circles) or 24 hr (open squares) in lO'3M cyanide, or T hr in lO'4M anti- mony (triangles) on longitudinal muscle tension changes induced by exposure to 60 mM potassium (upper graph) or lO‘4M carbachol (lower graph) in adult male S, mansoni- 7l Developmental changes in the effects of metabolic inhi- bition due to lO'3M cyanide (open bars) or 10‘ M anti- ‘ mony (shaded bars) on C0 (upper) or lactic aci (lower) produced during hr incubations in D-[I4CJ- glucose-containing media ------------------------------- 73 Schematic of apparatus used to record longitudinal muscle tension in adult male S, mansoni ---------------- 85 viii LIST OF FIGURES (continued) Figure ll 12 13 I4 15 16 I7 18 I9 20 Page Scanning and transmission electron micrographs obtained from adult male S. mansoni incubated 24 hr in 200 ug/ml pgromycin (A and B) and ixl0'5 M trifluoperazine (C and D Frequency of endogenous electrical potentials recorded from adult male S. mansoni showing absence of effects on this parameter due to drugs (open bars) or concur- rent exposure to drugs plus 5% polyclonal immune mouse serum and lO% fresh guinea pig complement (shaded bars) after 24 hr incubations -------------------------------- 95 Dose-dependent effects of 24-28 hr preincubations in poly-l-arginine on tegument potential recorded in adult male S, mansoni (closed circles) ----------------- 97 Acute effects of exposure (arrow) to lxlO'SM poly-l- arginine (closed circles) on tegument potential of adult male S, mansoni ---------------------------------- 99 Frequency of endogenous electrical potentials recorded from adult male S. mansoni showing dose-dependent effects of l— 2 hr incubations in poly- -l- -arginine----a-- lOl Dose-dependent effects of 24-28 hr preincubations in trifluoperazine on tegument potential recorded in adult male S, mansoni (closed circles) ----------------------- lO4 Frequency of endogenous electrical potentials recorded from adult male S. mansoni showing dose- -dependent effects of l- 2 hr in ncuEat1ons in trifluoperazine ------- l06 Acute effects of exposure (arrow) to lxlO'SM trifluo- perazine (closed circles) on tegument potential of adult male S, mansoni ---------------------------------- l08 Effects of preincubation for 24-28 hr in control medium (closed circles), lxlO‘5M cytochalasin B (closed tri- angles), lxlO'4M colchicine (open circles), or 200 ug/ml puromycin (open triangles) on longitudinal muscle tension changes produced by exgosure (arrows) to 60 mM potassium (upper graph) or l0 M carbachol (lower graph)- ------------------------------------------------ llO Dose-dependent effects of 2 hr preincubations in poly- l-arginine on longitudinal muscle tension changes in- duc d by exposure to 60 mM potassium (upper graph) or 10' M carbachol (lower graph) -------------------------- llZ ix LIST OF FIGURES (continued) Figure 21 22 Page Dose-dependent effects of 2 hr preincubations in tri- fluoperazine on longitudinal muscle tension chan es induced by exposure (arrows) to 60 mM potassium (upper graph) or lo-4M carbachol (lower graph) ---------------- ll4 Loss of covalently bound 1251 from adult male S, man- soni during incubations in RPMI-l64O alone (closed—— circles) or with 2xl0‘5M cytochalasin B (open circles), lxlO‘4M colchicine (open 5 uares), 200 ug/ml puromycin (open triangles), or lxlO' M trifluoperazine (closed triangles)--: ------------------------------------------ ll7 GENERAL INTRODUCTION The freshly transformed schistomulum represents a stage in the life- cycle of Schistosoma mansoni that is of extreme clinical importance. In addition to being the only mammalian stage that is not refractory to host immune effectors, the freshly transformed parasite is also charac- terized by a number of structural and biochemical features that could render it a distinct alternative target for chemotherapeutics. Despite its importance, little is known of the schistosomulum's physiological or biochemical properties. In light of this, I have developed an objective and highly quantitative electrophysiological assay for examining re- sponses of the immature parasite to a wide range of substances. In addition, correlations are obtained between the electrophysiological parameter and energy metabolism in various developmental stages of the schistosome. Schistosoma mansoni: Parasite, Life-Cycle and Disease Schistosoma mansoni is a digenetic trematode capable of infecting humans during one stage of its polymorphic life-cycle. The parasite is diecious, with male and female exhibiting extensive morphological and physiological differences at maturity within the definitive host. The life-cycle of schistosomes (Figure l) comprises the passing of ova from the gastrointestinal tract of a definitive host; their hatching Figure l. Life-cycle of S. mansoni. MAMMAI. FRESH WATER @egg (in feces) a A? paired adults ( mesenteries ) miracidium (free-living) {—G’L/ *‘ (in snails) 16 sparacysts lung stage A; schistosomulum \ cercaria @t skin (free- living) enetration skin stage p schistosomulum Figure l 4 in fresh water with the liberation of free-swimming miracidiae; the penetration of a suitable species of snail; the metamorphosis of the larvae into sporocysts then cercariae in the snail; the shedding of free-swimming cercariae into fresh water; the penetration of human skin by the cercariae; and the migration and growth of the immature worms in the liver and mesenteric vasculature (Noble and Noble, l976). Man is the most important definitive host for S, mansoni, although the parasite infects all primates and some rodents (Belding, l965). The agitation of infested water during such activities as bathing, planting, fishing, or recreation stimulates cercarial activity. Recent evidence indicates that fatty acids in the skin, particularly linolenic and linoleic acid attract the cercariae and promote penetration (Haas and Schmitt, 1982). During the penetration process, the cercaria attaches itself to the skin by its oral sucker, which everts while mucus is secreted from the postacetabular glands of the cercaria onto the skin of the host. This mucus substance appears to penetrate the layers of the squames and eventually swells, thereby forcing the epi- thelial cells apart and opening points of entry in the keratin layer along natural cleavage planes (Stirewalt and Dorsey, 1974). Bruce gt_ a1, (T970) describe fragmentation and granulation of the keratin layer, indicating that penetration may also be aided by chemical lysis. Stirewalt and Dorsey (1974) provide evidence that preacetabular gland secretions are released during the parasite's migration through subse- quent layers of the epidermis and dermis. The basement membrane of the dermo-epidermal junction appears to constitute the major barrier to migration, possibly requiring the concerted effort of many cercariae for successful penetration (Stirewalt and Dorsey, l974). 5 After penetrating the skin, the parasites enter the schistosomulum stage of development. They travel along the lymph ducts into the veins and lungs, and eventually the liver, where they mature into adult schistosomes within 5-6 weeks. The female normally lies enclosed within the ventrally located gynecophoric canal of the larger male parasite, leaving only for brief periods to penetrate the narrowest capillaries of the intestine and rectum during oviposition (Belding, l965). Most pathology associated with the disease, schistosomiasis, is due to the backflow of deposited ova which do not successfully pene- trate into the intestinal or rectal lumen for fecal expulsion. In the tissues, particularly the liver and spleen, the ova produce multiple foci of inflammatory cellular infiltration, ultimately producing exten- sive granulomatous pseudotubercles. These reactions are characterized by early eosinophilic and neutrophilic responses and late epithelioid cell and fibroblast involvement. The resulting granuloma formation ultimately leads to fibrosis of the liver and spleen, with accompanying extensive hydrodynamic impairment of hepatic-portal circulation. The number of persons suffering from schistosomiasis in various parts of the world today is estimated at 250-400 million, with approxi- mately l.5 billion at risk to the disease. It thus ranks second only to malaria in occurrence among infectious diseases. During recent decades, the disease has been steadily on the increase, particularly in areas where major efforts have been made to improve irrigation of the soil. As a result, regions in which sChistosomiasis had previously been endemic are showing a tendency to become hyperendemic zones. 