Journal of Chemical Ecology, Vol. 12, No. 8, 1986
INTERNAL CHEMICAL COMMUNICATION WITHIN FLATWORMS
P A U L F. BASCH Department of Family, Community & Preventive Medicine Stanford University School of Medicine Stanford University Medical Center Stanford, California 94305
(Received October 1, 1985; accepted February 10, 1986) Abstract--An understanding of reproductive function is important for control of parasitic helminths. In cestodes and trematodes virtually nothing is known about regulatory and coordinating mechanisms that control maturation, gamete formation, egg production, and related processes. Neurosecretory neurons have been reported in various species but specific modes of action of neurohormones have yet to be demonstrated. The role of ecdysone is being investigated. Key Words--Schistosomes, nerve-hormone interactions, platyhelminths, differentiation reproductive maturation, Schistosoma mansoni, ecdysteroids.
In all multicellular animal groups from sponges to vertebrates, hormonal and neural processes play an important role in control and coordination of reproduction and other somatic functions. Reproduction is almost the key to the existence of trematode parasites; maturation and egg production account for much of their metabolic activity. The egg is the basis o f transmission and distribution, and in schistosomes is also the agent of pathogenesis, yet little is known about the control or reproduction in these parasites. There is need for a broad understanding of the coordination of sexual development and reproduction in trematodes and cestodes, with comparisons to invertebrates in general. Such knowledge may aid in the development of methods to frustrate egg production and thus to minimize and control trematode diseases. A specific area of interest is the study of relationships between neural and hormonal conditions needed for the attainment and maintenance of reproductive maturity. Both o f the major groups of " h o r m o n a l " regulatory substances--steroids and peptides--are almost universally distributed in living organisms. Study of 1679 0098-0331/86/0800-1679505.00/0 @ 1986 Plenum Publishing Corporation
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these materials in parasitic helminths has barely begun and has so far been limited to a scattering of reports of their discovery in one or another species. Functional investigations are almost nonexistent. Knowledge of neurotransmitters, while still insufficient, is far more advanced (Hillman, 1983). In a larger context, the classical distinctions between the nervous and endocrine systems are, as reported by Bloom (1981) "now in disarray." It has been suggested, for example by Le Roith et al. (1982), that there is an early phylogenetic precursor common to both systems so that conventionally conceived neuronal and endocrine functions are manifestations of a single primitive coordinating mechanism. As discussed by Fujita (1983), Krieger (1983), and others, different roles have been assigned to (1) neurons that function through classical neurotransmitters for very local signals; (2) paracrine cells, for shortrange transfer of chemical messages; and (3) endocrine cells, which produce chemical substances generally active at a remote site and transmitted through body fluids. Many types of intermediate cells with blended functions are now coming to light. Moreover, it is now clear that insulin and various other polypeptide hormones, generally considered as endocrine substances, may also be products of neural tissues. Both classical brain and classical gut peptides are now known to be widely produced and distributed within vertebrates, so that the distinctions of site origin are largely blurred (Roth et al., 1982; Krieger, 1983). As relatively primitive acoelomate organisms lacking a circulatory system, platyhelminths would have no endocrine functions (as defined above) but may be good candidates for study of paracrine and nerve-hormone interactions. In their excellent review, Maddrell and Nordmann (1979) provide the following partial list of neurosecretory functions in invertebrates: growth, differentiation, regeneration, body water balance, pigment production, blood sugar levels, heart rate, molting, sexual development, ovulation, and oviposition. The regulatory substances are generally peptides varying from a few amino acid residues to about 10 kd in molecular weight. In starfish, Kanatani (1983) has identified a molecule as small as l-methyl adenine as a potent regulatory hormone inducing oocyte maturation and spawning in all species so far tested. Recent years have seen an explosion of interest in neurobiology and thousands of papers have dealt with neurohormones and neurotmnsmltters. Although the parasites in general have not yet shared in this informational wealth, it seems clear that future analyses will deal with basic biological phenomena such as metamorphic phase changes (e.g., from miracidium to sporocysl or cercaria to metacercaria to adult) as well as reproductive maturation. Metamorphosis and maturation are phenomena certainly not peculiar to parasites, but an understanding of their biological and hormonal bases could have practical application through pharmacologic development of specific inhibitors. An extraordinary phylogenetic conservation of functional peptide configurations is becoming apparent so that many of the peptide hormones once
INTERNAL CHEMICAL COMMUNICATION
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thought characteristic of mammals are being found in lower organisms. For example, thyrotropin-releasing hormone in gastropods (Grimm-Jorgensen and Connolly, 1983); cholecystokinin/gastrin activity in coelenterates, ectoprocts, crustaceans, insects, and annelids (Larson and Vigna, 1983); and neurophysins, ACTH, and angiotensin in planaria (Remy, 1982). Certain "invertebrate" peptides have also been found in mammalian brain. The phylogeny of neurosecretory functions has been reviewed by Scharrer (1976) and more comprehensively by Remy (1982), who described numerous relationships between vertebrate and invertebrate neuropeptides. Insulin, the subject of concentrated study, has been found in mollusks where it serves in regulation of carbohydrate metabolism (Fritsch et al., 1976; Plisetskaya et al., 1978), and also occurs in insects and annelids (Le Roith et al., 1983). A great number of papers, which cannot be reviewed here, have described neurohormonal regulation of growth, development, molting, and reproduction in arthropods, particularly insects and crustaceans. Ecdysteroids, best known as molting hormones of arthropods, have recently been reported in schistosomes by Torpier et al. (1982) and Nirde et al. (1983, 1984), and in the cestode Moniezia expansa by Mendis et al. (1984). Interrelations between peptide and steroid hormones are seen in such processes as neurosecretory control of production and release of ecdysone by the prothoracic gland, and it is not unlikely that similar functional cascades, feedback loops, and other control mechanisms are to be found in platyhelminths. In flatworms, work has concentrated on morphological description of neurosecretory cells and granules in planarians and other turbellarians (Grasso and Quaglia, 1970a,b, 1971; MacRae, 1967; Reuter, 1981; Reuter et al., 1980). Experimentally, Sakurai (1981) has demonstrated that feeding of sexually reproducing worms to a normally agamic strain that reproduced only by fission caused the latter to develop gonads. It was proposed by Grasso and Benazzi (1973) that sexualizing factors in planaria are neurosecretory in origin, as nerve plexi containing many neurosecretory cells innervate the gonads. Grasso et al. (1975) reported that sexuality could be induced in agamic planaria by feeding isolated neurosecretory granules concentrated by density gradient centrifugation from homogenized sexual planarians. In cestodes, Davey and Breckenridge (1967) demonstrated neurosecretory cells in the scolex of Hymenolepis diminuta and described a cycle of secretion related to development of the adult and to strobila formation. In Hymenotepis microstoma, Webb (1977) showed many neurosecretory cells in the neck region by electron microscopy and followed the release of the electron-dense neurosecretory granules into the intercellular spaces from sites resembling synapses. Gustafsson and Wikgren (1981a,b) and Gustafsson et al. (1983) demonstrated numerous neurosecretory cells in Diphyllobothrium dendriticum, showing that both peptidergic and aminergic nerves occur in this species. A rapid activation
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of the peptidergic NS system takes place when the plerocercoid larvae are transferred from poikilothermic fish to homeothermic environments in vivo and in vitro. After 1 hr of cultivation, large numbers of granules were evident. Among trematodes, Ude (1962) first demonstrated neurosecretory granules in the cerebral ganglia of Dierocoelium. Gresson and Threadgold (1964) reported that the beta neurons of adult Fasciola hepatica had a NS function, and Dixon and Mercer (1965) showed neurosecretory granules in the nervous system of its cercariae. Grasso and Quaglia (1972, 1974) and Shyamasundari and Rao (1975) suggested, but did not prove, a connection between neurosecretory cells and reproductive maturation in Fasciola. A scattering of morphological studies on neurosecretory cells and neurosecretory granules has appeared for other species of trematodes: Leucochloridiomorpha constantiae (Harris and Cheng, 1972); Gastrothylax sp. (Mehrotra and Bhutia, 1979); Proalarioides tropidonotus (Kalyankar and Kankal, 1981); and Opisthodiscus diplodiscoides by Matskasi (1970), who described a clear circadian cycle in the increase and reduction of neurosecretory granules within the neurosecretory cells. Sharrna and Sharma (1981), studying Ceylonocotyle scoliocoelium, stated that 'hour current investigations indicate that the activities of the neurosecretory cells in a trematode are responsible for initiating growth in juveniles and maturation in adults." An unpublished thesis (Steele, 1971) discussed neurosecretion and development in Acanthoparyphium spinulosum. Increased numbers of neurosecretory cells appeared just before large numbers of eggs and sperm were found in the reproductive system. In the author's opinion, neurosecretory cells were responsible for gametogenesis and possible differentiation of the entire reproductive system in this trematode. In the same year that Ude first described neurosecretory cells in a