6 Additionally, the wide and ever-increasing distribution of the disease is being accentuated by the increase in migration of infected people from endemic regions to the cities and other urban areas. Unfortunate- ly, currently available anthelmintics are either marginally effective or too expensive to be made available to third world inhabitants on a population-wide basis. While a vaccine for the disease has been the aim of intensive research during the past l5 years, its completion is probably many years away (Taylor, 1980). Anatomical and Physiological Characteristics Relevant to the resent Study The cercaria and early stage schistosomulum exhibit a body shape that is roughly pear-shaped with rounded ends. The cercaria also possesses a long tail with a terminal furca (Figure 2). The body dimensions of the parasites at these developmental stages are approxi- mately 20x50 um (Belding, l965). The mass of the cercaria minus its tail, or the early stage schistosomulum, is approximately 0.1% as great as the adult parasite's. Adult male S, mansoni are approximately 1 cm long and 0.2 cm wide, with a wet weight of 0.6 mg. Females are slightly longer than males, but only about l0% as wide and 25% as massive (Cornford §t_al,, l979). Both sexes possess rostrally located anterior and ventral suckers that are involved in locomotion and stabilization. The gross morphology of schistosomes is relatively simple. The tegument and underlying muscle bundles form a shell within which in- testinal cecae, protonephridia, and gonads are suspended in a paren- chymal matrix composed largely of lipid and glycogen particles and Figure 2. Micrographs of cercaria (left) and adult S, mansoni. The cercaria's tail is shed during transformation to the schistosomulum stage, however, the gross morphology of the body remains the same until after parasites reach the lungs on days 5—8. Calibration bars: 25 um (left), 500 pm (right). g”...,\-l.n ‘. ,u. .. s? it . fure 2 i F. 9 extracellular space. The nervous system is composed of two paired circumesophageal ganglia within which most nerve nuclei are located, and four lateral nerve trunks. From these trunks extend smaller fibers that synapse with muscle bundles, and may innervate putative sensory receptors located within the tegument (Morris and Threadgold, l967; Silk and Spence, l969a) (Figure 3). The outermost layer of the schistosome (i.e., the host-parasite interface) is a syncytium known as the tegument (Morris and Threadgold, 1968; Hockley, l977). In all stages of the parasite, the tegument contains: (1) spines that have thin filaments packed in a paracrystal- line array and extend from the surface membrane of the tegument to the basal lamina; (2) a few mitochondria; and (3) various membrane bound inclusions known as membranous, or multilaminate, or spherical bodies and elongate bodies (Morris and Threadgold, l968; Wilson and Barnes, 1974). The tegumental syncytium is connected by way of internuncial processes to cell bodies, called cytons, which contain nuclei, Golgi apparatus, and endoplasmic reticulum (Silk and Spence, l969a; Wilson and Barnes, l974). Internuncial processes pass through several layers, including: (T) a basal lamina, (2) an interstitial layer, and (3) an outer circularly arranged and an inner longitudinally arranged muscle layer in order to connect the tegumental syncytium with the cytons (Silk and Spence, l969a). The membranes bounding the tegument are complex. 0n the basal surface there is a classical trilaminate 8-l0 nm membrane which is continuous with the trilaminate membrane of the internuncial processes and the cytons (Hockley and McLaren, l973; Wilson and Barnes, l974). IO Figure 3. Schematic of tegument and muscle layers in S, mansoni. T, tegument; 0M, outer double membrane of tegument; BL, basal lamina; IM, internal membrane of the tegument; S, spine; MP membranous particles; EB, elongated bodies; IMa, interstitial matrix; CM,, circular muscle; LM, longitudinal muscle; TC, tegumental cyton; N, nucleus; CC, cyto- plasmic channel; JP, junctional process; L, lipid granule; ECS, extra- cellular space. ll - K, _ 'oé"“a;4T/" 'Y 7 a / O 0 cc ‘ ’ MP . , ‘\ L7.Io,.",,,,, a r“ f I]; (I ((IJrLo r h L! L, ' To 0‘ — w d _' m _ - 12 The morphology of the surface membrane depends on the stage of the 'life-cycle of the parasite (Smith et_gl,, l969; Hockley and McLaren, 1973). The cercarial membrane is trilaminate and is covered by a l pm thick extracellular coat which resembles the glycocalyx covering the brush border of intestinal epithelium (Hockley and McLaren, 1973). Upon penetration through the skin of the definitive host, most of this surface coat is lost (Stirewalt, 1963), but a few microvilli with some of the coat material remain on the surface of the freshly transformed schistosomulum (Brink gt_gl,, 1978). It has been suggested that the cercarial membrane is shed by means of the microvilli, but how much of the surface membrane of the cercaria is lost by this mechanism is unclear (McLaren and Hockley, 1976). Within 3-5 hr after penetration most of the trilaminate membrane is replaced by a heptalaminate mem- brane which consists of two adjacent lipid bilayers (Hockley gt_§l,, 1975). The surface membrane of the schistosome is important because it is the target of antibody-dependent cell mediated cytotoxic reactions which have been shown to kill the parasites jn_yjt§9_(5mithers gt_al,, 1977). The tegument of S, mansoni has also been implicated in several physiological processes. In addition to providing a barrier that is highly resistant to the host's degradative enzymes, it appears to be an important site of entry for glucose (Fripp, 1967) and several amino acids (Senft, 1968). A thin layer of material coats the outer surface of the tegument that is partially composed of acid mucopolysaccharides (Stein and Lumsden, 1973), and may contain enzymes involved in the absorption and digestion of nutrients (Lumsden, 1975; Pappas and Read, 1975; Ernst, 1977). There is also anatomical evidence suggesting that 13 innervated sensory structures are located in the tegument that may function to detect the direction of flow of the surrounding medium (Morris and Threadgold, 1968). Recent work conducted by Fetterer gt_al, (1980) has demonstrated that a well defined tegumental potential (E ) of about ~60 mV exists teg that can be altered by changing physical and/or chemical qualities of the parasite's environment. Elevated concentrations of external K+, re- duced Ca++, cold, and ouabain all induce depolarization of the tegu- ment, indicating that some form of active Na+-K+ transport must be involved in maintaining this potential (Fetterer gt_gl,, 1980). Fetterer gt_§l, (1980) employed horseradish peroxidase (HRP) as a marker for determining the location of the recording electrode while gathering these data. During all penetrations in which potentials in this range were recorded, HRP iontophoretically injected was subse- quently found to occupy only those compartments directly associated with the tegument (e.g., cytons cytoplasmic channels, and the outer tegument itself). Recordings made in the tegument of unanesthetized parasites re- vealed spontaneous depolarizations that ranged from 3 to 15 mV in amplitude with 20 to 100 msec