trematode, G6nnert (1962) reported on the histological structure of the egg-forming region (oogentop) in Fasciola hepatica. He discussed the reproductive coordination system as follows (freely translated). The oogentop i s . . . complicated in structure. Therefore, there must be a coordinating system, whose function it is to regulate the flow and the proper sequence of individual functions. To this question there are as yet no data available . . . . In my investigations I have seen nerve cells in the region of the Mehlis' gland. Precise examination of serial sections revealed the existence of two groups of nerve cells, which are called plexus I and plexus II. Plexus I consists of usually four and at most five nerve cells which lie in the region of the ovovitelline duct and presumably coordinate the functions of the oviduct, vitelline duct, ovovitelline duct, and glands of the upper ootype and Mehlis' gland . . . . Plexus II consists of only two nerve cells. These lie close to the upper uterus. One of these is found very near the ootype valve and appears to be connected to it by a process. The second cell lies further towards the uterus. I presume that plexus II inner-
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vates the lower ootype and the ootype valve as well as the upper uterus section, thus the region of eggshell formation. Specifically regarding schistosomes, much work has been done on neuropharmacology in connection with drug development and assessment (recent reviews by Hillman, 1983; Mellin et al., 1983), but this has little direct application to understanding the neurobiology of reproduction. The influence of host hormones on schistosomes has been reviewed by Knopf (1982), but the current state of knowledge contributes little to the study of schistosome reproductive biology. Numerous studies have been carded out on the neurobiology of freshwater mollusks, particularly Lymnaea, by a large group of Dutch investigators; however, the possible involvement of regulatory peptides in mollusk-trematode interactions does not appear to have been investigated and will most likely prove to be a fertile subject for research. The complexity of studies on trematode neurohormonal regulation should not be minimized: these organisms must function effectively within molluscan and vertebrate milieux at appropriate times, interacting with and perhaps obtaining regulatory chemical cues from each of their hosts, while at the same time retaining their independent internal coordinating systems. Despite the report and review by Dei-Cas et al. (1980) with diagrams of the neural ganglia and major nerve trunks, surprisingly little information is available on schistosome neuroanatomy. The few detailed studies made with the electron microscope (Silk and Spence, 1969; Reissig, 1970; Dei-Cas et al., 1980) agree in describing a variety of electron-dense granules, some of which were considered to be neurosecretory granules by all authors. However, no function was ascribed to these intraneuronal granules and no study has been made of their association with developmental states or with components of the reproductive system. We are now studying the presence and distribution of neurosecretory cells in various life history stages of schistosomes. The association between the polypeptide neurohormones and steroid regulatory substances, well established in arthropods, is likely to be found elsewhere. As mentioned above, ecdysteroids have been found by chemical analysis in S. mansoni. Accordingly, we have investigated the distribution of immunoreactive ecdysteroids in the life history stages of S. mansoni. A rabbit antiserum to beta-ecdysone 2-hemisuccinate (Soumoff et al., 1981) kindly provided by Dr. J.D. O'Connor of UCLA, was used for primary incubation, with fluorescein-conjugated goat anti-rabbit IgG as secondary antiserum. Whole cercariae and paraffin sections of sporocysts, unisexual, paired, and cultured adults were utilized. Immunoreactivity has been identified in all stages except miracidia. In intramolluscan sporocysts, activity is limited to portions with the maturing cercariae which, when liberated, show strong immunoreactivity in the median re-
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gion and postacetabular glands. In adult males and unisexual females activity is concentrated in an assemblage of cell bodies of unknown function within the parenchyma around the intestinal ceca. Interconnections are suggestive of a neuronal network, but this has not been demonstrated. They may also represent ducts of the excretory system. In mature paired females immunoreactive nuclei are found peripherally, just beneath the tegument. An area of strong reactivity in both unisexual and adult females is located in the epithelial lining in the ootype area. Despite a few tantalizing glimpses, the intricacies of maturational regulation in platyhelminthes remain almost completely unknown. However, the gradually emerging comprehension of generalized neuronal and hormonal control mechanisms in invertebrates will sooner or later be applied to this group. The resulting understanding of trematode and cestode reproductive biology will have profound consequences in new methods of control of these important parasites of animals and man.
REFERENCES
BLOOM,F.E. 1981. Neuropeptides. Sci. Am. 245:148-168. DAVEY, K.G., and BRECKENedD6E, W.R. 1967. Neurosecretory ceils in a cestode, Hymenolepis diminuta. Science 158:931-932. DEI-CAs, E., DnAINAUT-COtrRTOIS,N., and VERNES, A. 1980. Contribution a l'6tude du systeme nerveux des forrnes adultes et larvaires de Schistosoma rnansoni Sambon, 1907 (Trematoda Digenea) I. Aspects morphologiques: anatomie, histologie et ultrastructure chez la forme adulte. Ann. Parasitol. Hum. Comp. 55:69-86. DIXON,K.E., and MERCER,E.H. 1965. The fine structure of the nervous system of the cercaria of the liver fluke, Fasciola hepatica L. Parasitology 51:967-976. FRITSCH, H.A.R., VANNOORDEN,S., and PEARSE,A.G.E. 1976. Cytochemical and immunofluorescence investigations on insulin-like producing cells in the intestine of Mytiluis edulis L. (Bivalvia). Cell Tissue Res. 165:365-369. FUJITA,T. 1983. Messenger substances of neurons and paraneurons: their chemical nature and the routes and ranges of their transport to targets. Biomed. Res. 4:239-255. G(~NNERT, R. 1962. Histologische Untersuchungen uber den Feinbau der Eibildungsstatte (Oogentop) yon Fasciola hepatica, Z. Parasitenkd. 21:475-492. GRASSO,M., and BENAZZI,M. 1973. Genetic and physiologic control of fissioning and sexuality in planarians. Embryol. Exp. Morphol. 30:317-328. GRASSO, M., and QUAGLIA,A. 1970a. Studies on neurosecretion in planarians I. Neurosecretory fibers near the testes of Dugesia lugubris. Submicroscop. Cytol. 2:119-125. GRASSO,M., and QUAGLIA,A. 1970b. Studies on neurosecretion in planarians II. Observations on the ovaries of Dugesia lugubris. Submicroscop. Cytol. 2:127-132. GRASSO,M., and QUAGLIA,A. 1971. Studies on neurosecretion in planarians III. Neurosecretory fibers near the testes and ovaries of Polycelis nigra. Submicroscop. CytoL 3:171-180. GRASSO,M., and QUAGLIA,A. 1972. Ultrastructural studies in neurosecretion and gamete ripening in platyhelminthes. Gen. Comp. Endocrinol. 18:593-594. GRASSO, M., and QUAGLIA,A. 1974. Osservazionii al microscopio elettronico sui fenomeni di neurosecrezione in Fasciola hepatica. Parassitologia 16:113-115.
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GRASSO, M., MONTANARO, L., and QUAGLIA, A. 1975. Studies on the role of neurosecretion in the induction of sexuality in a planarian agamic strain. Ultrastruct. Res. 52:404-408. GRESSON, R.A.R., and THREADGOLD, L.T. 1964. The large neurons and interstitial material of Fasciola hepatica L. Proc. R. Soc. Edinb. 68:261-266. GRIMM-JORGENSEN,Y., and CONNOLLY, S.M. 1983. Effect of thyrotropin-releasing hormone (TRH) on the synthesis and secretion of polysaccharides by the integument of gastropods. Gen. Comp. Endocrinot. 52:23-29. GUSTAFSSON, M.K.S., JUKANEN, A.E., and W1K~R~N, M.C. 1983. Activation of the peptidergic neurosecretory system in DiphyUobothrium dendriticum (Cestoda) at suboptimal temperatures. Z. Parasitenkd. 69:279-282. GUSTAFSSON, M.K.S., and WIKGREN, M.C. 1981a. Activation of the peptidergic neurosecretory system in DiphyUobothrium dendriticum (Cestoda: Pseudophyllidea). Parasitology 83:243247. GUSTAFSSON, M.K.S., and WIKGREN, M.C. 1981b. Release of neurosecretory material by protrusions of bounding membranes extending through the axelemma, in Diphyllobothrium dendriticum (Cestoda). Cell Tissue Res. 220:473-479. GUSTAFSSON, M.K.S., JUKANEN~A.E., and WIKGREN, M.C. 1983. Activation of the peptidergic neurosecretory system in Diphyllobothrium dendriticum (Cestoda) at suboptimal temperatures. Z. Parasitenkd. 69:279-282. HARRIS, K.R., and CI-IENG, T.C. 1972. Presumptive neurosecretion in Leuchochloridiomorpha constantiae (Trematoda) and its possible role in governing maturation. Int. J. Parasitol. 2:361367. HILLMAN, G.R. 1983, The neuropharmacology of schistosomes. Pharmacol. Ther. 22:103-115. KALYANKAR, S.D., and KANKAL, N.C. 1981. Neurosecretion in Proalarioides tropidonotus Vidyarthi (1937) (Trematoda: Digenea). Indian J. Parasitol. 5:117-118. KANA'rANI, H. 1983. Nature and action of the mediators inducing maturation of the starfish oocyte. pp. 159-171, in R. Porter, and J. Wheldon (eds.). Molecular Biology of Egg Maturation. Ciba Foundation Symposium 98, Pitman, London. KNOPF, P.M. 1982. The role of host hormones in controlling survival and development of Schistosoma mansoni. Pharmacol. Ther. 15:293-311. KRIEGER, D.T. 1983. Brain peptides: What, where and why? Science 222:975-985. LARSON, B.A., and VIGNA, S.R. 1983. Species and tissue distribution of cholecystokinin/gastrinlike substances in some invertebrates. Gen. Comp. Endocrinol. 