durations. Most of this activity was abolished by anesthesia (Fetterer g§_al,, 1980). Other evidence sug- gesting that the tegument may actively propagate electrical activity has been reported in microelectrode (Thompson gt_gl,, 1982a) and sur- face electrical (Fetterer gt_gl,, 1977; Semeyn ggngl., 1982) record- ings. Alternatively, the transients recorded in these studies could be volume conducted depolarizations of underlying muscle elements which, 14 based on microelectrophysiological studies, are electrically coupled to the tegumental syncytium, with a coupling ratio of 0.85 (Thompson g3 31,, 1982a). A possible morphological substrate for electrical coup- ling between these tissues has been observed in electron micrographs. Plasma membranes of integumentary cells appear to form junctional complexes with neighboring muscle cells that closely resemble those connecting smooth muscle cells (Silk and Spence, l969a). The majority of the muscle system in S, mansoni is located imme- diately beneath the basement membrane of the tegument, where it is arranged in an outer circular and an inner longitudinal fashion (Silk and Spence, l969b). All muscle appears to be of the smooth variety with no striations, and in that sense it resembles most invertebrate muscle. Thick (180-400 3 diameter) myofilaments are surrounded by 8-14 thin (50 A diameter) filaments, a ratio very near that observed in vertebrate smooth muscle preparations (Perry and Grand, 1979). The thick filaments are arranged in parallel, while thin filaments exhibit a great deal of branching and cross-linking with other thin as well as thick filaments. Ovoid nuclei are normally separate from and deeper than the fiber bundles, and are connected to them by cytoplasmic pro- cesses (Silk and Spence, l969b). The sarcoplasmic reticulum in schistosomes is poorly developed, and appears only at scattered intervals. Mitochondria appear in sac- 1ike distensions of the sarcoplasm along myofibril bundles. Lipid globules as well as a and B glycogen particles are distributed through- out the muscle cells (Silk and Spence, 1969b). Junctional complexes are observed between the outer layers of adjoining sarcolemmas. These 15 junctions separate apposing cell membranes by about 7—9 nm, and are up to 400 pm long. They appear to be similar to those at the interface between cyton cells and neighboring muscle bundles (Silk and Spence, l969b). Fetterer gt_gl, (1977) have developed a technique for monitoring schistosome muscle activity directly, using suction electrodes in circuit with a force transducer. This method is, in some cases, as much as 100X more sensitive than those previously reported for measuring mechanical responses in the parasite. In addition to serving as a sensitive monitor for the effects of various pharmacological agents, this method has demonstrated that the contractile properties of the schistosome musculature are much like those reported for other inverte- brate as well as vertebrate muscle preparations. For example, elevated K+ (Fetterer gig” 1977) and hyperosmotic sucrose solutions (Pax gt 31,, 1981) both induce paralysis. Bricker gt_gl, (1982), using HRP as a marker, have recently identi- fied a second compartment of electrical potential in the schistosome as being endemic to muscle tissue. Resting membrane potentials recorded intramuscularly are generally about -30 mV, or about one-half as great as those recorded in overlying tegumental regions (Bricker g§_gl,, 1982; Thompson g§_gl,, 1982a). Electrophysiological recordings using multiple electrodes have revealed that the muscle compartment in schistosomes, like the tegument, is an electrical syncytium; and that low resistance pathways connect these two tissue compartments in a nonrectifying manner (Thompson _t_al,, 1982a). p '10 Developmental Changes in the Parasite within the Definitive Host Schistosomes undergo several important changes during the infec- tion process in man prior to their habitation of the liver and hepatic portal vasculature as mature organisms. There are three stages, in particular, during which the parasite undergoes marked changes that are easily identified: (1) during penetration of host skin, cercariae shed most of their surface coat and quickly adapt to new osmotic conditions (Stirewalt, 1963); (2) shortly after penetration, the parasite begins to show a decline in immunogenicity, so that by 24 hours it is refrac- tory to most immune effectors (Sher, 1982); concomitantly, the energy metabolism of the parasite begins to change from an aerobic to an anaerobic one (Coles, 1972a; Von Kruger g§_gl,, 1978); and (3) almost immediately upon entering the hepatic portal vasculature, the worms enter a period of exponential growth; this follows a period of 8 to 15 days of mitogenic dormancy which characterizes the lung stage of development (Lawson g£_al,, 1980). A number of theories have been advanced which attempt to define the conditions which precipitate the important changes that occur after transformation. Adaptation to the saline environment is thought to occur primarily as a result of physical changes cercariae undergo during penetration of host tissue. Three changes occur at the time of penetration that may be specifically related to osmoregulation in the new environment: (1) a rapid reduction in the carbohydrate-rich fila- mentous coat (glycocalyx) (Hockley gt_al,, 1972; Cousin gt 31,, 1981), (2) a transient appearance of microvilli at the surface of the tegument (McLaren g3_gl,, 1976; Brink gt_al,, 1977; Cousin §t_al,, 1981), and 17 (3) a rapid reduction of the bladder and collecting tubules (Cousin_33 31,, 1981). Because these changes are believed to be mediated by mechanical penetration itself, together with the osmotic pressure of the new environment, it is unlikely that they could be altered by chemotherapeutic intervention. Changes relevant to metabolism and the gradual loss of suscepti- bility to host immune reactions occur more gradually than the permeabi- lity changes associated with penetration. The precise timecourse as well as the mechanisms mediating these changes are not well understood. Previous 13_!113_studies have shown that damage mediated by antischisto- some antibody in the presence of complement or immune effector cells is possible only within the first day or two after a transcutaneous cer- carial infection (McLaren, 1982; Sher 3__31,, 1982). There are at least three processes that occur at the level of the tegument that could account, in part, for the development of immune refractoriness in schistosomes: (1) a transition in the outer tegumental membrane from a trilaminate to a heptalaminate (double membrane) formation (Hockley 31_ 31,, 1973), (2) the masking of endogenous surface antigens with host or host-like antigens (Clegg 31_31,, 1972; Dean 33_31,, 1977; Goldring 31_ 31,, 1977; Samuelson 31_31,, 1980), and (3) the rapid sloughing of tegumental membrane components from the surface of the parasite (Samuel- son 33_31,, 1982). Processes relevant to the development of the double outer tegu- mental membrane begin to occur immediately after the parasite enters the host. Multilaminate vesicles are transported from the cytons to the syncytium where they fuse with the outer tegumental membrane, gradually replacing the original trilaminate one. This process appears 18 to be completed within 3 to 5 hr after penetration (Hockley 33_31,, 1973). The fact that refractoriness to the cytotoxic effects of anti- body plus complement begins to increase within 4 hr after transforma- tion (Sher 33_31,, 1982) suggests that the double membrane structure may be important in conferring protection to the parasite. However, the parasite concomitantly shows reduced levels of antibody and C-3 binding (Samuelson 33_31,, 1980), and these factors may be more im- portant in the development of refractoriness. Furthermore, neutrophils will adhere to and phagocytise extensive areas of tegumental membrane in all stages of the schistosome, yet these effector cells are not cytotoxic to the parasites (Vadas 33_31,, 1979). Evidence supporting the theory that schistosomes evade host immune mechanisms by disguising their surface antigens is provided in a number of separate studies. Adult schistosomes share many surface antigens with their hosts (Smithers 31__1,, l969; Clegg 33_31,, 1970; Damian 33 31,, 1973). While some of these antigens appear to be produced by the parasite, others are apparently of host origin. This is supported by findings that schistosomula maintained 13_11133_and 13_3113_passively adsorb ABO bloodgroup (Goldring 31_31,, 1976), Forssman (Dean 31__1,. 