50:469-475. LE ROITH, D., SCHILOACH,J., and ROT~, J. 1982. Is there an earlier phylogenetic precursor that is common to both the nervous and endocrine systems? Peptides 3:211-215. LE ROITH, D., LESNIAK,M.A., and ROTH, J. 1983. Insulin in insects and annelids. Diabetes 30:7076. MACRAE, E.K. 1967. The fine structure of the sensory receptor processes in the auricular epithelium of the planarian, Dugesia tigrina. Z. Zellforsch. 82:479-494. MADDRELL, S.H.P., and NORDMANN, J.J. 1979. Neurosecretion. Blackie & Sons, Glasgow, 173 PP. MAa'SKASl,I. 1970. On the neurosecretory cells of Opisthodiscus diplodiseoides Cohn (trematodes), and their structural changes during the day. Folia Parasitol. 17:25-30. MEHROTRA, V., and BHtJTIA, P.T. 1979. Neurosecretory ceils in Gastrothylax sp. Indian J. ParasitoL 3:5-8. MELL1N, T.N., BUSCH, R.D., WANG, C.C., and KATH, G. 1983. Neuropharmacology of the parasitic trematode, Schistosoma mansoni. Am. J. Trop. Med. Hyg. 32:83-93. MENDIS, A.H.W., REES, H.H., and GOODWIN, T.W. 1984. The occurrence of ecdysteroids in the cestode, Moniezia expansa. Mol. Biochem. Parasitol. 10:123-138. NIRDE, P., TORPIER, G., DE REGG1, M.L., and CAPRON, A. 1983. Ecdysone and 20 hydroxyecdysone: new hormones for the human parasite Schistosoma mansoni. FEBS Lett. 151:223227.
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NIRDE,P., DE REGG/, J.L., TSOUPRAS,G., TORPIER,G., FRESSANCOURT,P., and CAPRON,A. 1984. Excretion of ecdysteroids by schistosomes as a marker of parasite infection. FEBS Lett. 168:235-240. PLISESTKAYA,E., KAZAKOV,V.K., SOLTITSKAYA,L., and LEIBSON,L.G. 1978. Insulin-producing cells in the gut of freshwater bivalve mollusks Anodonta cygnea and Unio pictorum and the role of insulin in the regulation of their carbohydrate metabolism. Gen. Comp. Endocrinol. 35:133-145. REISSIG, M. 1970. Characterization of cell types in the parenchyma of Schistosoma mansoni. Parasitology 60:273-279. REMY, C. 1982. Parentes immunochimiques entre produits de neurosecretion d'invertebres et neuropeptides de Vertebres. J. Physiol. 78:514-522. REUTER, M. 1981. The nervous system of Microstomum lineare (Turbellaria, Macrostomida) II. The ultrastructure of synapses and neurosecretory release sites. Cell Tissue Res. 218:375-387. REUTER, M., WIKGREN,M., and PALMBERG,I. 1980. The nervous system of Microstomum lineare (TurbeUaria, Macrostomida) I. A fluorescence and electron microscope study. Cell Tissue Res. 211:31-40. ROTH, J., LERoITH, D., SHILOACH,J., ROSENZWEIG,J.L., LESNIAK,M.A., and HAVRANKOVA,J. 1982. The evolutionary orgins of hormones, neurotransmitters, and other extraceUular chemical messengers. N. Engl. J. Med. 306:523-527. SAKURAI,T. 1981. Sexual induction by feeding in an asexual strain of the freshwater planafian, Dugesia japonica. Annot. Zool. Jpn. 54:103-112. SCHARRER,B. 1976. Neurosecretion--comparative and evolutionary aspects. Prog. Brain Res. 45:125-137. SHARMA,P.N., and SHARMA,A.N. 1981. Cytochemical characteristics of the neurosecretory cells of Ceylonocotyle scoliocoeliurn (Trematoda: Digenea), J. HelminthoL 55:223-229. SHYAMASUNDARI,K., and RAO, K.H. 1975. The structure and cytochemistry of the neurosecretory cells of Fasciola gigantica Cobbold and Fasciola hepatica L. Z. Parasitenkd. 47:103-109. S1LK, M.H., and SPENCE, I.M. 1969. Ultrastmctural studies of the blood fluke Schistosoma mansoni III. The nerve tissue and sensory structures. S. Afr. J. Med. Sci. 34:93-104. SOUMOFF, C., HORN, D.H.S., and O'CONNOR, I.D. 1981. Production of a new antiserum to arthropod molting hormone and comparison with two other antisera. J. Steroid Biochem. 14:429431. STEELE,D.F. 1970. Neurosecretion in the life cycle of the digenetic trematode, Acanthoparyphium spinulosum Johnston, 1917. Dissertation, University of Southern California, Los Angeles, CA. TORPmR, G., HmN, M., NIRt)E, P., DE REGGI, M.L., and CAPRON,A. 1982. Detection of ecdysteroids in the human trematode, Schistosoma mansoni. Parasitology 84:123-130. UDE, J. 1962. Neurosekretorische Zellen in Cerebralganglion von Dicrocoelium lanceatum St. u. H. (Trematoda-Digenea). Zool. Ariz. 169:455-457. WEBB, R.A. 1977. Evidence for neurosecretory cells in the cestode Hymenolepis microstoma. Can. Zool. 55:1726-1733.
INTERNAL CHEMICAL COMMUNICATION WITHIN FLATWORMS
P A U L F. BASCH Department of Family, Community & Preventive Medicine Stanford University School of Medicine Stanford University Medical Center Stanford, California 94305
(Received October 1, 1985; accepted February 10, 1986) Abstract--An understanding of reproductive function is important for control of parasitic helminths. In cestodes and trematodes virtually nothing is known about regulatory and coordinating mechanisms that control maturation, gamete formation, egg production, and related processes. Neurosecretory neurons have been reported in various species but specific modes of action of neurohormones have yet to be demonstrated. The role of ecdysone is being investigated. Key Words--Schistosomes, nerve-hormone interactions, platyhelminths, differentiation reproductive maturation, Schistosoma mansoni, ecdysteroids.
In all multicellular animal groups from sponges to vertebrates, hormonal and neural processes play an important role in control and coordination of reproduction and other somatic functions. Reproduction is almost the key to the existence of trematode parasites; maturation and egg production account for much of their metabolic activity. The egg is the basis o f transmission and distribution, and in schistosomes is also the agent of pathogenesis, yet little is known about the control or reproduction in these parasites. There is need for a broad understanding of the coordination of sexual development and reproduction in trematodes and cestodes, with comparisons to invertebrates in general. Such knowledge may aid in the development of methods to frustrate egg production and thus to minimize and control trematode diseases. A specific area of interest is the study of relationships between neural and hormonal conditions needed for the attainment and maintenance of reproductive maturity. Both o f the major groups of " h o r m o n a l " regulatory substances--steroids and peptides--are almost universally distributed in living organisms. Study of 1679 0098-0331/86/0800-1679505.00/0 @ 1986 Plenum Publishing Corporation
1680
BASCH
these materials in parasitic helminths has barely begun and has so far been limited to a scattering of reports of their discovery in one or another species. Functional investigations are almost nonexistent. Knowledge of neurotransmitters, while still insufficient, is far more advanced (Hillman, 1983). In a larger context, the classical distinctions between the nervous and endocrine systems are, as reported by Bloom (1981) "now in disarray." It has been suggested, for example by Le Roith et al. (1982), that there is an early phylogenetic precursor common to both systems so that conventionally conceived neuronal and endocrine functions are manifestations of a single primitive coordinating mechanism. As discussed by Fujita (1983), Krieger (1983), and others, different roles have been assigned to (1) neurons that function through classical neurotransmitters for very local signals; (2) paracrine cells, for shortrange transfer of chemical messages; and (3) endocrine cells, which produce chemical substances generally active at a remote site and transmitted through body fluids. Many types of intermediate cells with blended functions are now coming to light. Moreover, it is now clear that insulin and various other polypeptide hormones, generally considered as endocrine substances, may also be products of neural tissues. Both classical brain and classical gut peptides are now known to be widely produced and distributed within vertebrates, so that the distinctions of site origin are largely blurred (Roth et al., 1982; Krieger, 1983). As relatively primitive acoelomate organisms lacking a circulatory system, platyhelminths would have no endocrine functions (as defined above) but may be good candidates for study of paracrine and nerve-hormone interactions. In their excellent review, Maddrell and Nordmann (1979) provide the following partial list of neurosecretory functions in invertebrates: growth, differentiation, regeneration, body water balance, pigment production, blood sugar levels, heart rate, molting, sexual development, ovulation, and oviposition. The regulatory substances are generally peptides varying from a few amino acid residues to about 10 kd in molecular weight. In starfish, Kanatani (1983) has identified a molecule as small as l-methyl adenine as a potent regulatory hormone inducing oocyte maturation and spawning in all species so far tested. Recent years have seen an explosion of interest in neurobiology and thousands of papers have dealt with neurohormones and neurotmnsmltters. Although the parasites in general have not yet shared in this informational wealth, it seems clear that future analyses will deal with basic biological phenomena such as metamorphic phase changes (e.g., from miracidium to sporocysl or cercaria to metacercaria to adult) as well as reproductive maturation. Metamorphosis and maturation are phenomena certainly not peculiar to parasites, but an understanding of their biological and hormonal bases could have practical application through pharmacologic development of specific inhibitors. An extraordinary phylogenetic conservation of functional peptide configurations is becoming apparent so that many of the peptide hormones once