1972) and histocompatibility (Sher 31_31,, 1978) antigens. Further- more, these antigens are serologically identical to host antigens found on adult worms. Thus, incorporation of host or host-like material into the tegument may confer protection from the host immune system by disguising endogenous parasite antigens, thereby rendering them anti- genically inert (Clegg 33_31,, 1972). In addition, recent 13_vitro tests demonstrate that significant amounts of host cholesterol and l9 triglycerides are taken up by the schistosome during the transformation process (Rumjanek, 1982). These additional cytochemical changes in the tegument could also contribute to the development of refractoriness by disguising the parasite with host material. Other attempts to define more specifically the biochemical changes that occur in the tegument which impart protection to the parasite from immune attack have implicated inherent processes that may occur in addition to or independent of the formation of double membrane or the masking phenomena. Wilson and Barnes (1977) have demonstrated that the outer tegument of schistosomes is continuously replaced, with a half- life of 2-4 hours in control medium 13_!1133, Thus, through a process of continuous turnover of the tegumental membrane, the parasite may effectively avoid lysis by host complement by detaching itself from its own antibody-bound epitopes. Wilson and Barnes (1977) have further demonstrated that the plant lectin concanavalin A appears to stabilize the vesicular fusion process which continuously provides new membrane to the host-parasite interface of the schistosome. Because of this effect, it was thought that concanavalin A might somehow interfere with the normal repair process of the outer tegumental membrane, thus rendering the parasites more susceptible to immune attack. However, while concanavalin A as well as lectin from Ricinus communis do exert profound tegumental disruption in adult S, mansoni, the effects do not depend on the presence of immune substances, and freshly transformed schistosomula are unaffected by these treatments (Simpson 31_31,, 1982). In fact, incubation of freshly transformed schistosomula for 5- 6 hr in culture medium containing 4 ug/ml concanavalin A may render this stage of the parasite more resistant to the lethal effects of antibody 20 plus complement, even though binding of anti-parasite antibody does not change from control levels (Van Pijkeren 31_31,, 1982). In another study designed to determine the effects of inhibiting a tegumental process on the immune response, Kemp 31_31, (1978) showed that schisto- somes actively shed antischistosome antibody from their surface with a half-time of less than 20 min, and that this process is inhibited by 10'5M cytochalasin B, implicating a role for microfilaments in this process. No data are reported in their study, however, regarding possible changes in cytotoxicity brought about by drug-induced inhibi- tion of antibody elimination. It is also possible that changes in the distribution of tegumental integral membrane proteins (IMP) in schistosomes is important in the process of immune evasion (McLaren 31_31,, 1978). Freeze fracture studies indicate that the IMP of the outer tegumental membrane of schistosomes cultured 13_31133_or recovered from mice undergo dramatic changes in number and distribution during the early stages of trans- formation. During the first 4 hr, the outer bilayer is mostly lipid with few IMP. By 6 hours after penetration, the number of IMP in the outer tegumental membrane begins to increase, reaching a peak in the E2 face of the outer bilayer by day 4, after which they decline (McLaren 33_31,, 1978). The extent to which these IMP are targets for specific or nonspecific immune mechanisms has not been determined. Another important stage in the development of the schistosome occurs while the parasite occupies the lungs of the vertebrate host. After reaching the lungs, the young parasites slowly increase up to 4 times in length, but not at all in mass. This change results in a 50% 21 _ increase in the surface to volume ratio of the organism, and probably facilitates migration through the lumina of blood capillaries (Wilson _13 _1., 1977; Miller 31_31,, 1979). Also linked to mobility enhance- ment and occurring during the lung stage is the regression of tegu- mental spines and sensory papillae, so that only those at the rostral and caudal extremes of the parasite remain. These organs are observed to reappear immediately after the hepatic migration which begins to occur on day 8 or 9 (Miller 31_31,, 1979). Coincident with the above changes are a series of superficial tegumental enfoldings that appear to pinch off, incorporate, and de- generate surrounding host lung tissue (Bruce 31_31,, 1974). This process begins immediately upon the schistosomulum's arrival in the lungs, and is probably linked to the appearance of additional host antigens on the tegumental surface, which by day 5 completely obscure identifiable worm surface antigens (Goldring 33_31,, 1977). By this time, neither hyperimmune serum from a previous infection (Smithers 33 31,, 1965; McLaren 31_31,, 1975) nor cell mediated immune mechanisms (Sher 31_31,, 1982) exert significant effects on the schistosomula 13_ vitro or 13_vivo. Within 5 days after penetration, the schistosomula begin to migrate from the lungs to the liver and hepatic portal vasculature where they remain. This migration may require several trips through the systemic circulation. Most parasites will reach the liver by day 12 or 13 (Miller _3 31,, 1979). Prior to reaching the liver, immature worms appear to be in a semi-quiescent metabolic state during which: (1) no mitosis occurs, (2) their mass is slightly reduced and (3) no net N2 incorporation occurs (Lawson 31_31,, 1980). Immediately upon entering 22 the hepatic portal system, some unknown mechanism(s) signal the ini- tiation of a period of rapid growth and metabolic activity. Wilson 3t_ 31, (1978) have suggested that this process may be a direct response to the nutrient-enriched environment. Also apparent at this time is a doubling in the duration of the peristaltic contractions of the para- site (Wilson 31 31,, 1978), and an enhanced level of responsiveness to the drug hycanthone (Tomosky 31_31,, 1977), which stimulates mechanical activity. After the schistosomulum enters the liver, additional changes in its development appear to be primarily quantitative. However, Coles (1973) points out that several drugs tested in the past have exhibited a range of effectiveness on schistosomes at various developmental stages. Most thoroughly studied have been the antimonial derivatives, which are extremely effective at killing worms in the first week of infection (Schubert, 1948), but less so on 14-21 day old parasites (Standen, 1955). Stohler _1_31, (1963) demonstrated that the drug becomes effective once again by day 34. Lucanthone HCl also shows a biphasic pattern of effectiveness, being lethal to 0-24 hr worms (Stohler 33_31,, 1963) and worms older than 28 days. Other drugs tested showing a similar biphasic pattern of effectiveness include maleic acid-mono-4(3'-chloro-4'-methylpheny1)piperazide (Lammler, 1958), nitrothiazole derivative (Sadun 31_31,, 1966), 2-aminomethy1- tetrahydroquinolone.derivatives (Foster 33_31,, 1971), and nitrovinyl furane (Lennox 31__1,, 1972). It appears from these studies that worms in the 3rd and 4th weeks of an infection are particularly resistant to drugs. It has not yet been determined how much of this is due to 23 changes in worm metabolism, changes in position within the host, or to other developmental factors. Metabolic Changes Occurring in S. mansoni within the Definitive Host Coles (1973) has suggested that changes in the schistosome's energy metabolism may contribute to the observations that 13_3113, both antimony (Coles, 1972) and cyanide (Bueding 31_31,, 1953) show lower levels of depression when administered to 2—3 week old infection stages than when administered to mature stages. These changes are important because major differences in the metabolism of the parasite and its host could hold the key to the development of successful chemothera- peutics. A number of previous studies have examined energy metabolism in various stages of the parasite's life-cycle. Oliver 33_31, (1953) showed that the free-living cercaria of S, mansoni is an aerobic orga- nism with the potential to obtain some energy by fermentation. Results of inhibition studies suggest that a functional citric acid cycle is present in the cercaria (Bruce 31_31,, 1969), and a number of the enzymes of aerobic carbohydrate metabolism have been assayed quanti- tatively (Coles, 1972b; Shapiro 31_31,, 1983). Oxygen uptake in the cercaria has been measured using Clark electrodes by Coles (1972) and Von Kruger 31_31, (1978) and found to be on the order of 80-100 ul 4M cyanide showed an 83% Oz/mg protein/hr. Cercariae incubated in 2x10- inhibition of oxygen uptake (Coles, 1972). Furthermore, Coles (1972) has shown that cercariae made anaerobic or treated with 10-3M cyanide or 10'3M fluoroacetate excrete low levels of lactic acid and demon- strate a Pasteur effect; i.e. increased glycogen usage under anaerobic 24 conditions. Quantitative determinations of glycogen utilized (measured as glucose) and lactic acid formed after cercariae had been kept for 2 hours in 10'3M NaCN showed that all of the glucose used under these conditions could be accounted for by lactic acid formed; i.e. cercariae are homolactic fermenters under anaerobic conditions. However, Coles 3M cyanide (1972) further reported that exposure of the cercariae to 10- for 4 hours appeared to result in mortality, based on visual observa- tions of their motility levels. This effect was not attenuated by the addition of high levels of glucose to the incubation medium. After penetrating the mammalian skin, the newly transformed schistosomulum begins to excrete lactic acid under both aerobic and anaerobic conditions (Coles, 1972), although respiration continues to be of some importance in energy production (Coles, 1972). The time- course and the mechanism of the switch from the fully aerobic metabo- lism of the cercaria to the anaerobic one of the adult schistosome are unknown. Shiff (1972) has suggested that the trigger which initiates penetration of the cercaria, believed to be host fatty acids, may also initiate the metabolic change. Coles (1972) found that by raising the osmotic pressure of their incubation media, cercariae were induced to transiently excrete lactate, suggesting that the metabolic shift may be a response to elevated osmotic pressure. Alternatively, Von Kruger 33_ 31, (1978) have shown that mere separation of the cercarial body from its tail initiates lactate excretion. The freshly transformed schistosomulum takes up oxygen at a rate of 20-30 pl Oz/mg protein/hr (Von Kruger 31_31,, 1978), or about one- third of that recorded in the cercaria. This level is reduced by 85% in the presence of 2x10'4M cyanide (Coles, 1972). Other metabolic 25 inhibitors shown to depress respiration in the schistosomulum include rhotenone, sodium arsenite, and sodium malonate (Von Kruger 31_31,, 1978). Much of the reduction from cercarial levels in oxygen uptake recorded in the freshly transformed schistosomulum is attributed to the loss of the cercaria's tail, which accounts for 30-35% of its total protein content (Coles 31_31,, 1972). The outstanding feature of carbohydrate metabolism in the adult schistosome is the rapid rate of glucose uptake and utilization, and the subsequent high levels of lactic acid produced. The adult may use up to 26% of its dry weight in glucose per hour in lactate fermentation (Bueding, 1950). Most energy needs of the adult schistosome are met anaerobically, so the adult stage does not appear to have a strict requirement for oxygen in energy production. Evidence for the lack of a role for oxygen in the energy metabolism of adult schistosomes is presented primarily in the work of Bueding 33_31, (1950, 1959, 1969, 1972, 1982) and Schiller (1975). Their studies have shown that the survival of S, mansoni 13_31333_is not adversely affected by the ab- sence of oxygen (Schiller 31_31,, 1975; Bueding 31.31,, 1972). Glucose utilization and production of lactic acid has been shown to be similar under aerobic and anaerobic conditions, indicating the absence of a Pasteur effect. Furthermore, ATP levels in the parasite are not re- duced after incubation under nitrogen, even after periods of increased carbohydrate metabolism induced by exposure to 5x10'5M 5HT (Bueding, 1972). In previously tested vertebrate and invertebrate preparations that show a Pasteur effect, phosphofructokinase activity, a rate- 1imiting factor in glycolysis, is inhibited by citrate, an important 26 intermediate of respiration (Passenneau and Lowry, 1964). It is be- lieved that the mechanism underlying the Pasteur effect is based on the reduction of citrate brought about by anaerobiosis and the subsequent disinhibition of PFK. Unless the organism possesses a cyanide-insensi- tive terminal oxidase, a Pasteur effect is also brought about by inhi- bition of the terminal oxidase of electron transport by cyanide. Alternate oxidases have not been reported in schistosomes (Coles, 1972). Production of ATP by glycolysis is inhibited in schistosomes by the presence of trivalent antimonials (Coles, 1978). Schistosomes exposed 13_vitro or 1__vivo to trivalent antimonials show elevated levels of glucose-6-phosphate and fructose-6-phosphate and reduced levels of fructose-1,6-diphosphate. These sugar phosphate levels return to normal when the drug is withdrawn or the worms are trans- ferred to untreated hosts (Bueding and Fisher, 1966). This information has been used to argue that antimony compounds are toxic to schistosomes because of their inhibition of PFK (Bueding and Fisher, 1966; Bueding, 1959). More recent studies using worm extracts have corroborated this view (Coles and Chappell, 1979). Shen 33_31, (1959) have demonstrated that the antimonials also inhibit glycolysis in S, japonicum. Their studies, however, indicate that they may also inhibit schistosome glutamic-pyruvic transaminase, suggesting another possible mode of action for these compounds. OBJECTIVES The general goal of the present study is to gain a better under- standing of the biochemical and physiological properties of the schisto- somulum of Schistosoma mansoni. To date, most research on the parasite has focused on the adult stage. Because of their size and the diffi- culties involved in their recovery from host organisms, the schisto- somulum stage of the parasite has been studied on an extremely limited basis. Recent findings, however, indicating that the freshly trans- formed parasite is the most important and perhaps the only viable target for immune mechanisms 13_3113_(McLaren 31_31,, 1982; Sher 33 31,, 1982; Samuelson 31_31,, 1982) have stimulated a great deal of interest in the schistosomulum. Previous work on the schistosomulum stage has focused on the morphology of the parasite or its responses to various immune sub- stances. These studies have relied primarily on assays based on pat- terns of recovery (Periera 31_31,, 1975; Mamoud, 1979), microscopic evaluation (Torpier 31_31,, 1979; Kassis 31_31,, 1979), or release of 51Cr (David 31_31,, 1977); all of which are time-consuming, difficult to quantify, and of questionable validity (McLaren, 1982). A major objective of the present study is, therefore, to develop an objective and quantifiable assay for detecting physiologiccal responses in the immature stages.of S, mansoni. 