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thought characteristic of mammals are being found in lower organisms. For example, thyrotropin-releasing hormone in gastropods (Grimm-Jorgensen and Connolly, 1983); cholecystokinin/gastrin activity in coelenterates, ectoprocts, crustaceans, insects, and annelids (Larson and Vigna, 1983); and neurophysins, ACTH, and angiotensin in planaria (Remy, 1982). Certain "invertebrate" peptides have also been found in mammalian brain. The phylogeny of neurosecretory functions has been reviewed by Scharrer (1976) and more comprehensively by Remy (1982), who described numerous relationships between vertebrate and invertebrate neuropeptides. Insulin, the subject of concentrated study, has been found in mollusks where it serves in regulation of carbohydrate metabolism (Fritsch et al., 1976; Plisetskaya et al., 1978), and also occurs in insects and annelids (Le Roith et al., 1983). A great number of papers, which cannot be reviewed here, have described neurohormonal regulation of growth, development, molting, and reproduction in arthropods, particularly insects and crustaceans. Ecdysteroids, best known as molting hormones of arthropods, have recently been reported in schistosomes by Torpier et al. (1982) and Nirde et al. (1983, 1984), and in the cestode Moniezia expansa by Mendis et al. (1984). Interrelations between peptide and steroid hormones are seen in such processes as neurosecretory control of production and release of ecdysone by the prothoracic gland, and it is not unlikely that similar functional cascades, feedback loops, and other control mechanisms are to be found in platyhelminths. In flatworms, work has concentrated on morphological description of neurosecretory cells and granules in planarians and other turbellarians (Grasso and Quaglia, 1970a,b, 1971; MacRae, 1967; Reuter, 1981; Reuter et al., 1980). Experimentally, Sakurai (1981) has demonstrated that feeding of sexually reproducing worms to a normally agamic strain that reproduced only by fission caused the latter to develop gonads. It was proposed by Grasso and Benazzi (1973) that sexualizing factors in planaria are neurosecretory in origin, as nerve plexi containing many neurosecretory cells innervate the gonads. Grasso et al. (1975) reported that sexuality could be induced in agamic planaria by feeding isolated neurosecretory granules concentrated by density gradient centrifugation from homogenized sexual planarians. In cestodes, Davey and Breckenridge (1967) demonstrated neurosecretory cells in the scolex of Hymenolepis diminuta and described a cycle of secretion related to development of the adult and to strobila formation. In Hymenotepis microstoma, Webb (1977) showed many neurosecretory cells in the neck region by electron microscopy and followed the release of the electron-dense neurosecretory granules into the intercellular spaces from sites resembling synapses. Gustafsson and Wikgren (1981a,b) and Gustafsson et al. (1983) demonstrated numerous neurosecretory cells in Diphyllobothrium dendriticum, showing that both peptidergic and aminergic nerves occur in this species. A rapid activation
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of the peptidergic NS system takes place when the plerocercoid larvae are transferred from poikilothermic fish to homeothermic environments in vivo and in vitro. After 1 hr of cultivation, large numbers of granules were evident. Among trematodes, Ude (1962) first demonstrated neurosecretory granules in the cerebral ganglia of Dierocoelium. Gresson and Threadgold (1964) reported that the beta neurons of adult Fasciola hepatica had a NS function, and Dixon and Mercer (1965) showed neurosecretory granules in the nervous system of its cercariae. Grasso and Quaglia (1972, 1974) and Shyamasundari and Rao (1975) suggested, but did not prove, a connection between neurosecretory cells and reproductive maturation in Fasciola. A scattering of morphological studies on neurosecretory cells and neurosecretory granules has appeared for other species of trematodes: Leucochloridiomorpha constantiae (Harris and Cheng, 1972); Gastrothylax sp. (Mehrotra and Bhutia, 1979); Proalarioides tropidonotus (Kalyankar and Kankal, 1981); and Opisthodiscus diplodiscoides by Matskasi (1970), who described a clear circadian cycle in the increase and reduction of neurosecretory granules within the neurosecretory cells. Sharrna and Sharma (1981), studying Ceylonocotyle scoliocoelium, stated that 'hour current investigations indicate that the activities of the neurosecretory cells in a trematode are responsible for initiating growth in juveniles and maturation in adults." An unpublished thesis (Steele, 1971) discussed neurosecretion and development in Acanthoparyphium spinulosum. Increased numbers of neurosecretory cells appeared just before large numbers of eggs and sperm were found in the reproductive system. In the author's opinion, neurosecretory cells were responsible for gametogenesis and possible differentiation of the entire reproductive system in this trematode. In the same year that Ude first described neurosecretory cells in a trematode, G6nnert (1962) reported on the histological structure of the egg-forming