28 Studies on the immature stages of the schistosome have been aided by the development of techniques for artificially transforming large numbers of cercariae to the schistosomulum.stage by means of mechanical decaudation in serum-containing media (Ramalho-Pinto 31_31,, 1974). These organisms develop the outer double membrane in the same manner, and over a similar timecourse as schistosomula which have penetrated through the skin of a mouse (Brink 3331., 1977). Furthermore, the i_n_ 11133_development of artificially transformed schistosomula parallels that of skin penetrated organisms, and when the two forms are injected intravenously into mice, comparable percentages of the organisms develop to maturity. The only morphologically detectable differences appear to be the longer retention of acetabular gland contents and elements of the glycocalyx by the mechanically transformed schistosomula (Brink 31_31,, 1977). Both types of schistosomula bind antischistosome antibodies, as visualized by immunofluorescence, but the binding is slightly stronger in artificially transformed worms (Brink 33_31,, 1977; Bickle and Ford, 1982). Based on the above studies, it appears that the artificially prepared schistosomula fulfill all of the main criteria for transfor- mation from cercaria to schistosomulum (Stirewalt, 1974). Since they can be prepared in large numbers, uncontaminated by host material, they appear to constitute an excellent source of organisms for experimental analysis. It should be noted, however, that because the mechanically- transformed parasites do not have the opportunity to acquire surface molecules from the host skin they may present a significantly different surface configuration from that of the skin-transformed organisms. In 29 this context it is relevant to note that both Tavares 31_31, (1978) and McLaren (1982) have reported enhanced susceptibility of 13_11133_de- rived schistosomula to the effects of complement-dependent mouse anti- schistosome antibody in an immune assay system. However, because the artificial transformation techniques yield large quantities of orga- nisms with relative ease, it is likely that they will be used exten- sively in future studies that require large numbers of parasites. It will be extremely important, therefore, to determine more precisely the extent to which these organisms are suitable material for study. Thus, a second major objective of the present study is to determine the extent to which biochemical and physiological responses in the arti- ficially transformed schistosomulum parallel those of the skin pene- trated schistosomulum. A third major objective of the present study is to elucidate more precisely the timecourse of the metabolic conversion undergone by the parasite during its development from the cercarial stage, which is aerobic (Coles, 1972), to the adult stage, which is predominantly anaerobic (Bueding 31_31,, 1950, 1959, 1982). Because the adult schistosome exhibits an energy metabolism which differs significantly from that of its vertebrate host, this metabolic conversion is ex- tremely important, as enzymes and intermediates involved in energy formation are likely targets for chemotherapeutics (Coles, 1972). A final major objective of the present study is to obtain addi- tional information on the mechanisms by which the adult schistosome evades the complement mediated effects of anti-schistosome antibody 13_ 11133, If the principal mechanism(s) by which immune refractoriness is accomplished are linked to the parasite's capacity to insert new 30 membrane into its outer tegumental membrane (Hockley 31_31,, 1973) or to rapidly slough off anti-schistosome antibodies (Kemp 33_31,, 1980), then inhibition of these processes during 13_31133 incubations should render the parasites more susceptible to immune mediated damage. The present study is divided into three sections, each of which directly addresses one or more of the preceding objectives. Each section contains a Summary, Introduction, Materials and Methods, Results, and Discussion. It is hoped that the information obtained through these studies will make possible increasingly rational ap- proaches to the study of, and ultimately the clinical control of schistosomiasis. SECTION I Schistosoma mansoni: A Comparative Study of Schistosomula Transformed Mechanically and by Skin Penetration. Electrophysiological Responses to a Wide Range of Substances Summar Volume conducted electrical potentials were recorded from schisto- somula of Schistosoma mansoni transformed mechanically (MS) and by skin penetration (SS). The spontaneous electrical activity recorded con- sisted of bi- and triphasic transients ranging from 20-200 uV in ampli- tude and 10-300 msec in duration. Low amplitude potentials occurred at a much greater frequency than large amplitude potentials, which ap- peared to correlate with peristaltic-like contractions of the schisto- somulum's musculature. Electrical activity in the schistosomulum was highly sensitive to a number of agents believed to affect metabolic pathways, the neuromuscular system or the host/parasite interface of adult schistosomes. Among the most reactive substances were potassium antimony tartrate, eserine, poly-l-arginine and potassium cyanide. Over a wide range of experimental treatments, electrophysiological responses in schistosomula transformed from cercariae by mechanical decaudation and skin penetration were remarkably similar, supporting the notion that MS are suitable material for 13_11133_immunochemical, biochemical and physiological study. Some treatments, however, were 31 32 more or less effective in altering electrophysiological activity and .motility in the schistosomulum than in adult S, mansoni. This suggests that significant physiological atlerations may occur during develop- ment from skin stage to adult parasites concomitant with immunochemical and morphological changes already known to occur. Introduction Schistosomes undergo a number of structural and biochemical changes during transformation from cercaria to schistosomulum. Within three hours after host penetration, the glycocalyx is lost, the outer tegumental membrane becomes heptalaminate (Hockley and McLaren, 1973), the parasites become sensitive to hypo-osmotic conditions (Stirewalt, 1963), and produce lactic acid under aerobic conditions 13_11133. (Coles, 1972). In addition, the newly transformed schistosomula under- go a rapid, nearly linear decline in susceptibility to rejection by passively transferred immune serum (Sher, 1977), 13_111§3_cu1ture with antibody plus complement (Dean, 1977), or 13_11133_exposure to comple- ment plus eosinophils (Dessein, Samuelson, Butterworth, Hogan, Sherry, Vadas and David, 1981). This development of refractoriness to anti- body-dependent killing appears to be an inherent process that occurs in the absence of host macromolecules and is complete within 24 hours after transformation (Dean, 1977; Moser 33_31,, 1980; Samuelson 31_31,, 1980), although some recent studies suggest that the parasite becomes susceptible again between 6 and 14 days post-infection (Smithers and Gammage, 1980; Georgi 31_31,, 1983). Techniques for transforming large numbers of cercariae 13_11113_ using isolated skin penetration (Clegg and Smithers, 1972) or mechanical 33 decaudation (Ramalho-Pinto 33 31,, 1974) have made the schistosomulum readily accessible for study. Most studies indicate that parasites prepared by these methods fulfill the main criteria of transformation from cercaria to schistosomulum (Stirewalt, 1964), and are therefore appropriate material for immunochemical and physiological studies (Brink 31_31,, 1977). Some recent findings of Tavares 31_31, (1978) and McLaren and Incani (1982), however, suggest that schistosomula derived mechanically exhibit an enhanced susceptibility to the effects of complement-dependent anti-schistosome antibody. This difference could be due to the absence of host material on the surface of mecha- nically-transformed worms that is acquired by skin penetration worms during the penetration process (Smithers 33_31,, 1969). That is, host antigens specific for the glycocalyx of skin epidermis which are found on skin penetrated parasites (Smith and Kusel, 1979) could mask exposed epitopes of the schistosomulum. Alternatively, the differences in immune sensitivity could be due to changes in parasite lipid composi- tion which accompanies skin penetration (Rumjanek, 1982) or to exposure of the parasite to its own lytic substances secreted during the pene- tration process (Bruce 33_31,, 1970). Because the freshly transformed schistosome is the principal target for immunological control, any changes undergone by the parasite during this period are potentially important. While numerous transfor- mation-related changes in the parasite's structure and immunochemistry have been documented, very little is known about the physiological properties of the schistosomulum. In this paper, we describe an objec- tive and quantifiable technique for analyzing the short-term 13_vitro 34 effects of experimental substances on electrophysiological properties of the schistosomulum. Electrical responses to a wide range of sub- stances were measured in mechanical and skin transformed parasites in order to determine whether or not significant physiological difference exist between the two groups. Materials and Methods Preparation and culture of schistosomula. A Puerto Rican strain of S, mansoni was maintained by passage through Swiss Webster mice and Biomphalaria glabrata snails. Schistosomula, the larval or skin stage parasites, were prepared from the same batch of cercariae by 2 methods. Mechanically-transformed schistosomula (MS) were obtained by a modifica- tion of the method of Ramalho-Pinto 33 31, (1974). Cercariae were collected in dechlorinated water, placed in 15 ml centrifuge tubes and cooled to 4°C for 10 min to reduce motility. After centrifugation at low speed for 1 min the supernatant was decanted and replaced by a cold 50:50 mixture of RPMI-164O (Gibco, Grand Island, New York) and heat inactivated horse serum. The packed cercariae were resuspended and stirred for l min at moderate speed in a Vortex mixer. Following a 30 min incubation at 30°C, transformed parasites observed to be motile under a dissecting microscope were individually transferred by way of suction pipettes into vials containing RPMI-164O alone, and maintained at 37°C. Skin penetration schistosomula (SS) were prepared by a modi- fication of the method of Cousin 31_31, (1981). Approximately 1,000 cercariae in dechlorinated water were pipetted onto the inner surface of the ears of ether-anesthetized Swiss Webster mice and allowed to 35 penetrate for 45 min. The mice were killed by cervical dislocation; nonpenetrating cercariae were removed by cotton swabs; the ears were removed and finely minced in a solution of RPMI-l640. After mincing, motile schistosomula were individually transferred into vials contain- ing RPMI-164O and maintained at 37°C. For both groups, transformation was verified by means of the methylene blue dye exclusion test (Clegg and Smithers, 1968). Apparatus and recordingflprocedures. Endogenous electrical acti- vity was recorded from schistosomula between 3 and 6 hr after transfor- mation by a modification of the method described by Fetterer 33_31, (1977). Glass suction electrodes were manufactured from 1.25 mm capil- lary tubing (W.P. Instruments, New Haven, CT) that was pulled to a fine tip on a horizontal electrode puller (Narishige, Tokyo, Japan). The electrodes were modified by cutting back the tip with a diamond pencil so that the new tip was relatively flat, with an outside diameter of 10-15 pm. The electrode tip was then heated on a microforge (Aloe Corp., NY) which melted the glass from the inside. Heat was applied until the flattened tip of the electrode was smooth and the aperture was reduced to about 2 pm. Electrodes were firmly attached to the parasite's surface during electrical recordings by way of negative pressure created by a 10 m1 syringe (Figure 4). Prior to attachment of the parasite, Hanks' Balanced Salt solution (HBS) contained in a separate vial was drawn into the electrode until it made contact with a silver wire which led to an amplifier (Model P-15, Grass Instruments, Quincy, MA). Electri- cal signals from the schistosomula were filtered with the low pass set 36 Figure 4. Schematic of apparatus used for recording endogenous elec- trical transients from the surface of schistosomes. Volume conducted potentials were detected by way of a microforged glass suction elec- trode constructed from 1.25 mM capillary tubing. Electrical signals were filtered at the first amplifier with the low pass set at 0.3 Hz and the high pass set at l KHz. Signals were displayed on an oscillo- scope or a chart recorder and passed onto an analog-to-digital con- verter prior to computer analysis. t:;::>\\s o— CRO than recorder suction cleared. < computer ——_‘1 5525555 and converter prapara'ion ---I-- primer Figure 4 38 at 0.3 Hz and the high pass at 1 KHz, displayed on an oscilloscope (Model 5113, Tektronix Inc., Beaverton, OR) or a chart recorder (Narco- Biosystems Inc., Houston, TX), and passed onto an analog-to-digital converter prior to computer analysis (Alis I-10 Data Acquisition and Control System, Ecotech, Inc., Lansing, MI). After electrode attachment, a 2 min equilibration period was allowed prior to electrical recording. During this time the parasite was observed and its level of motility recorded as being above control level, (+); control level, (0); below control level, (-); or nomotile (=). The recorded electrical activity was quantified by counting all negative field potentials in excess of 20 uV over six lO-sec intervals. Data from these intervals were then averaged. Potentials under 20 uV were not counted as they were within the noise range of the system. Electrical responses were measured in at least 6 control parasites and 6 parasites exposed to each experimental substance. Statistical analy- sis for significance of differences between means used Student's E-test non-paired. Experimental treatments. Praziquantel (Bayer AG), oltipraz (Rhone-Poulenc) and cytochalasin 8 (Sigma) were initially dissolved in dimethylsulfoxide (DMSO, Mallinckrodt) at such concentrations that the final incubation media never contained more than 0.1% DMSO. An appro- priate amount of DMSO was placed in all controls. Concanavalin A (Pharmacia), potassium antimony tartrate (KSB, kindly supplied by Dr. E. Bueding, Johns Hopkins University, Baltimore, MD), aminophylline, carbachol, 2,4-dinitrophenol (2,4-DNP), dopamine (DA), eserine, 5- hydroxytryptamine (5HT), linolenic acid, ouabain, sodium pentobarbital 39 (Na-PB), poly-l-argninine, poly-l-lysine, potassium cyanide (KCN) and purOmycin (all from Sigma) were dissolved in double-distilled water. In all experiments RPMI-l640 alone, or with an appropriate amount of DMSO, served as the control medium. Most drugs were tested in the range of concentrations demonstrated to be most effective in altering endogenous electrical activity in adult parasites. The incubation period, in most cases, was 1 hr; exceptions included 5HT, linolenic acid (15 min), cytochalasin B, concanavalin A (3 hr) and puromycin (6 hr). In altered ion experiments, HBS served