region (oogentop) in Fasciola hepatica. He discussed the reproductive coordination system as follows (freely translated). The oogentop i s . . . complicated in structure. Therefore, there must be a coordinating system, whose function it is to regulate the flow and the proper sequence of individual functions. To this question there are as yet no data available . . . . In my investigations I have seen nerve cells in the region of the Mehlis' gland. Precise examination of serial sections revealed the existence of two groups of nerve cells, which are called plexus I and plexus II. Plexus I consists of usually four and at most five nerve cells which lie in the region of the ovovitelline duct and presumably coordinate the functions of the oviduct, vitelline duct, ovovitelline duct, and glands of the upper ootype and Mehlis' gland . . . . Plexus II consists of only two nerve cells. These lie close to the upper uterus. One of these is found very near the ootype valve and appears to be connected to it by a process. The second cell lies further towards the uterus. I presume that plexus II inner-
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vates the lower ootype and the ootype valve as well as the upper uterus section, thus the region of eggshell formation. Specifically regarding schistosomes, much work has been done on neuropharmacology in connection with drug development and assessment (recent reviews by Hillman, 1983; Mellin et al., 1983), but this has little direct application to understanding the neurobiology of reproduction. The influence of host hormones on schistosomes has been reviewed by Knopf (1982), but the current state of knowledge contributes little to the study of schistosome reproductive biology. Numerous studies have been carded out on the neurobiology of freshwater mollusks, particularly Lymnaea, by a large group of Dutch investigators; however, the possible involvement of regulatory peptides in mollusk-trematode interactions does not appear to have been investigated and will most likely prove to be a fertile subject for research. The complexity of studies on trematode neurohormonal regulation should not be minimized: these organisms must function effectively within molluscan and vertebrate milieux at appropriate times, interacting with and perhaps obtaining regulatory chemical cues from each of their hosts, while at the same time retaining their independent internal coordinating systems. Despite the report and review by Dei-Cas et al. (1980) with diagrams of the neural ganglia and major nerve trunks, surprisingly little information is available on schistosome neuroanatomy. The few detailed studies made with the electron microscope (Silk and Spence, 1969; Reissig, 1970; Dei-Cas et al., 1980) agree in describing a variety of electron-dense granules, some of which were considered to be neurosecretory granules by all authors. However, no function was ascribed to these intraneuronal granules and no study has been made of their association with developmental states or with components of the reproductive system. We are now studying the presence and distribution of neurosecretory cells in various life history stages of schistosomes. The association between the polypeptide neurohormones and steroid regulatory substances, well established in arthropods, is likely to be found elsewhere. As mentioned above, ecdysteroids have been found by chemical analysis in S. mansoni. Accordingly, we have investigated the distribution of immunoreactive ecdysteroids in the life history stages of S. mansoni. A rabbit antiserum to beta-ecdysone 2-hemisuccinate (Soumoff et al., 1981) kindly provided by Dr. J.D. O'Connor of UCLA, was used for primary incubation, with fluorescein-conjugated goat anti-rabbit IgG as secondary antiserum. Whole cercariae and paraffin sections of sporocysts, unisexual, paired, and cultured adults were utilized. Immunoreactivity has been identified in all stages except miracidia. In intramolluscan sporocysts, activity is limited to portions with the maturing cercariae which, when liberated, show strong immunoreactivity in the median re-
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gion and postacetabular glands. In adult males and unisexual females activity is concentrated in an assemblage of cell bodies of unknown function within the parenchyma around the intestinal ceca. Interconnections are suggestive of a neuronal network, but this has not been demonstrated. They may also represent ducts of the excretory system. In mature paired females immunoreactive nuclei are found peripherally, just beneath the tegument. An area of strong reactivity in both unisexual and adult females is located in the epithelial lining in the ootype area. Despite a few tantalizing glimpses, the intricacies of maturational regulation in platyhelminthes remain almost completely unknown. However, the gradually emerging comprehension of generalized neuronal and hormonal control mechanisms in invertebrates will sooner or later be applied to this group. The resulting understanding of trematode and cestode reproductive biology will have profound consequences in new methods of control of these important parasites of animals and man.
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