as the control medium. Elevated potassium HBS was made by replacing NaCl with KCl to bring the final potassium concentration to 60 mM. Ca++-free HBS was made by eliminating CaCl2 from the solution and adding 0.5 mM ethyleneglycol- bis(s-aminoethy1ester), N,N'-tetraacetic acid (EGTA, Sigma). Parasites were individually pipetted into HBS or altered ion media 15 min prior to recording. Monoclonal antischistosomulum antibody (kindly supplied by Dr. Michael Phillips, University of Pennsylvania) was prepared as described by Zodda and Phillips (1982). The monoclone was added to a solution also containing fresh guinea pig complement and a 50:50 mixture of RPMI-l640 plus heat inactivated horse serum (RPMI/HS) in a volume ratio of 1:3:30. Motile MS and SS were individually pipetted into vials containing the monoclonal solution 1 hr after the completion of trans- formation and incubated at 37°C for a period of 24 hr prior to testing. Controls consisted of schistosomula incubated for a similar period in solutions containing: (a) RPMI/HS alone, (b) RPMI/HS plus fresh 4O complement, or (c) RPMI/HS plus monoclonal antibody. Incubation media also contained 100 units/ml penicillin and 100 ug/ml streptomycin. Results Characteristics of spontaneous electrical activity. Endogenous electrical activity recorded from the surface of schistosomula of S, mansoni consisted of multi-phasic potentials ranging from 20-200 uV in amplitude and 10-300 msec in duration. Under control conditions (in RPMI-l640, 37°C) these potentials occurred at a rate of 31.511.3/sec for MS and 31.431.6/sec for SS with an average amplitude of 35.811.4 uV and 34.3:0.7 uV, respectively. Rise times for potentials in both MS and SS were on the order of 3-8 uV/msec. There was an approximately exponential decrease in the frequency of potentials with increasing amplitude (Figure 5). All features of electrical activity recorded from mechanical and skin transformed schistosomula under control condi- tions were essentially identical. After a brief (1-2 min) episode of enhanced activity which nor- mally followed electrode attachment, the frequency of low amplitude potentials was relatively constant over a period of up to 30 min of recording in both MS and SS. Large amplitude potentials (>50 uV) occurred more sporadically and appeared to correlate with gross peri- staltic-like contractions of the parasite, suggesting that the origin of these transients may be underlying muscle bundles. To verify that the electrical activity originated from within the parasite, controls were conducted by recording from worms before and after the addition of 50% ethanol to the recording medium. This treat- ment immediately immobilized the parasites and eliminated nearly all 41 Figure 5. Electrical activity recorded from the surface of mecha- nically-transformed 3 hr schistosomula under control conditions and after the addition of 50% ethyl alcohol to the recording medium. Upper: slow speed chart recording showing decline in electrical acti- vity after alcohol exposure. Middle right: high speed oscilloscope traces obtained from the same parasite before (upper) and after (lower) exposure to alcohol. Note the elimination of all large amplitude multiphasic potentials. Lower: computer-generated histogram showing the frequency distribution of endogenous electrical transients of various amplitudes. Open bars represent electrical activity recorded from schistosomula incubated in HS/RPMI; hatched bars represent elec- trical activity recorded from the same parasites 1 min after the addi- tion of alcohol. Vertical lines are i_l SE; N=8. Average No. Pealu I 10 sec 200 _- 00 50 10° Peak Amplitude (NV) MAW" 15_°HV 20 msec WWW, >150 Figure 5 43 electrical transients (Figure 5). No distinct regional variation in the frequency or amplitude of electrical activity was detected. The level of electrical activity was similar for schistosomula incubated in , RPMI-l640 alone or in a 1:1 mixture of RPMI-164O plus heat-inactivated horse serum. .After a 15 min incubation in HBS, however, the frequency of activity was reduced by about 25%, while the average amplitude of potentials was unchanged. Ion substitution experiments. Recordings obtained from schisto- somula incubated in HBS containing 60 mM potassium or 0 mM calcium plus 0.5 mM EGTA indicate that both ions are important for generation of the electrical transients recorded. The frequency of potentials recorded after 15 min in 0 mM Ca++-HBS plus 0.5 mM EGTA was reduced by 63% in MS and 53% in SS. The average amplitude of potentials was also reduced by this treatment; 25% in MS and 27% in SS. The frequency of potentials was also reduced in parasites exposed to elevated K+; 21% in MS and 49% in SS; average potential amplitude, however, was unaffected in both groups. Motility in the schistosomulum appeared to be unaffected by exposure to 60 mM K+-HBS, while the absence of Ca++ in the medium resulted in a gradual reduction in motility and a significant decrease in the frequency of large (>50 uV) amplitude potentials in both MS, down 75% and SS, down 94%. Drug responses. A number of substances tested exerted signifi- cant effects on the motility and electrical activity of the schisto- somulum (Table 1). MS and SS responded qualitatively in a similar fashion to all drugs which exerted significant effects on motility and endogenous electrical activity. Treatments producing highly signifi- cant (p<.01) changes in schistosomulum activity were: lxlO'SM 44 ....mwa=aa=ou A-v nemm- on gave. a: _ ze-c_x_ Pagumncau Auv aemm- Auv meme- a; P 2m-o_x_ apacpaap seoewpe< on as- Roy Nop+ 2; _ 2m-o_x_ a=__Fs;ao=_e< Auv afioo_- Auv mama- ewe N gem Focazum umscoemcmch umscowmcmch mesmoaxm umumop acmspmmch c_xm >p_muwcmgumz we me_h cowumgucmuzou _mgcwswcmaxm «Azuvpruozv mxuw>wpu< pmuwcuumpm & umpm>04 Focucou soc» mmcmgu mmucmumnzm Pmpcmswcmaxm An unon< uzmzocm m_:somoumw;um vmacowmcmchuc_xm can Iquwcmcomz cw zpwpwuoz can auw>wuu< qu_cuum—m :_ mmmzmgo p m4m<~ 45 .mpwuoscoc Anv upm>mp pagacou zepmn A-V mm_m>m_ Pocpcou on upm>wp Pocucoo m>onw A+v "mammgpcwgma cw Fm>mp xuwpwpoza mo.ovavpo.on _o.ovam on em_- on emm- a; «N A>\>v om", Aa=o_mv sconwuc< on em_+ on em+ a; em A>\>V omu_ Aa=o_av acmea_aeou A-V memo- A-v mean. an em A>\>V on .”m seonwp=< + pcmsmpasou Ace neme- on &_N- ewe m_ --- mm=-+¥ :5 on A-v name- A-v name- see m. -- mm:-++au o A+v QNNN+ A+V N__+ =_e m. zm-o_xp Apzmv cwcoaocmm on em_- on em- a: o _e\ms com etchedasa A-v.asee- A-v exec- a; _ zm-o_xp _aa=e=chmca A-v mesa- A-v aspm- a; _ ze-o~x_ a=_msa-S-s~oa A-V meme- .o.z a; P zo-o_x_ .werewma<-S-s_oa uwecoemcmch umEgowmcmcp mczmoaxm umammp pcmapmmcp cwxm appnuwcmcumz we wave cowumcucmucou —mucwewcmaxu sfisawpwpozv mspwsweu< .muwepuapm & ”mpw>m4 Focacou soc» mmcozo fleasespcoov _ msm50 uV) potentials (0.0150 uV) occurred more sporadi— cally and appeared to correlate with peristaltic-like contractions of the parasite, suggesting that these transients may originate in under- lying muscle bundles. To verify that the electrical activity origi- nated from within the parasite, controls were conducted by recording from worms before and after addition of 0.5 ml 50% ethanol to the recording medium. This treatment immediately immobilized the parasites and eliminated nearly all electrical transients (Figure 5). Developmental changes in substrate utilization. The results presented in Figure 6 demonstrate that the schistosome undergoes a pronounced metabolic alteration during development from the 3 hr schisto- somulum to the adult stage. Most of this change appears to be complete 65 Figure 6. Developmental changes in S, mansoni in the distribution of metabolic endproducts after 4 hr incubations in D-[14Cngucose-con- taining media under control conditions. Values presented are means of at least 3 separate experiments performed in triplicate (N39); vertical lines are 1 SE. 'IO 0 0 .3 Panza >an A:3\30 v3.93}; 0 0 1 200 5 A; \5295 uE\¢